WO2002040539A2 - Gpcr-like protein and nucleic acids encoding same - Google Patents

Abstract

Disclosed herein are nucleic acid sequences that encode G-coupled protein-receptor related polypeptides. Also disclosed are polypeptides encoded by these nucleic acid sequences, and antibodies, which immunospecifically-bind to the polypeptide, as well as derivatives, variants, mutants, or fragments of the aforementioned polypeptide, polynucleotide, or antibody. The invention further discloses therapeutic, diagnostic and research methods for diagnosis, treatment, and prevention of disorders involving any one of these novel human nucleic acids and proteins.

Description

NOVEL GPCR-LIKE PROTEIN AND NUCLEIC ACIDS ENCODING

SAME

FIELD OF THE INVENTION

The invention generally relates to novel GPCRl, GPCR2, GPCR3, GPCR4, GPCR5,

GPCR6, GPCR7, GPCR8, GPCR9, GPCR10, GPCRl 1 and GPCR12 nucleic acids and polypeptides encoded therefrom. More specifically, the invention relates to nucleic acids encoding novel polypeptides, as well as vectors, host cells, antibodies, and recombinant methods for producing these nucleic acids and polypeptides.

BACKGROUND OF THE INVENTION

The invention generally relates to nucleic acids and polypeptides. More particularly, the invention relates to nucleic acids encoding novel G-protein coupled receptor (GPCR) polypeptides, as well as vectors, host cells, antibodies, and recombinant methods for producing these nucleic acids and polypeptides.

SUMMARY OF THE INVENTION

The invention is based in part upon the discovery of nucleic acid sequences encoding novel polypeptides. The novel nucleic acids and polypeptides are referred to herein as GPCRl, GPCR2, GPCR3, GPCR4, GPCR5, GPCR6, GPCR7, GPCR8, GPCR9, GPCRl 0, GPCRl 1 and GPCRl 2 nucleic acids and polypeptides. These nucleic acids and polypeptides, as well as derivatives, homologs, analogs and fragments thereof, will hereinafter be collectively designated as "GPCRX" nucleic acid or polypeptide sequences.

In one aspect, the invention provides an isolated GPCRX nucleic acid molecule encoding a GPCRX polypeptide that includes a nucleic acid sequence that has identity to the nucleic acids disclosed in SEQ ID NOS:l, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96. In some embodiments, the GPCRX nucleic acid molecule will hybridize under stringent conditions to a nucleic acid sequence complementary to a nucleic acid molecule that includes a protein-coding sequence of a GPCRX nucleic acid sequence. The invention also includes an isolated nucleic acid that encodes a GPCRX polypeptide, or a fragment, homolog, analog or derivative thereof. For example, the nucleic acid can encode a polypeptide at least 80% identical to a polypeptide comprising the amino acid sequences of SEQ ID NOS:l, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96. The nucleic acid can be, for example, a genomic DNA fragment or a cDNA molecule that includes the nucleic acid sequence of any of SEQ ID NOS:l, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96.

Also included in the invention is an oligonucleotide, e.g., an oligonucleotide which includes at least 6 contiguous nucleotides of a GPCRX nucleic acid (e.g., SEQ ID NOS:l, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96) or a complement of said oligonucleotide. Also included in the invention are substantially purified GPCRX polypeptides (SEQ ID

NOS: 2, 4, 6, 8, 10, 12, 21, 28, 35, 37, 44, 51, 58, 60, 67, 74, 81, 83, 90 or 97). In certain embodiments, the GPCRX polypeptides include an amino acid sequence that is substantially identical to the amino acid sequence of a human GPCRX polypeptide.

The invention also features antibodies that immunoselectively bind to GPCRX polypeptides, or fragments, homologs, analogs or derivatives thereof.

In another aspect, the invention includes pharmaceutical compositions that include therapeutically- or prophylactically-effective amounts of a therapeutic and a pharmaceutically- acceptable carrier. The therapeutic can be, e.g., a GPCRX nucleic acid, a GPCRX polypeptide, or an antibody specific for a GPCRX polypeptide. In a further aspect, the invention includes, in one or more containers, a therapeutically- or prophylactically-effective amount of this pharmaceutical composition.

In a further aspect, the invention includes a method of producing a polypeptide by culturing a cell that includes a GPCRX nucleic acid, under conditions allowing for expression ofthe GPCRX polypeptide encoded by the DNA. If desired, the GPCRX polypeptide can then be recovered.

In another aspect, the invention includes a method of detecting the presence of a GPCRX polypeptide in a sample, hi the method, a sample is contacted with a compound that selectively binds to the polypeptide under conditions allowing for formation of a complex between the polypeptide and the compound. The complex is detected, if present, thereby identifying the GPCRX polypeptide within the sample.

The invention also includes methods to identify specific cell or tissue types based on their expression of a GPCRX. Also included in the invention is a method of detecting the presence of a GPCRX nucleic acid molecule in a sample by contacting the sample with a GPCRX nucleic acid probe or primer, and detecting whether the nucleic acid probe or primer bound to a GPCRX nucleic acid molecule in the sample. hi a further aspect, the invention provides a method for modulating the activity of a

GPCRX polypeptide by contacting a cell sample that includes the GPCRX polypeptide with a compound that binds to the GPCRX polypeptide in an amount sufficient to modulate the activity of said polypeptide. The compound can be, e.g., a small molecule, such as a nucleic acid, peptide, polypeptide, peptidomimetic, carbohydrate, lipid or other organic (carbon containing) or inorganic molecule, as further described herein.

Also within the scope ofthe invention is the use of a therapeutic in the manufacture of a medicament for treating or preventing disorders or syndromes including, e.g., developmental diseases; MHCII and III diseases (immune diseases); taste and scent detectability disorders; Burkitt's lymphoma; corticoneurogenic disease; signal transduction pathway disorders; metabolic pathway disorders; retinal diseases including those involving photoreception; cell growth rate disorders; cell shape disorders; metabolic disorders; feeding disorders; control of feeding; the metabolic syndrome X; wasting disorders associated with chronic diseases; obesity; potential obesity due to over-eating or metabolic disturbances; potential disorders due to starvation (lack of appetite); diabetes; noninsulin-dependent diabetes mellitus (NIDDM1); infectious disease; bacterial, fungal, protozoal and viral infections

(particularly infections caused by HIN-1 or HIN-2); pain; cancer (including but not limited to neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus cancer); cancer-associated cachexia; anorexia; bulimia; asthma; Parkinson's disease; acute heart failure; hypotension; hypertension; urinary retention; osteoporosis; Crohn's disease; multiple sclerosis; Albright Hereditary Ostoeodysfrophy; angina pectoris; myocardial infarction; ulcers; allergies; benign prostatic hypertrophy; and psychotic and neurological disorders; including anxiety; schizophrenia; manic depression; delirium; dementia; neurodegenerative disorders;

Alzheimer's disease; severe mental retardation; Dentatorubro-pallidoluysian atrophy (DRPLA); Hypophosphatemic rickets; autosomal dominant (2) Acrocallosal syndrome and dyskinesias, such as Huntington's disease or Gilles de la Tourette syndrome; immune disorders; Adrenoleukodystrophy; Congenital Adrenal Hyperplasia; Hemophilia; Hypercoagulation; Idiopathic thrombocytopenic purpura; autoimmume disease; immunodeficiencies; transplantation; Non Hippel-Lindau (NHL) syndrome; Stroke; Tuberous sclerosis; hypercalceimia; Cerebral palsy; Epilepsy; Lesch-Nyhan syndrome; Ataxia- telangiectasia; Leukodysfrophies; Behavioral disorders; Addiction; Neuroprotection; Cirrhosis; Transplantation; Systemic lupus erythematosus; Emphysema; Scleroderma; ARDS; Renal artery stenosis; Interstitial nephritis; Glomerulonephritis; Polycystic kidney disease; Systemic lupus erythematosus; Renal tubular acidosis; IgA nephropathy; Cardiomyopathy; Atherosclerosis; Congenital heart defects; Aortic stenosis ; Atrial septal defect (ASD); Atrioventricular (A-V) canal defect; Ductus arteriosus; Pulmonary stenosis ; Subaortic stenosis; Ventricular septal defect (VSD); valve diseases; Scleroderma; fertility; Pancreatitis; Endocrine dysfunctions; Growth and reproductive disorders; Inflammatory bowel disease; Diverticular disease; Leukodysfrophies; Graft vesus host; Hyperthyroidism; Endometriosis; hematopoietic disorders and/or other pathologies and disorders ofthe like. The therapeutic can be, e.g., a GPCRX nucleic acid, a GPCRX polypeptide, or a GPCRX-specific antibody, or biologically-active derivatives or fragments thereof.

For example, the compositions ofthe present invention will have efficacy for treatment of patients suffering from the diseases and disorders listed above and/or other pathologies and disorders.

The polypeptides can be used as immunogens to produce antibodies specific for the invention, and as vaccines. They can also be used to screen for potential agonist and antagonist compounds. For example, a cDNA encoding GPCRX may be useful in gene therapy, and GPCRX may be useful when administered to a subject in need thereof. By way of nonlimiting example, the compositions ofthe present invention will have efficacy for treatment of patients suffering the diseases and disorders listed above and/or other pathologies and disorders.

The invention further includes a method for screening for a modulator of disorders or syndromes including, e.g., diseases and disorders listed above and/or other pathologies and disorders and those disorders related to cell signal processing and metabolic pathway modulation. The method includes contacting a test compound with a GPCRX polypeptide and determining if the test compound binds to said GPCRX polypeptide. Binding ofthe test compound to the GPCRX polypeptide indicates the test compound is a modulator of activity, or of latency or predisposition to the aforementioned disorders or syndromes.

Also within the scope ofthe invention is a method for screening for a modulator of activity, or of latency or predisposition to an disorders or syndromes including the diseases and disorders listed above and/or other pathologies and disorders or other disorders related to cell signal processing and metabolic pathway modulation by administering a test compound to a test animal at increased risk for the aforementioned disorders or syndromes. The test animal expresses a recombinant polypeptide encoded by a GPCRX nucleic acid. Expression or activity of GPCRX polypeptide is then measured in the test animal, as is expression or activity ofthe protein in a control animal which recombinantly-expresses GPCRX polypeptide and is not at increased risk for the disorder or syndrome. Next, the expression of GPCRX polypeptide in both the test animal and the control animal is compared. A change in the activity of GPCRX polypeptide in the test animal relative to the control animal indicates the test compound is a modulator of latency ofthe disorder or syndrome. In yet another aspect, the invention includes a method for determining the presence of or predisposition to a disease associated with altered levels of a GPCRX polypeptide, a GPCRX nucleic acid, or both, in a subject (e.g., a human subject). The method includes measuring the amount ofthe GPCRX polypeptide in a test sample from the subject and comparing the amount ofthe polypeptide in the test sample to the amount ofthe GPCRX polypeptide present in a control sample. An alteration in the level ofthe GPCRX polypeptide in the test sample as compared to the control sample indicates the presence of or predisposition to a disease in the subject. Preferably, the predisposition includes diseases and disorders listed above and/or other pathologies and disorders. Also, the expression levels ofthe new polypeptides ofthe invention can be used in a method to screen for various cancers as well as to determine the stage of cancers.

In a further aspect, the invention includes a method of treating or preventing a pathological condition associated with a disorder in a mammal by administering to the subject a GPCRX polypeptide, a GPCRX nucleic acid, or a GPCRX-specific antibody to a subject (e.g., a human subject), in an amount sufficient to alleviate or prevent the pathological condition. In preferred embodiments, the disorder, includes the diseases and disorders listed above and/or other pathologies and disorders.

In yet another aspect, the invention can be used in a method to identity the cellular receptors and downstream effectors ofthe invention by any one of a number of techniques commonly employed in the art. These include but are not limited to the two-hybrid system, affinity purification, co-precipitation with antibodies or other specific-interacting molecules. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing ofthe present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Other features and advantages ofthe invention will be apparent from the following detailed description and claims.

DETAILED DESCRIPTION OF THE INVENTION

The invention is based, in part, upon the discovery of novel nucleic acid sequences that encode novel polypeptides. The novel nucleic acids and their encoded polypeptides are referred to individually as GPCRl, GPCR2, GPCR3, GPCR4, GPCR5, GPCR6, GPCR7, GPCR8, GPCR9, GPCRl 0, GPCRl 1 and GPCR12. The nucleic acids, and their encoded polypeptides, are collectively designated herein as "GPCRX".

The novel GPCRX nucleic acids ofthe invention include the GPCRl, GPCR2, GPCR3, GPCR4, GPCR5, GPCR6, GPCR7, GPCR8, GPCR9, GPCRl 0, GPCRl 1 and GPCRl 2 nucleic acids, or a fragment, derivative, analog or homolog thereof. The novel GPCRX proteins ofthe invention include the GPCRl, GPCR2, GPCR3, GPCR4, GPCR5, GPCR6, GPCR7, GPCR8, GPCR9, GPCR10, GPCRl 1 and GPCR12 proteins, or a , derivative, analog or homolog thereof. The individual GPCRX nucleic acids and proteins are described below. Within the scope of this invention is a method of using these nucleic acids and peptides in the treatment or prevention of a disorder related to cell signaling or metabolic pathway modulation.

The GPCRX proteins ofthe invention have a high homology to the 7tm_l domain (PFam Ace. No. pfamOOOOl). The 7tm_l domain is from the 7 transmembrane receptor family, which includes a number of different proteins, including, for example, serotonin receptors, dopamine receptors, histamine receptors, andrenergic receptors, cannabinoid receptors, angiotensin II receptors, chemokine receptors, opioid receptors, G-protein coupled receptor (GPCR) proteins, olfactory receptors (OR), and the like. Some proteins and the Protein Data Base Ids/gene indexes include, for example: 5-hydroxytryptamine receptors (See, e.g., PMIM 112821, 8488960, 112805, 231454, 1168221, 398971, 112806); rhodopsin (129209); G protein-coupled receptors (119130, 543823, 1730143, 132206, 137159, 6136153, 416926, 1169881, 136882, 134079); gustatory receptors (544463, 462208); c-x-c chemokine receptors (416718, 128999, 416802, 548703, 1352335); opsins (129193, 129197, 129203); and olfactory receptor-like proteins (129091, 1171893, 400672, 548417).

Because ofthe close homology among the members ofthe GPCRX family, proteins that are homologous to any one member ofthe family are also largely homologous to the other members, except where the sequences are different as shown below.

The similarity information for the GPCRX proteins and nucleic acids disclosed herein suggest that GPCRl -GPCR12 may have important structural and/or physiological functions characteristic ofthe Olfactory Receptor family and the GPCR family. Therefore, the nucleic acids and proteins ofthe invention are useful in potential diagnostic and therapeutic applications and as a research tool. These include serving as a specific or selective nucleic acid or protein diagnostic and/or prognostic marker, wherein the presence or amount ofthe nucleic acid or the protein are to be assessed, as well as potential therapeutic applications such as the following: (i) a protein therapeutic, (ii) a small molecule drug target, (iii) an antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), (iv) a nucleic acid useful in gene therapy (gene delivery/gene ablation), and (v) a composition promoting tissue regeneration in vitro and in vivo (vi) biological defense weapon.

G-Protein Coupled Receptor proteins ("GPCRs") have been identified as a large family of G protein-coupled receptors in a number of species. These receptors share a seven transmembrane domain structure with many neurotransmitter and hormone receptors, and are likely to underlie the recognition and G-protein-mediated fransduction of various signals.

Human GPCR generally do not contain introns and belong to four different gene subfamilies, displaying great sequence variability. These genes are dominantly expressed in olfactory epithelium. See, e.g., Ben-Arie et al., Hum. Mol. Genet. 1994 3:229-235; and, Online Mendelian Inheritance in Man ("OMIM") entry # 164342 (http://www.ncbi.nlm.nih.gov/ entrez/ dispomim.cgi?).

The olfactory receptor ("OR") gene family constitutes one ofthe largest GPCR multigene families and is distributed among many chromosomal sites in the human genome. See Rouquier et al., Hum. Mol. Genet. 7(9):1337-45 (1998); Malnic et al., Cell 96:713-23 (1999). Olfactory receptors constitute the largest family among G protein-coupled receptors, with up to 1000 members expected. See Nanderhaeghen et al., Genomics 39(3):239-46 (1997); Xie et al., Mamm. Genome 11 (12): 1070-78 (2000); Issel-Tarver et al., Proc. Natl. Acad. Sci. USA 93(20): 10897-902 (1996). The recognition of odorants by olfactory receptors is the first stage in odor discrimination. See Krautwurst et al., Cell 95(7):917-26 (1998); Buck et al., Cell 65(1): 175-87 (1991). Many ORs share some characteristic sequence motifs and have a central variable region corresponding to a putative ligand binding site. See Issel-Tarver et al, Proc. Natl. Acad. Sci. USA 93:10897-902 (1996).

Other examples of seven membrane spanning proteins that are related to GPCRs are chemoreceptors. See Thomas et al., Gene 178(1-2): 1-5 (1996). Chemoreceptors have been identified in taste, olfactory, and male reproductive tissues. See id.; Walensky et al., J. Biol.

Chem. 273(16):9378-87 (1998); Parmentier et al., Nature 355(6359):453-55 (1992); Asai et al., Biochem. Biophys. Res. Commun. 221(2):240-47 (1996).

The GPCRX nucleic acids ofthe invention encoding GPCR-like proteins include the nucleic acids whose sequences are provided herein, or fragments thereof. The invention also includes mutant or variant nucleic acids any of whose bases may be changed from the corresponding base shown herein while still encoding a protein that maintains its GPCR-like activities and physiological functions, or a fragment of such a nucleic acid. The invention further includes nucleic acids whose sequences are complementary to those just described, including nucleic acid fragments that are complementary to any ofthe nucleic acids just described. The invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications. Such modifications include, byway of nonlimiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability ofthe modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject.

The GPCRX proteins ofthe invention include the GPCR-like proteins whose sequences are provided herein. The invention also includes mutant or variant proteins any of whose residues may be changed from the corresponding residue shown herein while still encoding a protein that maintains its GPCR-like activities and physiological functions, or a functional fragment thereof. The invention further encompasses antibodies and antibody fragments, such as F_ab or (F_ab)₂, that bind immunospecifically to any ofthe proteins ofthe invention.

The GPCRX nucleic acids and proteins are useful in potential therapeutic applications implicated in various GPCR-related pathological disorders and/or OR-related pathological disorders, described further below. For example, a cDNA encoding the GPCR (or olfactory- receptor) like protein may be useful in gene therapy, and the receptor -like protein may be useful when administered to a subject in need thereof. The nucleic acids and proteins ofthe invention are also useful in potential therapeutic applications used in the treatment of developmental diseases; MHCII and III diseases (immune diseases); taste and scent detectability disorders; Burkitt's lymphoma; corticoneurogenic disease; signal fransduction pathway disorders; metabolic pathway disorders; retinal diseases including those involving photoreception; cell growth rate disorders; cell shape disorders; metabolic disorders; feeding disorders; control of feeding; the metabolic syndrome X; wasting disorders associated with chronic diseases; obesity; potential obesity due to over-eating or metabolic disturbances; potential disorders due to starvation (lack of appetite); diabetes; noninsulin-dependent diabetes mellitus (NIDDM1); infectious disease; bacterial, fungal, protozoal and viral infections (particularly infections caused by HIN-1 or HIN-2); pain; cancer (including but not limited to neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus cancer); cancer-associated cachexia; anorexia; bulimia; asthma; Parkinson's disease; acute heart failure; hypotension; hypertension; urinary retention; osteoporosis; Crohn's disease; multiple sclerosis; Albright Hereditary Ostoeodysfrophy; angina pectoris; myocardial infarction; ulcers; allergies; benign prostatic hypertrophy; and psychotic and neurological disorders; including anxiety; schizophrenia; manic depression; delirium; dementia; neurodegenerative disorders; Alzheimer's disease; severe mental retardation; Dentatorubro-pallidoluysian atrophy (DRPLA); Hypophosphatemic rickets; autosomal dominant (2) Acrocallosal syndrome and dyskinesias, such as Huntington's disease or Gilles de la Tourette syndrome; immune disorders; Adrenoleukodystrophy; Congenital Adrenal Hyperplasia; Hemophilia; Hypercoagulation; Idiopathic thrombocytopenic purpura; autoimmume disease; immunodeficiencies; transplantation; Non Hippel-Lindau (NHL) syndrome; Stroke; Tuberous sclerosis; hypercalceimia; Cerebral palsy; Epilepsy; Lesch-Νyhan syndrome; Ataxia- telangiectasia; Leukodysfrophies; Behavioral disorders; Addiction; Νeuroprotection; Cirrhosis; Transplantation; Systemic lupus erythematosus; Emphysema; Scleroderma; ARDS; Renal artery stenosis; Interstitial nephritis; Glomerulonephritis; Polycystic kidney disease; Systemic lupus erythematosus; Renal tubular acidosis; IgA nephropathy; Cardiomyopathy; Atherosclerosis; Congenital heart defects; Aortic stenosis ; Atrial septal defect (ASD); Atrioventricular (A-V) canal defect; Ductus arteriosus; Pulmonary stenosis ; Subaortic stenosis; Nentricular septal defect (NSD); valve diseases; Scleroderma; fertility; Pancreatitis; Endocrine dysfunctions; Growth and reproductive disorders; Infl--mmatory bowel disease; Diverticular disease; Leukodysfrophies; Graft vesus host; Hyperthyroidism; Endometriosis; hematopoietic disorders and/or other pathologies and disorders. Other GPCR-related diseases and disorders are contemplated.

The polypeptides can be used as immunogens to produce antibodies specific for the invention, and as vaccines. They can also be used to screen for potential agonist and antagonist compounds. For example, a cDNA encoding the GPCR-like protein may be useful in gene therapy, and the GPCR-like protein may be useful when administered to a subject in need thereof. By way of nonlimiting example, the anti-GPCRX antibody compositions ofthe present invention will have efficacy for treatment of patients suffering from the diseases and disorders listed above, as well as other related or associated pathologies. The novel nucleic acid encoding GPCR-like protein, and the GPCR-like protein ofthe invention, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed. These materials are further useful in the generation of antibodies that bind immunospecifically to the novel substances ofthe invention for use in therapeutic or diagnostic methods.

GPCRl

A GPCR-like protein ofthe invention, referred to herein as GPCRl, is an Olfactory Receptor ("OR")-like protein. Some members ofthe Olfactory Receptor-Like Protein Family end up localized at the cell surface, where they exhibit activity. Therefore it is likely that these novel GPCRl proteins are available at the appropriate sub-cellular localization and hence accessible for the therapeutic uses described in this application.

Six alternative novel GPCRl nucleic acids and encoded polypeptides are provided, namely GPCRla, GPCRlb, GPCRlc, GPCRld, GPCRle and GPCRlf. The GPCRl proteins are predicted to be a likely Type Illb membrane protein.

GPCRla In one embodiment, the disclosed GPCRl variant is the novel GPCRl a (alternatively referred to as GMAC073079_A), which includes the 964 nucleotide sequence (SEQ ID NO:l) shown in Table 1A. The disclosed GPCRla open reading frame ("ORF") begins at an ATG initiation codon at nucleotides 19-21 and terminates at a TGA codon at nucleotides 958-960. Putative untranslated regions upstream from the initiation codon and downstream from the termination codon are underlined in Table 1 A, and the start and stop codons are in bold letters. Table 1A. GPCRl nucleotide sequence (SEQ ID NO:l).

GGCCCCATACTGTGGATC GCAAATCTGAGCCAGCCCTCCGAATTTGTCCTCTTGGGCTTCTCCTCC TTTGGTGAGCTGCAGGCCCTTCTGTATGGCCCCTTCCTCATGCTTTATCTTCTCGCCTTCATGGGAAAC ACCATCATCATAGTTATGGTCATAGCTGACACCCACCTACATACACCCATGTACTTCTTCCTGGGCAAT TTTTCCCTGCTGGAGATCTTGGTAACCATGACTGCAGTGCCCAGGATGCTCTCAGACCTGTTGGTCCCC CACAAAGTCATTACCTTCACTGGCTGCATGGTCCAGTTCTACTTCCACTTTTCCCTGGGGTCCACCTCC TTCCTCATCCTGACAGACATGGCCCTTGATCGCTTTGTGGCCATCTGCCACCCACTGCGCTATGGCACT CTGATGAGCCGGGCTATGTGTGTCCAGCTGGCTGGGGCTGCCTGGGCAGCTCCTTTCCTAGCCATGGTA CCCACTGTCCTCTCCCGAGCTCATCTTGATTACTGCCATGGCGACGTCATCAACCACTTCTTCTGTGAC AATGAACCTCTCCTGCAGTTGTCATGCTCTGACACTCGCCTGTTGGAATTCTGGGACTTTCTGATGGCC TTGACCTTTGTCCTCAGCTCCTTCCTGGTGACCCTCATCTCCTATGGCTACATAGTGACCACTGTGCTG CGGATCCCCTCTGCCAGCAGCTGCCAGAAGGCTTTCTCCACTTGCGGGTCTCACCTCACACTGGTCTTC ATCGGCTACAGTAGTACCATCTTTCTGTATGTCAGGCCTGGCAAAGCTCACTCTGTGCAAGTCAGGAAG GTCGTGGCCTTGGTGACTTCAGTTCTCACCCCCTTTCTCAATCCCTTTATCCTTACCTTCTGCAATCAG ACAGTTAAAACAGTGCTACAGGGGCAGATGCAGAGGCTGAAAGGCCTTTGCAAGGCACAATGATGAG

In the present invention, the GPCRla target sequence was subjected to the exon linking process to confirm the sequence. These procedures provide the sequences reported below, which are designated GPCRlb (also refered to as AC073079_dal), GPCR2 (also refered to as AC073079_da2), and GPCRlc (also refered to as AC073079_da3).

The sequence of GPCRla was derived by laboratory cloning of cDNA fragments, by in silico prediction ofthe sequence. The cDNA fragments covering either the full length ofthe DNA sequence, or part ofthe sequence, or both, were cloned. In silico prediction was based on sequences available in CuraGen's proprietary sequence databases or in the public human sequence databases, and provided either the full length DNA sequence, or some portion thereof.

The disclosed GPCRl of this invention maps to chromosome 1. Chromosome localization information was assigned using OMIM, the electronic northern bioinfo-rmatic tool implemented by CuraGen Corporation, public ESTs, public literature references and/or genomic clone homologies. This was executed to derive the chromosomal mapping ofthe

SeqCalling assemblies, Genomic clones, literature references and/or EST sequences that were included in the invention.

The disclosed GPCRla polypeptide (SEQ ID NO:2) encoded by SEQ ID NO:l has 313 amino acid residues, has a molecular weight of 34900.65 Daltons, and is presented in Table IB using the one-letter amino acid code. The Signal P, Psort and/or Hydropathy results predict that GPCRla has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6000. In alternative embodiments, the GPCRla protein is localized to the Golgi body with a certainty of 0.4000, the endoplasmic reticulum (membrane) with a certainty of 0.3000, or the microbody (peroxisome) with a certainty of 0.3000. The most likely cleavage site for a GPCRla peptide is between amino acids 47 and 48, i.e., at the dash in the sequence VIA-DT.

Table IB. Encoded GPCRl protein sequence (SEQ ID NO:2).

MANLS_QPSEFVLLGFSSFGELQALLYGPFLMLYLLAFMGNTIIIVMVIADTHLHTP YFFLGNFSLLEI LVTMTAVPRMLSDLLVPHKVITFTGC VQFYFHFSLGSTSFLILTDMALDRFVAICHPLRYGTLMSRAM CVQLAGAAWAAPFLAMVPTVLSRAHLDYCHGDVINHFFCDNEPLLQLSCSDTRLLEF DFLMALTFVLS SFLVTLISYGYIVTTVLRIPSASSCQKAFSTCGSHLTLVFIGYSSTIFLYVRPGKAHSVQVRKVVALVT SVLTPFLNPFILTFCNQTVKTVLQGQMQRLKGLCKAQ

GPCRlb

In one embodiment, the disclosed GPCRl variant is the novel GPCRlb (alternatively referred to as AC073079_dal), which includes the 971 nucleotide sequence (SEQ ID NO:3) shown in Table IC. The disclosed GPCRlb open reading frame ("ORF") begins at an ATG initiation codon at nucleotides 30-32 and tenninates at a TGA codon at nucleotides 963-965. Putative untranslated regions upstream from the initiation codon and downstream from the termination codon are underlined in Table IC, and the start and stop codons are in bold letters.

Table IC. GPCRlb nucleotide sequence (SEQ ID NO:3).

CCCCATACTGTGGATCATGGCAAGGCACA ATGAGCCAGCCCTCCGAATTTGTCCTCTTGGGCTTCTCCT CCTTTGGTGAGCTGCAGGCCCTTCTGTATGGCCCCTTCCTCATGCTTTATCTTCTCGCCTTCATGGGAAACA CCATCATCATAGTTATGGTCATAGCTGACACCCACCTACATACACCCATGTACTTCTTCCTGGGCAATTTTT CCCTGCTGGAGATCTTGGTAACCATGACTGCAGTGCCCAGGATGCTCTCAGACCTGTTGGTCCCCCACAAAG TCATTACCTTCACTGGCTGCATGGTCCAGTTCTACTTCCACTTTTCCCTGGGGTCCACCTCCTTCCTCATCC TGACAGACATGGCCCTTGATCGCTTTGTGGCCATCTGCCACCCACTGCGCTATGGCACTCTGATGAGCCGGG CTATGTGTGTCCAGCTGGCTGGGGCTGCCTGGGCAGCTCCTTTCCTAGCCATGGTACCCACTGTCCTCTCCC GAGCTCATCTTGATTACTGCCATGGCGACGTCATCAACCACTTCTTCTGTGACAATGAACCTCTCCTGCAGT TGTCATGCTCTGACACTCGCCTGTTGGAATTCTGGGACTTTCTGATGGCCATGACCTTTGTCCTCAGCTCCT TCCTGGTGACCCTCATCTCATATGGCTACATAGTGACCACTGTGCTGCGGATCCCCTCTGCCAGCAGCTGCC AGAAGGCTTTCTCCACTTGCGGGTCTCACCTCACACTGGTCTTCATCGGCTACAGTAGTACCATCTTTCTGT ATGTCAGGCCTGGCAAAGCTCACTCTGTGCAAGTCAGGAAGGTCGTGGCCTTGGTGACTTCAGTTCTCACCC CCTTTCTCAATCCCTTTATCCTTACCTTCTGCAATCAGACAGTTAAAACAGTGCTACAGGGGCAGATGCAGA GGCTGAAAGGCCTTTGCAAGGCACAATGATGAGCC

The disclosed GPCRlb polypeptide (SEQ ID NO:4) encoded by SEQ ID NO:3 has 311 amino acid residues, a molecular weight of 34751.56 Daltons, and is presented in Table ID using the one-letter amino acid code. The Signal P, Psort and/or Hydropathy results predict that GPCRlb has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6000. In alternative embodiments, the GPCRlb protein is localized to the Golgi body with a certainty of 0.4000, the endoplasmic reticulum (membrane) with a certainty of 0.3000, or the microbody (peroxisome) with a certainty of 0.3000. The most likely cleavage site for a GPCRlb peptide is between amino acids 47 and 48, i.e., at the dash in the sequence NIA-DT.

Table ID. Encoded GPCRlb protein sequence (SEQ ID ΝO:4).

MMSQPSEFVLLGFSSFGELQALLYGPFLMLYLLAFMGNTIIIVMVIADTHLHTPMYFFLGNFSLLEILV TMTAVPRMLSDLLVPHKVITFTGCMVQFYFHFSLGSTSFLILTDMALDRFVAICHPLRYGTLMSRAMCV QLAGAAW-AAPFLAMVPTVLSRAHLDYCHGDVINHFFCDNEPLLQLSCSDTRLLEFWDFLM/-MTFVLSSF LVTLISYGYIVTTVLRIPSASSCQKAFSTCGSHLTLVFIGYSSTIFLYVRPGKAHSVQVRKVVALVTSV LTPFLNPFILTFCNQTVKTVLQGQMQRLKGLCKAQ

In a search of sequence databases, it was found, for example, that the GPCRlb nucleic acid sequence of this invention has 543 of 882 bases (61%) identical to a gb.-GENBANK- ID:AF102523| acc:AF102523.1 mRNA from Mus musculus (Mus musculus olfactory receptor C6 gene, complete eds). The full amino acid sequence ofthe protein ofthe invention was found to have 137 of 301 amino acid residues (45%) identical to, and 199 of 301 amino acid residues (66%) similar to, the 313 amino acid residue ptnr:SPTREMBL-ACC:Q9ZlN0 protein from Mus musculus (Mouse) (OLFACTORY RECEPTOR C6).

GPCRlc

In one embodiment, the disclosed GPCRl variant is the novel GPCRlc (alternatively referred to as AC073079_da3), which includes the 971 nucleotide sequence (SEQ ID ΝO:5) shown in Table IE. The disclosed GPCRlc open reading frame ("ORF") begins at an ATG initiation codon at nucleotides 30-32 and terminates at a TGA codon at nucleotides 963-965. Putative untranslated regions upstream from the initiation codon and downstream from the termination codon are underlined in Table IE, and the start and stop codons are in bold letters.

Table IE. GPCRlc nucleotide sequence (SEQ ID NO:5).

CCCCATACTGTGGATCATGGCAAGGCACA ATGAGCCAGCCCTCCGAATTTGTCCTCTTGGGCTTCTCCT CCTTTGGTGAGCTGCAGGCCCTTCTGTATGGCCCCTTCCTCATGCTTTATCTTCTCGCCTTCATGGGAAACA CCATCATCATAGTTATGGTCATAGCTGACACCCACCTACATACACCCATGTACTTCTTCCTGGGCAATTTTT CCCTGCTGGAGATCTTGGTAACCATGACTGCAGTGCCCAGGATGCTCTCAGACCTGTTGGTCCCCCACAAAG TCATTACCTTCACTGGCTGCATGGTCCAGTTCTACTTCCACTTTTCCCTGGGGTCCACCTCCTTCCTCATCC TGACAGACATGGCCCTTGATCGCTTTGTGGCCATCTGCCACCCACTGCGCTATGGCACTCTGATGAGCCGGG CTATGTGTGTCCAGCTGGCTGGGGCTGCCTGGGCAGCTCCTTTCCTAGCCATGGTACCCACTGTCCTCTCCC GAGCTCATCTTGATTACTGCCATGGCGACGTCATCAACCACTTCTTCTGTGACAATGAACCTCTCCTGCAGT TGTCATGCTCTGACACTCGCCTGTTGGAATTCTGGGACTTTCTGATGGCCTTGACCTTTGTCCTCAGCTCCT TCCTGGTGACCCTCATCTCCTATGGCTACATAGTGACCACTGTGCTGCGGATCCCCTCTGCCAGCAGCTGCC AGAAGGCTTTCTCCACTTGCGGGTCTCACCTCACACTGGTCTTCATCGGCTACAGTAGTACCATCTTTCTGT ATGTCAGGCCTGGCAAAGCTCACTCTGTGCAAGTCAGGAAGGTCGTGGCCTTGGTGACTTCAGTTCTCACCC CCTTTCTCAATCCCTTTATCCTTACCTTCTGCAATCAGACAGTTAAAACAGTGCTACAGGGGCAGATGCAGA GGCTGAAAGGCCTTTGCAAGGCACAA-CGATGAGCC

The disclosed GPCRlc polypeptide (SEQ ID NO:6) encoded by SEQ ID NO:5 has 311 amino acid residues, a molecular weight of 34733.52 Daltons, and is presented in Table IF using the one-letter amino acid code. The Signal P, Psort and/or Hydropathy results predict that GPCRlc has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6000. In alternative embodiments, the GPCRlc protein is localized to the Golgi body with a certainty of 0.4000, the endoplasmic reticulum (membrane) with a certainty of 0.3000, or the microbody (peroxisome) with a certainty of 0.3000. The most likely cleavage site for a GPCRlc peptide is between amino acids 47 and 48, i.e., at the dash in the sequence VIA-DT.

Table IF. Encoded GPCRlc protein sequence (SEQ ID NO:6).

MMSQPSEFVL GFSSFGELQALLYGPFLMLYLLAFMGNTIIIVMVIADTHLHTPMYFFLGNFSLLEILV TMTAVPRMLSDLLVPHKVITFTGCMVQFYFHFSLGSTSFLILTDMALDRFVAICHPLRYGTLMSRAMCV Q AGAAW7-APFLA VPTVLSRAHLDYCHGDVINHFFCDNEPLLQLSCSDTRLLEFWDFLMALTFVLSSF LVTLISYGYIVTTVLRIPSASSCQKAFSTCGSHLTLVFIGYSSTIFLYVRPGKAHSVQVRKVVALVTSV LTPFLNPFILTFCNQTVKTVLQGQMQRLKGLCKAQ

In a search of sequence databases, it was found, for example, that the GPCRlc nucleic acid sequence of this invention has 589 of 932 bases (63%) identical to a gb:GENBANK- ID:AF101760| acc.AFl 01760.1 mRNA from Gorilla gorilla (Gorilla gorilla isolate PPOR1E2 olfactory receptor gene, complete eds). The full amino acid sequence ofthe protein ofthe invention was found to have 137 of 301 amino acid residues (45%) identical to, and 199 of 301 amino acid residues (66%) similar to, the 313 amino acid residue ptnr:SPTREMBL- ACC:Q9Z1V0 protein from Mus musculus (Mouse) (OLFACTORY RECEPTOR C6). GPCRld

In one embodiment, the disclosed GPCRl variant is the novel GPCRld (alternatively referred to as BA113A10_B_dal), which includes the 992 nucleotide sequence (SEQ ID NO:7) shown in Table 1G. The disclosed GPCRld open reading frame ("ORF") begins at an GTC codon at nucleotides 3-5 and terminates at a TGA codon at nucleotides 987-989. Putative untranslated region downstream from the termination codon is underlined in Table 1G, and the initial and stop codons are in bold letters. Table 1G. GPCRl nucleotide sequence (SEQ ID NO:7).

CTGTCTTTTGTTTCTCTTGCATGCAGGGCCCCATACTGTGGATCATGGCAAATCTGAGCCAGCCCTCCGAAT TTGTCCTCTTGGGCTTCTCCTCCTTTGGTGAGCTGCAGGCCCTTCTGTATGGCCCCTTCCTCATGCTTTATC TTCTCGCCTTCATGGGAAACACCATCATCATAGTTATGGTCATAGCTGACACCCACCTACATACACCCATGT ACTTCTTCCTGGGCAATTTTTCCCTGCTGGAGATCTTGGTAACCATGACTGCAGTGCCCAGGATGCTCTCAG ACCTGTTGGTCCCCCACAAAGTCATTACCTTCACTGGCTGCATGGTCCAGTTCTACTTCCACTTTTCCCTGG GGTCCACCTCCTTCCTCATCCTGACAGACATGGCCCTTGATCGCTTTGTGGCCATCTGCCACCCACTGCGCT ATGGCACTCTGATGAGCCGGGCTATGTGTGTCCAGCTGGCTGGGGCTGCCTGGGGAGCTCCTTTCCTAGCCA TGGTACCCACTGTCCTCTCCCGAGCTCATCTTGATTACTGCCATGGCGACGTCATCAACCACTTCTTCTGTG ACAATGAACCTCTCCTGCAGTTGTCATGCTCTGACACTCGCCTGTTGGAATTCTGGGACTTTCTGATGGCCT TGACCTTTGTCCTCAGCTCCTTCCTGGTGACCCTCATCTCCTATGGCTACATAGTGACCACTGTGCTGCGGA TCCCCTCTGCCAGCAGCTGCCAGAAGGCTTTCTCCACTTGCGGGTCTCACCTCACACTGGTCTTCATCGGCT ACAGTAGTACCATCTTTCTGTATGTCAGGCCTGGCAAAGCTCACTCTGTGCAAGTCAGGAAGGTCGTGGCCT TGGTGACTTCAGTTCTCACCCCCTTTCTCAATCCCTTTATCCTTACCTTCTGCAATCAGACAGTTAAAACAG TGCTACAGGGGCAGATGCAGAGGCTGAAAGGCCTTTGCAAGGCACAATGATGAGCC

hi certain embodiments, the open reading frame ofthe disclosed GPCRld nucleic acid is an incomplete cDNA fragment, and it is contemplated that the ORF extends upstream (i.e., in the 5' direction) ofthe sequence provided in SEQ ID NO:7. It is further contemplated that a complete ORF would include an in-frame ATG codon as the start codon.

The disclosed GPCRld polypeptide (SEQ ID NO: 8) encoded by SEQ ID NO:7 has 327 amino acid residues, a molecular weight of 36526.60 Daltons, and is presented in Table IH using the one-letter amino acid code. The Signal P, Psort and/or Hydropathy results predict that GPCRld has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6400. In alternative embodiments, the GPCRld protein is localized to the Golgi body with a certainty of 0.4600, the endoplasmic reticulum (membrane) with a certainty of 0.3700, or the microbody (peroxisome) with a certainty of 0.1000. The most likely cleavage site for a GPCRld peptide is between amino acids 63 and 64, i.e., at the dash in the sequence VIA-DT.

Table IH. Encoded GPCRld protein sequence (SEQ ID NO:8).

VFCFSCMQGPILWIMANLSQPSEFVLLGFSSFGELQALLYGPFLMLYLLAFMGNTIIIVMVIADTHLHT PMYFFLGNFS LEILVTMTAVPRMLSD LVPHKVITFTGCMVQFYFHFSLGSTSFLILTDMALDRFVAI CHPLRYGTLMSRAMCVQLAGAA AAPFLAMVPTVLSRAHLDYCHGDVINHFFCDNEPLLQLSCSDTRLL EFWDFLMALTFVLSSFLVTLISYGYIVTTVLRIPSASSCQKAFSTCGSHLTLVFIGYSSTIFLYVRPGK AHSVQVRPVVALVTSVLTPFLNPFILTFCNQTVKTVLQGQMQRLKGLCΪAQ

hi a search of sequence databases, it was found, for example, that the GPCRld nucleic acid sequence of this invention has 555 of 904 bases (61%) identical to a gb:GENBANK- ID:AF 102523 |acc:AF 102523.1 mRNA from Mus musculus (Mus musculus olfactory receptor C6 gene, complete eds). The full amino acid sequence ofthe protein ofthe invention was found to have 140 of 308 amino acid residues (45%) identical to, and 203 of 308 amino acid residues (65%) similar to, the 313 amino acid residue ptnr:SPTREMBL-ACC:Q9ZlN0 protein from Mus musculus (Mouse) (OLFACTORY RECEPTOR C6).

GPCRl e

In one embodiment, the disclosed GPCRl variant is the novel GPCRl e (alternatively referred to as BA113A10_B_da3), which includes the 971 nucleotide sequence (SEQ ID ΝO:9) shown in Table II. The disclosed GPCRle open reading frame ("ORF") begins at an ATG initiation codon at nucleotides 24-26 and terminates at a TGA codon at nucleotides 936- 938. Putative untranslated regions upstream from the initiation codon and downstream from the termination codon are underlined in Table II, and the start and stop codons are in bold letters.

Table II. GPCRle nucleotide sequence (SEQ ID NO:9).

TGCAAGGCCCCATACTGTGGATC GCAAATCTGAGCCAGCCCTCCGAATTTGTCCTCTTGGGCTTCTCCTCCTTTGGTG AGCTGCAGGCCCTTCTGTATGGCCCCTTCCTCATGCTTTATCTTCTCGCCTTCATGGGAAACACCATCATCATAGTTATGG TCATAGCTGACACCCACCTACATACACCCATGTACTTCTTCCTGGGCAATTTTTCCCTGCTGGAGATCTTGGTAACCATGA CTGCAGTGCCCAGGATGCTCTCAGACCTGTTGGTCCCCCACAAAGTCATTACCTTCACTGGCTGCATGGTCCAGTTCTACT TCCACTTTTCCCTGGGGTCCACCTCCTTCCTCATCCTGACAGACATGGCCCTTGATCGCTTTGTGGCCATCTGCCACCCAC TGCGCTATGGCACTCTGATGAGCCGGGCTATGTGTGTCCAGCTGGCTGGGGCTGCCTGGGCAGCTCCTTTCCTAGCCATGG TACCCACTGTCCTCTCCCGAGCTCATCTTGATTACTGCCATGGCGACGTCATTAACCACTTCTTCTGTGACAATGAACCTC TCCTGCAGTTGTCATGCTCTGACACTCGCCTGTTGGAATTCTGGGACTTTCTGATGGTCTTGACCTTTGTCCTCAGCTCCT TCCTGGTGACCCTCATCTCCTATGGCTACATAGTGACCACTGTGCTGCGGATCCCCTCTGCCAGCAGCTGCCAGAAGGCTT TCTCCACTTGCGGGTCTCACCTCACACTGGTCTTCATCGGCTACAGTAGTACCATCTTTCTGTATGTCAGGCCTGGCAAAG CTCACTCTGTGCAAGTCAGGAAGGTCGTGGCCTTGGTGACTTCAGTTCTCACCCCCTTTCTCAATCCCTTTATCCTTACCT TCTGCAATCAGACAGTTAAAACAGTGCTACAGGGGCAGATGCAGAGGCTGAAAGGCCTTTGCAAGGCACAATGATGAGCC

The disclosed GPCRle polypeptide (SEQ ID NO: 10) encoded by SEQ ID NO:9 has 313 amino acid residues, a molecular weight of 34928.71 Daltons, and is presented in Table IJ using the one-letter amino acid code. The Signal P, Psort and/or Hydropathy results predict that GPCRle has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6000. In alternative embodiments, the GPCRle protein is localized to the Golgi body with a certainty of 0.4000, the endoplasmic reticulum (membrane) with a certainty of 0.3000, or the microbody (peroxisome) with a certainty of 0.3000. The most likely cleavage site for a GPCRle peptide is between amino acids 49 and 50, i.e., at the dash in the sequence VIA-DT. Table IJ. Encoded GPCRle protein sequence (SEQ ID NO:10).

MANLS_QP_SEFVLLGF_SSFGEL_QALLY_GPF MLY_LLAFMGNTIIIVMVIADTHLHTPMYFFLGNFSL EILV TMTAVPRMLSDLL_VP_HK_VIT_FT_GCMVQ_FYFHF_SL_GST_SFLILTDMALDRFVAICHPLRYGTLMSRAMCVQL A_GAAAAPFLAM_VPTVLS_RAHLDYC_HGDVINHFFCDNEPLLQLSCSDTRLLEF DFL VLTFVLSSFLVTL I_SYGYIVTTVLRIP_SASSCQKAF_STCGSHLTLVFIGYSSTIFLYVRPGKAHSVQVRKVVALVTSVLTPFLN PFILTFCNQTVKTVLQGQMQRKG CKAQ

In a search of sequence databases, it was found, for example, that the GPCRle nucleic acid sequence of this invention has 555 of 904 bases (61%) identical to a gb:GENBANK- ID:AF102523|acc:AF102523.1 mRNA from Mus musculus (Mus musculus olfactory receptor C6 gene, complete eds). The full amino acid sequence ofthe protein ofthe invention was found to have 139 of 308 amino acid residues (45%) identical to, and 202 of 308 amino acid residues (65%) similar to, the 313 amino acid residue ptnr:SPTREMBL-ACC:Q9ZlN0 protein from Mus musculus (Mouse) (OLFACTORY RECEPTOR C6).

GPCRlf In one embodiment, the disclosed GPCRl variant is the novel GPCRlf (alternatively referred to as CG50303_02), which includes the 992 nucleotide sequence (SEQ ID ΝO:l 1) shown in Table IK. The disclosed GPCRl open reading frame ("ORF") begins at an ATG initiation codon at nucleotides 21-23 and terminates at a TAG codon at nucleotides 954-956. Putative untranslated regions upstream from the initiation codon and downstream from the termination codon are underlined in Table IK, and the start and stop codons are in bold letters.

Table IK. GPCRl nucleotide sequence (SEQ ID NO:ll).

CTGTCTTTTGTTTCTCTTGC CAAGGCCCCATACTGTGGATCATGGCAAATCTGAGCCAGCCCTCCG AATTTGTCCTCTTGGGCTTCTCCTCCTTTGGTGAGCTGCAGGCCCTTCTGTATGGCCCCTTCCTCATGC TTTATCTTCTCGCCTTCATGGGAAACACCATCATCATAGTTATGGTCATAGCTGACACCCACCTACATA CACCCATGTACTTCTTCCTGGGCAATTTTTCCCTGCTGGAGATCTTGGTAACCATGACTGCAGTGCCCA GGATGCTCTCAGACCTGTTGGTCCCCCACAAAGTCATTACCTTCACTGGCTGCATGGTCCAGTTCTACT TCCACTTTTCCCTGGGGTCCACCTCCTTCCTCATCCTGACAGACATGGCCCTTGATCGCTTTGTGGCCA TCTGCCACCCACTGCGCTATGGCACTCTGATGAGCCGGGCTATGTGTGTCCAGCTGGCTGGGGCTGCCT GGGCAGCTCCTTTCCTAGCCATGGTACCCACTGTCCTCTCCCGAGCTCATCTTGATTACTGCCATGGCG ACGTCATTAACCACTTCTTCTGTGACAATGAACCTCTCCTGCAGTTGTCATGCTCTGACACTCGCCTGT TGGAATTCTGGGACTTTCTGATGGTCTTGACCTTTGTCCTCAGCTCCTTCCTGGTGACCCTCATCTCCT ATGGCTACATAGTGACCACTGTGCTGCGGATCCCCTCTGCCAGCAGCTGCCAGAAGGCTTTCTCCACTT GCGGGTCTCACCTCACACTGGTCTTCATCGGCTACAGTAGTACCATCTTTCTGTATGTCAGGCCTGGCA AAGCTCACTCTGTGCAAGTCAGGAAGGTCGTGGCCTTGGTGACTTCAGTTCTCACCCCCTTTCTCAATC CCTTTATCCTTACCTTCTGCAATCAGACAGTTAAAACAGTGCTACAGGGGCAGATGTAGAGGCTGAAAG GCCTTTGCAAGGCACAATGATGAGCC

The disclosed GPCRlf polypeptide (SEQ ID NO:12) encoded by SEQ ID NO:ll has 311 amino acid residues, a molecular weight of 34741.50 Daltons, and is presented in Table 1L using the one-letter amino acid code. The Signal P, Psort and/or Hydropathy results predict that GPCRlf has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6400. hi alternative embodiments, the GPCRlf protein is localized to the Golgi body with a certainty of 0.4600, the endoplasmic reticulum (membrane) with a certainty of 0.3700, or the microbody (peroxisome) with a certainty of 0.1000. The most likely cleavage site for a GPCRlf peptide is between amino acids 57 and 58, i.e., at the dash in the sequence NIA-DT.

Table 1L. Encoded GPCRlf protein sequence (SEQ ID ΝO:12).

MQGPI IMANLSQPSEFVLLGFSSFGELQALLYGPFLMLY LAFMGNTIIIVMVIADTHLHTPMYFFL GNFSLLEILVTMTAVPRMLSDLLVPHKVITFTGCMVQFYFHFSLGSTSFLILTDMALDRFVAICHPLRY GT SR-AMCVQLAGAA AAPFLAMVPTVLSRAHLDYCHGDVINHFFCDNEPLLQLSCSDTRLLEF DFL MVLTFVLSSFLVTLISYGYIVTTVLRIPSASSCQKAFSTCGSHLTLVFIGYSSTIFLYVRPGKAHSVQV RKVVALVTSVLTPFLNPFILTFCNQTVKTVLQGQM In a search of sequence databases, it was found, for example, that the GPCRlf nucleic acid sequence of this invention has 560 of 912 bases (61%) identical to a gb:GENBANK- ID:AF102523|acc:AF102523.1 mRNA from Mus musculus (Mus musculus olfactory receptor C6 gene, complete eds). The full amino acid sequence ofthe protein ofthe invention was found to have 139 of 301 amino acid residues (46%) identical to, and 199 of 301 amino acid residues (66%) similar to, the 313 amino acid residue ptnr:SPTREMBL-ACC:Q9ZlV0 protein from Mus musculus (Mouse) (OLFACTORY RECEPTOR C6).

GPCRl Clones

Unless specifically addressed as GPCRla, GPCRlb, GPCRlc, GPCRld, GPCRle or GPCRl , any reference to GPCRl is assumed to encompass all variants. Residue differences between any GPCRX variant sequences herein are written to show the residue in the "a" variant, the residue position with respect to the "a" variant, and the residue in any one ofthe "b" through "f ' variants.

Additional SNP variants of GPCRl are disclosed in Example 3. Sequence differences between the GPCRl clones are shown in the ClustalW alignment in Table 1L, with variant positions marked with a "o" above the variant sequence.

Public and proprietary sequence databases were searched for protein sequences with homology to GPCRl using BLASTP software. In all BLAST alignments herein, the "E-value" or "Expect" value is a numeric indication ofthe probability that the aligned sequences could have achieved their similarity to the BLAST query sequence by chance alone, within the database that was searched. For example, the probability that the subject sequence ("Sbjct"), e.g., patp ace no. AAG71691 Homo sapiens olfactory receptor polypeptide, retrieved from the GPCRl BLAST analysis ofthe proprietary PatP database matched the Query GPCRl sequence purely by chance is 7.2xl0^"166, as shown in Table 1M. The Expect value (E) is a parameter that describes the number of hits one can "expect" to see just by chance when searching a database of a particular size. It decreases exponentially with the Score (S) that is assigned to a match between two sequences of a database of comparable complexity. Essentially, the E value describes the random background noise that exists for matches between sequences. The E value is used as a convenient way to create a significance threshold for reporting results. The default value used for blasting is typically set to 0.0001. In BLAST 2.0, the E value is also used instead ofthe P value (probability) to report the significance of matches. For example, an E value of one assigned to a hit can be interpreted as meaning that in a database ofthe current size one might expect to see one match with a similar score simply by chance. An E value of zero means that one would not expect to see any matches with a similar score simply by chance. See, e.g., http://www.ncbi.nlm.mh.gov/Education BLASTinfo/. Occasionally, a string of X's or N's will result from a BLAST search. This is a result of automatic filtering ofthe query for low-complexity sequence that is performed to prevent artifactual hits. The filter substitutes any low-complexity sequence that it finds with the letter "N" in nucleotide sequence (e.g., "NN-NπN -N-NNN") or the letter "X" in protein sequences (e.g., "XXX"). Low-complexity regions can result in high scores that reflect compositional bias rather than significant position-by-position alignment (Wootton and Federhen, Methods Enzymol 266:554-511, 1996).

The disclosed GPCRl amino acid sequence has 210 of 314 amino acid residues (66%) identical to, and 250 of 314 residues (79%) positive with, the Mus musculus 315 amino acid residue olfactory receptor protein (ptnr: SPTREMBL-ACC:Q9QZ17)(E = 3.1e^"106).

The amino acid sequence of GPCRl had high homology to other proteins as shown in Table 1M.

Table 1M. BLASTX results from PatP database for GPCRl

Smallest Sum High prob Sequences producing High-scoring Segment Pairs : Score p _(N) patp :AAG71691 Human olfactory receptor polypeptide 1615 7 .2e-166 patp :AAG71685 Human olfactory receptor polypeptide 776 5. 8e-77 patp :AAG72507 Human OR-li e polypeptide query sequence 776 5 . 8e-77 patp :AAG71951 Human olfactory receptor polypeptide 743 1. 8e-73 patp :AAG72408 Human OR-like polypeptide query sequence 743 1.8e-73

In a search of sequence databases, it was found, for example, that the nucleic acid sequence of this invention has 555 of 904 bases (61%) identical to a gb:GENBANK- ID:AF102523|acc:AF102523.1 mRNA fxomMus musculus (Mus musculus olfactory receptor C6 gene, complete eds). The full amino acid sequence ofthe protein ofthe invention was found to have 140 of 308 amino acid residues (45%) identical to, and 203 of 308 amino acid residues (65%) similar to, the 313 amino acid residue ptnr:SPTREMBL-ACC:Q9ZlV0 protein from Mus musculus (Mouse) (Olfactory Receptor C6).

GPCRl also has homology to the proteins shown in the BLASTP data in Table IF.

Table IN. GPCRl BLASTP results

Gene Index / Protein / Organism Lengthl Identity Positives Expect

Identifier (aa) (%) (%)

TREMBLNEW- OLFACTORY RECEPTOR SDOLF 280 144/269 195/269 8.5e-7

ACC:AAK69553 Homo sapiens (53%) (72%)

TREMBLNEW- ODORANT RECEPTOR ORZ6 -

ACC:AAK70859 Mus musculus (Mouse) 314 139/307 203/307

(45%) (66%) 9.1e-7

140/308 203/308

SPTREMBL- OLFACTORY RECEPTOR C6 - ACC.-Q9Z1V0 Mus musculus (Mouse) 313 (45%) (65%) 6.6e-7

TREMBLNEW- (NOVEL 7 TRANSMEMBRANE 317 137/298 196/298 1.6e-6 ACC:CAC43444 RECEPTOR (RHODOPS1N (45%) (65%)

FAMILY) (OLFACTORY

RECEPTOR LIKE) PROTEIN

SIMILAR TO HUMAN HS6M1-21)

- Mus musculus (Mouse) SPTREMBL- M51 OLFACTORY RECEPTOR - , 132/298 188/298 ACC:Q9EPG2 _ Mus musculus (Mouse) (44%) (63%)

A multiple sequence alignment is given in Table 1O, with the GPCRl a-GPCRlf proteins being shown on lines 1 through 6, in a ClustalW analysis comparing the protein ofthe invention with the related protein sequences shown in Table IN. This BLASTP data is displayed graphically in the ClustalW in Table 10.

Table 10. ClustalW Analysis of GPCRl

1. SEQ ID NO: 2, GPCRla

2. SEQ ID NO: 4, GPCRlb

3. SEQ ID NO: 6, GPCRlc

4. SEQ ID NO: 8, GPCRld

5. SEQ ID NO: 10, GPCRle

6. SEQ ID NO: 12, GPCRlf

7. SEQ ID NO: 13, AAK70859 ODORANT RECEPTOR ORZ6

8. SEQ ID NO:14, Q9Z1V0 OLFACTORY RECEPTOR C6

9. SEQ ID NO:15, CAC43444

10. SEQ ID NO: 16, Q9EPG2 M51 OLFACTORY RECEPTOR 11 . SEQ ID NO : 17 , AAK69553 OLFACTORY RECEPTOR SDOLF oooooooooooooooooo

GPCRla 36 f-tPPR1h - 34 f-tPPRI n _ _ _ _ _ 34

GPCRld 50

GPCRle 36

GPCRlf 44

AATC7OR Q - - T 37

Q9Z1V0 I 36

C&CΔ' ΔΔA — — — — CT 38

OQ-TPf-!.? — — I 38

-

GPCRla 86

GPCRlb 84

GPCRlc 84

GPCRld 100

GPCRle 86

GPCRlf 94

AAK70859 87

Q9Z1V0 T- 85

CAC43444 SE 88

Q9EPG2 Q 88

AAK69553 ST 51

GPCRla 135

GPCRlb 133

GPCRlc 133

GPCRld 149

GPCRle 135

GPCRlf 143

AAK70859 G 137

Q9Z1V0 135

CAC43444 137

Q9EPG2 137

AAK69553 100

GPCRla 185

GPCRlb 183

GPCRlc 183

GPCRld 199

GPCRle 185

GPCRlf 193

AAK70859 187

Q9Z1V0 185

CAC43444 187

Q9EPG2 I 187

AAK69553

150 oo

GPCRla

235

GPCRlb 233

GPCRlc

233

GPCRld 249

GPCRle

235

GPCRlf

AAK70859 243

237

Q9Z1V0

235

oooooooooo

DOMAIN

The results indicate that the GPCRl protein contains the following protein domain (as defined by friterpro): domain name 7tm_l 7 transmembrane receptor (rhodopsin family). DOMAIN results for GPCRl were collected from the Conserved Domain Database (CDD) with Reverse Position Specific BLAST. This BLAST samples domains found in the Smart and Pfam collections.

As discussed below, all GPCRX proteins ofthe invention contain significant homology to the 7tm_l domain. This indicates that the GPCRX sequence has properties similar to those of other proteins known to contain this 7tm_l domain and similar to the properties of these domains. The 254 amino acid domain termed 7tm_l (SEQ ID NO:18)(Pfam ace. no. 00001), a seven transmembrane receptor (rhodopsin family), is shown in Table IP

Table IP. 7tm_l, 7 transmembrane receptor domain (SEQ ID NO-18)

GNLLVILVILRTKKLRTPTNIFLLNLAVADLLFLLTLPPWALYYLVGGDWVFGDALCKLVGALFWNGYASILLLTAISIDRYL AIVHPLRYRRIRTPRRAKVLILLVWVLALLLSLPPLLFSWLRTVEEGNTTVCLIDFPEESVKRSYVLLSTLVGFVLPLLVILVC YTRILRTLRKRARSQRSLKRRSSSERKAAKMLLWWVFVLCWLPYHIVLLLDSLCLLSIWRVLPTALLITLWLAYVNSCLNPI IY The encoded GPCRl polypeptide was identified as a member ofthe G protein receptor family due to the presence of a signature consensus sequence (SEQ ID NO: 19) shown in Table 1Q below.

Table 1Q. G-protein coupled receptors signature domain (SEQ ID NO: 19)

Entry Name G PROTEIN RECEPTOR

Entry Type PATTERN

Primary Accession Number PS00237

Created / Last Updated 01 -APR- 1990 / 01-IUL-1998

Description G-protein coupled receptors signature.

Pattern [GSTAL MFYWC]-[GSTANCPDE]-{EDPKRH}-x(2)-

[LIVMNQGA]-x(2)-[LINMFT]-[GSTANC]-

[LIVMFYWSTACl-[DENH]-R-[FYWCSH]-x(2)-[L-NM].

Table IR lists the domain description from DOMAIN analysis results against GPCRl. This indicates that the GPCRl sequence has properties similar to those of other proteins known to contain this domain as well as to the 254 amino acid 7tm domain (SEQ ID NO:39). For Table IR and all successive DOMAIN sequence alignments, fully conserved single residues are indicated by black shading and "strong" semi-conserved residues are indicated by grey shading. The "strong" group of conserved amino acid residues may be any one ofthe following groups of amino acids: STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, FYW. The DOMAIN results are listed in Table IR with the statistics and domain description.

An alignment of GPCRl residues 41-290 (SEQ ID NO:2) with the full 7tm_l domain, residues 1-254 (SEQ ID NO: 18), are shown in Table IR.

7 m_l 1 *->_GNlLVilvilrtkklrtptnifilNLAvADLLflltlppwalyylvg 47 GN ++i+ ++ +l+tp+++f++N ++ +L++ t +p +1+ 1+

GPCRl 53 GNTIIIVMVIADTHLHTPMYFFLGNFSLLEILVTMTAVPRMLSDLLV 99

7tm_l 48 gsed pfGsalCklvtaldvvnmyaSillLtalSiDRYlAIvhPlryrrr 97

++++ +C ++ ++ + + +S 1 Lt +++DR++AI+hPlry ++

GPCRl 100 —PHKVITFTGCMVQFYFHFSLGSTSFLILTDiMALDRFVAICHPLRYGTL 147

7tm_l 98 rtsprrAkvvillv vlalllslPpllfswvktveegngtlnvnvtvCli 147 ++ + ++ + +++ ++ +1+ +P ++s ++ + +++ +n+++C+

GPCRl 148 MS-RAMCVQLAGAAAAPFLAMVPT-VLSRAHLDYCHGDV—INHFFCDN 193

7tm_l 148 dfpeestasvstwlrsyvllstlvgFllPllvilvcYtrllrtlr 192

+ +4-S+ 1+++ +1 1 + 1 +lv 1+ Y+ 1+ t+ + ++

GPCRl 194 EPLLQLSCSDTRLLEF DFLMALTFVLSSFLVTLISYGYIVTTVLripsa 243

7tm_l 193 ... kaaktllvvvvvFvlC lPyfivllldtlc.lsiimsstCelervlp 239

++ + a+ ++ +++ v+ + i+l++++ + s ++

GPCRl 244 281

7 m_l 240 tallvtlwLayvNsclNPiIY<-* 261 + v+l+ +++ + 1NP+I

GPCRl 282 VRKVVALVTSVLTPFLNPFIL 302

The rhodopsin-like GPCRs themselves represent a widespread protein family that includes hormone, neurotransmitter and light receptors, all of which transduce extracellular signals through interaction with guanine nucleotide-binding (G) proteins. Although their activating ligands vary widely in structure and character, the amino acid sequences ofthe receptors are very similar and are believed to adopt a common structural framework comprising 7 transmembrane (TM) helices. G-protein-coupled receptors (GPCRs) constitute a vast protein family that encompasses a wide range of functions (including various autocrine, paracrine and endocrine processes). They show considerable diversity at the sequence level, on the basis of which they can be separated into distinct groups. The term clan is use to describe the GPCRs, as they embrace a group of families for which there are indications of evolutionary relationship, but between which there is no statistically significant similarity in sequence. The currently known clan members include the rhodopsin-like GPCRs, the secretin- like GPCRs, the cAMP receptors, the fungal mating pheromone receptors, and the metabotropic glutamate receptor family.

The homologies shown in the tables above indicates that the GPCRl sequences ofthe invention have properties similar to those of other proteins known to contain this/these domain(s) as well as properties similar to the properties of these domains. The Olfactory Receptor-like GPCRl disclosed in this invention is expressed in at least the following tissues: Apical microvilli ofthe retinal pigment epithelium, arterial (aortic), basal forebrain, brain, Burkitt lymphoma cell lines, coipus callosum, cardiac (atria and ventricle), caudate nucleus, CNS and peripheral tissue, cerebellum, cerebral cortex, colon, cortical neurogenic cells, endothelial (coronary artery and umbilical vein) cells, palate epithelia, eye, neonatal eye, frontal cortex, fetal hematopoietic cells, heart, hippocampus, hypothalamus, leukocytes, liver, fetal liver, lung, lung lymphoma cell lines, fetal lymphoid tissue, adult lymphoid tissue, those that express MHC II and III nervous, medulla, subthalamic nucleus, ovary, pancreas, pituitary, placenta, pons, prostate, putamen, serum, skeletal muscle, small intestine, smooth muscle (coronary artery in aortic) spinal cord, spleen, stomach, taste receptor cells ofthe tongue, testis, thalamus, and thymus tissue. This information was derived by determimng the tissue sources ofthe sequences that were included in the invention including but not limited to SeqCalling sources, Public EST sources, Literature sources, and/or RACE sources. Further expression data for GPCRl is provided in Example 2. The nucleic acids and proteins of GPCRl are useful in potential therapeutic applications implicated in various GPCR-related pathological disorders and/or OR-related pathological disorders, described above and further herein. The novel GPCRl nucleic acid encoding the GPCR-like protein ofthe invention, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount ofthe nucleic acid or the protein are to be assessed.

These materials are further useful in the generation of antibodies that bind immunospecifically to the novel substances ofthe invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-GPCRX Antibodies" section below. The disclosed GPCRl protein has multiple hydrophilic regions, each of which can be used as an immunogen.

GPCRla, GPCRlb and GPCRle have similar hydropathy plots, and hence are predicted to have similar epitope locations. In one embodiment, a contemplated GPCRl epitope for these variants is from about amino acids 1 to 20. hi additional embodiments, these GPCRl variants have epitopes that are from about amino acids 155 to 195, from about amino acids 230 to 240, from about amino acids 255 to 275 and from about amino acids 290 to the C- terminus. hi another embodiment, a contemplated GPCRlc epitope is from about amino acids 1 to 25, from about amino acids 50 to 65, from about amino acids 120 to 130, from about amino acids 155 to 190, from about amino acids 220 to 240, from about amino acids 250 to 275 and from about amino acids 280 to the C-terminus. In a further embodiment, a contemplated GPCRld epitope is from about amino acids 1 to 20, from about amino acids 170 to 210, from about amino acids 245 to 255, from about amino acids 270 to 285 and from about amino acids 305 to the C-terminus. In a final embodiment, a contemplated GPCRlf epitope is from about amino acids 1 to 20, from about amino acids 165 to 210, from about amino acids 235 to 250, from about amino acids 260 to 280 and from about amino acids 300 to the C-terminus.

GPCR2

The disclosed novel GPCR2 (alternatively referred to herein as AC073079_da2) includes the 990 nucleotide sequence (SEQ ID NO:20) shown in Table 2 A. A GPCR2 ORF begins with a Kozak consensus ATG initiation codon at nucleotides 7-9 and ends with a TAG codon at nucleotides 937-939. Putative untranslated regions upstream from the initiation codon and downstream from the termination codon are underlined in Table 2A, and the start and stop codons are in bold letters.

Table 2A. GPCR2 Nucleotide Sequence (SEQ ID NO:20)

GACTAAATGATGGACAACCACTCTAGTGCCACTGAATTCCACCTTCTAGGCTTCCCTGGGTCCCAAGG ACTACACCACATTCTTTTTGCTATATTCTTTTTCTTCTATTTAGTGACATTAATGGGAAACACGGTCA TCATTGTGATTGTCTGTGTGGATAAACGTCTGCAGTCCCCCATGTATTTCTTCCTCAGCCACCTCTCT ACCCTGGAGATCCTGGTCACAACCATAATTGTCCCCATGATGCTTTGGGGATTGCTCTTCCTGGGATG CAGACAGTATCTTTCTCTACATGTATCGCTCAACTTTTCCTGTGGGACCATGGAGTTTGCATTACTTG GAGTGATGGCTGTGGACCGTTATGTGGCTGTGTGTAACCCTTTGAGGTACAACATCATTATGAACAGC AGTACCTGTATTTGGGTGGTAATAGTGTCATGGGTGTTTGGATTTCTTTCTGAAATCTGGCCCATCTA TGCCACATTTCAGTTTACCTTCCGCAAATCAAATTCATTAGACCATTTTTACTGTGACCGAGGGCAAT TGCTCAAACTGTCCTGCGATAACACTCTTCTCACAGAGTTTATCCTTTTCTTAATGGCTGTTTTTATT CTCATTGGTTCTTTGATCCCTACGATTGTCTCCTACACCTACATTATCTCCACCATCCTCAAGATCCC GTCAGCCTCTGGCCGGAGGAAAGCCTTCTCCACTTTTGCCTCCCACTTCACCTGTGTTGTGATTGGCT ATGGCAGCTGCTTGTTTCTCTACGTGAAACCCAAGCAAACACAGGGAGTTGAGTACAATAAGATAGTT TCCTTGTTGGTTTCTGTGTTAACCCCCCTTCCTGAATCCTTTCATCTTTACTCTTCGGATGACAAAGT CAAAGAGGCCCTCCGAGATGGGATGAAACGCTGCTGTCAACTCCTGAAAGATTAGCTGTTCTGTAAGT CAGTTTTAGGTGGTCCAAGCCTCAGGGTTAATTATTAA

A GPCR-like protein ofthe invention, referred to herein as GPCR2, is an Olfactory Receptor ("OR")-like protein. Some members ofthe Olfactory Receptor-Like Protein Family end up localized at the cell surface, where they exhibit activity. Therefore it is likely that these novel GPCR2 proteins are available at the appropriate sub-cellular localization and hence accessible for the therapeutic uses described in this application. The GPCR2 polypeptide (SEQ ID NO:21) encoded by SEQ ID NO:20 is 310 amino acids in length, has a molecular weight of 35240.98 Daltons, and is presented using the one- letter amino acid code in Table 2B. The Signal P, Psort and/or Hydropathy results predict that GPCR2 has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6000. hi alternative embodiments, the GPCR2 protein is localized to the Golgi body with a certainty of 0.4000, the endoplasmic reticulum (membrane) with a certainty of 0.3000, or the microbody (peroxisome) with a certainty of 0.3000. The most likely cleavage site for a GPCRla peptide is between amino acids 40 and 41, i.e., at the dash in the sequence LMG-NT. GPCR2 is predicted to be a likely Type Illb membrane protein. Additional SNP variants of GPCR2 are disclosed in Example 3.

Table 2B. GPCR2 protein sequence (SEQ ID NO:21)

MMDNHSSATEFHLLGFPGSQGLHHILFAIFFFFYLVTLMGNTVIIVIVCVDKRLQSPMYFFLSHLST LEILVTTIIVPMMLWGLLFLGCRQYLSLHVSLNFSCGTMEFALLGVMAVDRYVAVCNPLRYNIIMNS STCI VVIVS VFGFLSEIWPIYATFQFTFRKSNSLDHFYCDRGQLLKLSCDNTLLTEFILFLMAVF ILIGSLIPTIVSYTYIISTILKIPSASGRRKAFSTFASHFTCVVIGYGSCLFLYVKPKQTQGVEYNK IVSLLVSVLTPLPESFHLYSSDDKVKEALRDGMKRCCQLLKD

In a search of sequence databases, it was found, for example, that the nucleic acid sequence of this invention has 302 of 343 bases (88%) identical to a gb:GENBANK- ID:HSHTPRX06| acc:X64982.1 mRNA from Homo sapiens (H.sapiens mRNA HTPCRX06 for olfactory receptor). The full amino acid sequence ofthe protein ofthe invention was found to have 134 of 306 amino acid residues (43%) identical to, and 189 of 306 amino acid residues (61%) similar to, the 313 amino acid residue ptnr: SPTREMBL- ACC.-Q9Z1 NO protein from Mus musculus (Mouse) (OLFACTORY RECEPTOR C6). The amino acid sequence of GPCR2 had high homology to other proteins as shown in

Table 2E.

Table 2E. BLASTX results from PatP database for GPCR2

Smallest Sum High Prob Sequences producing High-scoring Segment Pairs : Score P (Ν) patp:AAG71689 Human olfactory receptor polypeptide 1554 2, . le-159 patp :AAG71971 Human olfactory receptor polypeptide 1207 1. ,2e-122 patp :AAG71969 Human olfactory receptor polypeptide 1128 2. .9e-114 patp:AAG72438 Human OR-like polypeptide query sequence 1128 2. .9e-114 pat :AAG71813 Human olfactory receptor polypeptide 996 2. .8e-100

In a search of sequence databases, it was found, for example, that the nucleic acid sequence of this invention has 543 of 882 bases (61%) identical to a gb:GEΝBA-ΝK- ID:AF102523|acc:AF102523.1 mRNA from Mus musculus (Mus musculus olfactory receptor C6 gene, complete eds). The full amino acid sequence ofthe protein ofthe invention was found to have 137 of 301 amino acid residues (45%) identical to, and 199 of 301 amino acid _ι residues (66%) similar to, the 313 amino acid residue ptnr: SPTREMBL- ACC:Q9Z1V0 protein from Mus musculus (Mouse) (OLFACTORY RECEPTOR C6).

GPCR2 also has homology to the proteins shown in the BLASTP data in Table 2F.

A multiple sequence alignment is given in Table 2G, with the GPCR2 protein being shown on line 1, in a ClustalW analysis comparing the protein ofthe invention with the related protein sequences shown in Table 2F. This BLASTP data is displayed graphically in the ClustalW in Table 2G.

Table 2G. ClustalW Analysis of GPCR2

1. SEQ ID NO 21, GPCR2 2. SEQ ID NO 22, TREMBLNEW-ACC:AAK70859 ODORANT RECEPTOR ORZ6 3. SEQ ID NO 23, SPTREMBL-ACC:Q9Z1V0 OLFACTORY RECEPTOR C6 4. SEQ ID NO 24, SPTREMBL-ACC:Q9EPV0 M50 OLFACTORY RECEPTOR 5. SEQ ID NO 25, SPTREMBL-ACC:Q9EPG1 M50 OLFACTORY RECEPTOR 6. SEQ ID NO 26, SPTREMBL-ACC:Q9EPG2 M51 OLFACTORY RECEPTOR

Table 2H lists the domain description from DOMAIN analysis results against GPCR2. This indicates that the GPCR2 sequence has properties similar to those of other proteins known to contain this domain as well as to the 254 amino acid 7tm domain (SEQ ID NO: 18) itself.

7tm_l 1 *->GNlLVιlvilrtkklrtptnifilNLAvADLLflltlppwalyylvg 47

GN ++i++++ k+l++p+++f+ +L+ +L+++ +p++l 1++ GPCR2 40 GNTVIIVIVCVDKRLQSPMYFFLSHLSTLEILVTTIIVPMML GLLF 86

7tm_l 48 gsedWpfGsalCklvtaldvvnmyaSillLtalSiDRYlAIvhPlryrrr 97

++ ++ 1 ++1 + ++++ 1L ++++DRY+A+++Plry+ + GPCR2 87 LGCRQYLS LHVSLNFSCGTMEFALLGVMAVDRYVAVCNPLRYNII 131

7tm_l 98 rtsprrAkvvillvWvlall 117 ++ + v+++ Wv+++1 GPCR2 132 MN-SSTCI VVIVSWVFGFL 150

The Olfactory Receptor-like GPCR2 disclosed in this invention is expressed in at least the following tissues: Apical microvilli ofthe retinal pigment epithelium, arterial (aortic), basal forebrain, brain, Burkitt lymphoma cell lines, corpus callosum, cardiac (atria and ventricle), caudate nucleus, CNS and peripheral tissue, cerebellum, cerebral cortex, colon, cortical neurogenic cells, endothelial (coronary artery and umbilical vein) cells, palate epithelia, eye, neonatal eye, frontal cortex, fetal hematopoietic cells, heart, hippocampus, hypothalamus, leukocytes, liver, fetal liver, lung, lung lymphoma cell lines, fetal lymphoid tissue, adult lymphoid tissue, Those that express MHC II and III nervous, medulla, subthalamic nucleus, ovary, pancreas, pituitary, placenta, pons, prostate, putamen, serum, skeletal muscle, small intestine, smooth muscle (coronary artery in aortic) spinal cord, spleen, stomach, taste receptor cells ofthe tongue, testis, thalamus, and thymus tissue. This information was derived by determining the tissue sources ofthe sequences that were included in the invention including but not limited to SeqCalling sources, Public EST sources, Literature sources, and/or RACE sources. Further expression data for GPCR2 is provided in Example 2.

The nucleic acids and proteins of GPCR2 are useful in potential therapeutic applications implicated in various GPCR-related pathological disorders and/or OR-related pathological disorders, described further herein. The novel GPCR2 nucleic acid encoding the GPCR-like protein ofthe invention, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount ofthe nucleic acid or the protein are to be assessed. These materials are further useful in the generation of antibodies that bind immunospecifically to the novel substances ofthe invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-GPCRX Antibodies" section below. The disclosed GPCR2 protein has multiple hydrophilic regions, each of which can be used as an immunogen. In one embodiment, a contemplated GPCR2 epitope is from about amino acids 5 to 25. In additional embodiments, GPCR2 epitopes are from about amino acids 50 to 60, from about amino acids 80 to 100, from about amino acids 130 to 145, from about amino acids 230 to 240, from about amino acids 260 to 270 and from about amino acids 290 to 310. GPCR3

The disclosed novel GPCR3 (alternatively referred to herein as sggc_draft_ba656o22_ 2000073 l_da4) includes the 971 nucleotide sequence (SEQ ID NO:27) shown in Table 5 A. A GPCR3 ORF begins with a Kozak consensus ATG initiation codon at nucleotides 3-5 and ends with a TAG codon at nucleotides 963-965. Putative untranslated regions upstream from the initiation codon and downstream from the termination codon are underlined in Table 3A, and the start and stop codons are in bold letters.

Table 3A. GPCR3 Nucleotide Sequence (SEQ ID NO:27)

CA_ATGA_TG_GA_AATA_GCCAA_TGT_GA_GT_TCTCC_AGAAGTCTTTGTCCTCCTGGGCTTCTCCGCACGACC C_TCA_CTAGAA_ACTGT_CCT_CTTC_ATA_GTTGTCTTGAGTTTTTACATGGTATCGATCTTGGGCAATGGC AT_CA_TCATT_CT_GG_TCT_CC_CA_TAC_AGAT_GTGCACCTCCACACACCTATGTACTTCTTTCTTGCCAACC T_CTCCTTCC_TGGA_CAT_GAGCTTCACCACGAGCATTGTCCCACAGCTCCTGGCTAACCTCTGGGGACC A_CAGAA_AACCATAAG_CTA_TGGAGGGTG_TGTGGTCCAGTTCTATATCTCCCATTGGCTGGGGGCAACC G_AGT_GTGT_CCTGC_TG_GCCACCATGTCCTATGACCGCTACGCTGCCATCTGCAGGCCACTCCATTACA CT_GT_CA_TTAT_GCA_TC_CACAGCTTTGCCTTGGGCTAGCTTTGGCCTCCTGGCTGGGGGGTCTGACCAC CA_GCA_TGGT_GGG_CTC_CA_CGC_TCACCA_TGCTCCTACCGCTGTGTGGGAACAATTGCATCGACCACTTC TTTTGCGAGATGCCCCTCATTATGCAACTGGCTTGTGTGGATACCAGCCTCAATGAGATGGAGATGT A_CCTGGCCAGCTT_TGTCTTTGTTGTCCTGCCTCTGGGGCTCATCCTGGTCTCTTACGGCCACATTGC CCGGGCCGTGTTGAAGATCAGGTCAGCAGAAGGGCGGAGAAAGGCATTCAACACCTGTTCTTCCCAC GTGGCTGTGGTGTCTCTGTTTTACGGGAGCATCATCTTCATGTATCTCCAGCCAGCCAAGAGCACCT CCCATGAGCAGGGCAAGTTCATAGCTCTGTTCTACACCGTAGTCACTCCTGCGTTGAACCCAGTTAT TTACACCCTGAGGAACACGGAGGTGAAGAGCGCCCTCCGGCACATGGTATTAGAGAACTGCTGTGGC TCTGCAGGCAAGCTGGCGCAAATTTAGAGACTC

The GPCR3 polypeptide (SEQ ID NO:28) encoded by SEQ ID NO:27 is 320 amino acids in length, has a molecular weight of 35321.4 Daltons, and is presented using the one- letter amino acid code in Table 3B. The Psort profile for GPCR3 predicts that these sequences have a signal peptide and are likely to be localized at the endoplasmic reticulum (membrane) with a certainty of 0.6850. hi alternative embodiments, a GPCR3 polypeptide is located to the plasma membrane with a certainty of 0.6400, the Golgi body with a certainty of 0.4600, or the endoplasmic reticulum (lumen) with a certainty of 0.1000. The Signal P predicts a likely cleavage site for a GPCR3 peptide is between positions 44 and 45, i.e., at the dash in the sequence GNG-II.

Additional SNP variants of GPCR3 are disclosed in Example 3. The amino acid sequence of GPCR3 had high homology to other proteins as shown in Table 3C.

Table 3C. BLASTX results for GPCR3

Smallest Sum

High Prob

Sequences producing High-scoring Segment Pairs: Score P(N) patp:AAG71896 Human olfactory receptor polypeptide 319 aa 1638 2.5e-168 patp: AAG71912 Human olfactory receptor polypeptide 319 aa 1638 2.5e-168 patp:AAG71891 Human olfactory receptor polypeptide 309 aa 983 6.5e-99 patp:AAG72908 Human olfactory receptor 312 aa 982 8.3e-99

In a search of sequence databases, it was found, for example, that the nucleic acid sequence of this invention has 602 of 906 bases (66%) identical to a gb:GENBANK- ID:AF098664|acc:AF098664 mRNA from Homo sapiens [Homo sapiens olfactory receptorlike protein (OR2C1) gene, complete eds]. The full amino acid sequence ofthe protein ofthe invention was found to have 182 of 305 amino acid residues (59%) identical to, and 233 of 305 amino acid residues (76%) similar to, the 312 amino acid residue ptnr:SWISSPROT- ACC:P23275 protein from-M ϊ musculus (Mouse) [OLFACTORY RECEPTOR 15 (O-R3)].

Additional BLASTP results are shown in Table 3D.

A multiple sequence alignment is given in Table 3E, with the GPCR3 protein ofthe invention being shown on line 1, in a ClustalW analysis comparing GPCR3 with related protein sequences disclosed in Table 3D.

Table 3E. Information for the ClustalW proteins:

1 . SEQ ID NO : 28 , GPCR3 2 SEQ ID NO 29, P23275

3 SEQ ID NO 30, Q9GZK6

4 SEQ ID NO 31, Q9GZK1

5 SEQ ID NO 32, 076001

6 SEQ ID NO 33, Q9Y3N9

110 120 130 140 150

310 320

Table 3F lists the domain description from DOMAIN analysis results against GPCR3.

This protein contains domain IPR000276 at amino acid positions 42 to 291. This indicates that the GPCR3 sequence has properties similar to those of other proteins known to contain this domain as well as to the 254 amino acid 7tm domain (SEQ ID NO: 18) itself.

Table 3F Domain Analysis of GPCR3

PSSMs producing significant alignments: Score E

(bits) value gnl|Pfam|pfam00001 7tm_l , 7 transmembrane receptor (rhodopsin family) 144.2 9.9e-45

7tm_l GNLLVILVILRTKKLRTPTNIFILNLAVADLLFLLTLPP ALYYLVG

II+++III +I + H+++1+ ||++ |+ |+++ +) I 4-1 + 1 GPCR3 42 GNGIIILVSHTDVHLHTPMYFFLANLSFLDMSFTTSIVPQLLANLWG 88

GSED PFGSALCKLVTALDVVNMYASILLLTAISIDRYLAIVHPLRYRRR ++ +++ +| ++ ++ + + + + I |+ +| + | I I I I++I l + l ++ GPCR3 89 —PQKTISYGGCVVQFYISHWLGATECVLLATMSYDRYAAICRPLHYTVI 136

RTSPRRAKVVILLV VLALLLSLPPLLFS VKTVEEGNGTLNVNVTVCLI + I ++++++ |+ |+ +| |+ ++ ++ ++ +M+ +++++I + GPCR3 137 MH-PQLCLGLALASWLGGLTTSMVGSTL-TMLLPLCGNNC—IDHFFCEM 182

DFPEESTAS.VSTWLRSYVLLSTLVGFLLPLLVILVCYTRILRTLR.... + ++ ++|+ ++ ++| |++++ 4-| I I +||| |++| + |++ + ++ GPCR3 183 PLIMQLACVDTSLNEMEMYLASFVFV-VLPLGLILVSYGHIARAVLKIRS 231

....KAAKTLLVVVVVFVLCWLPYFIVLLLDTLC.LSIIMSSTCELERVL ++++ |+ ++ +|++| +++ |+++|++ +++| +

GPCR3 232 AEGRRKAFNTCSSHVAVVSLFYGSIIFMYLQPAKSTS H 269

PTALLVTLWLAYVNSCLNPIIY

+ ++++I++++I ++| I l+l I

GPCR3 270 EQGKFIALFYTVVTPALNPV1Y 291

A GPCR-like protein ofthe invention, referred to herein as GPCR3, is an Olfactory Receptor ("OR")-like protein. Some members ofthe Olfactory Receptor-Like Protein Family end up localized at the cell surface, where they exhibit activity. Therefore it is likely that these novel GPCR3 proteins are available at the appropriate sub-cellular localization and hence accessible for the therapeutic uses described in this application. The GPCR3 disclosed in this invention is expressed in at least some ofthe following tissues: adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea, uterus. This information was derived by determining the tissue sources ofthe sequences that were included in the invention. Further expression data for GPCR3 is provided in Example 2.

The nucleic acids and proteins of GPCR3 are useful in potential therapeutic applications implicated in various GPCR-related pathological disorders and/or OR-related pathological disorders, described further above.

The novel nucleic acid encoding the GPCR-like protein ofthe invention, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed. These materials are further useful in the generation of antibodies that bind immunospecifically to the novel substances ofthe invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-GPCRX Antibodies" section below. The disclosed GPCR3 protein has multiple hydrophilic regions, each of which can be used as an immunogen. In one embodiment, a contemplated GPCR3 epitope is from about amino acids 220 to 245. In another embodiment, a GPCR3 epitope is from about amino acids 255 to 270. In further specific embodiments, GPCR3 epitopes are from about amino acids 275 to 315.

GPCR4

A fourth GPCR-like protein ofthe invention, referred to herein as GPCR4, is an Olfactory Receptor ("OR")-Hke protein. Some members of the Olfactory Receptor-Like

Protein Family end up localized at the cell surface, where they exhibit activity. Therefore it is likely that these novel GPCR4 proteins are available at the appropriate sub-cellular localization and hence accessible for the therapeutic uses described in this application.

Two alternative novel GPCR4 nucleic acids and encoded polypeptides are provided, namely GPCR4a and GPCR4b. GPCR4a

In one embodiment, a GPCR4 variant is the novel GPCR4a (alternatively referred to herein as AC0170103_A_dal), which includes the 1025 nucleotide sequence (SEQ ID NO:34) shown in Table 4A. A GPCR4a ORF begins with a Kozak consensus ATG initiation codon at nucleotides 33-35 and ends with a TGA codon at nucleotides 969-971. Putative untranslated regions upstream from the initiation codon and downstream from the termination codon are underlined in Table 4A, and the start and stop codons are in bold letters.

Table 4A. GPCR4a Nucleotide Sequence (SEQ ID NO:34)

AGCTGTGGACCATCTCTTCAGAACTCTGCAGCATGGAGCCGCTCAACAGAACAGAGGTGTCCGAGTT CTTTCTGAAAGGATTTTCTGGCTACCCAGCCCTGGAGCATCTGCTCTTCCCTCTGTGCTCAGCCATG TACCTGGTGACCCTCCTGGGGAACACAGCCATCATGGCGGTGAGCGTGCTAGATATCCACCTGCACA CGCCCGTGTACTTCTTCCTGGGCAACCTCTCTACCCTGGACATCTGCTACACGCCCACCTTTGTGCC TCTGATGCTGGTCCACCTCCTGTCATCCCGGAAGACCATCTCCTTTGCTGTCTGTGCCATCCAGATG TGTCTGAGCCTGTCCACGGGCTCCACGGAGTGCCTGCTACTGGCCATCACGGCCTATGACCGCTACC TGGCCATCTGCCAGCCACTCAGGTACCACGTGCTCATGAGCCACCGGCTCTGCGTGCTGCTGATGGG AGCTGCCTGGGTCCTCTGCCTCCTCAAGTCGGTGACTGAGATGGTCATCTCCATGAGGCTGCCCTTC TGTGGCCACCACGTGGTCAGTCACTTCACCTGCAAGATCCTGGCAGTGCTGAAGCTGGCATGCGGCA ACACGTCGGTCAGCGAAGACTTCCTGCTGGCGGGCTCCATCCTGCTGCTGCCTGTACCCCTGGCATT CATCTGCCTGTCCTACTTGCTCATCCTGGCCACCATCCTGAGGGTGCCCTCGGCCGCCAGGTGCTGC AAAGCCTTCTCCACCTGCTTGGCACACCTGGCTGTAGTGCTGCTTTTCTACGGCACCATCATCTTCA TGTACTTGAAGCCCAAGAGTAAGGAAGCCCACATCTCTGATGAGGTCTTCACAGTCCTCTATGCCAT GGTCACGACCATGCTGAACCCCACCATCTACAGCCTGAGGAACAAGGAGGTGAAGGAGGCCGCCAGG AAGGTGTGGGGCAGGAGTCGGGCCTCCAGGTGAGGGAGGGCGGGGCTCTGTACAGACGCAGGTCTCA GGTTAGTAGCTGAGGCCATC

The sequence of GPCR4a was derived by laboratory cloning of cDNA fragments, by in silico prediction ofthe sequence. The cDNA fragments covering either the full length ofthe DNA sequence, or part ofthe sequence, or both, were cloned. In silico prediction was based on sequences available in CuraGen's proprietary sequence databases or in the public human sequence databases, and provided either the full length DNA sequence, or some portion thereof.

The cDNA coding for the GPCR4a sequence was cloned by the polymerase chain reaction (PCR). Primers were designed based on in silico predictions ofthe full length or some portion (one or more exons) ofthe cDNA/protein sequence ofthe invention. The DNA sequence and protein sequence for a novel Olfactory Receptor-like gene were obtained by exon linking and are reported here as GPCR4a.These primers and methods used to amplify GPCR4a cDNA are described in the Examples.

The GPCR4a polypeptide (SEQ ID NO:35) encoded by SEQ ID NO:34 is 312 amino acids in length, has a molecular weight of 34688.2 Daltons, and is presented using the one- letter amino acid code in Table 4B. The Psort profile for GPCR4a predicts that this sequence has a signal peptide and are likely to be localized at the plasma membrane with a certainty of 0.6000. hi alternative embodiments, a GPCR4a polypeptide is located to the Golgi body with a certainty of 0.4000, the endoplasmic reticulum (membrane) with a certainty of 0.3000, or a microbody (peroxisome) with a certainty of 0.3000. The Signal P predicts a likely cleavage site for a GPCR4a peptide is between positions 47 and 48, i.e., at the dash in the sequence LMANS.

Table 4B. GPCR4a protein sequence (SEQ ID ΝO:35)

MEPLNRTEVSEFFLKGFSGYPALEHLLFPLCSAMYLVTLLGNTAIMAVSVLDIHLHTPVYFFLGNLS TLDICYTPTFVPLMLVHLLSSRKTISFAVCAIQMCLSLSTGSTECLLLAITAYDRYLAICQPLRYHV LMSHRLCVLLMGAAWVLCLLKSVTEMVISMRLPFCGHHVVSHFTCKILAVLKLACGNTSVSEDFLLA GSILLLPVPLAFICLSYLLILATILRVPSAARCCKAFSTCLAHLAVVLLFYGTIIFMYLKPKSKEAH ISDEVFTVLYAMVTTMLNPTIYSLRNKEVKEAARKV GRSRASR

Additional SNP variants of GPCR4a are disclosed in Example 3. GPCR4b

In an alternative embodiment, a GPCR4 variant is the novel GPCR4b (alternatively referred to herein as CG54212-03), which includes the 917 nucleotide sequence (SEQ ID NO:36) shown in Table 4C. The GPCR4b ORF was identified at nucleotides 3-5 with a CTC codon and ends with a TGA codon at nucleotides 861-863. Putative untranslated regions upstream from the initiation codon and downsfream from the termination codon are underlined in Table 4C, and the start and stop codons are in bold letters.

Table 4C. GPCR4b Nucleotide Sequence (SEQ ID NO:36)

TGCTCTTCCCTCTGTGCTCAGCCATGTACCTGGTGACCCTCCTGGGGAACACAGCCATCATGGCGGT GAGCGTGCTAGATATCCACCTGCACACGCCCGTGTACTTCTTCCTGGGCAACCTCTCTACCCTGGAC ATCTGCTACACGCCCACCTTTGTGCCTCTGATGCTGGTCCACCTCCTGTCATCCCGGAAGACCATCT CCTTTGCTGTCTGTGCCATCCAGATGTGTCTGAGCCTGTCCACGGGCTCCACGGAGTGCCTGCTACT GGCCATCACGGCCTATGACCGCTACCTGGCCATCTGCCAGCCACTCAGGTACCACGTGCTCATGAGC CACCGGCTCTGCGTGCTGCTGATGGGAGCTGCCTGGGTCCTCTGCCTCCTCAAGTCGGTGACTGAGA TGGTCATCTCCATGAGGCTGCCCTTCTGTGGCCACCACGTGGTCAGTCACTTCACCTGCAAGATCCT GGCAGTGCTGAAGCTGGCATGCGGCAACACGTCGGTCAGCGAAGACTTCCTGCTGGCGGGCTCCATC CTGCTGCTGCCTGTACCCCTGGCATTCATCTGCCTGTCCTACTTGCTCATCCTGGCCACCATCCTGA GGGTGCCCTCGGCCGCCAGGTGCTGCAAAGCCTTCTCCACCTGCTTGGCACACCTGGCTGTAGTGCT GCTTTTCTACGGCACCATCATCTTCATGTACTTGAAGCCCAAGAGTAAGGAAGCCCACATCTCTGAT GAGGTCTTCACAGTCCTCTATGCCATGGTCACGACCATGCTGAACCCCACCATCTACAGCCTGAGGA ACAAGGAGGTGAAGGAGGCCGCCAGGAAGGTGTGGGGCAGGAGTCGGGCCTCCAGGTGAGGGAGGGC GGGGCTCTGTACAGACGCAGGTCTCAGGTTAGTAGCTGAGGCCATC

The GPCR4b protein (SEQ ID NO:37) encoded by SEQ ID NO:36 is 286 amino acids in length, has a molecular weight of 31693.8 Daltons, and is presented using the one-letter code in Table 4D. The Psort profile for GPCR4b predicts that this sequence has a signal peptide and is likely to be localized at the endoplasmic reticulum (membrane) with a certainty of 0.6850. In alternative embodiments, a GPCR4b polypeptide is located to the plasma membrane with a certainty of 0.6400, the Golgi body with a certainty of 0.4600, or to the endoplasmic reticulum (lumen) with a certainty of 0.1000. The Signal P predicts a likely cleavage site for a GPCR4a peptide is between positions 21 and 22, i.e., at the dash in the sequence MAN^'S.

Table 4D. GPCR4b protein sequence (SEQ ID ΝO-37)

LFPLCSAMYLVTLLGNTAIMAVSVLDIHLHTPVYFFLGNLSTLDICYTPTFVPLMLVHLLSSRKTISFAVC AIQMCLSLSTGSTECLLLAITAYDRYLAICQPLRYHVLMSHRLCVLLMGAA VLCLLKSVTEMVISMRLPF CGHHVVSHFTCKILAVLKLACGNTSVSEDFLLAGSILLLPVPLAFICLSYLLILATILRVPSAARCCKAFS TCLAHLAVVLLFYGTIIFMYLKPKSKEAHISDEVFTVLYAMVTTMLNPTIYSLRNKEVKEAARKV GRSRA SR

Additional SNP variants of GPCR4b are disclosed in Example 3.

GPCR4 Clones

Unless specifically addressed as GPCR4a or GPCR4b, any reference to GPCR4 is assumed to encompass all variants. Residue differences between any GPCRX variant sequences herein are written to show the residue in the "a" variant, the residue position with respect to the "a" variant, and the residue in the "b" variant.

The amino acid sequence of GPCR4 has high homology to other proteins as shown in Table 4E.

Table 4E. BLASTX results for GPCR4

Smallest

Sum

High Prob

Sequences producing High-scoring Segment Pairs : Score P (N) patp:AAG71899 Human olfactory receptor polypeptide, 312 aa 1603 1. 3e-164 patp : AAG71954 Human olfactory receptor polypeptide, 333 aa 883 2 . 6e-98 patp:AAG71946 Human olfactory receptor polypeptide, 316 aa 848 1.3e-84

In a search of sequence databases, it was found, for example, that the GPCR4a nucleic acid sequence has 852 of 1010 bases (84 %) identical to aMus musculus orl7c gene mRNA (GENBANK-ID: MMU133429|acc:AJl 33429). The full amino acid sequence ofthe GPCR4a protein ofthe invention was found to have 256 of 312 amino acid residues (82%) identical to, and 275 of 312 residues (88 %) positive with, the 317 amino acid residue OLFACTORY RECEPTOR-LIKE PROTEIN protein froml-β musculus, 312 aa (ptnr: SPTREMBL- ACC:Q9QZ18). Similarly, in a search of sequence databases, it was found, for example, that the GPCR4b nucleic acid sequence of this invention has 781 of 920 bases (84%) identical to a gb:GENBANK-ID:MMU133429|acc:AI133429.1 mRNA from. Mus musculus (Mus musculus orl7 gene). The full amino acid sequence ofthe GPCR4b protein ofthe invention was found to have 234 of 286 amino acid residues (81%) identical to, and 253 of 286 amino acid residues (88%) similar to, the 312 amino acid residue ptnr:SPTREMBL-ACC:Q9QZ18 protein from Mus musculus (Mouse) (OLFACTORY RECEPTOR).

Additional BLASTP results are shown in Table 4F.

A multiple sequence alignment is given in Table 4G, with the GPCR4 protein ofthe invention being shown on line 1 and 2, in a ClustalW analysis comparing GPCR4 with related protein sequences of Table 4F. The residue that differs between GPCR4a and GPCR4b is marked with the (o) symbol.

Table 4G. Information for the ClustalW proteins:

1. SEQ ID NO: 35, GPCR4a

2. SEQ ID NO: 37, GPCR4b

3. SEQ ID NO:38, Q9QZ18

4. SEQ ID NO:39, AAK95088

5. SEQ ID NO: 40, Q9QZ22

6. SEQ ID NO: 41, Q9QZ19

7. SEQ ID NO:42, Q9QZ21

60 70 90 100 I .. . I.. ■ • I

110 120 130 140 150

210 220 230 240 250

260 270 280 290 300

DOMAIN results for GPCR4 were collected from the Conserved Domain Database

(CDD) with Reverse Position Specific BLAST. This BLAST samples domains found in the Smart and Pfam collections. The results are listed in Table 4H with the statistics and domain description. The 7tm_l, a seven transmembrane receptor (rhodopsin family), was shown to have significant homology to GPCR4. An alignment of GPCR4b residues 40-290 (SEQ ID NO:X) with 7tm_l residues 1-254 (SEQ ID NO: 18) are shown in Table 4H.

Table 4H. DOMAIN results for GPCR4

PSSMs producing significant alignments: Score E

(bits) value gnl|Pfam|pfam00001 7tm_l, 7 transmembrane receptor (rhodopsin family) 123.1 4.6e-38

7tm_l GNLLVILVILRTKKLRTPTNIFILNLAVADLLFLLTLPPWALYYLVG

II +++| ++ +| + | I ++I++M+ I++++ I +1 +I++I + GPCR4 41 GNTAIMAVSVLDIHLHTPVYFFLGNLSTLDICYTPTFVPLMLVHLLS 87

GSEDWPFGSALCKLVTALDVVNMYASILLLTAISIDRYLAIVHPLRYRRR +++ ++ l + l ++++| + ++ + +| I |+ ++| I I I I |+ I I I I +++ GPCR4 88 —SRKTISFAVCAIQMCLSLSTGSTECLLLAITAYDRYLAICQPLRYHVL 135 RTSPRRAKVVILLV VLALLLSLPPLLFS VKTVEEGNGTLNVNVTVCLI

++ +I+++++ +++III+M |+ + +++++++ I +++ I +++1 I GPCR4 136 MS-HRLCVLLMGAAVLCLLKSVTE-MVISMRLPFCGHHV—VSHFTCKI 181

DFPEESTASVST LRSYVLLSTLVGFLLPLLVILVCYTRILRTLR + +++ ++++ + I +++ +|| +|+ I++M |+ + ++

GPCR4 182 LAVLKLACGNTSVSEDFLLAGSILLLPVPLAFICLSYLLILATILRVPSA 231

...KAAKTLLVVVVVFVLC LPYFIVLLLDTLC.LSIIMSSTCELERVLP + |+ +++ ++++M+++ I+++I + +++ + GPCR4 232 ARCCKAFSTCLAHLAVVLLFYGTIIFMYLKPKSKEA HI 269

TALLVTLWLAYVNSCLNPIIY +I+++I+I +IM || GPCR4 270 SDEVFTVLYAMVTTMLNPTIY 290

The cDNA coding for the GPCR4b sequence was cloned by the polymerase chain reaction (PCR). Primers were designed based on in silico predictions ofthe full length or some portion (one or more exons) ofthe cDNA protein sequence ofthe invention. The DNA sequence and protein sequence for a novel Olfactory Receptor-like gene were obtained by exon linking and are reported here as GPCR4b. These primers and methods used to amplify GPCR4b cDNA are described in the Examples.

The GPCR4 disclosed in this invention is expressed in at least the following tissues: Apical micro villi ofthe retinal pigment epithelium, arterial (aortic), basal forebrain, brain, Burkitt lymphoma cell lines, corpus callosum, cardiac (atria and ventricle), caudate nucleus, CNS and peripheral tissue, cerebellum, cerebral cortex, colon, cortical neurogenic cells, endothelial (coronary artery and umbilical vein) cells, palate epithelia, eye, neonatal eye, frontal cortex, fetal hematopoietic cells, heart, hippocampus, hypothalamus, leukocytes, liver, fetal liver, lung, lung lymphoma cell lines, fetal lymphoid tissue, adult lymphoid tissue, Those that express MHC II and III nervous, medulla, subthalamic nucleus, ovary, pancreas, pituitary, placenta, pons, prostate, putamen, serum, skeletal muscle, small intestine, smooth muscle (coronary artery in aortic) spinal cord, spleen, stomach, taste receptor cells ofthe tongue, testis, thalamus, and thymus tissue. This information was derived by determining the tissue sources ofthe sequences that were included in the invention including but not limited to SeqCalling sources, Public EST sources, Literature sources, and/or RACE sources. This is by no way limiting in that olfactory receptors are a class of G protein-coupled receptor which are known to be expressed in all tissue types. Further tissue expression analysis is provided in Example 2.

The nucleic acids and proteins of GPCR4 are useful in potential therapeutic applications implicated in various GPCR-related pathological disorders and/or OR-related pathological disorders, described further above.

The novel nucleic acid encoding the GPCR-like protein ofthe invention, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed. These materials are further useful in the generation of antibodies that bind immunospecifically to the novel substances ofthe invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-GPCRX Antibodies" section below. The disclosed GPCR4b protein has multiple hydrophilic regions, each of which can be used as an immunogen. In one embodiment, a contemplated GPCR4b epitope is from about amino acids 220 to 250. In other specific embodiments, GPCR4b epitopes are from about amino acids 255 to 290.

GPCR5 The disclosed novel GPCR5 (alternatively referred to herein as 21629632.0.20) includes the 2028 nucleotide sequence (SEQ ID NO:43) shown in Table 5A. A GPCR5 ORF begins with a Kozak consensus ATG initiation codon at nucleotides 469-471 and ends with a TAG codon at nucleotides 1447-1449. Putative untranslated regions upstream from the initiation codon and downstream from the termination codon are underlined in Table 5A, and the start and stop codons are in bold letters. Table 5A. GPCR5 Nucleotide Sequence (SEQ ID NO:43)

TGCTATAGCCCCAGCACTTGATACCTAGCACAGAATAGGTACTTAATAAATACTTAGTGGATGAATAAATCTG AAATACTATGGCCATAATTTGGTCACATGAAGCCGTAATGTAGAAAAGATGCTTCCTGTTAATGACCAAAAAC ACTTTGGATTCCAAACGATCATTTTAAACATGAATCTTTCTCTGCTGTCTCCTCTGACCCCATCCTGGGGAGA GCAGAGAGGAGCCTAGGGGACTAGAATGTGCCCCATCCTCCCCTCAGTGACGTCCACAGAACTGCAGCGCTGA GAAGGCCAGATTGCAGATCTGAAGTCCAACTCCCTCATTATACAGATGGTGAAACTAAATTCCAGAGAGGGAG GCTGACCTGCTGCAGCTCAGACATCAGGTCACTGGGCTCCCAGGCCAGTTGGAGCTTTTTCCAAAAAGCTGGG TGGTCCAGATGGAAAAGGAGAGAGAATGAGATGAAGTGGGCAAACCAGACAGCTGTGACGGAATACGTCCTGA TGGGGCTACACGAGCACTGTAACCTGGAGGTGGTCCTGTTTGTGTTCTGCCTGGGCATCTACTCCGTGAATGT GTTGGGGAACGCCCTCCTCATAGGGCTGAACGTGCTGCACCCTCGCCTGCACAACCCCATGTACTTCCTTCTC AGCAACCTCTCCCTCATGGACATCTGCGGCACCTCCTCCTTTGTGCCTCTCATGCTAGACAATTTCCTGGAAA CCCAGAGGACCATTTCCTTCCCTGGCTGTGCCCTGCAGATGTACCTGACCCTGGCGCTGGGATCAACGGAGTG CCTGCTGCTGGCTGTGATGGCATATGACCGTTATGTGGCTATCTGCCAGCCGCTTAGGTACCCAGAGCTCATG AGTGGGCAGACCTGCATGCAGATGGCAGCGCTGAGCTGGGGGACAGGCTTTGCCAACTCACTGCTACAGTCCA TCCTTGTCTGGCACCTCCCCTTCTGTGGCCACGTCATCAACTACTTCTATGAGATCTTGGCAGTGCTAAAACT GGCCTGTGGGGACATCTCCCTCAATGCGCTGGCATTAATGGTGGCCACAGCCGTCCTGACACTGGCCCCCCTC TTGCTCATCTGCCTGTCTTACCTTTTCATCCTGTCTGCCATCCTTAGGGTACCCTCTGCTGCAGGCCGGTGCA AAGCCTTCTCCACCTGCTCAGCCCACCGCACAGTGGTGGTGGTTTTTTATGGGACAATCTCCTTCATGTACTT CAAACCCAAGGCCAAGGATCCCAACGTGGATAAGACTGTCGCATTGTTCTACGGGGTTGTGACGCCCTCGCTG AACCCCATCATTTACAGCCTGAGGAATGCAGAGGTGAAAGCTGCCGTCCTAACTCTGCTGAGAGGAGGTTTGC TCTCCAGGAAAGCATCCCACTGCTACTGCTGCCCTCTGCCCCTGTCAGCTGGCATAGGCTAGGTTGTGCTGTG GTCATGACCTCAAACCTTGAGAGGCTTAAAGCCATTAAGGTTTGTTTCTTGCTCCTGATGCAGGTCCACCAGA GGCTGGTGGGGCTTCTGCTCCGCATCATGGTCTTCACCCCTCTGGGACTCAGGATGACAAAACAGCTACCATT GGGAACACTGCTGGTCACCATGACAAAAAGAAAAGGGAAAGTAACAAAGCCTACACTGACTCTTAAAGCTTCT ACTCAGAAGTGGCTGTGTTGCCTCCACCTACATTTCAGTGGCCAACACAATGGCAACAGGAAGGCACAGGACC ACACCTATTGTTAAGGGGGAAAAGCACACTATCGTGTGTCTGGATGGCAAACGAGAGGGACAGAGAGATTTGT GAATGGCCTAATGACTACCACACCAGCTGACAGTGTCAACCCAAGAGCTATGGGAGGTTTGGCTTTCTTTATC CTGACCATCTATCCTTCACGGGCTGCTGCCAAGTTAATCGTCCCAAGAAAGCTCTGGTTAGCTCACGTGTGGT AGCTTTATACTGAGTCAACCAAACTAGGCTAGAGGGTGTGGGTTAGGGTTGGCCACA

The GPCR5 polypeptide (SEQ ID NO:44) encoded by SEQ ID NO:43 is 326 amino acids in length, has a molecular weight of 35715.1 Daltons, and is presented using the one- letter amino acid code in Table 5B. The Psort profile for GPCR5 predicts that these sequences have a signal peptide and are likely to be localized at the plasma membrane with a certainty of 0.0.6000. hi alternative embodiments, a GPCR5 polypeptide is located to the Golgi body with a certainty of 0.0.4000, the endoplasmic reticulum (membrane) with a certainty of 0.3000, or the mitochondrial innpr membrane with a certainty of 0.3000.

Table 5B. GPCR5 protein sequence (SEQ ID NO:44)

MKWANQTAVTEYVLMGLHEHCNLEVVLFVFCLGIYSVNVLGNALLIGLNVLHPRLHNPMYFLLSNLS LMDICGTSSFVPLMLDNFLETQRTISFPGCALQMYLTLALGSTECLLLAVMAYDRYVAICQPLRYPE LMSG^QTCMQMAALS GTGFANSLLQSILVWHLPFCGHVINYFYEILAVLKLACGDISLNALALMVAT AVLTLAPLLLICLSYLFILSAILRVPSAAGRCKAFSTCSAHRTVVVVFYGTISFMYFKPKAKDPNVD KTVALFYGVVTPSLNPIIYSLRNAEVKAAVLTLLRGGLLSRKASHCYCCPLPLSAGIG

Additional SNP variants of GPCR5 are disclosed in Example 3. The amino acid sequence of GPCR5 had high homology to other proteins as shown in Table 5C. Table 5C. BLASTX results for GPCR5

Smallest Sum

High Prob

Sequences producing High-scoring Segment Pairs : Score P(N) patp:AAG71895 Human olfactory receptor polypeptide, 311 aa 1588 5.2e-163 patp:AAG71924 Human olfactory receptor polypeptide, 197 aa 987 2.5e-99 patp:AAG72651 Murine OR-like polypeptide, 356 aa 895 1.4e-89~

In a search of sequence databases, it was found, for example, that the nucleic acid sequence of this invention has 1024 of 1351 bases (75%) identical to a gb:GENBANK- ID:-yi-MU133430|acc:AJ133430 mRNA from Mus musculus (Mus musculus ox6 gene, Fig. 3A). The full amino acid sequence ofthe protein ofthe invention was found to have 230 of 314 amino acid residues (73%) identical to, and 261 of 314 amino acid residues (83%) similar to, the 315 amino acid residue ptnr:SPTREMBL-ACC:Q9QZ17 protein from Mus musculus (Mouse) (OLFACTORY RECEPTOR).

Additional BLASTP results are shown in Table 5D.

A multiple sequence alignment is given in Table 5E, with the GPCR5 protein ofthe invention being shown on line 1, in a ClustalW analysis comparing GPCR5 with related protein sequences disclosed in Table 5D.

Table 5E. Information for the ClustalW proteins:

SEQ ID NO:44, GPCR5 SEQ ID NO:45, Q9QZ17 Olfactory receptor- Mus muscul us SEQ ID NO: 46, Q9QZ20 Olfactory receptor- Mus musculus SEQ ID NO: 47, Q9QZ21 Olfactory receptor- Mus musculus SEQ ID NO:48, Q9QZ22 Olfactory receptor- Mus musculus SEQ ID NO:49, Q9QZ19 Olfactory receptor- Mus musculus

Table 5F lists the domain description from DOMAIN analysis results against GPCR5. This protein contains domain IPR000276 at amino acid positions 41 to 287. This indicates that the GPCR5 sequence has properties similar to those of other proteins known to contain this domain as well as to the 254 amino acid 7tm domain (SEQ ID NO: 18) itself. Table 5F Domain Analysis of GPCR5

PSSMs producing significant alignments: Score E

(bits) value gnl|Pfam|pfam00001 7tm_ 1 , 7 transmembrane receptor (rhodopsin family) 114 3.3 e-35

7tm_l *->GNLLVILVILRTKKLRTPTNIFILNLAVADLLFLLTLPPWALYYLVG I l+l I + +++ + I ++)+++++ ||++ |++ + + +| +| +++ GPCR5 41 GNALLIGLNVLHPRLHNPMYFLLSNLSLMDICGTSSFVPLMLDNFLE 87

GSED PFGSALCKLVTALDVVNMYASILLLTAISIDRYLAIVHPLRYRRR + ++ +| |+++| ++ + + +| I |+++++| II + M+ 1111+ +

GPCR5 88 —TQRTISFPGCALQMYLTLALGSTECLLLAVMAYDRYVAICQPLRYPEL 135

RTSPRRAKVVILLVWVLALLLSLPPLLFS VKTVEEGNGTLN VN

++ + ++ + +| I +++ I |++ ++ ++++++ I ++ + + I

GPCR5 136 MS-GQTCMQMAALS GTGFANSLLQSIL-V HLPFCGHVINYFYEILAVL 183

VTVCLIDFPEESTASVSTWLRSYVLLSTLVGFLLPLLVILVCYTRILRTL

+ +1+ + ++I I +1 I I 1 + 1+ I++I I ++

GPCR5 184 KLACGDISLNAL ALMVATAVLTLAPLLLICLSYLFILSAI 223

R KAAKTLLVVVVVFVLC LPYFIVLLLDTLC.LSIIMSSTCE

+ ++ ++ I + ++ + ++|+ ++ +++ + ++++ GPCR5 224 LRVPSAAGRCKAFSTCSAHRTVVVVFYGTISFMYFKPKAKDP 265

LERVLPTALLVTLWLAYVNSCLNPIIY<-* + + I + I++++I + 1 I I I M

GPCR5 266 NVDKTVALFYGVVTPSLNPIIY 287

A GPCR-like protein ofthe invention, referred to herein as GPCR5, is an Olfactory

Receptor ("OR")-like protein. Some members ofthe Olfactory Receptor-Like Protein Family end up localized at the cell surface, where they exhibit activity. Therefore it is likely that these novel GPCR5 proteins are available at the appropriate sub-cellular localization and hence accessible for the therapeutic uses described in this application. The GPCR disclosed in this invention is expressed in at least in some of the following tissues: adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea, uterus. This information was derived by determining the tissue sources ofthe sequences that were included in the invention. In addition, the sequence is predicted to be expressed in the following tissues because ofthe expression pattern of (GENBANK-ID: gb:GENBANK-ID:MMU133430|acc: AJ133430) a closely related or6 gene homolog in species Mus musculus: olfactory epithelium. Expression data for GPCR5 is provided in Example 2.

The nucleic acids and proteins of GPCR5 are useful in potential therapeutic applications implicated in various GPCR-related pathological disorders and/or OR-related pathological disorders, described further above.

The novel nucleic acid encoding the GPCR-like protein ofthe invention, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed. These materials are further useful in the generation of antibodies that bind immunospecifically to the novel substances ofthe invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-GPCRX Antibodies" section below. The disclosed GPCR5 protein has multiple hydrophilic regions, each of which can be used as an immunogen. hi one embodiment, a contemplated GPCR5 epitope is from about amino acids 1 to 25. In another embodiment, a GPCR5 epitope is from about amino acids 120 to 140. hi further specific embodiments,

GPCR5 epitopes are from about amino acids 230 to 245 and from about amino acids 252 to 280.

GPCR6

A sixth GPCR-like protein ofthe invention, referred to herein as GPCR6, is an Olfactory Receptor ("OR")-like protein. Some members ofthe Olfactory Receptor-Like

Protein Family end up localized at the cell surface, where they exhibit activity. Therefore it is likely that these novel GPCR6 proteins are available at the appropriate sub-cellular localization and hence accessible for the therapeutic uses described in this application.

The disclosed novel GPCR6 (alternatively referred to herein as CG 50177-01) includes the 766 nucleotide sequence (SEQ ID NO:50) shown in Table 6A. A GPCR6 ORF begins with a Kozak consensus ATG initiation codon at nucleotides 2-4 and ends with a TAG codon at nucleotides 761-763. Putative untranslated regions upstream from the initiation codon and downstream from the termination codon are underlined in Table 6A, and the start and stop codons are in bold letters.

Table 6A. GPCR6 Nucleotide Sequence (SEQ ID NO:50)

GTCAGCCTCCAATATCACCTTAACACATCCAACTGCCTTCTTGTTGGTGGGGATTCCAGGCCTGGAA CACCTGCACATCTGGATCTCCATCCCTTTCTGCTTAGCATGTACACTGGCCCTGCTTGGAAACTGCA CTCTCCTTCTCATCATCCAGGCTGATGCAGCCCTCCATGAACCCATGTACCTCTTTCTGGCCATGTT GGCAGCCATCGACCTGGTCCTTTCCTCCTCAGCACTGCCCAAGATGCTTGCCATATTCTGGTTCAGG GATCGGGAGATAAACTTCTTTGCCTGTCTGGCCCAGATGTTCTTCCTTCACTCCTTCTCCATCATGG AGTCAGCAGTGCTGCTGGCCATGGCCTTTGACCGCTATGTGGCTATCTGCAAGCCACTGCACTACAC CAAGGTCCTGACTGGGTCCCTCATCACCAAGATTTTTATTGTGGTGTTGGACCTGCTCCTTGTTATC CTGTCTTATATCTTTATTCTTCAGGCAGTTCTACTGCTTGCCTCTCAGGAGGCCCGCTACAAGGCAT TTGGGACATGTGTCTCTCATATAGGTGCCATCTTAGCCTTCTACACAACTGTGGTCATCTCTTCAGT CATGCACCGTGTAGCCCGCCATGCTGCCCCTCATGTCCACATCCTCCTTACCAATTTCTATCTGCTC TTCCCACCCATGGTCAATCCCATAATCTATGGTGTCAAGACCAAGCAAATCCGTGAGAGCATCTTGG GAGTATTCCCAAGAAAGGATATGTAGAGG

The GPCR6 protein (SEQ ID NO:51 encoded by SEQ ID NO50 is 253 aa in length, has a molecular weight of 28233.48 Daltons, and is presented using the one-letter amino acid code in Table 6B. The Psort profile for GPCR6 predicts that these sequences have a signal peptide and are likely to be localized at the plasma membrane with a certainty of 0.640. The Signal P predicts a likely cleavage site for a GPCR6 peptide is between positions 54 and 55, i.e., at the dash in the sequence ADA-AL.

Table 6B. Encoded GPCR6 protein sequence (SEQ ID NO:51)

SASNITLTHPTAFLLVGIPGLEHLHI ISIPFCLACTLALLGNCTLLLIIQADAALHEPMYLFLAMLA AIDLVLSSSALPKMLAIFWFRDREINFFACLAQMFFLHSFSIMESAVLLAMAFDRYVAICKPLHYTKV LTGSLITKIFIVVLDLLLVILSYIFILQAVLLLASQEARYKAFGTCVSHIGAILAFYTTWISSVMHR VARHAAPHVHILLTNFYLLFPPMVNPIIYGVKTKQIRESILGVFPRKDM

Additional SNP variants are disclosed in Example 3. The amino acid sequence of GPCR6 had high homology to other proteins as shown in

Table 6C.

Table 6C. BLASTX results for GPCR6

Smallest Sum

High Prob

Sequences producing High-scoring Segment Pairs: Score P(N) patp :AAG72254 Human olfactory receptor polypeptide, 313 a. a. 492 2.7e-75 patp:AAG71568 Human olfactory receptor polypeptide, 319 a. a. 473 5.5e-73 patp :AAG71839 Human olfactory receptor polypeptide, 314 a. a. 733 2.1e-72 patp:AAB85003 rat olefactory recep. 320 a. a. 437 2.4e-70 patp:AAG71643 Human olfactory receptor polypeptide, 313 a. a. 456 2.7e-69

In a search of sequence databases, it was found, for example, that the nucleic acid sequence of this invention has 278 of 406 bases (68%) identical to a gb:GENBANK-

ID:AF121975|acc:AFl 21975.1 mRNA from Mus musculus (Mus musculus odorant receptor S18 gene, complete eds). The full amino acid sequence ofthe protein ofthe invention was found to have 87 of 163 amino acid residues (53%) identical to, and 116 of 163 amino acid residues (71%) similar to, the 321 amino acid residue ptnr:SPTREMBL-ACC:Q9WU89 protein from Mus musculus (Mouse) (ODORANT RECEPTOR SI 8).

GPCR6 also has homology to the proteins shown in the BLASTP data in Table 6D.

A multiple sequence alignment is given in Table 6E, with the GPCR6 protein being shown on line 1 in Table 6E in a ClustalW analysis, and comparing the GPCR6 protein with the related protein sequences shown in Table 6D. This BLASTP data is displayed graphically in the ClustalW in Table 6E.

Table 6E. ClustalW Analysis of GPCR6

1. SEQ ID NO 51, GPCR6

2. SEQ ID NO 52, Q9WU89, Odorant receptor S18/mouse

3. SEQ ID NO 53, 088628, Olfactory receptor 51E2/rat

4. SEQ ID NO 54, Q9WVD9, MOR 3'BETAl/mouse 51E2/human

5. SEQ ID NO 55, Q9H255, Olfactory receptor

6. SEQ ID NO 56, Q9H346, Olfactory receptor 52Dl/human

GPCR6 YGgKTKQIR |ESI|JGV[ PRKDM 253

Q9WU89 YGAKTKQIB jDSMTR LSWWKS 321

088628 |τgv0AMHκlSCDKDIEAGGNT 320

Q9 VD9 QDj WFLHSSVSTCQHDSRC— 326

Q9H255 |T VfflAMHKp:SCDKDLQAVGGK 320

Q9H346 JSSLBKLLHLGKTSI 318

Table 6F lists the domain description from DOMAIN analysis results against GPCR6.

This indicates that the GPCR6 sequence has properties similar to those of other proteins known to contain this domain as well as to the 254 amino acid 7tm domain (SEQ ID NO: 18) itself.

Table 6F Domain Analysis of GPCR6

PSSMs producing significant alignments: Score E

(bits) value gnl|Pfam|pfam00001 7tm_l , 7 transmembrane receptor (rhodopsin family) 53.3 1 e-08

GPCR6: 56 LHEPMYLFLAMLAAIDLVLSSSALPKMLAIFWFRDREINFFACLAQMFFLHSFSIMESAV 115 7tm_l: 15 LRTPTNIFLLNLAVADLLFLLTLPPWALYYLVGGDWVFGDALCKLVGALFWNGYASILL 74

GPCR6: 116 LLAMAFDRYVAICKPLHYTKVLT 138 (SEQ ID NO.51) 7tm_l: 75 LTAISIDRYLAIVHPLRYRRIRT 97 (SEQ ID NO: 18)

The GPCR6 disclosed in this invention is expressed in at least the following tissues: Apical micro villi ofthe retinal pigment epithelium, arterial (aortic), basal forebrain, brain, Burkitt lymphoma cell lines, corpus callosum, cardiac (atria and ventricle), caudate nucleus, CNS and peripheral tissue, cerebellum, cerebral cortex, colon, cortical neurogenic cells, endothelial (coronary artery and umbilical vein) cells, palate epithelia, eye, neonatal eye, frontal cortex, fetal hematopoietic cells, heart, hippocampus, hypothalamus, leukocytes, liver, fetal liver, lung, lung lymphoma cell lines, fetal lymphoid tissue, adult lymphoid tissue, Those that express MHC II and III nervous, medulla, subthalamic nucleus, ovary, pancreas, pituitary, placenta, pons, prostate, putamen, serum, skeletal muscle, small intestine, smooth muscle (coronary artery in aortic) spinal cord, spleen, stomach, taste receptor cells ofthe tongue, testis, thalamus, and thymus tissue. This information was derived by determining the tissue sources ofthe sequences that were included in the invention including but not limited to SeqCalling sources, Public EST sources, Literature sources, and/or RACE sources. Further expression data for GPCR6 is provided in Example 2.

The nucleic acids and proteins of GPCR6 are useful in potential therapeutic applications implicated in various GPCR-related pathological disorders and/or OR-related pathological disorders, described further above. The novel nucleic acid encoding the GPCR-like protein ofthe invention, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed. These materials are further useful in the generation of antibodies that bind immunospecifically to the novel substances ofthe invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-GPCRX Antibodies" section below. The disclosed GPCR6 protein has multiple hydrophilic regions, each of which can be used as an immunogen. In one embodiment, a contemplated GPCR6 epitope is from about amino acids 2 to 50. In another embodiment, a GPCR6 epitope is from about amino acids 50 to 80. In further specific embodiments, GPCR6 epitopes are from about amino acids 90 to 100, from about amino acids 100 to 175, from about amino acids 180 to 210 and from about amino acids 220 to 230.

GPCR7

A further GPCR-like protein ofthe invention, referred to herein as GPCR7, is an Olfactory Receptor ("OR")-like protein. The novel GPCR7 nucleic acid sequences were identified on chromosome 9 as described in Example 1. Some members ofthe Olfactory Receptor-Like Protein Family end up localized at the cell surface, where they exhibit activity. Therefore it is likely that these novel GPCR7 proteins are available at the appropriate sub- cellular localization and hence accessible for the therapeutic uses described in this application. Two alternative novel GPCR7 nucleic acids and encoded polypeptides are provided, namely GPCR7a and GPCR7b. GPCR7a

In one embodiment, a GPCR7 variant is the novel GPCR7a (alternatively referred to herein as CG50201-01), which includes the 1000 nucleotide sequence (SEQ ID NO:57) shown in Table 7A. A GPCR7a ORF begins with a Kozak consensus ATG initiation codon at nucleotides 3-5 and ends with a TGA codon at nucleotides 951-953. Putative untranslated regions upstream from the initiation codon and downstream from the termination codon are underlined in Table 7 A, and the start and stop codons are in bold letters.

Table 7A. GPCR7a Nucleotide Sequence (SEQ ID NO:57-)

CCATGGAGGCTGCCAATGAGTCTTCAGAGGGAATCTCATTCGTTTTATTGGGACTGACAACAAGTCC TGGACAGCAGCGGCCTCTCTTTGTGCTGTTCTTGCTCTTGTATGTGGCCAGCCTCCTGGGCAATGGA CTCATTGTGGCTGCCATCCAGGCCAGTCCAGCCCTTCATGCACCCATGTACTTCCTGCTGGCCCACC TGTCCTTTGCTGACCTCTGCTTCGCCTCCGTCACTGTGCCCAAGATGTTGGCCAACTTGTTGGCCCA TGACCACTCCATCTCGCTGGCTGGCTGCCTGACCCAAATGTACTTCTTCTTTGCCCTGGGGGTAACT GATAGCTGTCTTCTGGCGGCCATGGCCTATGACTGCTACGTGGCCATCCGGCACCCCCTCCCCTATG CCACGAGGATGTCCCGGGCCATGTGCGCAGCCCTGGTGGGAATGGCATGGCTGGTGTCCCACGTCCA CTCCCTCCTGTATATCCTGCTCATGGCTCGCTTGTCCTTCTGTGCTTCCCACCAAGTGCCCCACTTC TTCTGTGACCACCAGCCTCTCTTAAGGCTCTCGTGCTCTGACACCCACCACATCCAGCTGCTCATCT TCACCGAGGGCGCCGCAGTGGTGGTCACTCCCTTCCTGCTCATCCTCGCCTCCTATGGGGCCATCGC AGCTGCCGTGCTCCAGCTGCCCTCAGCCTCTGGGAGGCTCCGGGCTGTGTCCACCTGTGGCTCCCAC CTGGCTGTGGTGAGCCTCTTCTATGGGACAGTCATTGCAGTCTACTTCCAGGCCACATCCCGACGCG AGGCAGAGTGGGGCCGTGTGGCCACTGTCATGTACACTGTAGTCACCCCCATGCTGAACCCCATCAT CTACAGCCTCTGGAATCGCGATGTACAGGGGGCACTCCGAGCCCTTCTCATTGGGCGAAGGATCTCA GCTAGTGACTCCTGAGGGCAGGACCCCACTGAGGACAGACTGCATCACCCACACTGGCAACT

The GPCR7 protein (SEQ ID NO:58) encoded by SEQ ID NO:57 has 316 amino acid residues and is presented using the one-letter code in Table 7B. The predicted molecular weight of GPCR7 protein is approximately 34266.42 Daltons. The Psort profile for GPCR7 predicts that this sequence has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.4600. The Signal P predicts a likely cleavage site between positions 42 and 43, i.e., at the dash in the sequence LLG-NG.

The DNA sequence and protein sequence of GPCR7a was obtained by exon linking as described in the Example 1.

Table 7B. Encoded GPCR7a protein sequence (SEQ ID NO:58)

MEAANESSEGISFVLLGLTTSPG^QQRPLFVLFLLLYVASLLGNGLIVAAIQASPALHAPMYFLLAHLSFA DLCFASVTVPKMLANLLAHDHSISLAGCLTQMYFFFALGVTDSCLLAAMAYDCYVAIRHPLPYATRMSRA MCAALVGMA LVSHVHSLLYILLMARLSFCASHQVPHFFCDHQPLLRLSCSDTHHIQLLIFTEGAAVVVT PFLLILASYGAIAAAVLQLPSASGRLRAVSTCGSHLAVVSLFYGTVIAVYFQATSRREAEWGRVATVMYT VVTPMLNPIIYSL NRDVQGALRALLIGRRISASDS Additional SNP variants are disclosed in Example 3. GPCR7b

In an alternative embodiment, a GPCR7 variant is the novel GPCR7b (alternatively referred to herein as CG50257-01 or CG40267-01), which includes the 991 nucleotide sequence (SEQ ID NO:59) shown in Table 7C. The GPCR7b ORF begins with a Kozak consensus ATG initiation codon at nucleotides 3-5 and ends with a TGA codon at nucleotides 951-953, which are in bold letters in Table 4C.

Table 7C. GPCR7b Nucleotide Sequence (SEQ ID NO:59)

CCATGGAGGCTGCCAATGAGTCTTCAGAGGGAATCTCATTCGTTTTATTGGGACTGACAACAAGTCC TGGACAGCAGCGGCCTCTCTTTGTGCTGTTCTTGCTCTTGTATGTGGCCAGCCTCCTGGGTAATGGA CTCATTGTGGCTGCCATCCAGGCCAGTCCAGCCCTTCATGCACCCATGTACTTCCTGCTGGCCCACC TGTCCTTTGCTGACCTCTGCTTCGCCTCCGTCACTGTGCCCAAGATGTTGGCCAACTTGTTGGCCCA TGACCACTCCATCTCGCTGGCTGGCTGCCTGACCCAAATGTACTTCTTCTTTGCCCTGGGGGTAACT GATAGCTGTCTTCTGGCGGCCATGGCCTATGACTGCTACGTGGCCATCCGGCACCCCCTCCCCTATG CCACGAGGATGTCCCGGGCCATGTGCGCAGCCCTGGTGGGAATGGCATGGCTGGTGTCCCACGTCCA CTCCCTCCTGTATATCCTGCTCATGGCTCGCTTGTCCTTCTGTGCTTCCCACCAAGTGCCCCACTTC TTCTGTGACCACCAGCCTCTCTTAAGGCTCTCGTGCTCTGACACCCACCACATCCAGCTGCTCATCT TCACCGAGGGCGCCGCAGTGGTGGTCACTCCCTTCCTGCTCATCCTCGCCTCCTATGGGGCCATCGC AGCTGCCGTGCTCCAGCTGCCCTCAGCCTCTGGGAGGCTCCGGGCTGTGTCCACCTGTGGCTCCCAC CTGGCTGTGGTGAGCCTCTTCTATGGGACAGTCATTGCAGTCTACTTCCAGGCCACATCCCGACGCG AGGCAGAGTGGGGCCGTGTGGCCACTGTCATGTACACTGTAGTCACCCCCATGCTGAACCCCATCAT CTACAGCCTCTGGAATCGCGATGTACAGGGGGCACTCCGAGCCCTTCTCATTGGGCGAAGGATCTCA GCTAGTGACTCCTGAGGGCAGGACCCCACTGAGGACAGACTGCATCACCCACA

The GPCR7b protein (SEQ ID NO:60) encoded by SEQ ID NO:59 is 316 amino acids in length, has a molecular weight of 34266.42 Daltons, and is presented using the one-letter code in Table 7D. As with GPCR7a, the most likely cleavage site for a GPCR7b peptide is between amino acids 42 and 43, i.e., at the dash in the sequence LLG-NG, based on the SignalP result. The DNA sequence and protein sequence of GPCR7a was obtained by exon linking as described in the Example 1.

Table 7D. GPCR7b protein sequence (SEQ ID NO:60 )

MEAANESSEGISFVLLGLTTSPGQQRPLFVLFLLLYVASLLGNGLIVAAIQASPALHAPMYFLLAHL SFADLCFASVTVPKMLANLLAHDHSISLAGCLTQMYFFFALGVTDSCLLAAMAYDCYVAIRHPLPYA TRMSRAMCAALVGMA LVSHVHSLLYILLMARLSFCASHQVPHFFCDHQPLLRLSCSDTHHIQLLIF TEGAAVVVTPFLLILASYGAIAAAVLQLPSASGRLRAVSTCGSHLAVVSLFYGTVIAVYFQATSRRE AE GRVATVMYTVVTPMLNPIIYSLWNRDVQGALRALLIGRRISASDS GPCR7 Clones

The amino acid sequence of GPCR7 had high homology to other proteins as shown in Table 7e.

Table 7e. BLASTX results for GPCR7

Smallest Sum

High Prob

Sequences producing High-scoring Segment Pairs: Score P(N) ptnr:TREMBLNE -ACC:AAK95107 OLFACTORY RECEPTOR - Homo sap. 1115 1.0e-112 ptnr:SWISSPR0T-ACC:P23266 OLFACTORY RECEPTOR-LIKE PROTEIN. 870 9.3e-87 ptnr:pir-id:B23701 olfactory receptor F5 - rat, 313 aa. 864 4.0e-86 ptnr:S ISSNEW-ACC:Q43749 Olfactory receptor 1F1 (Qlfactor. 859 1.4e-85 ptnr:SPTREMBL-ACC:Q9TUAl OLFACTORY RECEPTOR - Pan troglod. ■ ■ 801 1.9e-79

In a search of sequence databases, it was found, for example, that the nucleic acid sequence of this invention has 592 of 880 bases (67%) identical to a gb:GENBANK- ID:RATOLFPROC|acc:M64377.1 mRNA from Rattus norvegicus (Rat olfactory protein mRNA, complete eds). The full amino acid sequence ofthe GPCR7 was found to have 174 of 309 amino acid residues (56%) identical to, and 223 of 309 amino acid residues (72%) similar to, the 313 amino acid residue ptnr:pir-id:B23701 protein from rat (olfactory receptor F5).

GPCR7 also has homology to the proteins shown in the BLASTP data in Table 7F.

A multiple sequence alignment is given in Table 7G, with the GPCR7 protein being shown on line 1 in Table 7G in a ClustalW analysis, and comparing the GPCR7 protein with the related protein sequences shown in Table 7F. This BLASTP data is displayed graphically in the ClustalW in Table 7G. The amino acid residues of GPCR7a and GPCR7b are identical. Table 7G. ClustalW Analysis of GPCR7

1. SEQ ID NO 58 GPCR7a

2. SEQ ID NO 60 GPCR7b

3. SEQ ID NO 61 AAK95107

4. SEQ ID NO 62 P23266

5. SEQ ID NO 63 B23701

6. SEQ ID NO 64 AC 043749

7. SEQ ID NO 65 Q9TUA1

GPCR7a ΪRALLIGgRIR SDS 316 GPCR7b RALLIGSRIR -SDS 316 O 02/40539

AAK95107 216

P23266 iRKVLAMgFP >@KQ— 313 B23701 IRKVLAMHFP j KQ— 313

AC 043749 iKK-VVGRWFgV— 312 Q9TUA1 IERVIXKJSKNPFLL- 314

Table 7H lists the domain description from DOMAIN analysis results against GPCR7. This indicates that the GPCR7 sequence has properties similar to those of other proteins known to contain this domain as well as to the 254 amino acid 7tm domain (SEQ ID NO: 18) itself.

Table 7H Domain Analysis of GPCR7

PSSMs producing significant alignments : Score E

(bits ) value gnl|Pfam|pfam00001 7tm_l, 7 transmembrane receptor (rhodopsin family) 105 5e-24

GPCR7: 42 GNGLIV-AAIQASPALHAPMYFLLAHLSFADLCFASVTVPKMLANLLAHDHSISLAGCLTQ 101 7tm 1: 1 GNLLVILVILRTKKLRTPTNIFLLNLAVADLLFLLTLPPWALYYLVGGD VFGDALCKLV 60

GPCR7: 102 MYFFFALGVTDSCLLAAMAYDCYVAIRHPLPYATRMSRAMCAALVGMALVSHVHSLLYI 161

7tm_l: 61 GALFVVNGYASILLLTAISIDRYLAIVHPLRYRRIRTPRRAKVLILLV VLALLLSLPPL 120

GPCR7: 162 LLMARLSFCASHQVPHFFCDHQPLLRLSCSDTHHIQLLIFTEGAAWVTPFLLILASYGA 221

7tm 1: 121 LFSWLRTVEEGNTTVCLIDFPEESVKR SYVLLSTLVG FVLPLLVILVCYTR 171

GPCR7: 222 IAAAV LQLPSASGRLRAVSTCGSHLAWSLFYGTVIAVYFQA TSRRE 268 7tm 1: 172 ILRTLRKRARSQRSLKRRSSSERKAAKMLLVWVVFVLCWLPYHIVLLLDSLCLLSI RV 231

GPCR7: 269 AE GRVATVMYTWTP LNPIIY 291 (SEQ ID NO: 58, 60) 7tm 1: 232 LPTALLITLWLAYVNSCLNPIIY 254 (SEQ ID NO: 18)

The GPCR7 protein predicted here is similar to the "Olfactory Receptor-Like Protein Family", some members of which end up localized at the cell surface where they exhibit activity. Therefore, it is likely that this novel GPCR7 protein is available at the appropriate sub-cellular localization and hence accessible for the therapeutic uses described in this application.

The Olfactory Receptor-like GPCR7 proteins disclosed is expressed in at least the following tissues: Apical microvilli ofthe retinal pigment epithelium, arterial (aortic), basal forebrain, brain, Burkitt lymphoma cell lines, corpus callosum, cardiac (atria and ventricle), caudate nucleus, CNS and peripheral tissue, cerebellum, cerebral cortex, colon, cortical neurogenic cells, endothelial (coronary artery and umbilical vein) cells, palate epithelia, eye, neonatal eye, frontal cortex, fetal hematopoietic cells, heart, hippocampus, hypothalamus, leukocytes, liver, fetal liver, lung, lung lymphoma cell lines, fetal lymphoid tissue, adult lymphoid tissue, Those that express MHC II and III nervous, medulla, subthalamic nucleus, ovary, pancreas, pituitary, placenta, pons, prostate, putamen, serum, skeletal muscle, small intestine, smooth muscle (coronary artery in aortic) spinal cord, spleen, stomach, taste receptor cells ofthe tongue, testis, thalamus, and thymus tissue. This information was derived by determining the tissue sources ofthe sequences that were included in the invention including but not limited to SeqCalling sources, Public EST sources, Literature sources, and/or RACE sources. Further expression data for GPCR7 is provided in Example 2.

This is by no way limiting in that olfactory receptors are a class of G protein-coupled receptor which are known to be expressed in all tissue types. Further tissue expression analysis is provided in the Examples.

The nucleic acids and proteins of GPCR7 are useful in potential therapeutic applications implicated in various GPCR-related pathological disorders and/or OR-related pathological disorders, described further herein. These materials are further useful in the generation of antibodies that bind immunospecifically to the novel substances ofthe invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-GPCRX Antibodies" section below. The disclosed GPCR7 protein has multiple hydrophilic regions, each of which can be used as an immunogen. hi one embodiment, a contemplated GPCR7 epitope is from about amino acids 10 to 80. hi additional embodiments, GPCR7 epitopes are from about amino acids 100 to 125, from about amino acids 130 to 160, from about amino acids 180 to 240 and from about amino acids 270 to 285.

GPCR8 The disclosed GPCR8 (also referred to as CG50193-01) includes the 1022 nucleotide sequence (SEQ ID NO:66) shown in Table 8A. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 34-36 and ending with a TAG codon at nucleotides 1013-1015. Putative untranslated regions upstream from the initiation codon and downstream from the termination codon are underlined in Table 8 A, and the start and stop codons are in bold letters. O 02 4

Table 8A. GPCR8 Nucleotide Sequence (SEQ ID NO:66)

TCTCTGTTTCCTCAGGGATTGAGAAAGGGGACAATGTGGCAGAAGAATCAGACCTCTCTGGCAGACT TCATCCTTGAGGGGCTCTTCGATGACTCCCTTACCCACCTTTTCCTTTTCTCCTTGACCATGGTGGT CTTCCTTATTGCGGTGAGTGGCAACACCCTCACCATTCTCCTCATCTGCATTGATCCCCAACTTCAT ACACCAATGTATTTCCTGCTCAGCCAGCTCTCCCTCATGGATCTGATGCATGTCTCCACAATCATCC TGAAGATGGCTACCAACTACCTATCTGGCAAGAAATCTATCTCCTTTGTGGGCTGTGCAACCCAGCA CTTCCTCTATTTGTGTCTAGGTGGTGCTGAATGTTTTCTCTTAGCTGTCATGTCCTATGACCGCTAT GTTGCCATCTGTCATCCACTGCGCTATGCTGTGCTCATGAACAAGAAGGTGGGACTGATGATGGCTG TCATGTCATGGTTGGGGGCATCCGTGAACTCCCTAATTCACATGGCGATCTTGATGCACTTCCCTTT CTGTGGGCCTCGGAAAGTCTACCACTTCTACTGTGAGTTCCCAGCTGTTGTGAAGTTGGTATGTGGC GACATCACTGTGTATGAGACCACAGTGTACATCAGCAGCATTCTCCTCCTCCTCCCCATCTTCCTGA TTTCTACATCCTATGTCTTCATCCTTCAAAGTGTCATTCAGATGCGCTCATCTGGGAGCAAGAGAAA TGCCTTTGCCACTTGTGGCTCCCACCTCACGGTGGTTTCTCTTTGGTTTGGTGCCTGCATCTTCTCC TACATGAGACCCAGGTCCCAGTGCACTCTATTGCAGAACAAAGTTGGTTCTGTGTTCTACAGCATCA TTACGCCCACATTGAATTCTCTGATTTATACTCTCCGGAATAAAGATGTAGCTAAGGCTCTGAGAAG AGTGCTGAGGAGAGATGTTATCACCCAGTGCATTCAACGACTGCAATTGTGGTTGCCCCGAGTGTAG AGTGGAATAGGATAAGC

The disclosed GPCR8 nucleic acid sequence of this invention has 574 of 908 bases (63%) identical to a Canis familiaris olfactory receptor (CfOLF3) gene (gb:GENBANK- ID:CFU53681|acc:U53681.1)(E = 1.3e-45).

The GPCR8 protein (SEQ ID NO:67) encoded by SEQ ID NO:66 is 323 aa in length and is presented using the one-letter amino acid code in Table 8B. The Psort, SignalP and/or Hydropathy results predict that GPCR8 has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6000. In alternative embodiments, a GPCR8 polypeptide is located to the Golgi body with a certainty of 0.4000, the endoplasmic reticulum (membrane) with a certainty of 0.3000, or the microbody (peroxisome) with a certainty of 0.3000. The SignalP shows a signal sequence is coded for in the first 45 amino acids with a likely cleavage site at between positions 45 and 46, at TLT-IL.

Table 8B. Encoded GPCR8 protein sequence (SEQ ID NO:67)

MWQKNQTSLADFILEGLFDDSLTHLFLFSLTMWFLIAVSGNTLTILLICIDPQLHTPMYFLLSQLSL MDLMHVSTIILKMΆTNYLSGKKSISFVGCATQHFLYLCLGGAECFLLAVMSYDRYVAICHPLRYAVLM NKKVGLMMAVMS LGASVNSLIHMAILMHFPFCGPRKVYHFYCEFPAVVKLVCGDITVYETTVYISSI LLLLPIFLISTSYVFILQSVIQMRSSGSKRNAFATCGSHLTVVSL FGACIFSYMRPRSQCTLLQNKV GSVFYSIITPTLNSLIYTLRNKDVAKALRRVLRRDVITQCIQRLQL LPRV

The full amino acid sequence ofthe protein ofthe invention was found to have 137 of 305 amino acid residues (44%) identical to, and 205 of 305 amino acid residues (67%) similar to, the 313 amino acid residue ptnr:SPTREMBL-ACC.O76000 protein from Homo sapiens (Human) (DJ80I19.1 OLFACTORY RECEPTOR-LIKE PROTEIN (HS6M1-1)).

Additional SNP variants of GPCR8 are disclosed in Example 3. The amino acid sequence of GPCR3 has high homology to other proteins as shown in Table 8C. Table 8C. BLASTX results for GPCR8

Smallest Sum

High Prob

Sequences producing High-scoring Segment Pairs : Score P (N) ρatp :A--E04578 Human G-protein coupled receptor-34 protein, 3₂3 aa 1662 7 .3e-171 ρatp:AAG7₂019 Human olfactory receptor polypeptide, 318 aa 1636 4 . 1e-168 ρatp :AAG7 165 Human olfactory receptor polypeptide, 318 aa 1631 1.4e-167 patp :--AG72348 Human OR-like polypeptide query sequence, 318 aa 1631 1.4e-167

GPCR8 also has homology to the proteins shown in the BLASTP data in Table 8D.

A multiple sequence alignment is given in Table 8E, with the GPCR8 protein being shown on line 1, in a ClustalW analysis comparing GPCR8 with the related protein sequences disclosed in Table 8D.

Table 8E. ClustalW Analysis of GPCR8

1 SEQ ID NO 67 , GPCR8

2 SEQ ID NO 68 , 043869

3 SEQ ID NO 69 , CAC34662

4 SEQ ID NO 70 , 076000

5 SEQ ID NO 71 , Q9EPF6

6 SEQ ID NO 72 , Q95156

60 70 30 90 100

110 120 130 140 150

160 170 180 190 200

210 220 230 240 250

310 320

Table 8F lists the domain description from DOMAIN analysis results against GPCR8. The results indicate that GPCR8 contains the protein domain 7tm_l (InterPro)7 transmembrane receptor (rhodopsin family) at amino acid positions 41 to 289. This indicates that the GPCR8 sequence has properties similar to those ofotherproteins known to contain this domain as well as to the 254 amino acid 7tm domain (SEQ ID NO:18) itself.

GNLLVILVILRTKKLRTPTNIFILNLAVADLLFLLTLPPALYYLVG || I +++++ ++++++H-+++ |++ ||+ + + + ++++++

GPCR8 41 GNTLTILLICIDPQLHTPMYFLLSQLSLMDLMHVSTIILKMATNYLS 87

GSED PFGSALCKLVTALDVVNMYASILLLTAISIDRYLAIVHPLRYRRR + + ++ ++| ++++++++ + + +++ I +++ I + I I l + l |++|+++ ++

GPCR8 88 G—KKSISFVGCATQHFLYLCLGGAECFLLAVMSYDRYVAICHPLRYAVL 135

RTSPRRAKVVILLV VLALLLSLPPLLFSWVKTVEEGNGTLNVNVTVCLI

++ +++++ + + |+ + + ++++ + +++++++ +4- +++++I +

GPCR8 136 MN-KKVGLMMAVMS LGASVNSLIH-MAILMHFPFCGPRK—VYHFYCEF 181

DFPEESTASVST LRSYVLLSTLVGFLLPLLVILVCYTRILRTLR

+ +++ + + +++++ + ++ I ++++++ | +|+ + + +++

GPCR8 182 PAVVKLVCG-DITVYETTVYISSILLLLPIFLISTSYVFILQSVIQMRSS 230

...KAAKTLLVVVVVFVLCWLPYFIVLLLDTLC.LSIIMSSTCELERVLP ++++ ++ ++ +++++ +|+ ++ ++++ ++ +

GPCR8 231 GSKRNAFATCGSHLTVVSLWFGACIFSYMRPRSQCT LL 268

TALLVTLWLAYVNSCLNPIIY + +++++ + +++| +| I

GPCR8 269 QNKVGSVFYSIITPTLNSLIY 289

The GPCR8 disclosed in this invention is expressed in at least the following tissues: Apical microvilli ofthe retinal pigment epithelium, arterial (aortic), basal forebrain, brain, Burkitt lymphoma cell lines, corpus callosum, cardiac (atria and ventricle), caudate nucleus, CNS and peripheral tissue, cerebellum, cerebral cortex, colon, cortical neurogenic cells, endothelial (coronary artery and umbilical vein) cells, palate epithelia, eye, neonatal eye, frontal cortex, fetal hematopoietic cells, heart, hippocampus, hypothalamus, leukocytes, liver, fetal liver, lung, lung lymphoma cell lines, fetal lymphoid tissue, adult lymphoid tissue, those that express MHC II and III nervous, medulla, subthalamic nucleus, ovary, pancreas, pituitary, placenta, pons, prostate, putamen, serum, skeletal muscle, small intestine, smooth muscle (coronary artery in aortic) spinal cord, spleen, stomach, taste receptor cells ofthe tongue, testis, thalamus, and thymus tissue. This information was derived by determining the tissue sources ofthe sequences that were included in the invention including but not limited to SeqCalling sources, Public EST sources, Literature sources, and/or RACE sources. Further expression data for GPCR8 is provided in Example 2.

The nucleic acids and proteins of GPCR8 are useful in potential therapeutic applications implicated in various GPCR-related pathological disorders and/or OR-related pathological disorders, described further above.

The novel nucleic acid encoding the GPCR-like protein ofthe invention, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed. These materials are further useful in the generation of antibodies that bind immunospecifically to the novel substances ofthe invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-GPCRX Antibodies" section below. The disclosed GPCR8 protein has multiple hydrophilic regions, each of which can be used as an immunogen. hi one embodiment, a contemplated GPCR8 epitope is from about amino acids 5 to 15. In another embodiment, a GPCR8 epitope is from about amino acids 88 to 94. In further specific embodiments, GPCR8 epitopes are from about amino acids 130 to 135, from about amino acids 172 to 178, from about amino acids 220 to 235, from about amino acids 260 to 275 and from about amino acids 290 to 323.

GPCR9 The disclosed GPCR9 (also referred to as CG50203-01) includes the 932 nucleotide sequence (SEQ ID NO:73) shown in Table 9A. An open reading frame was identified beginning with an ACA which codes for the amino acid Threonine at nucleotides 3-5 and ending with a TGA codon at nucleotides 900-902. Putative untranslated regions, if any, are found upstream from the initiation codon and downstream from the termination codon and are underlined in Table 8A. The start and stop codons are in bold letters.

Table 9A. GPCR9 Nucleotide Sequence (SEQ ID NO:73)

TGACAGAATTCATTCTTCTTGGTCTGACTCAGTCTCAAGATGCTCAACTTCTGGTCTTTGTGCTAGT CTTAATTTTCTACCTTATCATCCTCCCTGGAAATTTCCTCATCATTTTCACCATAAAGTCAGACCCT GGGCTCACAGCCCCCCTCTATTTCTTTCTGGGCAACTTGGCCTTACTGGATGCATCCTACTCCTTCA TTGTGGTTCCCAGGATGTTGGTGGACTTCCTCTCTGAGAAGAAGGTAATCTCCTATAGAAGCTGCAT CACTCAGCTCTTTTTCTTGCATTTTCTTGGAGCGGGAGAGATGTTCCTCCTCGTTGTGATGGCCTTT GACCGCTACATCGCCATCTGCCGGCCTTTACACTATTCAACCATCATGAACCCTAGAGCCTGCTATG CATTATCGTTGGTTCTGTGGCTTGGGGGCTTTATCCATTCCATTGTACAAGTAGCCCTTATCCTGCA CTTGCCTTTCTGTGGCCCAAACCAGCTCGATAACTTCTTCTGTGATGTTCCACAGGTCATCAAGCTG GCCTGCACCAATACCTTTGTGGTGGAGCTTCTGATGGTCTCCAACAGTGGCCTGCTCAGCCTCCTGT O 02/40539

GCTTCCTGGGCCTTCTGGCCTCCTATGCAGTCATCCTCTGTCGTATAAGGGAGCACTCCTCTGAAGG AAAGAGCAAGGCTATTTCCACATGCACCACCCATATTATCATTATATTTCTCATGTTTGGACCTGCT ATTTTCATCTACACTTGCCCCTTCCAGGCTTTCCCAGCTGACAAGGTAGTTTCTCTTTTCCATACTG TCATCTTTCCTTTGATGAACCCTGTTATTTATACGCTTCGCAACCAGGAGGTGAAAGCTTCCATGAG GAAGTTGTTAAGTCAACATATGTTTTGCTGAATAGAAGAAAGAGAAAAGCAAGAACGGAGA

The disclosed GPCR9 nucleic acid sequence of this.invention has 550 of 848 bases (64%) identical to a Mus musculus gene for odorant receptor MOR10 (gb:GENBANK- ID:AB030893|acc:AB030893.1)(E = 5.4e-54).

The GPCR9 protein (SEQ ID NO:74) encoded by SEQ ID NO:73 is 299 aa in length and is presented using the one-letter amino acid code in Table 9B. The Psort, SignalP and/or Hydropathy results predict that GPCR9 has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6000. In alternative embodiments, a GPCR9 polypeptide is located to the Golgi body with a certainty of 0.4000, the endoplasmic reticulum (membrane) with a certainty of 0.3000, or the mitochondrial inner membrane with a certainty of 0.0300. The SignalP shows a signal sequence is coded for in the first 35 amino acids with a likely cleavage site at between positions 35 and 36, at NFL-II.

Table 9B. Encoded GPCR9 protein sequence (SEQ ID NO:74)

TEFILLGLTQSQDAQLLVFVLVLIFYLIILPGNFLIIFTIKSDPGLTAPLYFFLGNLALLDASYSFI VVPRMLVDFLSEKKVISYRSCITQLFFLHFLGAGEMFLLVVMAFDRYIAICRPLHYSTIMNPRACYA LSLVLWLGGFIHSIVQVALILHLPFCGPNQLDNFFCDVPQVIKLACTNTFVVELLMVSNSGLLSLLC FLGLLASYAVILCRIREHSSEGKSKAISTCTTHIIIIFLMFGPAIFIYTCPFQAFPADKVVSLFHTV IFPLMNPVIYTLRNQEVKASMRKLLSQHMFC

The amino acid sequence of GPCR9 had high homology to other proteins as shown in Table 9C.

Table 9C. BLASTX results for GPCR9

S allest Sum

High Prob patp Sequences producing High-scoring Segment Pairs: Score P(N)

AAG71733 Human olfactory receptor polypeptide, 308 aa 1536 1.6e- -157

AAY92364 Human G protein-coupled receptor protein 4, 307 aa 1336 2.6e- -136

AAE02496 Human C0N197 G protein-coupled receptor protein, 307 aa 1336 2.6e-136

AAG71483 Human olfactory receptor polypeptide, 317 aa 1335 3.3e- -136

AAG71692 Human olfactory receptor polypeptide, 316 aa 1335 6.8e- -136

GPCR9 also has homology to the proteins shown in the BLASTP data in Table 9D.

Table 9D. GPCR9 BLASTP results

Gene Index / Protein / Organism Length Identity Positives Expect Identifier (aa) (%) (%)

A multiple sequence alignment is given in Table 9E, with the GPCR9 protein being shown on line 1, in a ClustalW analysis comparing GPCR9 with the related protein sequences disclosed in Table 9D.

Table 9E. ClustalW Analysis of GPCR9

1. SEQ ID NO:74, GPCR9

2. SEQ ID NO:75, Q9R0K3 ODORANT RECEPTOR MOR83

3. SEQ ID NO:76, Q15615 Olfactory receptor 4D1 4. SEQ ID NO:77, P58180 Olfactory receptor 4D2

5. SEQ ID NO:78, Q9R0K4 ODORANT RECEPTOR MOR10

6. SEQ ID NO:79, Q9R0K5 ODORANT RECEPTOR MOR28

310 . . . . I . . . . I . . . GPCR9 MJgKJlLSQHMFC — Q9ROK3 MKHfflRQRRICS —

Q15615 V pi GKCLVICRE

P58180 VSRHGRHRLV—

Q9ROK4 IKRREK Q9R0K5 IRRKEGKEK

Table 9F lists the domain description from DOMAIN analysis results against GPCR9. This indicates that the GPCR9 sequence has properties similar to those of other proteins known to contain this domain as well as to the 254 amino acid 7tm domain (SEQ ID NO.T8) itself.

Table 9F Domain Analysis of GPCR9

PSSMs producing significant alignments: Score E

(bits) value gnl|Pfam|pfam00001 7tm_l, 7 transmembrane receptor (rhodopsin family) 110.9 3.1e-34

7tm_l GNLLVILVILRTKKLRTPTNIFILNLAVADLLFLLTLPPWALYYLVG | | | +++++ + + +++++ 1 I I + I +++ +++ +++ ++ GPCR9 32 GNFLII FTIKSDPGLTAPLYFFLGNLALLDASYSFIVVPRMLVDFLS 78

GSEDWPFGSALCKLVTALDVVNMYASILLLTAISI DRYLAIVHPLRYRRR + +++++ I ++ ++ + + +++ I +++++ I I I + I I ++ I +₄-++++

GPCR9 79 — EKKVISYRSCITQLFFLHFLGAGEMFLLVVMAFDRYIAICRPLHYSTI 126 RTSPRRAKVVILLV VLALLLSLPPLLFSWVKTVEEGNGTLN ++ ++ +++++++ |+ +++ ++ ++ + +++++++ +++++ + ++ GPCR9 127 MN-PRACYALSLVL LGGFIHSIVQVAL-ILHLPFCGPNQLDnffcdvpq 174

VNVTVCLIDFPEESTASVST LRSYVLLSTLVGFLLPLLVILVCYTRILR

+++ +|+ +++ + + ++ + + +++++ ++ I +|++

GPCR9 175 VIKLACTNTFVVEL LMVSNSGLLSLLCFLGLLASYAVILC 214

TLR KAAKTLLVVVVVFVLC LPYFIVLLLDTLC.LSIIMSSTC

++++++++++ + +++ +++ +++ + +++++ ++ ++

GPCR9 215 RIRehssegkSKAISTCTTHIIIIFLMFGPAIFIYTCPFQaFP 257

ELERVLPTALLVTL LAYVNSCLNPIIY +++ ++++ + + + +| l+l 1

GPCR9 258 ADKVVSLFHTVIF—PLMNPVIY 278

The GPCR9 disclosed in this invention is expressed in at least the following tissues: Apical microvilli ofthe retinal pigment epithelium, arterial (aortic), basal forebrain, brain, Burkitt lymphoma cell lines, corpus callosum, cardiac (atria and ventricle), caudate nucleus, CNS and peripheral tissue, cerebellum, cerebral cortex, colon, cortical neurogenic cells, endothelial (coronary artery and umbilical vein) cells, palate epithelia, eye, neonatal eye, frontal cortex, fetal hematopoietic cells, heart, hippocampus, hypothalamus, leukocytes, liver, fetal liver, lung, lung lymphoma cell lines, fetal lymphoid tissue, adult lymphoid tissue, Those that express MHC II and III nervous, medulla, subthalamic nucleus, ovary, pancreas, pituitary, placenta, pons, prostate, putamen, serum, skeletal muscle, small intestine, smooth muscle (coronary artery in aortic) spinal cord, spleen, stomach, taste receptor cells ofthe tongue, testis, thalamus, and thymus tissue. This information was derived by determining the tissue sources ofthe sequences that were included in the invention including but not limited to SeqCalling sources, Public EST sources, Literature sources, and/or RACE sources. Further expression data for GPCR9 is provided in Example 2.

In addition, the sequence is predicted to be expressed in the following tissues because ofthe expression pattern of (GENBANK-ID: gb:GENBANK-ID:AB030893lacc:AB030893.1) a closely related Mus musculus gene for odorant receptor MOR10, complete eds homolog in species Mus musculus: Brain. The nucleic acids and proteins of GPCR9 are useful in potential therapeutic applications implicated in various GPCR-related pathological disorders and/or OR-related pathological disorders, described further above.

The novel nucleic acid encoding the GPCR-like protein ofthe invention, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed. These materials are further useful in the O 02/40539 generation of antibodies that bind immunospecifically to the novel substances ofthe invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-GPCRX Antibodies" section below. The disclosed GPCR9 protein has multiple hydrophilic regions, each of which can be used as an immunogen. hi one embodiment, a contemplated GPCR9 epitope is from about amino acids 75 to 80. In another embodiment, a GPCR9 epitope is from about amino acids 118 to 123. hi further specific embodiments, GPCR9 epitopes are from about amino acids 127 to 132, from about amino acids 165 to 172, from about amino acids 215 to 230 and from about amino acids 280 to 299.

GPCR10

GPCRl 0 includes two GPCR proteins disclosed below. The disclosed proteins have been named GPCRl 0a and GPCRl 0b, and are related to olfactory receptors.

GPCRl 0a The disclosed GPCRlOa nucleic acid (SEQ ID NO:80) of 984 nucleotides (also referred to as CG50197-01) is shown in Table 10A. An open reading frame was identified beginning with an CTC initiation codon, which codes for leucine, at nucleotides 2-4 and ending with a TAA codon at nucleotides 941-943. Putative untranslated regions found upstream from the first codon and downstream from the termination codon are underlined in Table 10A, and the start and stop codons are in bold letters.

Table 10A. GPCRlOa Nucleotide Sequence (SEQ ID NO:80)

CCTCCAAAGAGCCACTTTCTTCCTGACGGGCTTCCAAGGTCTAGAAGGTCTCCATGGCTGGATCTCT ATTCCCTTCTGCTTCATCTACCTGACAGTTATCTTGGGGAACCTCACCATTCTCCACGTCATTTGTA CTGATGCCACTCTCCATGGACCCATGTACTATTTCTTGGGCATGCTAGCTGTCACAGACTTAGGCCT TTGCCTTTCCACACTGCCCACTGTGCTGGGCATTTTCTGGTTTGATACCAGAGAGATTGGCATCCCT GCCTGTTTCACTCAGCTCTTCTTCATCCACACCTTGTCTTCAATGGAGTCATCAGTTCTGTTATCCA TGTCCATTGACCGCTACGTGGCCGTCTGCAACCCACTGCATGACTCCACCGTCCTGACACCTGCATG TATTGTCAAGATGGGGCTAAGCTCAGTGCTTAGAAGTGCTCTCCTCATCCTCCCCTTGCCATTCCTC CTGAAGCGCTTCCAATACTGCCACTCCCATGTGCTGGCTCATGCTTATTGTCTTCACCTGGAGATCA TGAAGCTGGCCTGCTCTAGCATCATTGTCAATCACATCTATGGGCTCTTTGTTGTGGCCTGCACCGT GGGTGTGGACTCCCTGCTCATCTTTCTCTCATACGCCCTCATCCTTCGCACCGTGCTCAGCATTGCC TCCCACCAGGAGCGACTCCGAGCCCTCAACACCTGTGTCTCTCATATCTGTGCTGTACTGCTCTTCT ACATCCCCATGATTGGCTTGTCTCTTGTGCATCGCTTTGGTGAACATCTGCCCCGCGTTGTACACCT CTTCATGTCCTATGTGTATCTGCTGGTACCACCCCTTATGAACCCCATCATCTACAGCATCAAGACC AAGCAAATTCGCCAGCGCATCATTAAGAAGTTTCAGTTTATAAAGTCACTTAGGTGTTTTTGGAAGG ATTAAGTTAGAGTAAAGAGAGGAAGTTTTGGACATAAAGCCCACAG The disclosed GPCRlOa nucleic acid sequence has 593 of 888 bases (66%) identical to aMus musculus MOR 5'beta3, MOR 5'beta2, and MOR 5'betal mRNA; beta-globin (Hbb) gene, Hbb-D allele, locus control region (gb:GENBANK-ID:AF071080|acc:AF071080.2 ) (E

- Lie ⁶¹).

The disclosed GPCRlOa polypeptide (SEQ ID NO:81) encoded by SEQ ID NO:80 has 313 amino acid residues and is presented using the one-letter amino acid code in Table 10B. The SignalP, Psort and/or Hydropathy results predict that GPCRlOa has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6000. The most likely cleavage site for a GPCRlOa peptide is between amino acids 38 and 39, at: NTL-IL.

Table 10B. GPCRlOa protein sequence (SEQ ID NO:81)

LQRATFFLTGFQGLEGLHGWISIPFCFIYLTVILGNLTILHVICTDATLHGPMYYFLGMLAVTDLGLCL STLPTVLGIFWFDTREIGIPACFTQLFFIHTLSSMESSVLLSMSIDRYVAVCNPLHDSTVLTPACIVKM GLSSVLRSALLILPLPFLLKRFQYCHSHVLAHAYCLHLEIMKLACSSIIVNHIYGLFVVACTVGVDSLL IFLSYALILRTVLSIASHQERLRALNTCVSHICAVLLFYIPMIGLSLVHRFGEHLPRVVHLFMSYVYLL VPPLMNPIIYSIKTKQIRQRIIKKFQFIKSLRCFWKD

The disclosed GPCRlOa amino acid sequence has 161 of 301 amino acid residues (53%) identical to, and 223 of 301 amino acid residues (74%) similar to, the Mus musculus 315 amino acid residue MOR 5ΗETA3 protein (ptnr:SPTREMBL-ACC:Q9WVN6)(E = 7.2e^{" 89}).

GPCRl 0b

In the present invention, the target sequence identified previously, Accession Number CG50197-01, was subjected to the exon linking process to confirm the sequence. GPCRlOb was isolated as described in Example 1. GPCRlOb differs from the previously identified sequence (Accession Number CG50197-01) in having 2 extra amino acid at N-terminal and 1 aminoacid changed at position 117 Y->S.

The disclosed GPCRlOb nucleic acid (SEQ ID NO:82) of 1008 nucleotides (also refeired to as CG50197-02) is shown in Table IOC. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 20-22 and ending with a TAA codon at nucleotides 965-967. Putative untranslated regions found upstream from the initiation codon and downstream from the termination codon are underlined in Table 10C, and the start and stop codons are in bold letters. Table IOC. GPCRlOb Nucleotide Sequence (SEQ ID NO-82).

CTATGACAATTCTTCTTAATGCAGCCTCCAAAGAGCCACTTTCTTCCTGACGGGCTTCCAAGGTCTAGAA GGTCTCCATGGCTGGATCTCTATTCCCTTCTGCTTCATCTACCTGACAGTTATCTTGGGGAACCTCACCA TTCTCCACGTCATTTGTACTGATGCCACTCTCCATGGACCCATGTACTATTTCTTGGGCATGCTAGCTGT CACAGACTTAGGCCTTTGCCTTTCCACACTGCCCACTGTGCTGGGCATTTTCTGGTTTGATACCAGAGAG ATTGGCATCCCTGCCTGTTTCACTCAGCTCTTCTTCATCCACACCTTGTCTTCAATGGAGTCATCAGTTC TGTTATCCATGTCCATTGACCGCTCCGTGGCCGTCTGCAACCCACTGCATGACTCCACCGTCCTGACACC TGCATGTATTGTCAAGATGGGGCTAAGCTCAGTGCTTAGAAGTGCTCTCCTCATCCTCCCCTTGCCATTC CTCCTGAAGCGCTTCCAATACTGCCACTCCCATGTGCTGGCTCATGCTTATTGTCTTCACCTGGAGATCA TGAAGCTGGCCTGCTCTAGCATCATTGTCAATCACATCTATGGGCTCTTTGTTGTGGCCTGCACCGTGGG TGTGGACTCCCTGCTCATCTTTCTCTCATACGCCCTCATCCTTCGCACCGTGCTCAGCATTGCCTCCCAC CAGGAGCGACTCCGAGCCCTCAACACCTGTGTCTCTCATATCTGTGCTGTACTGCTCTTCTACATCCCCA TGATTGGCTTGTCTCTTGTGCATCGCTTTGGTGAACATCTGCCCCGCGTTGTACACCTCTTCATGTCCTA TGTGTATCTGCTGGTACCACCCCTTATGAACCCCATCATCTACAGCATCAAGACCAAGCAAATTCGCCAG CGCATCATTAAGAAGTTTCAGTTTATAAAGTCACTTAGGTGTTTTTGGAAGGATTAAGTTAGAGTAAAGA GAGGAAGTTTTGGACATAAAGCCCACAG

The disclosed GPCRlOb nucleic acid sequence has 564 of 880 bases (64%) identical to aMus musculus odorant receptor S19 mRNA (gb:GENBANK-ID:AF121976|acc:AF121976.2) (E = 1.9e^"51).

The disclosed GPCRlOb polypeptide (SEQ ID NO:83) encoded by SEQ ID NO:82 has 315 amino acid residues and is presented using the one-letter amino acid code in Table 10D. The SignalP, Psort and/or Hydropathy results predict that GPCRlOb has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6000. The most likely cleavage site for a GPCRlOb peptide is between amino acids 49 and 50, at: TDA-TL.

Table 10D. GPCRlOb protein sequence (SEQ ID NO:83)

CSLQRATFFLTGFQGLEGLHG ISIPFCFIYLTVILGNLTILHVICTDATLHGPMYYFLGMLAVTDLGL CLSTLPTVLGIF FDTREIGIPACFTQLFFIHTLSSMESSVLLSMSIDRSVAVCNPLHDSTVLTPACIV KMGLSSVLRSALLILPLPFLLKRFQYCHSHVLAHAYCLHLEIMKLACSSIIVNHIYGLFVVACTVGVDS LLIFLSYALILRTVLSIASHQERLRALNTCVSHICAVLLFYIPMIGLSLVHRFGEHLPRVVHLFMSYVY LLVPPLMNPIIYSIKTKQIRQRIIKKFQFIKSLRCF KD

The disclosed GPCRlOb amino acid sequence 164 of 295 amino acid residues (55%) identical to, and 223 of 295 amino acid residues (75%) similar to, the Mus musculus 319 amino acid residue protein; MOR 3ΗETA4 (ptnr:TREMBLNEW-ACC:AAG41684)(E = 8.1e^"

90

)^•

GPCRl 0 Family

The term GPCRl 0 is used to refer to all GPCRl 0 variants or members ofthe GPCRl 0 family disclosed herein unless we identify a specific family member or variant. GPCRl 0 is expressed in at least the following tissues: Apical micro villi ofthe retinal pigment epithelium, arterial (aortic), basal forebrain, brain, Burkitt lymphoma cell lines, corpus callosum, cardiac (atria and ventricle), caudate nucleus, CNS and peripheral tissue, cerebellum, cerebral cortex, colon, cortical neurogenic cells, endothelial (coronary artery and umbilical vein) cells, palate epithelia, eye, neonatal eye, frontal cortex, fetal hematopoietic cells, heart, hippocampus, hypothalamus, leukocytes, liver, fetal liver, lung, lung lymphoma cell lines, fetal lymphoid tissue, adult lymphoid tissue, those that express MHC II and III nervous, medulla, subthalamic nucleus, ovary, pancreas, pituitary, placenta, pons, prostate, putamen, serum, skeletal muscle, small intestine, smooth muscle (coronary artery in aortic) spinal cord, spleen, stomach, taste receptor cells ofthe tongue, testis, thalamus, and thymus tissue. This information was derived by determining the tissue sources ofthe sequences that were included in the invention including but not limited to SeqCalling sources, Public EST sources, Literature sources, and/or RACE sources. Further expression data for GPCRl 0 is provided in Example 2.

In addition, the GPCRl 0 sequence is predicted to be expressed in brain tissue because ofthe expression pattern of a closely related Mus musculus MOR 5'beta3, MOR 5'beta2, and MOR 5'betal genes, complete eds; beta-globin (Hbb) gene, Hbb-D allele, locus control region homolog (GENBANK-ID: gb:GENBANK-ID:AF071080|acc:AF071080.2).

Homologies between the GPCRl 0 variants is shown in a Clustal W in Table 10F.

Table 10F. Clustal W of GPCR10 Family

10 20 30 40 50 60 . . . . I . . . . | . . . . | |....|....|....|..

GPCRlOa

GPCRlOb CTATGACAATTCTTCTTAATGCAGl -T-maMBj-πw MHBH--II

70 80 90 100 110 120 ..| .. ..|..

GPCRlOa GPCRlOb -GGTCTAGAAGGTCTCCATGGCTGGATCTCTATTCCCTTCTGCTTCATCTACCTGACAG'-

130 140 150 160 170 180 ..|..

GPCRlOa GPCRlOb ATCTTGGGGAACCTCACCATTCTCCACGTCATTTGTACTGATGl

190 200 210 220 230 240

GPCRlOa -ATGTACTATTTCTTGGGCATGCTAGCTGTCACAGACTTAGGCCTTTGCC-.TTCCACAC-: GPCRlOb :ATGTACTATTTCTTGGGCATGCTAGCTGTCACAGACTTAGGCCTTTGCCTTTCCACACΪ

250 260 270 280 290 300 ..|....|....|

GPCRlOa GPCRlOb GCCCACTGTGCTGGGCATTTTCTGGTTTGATACCAGAGAGATTGGCATCCCTGCCTGTT-: 310 320 330 340 350 360 . . | . . . . | . . . . I

GPCRlOa :ACTCAGCTCTTCTTCATCCACACCTTGTCTTCAATGGAGTCATCAGTTCTGTTAT( GPCRlOb CACTCAGCTCTTCTTCATCCACACCTTGTCTTCAATGGAGTCATCAGTTCTGTTATCCA'

GPCRlOb

430 440 450 460 470 480 ..I

GPCRlOa ?GCATGTATTGTCAAGATGGGGCTAAGCTCAGTGCTTAGAAGTGCTCTCCTCHTCCTI GPCRlOb TGCATGTATTGTCAAGATGGGGCTAAGCTCAGTGCTTAGAAGTGCTCTCCTCATCCTCC

490 500 510 520 530 540 ..|.. ..|.. I....|..

GPCRlOa :TTGCCATTCCTCCTGAAGCGCTTCCAATACTGCCACTCCCATGTGCTGGCTCATGCTTA GPCRlOb :TTGCCATTCCTCCTGAAGCGCTTCCAATACTGCCACTCCCATGTGCTGGCTCATGCTTA

550 560 570 580 590 600

GPCRlOa rTGTCTTCACCTGGAGATCATGAAGCTGGCCTGCTCTAGCATCATTGTCAATCACATCT-. GPCRlOb rTGTCTTCACCTGGAGATCATGAAGCTGGCCTGCTCTAGCATCATTGTCAATCACATCTS

610 620 630 640 650 660 1....I

GPCRl

GPCRlOb .'GGCCTGCACCGTGGGTGTGGACTCCCTGCTCATCTTTCTCTCATA

670 680 690 700 710 720

GPCRlOa f-.-J«l^.-.^-.-.-feW-.-.-feW-,-.-<.-ι GPCRlOb -GCCCTCATCCTTCGCACCGTGCTCAGCATTGCCTCCCACCAGGAGCGACTCCGAGCCC

730 740 750 760 770 780 I .... I I I ....1.... I - ... I

GPCRlOa ------------------------------------------^^

GPCRlOb w--a-ft.----ι-.-M--ι-,ww

790 800 810 820 830 840

GPCRlOa₀ GPCRlOb Mifflffl--«-fflr-fflim

850 360 870 880 890 900 . I .. ..|..

GPCRlOa CTGGTACCACCCCTTATGAACCCCATCATCTACAGCATCAAGACCAAGC-- GPCRlOb CTGGTACCACCCCTTATGAACCCCATCATCTACAGCATCAAGACCAAGC-

910 920 930 940 950 960 ..|.. ..I..

GPCRlOa ----τ --3C ---,uι-ii-.--TCATTAAGAAGTTTCAGTTTATAAAGTCACTTAGGTGTTTTTGGA GPCRlOb &ATTCGCCAGCGCATCATTAAGAAGTTTCAGTTTATAAAGTCACTTAGGTG-.TTTTGGA-

970 980 990 1000 ..I.. ..|...

GPCRlOa GGATTAAGTTAGAGTAAAGAGAGGAAGTTTTGGACATAAAGCCCACAG GPCRlOb GGATTAAG-.TAGAGTAAAGAGAGGAAGTTTTGGACATAAAGCCCAC

The disclosed GPCRlOa has homology to the amino acid sequences shown in the BLASTP data listed in Table 10G.

The homology of these sequences is shown graphically in the ClustalW analysis shown in Table 10H.

Table 10H. ClustalW Analysis of GPCRlOa

1) GPCRlOa (SEQ ID NO: 81)

2) AF137396 (SEQ ID NO: 84)

3) AF133300 (SEQ ID NO: 85)

4) NP 038644.1 (SEQ ID NO:86)

5) NP 038646.1 (SEQ ID NO:87)

6) NP 038645.1 (SEQ ID NO:88)

250 260 270 280 290 300

310 320

The homologies shown above are shared by GPCRlOb insofar as GPCRlOa and GPCRlOb are homologous as shown in Table 10F. Table 101 lists the domain description from DOMAIN analysis results against

GPCRlOa. This indicates that the GPCRlOa sequence has properties similar to those of other proteins known to contain this domain as well as to the 377 amino acid 7tm domain (SEQ ID NO: 18) itself.

Table 101. Domain Analysis of GPCRlOa gnl I Pfaml farαOOOOl, 7tm_l, 7 transmembrane receptor

(rhodopsin family) . (SEQ ID NO:XXX)

Length = 254 residues, 100.0% aligned

Score = 58.5 bits (140), Expect = 5e-10

GPCRlOa 35 GNLTILHVICTDATLHGP YYF GMLAVTDLGLCLSTLPTVLGIFWFDTREIGIPACFTQ 94

I I I ++ I I I I I I I I I I I I + I I I I 00001 1 GNL VI VILRTKKLRTPTNIFLLNLAVADLLFL T PP ALYY VGGD VFGDA CK V 60

GPCRlOa 95 LFFIHTLSSMESSVL SMSIDRYVAVCNPLHDSTVLTPACIVKMGLSSV RS — LILP 152

+1 ++ 1 I I I I +1 + + 1 I + 11 + 1 + + I 00001 61 GA FVVNGYASILL TAISIDRYLAIVHP RYRRIRTPRRAKVLIL VWV ALL SLPPL 120

GPCRlOa 153 LPF LKRFQYCHSHVLAHAYCLH EIMKLACSSIIVNHIYGLFWACTVGVDSLLIF SY 212

I |+ + ++ | + l +l . | |+ +| |

00001 121 LFSW RTVEEGNTTVC IDFPEESVKRSYV LSTLVGFV PLLVI VCYTR—ILRTLRK 178

GPCRlOa 213 A ILRTVLSIASHQERLRALNTCVSHICAVLLFYIPMIGLS VHRFGEHLPRVVHLF SY 272

+ | l ll +l + + +I ++I + |+ I

00001 179 RARSQRSLKRRSSSER-KAAKML WVWFV C PYHIVLLLDS CL SI RV PTAX 237

GPCRlOa 273 VYL VP PLMNPIIY 286

+ I + +I1III 00001 238 ITLW AYVNSCLNPIIY 254

GPCRl 0 polypeptides are useful in the generation of antibodies that bind immunospecifically to the GPCRlO polypeptides ofthe invention. These antibodies maybe generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-GPCRX Antibodies" section below. The disclosed GPCRlO proteins have multiple hydrophilic regions, each of which can be used as an immunogen. h one embodiment, a contemplated GPCRlO epitope is from about amino acids 225 to 235. In another embodiment, a GPCRlO epitope is from about amino acids 280 to 305. The GPCRlO proteins also have value in the development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology ofthe disease and development of new drug targets for various disorders.

GPCR11 The disclosed GPCR11 nucleic acid (SEQ ID NO:89) of 922 nucleotides (also referred to as CG50199-01) is shown in Table 11A . An open reading frame was identified beginning with an AGT initiation codon, which codes for serine, at nucleotides 2-4 and ending with a TGA codon at nucleotides 920-922. A putative untranslated region upstream from the initiation codon is underlined in Table 11 A, and the start and stop codons are in bold letters.

Table 11A. GPCR11 Nucleotide Sequence (SEQ ID NO:89)

CAGTGAATTTGTTCTCGTGAGCTTCTCAGCCCTGTCCACTGAGCTTCAGGCTCTACTGTTTCTCCTTTT CTTGACCATTTACTTGGTTACTTTAATGGGCAATGTCCTCATCATCCTGGTCACTATAGCTGACTCTGC ACTACAAAGTCCTATGTACTTCTTCCTCAGAAACTTGTCCTTCCTGGAGATAGGTTTCAACTTGGTCAT TGTGTCCAAGATGCTGGGGACCCTGATCATTCAAGACACAACCATCTCCTTCCTTGGATGTGCCACTCA GATGTATTTCTTCTTCTTTTTTGGGGCTGCTGAGTGCTGCCTCCTGGCCACCATGGCATATGACCGCTA CGTGGCCATCTGTGACCCCTTGTACTACCCAGTCATCATGGGCCACATATCCTGTGCCCAGCTGGCAGC TGCCTCTTGGTTCTCAGGGTTTTCAGTGGCCACTGTGCAAACCACATGGATTTTCAGTTTCCCTTTTTG TGGCCCCAACAGGGTGAACCACTTCTTCTGTGACAGCCCTCCTGTTATTGCACTGGTCTGTGCTGACAC CTCTGTGTTTGAACTGGAGGCTCTGACAGCCACTGTCCTATTCATTCTCTTTCCTTTCTTGCTGATCCT GGGATCCTATGTCCGCATCCTCTCCACTATCTTCAGGATGCCGTCAGCTGAGGGGAAACATCAGGCATT CTCCACCTGTTCCGCCCACCTCTTGGTTGTCTCTCTCTTCTATAGCACTGCCATCCTCACGTATTTCCG ACCCCAATCCAGTGCCTCTTCTGAGAGCAAGAAGCTGCTGTCACTCTCTTCCACAGTGGTGACTCCCAT GTTGAACCCCATCATCTACAGCTCAAGGAATAAAGAAGTGAAGGCTGCACTGAAGCGGCTTATCCACAG GAACCTGGGCTCTCAGAAACTATGA

The disclosed GPCRl 1 nucleic acid sequence of this invention has 794 of 922 bases (86%) identical to a Mus musculus olfactory receptor P2 (Olfrl7) mRNA (gb:GENBANK- ID:AF247657|acc:AF247657.1) (E = 1.7e^"149). The disclosed GPCRl 1 polypeptide (SEQ ID NO:90) encoded by SEQ ID NO:89 has

306 amino acid residues and is presented using the one-letter code in Table 1 IB. The SignalP, Psort and/or Hydropathy results predict that GPCRl 1 has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6400. The most likely cleavage site for a GPCRl 1 peptide is between amino acids 45 and 46, at: ADS-AL.

Table 11B. Encoded GPCR11 protein sequence (SEQ ID NO:90).

SEFVLVSFSALSTELQALLFLLFLTIYLVTLMGNVLIILVTIADSALQSPMYFFLRNLSFLEIGFNLVI VSKMLGTLIIQDTTISFLGCATQMYFFFFFGAAECCLLATMAYDRYVAICDPLYYPVIMGHISCAQLAA ASWFSGFSVATVQTT IFSFPFCGPNRVNHFFCDSPPVIALVCADTSVFELEALTATVLFILFPFLLIL GSYVRILSTIFRMPSAEGKHQAFSTCSAHLLVVSLFYSTAILTYFRPQSSASSESKKLLSLSSTVVTPM LNPIIYSSRNKEVKAALKRLIHRNLGSQKL

The disclosed GPCRl 1 amino acid sequence has 268 of 305 amino acid residues

(87%) identical to, and 286 of 305 amino acid residues (93%) similar to, the Mus musculus 315 amino acid residue protein olfactory receptor P2 (ρtm:SPTREMBL-ACC:Q9JKA6) (E = 8.6e^"141).

GPCRl 1 is expressed in at least the following tissues: Apical microvilli ofthe retinal pigment epithelium, arterial (aortic), basal forebrain, brain, Burkitt lymphoma cell lines, corpus callosum, cardiac (atria and ventricle), caudate nucleus, CNS and peripheral tissue, cerebellum, cerebral cortex, colon, cortical neurogenic cells, endothelial (coronary artery and umbilical vein) cells, palate epithelia, eye, neonatal eye, frontal cortex, fetal hematopoietic cells, heart, hippocampus, hypothalamus, leukocytes, liver, fetal liver, lung, lung lymphoma cell lines, fetal lymphoid tissue, adult lymphoid tissue, Those that express MHC II and III nervous, medulla, subthalamic nucleus, ovary, pancreas, pituitary, placenta, pons, prostate, putamen, serum, skeletal muscle, small intestine, smooth muscle (coronary artery in aortic) spinal cord, spleen, stomach, taste receptor cells ofthe tongue, testis, thalamus, and thymus tissue. This information was derived by determining the tissue sources ofthe sequences that were included in the invention including but not limited to SeqCalling sources, Public EST sources, Literature sources, and/or RACE sources. Further expression data for GPCRl 1 is provided in Example 2.

In addition, the GPCRl 1 sequence is predicted to be expressed in brain tissue because ofthe expression pattern of a closely related Mus musculus olfactory receptor P2 (Olfrl7) gene homolog (GENBANK-ID: gb:GENBANK-ID:AF247657|acc:AF247657.1).

The disclosed GPCRl 1 has homology to the amino acid sequences shown in the BLASTP data listed in Table 1 IC.

The homology of these sequences is shown graphically in the ClustalW analysis shown in Table 1 ID.

Table 1 ID. ClustalW Analysis of GPCRl 1

1) GPCRll (SEQ ID NO: 90)

2) Q9H209 (SEQ ID NO: 91)

3) NP_065623.1 (SEQ ID NO:92) 4) Q9H207 (SEQ ID NO: 93)

5) AAG45186.1 (SEQ ID NO:94)

6) AAG45207.1 (SEQ ID NO:95)

130 140 150 160 170 180 I ■I....1....1....I....1....1

GPCRll B3-a^ΞB^DS!^γl |HISC0Q| a.qJ-JAlιιl«-ι-L-[fcιaιaa--ιawιaP RgN.[j

250 260 270 280 290 300

Table 1 IE lists the domain description from DOMAIN analysis results against GPCRl 1. This indicates that the GPCRl 1 sequence has properties similar to those of other proteins known to contain this domain as well as to the 377 amino acid 7tm domain (SEQ IS NO: 18) itself.

Table HE. Domain Analysis of GPCRll gnl I Pfaml famOOOOl, 7tm_l, 7 transmembrane receptor (rhodopsin family) . (SEQ ID NO:XXX) Length = 254 residues, 100.0% aligned Score = 85.5 bits (210), Expect = 4e-18

GPCRll 33 GNVLIILVTIADSALQSP YFFLRNLSFLEIGFNLVIVSK LGTLIIQDTTISFLGCATQ 92 11 + 1 + 111 + I ++ 1 I I I I + ++ I I + I I + I I

00001 1 GNLLVILVILRTK LRTPTNIFLLNLAVΛDLLFLLTLPP ALYYLVGGD VFGDALCKLV 60

GPCRll 93 MYFFFFFGAAECCLLATMAYDRYVAICDPLYYPVIMGHISCAQLAAASWFSGFSVATVQT 152

I I I 1 I ++ II l+ll II I I I I ++ 00001 61 GALFVVNGYASILLLTAISIDRYLAIVHPLRYRRIRTPRRAKVLILLVWVLALLLSLPPL 120

GPCRll 153 TWIFSFPFCGPNRVNHFFCDSPPVIALVCADTSVFELEALTATVLFILFPFLLILGSYVR 212 + + I + I I |+ + + +1 00001 121 LFSWLRTVEEGNTTVCLIDFPEESVKRSYVLLSTLVGFVLPLLVILVCYTRILRTLRKRA 180

GPCRll 213 ILSTIFR PSAEGKHQAFSTCSAHLLVVSLFYSTAIL TYFRPQSSASSESKKLLSL 268

+ | + + | +--- I + | + + + ] +--- 1

00001 181 RSQRSLKRRSSSERKAAKMLLVVWVFVLC LPYHIVLLLDSLCLLSI RVLPTALLITL 240

GPCRll 269 SSTWTP LNPIIY 282

I M U M

00001 241 WLAYVNSCLNPIIY 254

GPCRl 1 polypeptides are useful in the generation of antibodies that bind immunospecifically to the GPCRl 1 polypeptides ofthe invention. The antibodies are for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-GPCRX Antibodies" section below. The disclosed GPCRl 1 protein has multiple hydrophilic regions, each of which can be used as an immunogen. In one embodiment, a contemplated GPCRl 1 epitope is from about amino acids 160 to 170. In another embodiment, a GPCRl 1 epitope is from about amino acids 210 to 230. In additional embodiments, GPCRl 1 epitopes are from about amino acids 245 to 260 and from about amino acids 280 to 300. The GPCRl 1 protein also has value in the development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology ofthe disease and development of new drug targets for various disorders.

GPCR12

Yet another GPCR-like protein ofthe invention, referred to herein as GPCR12 (alternatively referred to as CG50217-01), is an Olfactory Receptor ("OR")-like protein. The novel GPCR12 nucleic acid (SEQ ID NO:96) of 1019 nucleotides encoding a novel Olfactory Receptor-like protein is shown in Table 12A. An open reading frame for the mature protein was identified beginning with a GTG codon which codes for the amino acid Valine at nucleotides 1-3 and ending with a TGA codon at nucleotides 943-45. Putative untranslated regions downstream from the termination codon are underlined in Table 12A, and the start and stop codons are in bold letters.

Table 12A. GPCR12 Nucleotide Sequence (SEQ ID NO:96)

GTGCTGGCTTCAGGGAACAGCTCTTCTCATCCTGTGTCCTTCATCCTGCTTGGAATCCCAGGCCTGGA GAGTTTCCAGTTGTGGATTGCCTTTCCGTTCTGTGCCACGTATGCTGTGGCTGTTGTTGGAAATATCA CTCTCCTCCATGTAATCAGAATTGACCACACCCTGCATGAGCCCATGTACCTCTTTCTGGCCATGCTG GCCATCACTGACCTGGTCCTCTCCTCCTCCACTCAACCTAAGATGTTGGCCATATTCTGGTTTCATGC TCATGAGATTCAGTACCATGCCTGCCTCATCCAGGTGTTCTTCATCCATGCCTTTTCTTCTGTGGAGT CTGGGGTGCTCATGGCTATGGCCCTGGACTGCTACGTGGCTATCTGCTTCCCACTCCGACACTCTAGC ATCCTGACCCCATCGGTCGTGATCAAACTGGGGACCATCGTGATGCTGAGAGGGCTGCTGTGGGTGAG CCCCTTCTGCTTCATGGTGTCTAGGATGCCCTTCTGCCAACACCAAGCCATTCCCCAGTCATACTGTG AGCACATGGCTGTGCTGAAGTTGGTGTGTGCTGATACAAGCATAAGTCGTGGGAATGGGCTCTTTGTG GCCTTCTCTGTGGCTGGCTTTGATATGATTGTCATTGGTATGTCATACGTGATGATTTTGAGAGCTGT GCTTCAGTTGCCCTCAGGTGAAGCCCGCCTCAAAGCTTTTAGCACACGTTCCTCCCATATCTGTGTCA TCTTGGCTCTTTATATCCCAGCCCTTTTTTCTTTCCTCACCTACCGCTTTGGCCATGATGTGCCCCGA GTTGTACACATCCTGTTTGCTAATCTCTATCTACTGATACCTCCCATGCTCAACCCCATCATTTATGG AGTTAGAACCAAACAGATCGGGGACAGGGTTATCCAAGGATGTTGTGGAAACATCCCCTGAGCAAΆGG GTCAGTGTATCCCCATCACTTACATTGCCCCACTAATGTGGGGACATTAATGAACATTTGACAGGCT

In a search of sequence databases, it was found that the disclosed GPCRl 2 nucleic acid sequence has 600 of 877 bases (68%) identical to a gb:GENBANK-ID:GGA012570| acc:AJ012570.1 mRNA from Gallus gallus (Gallus gallus DNA sequence downstream of beta- globin locus) (E = 1.4e^'73)).

The disclosed GPCR12 ploypeptide (SEQ ID NO: 97) encoded by SEQ ID NO: 96 has 314 amino acid residues using the one-letter code in Table 12B. The SignalP, Psort and/or Hydropathy results predict that GPCR12 has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6400. In alternative embodiments, a GPCR12 polypeptide is located to the Golgi body with a certainty of 0.4600, the endoplasmic reticulum (membrane) with a certainty of 0.3700, or the endoplasmic reticulum (lumen) with a certainty of 0.1000. The most likely cleavage site for a GPCR12 peptide is between amino acids 40 and 41, i.e. at the dash in the sequence ANA-W.

Table 12B. GPCR12 protein sequence (SEQ ID ΝO.97)

VLASGNSSSHPVSFILLGIPGLESFQLWIAFPFCATYAVAVVGNITLLHVIRIDHTLHEPMYLFLAMLAI TDLVLSSSTQPKMLAIFWFHAHEIQYHACLIQVFFIHAFSSVESGVLMAMALDCYVAICFPLRHSSILTP SVVIKLGTIVMLRGLL VSPFCFMVSRMPFCQHQAIPQSYCEHMAVLKLVCADTSISRGNGLFVAFSVAG FDMIVIGMSYVMILRAVLQLPSGEARLKAFSTRSSHICVILALYIPALFSFLTYRFGHDVPRVVHILFAN LYLLIPPMLNPIIYGVRTKQIGDRVIQGCCGNIP

Additional SNP variants of GPRC12 are disclosed in Example 3. Expression data for GPCR12 is provided in Example 2. hi a search of a proprietary PatP database, the amino acid sequence of GPCR12 was found to have high homology to other OR-like proteins, as shown in Table 12C.

Table 12C. BLASTX results for GPCR12

Smallest Sum High Prob

_ρatp: Sequences producing High-scoring Segment Pairs: Score P(N) -AG71726 Human olfactory receptor polypeptide, 314 aa 1607 5. le-165

J-AG71545 Human olfactory receptor polypeptide, 314 aa 924 1.2e-92 AAG72396 Human OR-like polypeptide query sequence, 314 aa 924 1 2e-92

AAG71651 Human olfactory receptor polypeptide, 321 aa 909 4 7e-91

AAE02491 Human CON193 G protein-coupled receptor protein, 321 aa 904 1 6e-90

The disclosed GCPR12 amino acid sequence has 163 of 285 amino acid residues (57%) identical to, and 210 of 285 amino acid residues (73%) similar to, the 339 amino acid residue ptnr: SPTREMBL- ACC:Q9WU90 protein from Mus musculus (Mouse) (ODORANT RECEPTOR SI 9) (E = 1.9e^"88).

Additional BLASTP results are shown in Table 12D.

A multiple sequence alignment is given in Table 12E, with the GPCRl 2 protein being shown on line 1 in Table 12E in a ClustalW analysis, and comparing the GPCR12 protein with the related protein sequences shown in Table 12D. This BLASTP data is displayed graphically in the ClustalW in Table 12E.

Table 12E. ClustalW Analysis of GPCR12

1. SEQ ID NO: 97, GPCRl2

2. SEQ ID NO:98, Q9 U90

3. SEQ ID NO: 99, Q9 VD9

4. SEQ ID NO: 100, Q9EQQ5

5. SEQ ID NO:101, Q9H346

6. SEQ ID NO:102, Q9UKL2

60 70 80 90 100

g g g g g -3 --- Q Q -30 g g g fl g g g g g α Q n n Q π

KD KD KD KD Ω KD KD KD KD KD Ω KD KD KD KD KD Ω KD KD KD KD Ω KD KD KD KD Ω

-) -> 10 io n 10 10 -i 10 -> n -⁾ 10 -no -> π ID -⁾ lO lO ID O so so so so Ώ

5. t- ≤ ≤ " Cj IB H -≤ -≤ HJ 5 K H 2 ≤ T. g a H 2 ≤ H) Cj S-- t?- 2j Sj *ti ω KD < c-J fθ w ω -0 < α !fl w ω D < 3 -fl « ω D 9 W « ωβ 3 m fc O D (O H r fcO io H H MO O IO H r iMO 0 -) μ H f> o ϋ 10 μ σi ϋi so 0 to to σs ui <--) 0 to to σ- in «D 0 to to σ- αi «D 0 to 10 0. 01 w 0 to

XX: Q9UKL2

Table 12F lists the domain description from DOMAIN analysis results against GPRC12. This protein contains domain 7tm_l, 7 transmembrane receptor at amino acid positions 43 to 255. This indicates that the GCPR12 sequence has properties similar to those of other proteins known to contain this domain as well as to the 255 amino acid 7tm domain (SEQ ID NO: 18) itself.

Table 12F Domain Analysis of GPCR12

PSSMs producing significant alignments: Score E

(bits) value gnl|Pfam)pfa--n00001 7tm_l, 7 transmembrane receptor (rhodopsin family) 44.7 2.1e-13

7 m_l GNLLVILVILRTKKLRTPTNIFILNLAVADLLFLLTLPP ALYYLVG I | + + ++ ++ ++++++ I | ++ | I ++ + + ++ ++++ GPCR12 43 GNITLLHVIRIDHTLHEPMYLF AMLAITDLVLSSSTQPKMLAIF F 89

GSEDWPFGSALCKLVTALDWNMYASI LTAISIDRY AIVHPLRYRRR + + ++ I ++++++ +++ + I ++++ 1 I + 1 I + 1 ++++++

GPCR12 90 —HAHEIQYHACLIQVFFIHAFSSVESGVLMAMALDCYVAICFPLRHSSI 137

RTSPRRAKVVILLV VLALLLSLPPLLFS VKTVEEGNGTLNV VTVCLI

+ + + ++ ++ + ++++ |+ ++ ++++++ ++ +4-4- +|

GPCR12 138 LT-PSVVIKLGTIVM RGL WVSPFCFM-VSRMPFCQHQ —IPQSYCEH 183

DFPEESTASVSTWLRSYVLLSTLVGFLLPLLVILVCYTRILRTLR

+ + ++ + + +++ ++ + + + ++++ I |++++ + 4-4-4-

GPCR12 184 MAVLKLVCADTSISRGNGLFVAFSVAGFD IVIG SYV ILRAVLQLPSG 233 KAAKT WV

+ + + ++++++++ + 4-4-4-4-4- GPCR12 234 EARLKAFSTRSSHICVI ALYI 255

The Olfactory Receptor-like GPCRl 2 disclosed in this invention is expressed in at least the following tissues: Apical micro villi ofthe retinal pigment epithelium, arterial (aortic), basal forebrain, brain, Burkitt lymphoma cell lines, corpus callosum, cardiac (atria and ventricle), caudate nucleus, CNS and peripheral tissue, cerebellum, cerebral cortex, colon, cortical neurogenic cells, endothelial (coronary artery and umbilical vein) cells, palate epithelia, eye, neonatal eye, frontal cortex, fetal hematopoietic cells, heart, hippocampus, hypothalamus, leukocytes, liver, fetal liver, lung, lung lymphoma cell lines, fetal lymphoid tissue, adult lymphoid tissue, Those that express MHC II and III nervous, medulla, subthalamic nucleus, ovary, pancreas, pituitary, placenta, pons, prostate, putamen, serum, skeletal muscle, small intestine, smooth muscle (coronary artery in aortic) spinal cord, spleen, stomach, taste receptor cells ofthe tongue, testis, thalamus, and thymus tissue. This information was derived by determining the tissue sources ofthe sequences that were included in the invention including but not limited to SeqCalling sources, Public EST sources, Literature sources, and/or RACE sources. Further expression data for GPCR12 is provided in Example 2. The nucleic acids and proteins of GPCRl 2 are useful in potential diagnostic and therapeutic applications implicated in various GPCR-related pathological diseases and/or disorders, and/or in various other pathologies, as described above.

The polypeptides can be used as immunogens to produce antibodies specific for the invention, and as vaccines. They can also be used to screen for potential agonist and antagonist compounds. For example, a cDNA encoding the OR -like protein may be useful in gene therapy, and the OR-like protein may be useful when administered to a subject in need thereof. The novel nucleic acid encoding OR-like protein, and the OR-like protein ofthe invention, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount ofthe nucleic acid or the protein are to be assessed. These materials are further useful in the generation of antibodies that bind immunospecifically to the novel substances ofthe invention for use in therapeutic or diagnostic methods and other diseases, disorders and conditions ofthe like.

These antibodies may be generated according to methods known in the art, using predictions from hydrophobicity charts, as described in the "Anti-GPCRX Antibodies" section below. The disclosed GPCRl 2 protein has multiple hydrophylhc regions, each of which can be used as an immunogen. In one embodiment, a contemplated GPCRl 2 epitope is from about amino acids 80 to 95. In another embodiment, a GPCR12 epitope is from about amino acids 170 to 185. In -further embodiments, a GCPR12 epitope is from about 230 to 247. In yet further specific embodiments, a GPCRl 2 epitope is from about amino acids 290 to 325.

GPCRX Nucleic Acids and Polypeptides

A summary ofthe GPCRX nucleic acids and proteins ofthe invention is provided in Table 13. TABLE 13: Summary Of Nucleic Acids And Proteins Of The Invention

One aspect ofthe invention pertains to isolated nucleic acid molecules that encode GPCRX polypeptides or biologically active portions thereof. Also included in the invention are nucleic acid fragments sufficient for use as hybridization probes to identify GPCRX- encoding nucleic acids (e.g., GPCRX mRNAs) and fragments for use as PCR primers for the amplification and/or mutation of GPCRX nucleic acid molecules. As used herein, the term "nucleic acid molecule" is intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g. , mRNA), analogs of the DNA or RNA generated using nucleotide analogs, and derivatives, fragments and homologs thereof. The nucleic acid molecule may be single-stranded or double-stranded, but preferably is comprised double- stranded DNA.

An GPCRX nucleic acid can encode a mature GPCRX polypeptide. As used herein, a "mature" form of a polypeptide or protein disclosed in the present invention is the product of a naturally occurring polypeptide or precursor form or proprotein. The naturally occurring polypeptide, precursor or proprotein includes, by way of nonlimiting example, the full-length gene product, encoded by the corresponding gene. Alternatively, it may be defined as the polypeptide, precursor or proprotein encoded by an ORF described herein. The product "mature" form arises, again by way of nonlimiting example, as a result of one or more naturally occurring processing steps as they may take place within the cell, or host cell, in which the gene product arises. Examples of such processing steps leading to a "mature" form of a polypeptide or protein include the cleavage ofthe N-terminal methionine residue encoded by the initiation codon of an ORF, or the proteolytic cleavage of a signal peptide or leader sequence. Thus a mature form arising from a precursor polypeptide or protein that has residues 1 to N, where residue 1 is the N-terminal methionine, would have residues 2 through N remaining after removal ofthe N-terminal methionine. Alternatively, a mature form arising from a precursor polypeptide or protein having residues 1 to N, in which an N-terminal signal sequence from residue 1 to residue M is cleaved, would have the residues from residue M+l to residue N remaining. Further as used herein,. a "mature" form of a polypeptide or protein may arise from a step of post-translational modification other than a proteolytic cleavage event. Such additional processes include, by way of non-limiting example, glycosylation, myristoylation or phosphorylation. In general, a mature polypeptide or protein may result from the operation of only one of these processes, or a combination of any of them.

The term "probes", as utilized herein, refers to nucleic acid sequences of variable length, preferably between at least about 10 nucleotides (nt), 100 nt, or as many as approximately, e.g., 6,000 nt, depending upon the specific use. Probes are used in the detection of identical, similar, or complementary nucleic acid sequences. Longer length probes are generally obtained from a natural or recombinant source, are highly specific, and much slower to hybridize than shorter-length oligomer probes. Probes may be single- or double-stranded and designed to have specificity in PCR, membrane-based hybridization technologies, or ELISA-like technologies.

The term "isolated" nucleic acid molecule, as utilized herein, is one, which is separated from other nucleic acid molecules which are present in the natural source ofthe nucleic acid. Preferably, an "isolated" nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5'- and 3'-termini ofthe nucleic acid) in the genomic DNA ofthe organism from which the nucleic acid is derived. For example, in various embodiments, the isolated GPCRX nucleic acid molecules can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA ofthe cell/tissue from which the nucleic acid is derived (e.g., brain, heart, liver, spleen, etc.). Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material or culture medium when produced by recombinant techniques, or of chemical precursors or other chemicals when chemically synthesized. A nucleic acid molecule ofthe invention, e.g., a nucleic acid molecule having the nucleotide sequence of SEQ ID NOS:l, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96, or a complement of this aforementioned nucleotide sequence, can be isolated using standard molecular biology techniques and the sequence information provided herein. Using all or a portion ofthe nucleic acid sequence of SEQ ID NOS:l, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96 as a hybridization probe, GPCRX molecules can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook, et al., (eds.), MOLECULAR CLONING: A LABORATORY MANUAL 2^nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Ausubel, et al, (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, NY, 1993.) A nucleic acid ofthe invention can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to GPCRX nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.

As used herein, the term "oligonucleotide" refers to a series of linked nucleotide residues, which oligonucleotide has a sufficient number of nucleotide bases to be used in a PCR reaction. A short oligonucleotide sequence may be based on, or designed from, a genomic or cDNA sequence and is used to amplify, confirm, or reveal the presence of an identical, similar or complementary DNA or RNA in a particular cell or tissue.

Oligonucleotides comprise portions of a nucleic acid sequence having about 10 nt, 50 nt, or 100 nt in length, preferably about 15 nt to 30 nt in length, hi one embodiment ofthe invention, an oligonucleotide comprising a nucleic acid molecule less than 100 nt in length would further comprise at least 6 contiguous nucleotides of SEQ ID NOS:l, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96, or a complement thereof.

Oligonucleotides may be chemically synthesized and may also be used as probes.

In another embodiment, an isolated nucleic acid molecule ofthe invention comprises a nucleic acid molecule that is a complement ofthe nucleotide sequence shown in SEQ ID NOS:l, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96, or a portion of this nucleotide sequence (e.g., a fragment that can be used as a probe or primer or a fragment encoding a biologically-active portion of an GPCRX polypeptide). A nucleic acid molecule that is complementary to the nucleotide sequence shown in SEQ ID NOS:l, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96 is one that is sufficiently complementary to the nucleotide sequence shown in SEQ ID NOS:l, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96 that it can hydrogen bond with little or no mismatches to the nucleotide sequence shown SEQ ID NOS:l, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96, thereby fonning a stable duplex. As used herein, the term "complementary" refers to Watson-Crick or Hoogsteen base pairing between nucleotides units of a nucleic acid molecule, and the term "binding" means the physical or chemical interaction between two polypeptides or compounds or associated polypeptides or compounds or combinations thereof. Binding includes ionic, non-ionic, van der Waals, hydrophobic interactions, and the like. A physical interaction can be either direct or indirect. Indirect interactions may be through or due to the effects of another polypeptide or compound. Direct binding refers to interactions that do not take place through, or due to, the effect of another polypeptide or compound, but instead are without other substantial chemical intermediates.

Fragments provided herein are defined as sequences of at least 6 (contiguous) nucleic acids or at least 4 (contiguous) amino acids, a length sufficient to allow for specific hybridization in the case of nucleic acids or for specific recognition of an epitope in the case of amino acids, respectively, and are at most some portion less than a full length sequence. Fragments may be derived from any contiguous portion of a nucleic acid or amino acid sequence of choice. Derivatives are nucleic acid sequences or amino acid sequences formed from the native compounds either directly or by modification or partial substitution. Analogs are nucleic acid sequences or amino acid sequences that have a structure similar to, but not identical to, the native compound but differs from it in respect to certain components or side chains. Analogs may be synthetic or from a different evolutionary origin and may have a similar or opposite metabolic activity compared to wild type. Homologs are nucleic acid sequences or amino acid sequences of a particular gene that are derived from different species. Derivatives and analogs maybe full length or other than full length, if the derivative or analog contains a modified nucleic acid or amino acid, as described below. Derivatives or analogs ofthe nucleic acids or proteins ofthe invention include, but are not limited to, molecules comprising regions that are substantially homologous to the nucleic acids or proteins ofthe invention, in various embodiments, by at least about 70%, 80%, or 95% identity (with a preferred identity of 80-95%) over a nucleic acid or amino acid sequence of identical size or when compared to an aligned sequence in which the alignment is done by a computer homology program known in the art, or whose encoding nucleic acid is capable of hybridizing to the complement of a sequence encoding the aforementioned proteins under stringent, moderately stringent, or low stringent conditions. See e.g. Ausubel, et ah, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, NY, 1993, and below. A "homologous nucleic acid sequence" or "homologous amino acid sequence," or variations thereof, refer to sequences characterized by a homology at the nucleotide level or amino acid level as discussed above. Homologous nucleotide sequences encode those sequences coding for isoforms of GPCRX polypeptides. Isoforms can be expressed in different tissues ofthe same organism as a result of, for example, alternative splicing of RNA. Alternatively, isoforms can be encoded by different genes. In the invention, homologous nucleotide sequences include nucleotide sequences encoding for an GPCRX polypeptide of species other than humans, including, but not limited to: vertebrates, and thus can include, e.g., frog, mouse, rat, rabbit, dog, cat cow, horse, and other organisms. Homologous nucleotide sequences also include, but are not limited to, naturally occurring allelic variations and mutations ofthe nucleotide sequences set forth herein. A homologous nucleotide sequence does not, however, include the exact nucleotide sequence encoding human GPCRX protein. Homologous nucleic acid sequences include those nucleic acid sequences that encode conservative amino acid substitutions (see below) in SEQ ID NOS: 2, 4, 6, 8, 10, 12, 21, 28, 35, 37, 44, 51, 58, 60, 67, 74, 81, 83, 90 or 97, as well as a polypeptide possessing GPCRX biological activity. Various biological activities ofthe GPCRX proteins are described below. As used herein, "identical" residues correspond to those residues in a comparison between two sequences where the equivalent nucleotide base or amino acid residue in an alignment of two sequences is the same residue. Residues are alternatively described as "similar" or "positive" when the comparisons between two sequences in an alignment show that residues in an equivalent position in a comparison are either the same amino acid or a conserved amino acid as defined below.

An GPCRX polypeptide is encoded by the open reading frame ("ORF") of an GPCRX nucleic acid. An ORF corresponds to a nucleotide sequence that could potentially be translated into a polypeptide. A stretch of nucleic acids comprising an ORF is uninterrupted by a stop codon. An ORF that represents the coding sequence for a full protein begins with an ATG "start" codon and terminates with one ofthe three "stop" codons, namely, TAA, TAG, or TGA. For the purposes of this invention, an ORF may be any part of a coding sequence, with or without a start codon, a stop codon, or both. For an ORF to be considered as a good candidate for coding for a bonafide cellular protein, a minimum size requirement is often set, e.g., a stretch of DNA that would encode a protein of 50 amino acids or more.

The nucleotide sequences determined from the cloning ofthe human GPCRX genes allows for the generation of probes and primers designed for use in identifying and/or cloning GPCRX homologues in other cell types, e.g. from other tissues, as well as GPCRX homologues from other vertebrates. The probe/primer typically comprises substantially purified oligonucleotide. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, 25, 50, 100, 150, 200, 250, 300, 350 or 400 consecutive sense strand nucleotide sequence of SEQ ID NOS:l, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96; or an anti-sense strand nucleotide sequence of SEQ ID NOS:l, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96; or of a naturally occurring mutant of SEQ ID NOS:l, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96.

Probes based on the human GPCRX nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins. In various embodiments, the probe further comprises a label group attached thereto, e.g. the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used as a part of a diagnostic test kit for identifying cells or tissues which mis- express an GPCRX protein, such as by measuring a level of an GPCRX-encoding nucleic acid in a sample of cells from a subject e.g., detecting GPCRX mRNA levels or determining whether a genomic GPCRX gene has been mutated or deleted.

"A polypeptide having a biologically-active portion of an GPCRX polypeptide" refers to polypeptides exhibiting activity similar, but not necessarily identical to, an activity of a polypeptide ofthe invention, including mature forms, as measured in a particular biological assay, with or without dose dependency. A nucleic acid fragment encoding a "biologically- active portion of GPCRX" can be prepared by isolating a portion SEQ ID NOS: 1, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96 that encodes a polypeptide having an GPCRX biological activity (the biological activities ofthe GPCRX proteins are described below), expressing the encoded portion of GPCRX protein (e.g., by recombinant expression in vitro) and assessing the activity ofthe encoded portion of GPCRX.

GPCRX Nucleic Acid and Polypeptide Variants

The invention further encompasses nucleic acid molecules that differ from the nucleotide sequences shown SEQ ID NOS:l, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96 due to degeneracy ofthe genetic code and thus encode the same GPCRX proteins as that encoded by the nucleotide sequences shown in SEQ ID NOS:l, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96. hi another embodiment, an isolated nucleic acid molecule ofthe invention has a nucleotide sequence encoding a protein having an amino acid sequence shown in SEQ ID NOS:2, 4, 6, 8, 10, 12, 21, 28, 35, 37, 44, 51, 58, 60, 67, 74, 81, 83, 90 and 97.

In addition to the human GPCRX nucleotide sequences shown in SEQ ID NOS:l, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96 it will be appreciated by those skilled in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequences of the GPCRX polypeptides may exist within a population (e.g. , the human population). Such genetic polymorphism in the GPCRX genes may exist among individuals within a population due to natural allelic variation. As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules comprising an open reading frame (ORF) encoding an GPCRX protein, preferably a vertebrate GPCRX protein. Such natural allelic variations can typically result in 1-5% variance in the nucleotide sequence ofthe GPCRX genes. Any and all such nucleotide variations and resulting amino acid polymorphisms in the GPCRX polypeptides, which are the result of natural allelic variation and that do not alter the functional activity ofthe GPCRX polypeptides, are intended to be within the scope ofthe invention. Moreover, nucleic acid molecules encoding GPCRX proteins from other species, and thus that have a nucleotide sequence that differs from the human sequence SEQ ID NOS:l, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96 are intended to be within the scope ofthe invention. Nucleic acid molecules corresponding to natural allelic variants and homologues ofthe GPCRX cDNAs ofthe invention can be isolated based on their homology to the human GPCRX nucleic acids disclosed herein using the human cDNAs, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions. Accordingly, in another embodiment, an isolated nucleic acid molecule ofthe invention is at least 6 nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOS:l, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96. hi another embodiment, the nucleic acid is at least 10, 25, 50, 100, 250, 500, 750, 1000, 1500, or 2000 or more nucleotides in length. In yet another embodiment, an isolated nucleic acid molecule ofthe invention hybridizes to the coding region. As used herein, the term "hybridizes under stringent conditions" is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% homologous to each other typically remain hybridized to each other.

Homologs (i.e., nucleic acids encoding GPCRX proteins derived from species other than human) or other related sequences (e.g., paralogs) can be obtained by low, moderate or high stringency hybridization with all or a portion ofthe particular human sequence as a probe using methods well known in the art for nucleic acid hybridization and cloning. As used herein, the phrase "stringent hybridization conditions" refers to conditions under which a probe, primer or oligonucleotide will hybridize to its target sequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures than shorter sequences. Generally, stringent conditions are selected to be about 5 °C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% ofthe probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50% ofthe probes are occupied at equilibrium. Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes, primers or oligonucleotides (e.g., 10 nt to 50 nt) and at least about 60°C for longer probes, primers and oligonucleotides. Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide. Stringent conditions are known to those skilled in the art and can be found in Ausubel, et al., (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Preferably, the conditions are such that sequences at least about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% homologous to each other typically remain hybridized to each other. A non-limiting example of stringent hybridization conditions are hybridization in a high salt buffer comprising 6X SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PNP, 0.02% FicoU, 0.02% BSA, and 500 mg/ml denatured salmon sperm DΝA at 65°C, followed by one or more washes in 0.2X SSC, 0.01% BSA at 50°C. An isolated nucleic acid molecule ofthe invention that hybridizes under stringent conditions to the sequences of SEQ ID ΝOS:l, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96 corresponds to a naturally-occurring nucleic acid molecule. As used herein, a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein). In a second embodiment, a nucleic acid sequence that is hybridizable to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOS:l, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96 or fragments, analogs or derivatives thereof, under conditions of moderate stringency is provided. A non-limiting example of moderate stringency hybridization conditions are hybridization in 6X SSC, 5X Denhardt's solution, 0.5% SDS and 100 mg/ml denatured salmon sperm DNA at 55°C, followed by one or more washes in IX SSC, 0.1% SDS at 37°C. Other conditions of moderate stringency that may be used are well-known within the art. See, e.g., Ausubel, et al. (eds.), 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY, and Kriegler, 1990; GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY. In a third embodiment, a nucleic acid that is hybridizable to the nucleic acid molecule comprising the nucleotide sequences of SEQ ID NOS:l, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96 or fragments, analogs or derivatives thereof, under conditions of low stringency, is provided. A non-limiting example of low stringency hybridization conditions are hybridization in 35% formamide, 5X SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PNP, 0.02% FicoU, 0.2% BSA, 100 mg/ml denatured salmon sperm DΝA, 10% (wt/vol) dextran sulfate at 40°C, followed by one or more washes in 2X SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS at 50°C. Other conditions of low stringency that may be used are well known in the art (e.g., as employed for cross-species hybridizations). See, e.g., Ausubel, et al. (eds.), 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY, and Kriegler, 1990, GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY; Shilo and Weinberg, 1981. Proc Natl Acad Sci USA 78: 6789-6792. Conservative Mutations

In addition to naturally-occurring allelic variants of GPCRX sequences that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequences of SEQ ID NOS:l, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96 thereby leading to changes in the amino acid sequences ofthe encoded GPCRX proteins, without altering the functional ability of said GPCRX proteins. For example, nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues can be made in the sequence of SEQ ID NOS:2, 4, 6, 8, 10, 12, 21, 28, 35, 37, 44, 51, 58, 60, 67, 74, 81, 83, 90 and 97. A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequences ofthe GPCRX proteins without altering their biological activity, whereas an "essential" amino acid residue is required for such biological activity. For example, amino acid residues that are conserved among the GPCRX proteins of the invention are predicted to be particularly non-amenable to alteration. Amino acids for which conservative substitutions can be made are well-known within the art. Another aspect ofthe invention pertains to nucleic acid molecules encoding GPCRX proteins that contain changes in amino acid residues that are not essential for activity. Such GPCRX proteins differ in amino acid sequence from SEQ ID NOS:2, 4, 6, 8, 10, 12, 21, 28, 35, 37, 44, 51, 58, 60, 61, 74, 81, 83, 90 and 97 yet retain biological activity. In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 45% homologous to the amino acid sequences of SEQ ID NOS:2, 4, 6, 8, 10, 12, 21, 28, 35, 37, 44, 51, 58, 60, 67, 74, 81, 83, 90 and 97. Preferably, the protein encoded by the nucleic acid molecule is at least about 60% homologous to SEQ ID NOS:2, 4, 6, 8, 10, 12, 21, 28, 35, 37, 44, 51, 58, 60, 67, 74, 81, 83, 90 and 97; more preferably at least about 70% homologous to SEQ ID NOS:2, 4, 6, 8, 10, 12, 21, 28, 35, 37, 44, 51, 58, 60, 67, 74, 81, 83, 90 and 97; still more preferably at least about 80% homologous to SEQ ID NOS:2, 4, 6, 8, 10, 12, 21, 28, 35, 37, 44, 51, 58, 60, 67, 74, 81, 83, 90 and 97; even more preferably at least about 90% homologous to SEQ ID NOS:2, 4, 6, 8, 10, 12, 21, 28, 35, 37, 44, 51, 58, 60, 67, 74, 81, 83, 90 and 97; and most preferably at least about 95% homologous to SEQ ID NOS:2, 4, 6, 8, 10, 12, 21, 28, 35, 37, 44, 51, 58, 60, 67, 74, 81, 83, 90 and 97.

An isolated nucleic acid molecule encoding an GPCRX protein homologous to the protein of SEQ ID NOS:2, 4, 6, 8, 10, 12, 21, 28, 35, 37, 44, 51, 58, 60, 67, 74, 81, 83, 90 and 97 can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NOS:l, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96 such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein.

Mutations can be introduced into SEQ ID NOS:2, 4, 6, 8, 10, 12, 21, 28, 35, 37, 44, 51, 58, 60, 67, 74, 81, 83, 90 and 97 by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted, non-essential amino acid residues. A "conservative amirio acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined within the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted non-essential amino acid residue in the GPCRX protein is replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of an GPCRX coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for GPCRX biological activity to identify mutants that retain activity. Following mutagenesis of SEQ ID NOS:l, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96, the encoded protein can be expressed by any recombinant technology known in the art and the activity ofthe protein can be determined.

The relatedness of amino acid families may also be determined based on side chain interactions. Substituted amino acids may be fully conserved "strong" residues or fully conserved "weak" residues. The "strong" group of conserved amino acid residues maybe any one ofthe following groups: STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, FYW, wherein the single letter amino acid codes are grouped by those amino acids that may be substituted for each other. Likewise, the "weak" group of conserved residues may be any one ofthe following: CSA, ATN, SAG, STΝK, STPA, SGΝD, SΝDEQK, ΝDEQHK, EQHRK, NLIM, HFY, wherein the letters within each group represent the single letter amino acid code. In one embodiment, a mutant GPCRX protein can be assayed for (i) the ability to form proteimprotein interactions with other GPCRX proteins, other cell-surface proteins, or biologically-active portions thereof, (ii) complex formation between a mutant GPCRX protein and an GPCRX ligand; or (in) the ability of a mutant GPCRX protein to bind to an intracellular target protein or biologically-active portion thereof; (e.g. avidin proteins). hi yet another embodiment, a mutant GPCRX protein can be assayed for the ability to regulate a specific biological function (e.g., regulation of insulin release).

Antisense Nucleic Acids

Another aspect ofthe invention pertains to isolated antisense nucleic acid molecules that are hybridizable to or complementary to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOS:l, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96, or fragments, analogs or derivatives thereof. An "antisense" nucleic acid comprises a nucleotide sequence that is complementary to a "sense" nucleic acid encoding a protein (e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence). In specific aspects, antisense nucleic acid molecules are provided that comprise a sequence complementary to at least about 10, 25, 50, 100, 250 or 500 nucleotides or an entire GPCRX coding strand, or to only a portion thereof. Nucleic acid molecules encoding fragments, homologs, derivatives and analogs of an GPCRX protein of SEQ ID NOS:2, 4, 6, 8, 10, 12, 21, 28, 35, 37, 44, 51, 58, 60, 67, 74, 81, 83, 90 and 97, or antisense nucleic acids complementary to an GPCRX nucleic acid sequence of SEQ ID NOS:l, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96, are additionally provided.

In one embodiment, an antisense nucleic acid molecule is antisense to a "coding region" ofthe coding strand of a nucleotide sequence encoding an GPCRX protein. The term "coding region" refers to the region ofthe nucleotide sequence comprising codons which are translated into amino acid residues. In another embodiment, the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence encoding the GPCRX protein. The term "noncoding region" refers to 5' and 3' sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5' and 3' untranslated regions).

Given the coding strand sequences encoding the GPCRX protein disclosed herein, antisense nucleic acids ofthe invention can be designed according to the rules of Watson and Crick or Hoogsteen base pairing. The antisense nucleic acid molecule can be complementary to the entire coding region of GPCRX mRNA, but more preferably is an oligonucleotide that is antisense to only a portion ofthe coding or noncoding region of GPCRX mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of GPCRX mRNA. An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleic acid of the invention can be constructed using chemical synthesis or enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally-occurring nucleotides or variously modified nucleotides designed to increase the biological stability ofthe molecules or to increase the physical stability ofthe duplex formed between the antisense and sense nucleic acids (e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used). Examples of modified nucleotides that can be used to generate the antisense nucleic acid include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl- 2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).

The antisense nucleic acid molecules ofthe invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding an GPCRX protein to thereby inhibit expression ofthe protein (e.g., by inhibiting transcription and/or translation). The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule that binds to DNA duplexes, through specific interactions in the major groove ofthe double helix. An example of a route of administration of antisense nucleic acid molecules ofthe invention includes direct injection at a tissue site. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For example, for systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface (e.g. , by linking the antisense nucleic acid molecules, to peptides or antibodies that bind to cell surface receptors or antigens). The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient nucleic acid molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred. In yet another embodiment, the antisense nucleic acid molecule ofthe invention is an α-anomeric nucleic acid molecule. An α-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual β-units, the strands run parallel to each other. See, e.g., Gaultier, et al., 1987. Nucl. Acids Res. 15: 6625-6641. The antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide (see, e.g., Inoue, et al. 1987. Nucl. Acids Res. 15: 6131-6148) or a chimeric RNA-DNA analogue (see, e.g., Inoue, et al., 1987. FEBSLett. 215: 327-330. Ribozymes and PNA Moieties

Nucleic acid modifications include, by way of non-limiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability ofthe modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject.

In one embodiment, an antisense nucleic acid ofthe invention is a ribozyme. Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes as described in Haselhoff and Gerlach 1988. Nature 334: 585-591) can be used to catalytically cleave GPCRX mRNA transcripts to thereby inhibit translation of GPCRX mRNA. A ribozyme having specificity for an GPCRX-encoding nucleic acid can be designed based upon the nucleotide sequence of an GPCRX cDNA disclosed herein (i.e., SEQ ID NOS:l, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96). For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence ofthe active site is complementary to the nucleotide sequence to be cleaved in an GPCRX-encoding mRNA. See, e.g., U.S. Patent 4,987,071 to Cech, et al. and U.S. Patent 5,116,742 to Cech, et al. GPCRX mRNA can also be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel etal, (1993) Science 261:1411-1418.

Alternatively, GPCRX gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region ofthe GPCRX nucleic acid (e.g., the

GPCRX promoter and/or enhancers) to form triple helical structures that prevent transcription ofthe GPCRX gene in target cells. See, e.g., Helene, 1991. Anticancer Drug Des. 6: 569-84; Helene, et al. 1992. Ann. NY. Acad. Sci. 660: 27-36; Maher, 1992. Bioassays 14: 807-15.

In various embodiments, the GPCRX nucleic acids can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility ofthe molecule. For example, the deoxyribose phosphate backbone ofthe nucleic acids can be modified to generate peptide nucleic acids. See, e.g., Hyrup, et al., 1996. Bioorg Med Chem 4: 5-23. As used herein, the terms "peptide nucleic acids" or "PNAs" refer to nucleic acid mimics (e.g., DNA mimics) in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup, et al., 1996. supra; Perry-O'Keefe, et al, 1996. Proc. Natl. Acad. Sci. USA 93: 14670-14675. PNAs of GPCRX can be used in therapeutic and diagnostic applications. For example,

PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g., inducing transcription or translation arrest or inhibiting replication. PNAs of GPCRX can also be used, for example, in the analysis of single base pair mutations in a gene (e.g., PNA directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, e.g., S_Ϊ nucleases (see, Hyrup, et al, 1996.supra); or as probes or primers for DNA sequence and hybridization (see, Hyrup, et al, 1996, supra; Perry-O'Keefe, et al, 1996. supra). m another embodiment, PNAs of GPCRX can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art. For example, PNA-DNA chimeras of GPCRX can be generated that may combine the advantageous properties of PNA and DNA. Such chimeras allow DNA recognition enzymes (e.g., RNase H and DNA polymerases) to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity. PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (see, Hyrup, et al., 1996. supra). The synthesis of PNA-DNA chimeras can be performed as described in Hyrup, et al, 1996. supra and Finn, et al, 1996. Nucl Acids Res 24: 3357-3363. For example, a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry, and modified nucleoside analogs, e.g., 5'-(4-methoxytrityl)amino-5'-deoxy-thymidine phosphoramidite, can be used between the PNA and the 5' end of DNA. See, e.g., Mag, et al, 1989. Nucl Acid Res 17: 5973-5988. PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5' PNA segment and a 3' DNA segment. See, e.g.,

Finn, et al, 1996. supra. Alternatively, chimeric molecules can be synthesized with a 5' DNA segment and a 3' PNA segment. See, e.g., Petersen, et al, 1975. Bioorg. Med. Chem. Lett. 5: 1119-11124.

In other embodiments, the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger, et al, 1989. Proc. Natl. Acad. Sci. U.S.A. 86: 6553-6556; Lemaitre, et al, 1987. Proc. Natl. Acad. Sci. 84: 648-652; PCT Publication No. WO88/09810) or the blood-brain barrier (see, e.g., PCT Publication No. WO 89/10134). In addition, oligonucleotides can be modified with hybridization triggered cleavage agents (see, e.g., Krol, et al, 1988. BioTechniques 6:958-976) or intercalating agents (see, e.g., Zon, 1988. Pharm. Res. 5: 539-549). To this end, the oligonucleotide maybe conjugated to another molecule, e.g., a peptide, a hybridization triggered cross-linking agent, a transport agent, a hybridization-triggered cleavage agent, and the like.

GPCRX Polypeptides A polypeptide according to the invention includes a polypeptide including the amino acid sequence of GPCRX polypeptides whose sequences are provided in SEQ ID NOS:2, 4, 6, 8, 10, 12, 21, 28, 35, 37, 44, 51, 58, 60, 61, 74, 81, 83, 90 and 97. The invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residues shown in SEQ ID NOS:2, 4, 6, 8, 10, 12, 21, 28, 35, 37, 44, 51, 58, 60, 67, 74, 81, 83, 90 and 97 while still encoding a protein that maintains its GPCRX activities and physiological functions, or a functional fragment thereof. In general, an GPCRX variant that preserves GPCRX-like function includes any variant in which residues at a particular position in the sequence have been substituted by other amino acids, and further include the possibility of inserting an additional residue or residues between two residues ofthe parent protein as well as the possibility of deleting one or more residues from the parent sequence. Any amino acid substitution, insertion, or deletion is encompassed by the invention. In favorable circumstances, the substitution is a conservative substitution as defined above.

One aspect ofthe invention pertains to isolated GPCRX proteins, and biologically- active portions thereof, or derivatives, fragments, analogs or homologs thereof. Also provided are polypeptide fragments suitable for use as immunogens to raise anti-GPCRX antibodies. In one embodiment, native GPCRX proteins can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques. In another embodiment, GPCRX proteins are produced by recombinant DNA techniques. Alternative to recombinant expression, an GPCRX protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques.

An "isolated" or "purified" polypeptide or protein or biologically-active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the GPCRX protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. The language "substantially free of cellular material" includes preparations of GPCRX proteins in which the protein is separated from cellular components ofthe cells from which it is isolated or recombinantly- produced. In one embodiment, the language "substantially free of cellular material" includes preparations of GPCRX proteins having less than about 30% (by dry weight) of non-GPCRX proteins (also referred to herein as a "contaminating protein"), more preferably less than about 20% of non-GPCRX proteins, still more preferably less than about 10% of non-GPCRX proteins, and most preferably less than about 5% of non-GPCRX proteins. When the GPCRX protein or biologically-active portion thereof is recombinantly-produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% ofthe volume of the GPCRX protein preparation.

The language "substantially free of chemical precursors or other chemicals" includes preparations of GPCRX proteins in which the protein is separated from chemical precursors or other chemicals that are involved in the synthesis ofthe protein, hi one embodiment, the language "substantially free of chemical precursors or other chemicals" includes preparations of GPCRX proteins having less than about 30% (by dry weight) of chemical precursors or non-GPCRX chemicals, more preferably less than about 20% chemical precursors or non-GPCRX chemicals, still more preferably less than about 10% chemical precursors or non-GPCRX chemicals, and most preferably less than about 5% chemical precursors or non-GPCRX chemicals.

Biologically-active portions of GPCRX proteins include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequences ofthe GPCRX proteins (e.g., the amino acid sequence shown in SEQ ID NOS:2, 4, 6, 8, 10, 12, 21, 28, 35, 37, 44, 51, 58, 60, 67, 74, 81, 83, 90 and 97) that include fewer amino acids than the full-length GPCRX proteins, and exhibit at least one activity of an GPCRX protein. Typically, biologically-active portions comprise a domain or motif with at least one activity ofthe GPCRX protein. A biologically-active portion of an GPCRX protein can be a polypeptide which is, for example, 10, 25, 50, 100 or more amino acid residues in length. Moreover, other biologically-active portions, in which other regions ofthe protein are deleted, can be prepared by recombinant techniques and evaluated for one or more ofthe functional activities of a native GPCRX protein. hi an embodiment, the GPCRX protein has an amino acid sequence shown in SEQ ID NOS:2, 4, 6, 8, 10, 12, 21, 28, 35, 37, 44, 51, 58, 60, 67, 74, 81, 83, 90 and 97. hi other embodiments, the GPCRX protein is substantially homologous to SEQ ID NOS:2, 4, 6, 8, 10, 12, 21, 28, 35, 37, 44, 51, 58, 60, 67, 74, 81, 83, 90 and 97, and retains the functional activity ofthe protein of SEQ ID NOS:2, 4, 6, 8, 10, 12, 21, 28, 35, 37, 44, 51, 58, 60, 67, 74, 81, 83, 90 and 97, yet differs in amino acid sequence due to natural allelic variation or mutagenesis, as described in detail, below. Accordingly, in another embodiment, the GPCRX protein is a protein that comprises an amino acid sequence at least about 45% homologous to the amino acid sequence SEQ ID NOS:2, 4, 6, 8, 10, 12, 21, 28, 35, 37, 44, 51, 58, 60, 67, 74, 81, 83, 90 and 97, and retains the functional activity ofthe GPCRX proteins of SEQ ID NOS:2, 4, 6, 8, 10, 12, 21, 28, 35, 37, 44, 51, 58, 60, 67, 74, 81, 83, 90 and 97.

Determining Homology Between Two or More Sequences To determine the percent homology of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are homologous at that position (i.e., as used herein amino acid or nucleic acid "homology" is equivalent to amino acid or nucleic acid "identity").

The nucleic acid sequence homology may be determined as the degree of identity between two sequences. The homology may be determined using computer programs known in the art, such as GAP software provided in the GCG program package. See, Needleman and Wunsch, 1970. JMol Biol 48: 443-453. Using GCG GAP software with the following settings for nucleic acid sequence comparison: GAP creation penalty of 5.0 and GAP extension penalty of 0.3, the coding region ofthe analogous nucleic acid sequences referred to above exhibits a degree of identity preferably of at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, with the CDS (encoding) part ofthe DNA sequence shown in SEQ ID NOS:l, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96. The term "sequence identity" refers to the degree to which two polynucleotide or polypeptide sequences are identical on a residue-by-residue basis over a particular region of comparison. The term "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over that region of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I, in the case of nucleic acids) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the region of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. The term "substantial identity" as used herein denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 80 percent sequence identity, preferably at least 85 percent identity and often 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison region.

Chimeric and Fusion Proteins

The invention also provides GPCRX chimeric or fusion proteins. As used herein, an GPCRX "chimeric protein" or "fusion protein" comprises an GPCRX polypeptide operatively- linked to a non-GPCRX polypeptide. An "GPCRX polypeptide" refers to a polypeptide having an amino acid sequence corresponding to an GPCRX protein (SEQ ID NOS:2, 4, 6, 8, 10, 12, 21, 28, 35, 37, 44, 51, 58, 60, 67, 74, 81, 83, 90 and 97), whereas a "non-GPCRX polypeptide" refers to a polypeptide having an amino acid sequence corresponding to a protein that is not substantially homologous to the GPCRX protein, e.g., a protein that is different from the GPCRX protein and that is derived from the same or a different organism. Within an GPCRX fusion protein the GPCRX polypeptide can correspond to all or a portion of an GPCRX protein. In one embodiment, an GPCRX fusion protein comprises at least one biologically-active portion of an GPCRX protein. In another embodiment, an GPCRX fusion protein comprises at least two biologically-active portions of an GPCRX protein, hi yet another embodiment, an GPCRX fusion protein comprises at least three biologically-active portions of an GPCRX protein. Within the fusion protein, the term "operatively-linked" is intended to indicate that the GPCRX polypeptide and the non-GPCRX polypeptide are fused in-frame with one another. The non-GPCRX polypeptide can be fused to the N-terminus or C-terminus ofthe GPCRX polypeptide.

In one embodiment, the fusion protein is a GST-GPCRX fusion protein in which the GPCRX sequences are fused to the C-terminus ofthe GST (glutathione S-transferase) sequences. Such fusion proteins can facilitate the purification of recombinant GPCRX polypeptides.

In another embodiment, the fusion protein is an GPCRX protein containing a heterologous signal sequence at its N-terminus. In certain host cells (e.g., mammalian host cells), expression and/or secretion of GPCRX can be increased through use of a heterologous signal sequence.

In yet another embodiment, the fusion protein is an GPCRX-immunoglobulin fusion protein in which the GPCRX sequences are fused to sequences derived from a member ofthe immunoglobulin protein family. The GPCRX-immunoglobulin fusion proteins ofthe invention can be incorporated into pharmaceutical compositions and administered to a subject to inhibit an interaction between an GPCRX ligand and an GPCRX protein on the surface of a cell, to thereby suppress GPCRX-mediated signal fransduction in vivo. The GPCRX- immunoglobulin fusion proteins can be used to affect the bioavailability of an GPCRX cognate ligand. Inhibition ofthe GPCRX ligand/GPCRX interaction may be useful therapeutically for both the treatment of proliferative and differentiative disorders, as well as modulating (e.g. promoting or inhibiting) cell survival. Moreover, the

GPCRX-immunoglobulin fusion proteins ofthe invention can be used as immunogens to produce anti-GPCRX antibodies in a subject, to purify GPCRX ligands, and in screening assays to identify molecules that inhibit the interaction of GPCRX with an GPCRX ligand. An GPCRX chimeric or fusion protein ofthe invention can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, e.g., by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, fiUing-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. hi another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, e.g., Ausubel, et al (eds.) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide). An GPCRX-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the GPCRX protein.

GPCRX Agonists and Antagonists

The invention also pertains to variants o the GPCRX proteins that function as either GPCRX agonists (i.e., mimetics) or as GPCRX antagonists. Variants ofthe GPCRX protein can be generated by mutagenesis (e.g., discrete point mutation or truncation ofthe GPCRX protein). An agonist ofthe GPCRX protein can retain substantially the same, or a subset of, the biological activities ofthe naturally occurring form ofthe GPCRX protein. An antagonist ofthe GPCRX protein can inhibit one or more ofthe activities ofthe naturally occurring form ofthe GPCRX protein by, for example, competitively binding to a downstream or upstream member of a cellular signaling cascade which includes the GPCRX protein. Thus, specific biological effects can be elicited by treatment with a variant of limited function. In one embodiment, treatment of a subject with a variant having a subset ofthe biological activities ofthe naturally occurring form ofthe protein has fewer side effects in a subject relative to treatment with the naturally occurring form ofthe GPCRX proteins.

Variants ofthe GPCRX proteins that function as either GPCRX agonists (i.e., mimetics) or as GPCRX antagonists can be identified by screening combinatorial libraries of mutants (e.g., truncation mutants) ofthe GPCRX proteins for GPCRX protein agonist or antagonist activity. In one embodiment, a variegated library of GPCRX variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library. A variegated library of GPCRX variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential GPCRX sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of GPCRX sequences therein. There are a variety of methods which can be used to produce libraries of potential GPCRX variants from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene then ligated into an appropriate expression vector. Use of a degenerate set of genes allows for the provision, in one mixture, of all ofthe sequences encoding the desired set of potential GPCRX sequences. Methods for synthesizing degenerate oligonucleotides are well-known within the art. See, e.g., Narang, 1983. Tetrahedron 39: 3; Itakura, et al, 1984. Annu. Rev. Biochem. 53: 323; Itakura, et al, 1984. Science 198: 1056; Ike, et al, 1983. Nucl. Acids Res. 11: 477.

Polypeptide Libraries In addition, libraries of fragments ofthe GPCRX protein coding sequences can be used to generate a variegated population of GPCRX fragments for screening and subsequent selection of variants of an GPCRX protein, hi one embodiment, a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of an GPCRX coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double-stranded DNA that can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with S_\ nuclease, and ligating the resulting fragment library into an expression vector. By this method, expression libraries can be derived which encodes N-terminal and internal fragments of various sizes ofthe GPCRX proteins.

Various techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property. Such techniques are adaptable for rapid screening ofthe gene libraries generated by the combinatorial mutagenesis of GPCRX proteins. The most widely used techniques, which are amenable to high throughput analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates O 02/40539 isolation ofthe vector encoding the gene whose product was detected. Recursive ensemble mutagenesis (REM), a new technique that enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify GPCRX variants. See, e.g., Arkin and Yourvan, 1992. Proc. Natl. Acad. Sci. USA 89: 7811-7815; Delgrave, et al, 1993. Protein Engineering 6:327-331.

Anti-GPCRX Antibodies

Also included in the invention are antibodies to GPCRX proteins, or fragments of GPCRX proteins. The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen. Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, F_a , F_{a '} and F_(ay₎₂ fragments, and an F_a expression library. In general, an antibody molecule obtained from humans relates to any ofthe classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature ofthe heavy chain present in the molecule. Certain classes have subclasses as well, such as IgG IgG₂, and others. Furthermore, in humans, the light chain may be a kappa chain or a lambda chain. Reference herein to antibodies includes a reference to all such classes, subclasses and types of human antibody species.

An isolated GPCRX-related protein ofthe invention may be intended to serve as an antigen, or a portion or fragment thereof, and additionally can be used as an immunogen to generate antibodies that immunospecifically bind the antigen, using standard techniques for polyclonal and monoclonal antibody preparation. The full-length protein can be used or, alternatively, the invention provides antigenic peptide fragments ofthe antigen for use as immunogens. An antigenic peptide fragment comprises at least 6 amino acid residues ofthe amino acid sequence ofthe full length protein and encompasses an epitope thereof such that an antibody raised against the peptide forms a specific immune complex with the full length protein or with any fragment that contains the epitope. Preferably, the antigenic peptide comprises at least 10 amino acid residues, or at least 15 amino acid residues, or at least 20 amino acid residues, or at least 30 amino acid residues. Preferred epitopes encompassed by the antigenic peptide are regions ofthe protein that are located on its surface; commonly these are hydrophilic regions.

In certain embodiments ofthe invention, at least one epitope encompassed by the antigenic peptide is a region of GPCRX-related protein that is located on the surface ofthe protein, e.g., a hydrophilic region. A hydrophobicity analysis ofthe human GPCRX-related protein sequence will indicate which regions of a GPCRX-related protein are particularly hydrophilic and, therefore, are likely to encode surface residues useful for targeting antibody production. As a means for targeting antibody production, hydropathy plots showing regions of hydrophilicity and hydrophobicity may be generated by any method well known in the art, including, for example, the Kyte Doolittle or the Hopp Woods methods, either with or without Fourier transformation. See, e.g., Hopp and Woods, 1981, Proc. Nat. Acad. Sci. USA 78: 3824-3828; Kyte and Doolittle 1982, J. Mol. Biol. 157: 105-142, each of which is incorporated herein by reference in its entirety. Antibodies that are specific for one or more domains within an antigenic protein, or derivatives, fragments, analogs or homologs thereof, are also provided herein.

A protein ofthe invention, or a derivative, fragment, analog, homolog or ortholog thereof, may be utilized as an immunogen in the generation of antibodies that immunospecifically bind these protein components. Various procedures known within the art may be used for the production of polyclonal or monoclonal antibodies directed against a protein ofthe invention, or against derivatives, fragments, analogs homologs or orthologs thereof (see, for example, Antibodies: A Laboratory Manual, Harlow and Lane, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference). Some of these antibodies are discussed below. Polyclonal Antibodies

For the production of polyclonal antibodies, various suitable host animals (e.g., rabbit, goat, mouse or other mammal) may be immunized by one or more injections with the native protein, a synthetic variant thereof, or a derivative ofthe foregoing. An appropriate immunogenic preparation can contain, for example, the naturally occurring immunogenic protein, a chemically synthesized polypeptide representing the immunogenic protein, or a recombinantly expressed immunogenic protein. Furthermore, the protein may be conjugated , to a second protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. The preparation can further include an adjuvant. Various adjuvants used to increase the immunological response include, but are not limited to, Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.), adjuvants usable in humans such as Bacille Calmette-Guerin and Corynebacterium parvum, or similar immunostimulatory agents. Additional examples of adjuvants which can be employed include MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).

The polyclonal antibody molecules directed against the immunogenic protein can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as affinity chromatography using protein A or protein G, which provide primarily the IgG fraction of immune serum. Subsequently, or alternatively, the specific antigen which is the target ofthe immunoglobulin sought, or an epitope thereof, may be immobilized on a column to purify the immune specific antibody by immunoaffinity chromatography. Purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia PA, Vol. 14, No. 8 (April 17, 2000), pp. 25-28). Monoclonal Antibodies

The term "monoclonal antibody" (MAb) or "monoclonal antibody composition", as used herein, refers to a population of antibody molecules that contain only one molecular species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product, hi particular, the complementarity determining regions (CDRs) of the monoclonal antibody are identical in all the molecules ofthe population. MAbs thus contain an antigen binding site capable of immunoreacting with a particular epitope ofthe antigen characterized by a unique binding affinity for it.

Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes can be immunized in vitro.

The immunizing agent will typically include the protein antigen, a fragment thereof or a fusion protein thereof. Generally, either peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell

(Goding, MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE, Academic Press, (1986) pp. 59-103). hnmortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival ofthe unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxantiiine, aminopterin, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol, 133:3001 (1984); Brodeur et al., MONOCLONAL ANTIBODY PRODUCTION TECHNIQUES AND APPLICATIONS, Marcel Dekker, Inc., New York, (1987) pp. 51-63).

The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity ofthe monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980). Preferably, antibodies having a high degree of specificity and a high binding affinity for the target antigen are isolated. After the desired hybridoma cells are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.

The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. The monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567. DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells ofthe invention serve as a preferred source of such DNA. Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA also can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place ofthe homologous murine sequences (U.S. Patent No. 4,816,567; Morrison, Nature 368, 812-13 (1994)) or by covalently joining to the immunoglobulin coding sequence all or part ofthe coding sequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody ofthe invention to create a chimeric bivalent antibody. Humanized Antibodies

The antibodies directed against the protein antigens ofthe invention can further comprise humanized antibodies or human antibodies. These antibodies are suitable for admimsfration to humans without engendering an immune response by the human against the administered immunoglobulin. Humanized forms of antibodies are chimeric immunoglobulins, immi oglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')₂ or other antigen- binding subsequences of antibodies) that are principally comprised ofthe sequence of a human immunoglobulin, and contain minimal sequence derived from a non-human immunoglobulin. Humanization can be performed following the method of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. (See also U.S. Patent No. 5,225,539.) In some instances, Fv framework residues ofthe human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies can also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all ofthe CDR regions correspond to those of a non-human immunoglobulin and all or substantially all ofthe framework regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., 1986; -Riechmann et al., 1988; and Presta, Curr. Op. Struct. Biol. , 2:593-596 (1992)). Human Antibodies

Fully human antibodies relate to antibody molecules in which essentially the entire sequences of both the light chain and the heavy chain, including the CDRs, arise from human genes. Such antibodies are termed "human antibodies", or "fully human antibodies" herein. Human monoclonal antibodies can be prepared by the trioma technique; the human B-cell hybridoma technique (see Kozbor, et al., 1983 Immunol Today 4: 72) and the EBV hybridoma technique to produce human monoclonal antibodies (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 11-96). Human monoclonal antibodies may be utilized in the practice ofthe present invention and may be produced by using human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80: 2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). hi addition, human antibodies can also be produced using additional techniques, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol, 227:381 (1991); Marks et al., J. Mol. Biol, 222:581 (1991)). Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825;

5,625,126; 5,633,425; 5,661,016, and in Marks et al. (Bio/Technology 10, 779-783 (1992)); Lonberg et al. (Nature 368 856-859 (1994)); Morrison ( Nature 368, 812-13 (1994)); Fishwild et al,( Nature Biotechnology 14, 845-51 (1996)); Νeuberger (Nature Biotechnology 14, 826 (1996)); and Lonberg and Huszar (Intern. Rev. Immunol 13 65-93 (1995)). Human antibodies may additionally be produced using transgenic nonhuman animals which are modified so as to produce fully human antibodies rather than the animal's endogenous antibodies in response to challenge by an antigen. (See PCT publication WO94/02602). The endogenous genes encoding the heavy and light immunoglobulin chains in O 02/40539 the nonhuman host have been incapacitated, and active loci encoding human heavy and light chain immunoglobulins are inserted into the host's genome. The human genes are incorporated, for example, using yeast artificial chromosomes containing the requisite human DNA segments. An animal which provides all the desired modifications is then obtained as progeny by crossbreeding intermediate transgenic animals containing fewer than the full complement ofthe modifications. The preferred embodiment of such a nonhuman animal is a mouse, and is termed the Xenomouse™ as disclosed in PCT publications WO 96/33735 and WO 96/34096. This animal produces B cells which secrete fully human immunoglobulins. The antibodies can be obtained directly from the animal after immunization with an immunogen of interest, as, for example, a preparation of a polyclonal antibody, or alternatively from immortalized B cells derived from the animal, such as hybridomas producing monoclonal antibodies. Additionally, the genes encoding the immunoglobulins with human variable regions can be recovered and expressed to obtain the antibodies directly, or can be further modified to obtain analogs of antibodies such as, for example, single chain Fv molecules.

An example of a method of producing a nonhuman host, exemplified as a mouse, lacking expression of an endogenous immunoglobulin heavy chain is disclosed in U.S. Patent No. 5,939,598. It can be obtained by a method including deleting the J segment genes from at least one endogenous heavy chain locus in an embryonic stem cell to prevent rearrangement of the locus and to prevent formation of a transcript of a rearranged immunoglobulin heavy chain locus, the deletion being effected by a targeting vector containing a gene encoding a selectable marker; and producing from the embryonic stem cell a transgenic mouse whose somatic and germ cells contain the gene encoding the selectable marker.

A method for producing an antibody of interest, such as a human antibody, is disclosed in U.S. Patent No. 5,916,771. It includes introducing an expression vector that contains a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell. The hybrid cell expresses an antibody containing the heavy chain and the light chain. In a further improvement on this procedure, a method for identifying a clinically relevant epitope on an immunogen, and a correlative method for selecting an antibody that binds immunospecifically to the relevant epitope with high affinity, are disclosed in PCT publication WO 99/53049. O 02/40539

F_ab Fragments and Single Chain Antibodies

According to the invention, techniques can be adapted for the production of single-chain antibodies specific to an antigenic protein ofthe invention (see e.g., U.S. Patent No. 4,946,778). In addition, methods can be adapted for the construction of F_ab expression libraries (see e.g., Huse, et al., 1989 Science 246: 1275-1281) to allow rapid and effective identification of monoclonal F_ab fragments with the desired specificity for a protein or derivatives, fragments, analogs or homologs thereof. Antibody fragments that contain the idiotypes to a protein antigen may be produced -by techniques known in the art including, but not limited to: (i) an F_{(a ')2} fragment produced by pepsin digestion of an antibody molecule; (ii) an F_ab fragment generated by reducing the disulfide bridges of an F_(ab') fragment; (iii) an F_ab fragment generated by the treatment ofthe antibody molecule with papain and a reducing agent and (iv) F_v fragments. Bispecific Antibodies

Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the present case, one ofthe binding specificities is for an antigenic protein ofthe invention. The second binding target is any other antigen, and advantageously is a cell-surface protein or receptor or receptor subunit.

Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 (1983)). Because ofthe random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture often different antibody molecules, of which only one has the correct bispecific structure. The purification ofthe correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al, 1991 EMB^' OJ., 10:3655-3659.

Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CHI) containing the site necessary for light-chain binding present in at least one ofthe fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co- O 02/40539 transfected into a suitable host organism. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986).

According to another approach described in WO 96/27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. The preferred interface comprises at least a part ofthe CH3 region of an antibody constant domain. In this method, one or more small amino acid side chains from the interface ofthe first antibody molecule are replaced with larger side chains (e.g. tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface ofthe second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). This provides a mechanism for increasing the yield ofthe heterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F(ab')₂ bispecific antibodies). Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science 229:81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab')₂ fragments. These fragments are reduced in the presence ofthe dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One ofthe Fab'-TNB derivatives is then reconverted to the Fab '-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount ofthe other Fab'-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes. Additionally, Fab' fragments can be directly recovered from E. coli and chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med. 175:217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab')₂ molecule. Each Fab' fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody. The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.

Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol. 148(5):1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy-chain variable domain (V_H) connected to a light-chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the V_H and V domains of one fragment are forced to pair with the complementary V_L and V_H domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See, Gruber et al., J. Immunol. 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60 (1991).

Exemplary bispecific antibodies can bind to two different epitopes, at least one of which originates in the protein antigen ofthe invention. Alternatively, an anti-antigenic arm of an immunoglobulin molecule can be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g. CD2, CD3, CD28, or B7), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CDl 6) so as to focus cellular defense mechanisms to the cell expressing the particular antigen. Bispecific antibodies can also be used to direct cytotoxic agents to cells which express a particular antigen. These antibodies possess an antigen-binding arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody of interest binds the protein antigen described herein and further binds tissue factor (TF). Heteroconjugate Antibodies

Heteroconjugate antibodies are also within the scope ofthe present invention. Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Patent No. 4,676,980), and for treatment of HIV infection (WO 91/00360; WO 92/200373; EP 03089). It is contemplated that the antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaρtobutyrimidate and those disclosed, for example, in U.S. Patent No. 4,676,980. Effector Function Engineering

It can be desirable to modify the antibody ofthe invention with respect to effector function, so as to enhance, e.g., the effectiveness ofthe antibody in treating cancer. For example, cysteine residue(s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated can have improved intemalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp Med., 176: 1191- 1195 (1992) and Shopes, J. Immunol., 148: 2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linkers as described in Wolff et al. Cancer Research, 53: 2560-2565 (1993). Alternatively, an antibody can be engineered that has dual Fc regions and can thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989). Immunoconjugates

The invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).

Chemotherapeutic agents useful in the generation of such immunoconjugates have been described above. Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y, and ¹⁸⁶Re.

Conjugates ofthe antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis- diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro- 2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al, Science, 238: 1098 (1987). Carbon- 14-labeled l-isothiocyanatobenzyl-3- methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO94/11026. h another embodiment, the antibody can be conjugated to a "receptor" (such streptavidin) for utilization in tumor pretargeting wherein the antibody-receptor conjugate is administered to the patient;, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a "ligand" (e.g., avidin) that is in turn conjugated to a cytotoxic agent.

hi one embodiment, methods for the screening of antibodies that possess the desired specificity include, but are not limited to, enzyme-linked immunosorbent assay (ELISA) and other immunologically-mediated techniques known within the art. In a specific embodiment, selection of antibodies that are specific to a particular domain of an GPCRX protein is facilitated by generation of hybridomas that bind to the fragment of an GPCRX protein possessing such a domain. Thus, antibodies that are specific for a desired domain within an GPCRX protein, or derivatives, fragments, analogs or homologs thereof, are also provided herein.

Anti-GPCRX antibodies may be used in methods known within the art relating to the localization and/or quantitation of an GPCRX protein (e.g., for use in measuring levels ofthe GPCRX protein within appropriate physiological samples, for use in diagnostic methods, for use in imaging the protein, and the like). In a given embodiment, antibodies for GPCRX proteins, or derivatives, fragments, analogs or homologs thereof, that contain the antibody derived binding domain, are utilized as pharmacologically-active compounds (hereinafter "Therapeutics"). An anti-GPCRX antibody (e.g. , monoclonal antibody) can be used to isolate an

GPCRX polypeptide by standard techniques, such as affinity chromatography or in munoprecipitation. An anti-GPCRX antibody can facilitate the purification of natural GPCRX polypeptide from cells and of recombinantly-produced GPCRX polypeptide expressed in host cells. Moreover, an anti-GPCRX antibody can be used to detect GPCRX protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression ofthe GPCRX protein. Anti-GPCRX antibodies can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include I, I, S or H. GPCRX Recombinant Expression Vectors and Host Cells

Another aspect ofthe invention pertains to vectors, preferably expression vectors, containing a nucleic acid encoding an GPCRX protein, or derivatives, fragments, analogs or homologs thereof. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a

"plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genomp. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively-linked. Such vectors are referred to herein as "expression vectors". In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions. The recombinant expression vectors ofthe invention comprise a nucleic acid ofthe invention in a form suitable for expression ofthe nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis ofthe host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, "operably-linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner that allows for expression ofthe nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).

The term "regulatory sequence" is intended to includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression ofthe nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design ofthe expression vector can depend on such factors as the choice ofthe host cell to be transformed, the level of expression of protein desired, etc. The expression vectors ofthe invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., GPCRX proteins, mutant foπns of GPCRX proteins, fusion proteins, etc.).

The recombinant expression vectors ofthe invention can be designed for expression of GPCRX proteins in prokaryotic or eukaryotic cells. For example, GPCRX proteins can be expressed in bacterial cells such as Escherichia coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in Escherichia coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus ofthe recombinant protein. Such fusion vectors typically serve three purposes: (i) to increase expression of recombinant protein; (ii) to increase the solubility ofthe recombinant protein; and (iii) to aid in the purification ofthe recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent to purification ofthe fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.

Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amrann et al, (1988) Gene 69:301-315) and pET 1 Id (Siudier et al, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89).

One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein. See, e.g., Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 119-128. Another strategy is to alter the nucleic acid sequence ofthe nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (see, e.g., Wada, et al, 1992. Nucl. Acids Res. 20: 2111-2118). Such alteration of nucleic acid sequences ofthe invention can be carried out by standard DNA synthesis techniques. In another embodiment, the GPCRX expression vector is a yeast expression vector.

Examples of vectors for expression in yeast Saccharomyces cerivisae include pYepSecl (Baldari, et al, 1987. EMBO J. 6: 229-234), pMFa (Kurjan and Herskowitz, 1982. Cell 30: 933-943), pJRY88 (Schultz et al, 1987. Gene 54: 113-123), pYES2 (Invifrogen Corporation, San Diego, Calif), and picZ (InNitrogen Corp, San Diego, Calif). Alternatively, GPCRX can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith, et al, 1983. Mol. Cell. Biol. 3: 2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170: 31-39). hi yet another embodiment, a nucleic acid ofthe invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM-8 (Seed, 1987. Nature 329: 840) and pMT2PC (Kaufinan, et al, 1987. EMBO J. 6: 187-195). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al, MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. In another embodiment, the recombinant mammalian expression vector is capable of directing expression ofthe nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert, et al, 1987. Genes Dev. 1: 268-277), lymphoid-specific promoters (Calame and Eaton, 1988. Adv. Immunol 43:

235-275), in particular promoters of T cell receptors (Winoto and Baltimore, 1989. EMBO J. 8: 729-733) and immunoglobulins (Banerji, et al, 1983. Cell 33: 729-740; Queen and Baltimore, 1983. Cell 33: 741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci. USA 86: 5473-5477), pancreas-specific promoters (Edlund, et al, 1985. Science 230: 912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, e.g., the murine hox promoters (Kessel and Gruss, 1990. Science 249: 374-379) and the α-fetoprotein promoter (Campes and Tilghman, 1989. Genes Dev. 3: 537-546). The invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively-linked to a regulatory sequence in a manner that allows for expression (by transcription ofthe DNA molecule) of an RNA molecule that is antisense to GPCRX mRNA. Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen that direct the continuous expression ofthe antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen that direct constitutive, tissue specific or cell type specific expression of antisense RNA. The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced. For a discussion ofthe regulation of gene expression using antisense genes see, e.g., Weintraub, et al, "Antisense RNA as a molecular tool for genetic analysis," Reviews-Trends in Genetics, Vol. 1(1) 1986.

Another aspect ofthe invention pertains to host cells into which a recombinant expression vector ofthe invention has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope ofthe term as used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example, GPCRX protein can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DΕAΕ-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and other laboratory manuals.

For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome, hi order to identify and select these integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Various selectable markers include those that confer resistance to drugs, such as G418, hygromycin and methotrexate. Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding GPCRX or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).

A host cell ofthe invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) GPCRX protein. Accordingly, the invention further provides methods for producing GPCRX protein using the host cells ofthe invention, hi one embodiment, the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding GPCRX protein has been introduced) in a suitable medium such that GPCRX protein is produced. In another embodiment, the method further comprises isolating GPCRX protein from the medium or the host cell.

Transgenic GPCRX Animals

The host cells ofthe invention can also be used to produce non-human transgenic animals. For example, in one embodiment, a host cell ofthe invention is a fertilized oocyte or an embryonic stem cell into which GPCRX protein-coding sequences have been introduced. Such host cells can then be used to create non-human transgenic animals in which exogenous GPCRX sequences have been introduced into their genome or homologous recombinant animals in which endogenous GPCRX sequences have been altered. Such animals are useful for studying the function and/or activity of GPCRX protein and for identifying and/or evaluating modulators of GPCRX protein activity. As used herein, a "transgenic animal" is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more ofthe cells ofthe animal includes a transgene. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, etc. A transgene is exogenous DNA that is integrated into the genome of a cell from which a transgenic animal develops and that remains in the genome ofthe mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues ofthe transgenic animal. As used herein, a "homologous recombinant animal" is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous GPCRX gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell ofthe animal, e.g., an embryonic cell ofthe animal, prior to development ofthe animal. A transgenic animal ofthe invention can be created by introducing GPCRX-encoding nucleic acid into the male pronuclei of a fertilized oocyte (e.g., by microinjection, retroviral infection) and allowing the oocyte to develop in a pseudopregnant female foster animal. The O 02/40539 human GPCRX cDNA sequences of SEQ ID NOS-1, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96 can be introduced as a transgene into the genome of a non-human animal. Alternatively, a non-human homologue ofthe human GPCRX gene, such as a mouse GPCRX gene, can be isolated based on hybridization to the human GPCRX cDNA (described further supra) and used as a transgene. hitronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression ofthe transgene. A tissue-specific regulatory sequence(s) can be operably-linked to the GPCRX transgene to direct expression of GPCRX protein to particular cells. Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Patent Nos. 4,736,866; 4,870,009; and 4,873,191; and Hogan, 1986. hi: MANIPULATING THE MOUSE EMBRYO, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. Similar methods are used for production of other transgenic animals. A transgenic founder animal can be identified based upon the presence ofthe GPCRX transgene in its genome and/or expression of GPCRX mRNA in tissues or cells ofthe animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene-encoding GPCRX protein can further be bred to other transgenic animals carrying other transgenes.

To create a homologous recombinant animal, a vector is prepared which contains at least a portion of an GPCRX gene into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the GPCRX gene. The GPCRX gene can be a human gene (e.g., the cDNA of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96), but more preferably, is a non-human homologue of a human GPCRX gene. For example, a mouse homologue of human GPCRX gene of SEQ ID NOS:l, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96 can be used to construct a homologous recombination vector suitable for altering an endogenous GPCRX gene in the mouse genome. In one embodiment, the vector is designed such that, upon homologous recombination, the endogenous GPCRX gene is functionally disrupted (i.e., no longer encodes a functional protein; also referred to as a "knock out" vector). Alternatively, the vector can be designed such that, upon homologous recombination, the endogenous GPCRX gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous GPCRX protein). In the homologous recombination vector, the altered portion ofthe GPCRX gene is flanked at its 5'- and 3 '-termini by additional nucleic acid ofthe GPCRX gene to allow for homologous recombination to occur between the exogenous GPCRX gene carried by the vector and an endogenous GPCRX gene in an embryonic stem cell. The additional flanking GPCRX nucleic acid is of sufficient length for successful homologous recombination with the endogenous gene. Typically, several kilobases of flanking DNA (both at the 5'- and 3'-termini) are included in the vector. See, e.g., Thomas, et al, 1987. Cell 51: 503 for a description of homologous recombination vectors. The vector is ten introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced GPCRX gene has homologously-recombined with the endogenous GPCRX gene are selected. See, e.g., Li, et al, 1992. Cell 69: 915.

The selected cells are then injected into ablastocyst of an animal (e.g., a mouse) to form aggregation chimeras. See, e.g., Bradley, 1987. In: TERATOCARCINOMAS AND EMBRYONIC STEM CELLS: A PRACTICAL APPROACH, Robertson, ed. IRL, Oxford, pp. 113-152. A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term. Progeny harboring the homologously-recombined DNA in their germ cells can be used to breed animals in which all cells ofthe animal contain the homologously-recombined DNA by germline transmission ofthe transgene. Methods for constructing homologous recombination vectors and homologous recombinant animals are described further in Bradley, 1991. Curr. Opin. Biotechnol 2: 823-829; PCT International Publication Nos.: WO 90/11354; WO 91/01140; WO 92/0968; and WO 93/04169. hi another embodiment, transgenic non-humans animals can be produced that contain selected systems that allow for regulated expression ofthe transgene. One example of such a system is the cre/loxP recombinase system of bacteriophage Pl. For a description ofthe cre/loxP recombinase system, See, e.g., Lakso, et al, 1992. Proc. Natl. Acad. Sci. USA 89: 6232-6236. Another example of a recombinase system is the FLP recombinase system of

Saccharomyces cerevisiae. See, O'Gorman, et al, 1991. Science 251:1351-1355. If a cre/loxP recombinase system is used to regulate expression ofthe transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein are required. Such animals can be provided through the construction of "double" transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.

Clones ofthe non-human transgenic animals described herein can also be produced according to the methods described in Wilmut, et al, 1997. Nature 385: 810-813. In brief, a cell (e.g., a somatic cell) from the transgenic animal can be isolated and induced to exit the growth cycle and enter G₀ phase. The quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal ofthe same species from which the quiescent cell is isolated. The reconstructed oocyte is then cultured such that it develops to morula or blastocyte and then transferred to pseudopregnant female foster animal. The offspring borne of this female foster animal will be a clone ofthe animal from which the cell (e.g., the somatic cell) is isolated.

Pharmaceutical Compositions

The GPCRX nucleic acid molecules, GPCRX proteins, and anti-GPCRX antibodies (also referred to herein as "active compounds") ofthe invention, and derivatives, fragments, analogs and homologs thereof, can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, finger's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions. A pharmaceutical composition ofthe invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. .

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use of surfactants. Prevention ofthe action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like, hi many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption ofthe injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin. Sterile injectable solutions can be prepared by incorporating the active compound (e.g., an GPCRX protein or anti-GPCRX antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part ofthe composition. The tablets, pills, capsules, troches and the like can contain any ofthe following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g. , a gas such as carbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811. It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on the unique characteristics ofthe active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

The nucleic acid molecules ofthe invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see, e.g., U.S. Patent No. 5,328,470) or by stereotactic injection (see, e.g., Chen, et al, 1994. Proc. Natl. Acad. Sci. USA 91: 3054-3057). The pharmaceutical preparation ofthe gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells that produce the gene delivery system.

The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

Screening and Detection Methods

The isolated nucleic acid molecules ofthe invention can be used to express GPCRX protein (e.g., via a recombinant expression vector in a host cell in gene therapy applications), to detect GPCRX m NA (e.g., in a biological sample) or a genetic lesion in an GPCRX gene, and to modulate GPCRX activity, as described further, below. In addition, the GPCRX proteins can be used to screen drugs or compounds that modulate the GPCRX protein activity or expression as well as to treat disorders characterized by insufficient or excessive production of GPCRX protein or production of GPCRX protein forms that have decreased or aberrant activity compared to GPCRX wild-type protein (e.g.; diabetes (regulates insulin release); obesity (binds and transport lipids); metabolic disturbances associated with obesity, the metabolic syndrome X as well as anorexia and wasting disorders associated with chronic diseases and various cancers, and infectious disease(possesses anti-microbial activity) and the various dyslipidemias. In addition, the anti-GPCRX antibodies ofthe invention can be used to detect and isolate GPCRX proteins and modulate GPCRX activity. In yet a further aspect, the invention can be used in methods to influence appetite, absorption of nutrients and the disposition of metabolic substrates in both a positive and negative fashion.

The invention further pertains to novel agents identified by the screening assays described herein and uses thereof for treatments as described, supra. Screening Assays

The invention provides a method (also referred to herein as a "screening assay") for identifying modulators, i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) that bind to GPCRX proteins or have a stimulatory or inhibitory effect on, e.g., GPCRX protein expression or GPCRX protein activity. The invention also includes compounds identified in the screening assays described herein.

In one embodiment, the invention provides assays for screening candidate or test compounds which bind to or modulate the activity ofthe membrane-bound form of an GPCRX protein or polypeptide or biologically-active portion thereof. The test compounds of the invention can be obtained using any ofthe numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound" library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds. See, e.g., Lam, 1997 ^'. Anticancer Drug Design 12: 145.

A "small molecule" as used herein, is meant to refer to a composition that has a molecular weight of less than about 5 kD and most preferably less than about 4 kD. Small molecules can be, e.g., nucleic acids,, peptides, polypeptides, peptidomimetics, carbohydrates, lipids or other organic or inorganic molecules. Libraries of chemical and/or biological mixtures, such as fungal, bacterial, or algal extracts, are known in the art and can be screened with any ofthe assays ofthe invention. Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt, et al, 1993. Proc. Natl. Acad. Sci. U.S.A. 90: 6909; Erb, et al, 1994. Proc. Natl. Acad. Sci. U.S.A. 91: 11422; Zuckermann, et al, 1994. J. Med. Chem. 37: 2678; Cho, et al, 1993. Science 261: 1303; Carrell, et al, 1994. Angew. Chem. Int. Ed. Engl. 33: 2059; Carell, et al, 1994. Angew. Chem. Int. Ed. Engl. 33: 2061; and Gallop, et al, 1994. J. Med. Chem. 37: 1233.

Libraries of compounds may be presented in solution (e.g., Houghten, 1992. Biotechniques 13: 412-421), or on beads (Lam, 1991. Nature 354: 82-84), on chips (Fodor, 1993. Nature 364: 555-556), bacteria (Ladner, U.S. Patent No. 5,223,409), spores (Ladner, U.S. Patent 5,233,409), plasmids (Cull, et al, 1992. Proc. Natl. Acad. Sci. USA 89:

1865-1869) or on phage (Scott and Smith, 1990. Science 249: 386-390; Devlin, 1990. Science 249: 404-406; Cwirla, et al, 1990. Proc. Natl. Acad. Sci. U.S.A. 87: 6378-6382; Felici, 1991. J. Mol. Biol. 222: 301-310; Ladner, U.S. Patent No. 5,233,409.).

In one embodiment, an assay is a cell-based assay in which a cell which expresses a membrane-bound form of GPCRX protein, or a biologically-active portion thereof, on the cell surface is contacted with a test compound and the ability ofthe test compound to bind to an GPCRX protein determined. The cell, for example, can of mammalian origin or a yeast cell. Determining the ability ofthe test compound to bind to the GPCRX protein can be accomplished, for example, by coupling the test compound with a radioisotope or enzymatic label such that binding ofthe test compound to the GPCRX protein or biologically-active portion thereof can be determined by detecting the labeled compound in a complex. For example, test compounds can be labeled with ¹²⁵1, ³⁵S, ¹⁴C, or ³H, either directly or indirectly, and the radioisotope detected by direct counting of radioemission or by scintillation counting. Alternatively, test compounds can be enzymatically-labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product. In one embodiment, the assay comprises contacting a cell which expresses a membrane-bound form of GPCRX protein, or a biologically-active portion thereof, on the cell surface with a known compound which binds GPCRX to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability ofthe test compound to interact with an GPCRX protein, wherein determining the ability ofthe test compound to interact with an GPCRX protein comprises determining the ability ofthe test compound to preferentially bind to GPCRX protein or a biologically-active portion thereof as compared to the known compound. In another embodiment, an assay is a cell-based assay comprising contacting a cell expressing a membrane-bound form of GPCRX protein, or a biologically-active portion thereof, on the cell surface with a test compound and determining the ability ofthe test compound to modulate (e.g., stimulate or inhibit) the activity ofthe GPCRX protein or biologically-active portion thereof. Determining the ability ofthe test compound to modulate the activity of GPCRX or a biologically-active portion thereof can be accomplished, for example, by determining the ability ofthe GPCRX protein to bind to or interact with an GPCRX target molecule. As used herein, a "target molecule" is a molecule with which an GPCRX protein binds or interacts in nature, for example, a molecule on the surface of a cell which expresses an GPCRX interacting protein, a molecule on the surface of a second cell, a molecule in the extracellular milieu, a molecule associated with the internal surface of a cell membrane or a cytoplasmic molecule. An GPCRX target molecule can be a non-GPCRX molecule or an GPCRX protein or polypeptide ofthe invention, hi one embodiment, an GPCRX target molecule is a component of a signal fransduction pathway that facilitates fransduction of an extracellular signal (e.g. a signal generated by binding of a compound to a membrane-bound GPCRX molecule) through the cell membrane and into the cell. The target, for example, can be a second intercellular protein that has catalytic activity or a protein that facilitates the association of downstream signaling molecules with GPCRX.

Determining the ability ofthe GPCRX protein to bind to or interact with an GPCRX target molecule can be accomplished by one ofthe methods described above for determining direct binding. In one embodiment, determining the ability ofthe GPCRX protein to bind to or interact with an GPCRX target molecule can be accomplished by determining the activity of the target molecule. For example, the activity ofthe target molecule can be determined by detecting induction of a cellular second messenger of the target (i. e. intracellular Ca²⁺, diacylglycerol, IP₃, etc.), detecting catalytic/enzymatic activity ofthe target an appropriate substrate, detecting the induction of a reporter gene (comprising an GPCRX-responsive regulatory element operatively linked to a nucleic acid encoding a detectable marker, e.g., luciferase), or detecting a cellular response, for example, cell survival, cellular differentiation, or cell proliferation. In yet another embodiment, an assay ofthe invention is a cell-free assay comprising contacting an GPCRX protein or biologically-active portion thereof with a test compound and determining the ability ofthe test compound to bind to the GPCRX protein or biologically- active portion thereof. Binding ofthe test compound to the GPCRX protein can be determined either directly or indirectly as described above. In one such embodiment, the assay comprises contacting the GPCRX protein or biologically-active portion thereof with a known compound which binds GPCRX to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability ofthe test compound to interact with an GPCRX protein, wherein determining the ability ofthe test compound to interact with an GPCRX protein comprises determining the ability ofthe test compound to preferentially bind to GPCRX or biologically-active portion thereof as compared to the known compound. h still another embodiment, an assay is a cell-free assay comprising contacting GPCRX protein or biologically-active portion thereof with a test compound and determining the ability ofthe test compound to modulate (e.g. stimulate or inhibit) the activity ofthe GPCRX protein or biologically-active portion thereof. Determining the ability ofthe test compound to modulate the activity of GPCRX can be accomplished, for example, by determining the ability of the GPCRX protein to bind to an GPCRX target molecule by one of the methods described above for determining direct binding. In an alternative embodiment, determining the ability of the test compound to modulate the activity of GPCRX protein can be accomplished by determining the ability ofthe GPCRX protein further modulate an GPCRX target molecule. For example, the catalytic/enzymatic activity ofthe target molecule on an appropriate substrate can be determined as described, supra.

In yet another embodiment, the cell- free assay comprises contacting the GPCRX protein or biologically-active portion thereof with a known compound which binds GPCRX protein to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability ofthe test compound to interact with an GPCRX protein, wherein determining the ability ofthe test compound to interact with an GPCRX protein comprises determining the ability ofthe GPCRX protein to preferentially bind to or modulate the activity of an GPCRX target molecule.

The cell-free assays ofthe invention are amenable to use of both the soluble form or the membrane-bound form of GPCRX protein, h the case of cell-free assays comprising the membrane-bound form of GPCRX protein, it may be desirable to utilize a solubilizing agent such that the membrane-bound form of GPCRX protein is maintained in solution. Examples of such solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton^® X-100, Triton^® X-l 14, Thesit^®, Isotridecypoly(ethylene glycol ether)_n, N-dodecyl~N,N-dimethyl-3-ammonio-l -propane sulfonate, 3-(3-cholamidopropyl) dimethylamminiol-1 -propane sulfonate (CHAPS), or 3-(3-cholamidopropyl)dimethylamminiol-2-hydroxy-l-propane sulfonate (CHAPSO).

In more than one embodiment ofthe above assay methods ofthe invention, it may be desirable to immobilize either GPCRX protein or its target molecule to facilitate separation of complexed from uncomplexed fonns of one or both ofthe proteins, as well as to accommodate automation ofthe assay. Binding of a test compound to GPCRX protein, or interaction of GPCRX protein with a target molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided that adds a domain that allows one or both ofthe proteins to be bound to a matrix. For example, GST-GPCRX fusion proteins or GST-target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtiter plates, that are then combined with the test compound or the test compound and either the non-adsorbed target protein or GPCRX protein, and the mixture is incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described, supra. Alternatively, the complexes can be dissociated from the matrix, and the level of GPCRX protein binding or activity determined using standard techniques.

Other techniques for immobilizing proteins on matrices can also be used in the screening assays ofthe invention. For example, either the GPCRX protein or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated GPCRX protein or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well-known within the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, 111.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies reactive with GPCRX protein or target molecules, but which do not interfere with binding ofthe GPCRX protein to its target molecule, can be derivatized to the wells ofthe plate, and unbound target or GPCRX protein trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the GPCRX protein or target molecule, as well as enzyme-linked assays that rely on detecting an enzymatic activity associated with the GPCRX protein or target molecule.

In another embodiment, modulators of GPCRX protein expression are identified in a method wherein a cell is contacted with a candidate compound and the expression of GPCRX mRNA or protein in the cell is determined. The level of expression of GPCRX mRNA or protein in the presence ofthe candidate compound is compared to the level of expression of GPCRX mRNA or protein in the absence ofthe candidate compound. The candidate compound can then be identified as a modulator of GPCRX mRNA or protein expression based upon this comparison. For example, when expression of GPCRX mRNA or protein is greater (i.e., statistically significantly greater) in the presence ofthe candidate compound than in its absence, the candidate compound is identified as a stimulator of GPCRX mRNA or protein expression. Alternatively, when expression of GPCRX mRNA or protein is less (statistically significantly less) in the presence ofthe candidate compound than in its absence, the candidate compound is identified as an inhibitor of GPCRX mRNA or protein expression. The level of GPCRX mRNA or protein expression in the cells can be determined by methods described herein for detecting GPCRX mRNA or protein.

In yet another aspect ofthe invention, the GPCRX proteins can be used as "bait proteins" in a two-hybrid assay or three hybrid assay (see, e.g., U.S. Patent No. 5,283,317; Zervos, et al, 1993. Cell 12: 223-232; Madura, et al, 1993. J. Biol. Chem. 268: 12046-12054; Bartel, et al, 1993. Biotechniques 14: 920-924; Iwabuchi, et al, 1993. Oncogene 8:

1693-1696; and Brent WO 94/10300), to identify other proteins that bind to or interact with GPCRX ("GPCRX-binding proteins" or "GPCRX-bp") and modulate GPCRX activity. Such GPCRX-binding proteins are also likely to be involved in the propagation of signals by the GPCRX proteins as, for example, upstream or downstream elements ofthe GPCRX pathway. The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for GPCRX is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein ("prey" or "sample") is fused to a gene that codes for the activation domain ofthe known transcription factor. If the "bait" and the "prey" proteins are able to interact, in vivo, forming an GPCRX-dependent complex, the DNA-binding and activation domains ofthe transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) that is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression ofthe reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene that encodes the protein which interacts with GPCRX. The invention further pertains to novel agents identified by the aforementioned screening assays and uses thereof for treatments as described herein.

Detection Assays

Portions or fragments ofthe cDNA sequences identified herein (and the corresponding complete gene sequences) can be used in numerous ways as polynucleotide reagents. By way of example, and not of limitation, these sequences can be used to: (i) map their respective genes on a chromosome; and, thus, locate gene regions associated with genetic disease; (ii) identify an individual from a minute biological sample (tissue typing); and (iii) aid in forensic identification of a biological sample. Some of these applications are described in the subsections, below. Chromosome Mapping

Once the sequence (or a portion ofthe sequence) of a gene has been isolated, this sequence can be used to map the location ofthe gene on a chromosome. This process is called chromosome mapping. Accordingly, portions or fragments ofthe GPCRX sequences, SEQ ID NOS:l, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96, or fragments or derivatives thereof, can be used to map the location ofthe GPCRX genes, respectively, on a chromosome. The mapping ofthe GPCRX sequences to chromosomes is an important first step in correlating these sequences with genes associated with disease.

Briefly, GPCRX genes can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp in length) from the GPCRX sequences. Computer analysis ofthe GPCRX, sequences can be used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers can then be used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the GPCRX sequences will yield an amplified fragment. Somatic cell hybrids are prepared by fusing somatic cells from different mammals

(e.g., human and mouse cells). As hybrids of human and mouse cells grow and divide, they gradually lose human chromosomes in random order, but retain the mouse chromosomes. By using media in which mouse cells cannot grow, because they lack a particular enzyme, but in which human cells can, the one human chromosome that contains the gene encoding the needed enzyme will be retained. By using various media, panels of hybrid cell lines can be established. Each cell line in a panel contains either a single human chromosome or a small number of human chromosomes, and a full set of mouse chromosomes, allowing easy mapping of individual genes to specific human chromosomes. See, e.g., D'Eustachio, et al, 1983. Science 220: 919-924. Somatic cell hybrids containing only fragments of human chromosomes can also be produced by using human chromosomes with translocations and deletions. PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular sequence to a particular cliromosome. Three or more sequences can be assigned per day using a single thermal cycler. Using the GPCRX sequences to design oligonucleotide primers, sub- localization can be achieved with panels of fragments from specific chromosomes. Fluorescence in situ hybridization (FISH) of a DNA sequence to a metaphase chromosomal spread can further be used to provide a precise chromosomal location in one step. Chromosome spreads can be made using cells whose division has been blocked in metaphase by a chemical like colcemid that disrupts the mitotic spindle. The chromosomes can be treated briefly with trypsin, and then stained with Giemsa. A pattern of light and dark bands develops on each chromosome, so that the chromosomes can be identified individually. The FISH technique can be used with a DNA sequence as short as 500 or 600 bases.

However, clones larger than 1,000 bases have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection. Preferably 1,000 bases, and more preferably 2,000 bases, will suffice to get good results at a reasonable amount of time. For a review of this technique, see, Verma, et al, HUMAN CHROMOSOMES: A MANUAL OF BASIC TECHNIQUES (Pergamon Press, New York 1988).

Reagents for chromosome mapping can be used individually to mark a single chromosome or a single site on that chromosome, or panels of reagents can be used for marking multiple sites and/or multiple chromosomes. Reagents corresponding to noncoding regions ofthe genes actually are preferred for mapping purposes. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping.

Once a sequence has been mapped to a precise chromosomal location, the physical position ofthe sequence on the chromosome can be correlated with genetic map data. Such data are found, e.g., in McKusick, MENDELIAN INHERITANCE IN MAN, available on-line through Johns Hopkins University Welch Medical Library). The relationship between genes and disease, mapped to the same chromosomal region, can then be identified through linkage analysis (co-inheritance of physically adjacent genes), described in, e.g., Egeland, et al, 1987. Nature, 325: 783-787.

Moreover, differences in the DNA sequences between individuals affected and unaffected with a disease associated with the GPCRX gene, can be determined. If a mutation is observed in some or all ofthe affected individuals but not in any unaffected individuals, then the mutation is likely to be the causative agent ofthe particular disease. Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes, such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that DNA sequence. Ultimately, complete sequencing of genes from several individuals can be performed to confirm the presence of a mutation and to distinguish mutations from polymorphisms. Tissue Typing

The GPCRX sequences ofthe invention can also be used to identify individuals from minute biological samples. In this technique, an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identification. The sequences ofthe invention are useful as additional DNA markers for RFLP ("restriction fragment length polymorphisms," described in U.S. Patent No. 5,272,057).

Furthermore, the sequences ofthe invention can be used to provide an alternative technique that determines the actual base-by-base DNA sequence of selected portions of an individual's genome. Thus, the GPCRX sequences described herein can be used to prepare two PCR primers from the 5'- and 3 '-termini ofthe sequences. These primers can then be used to amplify an individual's DNA and subsequently sequence it.

Panels of corresponding DNA sequences from individuals, prepared in this manner, can provide unique individual identifications, as each individual will have a unique set of such DNA sequences due to allelic differences. The sequences ofthe invention can be used to obtain such identification sequences from individuals and from tissue. The GPCRX sequences ofthe invention uniquely represent portions ofthe human genome. Allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions. It is estimated that allelic variation between individual humans occurs with a frequency of about once per each 500 bases. Much ofthe allelic variation is due to single nucleotide polymorphisms (SNPs), which include restriction fragment length polymorphisms (RFLPs).

Each ofthe sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification purposes. Because greater numbers of polymorphisms occur in the noncoding regions, fewer sequences are necessary to differentiate individuals. The noncoding sequences can comfortably provide positive individual identification with a panel of perhaps 10 to 1,000 primers that each yield a noncoding amplified sequence of 100 bases. If predicted coding sequences, such as those in SEQ 3D NOS-1, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96 are used, a more appropriate number of primers for positive individual identification would be 500-2,000. Predictive Medicine

The invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, pharmacogenomics, and monitoring clinical trials are used for prognostic (predictive) purposes to thereby treat an individual prophylactically. Accordingly, one aspect ofthe invention relates to diagnostic assays for determining GPCRX protein and/or nucleic acid expression as well as GPCRX activity, in the context of a biological sample (e.g., blood, serum, cells, tissue) to thereby determine whether an individual is afflicted with a disease or disorder, or is at risk of developing a disorder, associated with aberrant GPCRX expression or activity. The disorders include metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, and hematopoietic disorders, and the various dyslipidemias, metabolic disturbances associated with obesity, the metabolic syndrome X and wasting disorders associated with chronic diseases and various cancers. The invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a disorder associated with GPCRX protein, nucleic acid expression or activity. For example, mutations in an GPCRX gene can be assayed in a biological sample. Such assays can be used for prognostic or predictive purpose to thereby prophylactically treat an individual prior to the onset of a disorder characterized by or associated with GPCRX protein, nucleic acid expression, or biological activity.

Another aspect ofthe invention provides methods for determining GPCRX protein, nucleic acid expression or activity in an individual to thereby select appropriate therapeutic or prophylactic agents for that individual (referred to herein as "pharmacogenomics"). Pharmacogenomics allows for the selection of agents (e.g., drugs) for therapeutic or prophylactic treatment of an individual based on the genotype ofthe individual (e.g., the genotype ofthe individual examined to determine the ability ofthe individual to respond to a particular agent.) Yet another aspect ofthe invention pertains to monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of GPCRX in clinical trials.

These and other agents are described in further detail in the following sections.

Diagnostic Assays

An exemplary method for detecting the presence or absence of GPCRX in a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting GPCRX protein or nucleic acid (e.g., mRNA, genomic DNA) that encodes GPCRX protein such that the presence of GPCRX is detected in the biological sample. An agent for detecting GPCRX mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to GPCRX mRNA or genomic DNA. The nucleic acid probe can be, for example, a full-length GPCRX nucleic acid, such as the nucleic acid of SEQ ID NOS.l, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to GPCRX mRNA or genomic DNA. Other suitable probes for use in the diagnostic assays of the invention are described herein.

An agent for detecting GPCRX protein is an antibody capable of binding to GPCRX protein, preferably an antibody with a detectable label. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab')₂) can be used. The term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i. e. , physically linking) a detectable substance to the probe or antibody, as well as indirect labeling ofthe probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently-labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently- labeled streptavidin. The term "biological sample" is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. That is, the detection method ofthe invention can be used to detect GPCRX mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo. For example, in vitro techniques for detection of GPCRX mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detection of GPCRX protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and immunofluorescence. In vitro techniques for detection of GPCRX genomic DNA include Southern hybridizations. Furthermore, in vivo techniques for detection of GPCRX protein include introducing into a subject a labeled anti-GPCRX antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.

In one embodiment, the biological sample contains protein molecules from the test subject. Alternatively, the biological sample can contain mRNA molecules from the test subject or genomic DNA molecules from the test subject. A preferred biological sample is a peripheral blood leukocyte sample isolated by conventional means from a subject.

In another embodiment, the methods further involve obtaining a control biological sample from a control subject, contacting the control sample with a compound or agent capable of detecting GPCRX protein, mRNA, or genomic DNA, such that the presence of

GPCRX protein, mRNA or genomic DNA is detected in the biological sample, and comparing the presence of GPCRX protein, mRNA or genomic DNA in the control sample with the presence of GPCRX protein, mRNA or genomic DNA in the test sample.

The invention also encompasses kits for detecting the presence of GPCRX in a biological sample. For example, the kit can comprise: a labeled compound or agent capable of detecting GPCRX protein or mRNA in a biological sample; means for determining the amount of GPCRX in the sample; and means for comparing the amount of GPCRX in the sample with a standard. The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit to detect GPCRX protein or nucleic acid. Prognostic Assays

The diagnostic methods described herein can furthermore be utilized to identify subjects having or at risk of developing a disease or disorder associated with aberrant GPCRX expression or activity. For example, the assays described herein, such as the preceding diagnostic assays or the following assays, can be utilized to identify a subject having or at risk of developing a disorder associated with GPCRX protein, nucleic acid expression or activity. Alternatively, the prognostic assays can be utilized to identify a subject having or at risk for developing a disease or disorder. Thus, the invention provides a method for identifying a disease or disorder associated with aberrant GPCRX expression or activity in which a test sample is obtained from a subject and GPCRX protein or nucleic acid (e.g., mRNA, genomic DNA) is detected, wherein the presence of GPCRX protein or nucleic acid is diagnostic for a subject having or at risk of developing a disease or disorder associated with aberrant GPCRX expression or activity. As used herein, a "test sample" refers to a biological sample obtained from a subject of interest. For example, a test sample can be a biological fluid (e.g., serum), cell sample, or tissue.

Furthermore, the prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) to treat a disease or disorder associated with aberrant GPCRX expression or activity. For example, such methods can be used to determine whether a subject can be effectively treated with an agent for a disorder. Thus, the invention provides methods for determining whether a subject can be effectively treated with an agent for a disorder associated with aberrant GPCRX expression or activity in which a test sample is obtained and GPCRX protein or nucleic acid is detected (e.g., wherein the presence of GPCRX protein or nucleic acid is diagnostic for a subject that can be administered the agent to treat a disorder associated with aberrant GPCRX expression or activity).

The methods ofthe invention can also be used to detect genetic lesions in an GPCRX gene, thereby determining if a subject with the lesioned gene is at risk for a disorder characterized by aberrant cell proliferation and/or differentiation. In various embodiments, the methods include detecting, in a sample of cells from the subject, the presence or absence of a genetic lesion characterized by at least one of an alteration affecting the integrity of a gene encoding an GPCRX-protein, or the misexpression ofthe GPCRX gene. For example, such genetic lesions can be detected by ascertaining the existence of at least one of: (i) a deletion of one or more nucleotides from an GPCRX gene; (ii) an addition of one or more nucleotides to an GPCRX gene; (iii) a substitution of one or more nucleotides of an GPCRX gene, (iv) a chromosomal rearrangement of an GPCRX gene; (v) an alteration in the level of a messenger RNA transcript of an GPCRX gene, (vi) aberrant modification of an GPCRX gene, such as of the methylation pattern ofthe genomic DNA, (vii) the presence of a non-wild-type splicing pattern of a messenger RNA transcript of an GPCRX gene, (vώ) a non-wild-type level of an GPCRX protein, (ix) allelic loss of an GPCRX gene, and (x) inappropriate post-translational modification of an GPCRX protein. As described herein, there are a large number of assay techniques known in the art which can be used for detecting lesions in an GPCRX gene. A preferred biological sample is a peripheral blood leukocyte sample isolated by conventional means from a subject. However, any biological sample containing nucleated cells may be used, including, for example, buccal mucosal cells.

In certain embodiments, detection ofthe lesion involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Patent Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran, et al, 1988. Science 241 : 1077-1080; and Nakazawa, et al, 1994. Proc. Natl. Acad. Sci. USA 91 : 360-364), the latter of which can be particularly useful for detecting point mutations in the GPCRX-gene (see, Abravaya, et al, 1995. Nucl. Acids Res. 23: 675-682). This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells ofthe sample, contacting the nucleic acid sample with one or more primers that specifically hybridize to an GPCRX gene under conditions such that hybridization and amplification ofthe GPCRX gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size ofthe amplification product and comparing the length to a control sample. It is anticipated that PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any ofthe techniques used for detecting mutations described herein.

Alternative amplification methods include: self sustained sequence replication (see, Guatelli, et al, 1990. Proc. Natl. Acad. Sci. USA 87: 1874-1878), transcriptional amplification system (see, Kwoh, et al, 1989. Proc. Natl. Acad. Sci. USA 86: 1173-1177); Qβ Replicase (see, Lizardi, et al, 1988. BioTechnology 6: 1197), or any other nucleic acid amplification method, followed by the detection ofthe amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers. In an alternative embodiment, mutations in an GPCRX gene from a sample cell can be identified by alterations in restriction enzyme cleavage patterns. For example, sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA. Moreover, the use of sequence specific ribozymes (see, e.g., U.S. Patent

No. 5,493,531) can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site. h other embodiments, genetic mutations in GPCRX can be identified by hybridizing a sample and control nucleic acids, e.g., DNA or RNA, to high-density arrays containing hundreds or thousands of oligonucleotides probes. See, e.g., Cronin, et al, 1996. Human Mutation 7: 244-255; Kozal, et al, 1996. Nat. Med. 2: 753-759. For example, genetic mutations in GPCRX can be identified in two dimensional arrays containing light-generated DNA probes as described in Cronin, et al, supra. Briefly, a first hybridization array of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear arrays of sequential overlapping probes. This step allows the identification of point mutations. This is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected. Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.

In yet another embodiment, any of a variety of sequencing reactions known in the art can be used to directly sequence the GPCRX gene and detect mutations by comparing the sequence ofthe sample GPCRX with the corresponding wild-type (control) sequence. Examples of sequencing reactions include those based on techniques developed by Maxim and Gilbert, 1977. Proc. Natl. Acad. Sci. USA 74: 560 or Sanger, 1977. Proc. Natl. Acad. Sci. USA 74: 5463. It is also contemplated that any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays (see, e.g., Naeve, et al, 1995. Biotechniques 19: 448), including sequencing by mass specfromefry (see, e.g., PCT International Publication No. WO 94/16101; Cohen, et al, 1996. Adv. Chromatography 36: 127-162; and Griffin, et al, 1993. Appl. Biochem. Biotechnol 38: 147-159).

Other methods for detecting mutations in the GPCRX gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA RNA or

RNA/DNA heteroduplexes. See, e.g., Myers, et al, 1985. Science 230: 1242. hi general, the art technique of "mismatch cleavage" starts by providing heteroduplexes of formed by hybridizing (labeled) RNA or DNA containing the wild-type GPCRX sequence with potentially mutant RNA or DNA obtained from a tissue sample. The double-stranded duplexes are treated with an agent that cleaves single-stranded regions ofthe duplex such as which will exist due to basepair mismatches between the control and sample strands. For instance, RNA DNA duplexes can be treated with RNase and DNA DNA hybrids treated with nuclease to enzymatically digesting the mismatched regions. In other embodiments, either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion ofthe mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation. See, e.g., Cotton, et al, 1988. Proc. Natl. Acad. Sci. USA 85: 4397; Saleeba, et al, 1992. Methods Enzymol. 217: 286-295. In an embodiment, the control DNA or RNA can be labeled for detection.

In still another embodiment, the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called "DNA mismatch repair" enzymes) in defined systems for detecting and mapping point mutations in GPCRX cDNAs obtained from samples of cells. For example, the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches. See, e.g., Hsu, et al, 1994. Carcinogenesis 15: 1657-1662. According to an exemplary embodiment, a probe based on an GPCRX sequence, e.g., a wild-type GPCRX sequence, is hybridized to a cDNA or other DNA product from a test cell(s). The duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like. See, e.g., U.S. Patent No. 5,459,039.

In other embodiments, alterations in electrophoretic mobility will be used to identify mutations in GPCRX genes. For example, single strand conformation polymorphism (SSCP) may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids. See, e.g., Orita, et al, 1989. Proc. Natl. Acad. Sci. USA: 86: 2766; Cotton, 1993. Mutat. Res. 285: 125-144; Hayashi, 1992. Genet. Anal. Tech. Appl. 9: 13-19. Single-stranded DNA fragments of sample and control GPCRX nucleic acids will be denatured and allowed to renature. The secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change. The DNA fragments may be labeled or detected with labeled probes. The sensitivity ofthe assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence. In one embodiment, the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility. See, e.g., Keen, et al, 1991. Trends Genet. 1: 5.

In yet another embodiment, the movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGΕ). See, e.g., Myers, et al, 1985. Nature 313: 495. When DGGΕ is used as the method of analysis, DNA will be modified to insure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of high-melting GC-rich DNA by PCR. In a further embodiment, a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA. See, e.g., Rosenbaum and Reissner, 1987. Biophys. Chem. 265: 12753.

Examples of other techniques for detecting point mutations include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension. For example, oligonucleotide primers may be prepared in which the known mutation is placed centrally and then hybridized to target DNA under conditions that permit hybridization only if a perfect match is found. See, e.g., Saiki, et al, 1986. Nature 324: 163; Saiki, et al, 1989. Proc. Natl. Acad. Sci. USA 86: 6230. Such allele specific oligonucleotides are hybridized to PCR amplified target DNA or a number of different mutations when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA. Alternatively, allele specific amplification technology that depends on selective PCR amplification may be used in conjunction with the instant invention. Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center ofthe molecule (so that amplification depends on differential hybridization; see, e.g., Gibbs, et al, 1989. Nucl. Acids Res. 17: 2437-2448) or at the extreme 3'-terminus of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (see, e.g., Prossner, 1993. Tibtech. 11 : 238). In addition it may be desirable to introduce a novel restriction site in the region ofthe mutation to create cleavage-based detection. See, e.g., Gasparini, et al, 1992. Mol. Cell Probes 6: 1. It is anticipated that in certain embodiments amplification may also be performed using Taq ligase for amplification. See, e.g., Barany, 1991. Proc. Natl. Acad. Sci. USA 88: 189. In such cases, ligation will occur only if there is a perfect match at the 3 '-terminus ofthe 5' sequence, making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.

The methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which may be conveniently used, e.g., in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving an GPCRX gene. Furthermore, any cell type or tissue, preferably peripheral blood leukocytes, in which GPCRX is expressed may be utilized in the prognostic assays described herein. However, any biological sample containing nucleated cells may be used, including, for example, buccal mucosal cells. Pharmacogenomics

Agents, or modulators that have a stimulatory or inhibitory effect on GPCRX activity (e.g., GPCRX gene expression), as identified by a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) disorders (The disorders include metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer- associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, and hematopoietic disorders, and the various dyslipidemias, metabolic disturbances associated with obesity, the metabolic syndrome X and wasting disorders associated with chronic diseases and various cancers.) hi conjunction with such treatment, the pharmacogenomics (i.e., the study ofthe relationship between an individual's genotype and that individual's response to a foreign compound or drug) ofthe individual may be considered. Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration ofthe pharmacologically active drug. Thus, the pharmacogenomics ofthe individual permits the selection of effective agents (e.g., drugs) for prophylactic or therapeutic treatments based on a consideration ofthe individual's genotype. Such pharmacogenomics can further be used to determine appropriate dosages and therapeutic regimens. Accordingly, the activity of GPCRX protein, expression of GPCRX nucleic acid, or mutation content of GPCRX genes in an individual can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic treatment ofthe individual. Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See e.g., Eichelbaum, 1996. Clin. Exp. Pharmacol. Physiol, 23: 983-985; Linder, 1997. Clin. Chem., 43: 254-266. In general, two types of pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drugs act on the body (altered drug action) or genetic conditions transmitted as single factors altering the way the body acts on drugs (altered drug metabolism). These pharmacogenetic conditions can occur either as rare defects or as polymoφhisms. For example, glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common inherited enzymopathy in which the main clinical complication is hemolysis after ingestion of oxidant drugs (anti-malarials, sulfonamides, analgesics, nitrofurans) and consumption of fava beans.

As an illustrative embodiment, the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drug action. The discovery of genetic polymorphisms of drug metabolizing enzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymes CYP2D6 and CYP2C19) has provided an explanation as to why some patients do not obtain the expected drug effects or show exaggerated drug response and serious toxicity after taking the standard and safe dose of a drug. These polymorphisms are expressed in two phenotypes in the population, the extensive metabolizer (EM) and poor metabolizer (PM). The prevalence of PM is different among different populations. For example, the gene coding for CYP2D6 is highly polymorphic and several mutations have been identified in PM, which all lead to the absence of functional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quite frequently experience exaggerated drug response and side effects when they receive standard doses. If a metabolite is the active therapeutic moiety, PM show no therapeutic response, as demonstrated for the analgesic effect of codeine mediated by its CYP2D6-formed metabolite morphine. At the other extreme are the so called ultra-rapid metabolizers who do not respond to standard doses. Recently, the molecular basis of ultra-rapid metabolism has been identified to be due to CYP2D6 gene amplification.

Thus, the activity of GPCRX protein, expression of GPCRX nucleic acid, or mutation content of GPCRX genes in an individual can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic treatment ofthe individual. In addition, pharmacogenetic studies can be used to apply genotyping of polymorphic alleles encoding drug-metabolizing enzymes to the identification of an individual's drug responsiveness phenotype. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with an GPCRX modulator, such as a modulator identified by one ofthe exemplary screening assays described herein.

Monitoring of Effects During Clinical Trials

Monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of GPCRX (e.g. , the ability to modulate aberrant cell proliferation and/or differentiation) can be applied not only in basic drug screening, but also in clinical trials. For example, the effectiveness of an agent determined by a screening assay as described herein to increase GPCRX gene expression, protein levels, or upregulate GPCRX activity, can be monitored in clinical trails of subjects exhibiting decreased GPCRX gene expression, protein levels, or downregulated GPCRX activity. Alternatively, the effectiveness of an agent determined by a screening assay to decrease GPCRX gene expression, protein levels, or downregulate GPCRX activity, can be monitored in clinical trails of subjects exhibiting increased GPCRX gene expression, protein levels, or upregulated GPCRX activity. In such clinical trials, the expression or activity of GPCRX and, preferably, other genes that have been implicated in, for example, a cellular proliferation or immune disorder can be used as a "read out" or markers ofthe immune responsiveness of a particular cell.

Byway of example, and not of limitation, genes, including GPCRX, that are modulated in cells by treatment with an agent (e.g. , compound, drug or small molecule) that modulates GPCRX activity (e.g., identified in a screening assay as described herein) can be identified. Thus, to study the effect of agents on cellular proliferation disorders, for example, in a clinical trial, cells can be isolated and RNA prepared and analyzed for the levels of expression of GPCRX and other genes implicated in the disorder. The levels of gene expression (i.e., a gene expression pattern) can be quantified by Northern blot analysis or

RT-PCR, as described herein, or alternatively by measuring the amount of protein produced, by one ofthe methods as described herein, or by measuring the levels of activity of GPCRX or other genes, hi this manner, the gene expression pattern can serve as a marker, indicative of the physiological response ofthe cells to the agent. Accordingly, this response state may be determined before, and at various points during, treatment ofthe individual with the agent. In one embodiment, the invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, protein, peptide, peptidomimetic, nucleic acid, small molecule, or other drug candidate identified by the screening assays described herein) comprising the steps of (i) obtaining a pre-administration sample from a subject prior to administration ofthe agent; (ii) detecting the level of expression of an GPCRX protein, mRNA, or genomic DNA in the preadministration sample; (iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level of expression or activity ofthe GPCRX protein, mRNA, or genomic DNA in the post-administration samples; (v) comparing the level of expression or activity ofthe GPCRX protein, mRNA, or genomic DNA in the pre-administration sample with the GPCRX protein, mRNA, or genomic DNA in the post administration sample or samples; and (vi) altering the administration ofthe agent to the subject accordingly. For example, increased administration ofthe agent may be desirable to increase the expression or activity of GPCRX to higher levels than detected, i.e., to increase the effectiveness ofthe agent. Alternatively, decreased administration ofthe agent may be desirable to decrease expression or activity of GPCRX to lower levels than detected, i.e., to decrease the effectiveness ofthe agent.

Methods of Treatment The invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant GPCRX expression or activity. The disorders include cardiomyopathy, atherosclerosis, hypertension, congenital heart defects, aortic stenosis, atrial septal defect (ASD), atrioventricular (A-V) canal defect, ductus arteriosus, pulmonary stenosis, subaortic stenosis, ventricular septal defect (VSD), valve diseases, tuberous sclerosis, scleroderma, obesity, transplantation, adrenoleukodystrophy, congenital adrenal hyperplasia, prostate cancer, neoplasm; adenocarcinoma, lymphoma, uterus cancer, fertility, hemophilia, hypercoagulation, idiopathic thrombocytopenic purpura, immunodeficiencies, graft versus host disease, AIDS, bronchial asthma, Crohn's disease; multiple sclerosis, treatment of Albright Hereditary Ostoeodysfrophy, and other diseases, disorders and conditions ofthe like. These methods of treatment will be discussed more fully, below.

Disease and Disorders

Diseases and disorders that are characterized by increased (relative to a subject not suffering from the disease or disorder) levels or biological activity may be treated with Therapeutics that antagonize (i.e., reduce or inhibit) activity. Therapeutics that antagonize activity may be administered in a therapeutic or prophylactic manner. Therapeutics that may be utilized include, but are not limited to: (i) an aforementioned peptide, or analogs, derivatives, fragments or homologs thereof; (ii) antibodies to an aforementioned peptide; (iii) nucleic acids encoding an aforementioned peptide; (iv) administration of antisense nucleic acid and nucleic acids that are "dysfunctional" (i.e., due to a heterologous insertion within the coding sequences of coding sequences to an aforementioned peptide) that are utilized to "knockout" endoggenous function of an aforementioned peptide by homologous recombination (see, e.g., Capecchi, 1989. Science 244: 1288-1292); or (v) modulators ( i.e., inhibitors, agonists and antagonists, including additional peptide mimetic ofthe invention or antibodies specific to a peptide ofthe invention) that alter the interaction between an aforementioned peptide and its binding partner. Diseases and disorders that are characterized by decreased (relative to a subject not suffering from the disease or disorder) levels or biological activity may be treated with Therapeutics that increase (i.e., are agonists to) activity. Therapeutics that upregulate activity may be administered in a therapeutic or prophylactic manner. Therapeutics that may be utilized include, but are not limited to, an aforementioned peptide, or analogs, derivatives, fragments or homologs thereof; or an agonist that increases bioavailability.

Increased or decreased levels can be readily detected by quantifying peptide and/or RNA, by obtaining a patient tissue sample (e.g., from biopsy tissue) and assaying it in vitro for RNA or peptide levels, structure and or activity ofthe expressed peptides (or mRNAs of an aforementioned peptide). Methods that are well-known within the art include, but are not limited to, immunoassays (e.g., by Western blot analysis, immunoprecipitation followed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, immunocytochemistry, etc.) and/or hybridization assays to detect expression of mRNAs (e.g., Northern assays, dot blots, in situ hybridization, and the like). Prophylactic Methods hi one aspect, the invention provides a method for preventing, in a subject, a disease or condition associated with an aberrant GPCRX expression or activity, by administering to the subject an agent that modulates GPCRX expression or at least one GPCRX activity. Subjects at risk for a disease that is caused or contributed to by aberrant GPCRX expression or activity can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic ofthe GPCRX aberrancy, such that a disease or disorder is prevented or, alternatively, delayed in its progression. Depending upon the type of GPCRX aberrancy, for example, an GPCRX agonist or GPCRX antagonist agent can be used for treating the subject. The appropriate agent can be determined based on screening assays described herein. The prophylactic methods ofthe invention are further discussed in the following subsections. Therapeutic Methods

Another aspect ofthe invention pertains to methods of modulating GPCRX expression or activity for therapeutic purposes. The modulatory method ofthe invention involves contacting a cell with an agent that modulates one or more ofthe activities of GPCRX protein activity associated with the cell. An agent that modulates GPCRX protein activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring cognate ligand of an GPCRX protein, a peptide, an GPCRX peptidomimetic, or other small molecule. In one embodiment, the agent stimulates one or more GPCRX protein activity. Examples of such stimulatory agents include active GPCRX protein and a nucleic acid molecule encoding GPCRX that has been introduced into the cell. In another embodiment, the agent inhibits one or more GPCRX protein activity. Examples of such inhibitory agents include antisense

GPCRX nucleic acid molecules and anti-GPCRX antibodies. These modulatory methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject). As such, the invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant expression or activity of an GPCRX protein or nucleic acid molecule, hi one embodiment, the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., up-regulates or down-regulates) GPCRX expression or activity. In another embodiment, the method involves administering an GPCRX protein or nucleic acid molecule as therapy to compensate for reduced or aberrant GPCRX expression or activity.

Stimulation of GPCRX activity is desirable in situations in which GPCRX is abnormally downregulated and/or in which increased GPCRX activity is likely to have a beneficial effect. One example of such a situation is where a subject has a disorder characterized by aberrant cell proliferation and/or differentiation (e.g., cancer or immune associated disorders). Another example of such a situation is where the subject has a gestational disease (e.g., preclampsia).

Determination of the Biological Effect of the Therapeutic

In various embodiments ofthe invention, suitable in vitro or in vivo assays are performed to determine the effect of a specific Therapeutic and whether its administration is indicated for treatment ofthe affected tissue.

In various specific embodiments, in vitro assays may be performed with representative cells ofthe type(s) involved in the patient's disorder, to determine if a given Therapeutic exerts the desired effect upon the cell type(s). Compounds for use in therapy may be tested in suitable animal model systems including, but not limited to rats, mice, chicken, cows, monkeys, rabbits, and the like, prior to testing in human subjects. Similarly, for in vivo testing, any ofthe animal model system known in the art may be used prior to administration to human subjects. Prophylactic and Therapeutic Uses of the Compositions of the Invention

The GPCRX nucleic acids and proteins ofthe invention are useful in potential prophylactic and therapeutic applications implicated in a variety of disorders including, but not limited to: metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer- associated cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, hematopoietic disorders, and the various dyslipidemias, metabolic disturbances associated with obesity, the metabolic syndrome X and wasting disorders associated with chronic diseases and various cancers.

As an example, a cDNA encoding the GPCRX protein ofthe invention may be useful in gene therapy, and the protein may be useful when administered to a subject in need thereof. By way of non-limiting example, the compositions ofthe invention will have efficacy for treatment of patients suffering from: metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, hematopoietic disorders, and the various dyslipidemias.

Both the novel nucleic acid encoding the GPCRX protein, and the GPCRX protein of the invention, or fragments thereof, may also be useful in diagnostic applications, wherein the presence or amount ofthe nucleic acid or the protein are to be assessed. A further use could be as an anti-bacterial molecule (i.e., some peptides have been found to possess anti-bacterial properties). These materials are further useful in the generation of antibodies which immunospecifically-bind to the novel substances ofthe invention for use in therapeutic or diagnostic methods.

EXAMPLES

Example 1. Identification of GPCRX clones All novel GPCRX target sequences identified in the present invention were subjected to the exon linking process to confirm the sequence. PCR primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. Table 14A shows the sequences ofthe PCR primers used for obtaining different clones. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case ofthe reverse primer, until the stop codon was reached. Such primers were designed based on in silico predictions for the full length cDNA, part (one or more exons) ofthe DNA or protein sequence ofthe target sequence, or by translated homology ofthe predicted exons to closely related human sequences from other species. These primers were then employed in PCR amplification based on the following pool of human cDNAs: adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea, uterus. Usually the resulting amplicons were gel purified, cloned and sequenced to high redundancy. The PCR product derived from exon linking was cloned into the pCR2.1 vector from Invifrogen. The resulting bacterial clone has an insert covering the entire open reading frame cloned into the pCR2.1 vector. Table 14B shows a list of these bacterial clones. The resulting sequences from all clones were assembled with themselves, with other fragments in CuraGen Corporation's database and with public ESTs. Fragments and ESTs were included as components for an assembly when the extent of their identity with another component ofthe assembly was at least 95% over 50 bp. In addition, sequence traces were evaluated manually and edited for corrections if appropriate. These procedures provide the sequence reported herein.

Table 14A. PCR Primers for Exon Linking

GPCRld

GPCRle 111973: :AC073079.698284. L14

GPCRlf 111965: :AC073079.698320. D13

GPCR3 78355: : sggc draft ba656o22 20000731. 698081. GI FLC ELT

GPCR4a 93200: : sggc draft ball2j3 20000804.698247.N5

GPCR4b 93327: : sggc draft ball2j3 20000804.698200. B13

GPCR6 118118: :AC010930.698337. M3 and 118118 : :AC010930.698337.M5

GPCR7 119262: :bal63b6 A.698349.15

GPCR8 116713: :GMAC011904 A.698344.All; 116713 :: GMAC011904 A.698344.A23

GPCR9 115574: :GMAL163152 D.698322. B3; 115574 : :GMAL163152 D.698322. B21

GPCRll 116796: :G AC002555 A.698344. Gl; 116796 :: GMAC002555 A.698344. G5

GPCR12 114812: :AC011711.698329. F12; 114820: :AC026331.698329. 4

The protein [PF00001 7tm_l] OLFACTORY RECEPTOR 15 (OR3) - Mus musculus (Mouse), 312 amino acids, was used in a TBLASTN search against human genomic sequences. The genomic clone AC073079 was identified as containing a full length gene similar to this olfactory receptor. Primers were designed based on in silico these predictions for the full length or part (one or more exons) ofthe DNA/protein sequence ofthe invention.

Example 2. Quantitative expression analysis of clones in various cells and tissues

The quantitative expression of various clones was assessed using microtiter plates containing RNA samples from a variety of normal and pathology-derived cells, cell lines and tissues using real time quantitative PCR (RTQ PCR). RTQ PCR was performed on a Perkin- Elmer Biosystems ABI PRISM® 7700 Sequence Detection System. Various collections of samples are assembled on the plates, and referred to as Panel 1 (containing normal tissues and cancer cell lines), Panel 2 (containing samples derived from tissues from normal and cancer sources), Panel 3 (containing cancer cell lines), Panel 4 (containing cells and cell lines from normal tissues and cells related to inflammatory conditions), Panel 5D/5I (containing human tissues and cell lines with an emphasis on metabolic diseases), AI_comprehensive_panel (containing normal tissue and samples from autoinflammatory diseases), Panel CNSD.01 (containing samples from normal and diseased brains) and CNS_neurodegeneration_panel (containing samples from normal and diseased brains).

RNA integrity from all samples is controlled for quality by visual assessment of agarose gel electropherograms using 28S and 18S ribosomal RNA staining intensity ratio as a guide (2:1 to 2.5:1 28s: 18s) and the absence of low molecular weight RNAs that would be indicative of degradation products. Samples are controlled against genomic DNA contamination by RTQ PCR reactions run in the absence of reverse transcriptase using probe and primer sets designed to amplify across the span of a single exon.

First, the RNA samples were normalized to reference nucleic acids such as constitutively expressed genes (for example, β-actin and GAPDH). Normalized RNA (5 ul) was converted to cDNA and analyzed by RTQ-PCR using One Step RT-PCR Master Mix Reagents (PE Biosystems; Catalog No. 4309169) and gene-specific primers according to the manufacturer's instructions. Probes and primers were designed for each assay according to Perkin Elmer Biosystem's Primer Express Software package (version I for Apple Computer's Macintosh Power PC) or a similar algorithm using the target sequence as input. Default settings were used for reaction conditions and the following parameters were set before selecting primers: primer concentration = 250 nM, primer melting temperature (T_m) range = 58°-60° C, primer optimal Tm = 59° C, maximum primer difference = 2° C, probe does not have 5' G, probe T_m must be 10° C greater than primer T_m, amplicon size 75 bp to 100 bp. The probes and primers selected (see below) were synthesized by Synthegen (Houston, TX, USA). Probes were double purified by HPLC to remove uncoupled dye and evaluated by mass spectroscopy to verify coupling of reporter and quencher dyes to the 5' and 3' ends of the probe, respectively. Their final concentrations were: forward and reverse primers, 900 nM each, and probe, 200nM.

PCR conditions: Normalized RNA from each tissue and each cell line was spotted in each well of a 96 well PCR plate (Perkin Elmer Biosystems). PCR cocktails including two probes (a probe specific for the target clone and another gene-specific probe multiplexed with the target probe) were set up using IX TaqMan™ PCR Master Mix for the PE Biosystems 7700, with 5 mM MgC12, dNTPs (dA, G, C, U at 1 : 1 : 1 :2 ratios), 0.25 U/ml AmpliTaq Gold™ (PE Biosystems), and 0.4 U/μl RNase inhibitor, and 0.25 U/μl reverse transcriptase. Reverse transcription was performed at 48° C for 30 minutes followed by amplification/PCR cycles as follows: 95° C 10 min, then 40 cycles of 95° C for 15 seconds, 60° C for 1 minute. Results were recorded as CT values (cycle at which a given sample crosses a threshold level of fluorescence) using a log scale, with the difference in RNA concentration between a given sample and the sample with the lowest CT value being represented as 2 to the power of delta CT. The percent relative expression is then obtained by taking the reciprocal of this RNA difference and multiplying by 100. Panels 1, 1.1, 1.2, and 1.3D

The plates for Panels 1, 1.1, 1.2 and 1.3D include 2 control wells (genomic DNA control and chemistry control) and 94 wells containing cDNA from various samples. The samples in these panels are broken into 2 classes: samples derived from cultured cell lines and samples derived from primary normal tissues. The cell lines are derived from cancers of the following types: lung cancer, breast cancer, melanoma, colon cancer, prostate cancer, CNS cancer, squamous cell carcinoma, ovarian cancer, liver cancer, renal cancer, gastric cancer and pancreatic cancer. Cell lines used in these panels are widely available through the American Type Culture Collection (ATCC), a repository for cultured cell lines, and were cultured using the conditions recommended by the ATCC. The normal tissues found on these panels are comprised of samples derived from all major organ systems from single adult individuals or fetuses. These samples are derived from the following organs: adult skeletal muscle, fetal skeletal muscle, adult heart, fetal heart, adult kidney, fetal kidney, adult liver, fetal liver, adult lung, fetal lung, various regions ofthe brain, the spleen, bone marrow, lymph node, pancreas, salivary gland, pituitary gland, adrenal gland, spinal cord, thymus, stomach, small intestine, colon, bladder, trachea, breast, ovary, uterus, placenta, prostate, testis and adipose.

In the results for Panels 1, 1.1, 1.2 and 1.3D, the following abbreviations are used: ca. = carcinoma,

* = established from metastasis, met = metastasis, s cell var = small cell variant, non-s = non-sm = non-small, squam = squamous, pl. eff = pl effusion = pleural effusion, glio = glioma, astro = astrocytoma, and neuro = neuroblastoma.

General_screening_panel_vl-4

The plates for Panel 1.4 include 2 control wells (genomic DNA control and chemistry control) and 94 wells containing cDNA from various samples. The samples in Panel 1.4 are broken into 2 classes: samples derived from cultured cell lines and samples derived from primary normal tissues. The cell lines are derived from cancers ofthe following types: lung cancer, breast cancer, melanoma, colon cancer, prostate cancer, CNS cancer, squamous cell carcinoma, ovarian cancer, liver cancer, renal cancer, gastric cancer and pancreatic cancer. Cell lines used in Panel 1.4 are widely available through the American Type Culture

Collection (ATCC), a repository for cultured cell lines, and were cultured using the conditions recommended by the ATCC. The normal tissues found on Panel 1.4 are comprised of pools of samples derived from all major organ systems from 2 to 5 different adult individuals or fetuses. These samples are derived from the following organs: adult skeletal muscle, fetal skeletal muscle, adult heart, fetal heart, adult kidney, fetal kidney, adult liver, fetal liver, adult lung, fetal lung, various regions ofthe brain, the spleen, bone marrow, lymph node, pancreas, salivary gland, pituitary gland, adrenal gland, spinal cord, thymus, stomach, small intestine, colon, bladder, trachea, breast, ovary, uterus, placenta, prostate, testis and adipose.

Panels 2D and 2.2 The plates for Panels 2D and 2.2 generally include 2 control wells and 94 test samples composed of RNA or cDNA isolated from human tissue procured by surgeons working in close cooperation with the National Cancer Institute's Cooperative Human Tissue Network (CHTN) or the National Disease Research Initiative (NDRI). The tissues are derived from human malignancies and in cases where indicated many malignant tissues have "matched margins" obtained from noncancerous tissue just adjacent to the tumor. These are termed normal adjacent tissues and are denoted "NAT" in the results below. The tumor tissue and the "matched margins" are evaluated by two independent pathologists (the surgical pathologists and again by a pathologists at NDRI or CHTN). This analysis provides a gross histopathological assessment of tumor differentiation grade. Moreover, most samples include the original surgical pathology report that provides information regarding the clinical stage of the patient. These matched margins are taken from the tissue surrounding (i.e. immediately proximal) to the zone of surgery (designated "NAT", for normal adjacent tissue, in Table RR). In addition, RNA and cDNA samples were obtained from various human tissues derived from autopsies performed on elderly people or sudden death victims (accidents, etc.). These tissues were ascertained to be free of disease and were purchased from various commercial sources such as Clontech (Palo Alto, CA), Research Genetics, and hivitrogen. Panel 3D

The plates of Panel 3D are comprised of 94 cDNA samples and two control samples. Specifically, 92 of these samples are derived from cultured human cancer cell lines, 2 samples of human primary cerebellar tissue and 2 controls. The human cell lines are generally obtained from ATCC (American Type Culture Collection), NCI or the German tumor cell bank and fall into the following tissue groups: Squamous cell carcinoma ofthe tongue, breast cancer, prostate cancer, melanoma, epidermoid carcinoma, sarcomas, bladder carcinomas, pancreatic cancers, kidney cancers, leukemias/lymphomas, ovarian/uterine/cervical, gastric, colon, lung and CNS cancer cell lines. In addition, there are two independent samples of cerebellum. These cells are all cultured under standard recommended conditions and RNA extracted using the standard procedures. The cell lines in panel 3D and 1.3D are of the most common cell lines used in the scientific literature.

Panels 4D, 4R and 4.1D

Panel 4 includes samples on a 96 well plate (2 control wells, 94 test samples) composed of RNA (Panel 4R) or cDNA (Panels 4D/4.1D) isolated from various human cell lines or tissues related to inflammatory conditions. Total RNA from control normal tissues such as colon and lung (Stratagene, La Jolla, CA) and thymus and kidney (Clontech) were employed. Total RNA from liver tissue from cirrhosis patients and kidney from lupus patients was obtained from BioChain (Biochain Institute, hie, Hayward, CA). Intestinal tissue for RNA preparation from patients diagnosed as having Crohn's disease and ulcerative colitis was obtained from the National Disease Research Interchange (NDRI) (Philadelphia, PA).

Astrocytes, lung fibroblasts, dermal fibroblasts, coronary artery smooth muscle cells, small airway epithelium, bronchial epithelium, microvascular dermal endothelial cells, microvascular lung endothelial cells, human pulmonary aortic endothelial cells, human umbilical vein endothelial cells were all purchased from Clonetics (Walkersville, MD) and grown in the media supplied for these cell types by Clonetics. These primary cell types were activated with various cytokines or combinations of cytokines for 6 and/or 12-14 hours, as indicated. The following cytokines were used; IL-1 beta at approximately 1-5 ng/ml, TNF alpha at approximately 5-10 ng/ml, IFN gamma at approximately 20-50 ng/ml, IL-4 at approximately 5-10 ng/ml, IL-9 at approximately 5-10 ng/ml, IL-13 at approximately 5-10 ng/ml. Endothelial cells were sometimes starved for various times by culture in the basal media from Clonetics with 0.1% serum. Mononuclear cells were prepared from blood of employees at CuraGen Corporation, using FicoU. LAK cells were prepared from these cells by culture in DMEM 5% FCS (Hyclone), 100 μM non essential amino acids (Gibco/Life Technologies, Rockville, MD), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5 x 10^"5 M (Gibco), and 10 mM Hepes (Gibco) and Interleukin 2 for 4-6 days. Cells were then either activated with 10-20 ng/ml

PMA and 1-2 μg/ml ionomycin, IL-12 at 5-10 ng/ml, IFN gamma at 20-50 ng/ml and IL-18 at 5-10 ng/ml for 6 hours. In some cases, mononuclear cells were cultured for 4-5 days in DMEM 5% FCS (Hyclone), 100 μM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5 x 10^"5 M (Gibco), and 10 mM Hepes (Gibco) with PHA (phytohemagglutinin) or PWM (pokeweed mitogen) at approximately 5 μg/ml. Samples were taken at 24, 48 and 72 hours for RNA preparation. MLR (mixed lymphocyte reaction) samples were obtained by taking blood from two donors, isolating the mononuclear cells using FicoU and mixing the isolated mononuclear cells 1:1 at a final concentration of approximately 2x10⁶ cells/ml in DMEM 5% FCS (Hyclone), 100 μM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol (5.5 x 10^"5 M) (Gibco), and 10 mM Hepes (Gibco). The MLR was cultured and samples taken at various time points ranging from 1- 7 days for RNA preparation.

Monocytes were isolated from mononuclear cells using CD 14 Miltenyi Beads, +ve VS selection columns and a Vario Magnet according to the manufacturer's instructions. Monocytes were differentiated into dendritic cells by culture in DMEM 5% fetal calf serum (FCS) (Hyclone, Logan, UT), 100 μM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5 x 10^"5 M (Gibco), and 10 mM Hepes (Gibco), 50 ng/ml GMCSF and 5 ng/ml IL-4 for 5-7 days. Macrophages were prepared by culture of monocytes for 5-7 days in DMEM 5% FCS (Hyclone), 100 μM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5 x 10^"5 M (Gibco), 10 mM Hepes (Gibco) and 10% AB Human Serum or MCSF at approximately 50 ng/ml. Monocytes, macrophages and dendritic cells were stimulated for 6 and 12-14 hours with lipopolysaccharide (LPS) at 100 ng/ml. Dendritic cells were also stimulated with anti-CD40 monoclonal antibody (Pharmingen) at 10 μg/ml for 6 and 12-14 hours. CD4 lymphocytes, CD 8 lymphocytes and NK cells were also isolated from mononuclear cells using CD4, CD8 and CD56 Miltenyi beads, positive VS selection columns and a Vario Magnet according to the manufacturer's instructions. CD45RA and CD45RO CD4 lymphocytes were isolated by depleting mononuclear cells of CD8, CD56, CD14 and CD19 cells using CD8, CD56, CD14 and CD19 Miltenyi beads and positive selection. Then CD45RO beads were used to isolate the CD45RO CD4 lymphocytes with the remaining cells being CD45RA CD4 lymphocytes. CD45RA CD4, CD45RO CD4 and CD8 lymphocytes were placed in DMEM 5% FCS (Hyclone), 100 μM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5 x 10^"5 M (Gibco), and 10 mM Hepes (Gibco) and plated at 10⁶ cells/ml onto Falcon 6 well tissue culture plates that had been coated overnight with 0.5 μg/ml anti-CD28 (Pharmingen) and 3 ug/ml anti-CD3 (OKT3, ATCC) in PBS. After 6 and 24 hours, the cells were harvested for RNA preparation. To prepare chronically activated CD8 lymphocytes, we activated the isolated CD8 lymphocytes for 4 days on anti-CD28 and anti-CD3 coated plates and then harvested the cells and expanded them in DMEM 5% FCS (Hyclone), 100 μM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5 x 10^"5 M (Gibco), and 10 mM Hepes (Gibco) and IL-2. The expanded CD8 cells were then activated again with plate bound anti-CD3 and anti-CD28 for 4 days and expanded as before. RNA was isolated 6 and 24 hours after the second activation and after 4 days ofthe second expansion culture. The isolated NK cells were cultured in DMEM 5% FCS (Hyclone), 100 μM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5 x 10^"5 M (Gibco), and 10 mM Hepes (Gibco) and IL-2 for 4-6 days before RNA was prepared. To obtain B cells, tonsils were procured from NDRI. The tonsil was cut up with sterile dissecting scissors and then passed through a sieve. Tonsil cells were then spun down and resupended at 10⁶ cells/ml in DMEM 5% FCS (Hyclone), 100 μM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5 x 10^"5 M (Gibco), and 10 mM Hepes (Gibco). To activate the cells, we used PWM at 5 μg/ml or anti-CD40 (Pharmingen) at approximately 10 μg/ml and IL-4 at 5-10 ng/ml. Cells were harvested for RNA preparation at 24,48 and 72 hours.

To prepare the primary and secondary Thl/Th2 and Trl cells, six-well Falcon plates were coated overnight with 10 μg/ml anti-CD28 (Pharmingen) and 2 μg/ml OKT3 (ATCC), and then washed twice with PBS. Umbilical cord blood CD4 lymphocytes (Poietic Systems,

5 6 German Town, MD) were cultured at 10 -10 cells/ml in DMEM 5% FCS (Hyclone), 100 μM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5 x 10^"

5

M (Gibco), 10 mM Hepes (Gibco) and IL-2 (4 ng/ml). IL-12 (5 ng/ml) and anti-IL4 (1 Dg/ml) were used to direct to Thl, while IL-4 (5 ng/ml) and anti-IFN gamma (1 Dg/ml) were used to direct to Th2 and IL-10 at 5 ng/ml was used to direct to Trl. After 4-5 days, the activated Thl, Th2 and Trl lymphocytes were washed once in DMEM and expanded for 4-7 days in DMEM 5% FCS (Hyclone), 100 μM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5 x 10^"5 M (Gibco), 10 mM Hepes (Gibco) and IL-2 (1 ng/ml). Following this, the activated Thl, Th2 and Trl lymphocytes were re-stimulated for 5 days with anti-CD28/OKT3 and cytokines as described above, but with the addition of anti- CD95L (1 Dg/ml) to prevent apoptosis. After 4-5 days, the Thl, Th2 and Trl lymphocytes were washed and then expanded again with IL-2 for 4-7 days. Activated Thl and Th2 lymphocytes were maintained in this way for a maximum of three cycles. RNA was prepared from primary and secondary Thl, Th2 and Trl after 6 and 24 hours following the second and third activations with plate bound anti-CD3 and anti-CD28 mAbs and 4 days into the second and third expansion cultures in Interleukin 2.

The following leukocyte cells lines were obtained from the ATCC: Ramos, EOL-1, KU-812. EOL cells were further differentiated by culture in 0.1 mM dbcAMP at 5 xl 0⁵ cells/ml for 8 days, changing the media every 3 days and adjusting the cell concentration to 5 xlO⁵ cells/ml. For the culture of these cells, we used DMEM or RPMI (as recommended by the ATCC), with the addition of 5% FCS (Hyclone), 100 μM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5 x 10^"5 M (Gibco), 10 mM Hepes (Gibco). RNA was either prepared from resting cells or cells activated with PMA at 10 ng/ml and ionomycin at 1 μg/ml for 6 and 14 hours. Keratinocyte line CCD 106 and an airway epithelial tumor line NCI-H292 were also obtained from the ATCC. Both were cultured in DMEM 5% FCS (Hyclone), 100 μM non essential amino acids (Gibco), 1 mM sodium , pyruvate (Gibco), mercaptoethanol 5.5 x 10^"5 M (Gibco), and 10 mM Hepes (Gibco). CCD 1106 cells were activated for 6 and 14 hours with approximately 5 ng/ml TNF alpha and 1 ng/ml IL-1 beta, while NCI-H292 cells were activated for 6 and 14 hours with the following cytokines: 5 ng/ml IL-4, 5 ng/ml IL-9, 5 ng/ml IL-13 and 25 ng/ml IFN gamma.

For these cell lines and blood cells, RNA was prepared by lysing approximately 10⁷ cells/ml using Trizol (Gibco BRL). Briefly, 1/10 volume of bromochloropropane (Molecular Research Corporation) was added to the RNA sample, vortexed and after 10 minutes at room temperature, the tubes were spun at 14,000 rpm in a Sorvall SS34 rotor. The aqueous phase was removed and placed in a 15 ml Falcon Tube. An equal volume of isopropanol was added and left at -20 degrees C overnight. The precipitated RNA was spun down at 9,000 rpm for 15 min in a Sorvall SS34 rotor and washed in 70% ethanol. The pellet was redissolved in 300 μl of RNAse-free water and 35 μl buffer (Promega) 5 μl DTT, 7 μl RNAsin and 8 μl DNAse were added. The tube was incubated at 37 degrees C for 30 minutes to remove contaminating genomic DNA, extracted once with phenol chloroform and re-precipitated with 1/10 volume of 3 M sodium acetate and 2 volumes of 100% ethanol. The RNA was spun down and placed in RNAse free water. RNA was stored at -80 degrees C.

Panel CNSD.01

The plates for Panel CNSD.01 include two control wells and 94 test samples comprised of cDNA isolated from postmortem human brain tissue obtained from the Harvard Brain Tissue Resource Center. Brains are removed from calvaria of donors between 4 and 24 hours after death, sectioned by neuroanatomists, and frozen at -80°C in liquid nitrogen vapor. All brains are sectioned and examined by neuropathologists to confirm diagnoses with clear associated neuropathology.

Disease diagnoses are taken from patient records. The panel contains two brains from each ofthe following diagnoses: Alzheimer's disease, Parkinson's disease, Huntington's disease, Progressive Supernuclear Palsy, Depression, and "Normal controls". Within each of these brains, the following regions are represented: cingulate gyrus, temporal pole, globus palladus, substantia nigra, Brodman Area 4 (primary motor strip), Brodman Area 7 (parietal cortex), Brodman Area 9 (prefrontal cortex), and Brodman area 17 (occipital cortex). Not all brain regions are represented in all cases; e.g., Huntington's disease is characterized in part by neurodegeneration in the globus palladus, thus this region is impossible to obtain from confirmed Huntington's cases. Likewise Parkinson's disease is characterized by degeneration ofthe substantia nigra making this region more difficult to obtain. Normal control brains were examined for neuropathology and found to be free of any pathology consistent with neurodegeneration. hi the labels employed to identify tissues in the CNS panel, the following abbreviations are used:

PSP = Progressive supranuclear palsy

Sub Nigra = Substantia nigra Glob Palladus= Globus palladus

Temp Pole = Temporal pole

Cing Gyr = Cingulate gyrus

BA 4 = Brodman Area 4 Panel CNS_Neurodegeneration_N1.0

The plates for Panel CΝS_Νeurodegeneration_V1.0 include two control wells and 47 test samples comprised of cDNA isolated from postmortem human brain tissue obtained from the Harvard Brain Tissue Resource Center (McLean Hospital) and the Human Brain and Spinal Fluid Resource Center (VA Greater Los Angeles Healthcare System). Brains are removed from calvaria of donors between 4 and 24 hours after death, sectioned by neuroanatomists, and frozen at -80°C in liquid nitrogen vapor. All brains are sectioned and examined by neuropathologists to confirm diagnoses with clear associated neuropathology. Disease diagnoses are taken from patient records. The panel contains six brains from Alzheimer's disease (AD) pateins, and eight brains from "Normal controls" who showed no evidence of dementia prior to death. The eight normal control brains are divided into two categories: Controls with no dementia and no Alzheimer's like pathology (Controls) and controls with no dementia but evidence of severe Alzheimer's like pathology, (specifically senile plaque load rated as level 3 on a scale of 0-3; 0 = no evidence of plaques, 3 = severe AD senile plaque load). Within each of these brains, the following regions are represented: hippocampus, temporal cortex (Broddmaim Area 21), parietal cortex (Broddmann area 7), and occipital cortex (Brodmann area 17). These regions were chosen to encompass all levels of neurodegeneration in AD. The hippocampus is a region of early and severe neuronal loss in AD; the temporal cortex is known to show neurodegeneration in AD after the hippocampus; the parietal cortex shows moderate neuronal death in the late stages ofthe disease; the occipital cortex is spared in AD and therefore acts as a "control" region within AD patients. Not all brain regions are represented in all cases.

In the labels employed to identify tissues in the CNS_Neurodegeneration_V1.0 panel, the following abbreviations are used: AD = Alzheimer's disease brain; patient was demented and showed AD-like pathology upon autopsy

Control = Control brains; patient not demented, showing no_. neuropathology Control (Path) = Control brains; pateint not demented but showing sever AD-like pathology SupTemporal Ctx = Superior Temporal Cortex frif Temporal Ctx = Inferior Temporal Cortex GPCRl (also referred to as GMAC073079_A, AC073079_dal, AC073079_da3 / CG109854-01, BA113A10_B_dal, BA113A10_B_da3 and CG50303-02)

Expression ofthe GPCRl gene (i.e., GMAC073079_A and variants AC073079_dal, AC073079_da3, BA113A10_B_dal, BA113A10_B_da3, and CG50303-02) was assessed using the primer-probe sets Ag2610, Ag2607, Agl585, Agl501, Ag2545, and Ag2377 described in Tables 15 A, 15B, 15C, 15D, and 15E. Please note that Ag2545 contains a single mismatch in the forward primer relative to the GMAC073079_A, AC073079_dal, AC073079_da3, BA113A10_B_dal, and BA113A10_B_da3 sequences. These mismatches are not predicted to alter the RTQ-PCR results. Results from RTQ-PCR runs are shown in Tables 15F, 15G, 15H, 151, 15J, 15K, and 15L.

Table 15 A. Probe Name Ag2610 (SEQ ID NO: 126, 127, 128)

Table 1SF. Panel 1.2

Table 15G. Panel 1.3D

Table 15H. Panel 2.2

Table 15L. Panel CNS_Neurodegeneration_vl .0

Panel 1.2 Summary Aεl501 The GMAC073079_A gene is expressed at moderate levels throughout many of the samples in this panel. Highest expression is detected in an ovarian cancer cell line (CT=30.7). In addition, this gene is overexpressed in all six ovarian cancer cell lines present in this panel when compared to expression in normal ovary. The GMAC073079_A gene is also moderately expressed in cell lines derived from melanoma, breast cancer, and lung cancer. Thus, the expression of this gene could be used to distinguish these cell lines from other tissue samples. In addition, therapeutic modulation of the GMAC073079_A gene or its protein product, through the use of small molecule drugs or antibodies, might be useful in the treatment of ovarian cancer, breast cancer, lung cancer or melanoma.

Among tissues involved in metabolic function, the GMAC073079_A gene is moderately expressed in the adrenal gland, heart, skeletal muscle, and adult liver. Interestingly, GMAC073079_A gene expression is much lower in fetal liver and heart tissues than in the corresponding adult tissues. Thus, expression ofthe GMAC073079_A gene could be used to differentiate between adult and fetal tissues derived from the heart and hver. Furthermore, this gene or its protein product may be important in the pathogenesis and/or treatment of disease in any or all ofthe above-named tissues. There is widespread moderate expression ofthe GMAC073079_A gene across many of the samples derived from the CNS, including the amygdala, cerebellum, hippocampus, thalamus, cerebral cortex, and spinal cord. Please see CNS_neurodegeneration_panel_vl.O summary for description of potential utility in the treatment of CNS disorders.

Panel 1.3D Summary Aε2610/Aε2607/Aεl 585/Aε2377 Expression ofthe GMAC073079_A gene appears to be limited to tissues involved in central nervous system function on this panel.

Specifically, low but significant expression is detected in the thalamus, substantia nigra, spinal cord and fetal brain. Aε2545 Expression of the GMAC073079_A gene is low/undetectable (Ct values >35) in all samples on this panel (data not shown).

Panel 2.2 Summary Ag2377 Expression ofthe GMAC073079_A gene is highest in a sample derived from a breast cancer sample (CT=34.7). Thus, the expression of this gene could be used to distinguish breast cancer samples from other samples and as a diagnostic marker for the presence of breast cancer. Furthermore, therapeutic modulation ofthe GMAC073079_A gene or the activity of its protein product, through the use of small molecule drugs or antibodies, might be effective in the treatment of breast cancer. Ag2610/Ag2607/Agl585 Expression of the GMAC073079_A gene is low/undetectable (Ct values >35) in all samples on this panel (data not shown).

Panel 4D Summary Ag2607/Agl585/Ag2377 Experiments using three different probe/primer sets show disparate results and are uninterpretable.

Panel CNS_1 Summary Ag2377 Two experiments with the same probe and primer set produce results that are in very good agreement. Expression ofthe GMAC073079_A gene is highest in the substantia nigra of a Huntington's disease patient, indicating that this gene may participate in the genetic dysregulation associated with the neurodegeneration that occurs in this brain region. The substantia nigra is also critical to the progression of Parkinson's disease neurodegeneration. Thus, pharmacological targeting ofthe GPCR encoded by the GMAC073079_A gene may help counter this genetic dysregulation and contribute to the restoration of normal function in Huntington's disease as well as potentially Parkinson's disease patients. Pharmacological modulation of GPCR signaling systems is the mechanism by which powerful depression therapies, such as SSRIs, exert their effect. Panel CNSJL.l Summary Ag2377 In two experiments using the same probe and primer, highest expression is seen in the cingulate gyrus of patients with para supranuclear palsy PSP (CTs = 32) and depression. This observation indicates that targeting this GPCR could have therapeutic value in the treatment of these diseases.

Panel CNS_neurodegeneration_vl.0 Summary Aε2610/Aε2607/Aε2377/Aε2545 The GMAC073079_A gene is expressed more highly in the temporal cortex of Alzheimer's diseased brain than in control brain without amyloid plaques, which are diagnostic and potentially causative of Alzheimer's disease. The GMAC073079_A gene encodes a protein with homology to GPCRs. GPCRs are readily targetable with drugs, and regulate many specific brain processes, including signaling processes, that are currently the target of FDA-approved pharmaceuticals that treat Alzheimer's disease, such as the cholinergic system.

In Alzheimer's disease abnormal proteolytic processing of amyloid precursor protein (APP) is the central step that leads to formation of amyloid plaque, neurofibrillary tangles, and neuronal loss. The plaques, which accumulate extracellularly in the brain, are composed of aggregates and cause direct neurotoxic effects and/or increase neuronal vulnerability to excitotoxic insults. The aggregates consist of soluble pathologic amyloid beta peptides AbetaP[l-42] and AbetaP[l-43] and soluble nonpathologic AbetaP [1-40]. Both APP and AbetaP interact with ion transport systems. The major mechanisms proposed for AbetaP-induced cytotoxicity involve the loss of Ca2+ homeostasis and the generation of reactive oxygen species (ROS). Kourie, J.I., Cell Mol Neurobiol 2001 Jun;21(3):173- 213. The changes in Ca2+ homeostasis could be the result of changes in G-protein-driven releases of second messengers. Thus, targeting this class of molecule can have therapeutic potential in Alzheimer's disease treatment. In particular, the increased GMAC073079_A gene expression in brains affected by Alzheimer's indicates potential therapeutic value to drugs that target this GPCR.

GPCR2 (also refered to as AC073079_da2) Expression of gene AC073079_da2 was assessed using the primer-probe sets Ag2611,

Ag2609, and Agl500, described in Tables 16A and 16B. Results from RTQ-PCR runs are shown in Tables 16C, 16D, 16E, 16F, 16G, and 16H.

Table 16A. Probe Name Ag2611/Agl500 (identical sequences)

Primers Sequences TM Length Start

Table 16B. Probe Name Ag2609

Table 16C. Panel 1.2

Table 16D. Panel 1.3D

Table 16E. Panel 2.2 O 02/40539

Table 16F. Panel 4D

Table 16G. Panel CNS 1

Panel 1.2 Summary Agl500 Highest expression ofthe AC073079_da2 gene is seen in the cerebral cortex (CT=30.4). Among tissues active in the central nervous system, the AC073079_da2 gene is also moderately expressed in the cerebellum, hippocampus, thalamus and spinal cord. Please see CNS_neurodegeneration_panel_vl.0 summary for description of the potential utility of this gene in the treatment of CNS diseases.

Among tissues with metabolic function, the AC073079_da2 gene is expressed at low but significant levels in samples derived from the adrenal gland, heart and skeletal muscle. Therefore, the protem encoded by the AC073079_da2 gene may be important in the pathogenesis and/or treatment of disease in any or all ofthe above-named tissues. ,

The AC073079_da2 gene also shows an association with cancerous cell lines and is expressed in clusters of samples derived from breast, ovarian, melanoma and lung cancer cell lines. Thus, the expression of this gene could be used to distinguish samples derived from cell lines when compared to tissues. In addition, therapeutic modulation ofthe AC073079_da2 gene or its protein product, through the use of small molecule drugs or antibodies, might be beneficial in the treatment of ovarian cancer, breast cancer, lung cancer or melanoma.

Panel 1.3D Summary Ag2611/Ag2609 Two experiments with two different probe/primer sets both show preferential expression ofthe AC073079_da2 gene in tissues originating in the central nervous system, with expression seen in the spinal cord (CT=33.1) and thalamus (CT=34.1). Please see CNS_neurodegeneration_panel_vl .0 summary for description ofthe potential utility of this gene in the treatment of CNS diseases.

Panel 2.2 Summary Aε2611/Aε2609 In two experiments using two different probe and primer sets, expression of the AC073079_da2 gene is limited to a sample derived from a breast cancer (CT=33.2) and appears to be overexpressed in breast cancer as compared to normal adjacent tissue. This suggests that the AC073079_da2 gene could be used to distinguish breast cancer samples from other samples and for the detection of breast cancer. Moreover, therapeutic inhibition of this gene, through the use of small molecule drugs or antibodies might be of use in the treatment of breast cancer.

Panel 4D Summary Ag2611/Ag2609 The AC073079_da2 gene is expressed at moderate levels in LPS-activated monocytes but not in resting monocytes. Conversely, the AC073079_da2 gene is expressed at moderate levels in resting macrophages, but at low levels in activated macrophages. This pattern is evident in experiments using two different probe and primer sets that match the AC073079_da2 sequence. Since circulating monocytes and tissue macrophages are both developmental^ related cell types, the AC073079_da2 gene could serve as a useful target for the development of small molecule drugs as well as therapeutic antibodies. Therapeutic antibodies and small molecule inhibitors that block the function ofthe protein encoded by the AC073079_da2 gene may be useful in reducing inflammation and autoimmune disease symptoms in patients with Crohn's disease, inflammatory bowel disease, asthma, psoriasis, and rheumatoid arthritis.

Panel CNS_1 Summary Ag2609 Expression ofthe AC073079_da2 gene is highest in the substantia nigra of a Huntington's disease patient, indicating that this gene may participate in the genetic dysregulation associated with the neurodegeneration that occurs in this brain region. The substantia nigra is also critical to the progression of Parkinson's disease neurodegeneration. Thus, pharmacological targeting ofthe GPCR encoded by the AC073079_da2 gene may help counter this genetic dysregulation and contribute to the restoration of normal function in Huntington's disease as well as potentially Parkinson's disease patients. Pharmacological modulation of GPCR signaling systems is the mechanism by which powerful depression therapies, such as SSRIs, exert their effect.

Panel CNS_neurodegeneration_vl.0 Summary Ag2611/A^g2609 The AC073079_da2 gene is expressed more highly in the temporal cortex of Alzheimer's diseased brain than in control brain without amyloid plaques, which are diagnostic and potentially causative of Alzheimer's disease. The AC073079_da2 gene encodes a protein with homology to GPCRs. GPCRs are readily targetable with drugs, and regulate many specific brain processes, including signaling processes, that are currently the target of FDA-approved pharmaceuticals that treat Alzheimer's disease, such as the cholinergic system.

The major mechanisms proposed for AbetaP-induced cytotoxicity involve the loss of Ca2+ homeostasis and the generation of reactive oxygen species (ROS). The changes in Ca2+ homeostasis could be the result of changes in G-protein-driven releases of second messengers. Thus, targeting this class of molecule can have therapeutic potential in Alzheimer's disease treatment. In particular, the increased AC073079_da2 gene expression in brains affected by Alzheimer's indicates potential therapeutic value to drugs that target this GPCR.

GPCR3 (also refered to as sggc_draft_ba656o22_ 20000731_da4/ CG55881-02)

Expression of gene sggc_draft_ba656o22_ 2000073 l_da4 was assessed using the primer-probe sets Agl898 and Agl523, described in Tables 17A and 17B. Please note that Agl523 contains a single mismatch in the probe relative to the sggc_draft_ba656o22_ 2000073 l_da4 sequence. This mismatch is not predicted to alter the RTQ-PCR results. Results from RTQ-PCR runs are shown in Tables 17C, 17D, and 17E.

Table 17A Probe Name Agl 898

Table 17C. Panel 1.2

Table 17D. Panel 1.3D

Table 17E. Panel 2D

Panel 1.2 Summary Aεl523 Expression of the sggc_draft_ba656o22 2000073 l_da4 gene is highest in a melanoma cancer cell line (CT=26.5), with expression detected in a cluster of melanoma cell lines. Thus, the expression of this gene could be used to distinguish samples derived from melanoma cell lines from other samples. In addition, therapeutic modulation of the expression or function of this gene, through the use of small molecule drugs or antibodies might be of use in the treatment of melanoma. In addition, the sggc_draft_ba656o22_ 2000073 l_da4 gene is expressed in healthy prostate tissue but not in the prostate cancer cell line.

The sggc_draft_ba656o22_ 2000073 l_da4 gene is also expressed differentially in the cerebellum. A number of neurotransmitter effector systems are defective in the Alzheimer's disease brain including: defective G protein and protein kinase C function, drastically reduced level of receptors for the second messenger Ins(l,4,5) P3 and widespread impairment of G protein-stimulated adenylyl cyclase activity in Alzheimer's disease brain. Fowler C . et al., Ann N Y Acad Sci. 786:294-304 (1996); Cowburn R.F. et al., JNeurochem. 58:1409-19 (1992). Thus, cerebellum-preferential GPCR has utility as a drug target to counter the G- protein signaling deficit in Alzheimer's disease.

Panel - 1.3D Summary Agl898Highest expression of the sggc_draft_ba656o22_ 2000073 l_da4 gene is detected in a melanoma cell line (CT=31) as is seen in Panel 1.2, with low but significant expression also seen in the cerebellum and testis. Thus, the expression of this gene could be used to distinguish samples derived from this melanoma cell line from other samples. In addition, therapeutic modulation of the expression or function of this gene, through the use of small molecule drugs or antibodies, might be of use in the treatment of melanoma. Please see Panel 1.2 summary for potential relevance of expression in cerebellum to the treatment of CNS disorders. Panel 2D Summary Agl523 Two experiments with the same probe and primer set show expression of the sggc_draft_ba656o22_20000731_da4 gene to be highest in a normal prostate in one run and a prostate cancer in the second run. In addition, the expression seen in both runs on panel 2D is specific to prostate derived samples. Thus, expression of the sggc_draft_ba656o22_ 2000073 l_da4 gene gene could be used to distinguish samples derived from prostate tissue from other samples. Furthermore, since there is substantial over expression observed in a sample derived from prostate cancer when compared to a sample derived from its normal adjacent tissue, therapeutic modulation of the expression or function of the sggc_draft_ba656o22_20000731_da4 gene product, through the use of small molecule drugs or antibodies, may be useful in the treatment of prostate cancer.

Panel 4D Summary Agl 898 Expression ofthe sggc_draft_ba656o22_20000731_da4 gene is low/undetectable (Ct values >35) in all samples on this panel (data not shown).

GPCR4 (also refered to as AC0170103A_dal/CG54212-04 and CG54212-03)

Expression of gene AC0170103A_dal and variant CG54212-03 was assessed using the primer-probe set Ag431, described in Table 18 A. Results from RTQ-PCR runs are shown in Tables 18B, 18C, 18D, 18E, and 18F.

Table 18A Probe Name Ag431

Table 18B. Panel 1

Table 18C. Panel 1.3D

Panel 1 Summary Ag431 Expression ofthe AC0170103A_dal gene is highest in a melanoma cell line (CT=27.1). Significant expression is also detected in ovarian, lung, and colon cancer cell lines. Moreover, therapeutic modulation of the expression or function of this gene, through the use of small molecule drugs or antibodies, might be of use in the treatment of melanoma and lung, colon or ovarian cancer.

Among tissues with metabolic function, the AC0170103A_dal gene is expressed in the pituitary and adrenal glands, the hypothalamus, heart and skeletal muscle. Thus, the AC0170103A_dal gene could be important in the pathogenesis and/or treatment of disease in any of those tissues.

The AC0170103A_dal gene is also expressed at moderate levels in all ofthe tissues samples originating from the central nervous system including the fetal brain, cerebellum, amygdala, hippocampus, substantia nigra, thalamus, hypothalamus, and spinal cord. The protein encoded by the AC0170103A_dal gene has homology to the GPCR family of receptors, to which several neurotransmitter receptors belong. Thus, this protein may represent a novel neurotransmitter receptor. Neurotransmitter receptors that are GPCRs include the dopamine receptor family, the serotonin receptor family, the GABAB receptor, and muscarinic acetylcholine receptors. The selected targeting of dopamine and serotonin receptors has proven to be effective in the treatement of psychiatric illnesses such as schizophrenia, bipolar disorder and depression. Furthermore, the cerebral cortex and hippocampus regions ofthe brain are known to play critical roles in Alzheimer's disease, seizure disorders, and in the normal process of memory formation. Therefore, therapeutic modulation ofthe AC0170103A_dal gene or its protein product may be beneficial in the treatment of any of these diseases.

Panel 1.3D Summary Ag431 Expression ofthe AC0170103A_dal gene occurs exclusively in fetal skeletal muscle in this panel (CT=34.5). Interestingly, this gene is not significantly expressed in adult skeletal tissue (CT=38.5), suggesting that AC0170103A_dal gene expression could be used to distinguish between the two types of tissues. In addition, the relative overexpression ofthe AC0170103A_dal gene in fetal skeletal muscle suggests that the protein product may enhance muscular growth or development in the fetus and thus may also act in a regenerative capacity in the adult. Therefore, therapeutic modulation of the ACO 170103 A_dal gene could be useful in treatment of muscle related diseases. More specifically, treatment of weak or dystrophic muscle with the protein encoded by this gene could restore muscle mass or function.

Panel 2D Summary Ag431 Expression of the AC0170103A_dal gene is highest in breast cancer (CT=32.3) and is not detected at significant levels in normal breast tissue. In addition, there is substantial expression in other samples derived from breast cancers. Thus, the expression of the AC0170103A_dal gene could be used to distinguish breast cancer tissue from other samples. Moreover, therapeutic modulation ofthe protein encoded by the AC0170103A_dal gene, through the use of small molecule drugs or antibodies, might be of use in the treatment of breast cancer.

Panel 4D Summary Ag431 The AC0170103A_dal gene is expressed in --FN-gamma- stimulated mucoepidermoid (mucus-producing) NCI-H292 cells, but not in resting NCI-H292 cells, or in IL-4-, IL-9-, or IL-13 -stimulated NCI-H292 cells. The gene is also expressed at low but significant levels at the three-day time point in a two-way mixed lymphocyte reaction with cells from normal human donors. Thus, inhibition of the function of the AC0170103A_dal gene product with antagonistic antibody or small molecule therapeutic may reduce or eliminate inflammation in colitis. Panel CNS_neurodegeneration_vl.0 Summary Ag431 Expression ofthe

AC0170103A_dal gene in this panel is low/undetectable (CT values >35) in all samples (data not shown). GPCR5 (also refered to as 21629632.0.20)

Expression of gene 21629632.0.20 was assessed using the primer-probe set Agl 284, described in Tables 19A and 19B. Results from RTQ-PCR runs are shown in Tables 19C, 19D, 19E, 19F, 19G, 19H, and 191.

Table 19A Probe Name Agl284

Table 19B. Probe Name 1539

Table 19C. Panel 1.2

Table 19D. Panel 1.3D

Table 19G. Panel 4. ID

Table 19H. Panel CNSD.01

Panel 1.2 Summary Agl539 The 21629632.0.2 gene shows rather ubiquitous expression across the samples on this panel, with highest expression in cerebral cortex (CT=25) and hippocampus. See Panel 1.3D summary for explanation.

Panel 1.3D Summary Aεl539 The expression of the 21629632.0.2 εene is highest in the samples of brain tissue and fetal muscle. The latter profile is of particular interest in that it differs significantly from that of the adult skeletal muscle. This difference implies that this protein may function to enhance muscular growth or development in the fetus and thus may also act in a regenerative capacity in the adult. Thus, therapeutic modulation of this gene could be useful in treatment of muscular related disease. For instance treatment of weak or dystrophic muscle with the protein encoded by this gene could restore muscle mass or function. The 21629632.0.2 transcript also shows highly preferential expression in brain, especially in the hippocampus and cerebral cortex where the expression is fairly high (CT = 29.5). The protein encoded by the 21629632.0.2 gene appears to be a GPCR, making it an excellent small molecule target. Both the hippocampus and cerebral cortex are affected by neurodegeneration in Alzheimer's disease; thus this molecule is an excellent candidate for a drug target for the treatment/prevention of Alzheimer's disease, and may also be useful for memory enhancement/processing in healthy subjects.

Panel 2D Summary Agl539/Agl284 Two experiments with two different probe and primer sets show siεnificant expression of the 21629632.0.2 gene in breast cancer and prostate cancer. Thus, the expression of this gene could be used to distinguish a sample of breast cancer form other tissues. Moreover, therapeutic modulation of the expression or function of the 21629632.0.2 gene product, through the use of small molecule drugs or antibodies, may be useful in the treatment of breast cancer.

Panel 4.1D Summary Agl539 The 21629632.0.2 gene is expressed at high levels in the kidney and at somewhat lower levels in the thymus. The 21629632.0.2 gene, the protein encoded for by the gene, or antibodies designed with the protein could be used to identify kidney and thymus tissue.

Panel CNSD.01 Summary Aεl539 An examination of the 21629632.0.2 gene expression in 8 brain regions across 12 individuals confirms that this protein is expressed in the brain of most, if not all, individuals including those suffering from neurologic/psychiatric disease. Utility as a drug target would benefit from likely expression in most disease states.

Panel CNS_neurodegeneration_vl.O Aεl539 The 21629632.0.2 gene encodes a protein with homology to the GPCR family of receptors. In this panel, the gene is expressed in the brain, although no association of gene expression level with Alzheimer's disease was detected. Several neurotransmitter receptors are GPCRs, including the dopamine receptor family, the serotonin receptor family, the GABAB receptor, muscarinic acetylcholine receptors, and others; thus this GPCR may represent a novel neurotransmitter receptor. Targeting various neurotransmitter receptors (dopamine, serotonin) has proven to be an effective therapy in psychiatric illnesses such as schizophrenia, bipolar disorder and depression. Furthermore the cerebral cortex and hippocampus are regions ofthe brain that are known to play critical roles in Alzheimer's disease, seizure disorders, and in the normal process of memory formation. Therefore, therapeutic modulation of the 21629632.0.2 gene or its protein product may be beneficial in the treatment of one or more of these diseases, as may stimulation of the receptor coded for by the gene.

GPCR6 (also refered to as CG50177-01)

Expression of gene CG50177-01 was assessed using the primer-probe sets Ag2385, Agl609, Agl223, and Ag2320, described in Tables 20A, 20B, and 20C. Results firom RTQ- PCR runs are shown in Tables 20D, 20E, 20F, and 20G.

Table 20A Probe Name Ag2385

Table 20B Probe Name Agl 609/1223 (identical sequences)

Table 20C Probe Name Ag2320

Table 20D. Panel 1.3D

Table 20E. Panel 2D

Table 20F. Panel 4D

Table 20G. Panel CNS_neurodegeneration_vl.O

Panel 1.2 Summary Agl223 Expression ofthe CG50177-01 gene in this panel is low/undetectable (CT values >35) across all samples (data not shown).

PANEL 1.3D SUMMARY: AG1223/1609/AG2320 EXPRESSION OF THE CG50177-01 GENE IS HIGHEST P AND EXCLUSIVE TO A SAMPLE DERIVED FROM GASTRIC CANCER CELL LINE NCI-N87 (CT=31-34). APPARENT EXPRESSION P OTHER SAMPLES IS BELOW THE THRESHOLD OF RELIABILITY. THUS THE EXPRESSION OF THIS GENE COULD BE USED TO DISTINGUISH SAMPLES DERIVED FROM GASTRIC CANCER CELL LINES FROM OTHER SAMPLES. FURTHERMORE, THERAPEUTIC MODULATION OF THE CG50177-01 GENE OR ITS GENE PRODUCT, THROUGH THE APPLICATION OF SMALL MOLECULE OR SPECIFIC ANTIBODIES, COULD BE USEFUL IN THE TREATMENT OF GASTRIC CANCER. AG2385 IN THIS EXPERIMENT, EXPRESSION OF THE CG50177-01 GENE IS LIMITED TO A CLUSTER OF SAMPLES FROM LUNG CANCER CELL LINES. THE EXPRESSION OF THIS GENE APPEARS TO BE HIGHEST IN A SAMPLE DERIVED FROM A LUNG CANCER CELL LINE (SHP-77). IN ADDITION THERE IS SUBSTANTIAL EXPRESSION P OTHER SAMPLES DERIVED FROM LUNG CANCER CELL Ll-NES (5 OF 10). THUS, THE EXPRESSION OF THE CG50177-01 GENE COULD BE USED TO DIST-NGUISH LUNG CANCER CELL LINE DERIVED SAMPLES FROM OTHER SAMPLES. MOREOVER, THERAPEUTIC MODULATION OF THE EXPRESSION OR FUNCTION OF THIS GENE, THROUGH THE USE OF SMALL MOLECULE DRUGS OR ANTIBODIES, MIGHT BE OF USE IN THE TREATMENT OF LUNG CANCER.

Panel 2D Summary Agl 223 The expression profile ofthe CG50177-01 gene reveals significant levels of expression in a number of tissue samples. Specifically there appear to be clusters of expression in breast, gastric and renal cancers when compared to adjacent normal tissues. This expression profile indicates that therapeutic modulation ofthe CG50177-01 gene or its protein product could be effective in the treatment of breast, gastric and renal cancers.

Panel 4D Summary Agl609/Agl223/Ag2385/Ag2320 Multiple experiments show that expression ofthe CG50177-01 gene is high in resting monocytes and LPS activated macrophages. This expression profile suggests that the CG50177-01 gene may encode a monocyte differentation antigen and a macrophage activation antigen. Signalling through this molecule may stimulate differentiation of monocytes to macrophages and macrophages may upregulate this molecule after LPS activation. Therefore, agonistic small molecule therapuetics to the antigen encoded for by this gene could be useful in increasing immune responsiveness during gram negative bacterial infections. Alternatively, antagonistic antibody or small molecule therapuetics could reduce or eliminate inflammation in autoimmune diseases such as asthma/allergy, emphysema, psoriasis, arthritis or other acute or chronic diseses in which activated macrophages play a detrimental role.

Panel CNS_neurodegeneration_vl.0 Summary Aε2320 The CG50177-01 gene encodes a protein with homology to the GPCR family of receptors. This gene is expressed in the brain in this panel, although no clear association of gene expression level with Alzheimer's disease was detected once samples were normalized for RNA loading. Several neurotransmitter receptors are GPCRs, including the dopamine receptor family, the serotonin receptor family, the GABAB receptor, muscarinic acetylcholine receptors, and others; thus this GPCR may represent a novel neurotransmitter receptor. Targeting various neurotransmitter receptors (dopamine, serotonin) has proven to be an effective therapy in psychiatric illnesses such as schizophrenia, bipolar disorder and depression. Furthermore, the cerebral cortex and hippocampus are regions of the brain that are known to play critical roles in Alzheimer's disease, seizure disorders, and in the normal process of memory formation. Therefore, therapeutic modulation ofthe CG50177-01 gene or its protein product may be beneficial in the treatment of one or more of these diseases, as may blockade ofthe receptor coded for by the gene.

GPCR7 (also refered to as CG50201-01 and CG50257-01)

Expression of gene CG50201-01 and variant CG50257-01 was assessed using the primer-probe sets Ag2254, Agl916, Ag2485, and Ag2554, described in Tables 21A and 21B. Results from RTQ-PCR runs are shown in Tables 21C, 21D, 21E and 21F.

Table 21A Probe Name Ag2254/Agl916 (identical sequences)

Table 2 IB Probe Name Ag2485/Ag2554 (identical sequences)

Table 2 IC. Panel 1.3D

Table 2 ID. Panel 2.2

Table 21E. Panel 4D

Table 2 IF. Panel CNS_neurodegeneration_vl.O

Relative Relative Relative

Tissue Name Expression(%] )|Expression(%) Expression(%)

Control (Path) 1 Parietal Ctx 37.1 51.8 54.5

Control (Path) 2 Parietal Ctx 15.4 27.2 26.8

Control (Path) 3 Parietal Ctx 0.0 0.0 6.2

Control (Path) 4 Parietal Ctx 53.6 100.0 46.9

Panel 1.3D Summary Four experiments using two different probe/primer sets show somewhat disparate results, most likely due to the low level of CG50201-01 gene expression in this panel. Ag2554 The CG50201-01 gene is most highly expressed in a sample derived from renal cancer cell line A498 (CT=32.7). Thus, the expression of this gene could be used to distinguish samples derived from the renal cancer cell line from other samples. Moreover, therapeutic modulation ofthe expression or function of the protein encoded by the CG50201-01 gene, through the use of small molecule drugs or antibodies, may be useful in the treatment of renal cancer. Aεl916 In this experiment, expression of the CG50201-01 gene is limited to testis (CT=34.6). Ag2485 In this experiment, low but significant expression of the CG50201-01 gene is limited to amygdala (CT=34.6) and hippocampus (CT=34.9). This result suggests a potential role for the CG50201-01 gene in central nervous system function and is consistent with what is seen in CNS_neurodegeneration_panel_vl.O. Aε2254 In this experiment, low but significant expression of the CG50201-01 gene is limited to amygdala (CT=34.4) and spleen (CT=33.2).

Panel 2.2 Summary Aε2254 The CG50201-01 gene is most highly expressed in a sample from normal colon tissue adjacent to a tumor (CT=27). Thus, the expression of this gene could be used to distinguish samples derived from normal colon tissue adjacent to colon cancers. Since there appears to be differential expression of the CG50201-01 gene between normal colon tissue and colon cancer, therapeutic upregulation of the protein encoded by this gene may be effective in the treatment of colon cancer.

Panel 4D Summary Aεl 916/Aε2554/Ag2485/Aε2254 Four experiments using two different probe/primer sets show somewhat disparate results, most likely due to the low level of CG50201-01 gene expression in this panel. This gene is reproducibly found at background levels or slightly higher in most ofthe samples on this panel.

Panel CNS_neurodegeneration_vl.0 Summary Aε2554/Aε2485/Aε2254 In this panel, the CG50201-01 gene is expressed more highly in the hippocampus of some patients with Alzheimer's disease than in control brain without amyloid plaques, which are diagnostic and potentially causative of

Alzheimer's disease. GPCRs are readily targetable with drugs, and regulate many specific brain processes, including signaling processes that are currently the target of FDA-approved pharmaceuticals that treat Alzheimer's disease, such as the cholinergic system. The major mechanisms proposed for amyloid beta-induced cytotoxicity involve the loss of Ca2+ homeostasis and the generation of reactive oxygen species (ROS). The changes in Ca2+ homeostasis could be the result of changes in G-protein- driven releases of second messengers. Thus, targeting this class of molecule can have therapeutic potential in Alzheimer's disease treatment. In particular, the increased expression of the CG50201-01 gene in brains affected by Alzheimer's indicates potential therapeutic value to drugs that target this GPCR.

GPCR8 (also refered to as CG50193-01) :

Expression of gene CG50193-01 was assessed using the primer-probe sets Ag2537, Ag2479, Ag2201 , and Ag2433 described in Tables 22A, 22B, and 22C. Results from RTQ- PCR runs are shown in Tables 22D, 22E, 22F, 22G, 22H, and 221.

Table 22A Probe Name Ag2537/2479

Table 22B. Probe Name Ag2201

Table 22C. Probe Name Ag2433

Table 22D. Panel 1.3D

Table 22E. Panel 2.2

Table 22F. Panel 2D

Table 221. Panel CNS_neurodegeneration_ l.O

Panel 1.3D Summary Ag2479/Ag2537/Ag2201 Results from three experiments using different probe/primer sets show somewhat disparate results, most likely because the levels of gene expression are very low in this panel. Using Ag2201 and Ag2537, expression ofthe CG50193-01 gene is highest in a brain cancer cell line (CT=34). In addition, there is low but significant expression in an additional sample derived from a brain cancer cell line. Other apparent expression is below the level of reliable evaluation. Of note is the observation that both ofthe cell lines showing substantial CG50193-01 gene expression are derived from a type of brain cancer called glioblastoma. Thus, the expression of this gene could be used to distinguish between glioblastoma derived samples and other samples. Moreover, therapeutic modulation ofthe expression or function ofthe CG50193-01 gene or its protein product, through the use of small molecule drugs, antibodies or protein therapeutics might be of use in the treatment of glioblastoma. Using Ag2479, expression is highest in spleen (CT=34), with low but significant expression also seen in a melanoma cell line as well as a brain cancer cell line. Other apparent expression is below the level of reliable evaluation. Thus, therapeutic modulation ofthe expression or function of this gene, through the use of small molecule drugs, antibodies or protein therapeutics might be of use in the treatment of brain cancer or melanoma. Ag2433 Expression of this gene in panel 1.3D is low/undetectable (Ct values >35) in all samples (data not shown).

Panel 2.2 Summary Ag2479/Ag2433 Two experiments with two different probe and primer sets produce results that are in excellent agreement, h both runs, expression is limited to samples derived from breast cancer. Thus, expression ofthe CG50193-01 gene could be used to distinguish sample derived from breast cancer from other samples. Moreover, therapeutic modulation ofthe expression or function of this gene, through the use of small molecule drugs or antibodies, might be of use in the treatment of breast cancer.

Panel 2D Summary Ag2201 Expression ofthe CG50193-01 gene is highest in a sample derived from a breast cancer. In addition, a number of other breast cancer samples show substantial expression, including samples of cancer tissue with matched samples derived from normal adjacent tissue. In all these samples, the CG50193-01 gene appears to be over- expressed in the cancerous tissue. This result agrees with the expression profile detected in Panel 2.2 and suggests that expression of this gene could be used to distinguish breast cancer tissue from other tissues, perhaps as a diagnostic marker for the presence of breast cancer. Moreover, therapeutic inhibition ofthe protein encoded by the CG50193-01 gene may be effective in the treatment of breast cancer.

Panel 4D Summary Aε2479/Aε2537/Aε2201/Aε2433 The CG50193-01 gene is expressed in Panel 4D at moderate to low levels in numerous independent preparations of activated B cells, resting and activated T cells, and activated lymphokine-activated killer cells. This pattern of restricted expression suggests that specific antibodies and small molecule drugs that inhibit the function of the protein encoded by the CG50193-01 gene may be useful in reducing or eliminating inflammation and autoimmune disease symptoms in patients with Crohn's disease, inflammatory bowel disease, asthma, psoriasis, and rheumatoid arthritis.

Panel CNS_neurodegeneration_vl.0 Summary Ag2479/Ag2537/Ag2201 The CG50193-01 gene is expressed primarily in the cerebellum and also shows increased expression in the hippocampus and inferior temporal cortex of some brains affected with Alzheimer's disease when compared to normal baseline expression in unaffected brains. The hippocampus is an important anatomical focus of Alzheimer's pathology, indicating that the CG50193-01 gene product may be an important biochemical component ofthe disease. GPCRs are readily targetable with drugs, and regulate many specific brain processes, including signaling processes that are currently the target of FDA-approved pharmaceuticals that treat Alzheimer's disease, such as the cholinergic system. The major mechanisms proposed for Abeta-induced cytotoxicity involve the loss of Ca2+ homeostasis and the generation of reactive oxygen species (ROS). The changes in Ca2+ homeostasis could be the result of changes in G-protein-driven releases of second messengers. Thus, targeting this class of molecule can have therapeutic potential in Alzheimer's disease treatment. In particular, the increased expression ofthe CG50193-01 gene in some brains affected by Alzheimer's indicates potential therapeutic value to drugs that target this GPCR. Normal expression of this gene in the cerebellum suggests that this GPCR may also be effectively targeted to treat diseases involving the cerebellum, including spinocerebellar ataxias, batten disease, and Niemann-Pick disease.

GPCR9 (also refered to as CG50203-01)

Expression of gene CG50203-01 was assessed using the primer-probe sets Ag2484, Agl783, Agl 781, and Agl583 described in Tables 23A and 23B. Results from RTQ-PCR runs are shown in Tables 23C and 23D.

Table 23A Probe Name Ag2484

Table 23B Probe Name Agl783/1781/1583 (identical sequences)

Table 23C. Panel 1.3D

Table 23D. Panel 4D

Panel 1.3D Summary Agl783/Agl781 Two experiments with the same probe and primer set are in good agreement, with significant expression of the CG50203-01 gene is limited to the spleen. This suggests that expression of the gene could be used to differentiate spleen tissue from other tissue types. A^g2484/Agl583 Expression of the CG50203-01 gene in panel 1.3D is low/undetectable (CT values >35) in all samples (data not shown).

Panel 2.2 Summary Agl783/Agl781/Agl583 Expression of the CG50203-01 gene in panel 2.2 is low/undetectable (CT values >35) in all samples (data not shown).

Panel 4D Summary Agl783/Agl781 Two experiments using the same probe and primer set are in very good agreement, with significant expression of the CG50203-01 gene limited to a sample from liver cirrhosis. This result suggests that the protein encoded by the CG50203-01 gene may play a role in liver cirrhosis. Thus, small molecule drugs or specific antibodies to the CG50203-01 gene product may be effective in the treatment of liver cirrhosis. Agl583/Ag2484 Expression of this gene in panel 4D is low/undetectable (CT values >35) in all samples (data not shown). Panel CNS_neurodegeneration_vl.O Summary Ag2484 Expression of the CG50203-01 gene in panel CNS_jneurodegeneration is low/undetectable (CT values >35) in all samples (data not shown).

GPCRlO (also refered to as CG50197-01 and CG50197-02)

Expression of gene CG50197-01 and variant CG50197-02 was assessed using the primer-probe sets Ag2482 and Ag2347, described in Tables 24A, and 24B. Results from RTQ- PCR runs are shown in Tables 24C, 24D, 24E, and 24F.

Table 24A Probe Name Ag2482

Table 24B Probe Name Ag2347

Table 24C. Panel 1.3D

Table 24D. Panel 2.2

Table 24E. Panel 2D

Table 24F. Panel 3D

Relative Relative Expression(%) Expression(%)

3dx4tm5085f_ 3dx4tm5085f_

Tissue Name ag2482 b2 Tissue Name ag2482 b2

Panel 1.3D Summary Ag2482 Expression ofthe CG50197-01 gene in two independent runs is highest in a sample derived from a lung cancer cell line (SHP-77). Its expression in this panel is almost exclusive to this sample. Thus, the expression the CG50197-01 gene could be used to distinguish samples derived from this cell line and other samples. Furthemore, therapeutic modulation of the expression or function of the protein encoded by the CG50197-01 gene, through the use of small molecule drugs or antibodies, may be useful in the treatment of lung cancer. Ag2347 Expression ofthe CG50197-01 gene in panel 1.3D is low/undetectable (CT values >35) in all samples (data not shown).

Panel 2.2 Summary Ag2347 Expression ofthe CG50197-01 gene is limited to a sample derived from a colon cancer (CT=31.4). This result suggests that expression of the CG50197-01 gene could be used as a diagnostic marker for the presence of colon cancer. Furthermore, therapeutic modulation of the expression or function of the CG50197-01 gene product, through the use of small molecule drugs or antibodies, may be effective in the treatment of colon cancer. Ag2482 Expression of the CG50197-01 gene in Panel 2.2 is low/undetectable (CT values >35) in all samples (data not shown).

Panel 2D Summary Ag2482 Expression ofthe CG50197-01 gene is highest in a sample derived from a prostate cancer and overall, its expression appears to be specific for prostate tissue, hi addition, one sample derived from prostate cancer shows substantial over expression when compared to a matched sample derived from normal adjacent tissue. Thus, the expression of this gene could be used to distinguish prostate derived tissue from other tissues. Moreover, therapeutic modulation of the expression or function of the CG50197-01 gene or its protein product, through the use of small molecule drugs, antibodies or protein therapeutics, might be of use in the treatment of prostate cancer.

Panel 3D Summary Ag2482 Significant expression of the CG50197-01 gene is limited to a sample derived from a lung cancer cell line. This preferential expression in lung cancer is also seen in the expression profiles from Panel 1.3D. This result suggests that expression of the CG50197-01 gene could be used to distinguish this cell line from other samples. Furthermore, therapeutic modulation of the gene or its protein product could potentially be useful in the treatment of lung cancer.

Panel 4D Summary Ag2482/Ag2347 Expression of the CG50197-01 gene in this panel is low/undetectable (CT values >35) in all samples (data not shown).

Panel CNS_neurodegeneration_vl.0 Ag2347 Expression of the CG50197-01 gene in panel CNS_neurodegeneration_vl.O is low/undetectable (CT values >35) in all samples (data not shown). GPCRll (also refered to as CG50199-01)

Expression of gene CG50199-01 was assessed using the primer-probe sets Ag2483 and Agl729, described in Tables 25A and 25B. Results from RTQ-PCR runs are shown in Tables 25C, 25D, and 25E.

Table 25 A Probe Name Ag2483

Table 25B. Probe Name Agl 729

Table 25C. Panel 1.3D

Table 25D. Panel 4D

Panel 1.3D Summary Ag2483 Significant expression of the CG50199-01 gene is limited to a sample derived from a breast cancer (CT=34.9). Thus, the expression of this gene could be used to distinguish this cell line from other samples. Furthermore, therapeutic modulation ofthe expression or function ofthe protein encoded by the CG50199-01 gene, through the use of small molecule drugs or antibodies, might be beneficial in the treatment of breast cancer.

Panel 4D Summary Ag2483/Agl729 Two experiments using two different probe and primer sets show highest expression ofthe CG50199-01 gene in ionomycin-activated Ramos B lymphblastoid cells (CT=29.4) in one run and in activated Trl cells (CT=32.2) in the second run. Moderate expression is also detected in activated Thl and Th2 cells, resting and cytokine-activated dermal fibroblasts, and in resting and cytokine activated mucoepidermoid cells. Inhibition ofthe function ofthe protein encoded by the CG50199-01 gene by a specific antibody or small molecule drug may reduce inflammation and autoimmunity that result from asthma, psoriasis, and inflammatory bowel disease.

Panel CNS neurodegeneration vl.O Summary Ag2483 The protein encoded by the CG50199-01 gene contains homology to the GPCR family of receptors, and is shown by panel CNS_Neurodegeneration_Vl .0 to be expressed in the brain, with gene expression down regulated in the temporal cortex ofthe Alzheimer's diseased brain. The temporal cortex is a region that specifically shows neurodegeneration in Alzheimer's disease. Several neurotransmitter receptors are GPCRs, including the dopamine receptor family, the serotonin receptor family, the GABAB receptor, and the muscarinic acetylcholine receptors. Thus, the GPCR encoded by the CG50199-01 gene may represent a novel neurotransmitter receptor. Targeting various neurotransmitter receptors, such as dopamine, serotonin receptors, has proven to be an effective therapy in psychiatric illnesses such as schizophrenia, bipolar disorder and depression. Furthermore the cerebral cortex and hippocampus are regions ofthe brain that are known to play critical roles in Alzheimer's disease, seizure disorders, and in the normal process of memory formation. Thus, therapeutic modulation of this gene or its protein product may be beneficial in one or more of these diseases, as may stimulation ofthe receptor coded for by the gene.

GPCR12 (also refered to as CG50217-01)

Expression of gene CG50217-01 was assessed using the primer-probe sets Ag2494 and Ag2345, described in Tables 26A and 26B. Results from RTQ-PCR runs are shown in Tables 26C and 26D.

Table 26A Probe Name Ag2494

Table 26B. Probe Name Ag2345 (contains a single base mismatch within the probe sequence)

Table 26C. Panel 1.3D

Table 26D. Panel 2.2

Panel 1.3D Summary Ag2494/Ag2345 Two experiments with two different probe and primer sets show highest expression ofthe CG50217-01 gene in a sample derived from lung cancer cell line and a gastric cancer cell line. This result suggests that expression ofthe CG50217-01 gene could be used to differentiate between lung and gastric cancer cell lines and other tissues and to detect the presence of gastric and lung cancers. Moreover, therapeutic modulation ofthe expression or function of this gene, through the use of small molecule drugs or antibodies, might be of use in the treatment of gastric and lung cancers.

Panel 2.2 Summary Ag2345 Significant expression of the CG50217-01 gene is limited to normal prostate tissue adjacent to a prostate cancer (CT=33.5). The gene appears to be overexpressed in normal prostate tissue when compared to the adjacent tumor. Thus, therapeutic upregulation of the activity of the CG50217-01 gene product, through the application of the protein product or agonists might be of use in the treatment of prostate cancer.

Panel 4D Summary Ag2494/Ag2345 Expression ofthe CG50217-0 in this panel is low/undetectable (CT values >35) in all samples (data not shown). Example 3. SNP analysis of GPCRX clones

SeqCalling™ Technology: cDNA was derived from various human samples representing multiple tissue types, normal and diseased states, physiological states, and developmental states from different donors. Samples were obtained as whole tissue, cell lines, primary cells or tissue cultured primary cells and cell lines. Cells and cell lines may have been treated with biological or chemical agents that regulate gene expression for example, growth factors, chemokines, steroids. The cDNA thus derived was then sequenced using CuraGen's proprietary SeqCalling technology. Sequence traces were evaluated manually and edited for corrections if appropriate. cDNA sequences from all samples were assembled with themselves and with public ESTs using bioinformatics programs to generate CuraGen's human SeqCalling database of SeqCalling assemblies. Each assembly contains one or more overlapping cDNA sequences derived from one or more human samples. Fragments and ESTs were included as components for an assembly when the extent of identity with another component ofthe assembly was at least 95% over 50 bp. Each assembly can represent a gene and/or its variants such as splice forms and/or single nucleotide polymorphisms (SNPs) and their combinations. Variant sequences are included in this application. A variant sequence can include a single nucleotide polymoφhism (SNP). A SNP can, in some instances, be referred to as a "cSNP" to denote that the nucleotide sequence containing the SNP originates as a cDNA. A SNP can arise in several ways. For example, a SNP may be due to a substitution of one nucleotide for another at the polymorphic site. Such a substitution can be either a transition or a transversion. A SNP can also arise from a deletion of a nucleotide or an insertion of a nucleotide, relative to a reference allele. In this case, the polymorphic site is a site at which one allele bears a gap with respect to a particular nucleotide in another allele. SNPs occurring within genes may result in an alteration ofthe amino acid encoded by the gene at the position ofthe SNP. Intragenic SNPs may also be silent, however, in the case that a codon including a SNP encodes the same amino acid as a result ofthe redundancy ofthe genetic code. SNPs occurring outside the region of a gene, or in an intron within a gene, do not result in changes in any amino acid sequence of a protein but may result in altered regulation ofthe expression pattern for example, alteration in temporal expression, physiological response regulation, cell type expression regulation, intensity of expression, stability of transcribed message.

Method of novel SNP Identification: SNPs are identified by analyzing sequence assemblies using CuraGen's proprietary SNPTool algorithm. SNPTool identifies variation in assemblies with the following criteria: SNPs are not analyzed within 10 base pairs on both ends of an alignment; Window size (number of bases in a view) is 10; The allowed number of mismatches in a window is 2; Minimum SNP base quality (PHRED score) is 23; Minimum number of changes to score an SNP is 2/assembly position. SNPTool analyzes the assembly and displays SNP positions, associated individual variant sequences in the assembly, the depth ofthe assembly at that given position, the putative assembly allele frequency, and the SNP sequence variation. Sequence traces are then selected and brought into view for manual validation. The consensus assembly sequence is imported into CuraTools along with variant sequence changes to identify potential amino acid changes resulting from the SNP sequence variation. Comprehensive SNP data analysis is then exported into the SNPCalling database. Method of novel SNP Confirmation: SNPs are confirmed employing a validated method know as Pyrosequencing (Pyrosequencing, Westborough, MA). Detailed protocols for Pyrosequencing can be found in: Alderborn et al. Determination of Single Nucleotide Polymorphisms by Real-time Pyrophosphate DNA Sequencing. (2000). Genome Research. 10, Issue 8, August. 1249-1265. In brief, Pyrosequencing is a real time primer extension process of genotyping. This protocol takes double-stranded, biotinylated PCR products from genomic DNA samples and binds them to streptavidin beads. These beads are then denatured producing single stranded bound DNA. SNPs are characterized utilizing a technique based on an indirect bioluminometric assay of pyrophosphate (PPi) that is released from each dNTP upon DNA chain elongation. Following Klenow polymerase-mediated base incorporation, PPi is released and used as a substrate, together with adenosine 5'-phosphosulfate (APS), for ATP sulfurylase, which results in the formation of ATP. Subsequently, the ATP accomplishes the conversion of luciferin to its oxi-derivative by the action of luciferase. The ensuing light output becomes proportional to the number of added bases, up to about four bases. To allow processivity ofthe method dNTP excess is degraded by apyrase, which is also present in the starting reaction mixture, so that only dNTPs are added to the template during the sequencing. The process has been fully automated and adapted to a 96-well format, which allows rapid screening of large SNP panels. The DNA and protein sequences for the novel single nucleotide polymorphic variants are reported. Variants are reported individually but any combination of all or a select subset of variants are also included. In addition, the positions ofthe variant bases and the variant amino acid residues are underlined. GPCRl Results

GPCRla

The DNA and protein sequences for the novel single nucleotide polymoφhic variants ofthe GPCR-like gene of CuraGen Ace. No. GMAC073079_A_ are reported. In summary, there are 13 variants. Variant 13373946 is a C to T SNP at 100 bp ofthe nucleotide sequence that results in a Pro to Ser change at amino acid 28 of protem sequence, variant 13374313 is an A to G SNP at 221 bp ofthe nucleotide sequence that results in a Glu to Gly change at amino acid 68 of protein sequence, variant 13373944 is a T to C SNP at 268 bp ofthe nucleotide sequence that results in no change in the protein sequence (silent), variant 13374312 is a T to C SNP at 272 bp ofthe nucleotide sequence that results in a Val to Ala change at amino acid 85 of protein sequence, variant 13374311 is a C to T SNP at 318 bp ofthe nucleotide sequence that results in no change in the protein sequence (silent), variant 13373942 is a T to C SNP at 346 bp ofthe nucleotide sequence that results in a Phe to Leu change at amino acid 110 of protein sequence, variant 13374001 is a G to A SNP at 444 bp ofthe nucleotide sequence that results in no change in the protein sequence (silent), variant 13374309 is a C to T SNP at 472 bp ofthe nucleotide sequence that results in no change in the protein sequence (silent), variant 13374307 is a C to T SNP at 534 bp ofthe nucleotide sequence that results in no change in the protein sequence (silent), variant 13374306 is a T to C SNP at 563 bp ofthe nucleotide sequence that results in a Leu to Pro change at amino acid 182 of protein sequence, variant 13374305 is a C to A SNP at 663 bp ofthe nucleotide sequence that results in no change in the protein sequence (silent), variant 13373937 is a C to T SNP at 728 bp ofthe nucleotide sequence that results in a Ser to Phe change at amino acid 237 of protein sequence, and variant 13373936 is a C to T SNP at 928 bp ofthe nucleotide sequence that results in a Gin to Stop at amino acid 304 of protein sequence. GPCRlb

There are 14 variants reported for GPCRlb. Variant 13374122 is a C to T SNP at 105 bp ofthe nucleotide sequence that results in a Pro to Ser change at amino acid 26 of protein sequence, variant 13374313 is an A to G SNP at 226 bp ofthe nucleotide sequence that results in a Glu to Gly change at amino acid 66 of protein sequence, variant 13373944 is a T to C SNP at 273 bp ofthe nucleotide sequence that results in no change in the protein sequence

(silent), variant 13374312 is a T to C SNP at 277 bp ofthe nucleotide sequence that results in a Val to Ala change at amino acid 83 of protein sequence, variant 13374311 is a C to T SNP at 323 bp ofthe nucleotide sequence that results in no change in the protein sequence (silent), variant 13373942 is a T to C SNP at 351 bp ofthe nucleotide sequence that results in a Phe to Leu change at amino acid 108 of protein sequence, variant 13374001 is a G to A SNP at 449 bp ofthe nucleotide sequence that results in no change in the protein sequence (silent), variant 13374309 is a C to T SNP at 477 bp ofthe nucleotide sequence that results in no change in the protem sequence (silent), variant 13373940 is an insertion of nucleotide T after 514 bp ofthe nucleotide sequence that results in a frameshift with all amino acids after 162 being discordant with the original protein sequence, variant 13374307 is a C to T SNP at 539 bp ofthe nucleotide sequence that results in no change in the protein sequence (silent), variant 13374306 is a T to C SNP at 568 bp ofthe nucleotide sequence that results in a Leu to Pro change at amino acid 180 of protein sequence, variant 13373938 is an A to C SNP at 668 bp of the nucleotide sequence that results in no change in the protein sequence (silent), variant 13373937 is a C to T SNP at 733 bp ofthe nucleotide sequence that results in a Ser to Phe change at amino acid 235 of protein sequence, and variant 13373936 is a C to T SNP at 933 bp ofthe nucleotide sequence that results in a Gin to Stop at amino acid 302 of protein sequence. Additional GPCRl SNPs are provided in Table 27A below.

Table 27A: Additional GPCRl SNPs

GPCRlb SNPs:

Cons . Pos. : 166 Depth: 23 Change : C > T Putative Allele Freq. : 0 .087

Cons . Pos. : 172 Depth: 23 Change : T > C Putative Allele Freq. : 0 .217

GPCRlc SNPs:

Cons .Pos. 33 Depth: 18 Change : C > A Putative Allele Freq. : 0 .111

Cons Pos . 279 Depth: 31 Change : T > C Putative Allele Freq. : 0 .065

Cons Pos. 447 Depth: 41 Change : c > - Putative Allele Freq. : 0

Cons Pos. 453 Depth: 40 Change : G > A Putative Allele Freq. : 0 .050

Cons Pos. 673 Depth: 25 Change: C > A Putative Allele Freq. : 0 080

GPCRld SNPs:

Cons Pos. 40 Depth: 24 Change: G > A Putative Allele Freq. : 0 083

Cons Pos. 63 Depth: 51 Change: T > - Putative Allele Freq. 0 039

Cons Pos. 66 Depth: 51 Change : T > - Putative Allele Freq. 0 039

Cons Pos. 250 Depth: 51 Change : G > A Putative Allele Freq. 0 098

Cons Pos. 315 Depth: 51 Change : G > T Putative Allele Freq. 0 039

Cons Pos. 415 Depth: 58 Change : A > G Putative Allele Freq. 0 034

Cons Pos. 434 Depth: 65 Change: A > - Putative Allele Freq. 0 031

Cons Pos. 443 Depth: 66 Change: T > - Putative Allele Freq. 0 030

Cons Pos . 444 Depth: 66 Change : G > - Putative Allele Freq. 0 030

Cons Pos. 444 Depth: 66 Change : G > A Putative Allele Freq. 0 091

Cons Pos . 470 Depth: 75 Change : A > G Putative Allele Freq. 0 027

Cons Pos. 495 Depth: 78 Change : G > - Putative Allele Freq. 0 026

Cons Pos. 507 Depth: 79 Change : G > A Putative Allele Freq. 0. 025

Cons. Pos. 535 Depth: 77 Change : C > T Putative Allele Freq. 0. 026 Cons . Pos . : 663 Depth 50 Change : G > A Putative Allele Freq : 0.060

Cons. Pos. : 709 Depth 51 Change : A > G. Putative Allele Freq : 0.039

Cons. Pos.: 760 Depth 51 Change : T > C Putative Allele Freq : 0.039

Cons. Pos.: 886 Depth 51 Change: G > A Putative Allele Freq : 0.059

Cons. Pos. : 959 Depth 25 Change: G > T Putative Allele Freq : 0.080

GPCRle SNPs:

Cons . Pos . : 33 Depth 39 Change: C > A Putative Allele Freq. 0.051

Cons. Pos. : 105 Depth 65 Change : C > T Putative Allele Freq. 0.046

Cons. Pos.: 226 Depth 65 Change : A > G Putative Allele Freq. 0.031

Cons. Pos. : 277 Depth 65 Change: T > C Putative Allele Freq. 0.031

Cons . Pos . : 323 Depth 64 Change : C > T Putative Allele Freq. 0.047

Cons. Pos. : 449 Depth 88 Change : G > A Putative Allele Freq. 0.023

Cons. Pos. : 477 Depth 86 Change: C > T Putative Allele Freq. 0.023

Cons. Pos. : 514 Depth 82 Change: T > C Putative Allele Freq. 0.02

Cons. Pos. : 539 Depth 75 Change : C > T Putative Allele Freq. 0.107

Cons . Pos. : 568 Depth 67 Change: T > C Putative Allele Freq. 0.030

Cons . Pos . : 668 Depth 60 Change : C > A Putative Allele Freq. 0.033

Cons . Pos. : 733 Depth 61 Change: C > T Putative Allele Freq. 0.115

GPCRlf SNPs:

Cons. Pos. : 54 Depth 39 Change : C > A Putative Allele Freq : 0.051

AA translation view alpha)

Fragment Listing:

-> 145919905(+,i, 119650936) Fpos: 134

-> 145919950(+, 1,119650936) Fpos: 110

Cons. Pos. : 127 Depth 65 Change: c > T Putative Allele Freq : 0.046

AA translation view alpha)

Fragment Listing:

-> 146985611(+,i, 119650936) Fpos: 122

-> 146985939(+, 1,119650936) Fpos: 118

-> 146990206(+,i, 119650936) Fpos: 116

Cons. Pos. : 253 Depth 65 Change : A > G Putative Allele Freq : 0.031

AA translation view alpha)

Fragment Listing:

-> 147090582(+,i, 119650936) Fpos: 282

-> 147090676(+,i, 119650936) Fpo. 274

Cons. Pos.: 304 Depth 65 Change : T > C Putative Allele Freq : 0.031

AA translation view alpha)

Fragment Listing:

-> 146170427 (+,i, 119650936) Fpo. 3: 305

-> 146170442(+, 1,119650936) Fpos: 299

Cons. Pos. : 350 Depth: 64 Change : C > T Putative Allele Freq : 0.047

AA translation view alpha)

Fragment Listing:

-> 146985893(+,i, 119650936) Fpos 340

-> 146985897 (+,i, 119650936) Fpo. 344

-> 147090633 (+,i, 119650936) Fpos: 384

Cons. Pos. : 472 Depth: 90 Change : C > - Putative Allele Freq. : 0.022

AA translation view ( alpha)

Fragment Listing:

-> 146170397 (+,1,119650936) Fpos: 467 -> 146170474 (+,i, 119650936) Fpos: 467

Cons. Pos.: 478 Depth: 88 Change: G > A Putative Allele Freq. : 0 023

AA translation view (alpha)

Fragment Listing:

-> 145919950(+,i, 119650936) Fpos: 526

-> 146115328 (-,1,119650936) Fpos: 580

Cons. Pos.: 506 Depth: 86 Change: C > - Putative Allele Freq. : 0 023

AA translation view (alpha)

Fragment Listing:

-> 146170474 (+,i, 119650936) Fpos: 500

-> 147090702(-, 1,119650936) Fpos: 541

Cons. Pos.: 506 Depth: 86 Change: C > T Putative Allele Freq. : 0 023

AA translation view (alpha)

Fragment Listing:

-> 146952987(+,i, 119650936) Fpos: 519

-> 146985961(-,i, 119650936) Fpos: 512

Cons. Pos.: 518 Depth: 84 Change: C > - Putative Allele Freq. : 0 024

AA translation view (alpha)

Fragment Listing:

-> 146170540(+,i, 119650936) Fpos: 519

-> 147191380(+,i, 119650936) Fpos: 587

Cons. Pos.: 544 Depth: 82 Change: T > C Putative Allele Freq. : 0 024

AA translation view (alpha)

Fragment Listing:

-> 146952753(-,i, 119650936) Fpos: 487

-> 146985831(+, 1,119650936) Fpos: 528

Cons. Pos.: 570 Depth: 75 Change: C > T Putative Allele Freq. : 0 107

AA translation view (alpha)

Fragment Listing:

-> 146115375(-,i, 119650936) Fpos: 489

-> 146952753(-,i, 119650936) Fpos: 462

-> 146952861(-,i, 119650936) Fpos: 484

-> 146952886(-, 1,119650936) Fpos: 484

-> 146985831(+,i, 119650936) Fpos: 553

-> 146985913(-,i, 119650936) Fpos: 464

-> 147191402 (-,i, 119650936) Fpos: 506

-> 147191417 (-,i, 119650936) Fpos: 512

Cons. Pos.: 572 Depth: 75 Change: A > - Putative Allele Freq. : 0 053

AA translation view (alpha)

Fragment Listing:

-> 146115265(+, 1,119650936) Fpos: 594

-> 146952642(+,i, 119650936) Fpos: 574

-> 146985893(+,i, 119650936) Fpos: 557

-> 146985897 (+,i, 119650936) Fpos: 561

Cons. Pos.: 581 Depth: 74 Change: T > - Putative Allele Freq. : 0 027

AA translation view (alpha)

Fragment Listing:

-> 146952642 (+,i, 119650936) Fpos: 582 ,

-> 146985897 (+,i, 119650936) Fpos: 569

Cons. Pos.: 599 Depth: 67 Change: T > C Putative Allele Freq. : 0. 030

AA translation view (alpha) Fragment Listing:

-> 146985611(+,i, 119650936) Fpos: 585

-> 146990206(+,i, 119650936) Fpos : 578

Cons. Pos.: 699 Depth: 60 Change: C > A Putative Allele Freq. : 0.033

AA translation view (alphca)

Fragment Listing:

-> 145919890(-,i, 119650936) Fpos : 389

-> 146115328(-,i, 119650936) Fpos: 361

Cons. Pos.: 764 Depth: 61 Change: C > T Putative Allele Freq. : 0.115

AA translation view (alphca)

Fragment Listing:

-> 146952735(-,i, 119650936) Fpos : 287

-> 146952816(-,i, 119650936) Fpos: 286

-> 146952861(-,i, 119650936) Fpos : 290

-> 146952886(-,i, 119650936) Fpos: 290

-> 146985913(-,i, 119650936) Fpos : 270

-> 147191417(-,i, 119650936) Fpos: 318

-> 147191539(-,i, 119650936) Fpos: 311

Cons. Pos.: 948 Depth: 61 Change: A > - Putative Allele Freq. : 0.049

AA translation view (alph<a)

Fragment Listing

-> 146952735(-,i, 119650936) Fpos: 112

-> 147090580(-,i, 119650936) Fpos : 112

-> 147090702(-,i, 119650936) Fpos : 114

Cons. Pos.: 974 Depth: 34 Change: C > T Putative Allele Freq. : 0.118

AA translation view (alph<a)

Fragment Listing

-> 147090647(-,i, 119650936) Fpos: 89

-> 147090662(-,i, 119650936) Fpos : 101

-> 147191500(-,i, 119650936) Fpos : 104

-> 147191579(-,i, 119650936) Fpos : 115

GPCR2

Variants are reported individually but any combination of all or a select subset of variants are also included.

Cons Pos. 460 Depth: 27 Change: T > - Putative Allele Freq. : 0 111

Cons Pos. 496 Depth: 29 Change: T > - Putative Allele Freq. : 0 069

Cons Pos. 623 Depth: 19 Change : T > - Putative Allele Freq. : 0 158

Cons Pos. 638 Depth: 18 Change : T > - Putative Allele Freq. : 0 111

Cons Pos. 834 Depth: 14 Change: C > T Putative Allele Freq. : 0 143

Cons Pos. 854 Depth: 14 Change: C > - Putative Allele Freq. : 0 429

Cons Pos. 859 Depth: 14 Change: C > - Putative Allele Freq. : 0 429

GPCR3 A summarized below in Tables XX and XX, there are multiple variants reported for

GPCR3. As shown in Table XX, variant 13375479 is an A to G SNP at 71 bp ofthe nucleotide sequence that results in no change in the protein sequence (silent), variant 13375480 is a C to T SNP at 185 bp ofthe nucleotide sequence that results in no change in the protem sequence (silent), variant 13375481 is a G to C SNP at 389 bp ofthe nucleotide sequence that results in an Arg to Ser change at amino acid 129 of protein sequence, variant 13375482 is a G to A SNP at 541 bp ofthe nucleotide sequence that results in a Cys to Tyr change at amino acid 180 of protein sequence, variant 13375483 is a T to C SNP at 780 bp of the nucleotide sequence that results in a Tyr to His change at amino acid 260 of protein sequence, variant 13375484 is a T to C SNP at 858 bp ofthe nucleotide sequence that results in no change in the protein sequence (silent), and variant 13375485 is a G to C SNP at 867 bp ofthe nucleotide sequence that results in a Val to Leu change at amino acid 289 of protein sequence.

GPCR4

A summarized below in Tables XX and XX, there are multiple variants reported for GPCR4a. As shown in Table XX, variant 13373774 is a C to T SNP at 206 bp ofthe nucleotide sequence that results in no change in the protein sequence (silent), variant 13019736 is a T to C SNP at 210 bp ofthe nucleotide sequence that results in a Tyr to His change at amino acid 60 of protein sequence, variant 13373983 is a C to G SNP at 389 bp of the nucleotide sequence that results in no change in the protein sequence (silent), variant 13374293 is a T to C SNP at 390 bp ofthe nucleotide sequence that results in a Tyr to His change at amino acid 120 of protein sequence, variant 13374292 is an A to G SNP at 430 bp ofthe nucleotide sequence that results in a His to Arg change at amino acid 133 of protein sequence, variant 13374291 is a T to C SNP at 523 bp ofthe nucleotide sequence that results in a Met to Thr change at amino acid 164 of protein sequence, variant 13375488 is a C to T SNP at 536 bp ofthe nucleotide sequence that results in no change in the protein sequence (silent), variant 13374290 is a C to T SNP at 542 bp ofthe nucleotide sequence that results in no change in the protein sequence (silent), variant 13373986 is a G to A SNP at 551 bp ofthe nucleotide sequence that results in no change in the protein sequence (silent), variant 13373987 is a G to A SNP at 579 bp ofthe nucleotide sequence that results in an Ala to Thr change at amino acid 183 of protein sequence, variant 13375487 is a C to T SNP at 630 bp of the nucleotide sequence that results in no change in the protein sequence (silent), variant 13374288 is an A to G SNP at 702 bp ofthe nucleotide sequence that results in a Thr to Ala change at amino acid 224 of protein sequence, variant 13373989 is a C to T SNP at 760 bp of the nucleotide sequence that results in an Ala to Val change at amino acid 243 of protein sequence, and variant 13373990 is a G to C SNP at 960 bp ofthe nucleotide sequence that results in an Ala to Pro change at amino acid 310 of protein sequence.

A summarized below in Table 27G, there are multiple variants reported for GPCR4b.

GPCR5

A summarized below in Table XX, there are several variants reported for GPCR5. As shown in Table XX, variant 13375490 is an A to G SNP at 1380 bp ofthe nucleotide sequence that results in no change in the protein sequence (silent), and variant 13375489 is a C to T SNP at 1832 bp ofthe nucleotide sequence that results in no change in the protein sequence since he SNP is not in the amino acid coding region.

GPCR6

Variant 13373922 is a G to A SNP at 299 bp ofthe nucleotide sequence that results in an Ala to Thr change at amino acid 100 of protein sequence, variant 13373923 is an A to G SNP at 401 bp ofthe nucleotide sequence that results in a Thr to Ala change at amino acid 134 of protein sequence, and variant 13373924 is a T to A SNP at 459 bp ofthe nucleotide sequence that results in a Leu to His change at amino acid 153 of protein sequence.

GPCR7

Variant 13373933 is a C to T SNP at 128 bp ofthe nucleotide sequence that results in no change in the protein sequence (silent), variant 13373934 is a C to G SNP at 383 bp ofthe nucleotide sequence that results in an He to Met change at amino acid 127 of protein sequence, and variant 13373935 is a T to C SNP at 813 bp ofthe nucleotide sequence that results in a Tφ to Arg change at amino acid 271 of protein sequence.

GPCR8 As summarized below in Table XX, there is one variant reported for GPCR8. As shown in Table XX, variant 13375497 is a T to C SNP at 263 bp ofthe nucleotide sequence that results in an He to Thr change at amino acid 77 ofthe protein sequence.

GPCR9

As summarized below in Table XX, there is one variant reported for GPCR9. As shown in Table XX, variant 13375498 is a G to A SNP at 839 bp ofthe nucleotide sequence that results in no change in the protein sequence (silent).

GPCRlO

Possible SNPs found for GPCRlOa are listed in Table 27M. Putative Allele Frequency (PAF) is shown in column four.

GPCRll

Cons.Pos.: 505 Depth: 39 Change: G > A Putative Allele Freq.: 0.051

Cons.Pos.: 811 Depth: 33 Change: G > A Putative Allele Freq.: 0.091

GPCR12

As summarized below in Tables 27N and 270, there are multiple variants reported for GPCR12. As shown in Table 27N, variant 13373913 is a C to T SNP at 223 bp ofthe nucleotide sequence that results in a Leu to Phe change at amino acid 75 of protein sequence; variant 13373912 is a T to G SNP at 730 bp ofthe nucleotide sequence that results in a Ser to Ala change at amino acid 244 of protein sequence; and variant 13373911 is a G to A SNP at 805 bp ofthe nucleotide sequence that results in an Asp to Asn change at amino acid 269 of protein sequence.

EQUIVALENTS

Although particular embodiments have been disclosed herein in detail, this has been done by way of example for pmposes of illustration only, and is not intended to be limiting with respect to the scope ofthe appended claims, which follow. In particular, it is contemplated by the inventors that various substitutions, alterations, and modifications may be made to the invention without departing from the spirit and scope ofthe invention as defined by the claims. The choice of nucleic acid starting material, clone of interest, or library type is believed to be a matter of routine for a person of ordinary skill in the art with knowledge ofthe embodiments described herein. Other aspects, advantages, and modifications considered to be within the scope ofthe following claims.

Claims

WHAT IS CLAIMED IS:

1. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of:

(a) a mature form of an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 4, 6, 8, 10, 12, 21, 28, 35, 37, 44, 51, 58, 60, 67, 74, 81, 83, 90 and 97;

(b) a variant of a mature form of an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 4, 6, 8, 10, 12, 21, 28, 35, 37, 44, 51, 58, 60, 67, 74, 81, 83, 90 and 97, wherein one or more amino acid residues in said variant differs from the amino acid sequence of said mature form, provided that said variant differs in no more than 15% ofthe amino acid residues from the amino acid sequence of said mature form;

(c) an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 4, 6, 8, 10, 12, 21, 28, 35, 37, 44, 51, 58, 60, 67, 74, 81, 83, 90 and 97; and

(d) a variant of an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 4, 6, 8, 10, 12, 21, 28, 35, 37, 44, 51, 58, 60, 67, 74, 81, 83, 90 and 97, wherein one or more amino acid residues in said variant differs from the amino acid sequence of said mature form, provided that said variant differs in no more than 15% of amino acid residues from said amino acid sequence.

2 The polypeptide of claim 1, wherein said polypeptide comprises the amino acid sequence of a naturally-occurring allelic variant of an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 4, 6, 8, 10, 12, 21, 28, 35, 37, 44, 51, 58, 60, 67, 74, 81, 83, 90 and 97.

3. The polypeptide of claim 2, wherein said allelic variant comprises an amino acid sequence that is the translation of a nucleic acid sequence differing by a single nucleotide from a nucleic acid sequence selected from the group consisting of SEQ ID NOS:l, 3, 5, 1, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96.

4. The polypeptide of claim 1, wherein the amino acid sequence of said variant comprises a conservative amino acid substitution.

5. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence selected from the group consisting of:

(a) a mature form of an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 4, 6, 8, 10, 12, 21, 28, 35, 37, 44, 51, 58, 60, 67, 74, 81, 83, 90 and 97;

(b) a variant of a mature form of an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 4, 6, 8, 10, 12, 21, 28, 35, 37, 44, 51, 58, 60, 67, 74, 81, 83, 90 and 97, wherein one or more amino acid residues in said variant differs from the amino acid sequence of said mature form, provided that said variant differs in no more than 15% ofthe amino acid residues from the amino acid sequence of said mature form;

(c) an amino acid sequence selected from the group consisting of SEQ ID NOS :2, 4, 6, 8, 10, 12, 21, 28, 35, 37, 44, 51, 58, 60, 67, 74, 81, 83, 90 and 97;

(d) a variant of an amino acid sequence selected from the group consisting SEQ ID NOS:2, 4, 6, 8, 10, 12, 21, 28, 35, 37, 44, 51, 58, 60, 67, 74, 81, 83, 90 and 97, wherein one or more amino acid residues in said variant differs from the amino acid sequence of said mature form, provided that said variant differs in no more than 15% of amino acid residues from said amino acid sequence;

(e) a nucleic acid fragment encoding at least a portion of a polypeptide comprising an amino acid sequence chosen from the group consisting of SEQ ID NOS:2, 4, 6, 8, 10, 12, 21, 28, 35, 37, 44, 51, 58, 60, 67, 74, 81, 83, 90 and 97, or a variant of said polypeptide, wherein one or more amino acid residues in said variant differs from the amino acid sequence of said mature form, provided that said variant differs in no more than 15% of amino acid residues from said amino acid sequence; and

(f) a nucleic acid molecule comprising the complement of (a), (b), (c), (d) or (e).

6. The nucleic acid molecule of claim 5, wherein the nucleic acid molecule comprises the nucleotide sequence of a naturally-occurring allelic nucleic acid variant.

7. The nucleic acid molecule of claim 5, wherein the nucleic acid molecule encodes a polypeptide comprising the amino acid sequence of a naturally-occurring polypeptide variant.

8. The nucleic acid molecule of claim 5, wherein the nucleic acid molecule differs by a single nucleotide from a nucleic acid sequence selected from the group consisting of SEQ ID NOS:l, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96.

9. The nucleic acid molecule of claim 5, wherein said nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of:

(a) a nucleotide sequence selected from the group consisting of SEQ ID NOS:l, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96;

(b) a nucleotide sequence differing by one or more nucleotides from a nucleotide sequence selected from the group consisting of SEQ ID NOS:l, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96, provided that no more than 20% ofthe nucleotides differ from said nucleotide sequence;

(c) a nucleic acid fragment of (a); and

(d) a nucleic acid fragment of (b).

10. The nucleic acid molecule of claim 5, wherein said nucleic acid molecule hybridizes under stringent conditions to a nucleotide sequence chosen from the group consisting of SEQ ID NOS:l, 3, 5, 7, 9, 11, 20, 27, 34, 36, 43, 50, 57, 59, 66, 73, 80, 82, 89 and 96, or a complement of said nucleotide sequence.

11. The nucleic acid molecule of claim 5, wherein the nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of:

(a) a first nucleotide sequence comprising a coding sequence differing by one or more nucleotide sequences from a coding sequence encoding said amino acid sequence, provided that no more than 20% ofthe nucleotides in the coding sequence in said first nucleotide sequence differ from said coding sequence;

(b) an isolated second polynucleotide that is a complement ofthe first polynucleotide; and

(c) a nucleic acid fragment of (a) or (b).

12. A vector comprising the nucleic acid molecule of claim 11.

13. The vector of claim 12, further comprising a promoter operably-linked to said nucleic acid molecule.

14. A cell comprising the vector of claim 12.

15. An antibody that binds immunospecifically to the polypeptide of claim 1.

16. The antibody of claim 15, wherein said antibody is a monoclonal antibody.

17. The antibody of claim 15, wherein the antibody is a humanized antibody.

18. A method for determining the presence or amount of the polypeptide of claim 1 in a sample, the method comprising:

(a) providing the sample;

(b) contacting the sample with an antibody that binds immunospecifically to the polypeptide; and

(c) determining the presence or amount of antibody bound to said polypeptide, thereby determining the presence or amount of polypeptide in said sample.

19. A method for determining the presence or amount ofthe nucleic acid molecule of claim 5 in a sample, the method comprising:

(a) providing the sample;

(b) contacting the sample with a probe that binds to said nucleic acid molecule; and

(c) determining the presence or amount ofthe probe bound to said nucleic acid molecule, thereby determining the presence or amount ofthe nucleic acid molecule in said sample.

20. The method of claim 19 wherein presence or amount ofthe nucleic acid molecule is used as a marker for cell or tissue type.

21. The method of claim 20 wherein the cell or tissue type is cancerous.

22. A method of identifying an agent that binds to a polypeptide of claim 1, the method comprising:

(a) contacting said polypeptide with said agent; and

(b) determining whether said agent binds to said polypeptide.

23. The method of claim 22 wherein the agent is a cellular receptor or a downstream effector.

24. A method for identifying an agent that modulates the expression or activity of the polypeptide of claim 1, the method comprising:

(a) providing a cell expressing said polypeptide;

(b) contacting the cell with said agent, and

(c) determining whether the agent modulates expression or activity of said polypeptide, whereby an alteration in expression or activity of said peptide indicates said agent modulates expression or activity of said polypeptide.

25. A method for modulating the activity of the polypeptide of claim 1 , the method comprising contacting a cell sample expressing the polypeptide of said claim with a compound that binds to said polypeptide in an amount sufficient to modulate the activity ofthe polypeptide.

26. A method of treating or preventing a GPCRX-associated disorder, said method comprising administering to a subject in which such treatment or prevention is desired the polypeptide of claim 1 in an amount sufficient to treat or prevent said GPCRX-associated disorder in said subject.

27. The method of claim 26 wherein the disorder is selected from the group consisting of cardiomyopathy and atherosclerosis.

28. The method of claim 26 wherein the disorder is related to cell signal processing and metabolic pathway modulation.

29. The method of claim 26, wherein said subject is a human.

30. A method of treating or preventing a GPCRX-associated disorder, said method comprising administering to a subject in which such treatment or prevention is desired the nucleic acid of claim 5 in an amount sufficient to treat or prevent said GPCRX-associated disorder in said subject.

31. The method of claim 30 wherein the disorder is selected from the group consisting of cardiomyopathy and atherosclerosis.

32. The method of claim 30 wherein the disorder is related to cell signal processing and metabolic pathway modulation.

33. The method of claim 30, wherein said subject is a human.

34. A method of treating or preventing a GPCRX-associated disorder, said method comprising administering to a subject in which such treatment or prevention is desired the antibody of claim 15 in an amount sufficient to treat or prevent said GPCRX-associated disorder in said subject.

35. The method of claim 34 wherein the disorder is diabetes.

36. The method of claim 34 wherein the disorder is related to cell signal processing and metabolic pathway modulation.

37. The method of claim 34, wherein the subject is a human.

38. A pharmaceutical composition comprising the polypeptide of claim 1 and a pharmaceutically-acceptable carrier.

39. A pharmaceutical composition comprising the nucleic acid molecule of claim 5 and a pharmaceutically-acceptable carrier.

40. A pharmaceutical composition comprising the antibody of claim 15 and a pharmaceutically-acceptable carrier.

41. A kit comprising in one or more containers, the pharmaceutical composition of claim 38.

42. A kit comprising in one or more containers, the pharmaceutical composition of claim 39.

43. A kit comprising in one or more containers, the pharmaceutical composition of claim 40.

44. A method for determining the presence of or predisposition to a disease associated with altered levels ofthe polypeptide of claim 1 in a first mammalian subject, the method comprising:

(a) measuring the level of expression ofthe polypeptide in a sample from the first mammalian subject; and

(b) comparing the amount of said polypeptide in the sample of step (a) to the amount of the polypeptide present in a control sample from a second mammalian subject known not to have, or not to be predisposed to, said disease; wherein an alteration in the expression level ofthe polypeptide in the first subject as compared to the control sample indicates the presence of or predisposition to said disease.

45. The method of claim 44 wherein the predisposition is to cancers.

46. A method for determining the presence of or predisposition to a disease associated with altered levels ofthe nucleic acid molecule of claim 5 in a first mammalian subject, the method comprising:

(a) measuring the amount ofthe nucleic acid in a sample from the first mammalian subject; and

(b) comparing the amount of said nucleic acid in the sample of step (a) to the amount of the nucleic acid present in a control sample from a second mammalian subject known not to have or not be predisposed to, the disease; wherein an alteration in the level ofthe nucleic acid in the first subject as compared to the control sample indicates the presence of or predisposition to the disease.

47. The method of claim 46 wherein the predisposition is to a cancer.

48. A method of treating a pathological state in a mammal, the method comprising administering to the mammal a polypeptide in an amount that is sufficient to alleviate the pathological state, wherein the polypeptide is a polypeptide having an amino acid sequence at least 95%o identical to a polypeptide comprising an amino acid sequence of at least one of SEQ ID NOS:2, 4, 6, 8, 10, 12, 21, 28, 35, 37, 44, 51, 58, 60, 67, 74, 81, 83, 90 and 97, or a biologically active fragment thereof.

49. A method of treating a pathological state in a mammal, the method comprising administering to the mammal the antibody of claim 15 in an amount sufficient to alleviate the pathological state.

50. A method for the screening of a candidate substance interacting with an olfactory receptor polypeptide selected from the group consisting of SEQ ID NOS:2, 4, 6, 8, 10, 12, 21, 28, 35, 37, 44, 51, 58, 60, 67, 74, 81, 83, 90 and 97, or fragments or variants thereof, comprises the following steps: a) providing a polypeptide selected from the group consisting ofthe sequences of SEQ ID NOS:2, 4, 6, 8, 10, 12, 21, 28, 35, 37, 44, 51, 58, 60, 67, 74, 81, 83, 90 and 97, or a peptide fragment or a variant thereof; b) obtaining a candidate substance; c) bringing into contact said polypeptide with said candidate substance; and d) detecting the complexes formed between said polypeptide and said candidate substance.

51. A method for the screening of ligand molecules interacting with an olfactory receptor polypeptide selected from the group consisting of SEQ ID NOS:2, 4, 6, 8, 10, 12, 21, 28, 35, 37, 44, 51, 58, 60, 67, 74, 81, 83, 90 and 97, wherein said method comprises: a) providing a recombinant eukaryotic host cell containing a nucleic acid encoding a polypeptide selected from the group consisting ofthe polypeptides comprising the amino acid sequences SEQ ID NOS:2, 4, 6, 8, 10, 12, 21, 28, 35, 37, 44, 51, 58, 60, 67, 74, 81, 83, 90 and 97; b) preparing membrane extracts of said recombinant eukaryotic host cell; c) bringing into contact the membrane extracts prepared at step b) with a selected ligand molecule; and d) detecting the production level of second messengers metabolites.

52. A method for the screening of ligand molecules interacting with an olfactory receptor polypeptide selected from the group consisting of SEQ ID NOS:2, 4, 6, 8, 10, 12, 21, 28, 35, 37, 44, 51, 58, 60, 67, 74, 81, 83, 90 and 97, wherein said method comprises: a) providing an adenovirus containing a nucleic acid encoding a polypeptide selected from the group consisting of polypeptides comprising the amino acid sequences SEQ ID NOS:2, 4, 6, 8, 10, 12, 21, 28, 35, 37, 44, 51, 58, 60, 67, 74, 81, 83, 90 and 97; b) infecting an olfactory epithelium with said adenovirus; c) bringing into contact the olfactory epithelium b) with a selected ligand molecule; and d) detecting the increase ofthe response to said ligand molecule.