CA2425897A1 - Isolated human g-protein coupled receptors, nucleic acid molecules encoding human gpcr proteins, and uses thereof - Google Patents

Abstract

The present invention provides amino acid sequences of peptides that are encoded by genes within the Human genome, the GPCR peptides of the present invention. The present invention specifically provides isolated peptide and nucleic acid molecules, methods of identifying orthologs and paralogs of the GPCR peptides and methods of identifying modulators of the GPCR peptides.

Description

ISOLATED HUMAN G-PROTEIN COLiPLED RECEPTORS, NUCLEIC ACID
MOLECDLES ENCODING HUMAN GPCR PROTEINS, AND USES THEREOF
RELATED APPLICATIONS
S The present application claims priority to U.S. Application No. 09,G9d,821, tiled October 2~, 2000 (CIj000$99) and U.S. Serial No. 091781,559, filed February 13, 2001 (CL000899-CIP) FIELD OF THE INVENTION
The present invention is in the field of G-Protein coupled receptors (GPCRs) that are involved in cell signaling, particularly neurotransmitter signaling, recombinant DNA molecules, and protein production. The present invention specifically provides novel GPCR
peptides and proteins and nucleic acid molecules encoding such peptide and protein molecules, all of which are useful in the development of human therapeutics and diagnostic compositions and methods.
BACKGROUND OF THE INVENTION
G-protein coupled receptors G-pratein caupled receptors (GPCRs) constitute a major class oFproteins responsible For transducing a signal within a cell. GPCRs have three structural domains: an amino terminal extracellular domain, a transmembrane domain containing seven transmembrane segments, three extracellular loops, and three intracellular loops, and a carboxy ternlinal intracellular domain. Upon binding of a ligand to an extracellular portion of a GPCR, a signal is transduced within the cell that results in a change in a biological or physiological property of the cell.
GPCRs, along with G-proteins and eFfectors (intracellular enzymes and channels modulated by G-proteins), are the components of a modular signaling system that connects the state of intracellular second messengers to extracellulal- inputs.
GPCR genes and gene-products are potential causative agents of disease (Spiegel et rrl., J
C'lil~, Il~ai,sl, 93;1 I 19-1 125 ( 1993); McItusick c~l crl., J. Iblr?cl Gc~)ml, 3(J:1-2G (1993)). Specific defects in the rhodopsin gene and the V2 vasopressin receptor gene have been shown to cause various forms of retinitis pigmentosum (Nathans ct crl" ft)l)nl. Rcu. Gel~~tl.
?6:103-~I?~l( 1992)), and nephl'ogeIllC dlabeteS Ill~lpldLl~ ~)'IOItIIll~ill t'I Cll., Ihll)l. r1~(ll.
CiC'r?C'h ?:I~UI-1~0~11993))_ These receptors are of critical importance to both the central nervous system and peripheral physiological processes. Evolutionary analyses suggest that the ancestor of these proteins originally developed in concert with complex body plans and nervous systems.
'The GPCR protein superfamily can be divided into Five families: I~Aamily I, receptors typified by rhodopsin and the (32-purinergic receptor and currently represented by over 2DD unique members (Dohlman e! al.,~lr~nzi. Rev. Biochc~rn. «1;653-68$ (1991)); Family II, the parathyroid hotmonelcalcitoninlsecretin receptor family (Juppner et al., Science 25-l:1 D24-1 D26 ( 1991 ); Lin e!
al., Scier2ce 25:1:1D22-ID24 (1991)); Family III, the metabotropic glutamate receptor family (Nakanishi, Science 258 597:6D3 (1992)); Family IV, the cAMP receptor family, important in the 1D chemotaxis and development of D. di,scoideZrrrZ (Klein e! al., Science 2-11:1467-1172 (1988)); and Family V, the fungal mating pheromone receptors such as STE2 (Kurjan, ~ln~zt.
Rev. Biochenz.
61:1097-1129 ( 1992)).
There are also a small number of other proteins that present seven putative hydrophobic segments and appear to be unrelated to GPCRs; they have not been shown to couple to G-proteins.
I 5 Dnosophila expresses a photoreceptor-specific protein, bride of sevenless (boss), a seven-transmembrane-segment protein that has been extensively studied and does not show evidence of being a GPCR (I-Iart ~t al., Pnoc. Ncrtl. ~lcad Sci. LISA 90:SD47-SD51 ( 1993)). The geneyi~~led (fz) in Drosophila is also thought to be a protein with seven transmembrme segments. bike boss, fz has not been shown to couple to G-proteins (Vinson e1 crl., NalZn°e 338:263-264 (1989)).
2D G proteins represent a family of heterotrimeric proteins composed of a, (3 and y subunits, that bind guanine nucleotides. These proteins are usually linked to cell surface receptors, e.g., receptors containing seven transmembrane segments. Following ligand binding to the GPCR, a conformational change is transmitted to the G protein, which causes the a-subunit to exchange a bound GDP molecule for a GTP molecule and to dissociate from the (3y-subunits.
The GTP-bound 25 form of the a-subunit typically functions as an eFfector-modulating moiety, leading to the production of second messengers, such as cAMP (e.g., by activation of adenyl cyclase), diacylglycerol or inositol phosphates. Greater than 2D different types of a-subunits are known in humans. These subunits associate with a smaller pool of (3 and y suhunits.
Examples of mammalian G proteins include Gi, Go, Gq, Gs and Gt. G proteins are described ehtensively in 3D Lodish of al., ~t=Itrlr~catlcw C'cll Bioky~~y. (~cientiluc American gooks lnc., New York, N'.Y., 1995), the contents of which are incorporated herein by reference. GPCRs, G proteins and G protein-linked effector and second messenger s~°stems hove been reviewed in Thi.W-Pmolc~ir~ 1 irTked R~co~p!cu~ Fcrc~!
l3o~k, Watson cn al., eds.. Academic Press ( 1994).

Aminer~c GPCRs One Family oFthe GPCRS, Family II. contains receptors For acetylcholine, catccholanline, and indoleamine ligands (hereaFter reFerred to as biogenic amines). The bio~~~nic amine receptors (amincrgic GPCRs) represent a large group oFGPCRs that share a common evolutionary ancestor and which are present in both vertebrate (deuterostonle), and invertebrate (protostome) lineages. This Family oFGPCRs includes, but is not limited to the 5-HT-like, the dopamine-like, the acetylcholine-like, the adrenaline-like and the melatonin-like GPCRs.
Dopamine receptors The understanding oFthe dopaminergic system relevance in brain Function and disease 1 D developed several decades ago from three diverse observations following drug treatments. These were the observations that dopamine replacement therapy improved Parkinson's disease symptoms, depletion of dopamine and other catecholamines by reserpine caused depression and antipsychotic drugs blocked dopamine receptors. The Ending that the dopamine receptor binding affinities of typical antipsychotic drugs correlate with their clinical potency led to the dopamine overactivity hypothesis of schizophrenia (Snyder, S.H., ,~ni J PSyGhiaby 133, 197-2D2 (1976); Seeman, P. and Lee, T., Science I ~3c~, 1217-9 (1975)). Today, dopamine receptors aI°e crucial targets in the pharmacological therapy of schizophrenia, Parkinson's disease, Tourette's syndrome, tardive dyskinesia and Huntington's disease. The dopaminergic system includes the nigrostriatal, mesocorticolimbic and tuberoinfi.lndibular pathways. The nigrostriatal pathway is part of the striatal motor system and its degeneration leads to Parkinson's disease; the mesocorticolimbic pathway plays a key role in reinforcement and in emotional expression and is the desired site of action of antipsychotic drugs; the tuberoinfundibular pathways regulates prolactin secretion from the pituitary.
Dopamine receptors are members of the G protein coupled receptor superfamily, a large group proteins that share a seven helical membrane-spanning structure and transduce signals through coupling to heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins).
Dopamine receptors are classified into subfamilies: Dl-like (Dl and DS) and D'~-like (D2, D3 and D4) based on their different ligand binding profiles, signal transduction properties, sequence homologies and genomic organizations (Civelli, O~, F~unzow, .1R, and Grandy, D.K., ,~Irznu Reu I'hcrrrncrcol Toxfcml33, 281-307 (1993)). The Dl-like receptors, D1 and D5, stimulate cAMP
synthesis through coupling with Gs-like proteins and their Belles do not contain intron s within their pl'otelll Codlng CeglOnS. OIl the otheI' hatld, the D~-llkf= reCeptol'S, D?.
D3 alld Dzl, lllhlblt Cf~IVIP

synthesis through their interaction with Gi-like proteins and share a similar genomic organization which includes introns within their protein coding regions.
Sel'otolllll I'eCeptorS
Serotonin (5-I-Iydroxytryptamine; 5-HT) was first isolated from blood serum, where if was shown to promote vasoconstriction (Rapport, M.M., Green, A.A. and Page, LI-I., J Biol C'hern 17<, 1213-1251 (148). Interest on a possible relationship between 5-HT and psychiatric disease was spurred by the observations that hallucinogens such as LSD and psilocybin inhibit the actions of 5-HT on smooth muscle preparations {Gaddum, J.H. and Hameed, K.A., Br J
Pharmucol 9, 240-248 (1954)). This observation lead to the hypothesis that brain 5-HT activity might be altered in psychiatric disorders (Wooley, D.W. and Shaw, E., Proc Nail Accld Sci LIS~I -10, 228-231 (1951);
Gaddum, J.H, and Picarelli, Z.P., Bf~ J Phay~nzacol 12, 323-328 (1957)). This hypothesis was strengthened by the introduction of tricyclic antidepressants and monoasnine oxidase inhibitors For the treatment of major depression and the observation that those drugs affected noradrenaline and 5-HT metabolism. Today, drugs acting on the serotoninergic system have been proved to be effective I 5 in the pharmacotherapy of psychiatric diseases such as depression, schizophrenia, obsessive-compulsive disorder, panic disorder, generalized anxiety disorder and social phobia as well as migraine, vomiting induced by cancer chemotherapy and gastric motility disorders.
Serotonin receptors represent a very large and diverse family of neurotransmitter receptors.
To date thirteen 5-HT receptor proteins coupled to G proteins plus one ligand-gated ion channel receptor (5-HT3) have been described in mammals. This receptor diversity is thought to reflect serotonin's ancient origin as a neurotransmitter and a hormone as well as the many different roles of 5-HT in mammals. The 5-HT receptors have been classified into seven subfamilies or groups according to their different ligand-binding affinity profiles, molecular structure and intracellular transduction mechanisms (Hoyer, D. et al., Phcrrrrurcol. Rev. -16, 157-203 (1991)).
Adrener~ic GPCRs The adrenergic receptors comprise one o~the largest and most extensively characterized families within the G-protein coupled receptor "superFamily". This superfamily includes not only adrenergic receptors, but also muscarinic, cholinergic, dopaminergic, serotonergic, and histaminergic receptors. Numerous peptide receptors include glucagon, somatostatin, and vasopressin receptors, as well as sensory receptors for vision (rhodopsin), taste, and olfaction, also belong to this growing Family. Despite the diversity of signalling molecules, G-protein coupled receptors all possess a similar overall primary structure, characterized by 7 putative membrane-spanning .alpha, helices (Probst et al., 199?). In the most basic sense, the adrenergic receptors are the physiological sites of action of the catecholamines, epinephrine and norepinephrine. Adrencrgic receptors were initially classil7ed as either .alpha. or .beta. by Ahlquist, who demonstrated that the order of potency For a series of agonists to evolve a physiological response was distinctly different at the 2 receptor subtypes (Ahlquist, 1918).
functionally, .alpha, adrenergie receptors were shown to control vasoconstriction, pupil dilation and uterine inhibition, while .beta. adrenergic receptors were implicated in vasorelaxation, myocardial stimulation and bronchodilation (Regan et al., 1990). Eventually, pharmacalogists realized that these responses resulted from activation of several distinct adrenergic receptor subtypes. .beta. adrenergic receptors in the heart were defined as .beta._l, while those in the lung and vasculature were termed .beta.₂ (Lands et al., 1967).
.alpha. Adrenergic receptors, meanwhile, were first classified based on their anatomical location, as either pre or post-synaptic (.alpha.₂ and .alpha._l, respectively) (Langer et al., 1970. This classification scheme was confounded, however, by the presence of .alpha.₂ receptors in distinctly non-synaptic locations, such as platelets (Berthelsen and Pettinger, 1977).
With the development of radioligand binding techniques, .alpha. adrenergic receptors could be distinguished pharmacologically based on their affinities for the antagonists prazosin or yohimbine {Stark, 1981 ). Definitive evidence for adrenergic receptor subtypes, however, awaited purification and molecular cloning of adrenergic receptor subtypes. In 19$6, the genes For the hamster .beta.₂ (Dickson et al., 19$6) and turkey .beta._l adrenergic receptors {Yarden et al., 19$6) were cloned and sequenced. I-Iydropathy analysis revealed that these proteins contain 7 hydrophobic domains similar to rhodopsin, the receptor for light.
Since that time the adrenergic receptor family has expanded to include 3 subtypes of .beta.
receptors (Emorine et al,, 1989), 3 subtypes of .alpha. l receptors (Schwinn et al., 1990), and 3 distinct types of ,beta.₂ receptors (Lomasney et al., 1990).
The cloning, sequencing and expression of alpha receptor subtypes from animal tissues has led to the subclassitication of the alpha 1 receptors into alpha 1d (formerly known as alpha 1 a or 1 al l d), alpha 1 b and alpha 1 a (Formerly known as alpha 1 c) subtypes. Each alpha 1 receptor subtype exhibits its own pharmacologic and tissue speciFtcities. The designation "alpha la" is the appellation recently approved by the IUPI-IARN~omenclature Committee Far the previously designated "alpha 1 c" cloned subtype as outlined in the 1995 Receptor and Ion Channel l~lomenclature Supplement ( Watson and Girdlestone, 1995). The designation alpha 1 a is used throughout this application to reFer to this subtype. At the same time, the receptor Formerly t designated alpha 1 a was renamed alpha 1 d. The new nomenclature is used throughout this application. Stable cell lines expressing these alpha 1 receptor subtypes are referred to herein;
however, these cell lives were deposited with the American Tfype Culture Collection (ATCC) under the old nomenclature. For a review of the classification ol~alpha 1 adrenoceptor subtypes, see, Martin C. Michel, et al., Naunyn-Schmiedeberg's Arch. Pharmacol. (1995) 352;1-10.
The differences in the alpha adrenergic receptor subtypes have relevance in pathophysiologic conditions. Benign prostatic hyperplasia, also known as benign prostatic hypertrophy or BPH, is an illness typically affecting men over f fty years of age, increasing in severity with increasing age. The symptoms of the condition include, but are not limited to, increased difficulty in urination and sexual dysfunction. These symptoms are induced by enlargement, or hyperplasia, of the prostate gland. As the prostate increases in size, it impinges on free-flow of fluids through the male urethra. Concommitantly, the increased noradrenergic innervation of the enlarged prostate leads to an increased adrenergic tone of the bladder neck and urethra, further restricting the flow of urine through the urethra.
The .alpha.₂ receptors appear to have diverged rather early from either .beta. or .alpha._l receptors. The .alpha.₂ receptors have been broken down into 3 molecularly distinct subtypes termed .alpha.₂ C2, .alpha.₂ C4, and .alpha.₂ C10 based on their chromosomal location. These subtypes appear to correspond to the pharmacologically defined .alpha._2B, .alpha._2C, and .alpha._2A subtypes, respectively (Bylund et al., 1992).
While all the receptors of the adrenergic type are recognized by epinephrine, they are pharmacologically distinct and are encoded by separate genes. These receptors are generally coupled to different second messenger pathways that are linked through G-proteins. Among the adrenergic receptors, .beta._l and .beta.₂ receptors activate the adenylate cyclase, .alpha.₂ receptors inhibit adenylate cyclase and .alpha. l receptors activate phospholipase C pathways, stimulating breakdown of polyphosphoinositides (Chung, F. Z. et al., J. Biol. Chem., 263:1052 (1988)). .alpha._l and .alpha.₂ adrenergic receptors differ in their cell activity for drugs.
Issued US patent that disclose the utility of members of this family of proteins include, but are not limited to, 6,063,785 Phthalimido arylpiperazines useful in the treatment of benign prostatic hyperplasia; 6,060,492 Selective .beta.3 adrenergic agonists;
6,057,350 Alpha 1 a adrenergic receptor antagonists; 6,06,192 Phenylethanolaminotetralincarboxamide derivatives;
6,016,183 Method of synergistic treatment for benign prostatic hyperplasia;
6,013,253 Fused piperldine substituted arylsulfonamides as .beta. 3-a~.~onists; G.0~3.22~1 Compositions and methods for treatment of neurological disorders and neurodegenerative diseases; 6,037,354 Alpha la adrenergic receptor antagonists; 6,034,106 Oxadiazole benzenesulfonamides as selective .beta.₃ Agonist for the treatment of Diabetes and Obesity; 6,01 I ,048 Thiarole benzenesulfonamicles as .beta.3 agonists for treatment of diabetes and obesity; 6,008,361 5,994,506 Adrenergic receptor; 5,994,294 Nitrosated and nitrosylated .alpha.-adrenergic receptor antagonist compounds, compositions and their uses; 5,990,128 .alpha. l C
specific compounds to treat benign prostatic hyperplasia; 5,977,151 Selective .beta.3 adrenergic agonist;
5,977,1 15 Alpha I a adrenergic receptor antagonists; 5,939,443 Selective .beta,3 adrenergic agonists; 5,932,538 Nitrosated and nitrosylated ,alpha.-adrenergic receptor antagonist compounds, compositions and their uses; 5,922,722 Alpha 1 a adrenergic receptor antagonists 26 5,908,830 and 5,861,309 DNA endoding human alpha 1 adrenergic receptors.
Purinergic GPCRs Purinoceptor P2Y 1 P2 purinoceptors have been broadly classified as P2X receptors which are ATP-gated channels; P2Y receptors, a family of G protein-coupled receptors, and P2Z
receptors, which mediate nonselective pores in mast cells. Numerous subtypes have been identified for each of the P2 receptor classes. P2Y receptors are characterized by their selective responsiveness towards ATP
and its analogs. Some respond also to UTP. Based on the recommendation for nomenclature of P2 purinoceptors, the P2Y purinoceptors were numbered in the order of cloning.
P2Y1, P2Y2 and P2Y3 have been cloned from a variety of species. P2Y 1 responds to both ADP
and ATP. Analysis of P2Y receptor subtype expression in human bone and 2 osteoblastic cell lines by RT-PCR showed that all known human P2Y receptor subtypes were expressed: P2Y1, P2Y2, P2Y4, P2Y6, and P2Y7 (Maier et al. 1997). In contrast, analysis of brain-derived cell lines suggested that a selective expression of P2Y receptor subtypes occurs in brain tissue, Leon et al. generated P2Y I -null mice to define the physiologic role of the P2Y 1 receptor (J.
Clin. Invest. 104: 1731-1737(1999)). These mice were viable with no apparent abnormalities affecting their development, survival, reproduction, or morphology of platelets, and the platelet count in these animals was identical to that of wildtype mice. However, platelets Frotn P2Y1-deficient mice were unable to aggregate in response to usual concentrations of ADP and displayed impaired aggregation to other agonists, while high concentrations oFADP
induced platelet aggregation withou t shape change. In addition, ADP-induced inhibition of adenylyl cyelase still occurred, demonstrating the existence ofan ADf receptor distinct from P2Y1.
PZY1-null mice had no spontaneous bleeding tendency but were resistant to thromboemholism induced by intravenous inic:ction ofADI' or collagen and adi°enaline. f-Ience, the P?Yl recoptor plays an essential role in thrambotic states and represents a potential target for antithrombo tic drugs.
Somers et al. mapped the P2RY 1 gen a between Ranking markers D3S 1279 and D3S 1280 at a position 173 to 174 cM
from the most telomeric markers on the short arm of chromosome 3. (Genomics ~l~f: 127-130 1997)).
Purinoc~tor P2Y2 The chloride ion secretory pathway that is defective in cystic fibrosis (CF) can be bypassed by an alternative pathway for chloride ion transport that is activated by extracellular nucleotides.
Accordingly, the P2 receptor that mediates this effect is a therapeutic target For improving chloride secretion in CF patients. Parr et al. reported the sequence and functional expression of a cDNA
cloned from human airway epithelial cells that encodes a protein with properties of a P2Y
nucleotide receptor. (Proc. Nat. Acad. Sci. 91: 3275-3279 (1994)) The human P2RY2 gene was mapped to chromosome 11 q 13.5-q 1 ~.1.
Purinoceptor P2RY~I
The P2RY~1 receptor appears to be activated specifically by UTP and LJDP, but not by ATP
and ADP. Activation ofthis uridine nucleotide receptor resulted in increased inositol phosphate fomlation and calcium mobilization. The LJNR gene is located on chromosome Xql3.
Purinoceptor P2 Y6 Somers et al. mapped the P2RY6 gene to 11 q 13.5, between palymorphic markers D 11 S 131 ~I and D 115916, and P2RY2 maps within less than 4 cM of P2RY6.
(Genomics ~~: 127-130 (1997)) This was fhe first chromosomal clustering of this gene family to be described.
Adenine and uridine nucleotides, in addition to their well established role in intracellular energy metabolism, phosphorylation, and nucleic acid synthesis, also are important extracellular signaling molecules. P2Y metabotropic receptors are GPCRs that mediate the effects of extracellular nucleotides fo regulate a wide variety of physiological processes. At least ten subfamilies of P2Y receptors have been identified, Flhese receptor subfamilies differ greatly in their sequences and in their nucleotide agonist selectivities and eFticacies.
It has been demonstrated that the P2Y1 receptors are strongly expressed in the brain, but the P2Y2, P?Y~1 and P?Y6 receptors are also present. -Ihc localisation oCone or more of these subtypes on neurons, on glia cells. on brain vasculature or on vEntricle ependimal cells was found by in situ mRNA hybridisation and studies on thosE cells in culture. 'I"he P2Y 1 receptors are prt7minent on 17eu1"oIlS. ~hhe Coupling ol'Cel'ta117 P2Y reCeptD1' SLIbtypeS t0 N-type Ca?~' Chanl7ElS C>I'to partlCLlla1' k.+ channels was also demonstrated.
It has also been demonstrated that several P2Y receptors mediate potent growth stimulatory effects on sn7ooth n7uselE cells by stimulating intracellular pathways including Gq-proteins, protein kinase C and tyrosine phosphorylation, leading to increased immediate early gene expression, cell number, DNA and protein synthesis. It has been further demonstrated that P2Y
regulation plays a mitogenic rale in response to the development of artherosclerosis.
It has Further been demonstrated that P2Y receptors play a critical role in cystic fibrosis.
The volume and composition of the liquid that lines the airway surface is modulated by active transport of ions across the airway epithelium. This in turn is regulated both by autonomic agonists acting on basolateral receptors and by agonists acting on luminal receptors.
Specifically, extracellular nucleotides present in the airway surface liquid act on luminal P2Y receptors to control both Cl- secretion and Na+ absorption. Since nucleotides are released in a regulated manner from airway epithelial cells, it is likely that their control over airway ion transport forms part of an autocrine regulatory system localised to the luminal surface ofairway epithelia. In addition to this physiological role, P2Y receptor agonists have the potential to be of crucial benefit in the treatment of CF, a disorder of epithelial ion transport. The airways of people with CF
have defective C1-secretion and abnormally high rates of Na+ absorption. Since P2Y receptor agonists can regulate both these ion transport pathways they have the potential to pharmacologically bypass the ion transport defects in CF.
For a detailed description of a putative neurotransmitter GPCR involved in neurotransmitter signaling, see Zeng el al., Bioche~rz. Biophya". Res. ConZnzatn. 242 (3), 575-578 (1998).
GPCRs, particularly GPCRs involved in neurotransmitter signaling, are a major target far drug action and development. Accordingly, it is valuable to the field of pharmaceutical development to identify and characterize previously unknown GPCRs. The present invention advances the state of the art by providing a previously unidentified human GPCR.
SUM1V><ARY O>F THE INVENTION
The present invention is based in part on the identiFcation of nucleic acid sequences that encode an7ino acid sequences of human GPC"R peptides and proteins that are involved in cell c~

signaling. particularly neurotransmitter signaling, allelic variants thereof and other mammalian orthologs thereof. These unique peptide sequences, and nucleic acid sequences that encode these peptides. Can be Llsed a5 Illod(:1S Iol'the develOplnent Ul'hL1171a17 t17e1'apeLltIC targets, aid 111 the identification oh therapeutic proteins, and serve as targets For the development of human therapeutic agEnts.
The proteins of the present inventions are GPCRs that participate in signaling pathways, particularly neurotransmitter signaling pathways, in cells that express these proteins. Experimental data as provided in Figure 1 indicates expression in humans in the stomach, placenta, kidney, skeletal muscle, liver, bone marrow, and thymus. As used herein, a "signaling pathway" refers to I 0 the modulation (e.g., stimulation or inhibition) of a cellular functionlactivity upon the binding of a ligand to the GPCR protein. Examples of sLlch functions include mobilization of intracellular molecules that participate in a signal transduction pathway, e.g., phosphatidylinositol 4,5-bisphosphate (PIPz), inositol 1,~I,S-triphosphate (IP3) and adenylate cyclase;
polarization of the plasma membrane; production or secretion of molecules; alteration in the structure oFa cellular component; cell proliferation, e.g., synthesis of DNA; cell migration; cell differentiation; and cell survival The response mediated by the receptor protein depends on the type of cell it is expressed on.
Some information regarding the types of cells that express other members of the subfamily of GPCRs of the present invention is already known in the art (see references cited in Background and information regarding closest homologous protein provided in Figure 2;
Experimental data as provided in Figure 1 indicates expression in humans in the stomach, placenta, kidney, skeletal muscle, liver, bone marrow, and thymus). For example, in some cells, binding of a ligand to the receptor protein may stimulate an activity such as release of compounds, gating of a channel, cellular adhesion, migration, differentiation, etc., through phosphatidylinositol or cyclic AMP
metabolism and turnover while in other cells, the binding of the ligand will produce a diFferent result, Regardless of the cellular activityhesponse modulated by the particular GPCR of the present invention, a skilled artisan will clearly know that the receptor protein is a GPCR and interacts with G proteins to praduce one or more secondary signals, in a variety of intracellular signal trap sduction pathways, e.g., through phosphatidylinositol or cyclic AMP metabolism and turnover, in a cell thus participating in a biological process in the cells or tissues that express the GPCR. Experimental data as provided in Figure 1 indicates that GPCR proteins of the present invEntion are Expressed in humans in the stomach, placenta, Icidn Ey. sl:elctal muscle, liver. bone marrow, and thymus.
SpeciFically, a virtual northern blot shows Expression in the stomach. In addition, PCR-bawd tissue screening panels indicate expression in placenta. kidney, skeletal muscle, liver, bone marrow. and thymus tissue, As used herein, "phosphatidylinositol turnover and metabolism" refers to the molecules involved in the turnover and metabolism oFphosphatidylinositol X1,5-bisphosphate (PIPZ) as well as to the activities oFthese molecules. PIPS is a phospholipid Found in the cytosolic leaflet of the plasma membrane. Binding of ligand to the receptor activates, in some cells, the plasma-membrane enzyme phospholipase C that in turn can hydrolyze PIPS to produce 1,2-diacylglycerol (DAG} and inositol 1,4,5-triphosphate (IP;). Once formed IPA can diffuse to the endoplasmic reticulum surface where it can bind an IP3 receptor, e.g., a calcium channel protein containing an IP3 binding site. IP3 binding can induce opening of the channel, allowing calcium ions to be released into the cytoplasm.
IPA can also be phosphorylated by a specific kinase to Form inositol 1,3,1,5-tetraphosphate (IP,~}, a molecule that can cause calcium entry into the cytoplasm from the extracellular medium. IP,; and IPA can subsequently be hydrolyzed very rapidly to the inactive products inositol 1,~-biphosphate (IPA} and inositol 1,3,4-triphosphate, respectively. These inactive products can be recycled by the cell to synthesize PIPS. The other second messenger produced by the hydrolysis of PIPZ, namely 1,2-diacylglycerol (DAG), remains in the cell membrane where it can serve to activate the enzyme protein kinase C. Protein kinase C is usually found soluble in the cytoplasm of the cell, but upon an increase in the intracellular calcium concentration, this enzyme can move to the plasma membrane where it can be activated by DAG. The activation of protein kinase C in different cells results in various cellular responses such as the phosphorylation of glycogen synthase, or the phosphorylation of various transcription factors, e.g., NF-kB. The language "phosphatidylinositol activity", as used herein, refers to an activity of PIPZ or one of its metabolites.
Another signaling pathway in which the receptor may participate is the cAMP
turnover pathway. As used herein, "cyclic AMP turnover and metabolism" refers to the molecules involved in the turnover and metabolism oFcyclic AMP (CAMP) as well as to the activities of these molecules. Cyclic AMP is a second messenger produced in response to ligand-induced stimulation of certain G protein coupled receptors. In the cAMP signaling pathway, binding of a ligand to a GPCR can lead to the activation of the enzyme adenyl cyclase, which catalyzes the synthesis of cAMP. The newly synthesized CAMP can in turn activate a cAMP-dependent protein kinase. This activated kinase can phosphorylate a voltage-gated potassium channel protein, or an associated protein, and lead to the inability ofthe potassium channel to open during an action potential. The inability oFthe potassium channel to open results in a decrease in the outward Flow oFpotassium, which normally repolarizes the membrane oFa neuron. leading to prolonged membrane depolarization, f3y targeting an agent to modulate a GPCR, the signaling activity and biological process mediated by the receptor can be agonised or antagonised iv specific cells and tissues.
Experimental data as provided in F figure 1 indicates expression in humans in the stomach, placenta, kidney, skeletal muscle, liver, bone marrow, and thymus. Such agonistn and antagonism serves as a basis for modulating a biological activity in a therapeutic context (mammalian therapy) or toxic context (anti-cell therapy, e.g. anti-cancer agent).
DESCRIPTION OF THE FIGURE SHEETS
FIGURE 1 provides the nucleotide sequence of a cDNA molecule that encodes the GPCR
oFthe present invention. (SEQ ID NO: I) In addition, structure and functional information is provided, such as ATG start, stop and tissue distribution, where available, that allows one to readily determine specific uses of inventions based on this molecular sequence. Experimental data as provided in Figure 1 indicates expression in humans in the stomach, placenta, kidney, I S skeletal muscle, liver, bone marrow, and thymus.
FIGURE 2 provides the predicted amino acid sequence of the GPCR of the present invention. (SEQ ID N0:2) In addition structure and Functional information such as protein family, function, and modification sites is provided where available, allowing one to readily determine specific uses of inventions based on this molecular sequence.
FIGURE 3 provides genamie sequences that span the gene encoding the GPCR
protein of the present invention. (SEQ ID N0:3) In addition, structure and functional information, such as intron/exon structure, promoter location, etc., is provided where available, allowing one to readily determine specific uses of inventions based on this molecular sequence. As illustrated in Figure 3, G 171 I T is a known SNP variant.
DETAILED DESCRIPTION OF THE INVENTION
General Description The present invention is based on the sequencing of the human genome. During the sequencing and assembly of the human genome, analysis of the sequence information revealed previously unidentified fragments of the human genome that encode peptides that share structural and/or sequence homolof~y to protein/peptitieldomains identified and characterised 1?

within the art as being a GPCR protein or part of a GPCR protein, that are involved in cell signaling, particularly neurotransmitter signaling. Utilising these sequences, additional genomic sequences were assembled and transcript and/or cDNA sequences were isolated and characterized. Based on this analysis, the present invention provides amino acid sequences of human GPCR peptides and proteins that are involved in cell signaling, particularly neurotransmitter signaling, nucleic acid sequences in the farm of transcript sequences, cDNA
sequences andlor genomic sequences that encode these GPCR peptides and proteins. nucleic acid variation (allelic information), tissue distribution of expression, and information about the closest art known proteiupeptide/domain that has structural or sequence homology to the GPCR of the present invention.
In addition to being previously unknown, the peptides that are provided in the present invention are selected based on their ability to be used for the development of commercially important products and services. Specifically, the present peptides are selected based on homology andlor structural relatedness to known GPCR proteins involved in cell signaling, particularly neurotransmitter signaling, and the expression pattern observed.
Experimental data as provided in Figure 1 indicates expression in humans in the stomach, placenta, kidney, skeletal muscle, liver, bone marrow, and thymus. The art has clearly established the commercial importance of members of this family of proteins and proteins that have expression patterns similar to that of the present gene. Some of the more specific features of the peptides of the present invention, and the uses thereof, are described herein, particularly in the Background of the Invention and in the annotation provided in the Figures, and/or are knawn within the art for each of the known GPCR proteins involved in cell signaling, particularly neurotransmitter signaling.
Specific Embodiments Peptide Molecules The present invention provides nucleic acid sequences that encode protein molecules that have been identified as being members of the GPCR family of proteins and are involved in cell signaling. particularly neurotransmitter si~,~naling (protein sequences are provided in Figure ?, cDNA sequences are provided in Figure 1 and genomic sequences are provided in Figure 3).
The peptide sequences provided in higure ?. as well as the obvious variants described herein.
particularly allelic variants as ideniiCaed herein and u,sin~~ the information in Figure 3, will be I, reFerred herein as the GPCR peptides of the present invention, GPCR peptides, or peptides/proteins of the present invention, The present invention provides isolated peptide and protein molecules that consist o(~, consist essentially of, or comprise the amino acid seduences of the GPCR
peptides disclosed in figure 2, (encoded by the nucleic acid molecule shown in figure I, cDNA
sequence. or Figure 3, genomic sequence), as well as all obvious variants of these peptides that are within the art to make and use. Some of these variants are described in detail below.
As used herein, a peptide is said to be "isolated" or "purified" when it is substantially free of cellular material or free of chemical precursors or other chemicals. The peptides of the present invention can be purified to homogeneity or other degrees of purity. The level of purification will be based on the intended use. The critical feature is that the preparation allows for the desired function of the peptide, even if in the presence of considerable amounts of other components (the features of an isolated nucleic acid molecule is discussed below).
In some uses, "substantially free of cellular material" includes preparations of the peptide I 5 having less than about 30°~'0 (by dry weight} other proteins (i.e., contaminating protein), less than about 20°~'0 other proteins, less than about 10°r'o other proteins, or less than about 5°r'° other proteins.
When the peptide is recombinantly produced, it can also be substantially free of culture medium, i.e" culture medium represents less than about 20°~'° of the volume of the protein preparation.
The language "substantially free of chemical precursors or other chemicals"
includes preparations of the peptide in which it is separated From chemical precursors or other chemicals that are involved in its synthesis. In one embodiment, the language "substantially free of chemical precursors or other chemicals" includes preparations of the GPCR peptide having less than about 30°r'° (by dry weight) chemical precursors or other chemicals, less than about 20°r'° chemical precursors or other chemicals, less than about 10°l° chemical precursors or other chemicals, or less than about 5°r°° chemical precursors or other chemicals.
The isolated GPCR peptide can be puril7ed From cells that naturally express it, purified from cells that have been altered to express it (recombinant}, or synthesized using known protein synthesis methods. Experimental data as provided in Figure 1 indicates expression in humans in the stomach, placenta, kidney, skeletal muscle, liver, bone marrow, and thymus.
For example, a nucleic acid molecule encoding the GPCR peptide is cloned into an expression vector.
the expression vector introduced into a host cell and the protein expressed in the host cell. 'hhe protein can then be isolated from the cells by an appropriate purification scheme using standard protein purification tochuiques_ Many of these techniques are described in detail below.
1~

Accordingly, the present invention provides protein s that Consist of the amino acid SeqlleIlCeS pl'oVlCled I11 I"lgLlI'e 2 (S~';Q ID NO;2), loI' eXalnple, pl'Ote111S ellCOded by the CDNA
llLlClelC aCICI SCqLlenGeS SllQWl1 In flglll'e 1 (S~'rQ ID NO; 1 ~ and tile genoLIllC SeqLlenGeS pl'OVIdCLI In Figure 3 (ShQ ID N0:3). 'IAhe amino acid Sequence oFsuch a protein is provided in figure 2. A
protein consists ofan amino acid sequence when the amino acid sequence is the final amino acid sequence of the protein.
The present invention Further provides proteins that consist essentially of the amino acid sequences provided in Figure 2 (SEQ ID N0:2), for example, proteins encoded by the cDNA
nucleic acid sequences shown in Figure I (SEQ ID NO:1 ) and the genomic sequences provided in Figure 3 (SEQ ID N0:3). A protein consists essentially of an amino acid sequence when such an amino acid sequence is present with only a few additional amino acid residues, for example from about I to about 100 or so additional residues, typically From 1 to about 20 additional residues in the final protein.
The present invention filrther provides proteins that comprise the amino acid sequences I S provided in Figure 2 (SEQ ID N0:2), for example, proteins encoded by the cDNA nucleic acid sequences shown in Figure 1 (SEQ ID NO: l ) and the genomic sequences provided in Figure 3 (SEQ ID N0:3). A protein comprises an amino acid sequence when the amino acid sequence is at least part ofthe final amino acid sequence of the protein. In such a fashion, the protein can be only the peptide or have additional amino acid molecules, such as amino acid residues (Contiguous encoded sequence) that are naturally associated with it or heterologous amino acid residues/peptide sequences. Such a protein can have a few additional amino acid residues or can comprise several hundred or more additional amino acids. The preferred classes of proteins that are comprised of the GPCR peptides oFthe present invention are the naturally occurring mature proteins. A brief description of how various types of these proteins can be madelisolated is provided below.
The GPCR peptides of the present invention can be attached to heterologous sequences to Form chimeric or fusion proteins. Such Ghimeric and Fusion proteins comprise a GPCR peptide operatively linked fo a heterologous protein having an amino acid sequence not substantially homologous to the GPCR peptide. "Operatively linked'' indicates that the GPCR
peptide and the heterologous protein arc Fused in-Ii'anle. The heterologous protein can be fused to the N'-terminus or C-terminus of the GPCR peptide.
In some uses, the fusion protein does not aFFect thL: activity of the GPCR
peptide pc~r.ve. For example, the Fusion protein can include, but is not limited to, en~,ymatic fusion proteins, for example beta-galactosidase I~usions, yeast two-hybrid GAL Fusions, poly-I-Its fusions, MYC-tagged, Hl-l~

tagged and Ig I'usions. Such fusion proteins, particularly poly-His fusions, can facilitate the purification ofrecombinasit GPCR peptide, In certain host cells (e.g., mammalian host cells), expression andior secretion of a protein can be increased by using a heterologous signal sequence.
A chimeric or fusion protein can be produced by standard recombinant DN'A
technidues, F"or example, DNA fragments coding for the different protein sequences are ligated together in-frame in accordance with conventional techniques. In 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 which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be I 0 annealed and re-amplified to generate a ehimerie gene sequence (see Ausubel et al., Current Protocols irr tllolecatlar Biology, 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a CST protein). A GPCR
peptide-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the GPCR peptide.
As mentioned above, the present invention also provides and enables obvious variants of the amino acid sequence of the proteins of the present invention, such as naturally occurring mature forms of the peptide, alleliclsequenee variants of the peptides, non-naturally occurring recombinantly derived variants ofthe peptides, and orthologs and paralogs ofthe peptides. Such variants can readily be generated using art-known techniques in the fields of recombinant nucleic acid technology and protein biochemistry. It is understood, however, that variants exclude any amino acid sequences disclosed prior to the invention.
Such variants can readily be identifiedlmade using molecular techniques and the sequence information disclosed herein. Further, such variants can readily be distinguished from other peptides based on sequence and/or structural homology to the GPCR peptides of the present ?S invention. The degree ofhomologylidentity present will be based primarily on whether the peptide is a functional variant or non-functional variant, the amount of divergence present in the paralog family and the evolutionary distance between the ortholags.
To determine the percent identity oftwo amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amigo acid or nucleic acid sequence for optimal alignment and non-homologous sequences can brr disregarded for comparison purposes). In a preferred embodiment, the length of~a ref~:rence sequence aligned for comparison purposes is at least 30%, ~0°~'0, Sp°r'°, GO°~o, 70%, 80°
o, or c)()°,~o or more: of the length of the reference sequence.

~lAhe amino acid residues or nucleotides at corresponding amino acid positions or nucleotide poSltIOIIS al'C then COIllpat'ed. Whell a posltlOn 117 the llCSt SeqUeIlCe lS
OCCLlpled by the Sallle an7111C) aCld 1'CSldlle Or 1lLICleOtlde aS the GQI'reSpondlllg pOS1t1011 In the $eC011d SeqllCIlCe, tllell the InOleCUIeS al'e IdentICal at that pOSltl011 laS LlSed lleCeln alnlllo aCICl Or nLlClelC aCld "Idelltlty" 1S
equivalent to amino acid or Nucleic acid "homology"). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
The comparison of sequences and determination of percent identity and similarity between two sequences can be accomplished using a mathematical algorithm.
(C~rr~pattational Molecatlcrr Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988;
Bioc'orraprttir2g.' Infor'malic,~~ and Genoryre Pr'oject,s, Smith, D.W., ed., Academic Press, New York, 1993; C'omprtter' Analysis of SedZter?ce Dala, Par'/ l, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994 S~grtence Analysis in Molecztlar- Biology, van Eleinje, G., Academic Press, 1987; and Sec7Ztence Ar2alysis Pr'iryzer~, Gribskov, M. and Devereux, J., eds., M
Stockton Press, New York, 1991 ). In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J, tt~lol, f3iol. (~18):4~1~1-X53 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at http:l/www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of l, 2, 3, ~l, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (Devereux, J., et crl., Natclc~ic~4cicls Ro,r. l2(1):387 (19$x)) (available at http:l/www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, ~, 5, or 6. In another embodiment, the percent identity between two amino acid or nucleotide sequences is determined using the algorithm of >f. Meyers and W. Miller (CABIOS, X1:1 1-17 ( 1989)) which has been incorporated into the ALIGN pragram Cversion 2.0), using a PAM I 20 weight residue table, a gap length penalty of 12 and a gap penalty of ~.
'fhe nucleic acid and protein sequences ofthe present invention can further be used as a "query sequence" to perform a search against sequence databases to, for example, identify other Family members or related sequences. Such searches can be performed using the NBLAST and XBLAS~f programs (version 2.0) of Altschul, et al. (.l, ~tlol. 73io1. 215:403-10 ( 1990)). BLAS'f nucleotide searches cal be performed with the NBLAS°IT program, score =
1 Op, wordlength = 12 t0 Obtalll (lllCleOtlde SeqllellCeS 110111010~O11S tC? the lluCIC:IC acid 17101eCL11eS OI'the lIlVeIltlOn.
BLAS~C pri>tein searches can be performed with the ~BI~AST program, score =
50. wordlength =
3 t0 Obtalll am111O aClC1 SeqllellCeS 1101nOlOgOLIS t0 the prOtelnS 01' the lnvellt1011. ~1'l) Ob ti1111 gapped alignmelets for comparison purposes, <iappcd BLA~S~h can be utilized rls described in Altschul et al. (lVzac~lc~ic~ ~lc~icl.~ Rc.~,S~. 25( 17):3389-3102 ( 1997)). When utilizing BLAS'C and gapped BLAST
programs, the default parameters of the respective programs (e.g., XBLASlI' and NBLAS'C) can be used.
Full-length pre-processed forms, as well as mature processed forms, of proteins that comprise one of the peptides of the present invention can readily be identified as having complete sequence identity to one of the GPCR peptides of the present invention as well as being encoded by the same genetic locus as the GPCR peptide provided herein. As indicated by tile data presented in Figure 3, the map position was determined to be all chromosome 6 by radiation hybrid mapping.
Allelic variants of a GPCR peptide can readily be identified as being a human protein having a high degree (significant) of sequence homologylidentity to at least a portion of the GPCR
peptide as well as being encoded by the same genetic locus as the GPCR peptide provided herein.
Genetic locus can readily be determined based on the genomic information provided in Figure 3, such as the genomie sequence mapped to the reference human. As indicated by the data presented in Figure 3, the map position was determined to be on chromosome 6 by radiation hybrid mapping.
As used herein, two proteins (or a region of the proteins) have significant homology when the amino acid sequences are typically at least about 70-80°l°, 80-90%, and more typically at least about 90-95°r'° or more homologous. A significantly homologous amino acid sequence, according to the present invention, will be encoded by a nucleic acid sequence that will hybridize to a GPCR peptide encoding nucleic acid molecule under stringent conditions as more fully described below.
Figure 3 provides information on a SNP variant, 6171 IT, which has been found in a gene encoding the GPCR protein of the present invention. The change in the amino acid sequence caused by this SNP is indicated in Figure 3 and can readily be determined using the universal genetic code and the protein sequence provided in Figure ? as a reference.
Paralogs ofa GPCR peptide can readily be identified as leaving some degree of significant sequence homolo~y/identify fo at least a portion of~ihe GPCR peptide. as being encoded by a gene fram humans, and as having similar activity or function. Two proteins will typically be eon sidered paralogs when the amino acid sequences are typically at least about 60% or greater, and more typically at least about 70°I° or greater homology through a given region or domain. Such paralogs will be encoded by a nucleic acid sequence that will hybridize to a GfCR peptide encoding nucleic acid molecule under moderate to stringent conditions as more (lolly described below.
Urtholags of a GfCR peptide can readily be identit7ed as having some degree of significant sequence homologylidentity to at least a portion of the GPCR peptide as well as being encoded by a gene from another organism. Preferred orthologs will be isolated from mammals, preferably primates, for the development of human therapeutic targets and agents. Such orthologs will be encoded by a nucleic acid sequence that will hybridize to a GPCR peptide encoding nucleic acid molecule under moderate to stringent conditions, as more fully described below, depending on the degree of relatedness of the two organisms yielding the proteins.
hlon-naturally occurring variants of the GPCR peptides of the present invention can readily be generated using recombinant techniques. Such variants include, but are not limited to deletions, additions and substitutions in the amino acid sequence of the GPCR peptide.
For example, on a class of substitutions are conserved amino acid substitution. Such substitutions are chose that substitute a given amino acid in a GPCR peptide by another amino acid of like characteristics.
Typically seen as conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu, and Ile; interchange of the hydroxyl residues Ser and °fhr;
exchange of the acidic residues Asp and Glu; substitution between the amide residues Asn and Gln;
exchange of the basic residues I,ys and Arg; and replacements among the aromatic residues Phe and Tyr. Guidance concerning which amino acid changes are likely to be phenotypically silent are found in Bowie et crl., Science 217:1306-1310 (1990).
Variant GPCR peptides can be fully functional or can lack function in one or more activities, e.g. ability to bind ligand, ability to bind G-protein, ability to mediate signaling, etc.
Fully functional variants typically contain only conservative variation or variation in non-critical residues or in non-critical regions. Figure 2 provides the result of protein analysis that identifies critical domains/regions. Functional variants can also contain substitution of similar amino acids that result in no change or an insignificant change in function.
Alternatively, such substitutions may positively or negatively affect function to some degree.
Non-functional variants typically contain one or more nan-conservative amino acid substitutions, deletions. insertions. inversions, or truncation or a substitution. insertion, inversion, or deletion in a critical residue or critical region.
Amino acids that are essential for function can be identit7ed by methods known in the art, such as site-directed mutagenesis or alanine:-scanning mutageneSis (Cunninghatn ei crl , ,Sc~ic~nc~c~
1 ~) 2-I-1:108 i-1 (1$5 ~ 1989)), particularly using the results provided in Figure 2. 'IAhe latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity such as ligand/eF(octor molecule binding or in assays such as an in vitro prolil~rative activity. ~it~s that are critical For ligand-receptor binding can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoahlinity labeling (Smith e/ al.. J. tt~lol_ Biul. 2?-1:899-904 (1992); de Vos e/ crl. ~Sciejzoe 2»:306-312 (1992)).
The present invention further provides fragments of the GPCR peptides, in addition to proteins and peptides that comprise and consist of such fragments, particularly those comprising the residues identified in Figm-e 2. The fragments to which the invention pertains, however, are not to be construed as encompassing fragments that may be disclosed publicly prior to the present invention.
As used herein, a fragment comprises at least 8, 10, 12, I ~l, 16, or more contiguous amino acid residues from a GPCR peptide. Such fragments can be chosen based on the ability to retain I S one or more of the biological activities of the GPCR peptide or could be chosen for the ability to perform a function, e.g. ability to bind ligand or effector molecule or act as an immunogen.
Particularly important fragments are biologically active fragments, peptides which are, for example, about 8 or more amino acids in length. Such Fragments will typically comprise a domain or motif of the GPCR peptide, e.g., active site, a G-protein binding site, a transmembrane domain or a ligand-binding domain. Further, possible fragments include, but are not limited to, domain or motif containing fragments, soluble peptide fragments, and fragments containing immunogenic structures.
Predicted domains and functional sites are readily identifiable by computer programs well-known and readily available to those of skill in the art (e.g., PROSITE analysis).
The results of one such analysis are provided in Figure 2.
Polypeptides often contain amino acids other than the 20 amino acids commonly referred to as the 20 naturally occurring amino acids. Further, many amino acids, including the terminal amino acids, may be modified by natural processes, such as processing and other post-translational modifications, or by chemical modification technidues well known in the art.
Common modifications that occur naturally in GPCR peptides are described in basic texts, detailed ip monographs. and the research literature, and they are well known to those ofskill in the ari(some o~
these features are identified in Figure 2).
Known modifications include, but are not limited to, acetylation, acylation, ADN-ribosylatfon, amidation, covalent attachment of Ilavin, covalent attachment of a heme moiety, '' 0 covalent attachment ol~a nucleotide ar nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment ofphosphotidylinositol. cross-linking, cyclization, disulfide bond lbrmation, demethylation, formation ofcovaleni crosslinks, formation ofcystinc, formation of pyroglutamate, fo~7mylation, gamma carboxylation, glycosylation, Gf I anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.
Such modifications are well-knomm to those of skill in the art and have been described in great detail in the scientific literature. Several particularly common modifications, glycosylation, lipid attachment, sulfation, gamma-carboxylation ofglutamic acid residues, hydroxylation and ADP-ribosylation, For instance, are described in most basic texts, such as Py-oteins - SlrztclZtre and Molecztlar Pr~operlie,s, 2nd >~d., T.E. Creighton, W. H. Freeman and Company, New York (1993).
Many detailed reviews are available on this subject, such as by Wold, F., Po.stIrarZ.slalional Covalent ~~odificcrlion o~Py~oleins, B.C. Johnson, Ed., Academic Press, New York 1-12 (1983); Seifter el al.
(Melh. Enynlol. 182: 626-616 ( 1990)} and Rattan et al. (~l nn. N. ~'. ~I cad, Sci. 663:8-62 ( 1992)).
Accordingly, the GPCR peptides of the present invention also encompass derivatives or analogs in which a substituted amino acid residue is nat one encoded by the genetic code, in which a substituent group is included, in which the mature GPCR peptide is fused with another compound, such as a compound to increase the half=life of the GPCR peptide (for example, polyethylene glycol), or in which the additional amino acids are fused to the mature GPCR
peptide, such as a leader or secretory sequence or a sequence for purification of the mature GPCR
peptide or a pro-protein sequence.
ProteinlPeptide Uses The proteins of the present invention can be used in substantial and specific assays related to the functional information provided in the Figures and Back Ground Section; to raise antibodies or to elicit another immune response: as a reagent (including the labeled reagent} in assays designed to quantitatively determine levels of the protein (or its binding partner or receptor) in biological fluids; and as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in a disease state). Where the preat~:in binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction j, the protein can be used to identify ~' I

the binding partner so as to develop a system to identify inhibitors of the binding interaction.
Any or all of these research utilities are capable of being developed into reagent grade or kit lbrmat for commercialisation as commercial products.
Methods for performing the uses listed above are well known to those skilled in the art.
References disclosing such methods include "Molecular Cloning: A Laboratory Manual", 2d ed..
Cold Spring Harbor Laboratory Press, Sambrook, J., E. F. Fritsch and T.
Maniatis eds., 1989, and "Methods in En~ymology: Guide to Molecular Cloning Techniques'', Academic PreSS, Bergen S. L. and A. R. I~immel eds., 19$7.
The potential uses of the peptides of the present invention are based primarily on the source of the protein as well as the classjaction of the protein. For example, GPCRs isolated from humans and their human/mammalian orthologs serve as targets for identifying agents For use in mammalian therapeutic applications, e.g. a human drug, particularly in modulating a biological or pathological response in a cell or tissue that expresses the GPCR. Experimental data as provided in Figure 1 indicates that GPCR proteins of the present invention are expressed 1 S in humans in the stomach, placenta, kidney, skeletal muscle, liver, bone marrow, and thymus.
Specifically, a virtual northern blot shows expression in the stomach. In addition, PCR-based tissue screening panels indicate expression in placenta, kidney, skeletal muscle, liver, bone marrow, and thymus tissue. Approximately 70°r~° ofall pharmaceutical agents modulate the activity of a GPCR. A combination of the invertebrate and mammalian ortholog can be used in selective screening methods to find agents specific for invertebrates. The structural and functional information provided in the Background and Figures provide specific and substantial uses for the molecules of the present invention, particularly in combination with the expression information provided in Figure 1. Experimental data as provided in Figure 1 indicates expression in humans in the stomach, placenta, kidney, skeletal muscle, liver, bone marrow, and thymus.
Such uses can readily be determined using the information provided herein, that known in the art and routine experimentation.
The proteins of the present invention (including variants and fragments that may have been disclosed prior to the present invention) are useful far biological assays related to GPCRs that are involved in cell signaling, particularly n eurotransmitter signaling. Such assays involve any of the known GPCR functions or activities or properties useful for diagnosis and treatment of GPCR-related conditions that are specituc For the subfamily of GPCRs that the one ofthe present invention belongs to, particularly in cells and tissues that express this receptor.
Experimental data as provided iv figure 1 indicates that GPCR proteins ofthe present invention are expressed in human s in the stomach, placenta, kidney, skeletal muscle, liver, hone marrow, and thymus.
Specifically, a virtual northern blot shows expression in the stomach. In addition, PCR-based tissue screening panels indicate expression ill placenta, kidney, skeletal muscle, liver, bone marrow, and thymus tissue ~hhe pl'Ote111S Rf' the prGSf'.nt IIlVellt1011 al'e al SO LISefUI 111 tll'L!g SCI'Et;llln~ aSSayS, IIl Cell-baSC;d or cell-free systems. Cell-based systems can he native, i.e., cells that normally express the receptor protein, as a biopsy or expanded in cell culture, Experimental data as provided in Figure 1 indicates expression in humans in the stomach, placenta, kidney, skeletal muscle, liver, bone marrow, and thymus. In an alternate embodiment, cell-based assays involve recombinant host cells expressing the receptor protein.
The polypeptides can be used to identify compounds that modulate receptor activity of the protein in its natural state, or an altered form that causes a specific disease or pathology associated with the receptor. Both the GPCRs of the present invention and appropriate variants and fragments can be used in high-throughput screens to assay candidate compounds for the ability to bind to the receptor. These compounds can be ftlrther screened against a functional receptor to deternline the effect of the compound on the receptor activity. hurther, these compounds can be tested in animal or invertebrate systems to determine activityleffectiveness. Compounds can be identified that activate (agonist) or inactivate (antagonist) the receptor to a desired degree.
Further, the proteins of the present invention can be used to screen a compound for the ability to stimulate or inhibit interaction between the receptor protein and a molecule that normally interacts with the receptor protein, e.g. a ligand or a component ofthe signal pathway that the receptor protein normally interacts (for example, a G-protein or other interactor involved in cAMP
or phosphatidylinositol turnover andlor adenylate cyclase, or phospholipase C
activation). Such assays typically include the steps of combining the receptor protein with a candidate compound under conditions that allow the receptor protein, or Fragment, to interact with the target molecule, and to detect the formation of a complex between the protein and the target or to detect the biochemical consequence of the interaction with the receptor protein and the target, such as any of the associated effects of signal transduction such as G-protein phosphorylation, cAMP or phosphatidylinositol turnover, and adenylate cyclase or phospholipase C
activation.
Candidate compounds include, for example, 1 ) peptides such as soluble peptides, including Ig-tailed fusion peptides and members ofrandom peptide libraries (see, e.g., Lam et ul., tV'crlm~e 3~-l:8?-$~l ( 1991 ); I-IoLlghten ct ul" r'~crlm~~a 35-1:81-$6 ( 1 ~q 1 )) and combinatorial chemistry-derived molecular libraries made ol'I~- andlor L- conligurafion amino acids: ?) phosphopeptides Ie.g., members of random and partially degenerate, direcfed phosphopeptide:
libraries, see, e.g" Son gyang m ttl,, C'c~ll 7?:767-778 ( 1993 )); 3 ) antibodies (e.g.. polyclonal.
monoclonal, humanised, anti-idit>typic. chimerie, and single chain antibodies as well as Fah, Iv'~ab')?, Fab expression library fragments, and epitope-binding fragments of antibodies); and ~I) small organic and inorganic molecules (e.g., molecules obtained (rote combinatorial and natural product libraries), One candidate compound is a soluble fragment of the receptor that competes for ligand binding. Other candidate compounds include mutaait receptors or appropriate fragments containing mutations that affect receptor Function and thus compete for ligand.
Accordingly, a Fragment that competes for ligand, for example with a higher aF~nity, or a fragment that binds ligand but does not allow release, is encompassed by the invention.
The invention further includes other end point assays to identify compounds that modulate (stimulate or inhibit) receptor activity. The assays typically involve an assay of events in the signal transduction pathway that indicate receptor activity. Thus, a cellular process such as proliferation, the expression of genes that are up- or down-regulated in response to the receptor protein dependent signal cascade, can be assayed. In one embodiment, the regulatory region of such genes can be operably linked to a marker that is easily detectable, such as luciferase.
Any of the biological or biochemical functions mediated by the receptor can be used as an endpoint assay. These include all of the biochemical or biochemicallbiological events described herein, in the references cited herein, incorporated by reference for these endpoint assay targets, and other Functions known to those of ordinary skill in the art or that can be readily identified using the information provided in the Figures, particularly Figure 2. Specifically, a biological Function of a cell or tissues that expresses the receptor can be assayed. Experimental data as provided in Figure I
indicates that GPCR proteins of the present invention are expressed in humans in the stomach, placenta, kidney, skeletal muscle, liver, bone marrow, and thymus, Specifically, a virtual northern blot shows expression in the stomach. In addition, PCR-based tissue screening panels indicate expression in placenta, kidney, skeletal muscle, liver, bone marrow, and thymus tissue.
Binding and/or activating compounds can also be screened by using chimeric receptor proteins in which the amino terminal extracellular domain, as parts thereof, the entire transmembrane domain or subregions, such as any of the seven transmembrane segments or any of the intracellular or extracellular loops and the carboxy terminal intracellular domain, or parts thereof, can be replaced by heterologou s domains or subregions. For example, a G-protein-binding region can be used that interacts with a diFferent G-protein then that which is recognized by the native receptor. Accordingly, a different set of signal transduction components is available as an end-point assay For activation. Alternatively, the entire transmembrane portion or subregions Csuch ~ ~-I

as transtnembrane segments or intracellular or extracellular loops) can be replaced with the entire transmembrane portion or subregicans specific to a host cell that is different from the bast cell ti~om which th a amino terminal exiracellular domain and/or the G-protein-binding regian are derived.
IAhis allows for assays to be performed in alher than the specific bast cell from which the receptor is derived. Alternatively, the amino terminal extracellular domain {andlor other ligand-binding regions) could be replaced by a domain (and/or other binding region) binding a different ligand, thus, providing an assay for test compounds that interact with the heterologous amino terminal extracellular domain (or region) but still cause signal transduction. Finally, activation can be detected by a reporter gene containing an easily detectable coding region operably linked to a transcriptional regulatory sequence that is part of the native signal transduction pathway.
The proteins of the present invention are also useful in competition binding assays in methods designed to discover compounds that interact with the receptor. Thus, a compound is exposed to a receptor polypeptide under conditions that allow the compound to bind or to otherwise interact with the polypeptide {Hodgson, Bioltechnology, 1992, Sept 10(9);973-80). Soluble receptor polypeptide is also added to the mixture. If the test compound interacts with the soluble receptor polypeptide, it decreases the amount of complex formed or activity from the receptor target. This type of assay is particularly useful in eases in which compounds are sought that interact with specific regions of the receptor. Thus, the soluble polypeptide that competes with the target receptor region is designed to contain peptide sequences corresponding to the region of interest.
To perform cell free drug screening assays, it is sometimes desirable to immobilize either the receptor protein, or fragment, or its target molecule to facilitate separation of complexes from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay.
Techniques for immobilizing proteins on matrices can be used in the drug screening assays.
In one embodiment, a fission protein can be provided which adds a domain that allows the protein to be bound to a matrix. For example, glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads {Sigma Chemical, St. Louis, MO) or glutathione derivatized microtitre plates, which are then combined with the cell lysates {e.g.,''S-labeled) and the candidate compound, and the mixture incubated under conditions conducive to complex Formation {e.g., at physiological conditions for salt and pf-I). Following incubation, the beads are washed to remove any unbound label, and the matrix immobilized and radiolabel determined directly, or in the supernatant alter the complexes are dissociated. Alternatively, the complexes can be dissociated from the matrix, separated by ~D~-PAGC~. and the level of receptor-binding protein found in the ~i bead fraction quantitated from the gel Using standard electrophoretic technidues. laor example, either the polypeptide or its target molecule can be inlmobili~ed utilizing conjugation of biotin and streptavidin using techniques well known in the art. Alternatively, antibodies reactive with the protein but which do not interfere with binding of the protein to its target molecule can be derivati~ed to the wells of the plate, and the protein trapped in the wells by antibody conjugation.
Preparations of a receptor-binding protein and a candidate compound are incubated in the receptor protein-presenting wells and the amount of complex trapped in the well can be quantitated.
Methods for detecting such complexes, in addition to those described above for the GS'I°-immobilized complexes, include immunodeteetion of complexes using antibodies reactive with the receptor protein target molecule, or which are reactive with receptor protein and compete with the target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the target molecule.
Agents that modulate one of the GPCRs of the present invention can be identified using one or more of the above assays, alone or in combination. It is generally preferable to use a cell-based I 5 or cell free system first and then confirm activity in an animal or other model system. Such model systems are well known in the art and can readily be employed in this context.
Modulators of receptor protein activity identified according to these drug screening assays can be used to treat a subject with a disorder mediated by the receptor pathway, by treating cells or tissues that express the GPCR. Experimental data as provided in Figure 1 indicates expression in humans in the stomach, placenta, kidney, skeletal muscle, liver, bone marrow, and thymus. These methods of treatment include the steps of administering a modulator of the GPCR's activity in a pharmaceutical composition to a subject in need of such treatment, the modulator being identified as described herein.
In yet another aspect of the invention, the GPCR 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 72:223-232; Madura et al. (1993) J. Biol. C'hc~m4 268:12016-12051; Bartel et al.
( 1993 ) l3iolec~hniqite,r I 1:920-92~; Iwabuchi et al. ( 1993) ()f~c~o~crTt' 8:1693-1696; and Brent W094/10300), to identify other proteins, which bind to or interacf with the GPCR and are involved in GPCR activity. Such GPCR-binding proteins are also likely to be involved in the propagation of signals by the GPCR proteins or GfCR targets as, for example, downstream elements of a GPCR-mediated signaling pathway. Alternatively. such GPCR-binding proteins are likely to be GPCR inhibitors.
FI'he tWO-h ybl'Id SySt~:ln 1S based 011 the Illotllllal' nature ofInOSt tl'aIlSC1'1pt1011 IaCtol'S, 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 a GPCR
protein is Fused to a gene encoding the DNA binding domain oFa known transcription factor (e.g.. GAG-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 of the known transcription Factor. If the "bait" and the "prey" proteins are able to interact, ij~ vivo, forming a GPCR-dependent complex, the DNA-binding and activation domains of the transcription Factor are braught into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. l;xpression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the protein which interacts with the GPCR protein.
This invention Further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein in an appropriate animal model. For example, an agent identified as described herein (e.g., a GPCR modulating agent, an antisense GPCR nucleic acid molecule, a GPCR-specific antibody, or a GPCR-binding partner) can be used in an animal or other model to determine the efficacy, toxicity, or side effects of treatment with such an agent. Alternatively, an agent identified as described herein can be used in an animal or other model to determine the mechanism of action of such an agent. Furthermore, this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein.
The GPCR proteins of the present invention are also useful to provide a target for diagnosing a disease or predisposition to disease mediated by the peptide.
Accordingly, the invention provides methods For detecting the presence, or levels of the protein (or encoding mRl~f'A) in a cell, tissue, or organism. Experimental data as provided in Figure 1 indicates expression in humans in the stomach, placenta, kidney, skeletal muscle, Liver, bone marrow, and thymus. 'hhe method involves contacting a biological sample with a compound capable of interacting with the receptor protein such that the interaction can be detected. Such an assay can be provided in a single detection format or a mufti-detection format such as an antibody chip array.
One agent for detecting a protein in a sample is an antibody capable of selectively binding to protein. A biological sample includes tissues, cells and biological Iluids isolated from a subject, as well as tissues, cells and fluids present within a subject.
~hhe peptides oFihe present invention also provide targets Cor diagnosing, active protein ~7 activity, disease, or predisposition to disease. in a patient having a variant peptide, particularly activities and conditions that are known For other members ohthe Family of proteins to which the present one bilongs_ ~fhus, the peptide can be isolated From a biological sample and assayed far the presence of a genetic mutation that results in aberrant peptide. Al~his includes amino acid substitution, deletion, insertion, rearrangement, (as the result of aberrant splicing events), and inapprapriate post-translational modification. Analytic methods include altered electrophm°etic mobility, altered tryptic peptide digest, altered receptor activity in cell-based or cell-free assay, alteration in ligand or antibody-binding pattern, altered isoelectric point, direct amino acid sequencing, and any other of the known assay techniques useful for detecting mutations in a protein.
Such an assay can be provided in a single detection format or a multi-detection format such as an antibody chip array.
In vitro techniques for detection of peptide include enzyme linked immunosorbent assays {ELISAs), Western blots. immunoprecipitations and immunofluorescence using a detection reagent, such as an antibody or protein binding agent. Alternatively, the peptide can be detected in vioo in a subject by introducing into the subject a labeled anti-peptide antibody or other types of detection agent. 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.
Particularly useful are methods that detect the allelic variant of a peptide expressed in a subject and methods which detect Ii-agments of a peptide in a sample.
The peptides are also useful in pharmacogenomic analysis. Pharmacogenomics deal with clinically significant hereditary variations in the response to dnlgs due to altered drug disposition and abnarmal action in affected persons. See, e.g., Eichelbaum, M. (Clip. Exp.
Phanmacol. Phvsiol.
23(10-11):983-985 (1996)), and Linden M.W. (Clira. Chern. ~13(2):25~-266 (1997)). The clinical outcomes of these variations result in severe toxicity of therapeutic drugs in certain individuals or therapeutic failure of drugs in certain individuals as a result of individual variation in metabolism.
Thus, the genotype of the individual can determine the way a therapeutic compound acts on the body or the way the body metabolizes the compound. Further, the activity of drug metabolizing enzymes effects both the intensity and duration ofdrug action, Thus, the phamiacogenomics of the individual permit the selection of effective compounds and effective dosages of such compounds Ior prophylactic or therapeutic treatment based on the individual's genotype. The discovery oI'genetic polymorphisms in some drug metabolizing enzymes has explained why some patients do not obtain the expected drug elTects, show an exaggerated drug effect, or experience serious toxicity From standard dru~~ dosages. folymorphisms can be expressed in the phenotype ol~th~
extensive metaboli~er and the phenotype oFihe poor metaholizer. Accordingly, genetic polymorphism may lead to allelic protein variants oFihe receptor protein in which one or more of the receptor tlmctions in one population is diFFerent From those in another population. The peptides thus allow a target to ascertain a genetic predisposition that can alT ect treatment modality, 'Thus, in a ligand-based treatment, polymorphism may give rise to amino terminal extracellular domains and/or other ligand-binding regions that are more or less active in ligand binding, and receptor activation. Accordingly, ligand dosage would necessarily be modified to maximize the therapeutic effect within a given population containing a polymorphism. As an alternative to genotyping, specific polymorphic peptides could be identified.
The peptides are also useful for treating a disorder characterized by an absence of, inappropriate, or unwanted expression of the protein. Experimental data as provided in Figure 1 indicates expression in humans in the stomach, placenta, kidney, skeletal muscle, liver, bone marrow, and thymus. Accordingly, methods for treatment include the use of the GPCR protein or fragments.
Antibodies The invention also provides antibodies that selectively bind to one of the peptides of the present invention, a protein comprising such a peptide, as well as variants and fragments thereof, As used herein, an antibody selectively binds a target peptide when it binds the target peptide and ?0 does not significantly bind to unrelated proteins. An antibody is still considered to selectively bind a peptide even if it also binds to other proteins that are not substantially homologous with the target peptide so long as such proteins share homology with a fragment or domain of the peptide target of the antibody. In this ease, it would be understood that antibody binding to the peptide is still selective despite some degree oFcross-reactivity.
As used herein, an antibody is defined in terms consistent with that recognized within the art: they are multi-subunit proteins produced by a mammalian organism in response to an antigen challenge. The antibodies of the present invention include polyclonal antibodies and monoclonal antibodies, as well as fragments ofsuch antibodies, including, but not limited to, I~ab or IM(ab')~, and Fv fragments.
Many methods arc known for generating and/or identiFyint~ antibodies to a given target peptide. Several such methods are described by° I-Iarlow, Antibodies, Cold Spring Harbor Press.
( 1989)_ '' 9 In general. to generate antibodies, an isolated peptide is used as an inltllunogen and is administered to a mammalian organism, such as a rat, rabbit or mouse. The fLlll-length protein, an antigenic peptide fragment or a fusion protein can he used. fal-ticularly important fragments are tlloSe COVeI'lllg tullCtlollal dC)Illalns, SLICK as the CIoIl1a111S ldentlhed 111 I~1gL11'e ?, alld dOmaln 01' sequence homology or divergence amongst the family, such as those that can readily be identified using protein alignment methods and as presented in the Figures.
Antibodies are preferably prepared from regions or discrete fragments of the proteins. Antibodies can be prepared from any region of the peptide as described herein.
However, preferred regions will include those involved in function/activity and/or receptorlbinding partner interaction. Figure 2 can be used to identify particularly important regions while sequence alignment can be used to identify conserved and unique sequence Fragments.
An antigenic fragment will typically comprise at least 8 contiguous amino acid residues.
The antigenic peptide can comprise, however, at least 10, 12, 14, 16 or more amino acid residues.
Such fragments can be selected on a physical property, such as fragments con-espond to regions that are located on the surface of the protein, e.g., hydrophilic regions or can be selected based an sequence uniqueness (see Figure 2).
Detection on an antibody of the present invention 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, (3-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidinlbiotin and avidilllbiotin; 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, III, ~'S or ~H.
Antibody L.Fses ~() Ahhe antibodies can be used to isolate one oFthe proteins of the present invention by standard techniLlues, such as affinity chromatography or immunoprecipitation. The antibodies can facilitate the pLlrIflGat1011 of the llatLlI'al prote111 fl'om CClls allCl rCCO
I11b111aTltly p1'odLICed protein eXpl'eSSed In i() hOSt Cells. 111 CldC1lt10n, SLICK alltlbodlES are LlSef'Lll t0 dEteCt the preSC'.IlCe Of otle oI the pl"OtelllS Of the pI'eSEllt lnVen tlon lIl Cells of tISSLIe$ t0 deten11111e the pattern ol'expl'eSS10I1 ol~tll(: protElll alTlong val'louS tlSSIIC:S In all ol'gr11115n1 and ovCa' the Coul'Se Of'1101-111a1 C1L'Velopmellt. I>xpel'11T1elltal data as provided in Figure 1 indicates that UPC'IZ proteins of the present invention are expressed in humans in the stomach, placenta, kidney, skeletal mLlscle, livEr, bon a marrow, and thymus. Specifically, a virtual narth em blot shows expression in the stomach. In addition, PCIZ-based tissue screening panels indicate expression in placenta, kidney, skeletal muscle, liver, bone marrow, and thymus tissue. FuI-ther, such antibodies can be used to detect protein in suet, ijZ
uitro, or in a cell lysate or supernatant in order to evaluate the abundance and pattern of expression.
Also, such antibodies can be used to assess abnormal tissue distribution or abnormal expression during development or progression of a biological condition. Antibody detection of circulating fragments of the fidl length protein can be used to identify turnover.
Further, the antibodies can be used to assess expression in disease states such as in active stages of the disease or in an individual with a predisposition toward disease related to the protein's function. When a disorder is caused by an inappropriate tissue distribution, developmental expression, level of expression of the protein, or expressed/processed form, the antibody can be prepared against the normal protein. Experimental data as provided in Figure 1 indicates expression in humans in the stomach, placenta, kidney, skeletal muscle, liver, bone marrow, and thymus. If a disorder is characterized by a specific mutation in the protein, antibodies specific for this mutant protein can be used to assay for the presence ofthe specific mutant protein.
The antibodies can also be used to assess normal and aberrant subcellular localization of cells in the various tlSSUeS in an organism. Experimental data as provided in Figure 1 indicates expression in humans in the stomach, placenta, kidney, skeletal muscle, liver, bone marrow, and thymus. The diagnostic uses can be applied, not only in genetic testing, but also in monitoring a treatment modality. Accordingly, where treatment is ultimately aimed at correcting expression level or the presence of aben-ant sequence and aberrant tissue distribution or developmental expression, antibodies directed against the protein or relevant fragments can be used to monitor therapeutic efficacy.
Additionally, antibodies are useful in pharmacogenomic analysis. Thus, antibodies prepared a0 against polylnorphic proteins can be used to identify individuals that require modified treatment modalities. The antibodies are also useful as diagnostic tools as an immunological marker fi)r aberrant protein analyzed by electrophoretic mobility, isoele:ctric point, Cryptic peptide digest. and other physfcal assays known to those ill the art.
;l Fl~he antibodies are also useFul far tissue typing. IJxperimental data as provided in Figure 1 indicates expression in humans in the stomach, placenta, kidney, skeletal muscle, liver, bone marrow, and thymus. 'hhus, where a specific protein has been correlated with expression in a specific tissue, antibodies that are specific For this protein can be used to ir(entity a tissue type.
'fhe antibodies are also useful for inhibiting protein function, For example, blocking the binding of the GPCR peptide to a binding partner such as a ligand. 'these uses can also be applied in a therapeutic context in which treatment involves inhibiting the protein's Function. An antibody can be used, for example, to block binding, thus modulating (agonizing or antagonizing) the peptides activity. Antibodies can be prepared against specific fragments containing sites required for function or against intact protein that is associated with a cell or cell membrane. See Figure 2 for structural information relating to the proteins of the present invention.
The invention also encompasses kits for using antibodies to detect the presence of a protein in a biological sample. The kit can comprise antibodies such as a labeled or labelable antibody and a compound or agent for detecting protein in a biological sample; means for determining the amount of protein in the sample; means for comparing the amount of protein in the sample with a standard;
and instructions for use. Such a kit can be supplied to detect a single protein or epitope or can be configured to detect one of a multitude of epitopes, such as in an antibody detection array. Arrays are described in detail below for nucleic acid arrays and similar methods have been developed for antibody arrays.
ucleic Acid Molecules The present invention fm-ther provides isolated nucleic acid molecules that encode a GPCR
peptide or protein of the present invention (cDNA, transcript and genomic sequence). Such nucleic acid molecules will consist of, consist essentially of, or comprise a nucleotide sequence that encodes one of the GfCR peptides of the present invention, an allelic variant thereoF
or an ortholog or paralog thereof.
As used herein, an "isolated" nucleic acid molecule is one that is separated from other nucleic acid present in the natural source of the 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' ends of the nucleic acid) in the genomic D~IA of the organism from which the nucleic acid is derived. I-lowever, there can be some flanking nucleotide seque.races, for example up to about SKB, ~1K13, iKl3, ?KI3, or 1 KI3 or less, particularly contiguous peptide encoding sequences and peptide encoding sequences within the same gene but separated by introns in the ~ellomic sequence. The important point is that the nucleic acid is isolated from remote and unimportant tlanl:ing sequences SLICK theft It 0111 be SLIb~eCtCd t0 the SpeCIflC I11an1pLilat1011s deSCl'lbed llerelll SLICK a5 CeCOITlblllallt expression, preparation of probes and primers, and other uses specific to the nucleic acid sequences.
S Moreover, an "isolated" nucleic acid molecule, such as a transcript/cDNA
molecule, can be substantially free of ocher cellular material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized. However, the nucleic acid molecule can be fused to other coding or regulatory sequences and still be considered isolated.
For e~cample, recombinant DNA molecules contained in a vector are considered isolated.
Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution. Isolated RNA molecules include irr vivo or in vitro RNA transcripts of the isolated DNA
molecules of the present invention. Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically.
Accordingly, the present invention provides nucleic acid molecules that consist of the nucleotide sequence shown in Figure 1 or 3 (SEQ ID NO:I, cDNA sequence and SEQ
ID N0:3, genomic sequence), or any nucleic acid molecule that encodes the protein provided in Figure 2, SEQ ID N0:2. A nucleic acid molecule consists of a nucleotide sequence when the nucleotide sequence is the complete nucleotide sequence oFthe nucleic acid molecule.
The present invention further provides nucleic acid molecules that consist essentially of the nucleotide sequence shown in Figure I or 3 (SEQ ID NO:I, cDNA sequence and SEQ
ID N0:3, genomic sequence), or any nucleic acid molecule that encodes the protein provided in Figure 2, SEQ LD N0:2. A nucleic acid molecule consists essentially of a nucleotide sequence when such a nucleotide sequence is present with only a few additional nucleic acid residues in the final nucleic acid molecule.
The present invention further provides nucleic acid molecules that comprise the nucleotide sequences shown in Figure 1 or 3 (SEQ ID NO:I, cDNA sequence and SEQ ID N0:3, genonlic sequence), or any nucleic acid molecule that encodes the protein provided in Figure 2, SCQ ID
NO:2. A nucleic acid molecule comprises a nucleotide sequence when the nucleotide sequence is at leasf part of the final nucleotide seduence of the nucleic acid Molecule. In such a fashion, the nucleic acid molecule can be only the nucleotide sequence or have additional nucleic acid residues, such as nucleic acid resiclucs that arc naturally associated with it or lveterolof~ous nucleotide "

sequences. Such a nucleic acid molecule can have a few additional nucleotides or can comprises several hundred or more additional nucleotides. A brief description of how various types of these nucleic acid molecules can be readily madelisolated is provided below.
In Figures 1 and 3, both coding and non-coding sequences are provided, Because of the source ofthe present invention, human genomic sequences (Figure a) and cDNA
sequences (Figure I ), the nucleic acid molecules in the Figures will contain genomic intronic sequences, 5' and 3' non-coding sequences, gene regulatory regions and non-coding intergenic sequences. In general such sequence features are either noted in Figures 1 and 3 or can readily be iden tiFed using computational tools known in the art. As discussed below, some of the non-coding regions, particularly gene regulatory elements such as promoters, are useful for a variety of purposes, e.g. control of heterologous gene expression, target for identifying gene activity modulating compounds, and are particularly claimed as fragments of the genomic sequence provided herein.
The isolated nucleic acid molecules can encode the mature protein plus additional amino or I 5 carboxyl-terminal amino acids, or amino acids interior to the mature peptide (when the mature form has more than one peptide chain, for instance). Such sequences may play a role in processing of a protein from precursor to a mature form, facilitate protein trafficking, prolong or shorten protein half life or facilitate manipulation of a protein for assay or production, among other things. As generally is the case in situ, the additional amino acids may be processed away from the mature protein by cellular enzymes.
As mentioned above, the isolated nucleic acid molecules include, but are not limited to, the sequence encoding the GPCR peptide alone, the sequence encoding the mature peptide and additional coding sequences, such as a leader or secretory sequence (e.g., a pre-pro or pro-protein sequence), the sequence encoding the mature peptide, with or without the additional coding ?5 sequences, plus additional non-coding sequences, far example introns and non-coding 5' and 3' sequences such as transcribed but non-translated sequences that play a role in transcription, mRNA
processing (including splicing and polyadenylation signals), ribosome binding and stability of mRNA. In addition, the nucleic acid molecule may be fused to a marker sequence encoding, for Example, a peptide that facilitates purification _ Isolated nucleic acid molecules can be in the Form of RNA, such as mRNA, or in the form DNA, including cDNA and genomic DNA obtained by cloning or produced by chemical synthetic techniqu es or by a combination thereof The nucleic acid, especially DNA, can be double-stranded or single-stranded. Single-stranded nucleic acid can be the codin~~ strand (sense strand) or the non-=I

coding strand (anti-sense strand).
The invention fuI-ther provides nucleic acid molecules that encode fragments of the peptides of the present invention as well as nucleic acid molecules that encodE obvious variants of the GI'CR
proteins of the present invention that are described above. Such nucleic acid molecules may be naturally occul-ring, such as allelic variants (same locus), paralogs (different locus), and orthologs (different oI°ganism), ar may be constructed by recombinant DNA methods or by chemical synthesis. Such non-naturally occurring variants may be made by mutagenesis techniques, including those applied to nucleic acid molecules, cells, ar organisms.
Accordingly, as discussed above, the variants can contain nucleotide substitutions, deletions, inversions and insertions.
Variation can occur in either or both the coding and non-coding regions. The variations can produce both conservative and non-conservative amino acid substitutions.
The present invention further provides non-coding fragments of the nucleic acid molecules provided in Figures 1 and 3. Preferred non-coding fragments include, but are not limited to, promoter sequences, enhancer sequences, gene modulating sequences and gene termination I S sequences. Such fragments are useful in controlling heterologous gene expression and in developing screens to identify gene-modulating agents. A promoter can readily be identified as being 5' to the ATG stal-t site in the genomic sequence provided in Figure 3, A fragment comprises a contiguous nucleotide sequence greater than 12 or more nucleotides. Further, a fragment could at least 30, X10, 50, 100, 2~0 or 500 nucleotides in length.
The length of the fragment will be based on its intended use. For example, the fragment can encode epitope bearing regions of the peptide, or can be usefid as DNA probes and primers. Such fragments can be isolated using the known nucleotide sequence to synthesize an oligonucleotide probe. A labeled probe can then be used to screen a cDNA library, genomie DNA
library, or mRNA to isolate nucleic acid corresponding to the coding region. Further, primers can be used in PCR reactions to clone speciFic regions of gene.
A probelprimer typically comprises substantially a purified oligonucleotide or oligonucleotide pair. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, 20, ?5, X10, s0 or more consecutive I7LlcleOtldeS.
Orthologs, homologs, and allelic variants can be identified using methods well known in the art. As described in the Peptide Section, these variants comprise a nucleoside sequence encoding a peptide that is typically 60-70°~0, 70-80°%0, 80-90°~°0, avd more typically at least about 90-95a~"o or more homolo4~oLls to the nucleotide seduencc shown in the Figure sheets or a IragmEnt of this ;s sequence. Such nucleic acid molecules can readily be identified as being able to hybridi2e under moderate to stringent conditions, to the nucleotide sequence shown in the Figure sheets or a f~raglnent ol~the sequence. Allelic variants can readily be. determined by genetic locus of"the encoding gene. As indicated by the data presented in Figure 3, the map position was detel-Inined to be on clwomosorne 6 by radiation hybrid mapping.
Figure 3 provides information on a SNP variant, G 171 1 T, which has been found in a gene encoding the GPCR protein of the present invention. The change in the amino acid sequence caused by this SNP is indicated in Figure 3 and can readily be determined using the universal genetic code and the protein sequence provided in Figure 2 as a reference.
As used herein, the term "hybridizes under stringent conditions" is intended to describe conditions for hybridization and washing under which nucleotide sequences encoding a peptide at least 6p-70% homologous to each other typically remain hybridized to each other. The conditions can be such that sequences at least about 60°~'0, at least about 70%, or at least about 80% or more homologous to each other typically remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in Czts°renl Protocols in Molecztlar~ Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. One example of stringent hybridization conditions are hybridization in 6~ sodium chloride/sodium citrate (SSC) at about ~fSC, followed by one or more washes in 0.2 X SSC, 0.1°~'o SDS at 50-65C. Examples of moderate to low stringency hybridization conditions are well known in the art.
Nucleic Acid Molecule Uses The nucleic acid molecules of the present invention are useful for probes, primers, chemical intermediates, and in biological assays. The nucleic acid molecules are useful as a hybridization probe for messenger RNA, transcripUcDNA and genomic DNA to isolate full-length cDNA and genomic clones encoding the peptide described in Figure 2 and to isolate cDNA
and genomic clones that correspond to variants (alleles, orthologs, etc.) producing the same or related peptides shown in Figure 2. As illustrated in Figure' 3, 6171 1T is a known SNP
variant.
'I'he probe can correspond to any sequence along the entire length of"the nucleic acid mOleCUIeS pl'OVlded 111 the Flgttres. ACCOI"dlllgly, It COtild be deI'ived frOITl ~~ IlOnCOdIIl~ CegIOIIS, the COdlng reg1O11, alld 3~ nOncOd111g reglOnS. I'IOW(:Vel'" aS dlSGLlSSed, fCagIT7eI7tS are nOt t0 be COIIStI'tled as encompassing Fragments disclosed prior to the present invention.
'I"he nucleic acid molecules arc also useful as primers For PCR to amplify any given region ,6 of a nucleic acid molecule and are useful to synthesize antisense molecules of desired length and sequence.
Al~he nucleic acid molecules are also useful for constructing recombinant vectors. Such vectors include expression vectors that express a portion of, or all ol~, the peptide sequences.
Vectors also include insertion vectors, used to integrate into another nucleic acid molecule sequence, such as into the cellular genome, to alter in ,S~ilzt Expression of a gene andlor gene product.
For example, an endogenous coding sequence can be replaced via homologous recombination with all or part of the coding region containing one or more specifically introduced mutations.
The nucleic acid molecules are also useful for expressing antigenic portions of the proteins.
The nucleic acid molecules are also useful as probes for determining the chromosomal positions of the nucleic acid molecules by means of in sitzt hybridization methods. As indicated by the data presented in Figure 3, the map position was determined to be on chromosome 6 by radiation hybrid mapping.
The nucleic acid molecules are also useful in making vectors containing the gene regulatory I 5 regions of the nucleic acid molecules of the present invention.
The nucleic acid molecules are also useful for designing ribozymes corresponding to all, or a part, of the mRNA produced from the nucleic acid molecules described herein.
The nucleic acid molecules are also useful for making vectors that express part, or all, of the peptides.
The nucleic acid molecules are also useful for constructing host cells expressing a part, or all, of the nucleic acid molecules and peptides.
The nucleic acid molecules are also useful for constructing transgenic animals expressing all, or a part, of the nucleic acid molecules and pepfides.
The nucleic acid molecules are also useful as hybridization probes for determining the presence, level, form and distributian ofnucleic acid expression. Experimental data as provided in Figure 1 indicates that GPCR proteins of the present invention are expressed in humans in the stomach, placenta, kidney, skeletal muscle, liver, bone marrow, and thymus.
Specifically, a virtual northern blot shows expression in the stomach. In addition, PCR-based tissue screening panels indicate expression in placenta, kidney, skeletal muscle. liver, bone marrow, and thymus tissue.
Accordingly, the probes can be used to detect the presence of, or to determine levels o1; a specific nucleic acid molecule in cells, tissues, and in organisms. The nucleic acid whose level is determined can be DNA or RNA. Accordingly, probes corresponding to the peptides described herein can be used to assess expression andlor gene copy number iv a given cell, tissue, or ~7 organism. 'hh ese uses are relevant For diagnosis of disorders involving an increase or decrease in GPCR protein expression relative to normal results.
In uiiro techniques for detection of mRNA include Northern hybridizations and fn ,~~itn hybridizations. Ire ollrr~ techniques for detecting DNA include Southern hybridizations and in .~'ilu hybridization.
Probes can be used as a part oFa diagnostic test kit for identifying cells or tissues that express a GPCR protein, such as by measuring a level of a receptor-encoding nucleic acid in a sample of cells from a subject e.g., mRNA or genomie DNA, or determining if a receptor gene has been mutated. Experimental data as provided in Figure I indicates that GPCR
proteins of the present invention are expressed in humans in the stomach, placenta, kidney, skeletal muscle, liver, bane marrow, and thymus. Specifically, a virtual northern blot shows expression in the stomach. In addition, PCR-based tissue screening panels indicate expression in placenta, kidney, skeletal muscle, liver, bone marrow, and thymus tissue.
Nucleic acid expression assays are useful for drug screening to identify compounds that modulate GPCR nucleic acid expression.
The invention thus provides a method for identifying a compound that can be used to treat a disorder associated with nucleic acid expression of the GPCR gene, particularly biological and pathological processes that are mediated by the GPCR in cells and tissues that express it.
Experimental data as provided in Figure 1 indicates expression in humans in the stomach, placenta, kidney, skeletal muscle, liver, bone marrow, and thymus. The method typically includes assaying the ability of the compound to modulate the expression of the GPCR nucleic acid and thus identifying a compound that can be used to treat a disorder characterized by undesired GPCR
nucleic acid expression. The assays can be performed in cell-based and cell-free systems. Cell-based assays include cells naturally expressing the GPCR nucleic acid or recombinant cells genetically engineered to express specific nucleic acid sequences.
The assay for GPCR nucleic acid expression can involve direct assay of nucleic acid levels, such as mRNA levels, or on collateral compounds involved in the signal pathway. Further, the expression of genes that are up- or down-regulated in response to the GPCR
protein signal pathway can also be assayed. In this embodiment the regulatory regions of these genes can be operably linked to a reporter gene such as luciferase.
Thus, madulators of GPCR gene etpression can be identified in a method wherein a cell is contacted with a candidate compound and the expression of mRNA determined.
'The level of expression of GPCR mRNA in the presence of the candidate compound is compared to the level of s Expression oFGPCR mRNA in the absence ofthe candidate compound, 'fhe candidate compound can then he identified as a modulator of nucleic acid expression based on this comparison and he used, for example to treat a disorder characterioed by aber i°ant nucleic acid Expression. When expression of mRNA is statistically significantly greater in the presence oFthe candidate compound than in its absence, the candidate compoLmd is identified as a stimulator of nucleic acid expression.
When nucleic acid expression is statistically significantly less in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of nucleic acid expression.
The invention further provides methods of treatment, with the nucleic acid as a target, using a compound identified through drug screening as a gene modulator to modulate GPCR nucleic acid expression, particularly to modulate activities within a cell or tissue that expresses the proteins.
Experimental data as provided in Figure 1 indicates that GPCR proteins of the present invention are expressed in humans in the stomach, placenta, kidney, skeletal muscle, liver, bone marrow, and thymus. Specifically, a virtual northern blot shows expression in the stomach.
In addition, PCR-based tissue screening panels indicate expression in placenta, kidney, skeletal muscle, liver, bone marrow, and thymus tissue. Modulation includes both up-regulation (i.e.
activation or agonization) or down-regulation (suppression or antagonization) or nucleic acid expression.
Alternatively, a modulator for GPCR nucleic acid expression can be a small molecule or drug identified using the screening assays described herein as long as the drug or small molecule inhibits the GPCR nucleic acid expression in the cells and tissues that express the protein.
Experimental data as provided in Figure 1 indicates expression in humans in the stomach, placenta, kidney, skeletal muscle, liver, bone marrow, and thymus.
The nucleic acid molecules are also useful for monitoring the effectiveness of modulating compounds on the expression or activity of the GPCR gene in clinical trials or in a treatment regimen. 'thus, the gene expression pattern can serve as a barometer for the continuing effectiveness of treatment with the compound, particularly with compounds to which a patient can develop resistance. The gene expression pattern can also serve as a marker indicative of a physiological response of the aFfected cells to the compound. Accordingly, such monitoring would allow either increased administration of the compound or the administration of alternative compounds to which the patient has not become resistant. Similarly, iFthe level ol~nucleic acid expression Falls below a clesirable level, administration of the compound could be commensurately decreased.
'Fhe nucleic acid molecules are also useCl~l in diaf~nostic assays Ior qualitative: changes in GPCR nucleic acid, and particularly in qualitative changes that lead to pathology. The nucleic acid molecules can be used to detect mutations in GPCR genes and gen a expression products such as mRNA. The nucleic acid molecules can be used as hybridization probes to detect naturally-occurring genetic mutation s in the GPCR gone and thereby to determine whether a subject with the mutation is at risk for a disorder caused by the mutation. Mutations include deletion, addition, or substitution of one or more nucleotides in the gone, chromosomal rean-angement, such as inversion or transposition, modification of genomic DNA, such as aberrant methylation patterns or changes in gone copy number, such as amplification. Detection of a mutated form of the GPGR gone associated with a dysfunction provides a diagnostic tool for an active disease or susceptibility to disease when the disease results from overexpression, underexpression, or altered expression of a GPCR protein.
Individuals carrying mutations in the GPCR gene can be detected at the nucleic acid level by a variety of techniques. Figure 3 provides information on a SNP variant, G
1711 T, which has been found in a gene encoding the GPCR protein of the present invention. The change in the amino acid sequence caused by this SNP is indicated in Figure 3 and can readily be determined using the universal genetic code and the protein sequence provided in Figure 2 as a reference. As indicated by the data presented in Figure 3, the map position was determined to be on chromosome 6 by radiation hybrid mapping. Genomic DNA can be analyzed directly or can be amplified by using PCR prior to analysis. RNA or cDNA can be used in the same way. In some uses, detection of the mutation 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 el nl., Scierzce 2-11:10'77-1080 (1988); and Nakazawa et al., PIVAS 91:360-364 (1994)), the latter of which can be particularly useful far detecting point mutations in the gene (see Abravaya et girl., Nzxcleic.~cic~s Res~ 23:675-682 ( I 995)).
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 of the sample, contacting the nucleic acid sample with one or mare primers which speciFcally hybridize to a gene under conditions such that hybridization and amplification of the gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample, Deletions and insertions can be detected by a change in sine ofthe amplified product compared to the narnlal genotype. faint mutations can be identifed toy hybridizing amplified DNA to normal RNA or antisense DNA sequences.
Alternatively, mutations in a GPC'R gene can be directly iclentificd, for example, by alterations in restriction enzyrtne digestion patterns determined by gel electrophoresis.
Further, sequence-specihic ribozymes ~U.S. Patent No. 5,198,531 ) can be used to score for tho presence of specific mutations by development or loss of a ribozyme cleavage site. Perfectly matched sequences can be distinguished from mismatched sequences by nuclease cleavage digestion assays or by differences in melting temperature.
Sequence changes at specific locations can also be assessed by nuclease protection assays such as RNase and S I protection or the chemical cleavage method. Furthermore, sequence differences between a mutant GPCR gene and a wild-type gene can be determined by direct DNA
sequencing. A variety of automated sequencing procedures can be utilized when performing the diagnostic assays (Naeve, C.W., (1995) BiotechzZigzres 79:418), including sequencing by mass spectrometry (see, e.g., PCT International Publication No. WO 91/16101; Cohen et al., .~dv.
C"hroznatogr. 36:127-162 ( 1996); and Griffin et al.,,~lppl. BioclZem.
Biol~chzzol. 38:17-159 (1993)).
Other methods for detecting mutations in the gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNAIRNA or RNAIDNA
duplexes I S (Myers et al., Sciezzce 23a:12~12 (1985)); Cotton et czl., PNAS 8~:~1397 (1988); Saleeba et al., Meth.
Erzymol. 217:286-295 ( 1992)), electrophoretic mobility of mutant and wild type nucleic acid is compared (Orita et al., Plf~l~S' c~6:2766 (1989); Cotton et al., MZttal.
Rc,S~. 285:125-1 ~I~l ( 1993); and Hayashi e1 al., Genel. anal, Tech. ~lppl. 9:73-79 (1992)), and movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (Myers el al., Natazre 313:95 (1985)). Examples of other techniques for detecting point mutations include selective oligonucleotide hybridization, selective amplif canon, and selective primer extension.
The nucleic acid molecules are also useful for testing an individual for a genotype that while not necessarily causing the disease, nevertheless affects the treatment modality. Thus, the nucleic acid molecules can be used to study the relationship between an individual's genotype and the individual's response to a compound used for treatment {pharmacogenomic relationship).
Accordingly, the nucleic acid molecules described herein can be used to assess the mutation content of the GPCR gene in an individual in order to select an appropriate compound or dosage regimen for treatment. As illustrated in Figure 3, 0,171 1T is a known SNP variant.
Thu s nucleic acid molecules displaying genetic variations that affect treatment provide a diagnostic target that can be used to tailor treatment in an individual.
Accordingly, the production of recombinant cells and animals containing these polymorphisms allow effective clinical design of treatment compounds and dosage re«imens.
~l The nucleic acid molecules are thus useful as an tiscnse constructs to control GP CR gene expression in cells, tissues, and organisms. A DNA antisen se nucleic acid molecule is designed to be complementary to a region oFthe gene involved in transcription, preventing transcription and hence production of GPCR protein. An antisense RNA or DNA nucleic acid molecule would hybridize to the mRNA and thus block translation of mRNA into GPCR protein.
Alternatively, a class of a~~tisense molecules can be used to inactivate mRNA
in order to decrease expression of GPCR nucleic acid. Accordingly, these molecules can treat a disorder characterized by abnormal or undesired GPCR nucleic acid expression. This technique involves cleavage by means of ribozymes containing nucleotide sequences complementary to one or more regions in the mRNA that attenuate the ability of the mRNA to be translated.
Possible regions include coding regions and particularly coding regions corresponding to the catalytic and other functional activities of the GPCR protein, such as ligand binding.
The nucleic acid molecules also provide vectors For gene therapy in patients containing cells that are aberrant in GPCR gene expression. Thus, recombinant cells, which include the patient's cells that have been engineered ex vivo and returned to the patient, are introduced into an individual where the cells produce the desired GPCR protein to treat the individual.
The invention also encompasses kits ~or detecting the presence of a GPCR
nucleic acid in a biological sample. Experimental data as provided in Figure 1 indicates that GPCR proteins of the present invention are expressed in humans in the stomach, placenta, kidney, skeletal muscle, liver, bone marrow, and thymus. Specifically, a virtual northern blot shows expression in the stomach, In addition, PCR-based tissue screening panels indicate expression in placenta, kidney, skeletal muscle, liver, bone marrow, and thymus tissue. For example, the kit can comprise reagents such as a labeled or labelable nucleic acid or agent capable of detecting GPCR nucleic acid in a biological sampled means for determining the amount of GPCR nucleic acid in the sample;
and means For comparing the amount oFGPCR nucleic acid 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 GPCR protein mRNA or DNA.
Nucleic Acid Arrays l~he present invention Further provides nucleic acid detection kits, such as arrays or microarrays of nucleic acid molecules that are based on the sequence inFormation provided in higures 1 and 3 (SEQ ID NUS:I and i j~

As used herein "Arrays' or "Microarrays" refers to an array oFdistinet polynucleotides or oligonucleotides synthesized on a substrate, such as paper. nylon or other type oFmembrane, Intel', Glllp, glaSS Slide, Or any Othel' SLlltable SOlICi SLIpp01't. Ill 017e e171bOd1111ent, the 1711C1'Oal'1'ay IS
prepared an~i used according to the methods described in U'S Patent 5,837,832, Chee et al., fC"F
application W09511 1905 (Chee et al.), Lockhart, D. .l. et al. ( 1996; Nat.
Biotech. l~: I675-1680) and Schena, M. et al. (1996; Nroc. Natl. Acad. Sci. 93: 1061-10619), all of which are incorporated herein in their entirety by reference. In other embodiments, such arrays are produced by the methods described by Brown et. al., US Patent N'o. 5,807,522.
The microarray or detection kit is preferably composed of a large number of unique, single-stranded nucleic acid sequences, usually either synthetic antisense oligonucleotides or fragments of cDNAs, fixed to a solid support. The oligonucleotides are preferably about 6-60 nucleotides in length, more preferably 15-30 nucleotides in length, and most preFerably about 20-25 nucleotides in length. Far a certain type of microarray or detection kit, it may be preferable to use oligonucleotides that are only 7-20 nucleotides in length. The microarray or detection kit may contain oligonucleotides that cover the known 5', or 3', sequence, sequential oligonucleotides which cover the full length sequence; or unique oligonucleotides selected from particular areas along the length of the sequence. Polynucleotides used in the microarray or detection kit may be oligonucleotides that are specific to a gene or genes of interest.
In order to produce oligonueleotides to a known sequence for a microarray or detection kit, the genes) of interest (or an ORF identified from the contigs of the present invention) is typically exan7ined using a computer algorithm which starts at the 5' or at the 3' end of the nucleotide sequence. Typical algorithms will then identify aligomers of defined length that are unique to the gene, have a GC content within a range suitable for hybridization, and lack predicted secondary structure that may interfere with hybridisation. In certain situations it may be appropriate to use pairs oFoligonucleotides on a microarray or detection kit, The "pairs" will be identical, except for one nucleotide that preferably is located in the center of the sequence, The second oligonucleotide in the pair (mismatched by one) serves as a control. The number of oligonucleotide pairs may range from two to one million. The oligomers are synthesized at designated areas on a substrate using a light-directed chemical process. The substrate may be paper, nylon or other type oFmembrane, Filter, chip. glass slide or any other suitable solid support.
In another aspect. an oligonucleotide may be synthesized on the surface ol~
the substrate by using a chemical coupling procedure and an inkaet application apparatus, as described in PC'h application W095/251 1 16 (Baldescllweiler et al.) which is incorporated herein in its entirety by reference. In another aspect, a "gridded" array analogous to a dot (or slot) blot may be used to arrange and linl. cDNA fragments or oligonucleotides to the surlace ofa substrate using a vacuum system, thermal, UV, mechanical or chemical bonding procedurta. An array, such as those described above, may be produced by hand or by using available devices slot blot or dot blot apparatus), materials (any suitable solid support), and machines (including robotic instruments), and may contain 8, 24, 96, 381, 1536, 6141 or more oligonucleotides, or any other number between two and one million which lends itself to the eCFicient use of commercially available instrumentation.
In order to conduct sample analysis using a mieroarray or detection kit, the RNA or DNA
from a biological sample is made into hybridization probes. The mRNA is isolated, and eDNA is produced and used as a template to make antisense RNA (aRNA). The aRNA is amplified in the presence of fluorescent nucleotides, and labeled probes are incubated with the microarray or detection kit so that the probe sequences hybridize to complementary oligonucleotides of the mieroarray or detection kit. Incubation conditions are adjusted so that hybridization occurs with precise complementary matches or with various degrees of less complementarity.
After removal of nonhybridized probes, a scanner is used to determine the levels and patterns of fluorescence.
The scanned images are examined to determine degree of complementarity and the relative abundance of each oligonucleotide sequence on the microarray or detection kit.
The biological samples may be obtained from any bodily fluids (such as blood, urine, saliva, phlegm, gastric juices, etc.), cultured cells, biopsies, or other tissue preparations. A
detection system may be used to measure the absence, presence, and amount of hybridization for all of the distinct sequences simultaneously. This data may be used for large scale correlation studies on the sequences, expression patterns, mutations, variants, or polymorphisms among samples.
Using such arrays, the present invention provides methods to identify the expression of the GPCR proteins/peptides of the present invention. In detail, such methods comprise incubating a test sample with one or more nucleic acid molecules and assaying for binding of the nucleic acid molecule with components within the test sample. Such assays will typically involve arrays comprising many genes, at least one ofwhich is a gene of the present invention and or alleles of the GPCR gene of the present invention. H figure 3 provides information on a SNP variant, G 171 l~I~, which has been found in a gent' encoding the GPCR
protein of the present invention. 'I°he change in the amino acid sequence caused by this SNP
is indicated in Figure 3 anti can readily be determined using the universal genetic code and the protein sequence ~l~l provided in higure ? ~ts a reference.
conditions Cor incubating a nucleic acid molecule with a test sample vary.
Incubation conditions depend on the Format employed in the assay, the detection methods employed, and the type and nature ol'the nucleic acid molecule used in the assay, One skilled in the ~trt will recognize that any one of the commonly available hybridization, amplification ar array assay formats can readily be adapted to employ the novel fragments of the 1-Iuman genome disclosed herein. Examples of such assays can be found in shard, T> ~ln Introdz~r~lion to Radioitnmztzzoas,scrv rrr~d Related Techniqztes, Elsevier Science Publishers, Amsterdam, The Netherlands ( 1986); Bullock, G. R. et al., I'echniqztes in Imtzzunoc~lochemi,slz_la, Academic Press, Orlando, FL Vol. 1 (1 982), Vol. 2 (1983), Vol. 3 (1985); Tijssen, P., Pz°aclice czvtd Theozy of'Enzynae Inamztnoassays~ Lal~onalo~y Techniques itz Biochenaistjy crud tLlolecztlar Biology, Elsevier Science Publishers, Amsterdam, The Netherlands ( 1985).
The test samples of the present invention include cells, protein or membrane extracts of cells. The test sample used in the above-described method will vary based on the assay format, nature of the detection method and the tissues, cells or extracts used as the sample to be assayed.
Methods for preparing nucleic acid extracts or of cells are well known in the art and can be readily be adapted in order to obtain a sample that is compatible with the system utilized.
In another embodiment of the present invention, kits are provided which contain the necessary reagents to carry out the assays of the present invention.
SpeciFcally, the invention provides a compartmentalized kit to receive, in close confnement, one or more containers which comprises: (a) a first container comprising one of the nucleic acid molecules that can bind to a fragment of the Human genome disclosed herein; and (b) one or more other containers comprising one or more of the following: wash reagents, reagents capable of detecting presence of a bound nucleic acid.
2S In detail. a compartmentalized kit includes any kit in which reagents are contained in separate containers. Such containers include small glass containers, plastic containers, strips of plastic, glass or paper, or arraying material such as silica. Such containers allows one to efFtciently transfer reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated, and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another. Such containers will include a container which will accept the test sample, a container which contains the nucleic acid probe. containers which contain wash reagents (such as phosphate buffered saline, ~I'ris-buffers, etc.j, and containers which contain the reagents used to detect the bound probe. Une skilled in =lj the art will readily recognize that the previously unidentified GPCR genes of the present invention can be routinely identitied using the sequence information disclosed herein can be readily incorporated into one of the established kit formats which are well known in the art, particularly expression arrays.
Vectorslhost cells The invention also provides vectors containing the nucleic acid molecules described herein.
The term "vector" refers to a vehicle, preferably a nucleic acid molecule, which can transport the nucleic acid molecules. When the vector is a nucleic acid molecule, the nucleic acid molecules are covalently linked to the vector nucleic acid. With this aspect of the invention, the vector includes a plasmid, single or double stranded phage, a single or double stranded RNA or DNA viral vector. or artificial chromosome, such as a BAC, PAC, YAC, OR MAC.
A vector can be maintained in the host cell as an extrachromosomal element where it replicates and produces additional copies of the nucleic acid molecules.
Alternatively, the vector may integrate into the host cell genome and produce additional copies of the nucleic acid molecules when the host cell replicates.
The invention provides vectors for the maintenance (cloning vectors) or vectors for expression (expression vectors) ofthe nucleic acid molecules. The vectors can function in procaryotic or eulcaryotic cells or in both (shuttle vectors).
Expression vectors contain cis-acting regulatory regions that are operably linked in the vector to the nucleic acid molecules such that transcription of the nucleic acid molecules is allowed in a host cell. The nucleic acid molecules can be introduced into the host cell with a separate nucleic acid molecule capable of affecting transcription. Thus, the second nucleic acid molecule may provide a trans-acting factor interacting with the cis-regulatory control region to allow transcription of the nucleic acid molecules from the vector. Alternatively, a trans-acting factor may be supplied by the host cell. Finally, a trans-acting factor can be produced from the vector itself. It is understood, however, that in some embodiments, transcription and/or translation of the nucleic acid molecules can occur in a cell-free system.
The regulatory sequence to which the nucleic acid molecules described herein can be operably linked include promoters for directing mRNA transcription. These include, but are not limited to. the left promoter from bacteriophage 7~, ih a lac, TRP, and TAC
promoters from l;. c~~~ll, the early and late promoters from SV~10, the CMV iwmediate early promoter, the ndeno virus early -I(>

and late promoters, and retrovirus long-terminal repeats.
In addition to control regions that promote transcription, expression vectors may also include regions that modulate transcription. such as repressor binding sites and enhancers.
Examples include the SV~IO enhances, the cytomegalovirus immediate early enhances, polyoma enhances, adenovirus enhancers, and retrovirus LTR enhanoers.
In addition to containing sites Far transcription initiation and control, expression vectors can also contain sequences necessary for transcription termination and, in the transcribed region a ribosome binding site for translation. Other regulatory control elements For expression include initiation and termination colons as well as polyadenylation signals. The person of ordinary skill in the art would be aware of the numerous regulatory sequences that are useful in expression vectors.
Such regulatory sequences are described, for example, in Sambrook el al., Molecatlaz~ Cloz2iz7g: ,~
Labouatoz~ml~lcrzzztal. Zna', el., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1989).
A variety of expression vectors can be used to express a nucleic acid molecule. Such vectors include chromosomal, episomal, and virus-derived vectors, for example vectors derived from bacterial plasmids, from bacteriophage, from yeast episomes, from yeast chromosomal elements, including yeast artificial chromosomes. from viruses such as baculoviruses, papovavin.tses such as SV40, Vaccinia viruses, adenoviruses, poxvimses, pseudorabies viruses, and retroviruses. Vectors may also be derived from combinations of these sources such as those derived from plasmid and bacteriophage genetic elements, eg. cosmids and phagemids.
Appropriate cloning and expression vectors for prokaryotic and eukaryotic hosts are described in Sambrook et czl., tl~lolecazlar Clonizzg: ~I Laboz~atozy ~Iflcznz~czl. ?zzd. el., Cold Spring Harbor Laboratory Press, Gold Spring Harbor, NY, ( I 989).
The regulatory sequence may provide constitutive expression in one or more host cells (i.e.
tissue specific) or may provide for inducible expression in one or more cell types such as by temperature, nutrient additive, or exogenous factor such as a hormone or other Iigand. A variety of vectors providing for constitutive and inducible expression in prokaryotic and eukatyotic hosts are well known to those of ordinary skill in the art.
The nucleic acid molecules can be inserted into the vector nucleic acid by well-known methodology. Generally, the DNA sequence that will ultimately be expressed is joined to an expression vector by cleaving the DNA sequence and the expression vector with one or more restriction enzymes and then ligating the fragments together. Procedures for restriction enzyme di~,estion and ligation are well known to those of~ordinary skill in the art.
~17 The vector containing the appropriate nucleic acid molecule can be introduced into an appropriate host cell Cor propagation or expression using well-known techniques. Bacterial ells include, but are not limited to, E. coli, ~S'lrc~hlona~~oc~.v, and ,~'ulmor~~lla llaphimurimn. I=,ukasyo tic cells include. but are not limited to, yeast, insect cells such as l~rr~.~~ohl7ilcr, animal culls such as COS and CI-10 cells, and plant cells.
As described herein, it may be desirable to express the peptide as a fusion protein.
Accordingly, the invention provides Fusion vectors that allow for the producfion of the peptides.
Fusion vectors can increase the expression of a recombinant protein, increase the solubility of the recombinant protein, and aid in the purification of the protein by acting for example as a ligand for affinity purification. A proteolytic cleavage site may be introduced at the junction ofthe fusion moiety so that the desired peptide can ultimately be separated from the fusion moiety. Proteolytic enzymes include, but are not limited to, factor Xa, tl-trombin, and enterokinase. Typical fusion expression vectors include pGEX (Smith et czl., Gerac~ 67:31-40 (1988)), pMAL
(New England Biolabs, Beverly, MA) and pRITS (Phatmacia, Piscataway, N.1) which fssse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein. Examples of suitable inducible non-fission E. coli expression vectors include pTrc (Amann e! al., GeraL 6:301-315 (19$8)) and pET 1 Id (Studier et al., Genre Expressiorz Technology; MeClZOCIs if2 E>?zynzology 18.5:60-89 ( 1990)).
Recombinant protein expression can be maximized in a host bacteria by providing a genetic background wherein the host cell has an impaired capacity to proteolytically cleave the recombinant protein. (Gottesman, S., Gene Expres.~ion Technolog~~= ~l~lelhocls in EnzJmzolo~~ 185, Academic Press, San Diego, California (1990) 1 19-128). Alternatively, the sequence of the nucleic acid molecule of interest can be altered to provide preferential codon usage for a specific host cell, for example E. ioli. (Wada et crl., Nztclei~.~lcicls Rr~s. X0;2111-.2118 (1992)).
The nucleic acid molecules can alsa be expressed by expression vectors that are operative in yeast. Examples of vectors far expression in yeast e.g., ~.S'. oer~eui,~~iae include pYepSecl (Baldari, et crl., E~'IrIB(),1 6:229-23d (1987)), pMFa (Kurjan e! crl., Cell 3l):933-93(1982)), pJRY88 (Scht,sltz ei al., Ger~c 5-l:1 13-123 ( 1987)), and pYES2 (Invitrogen Cot~aoration, San Diego, CA).
The nucleic acid molecules can also be expressed in insect cells using, for example, baculovirus expression vectors. Baculovirus vectors availahle for expression of proteins in cultured insect cells (e.g., Sf 9 cells) include the pAc series (Smith r.?l rrl., ~
lal. C'i.~ll Biol. 3;21 ~6-? 1 d5 (1983)) and the pVI. series (Irucklowcalrrl" ln~r~lpv I ~C1:31-39 X19$9)).
In certain embodiments ofthe invention, the nuclefc acid molecules described herein are expreSSed 111 Illilllllllallall Ce115 LIS111g 111a1171Tlallan expl'e5S1o11 veCtOT'S. I;XanlpleS of'lTIamI7lallal7 expression vectors include pCDM$ Geed. B. JVolan'e 329:84( 1987)) and plvl'I"?PC (Kaufman t~~l crl..
~~113(),I. x;187-l~)5 (1987)).
ThL: etpression vectors listed herein are provided by way of example only ofthe well-known vectors Available to those of ordinary skill in the art that would be useful to express the nucleic acid molecules. '1"he person of ordinary skill in the art would be aware ofother vectors suitable for maintenance propagation or expression of the nucleic acid molecules described herein.
These are found for example in San'lbrook, J., Fritsh, E. F., and Maniatis, T.
Moleczzlar Cloning; A
Laboratory thlcrnztcrl. 2nd, eo', Cold Spring harbor Laborafozy, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.
The invention also encompasses vectors in which the nucleic acid sequences described herein are cloned into the vector in reverse orientation, but operably linked to a regulatory sequence that permits transcription of antisense RNA. Thus, an antisense transcript can be produced to all, or to a portion, of the nucleic acid molecule sequences described herein, including bath coding and non-coding regions. Expression of this antisense RNA is subject to each of the parameters described above in relation to expression of fhe sense RNA (regulatory sequences, constitutive or indueible expression, tissue-specific expression).
The invention also relates to recombinant host cells containing the vectors described herein.
Host cells therefore include prokaryotic cells, lower eukaryotic cells such as yeast, other eukaryotic cells such as insect cells, and higher eukaryotic cells such as mammalian cells.
The recombinant host cells are prepared by introducing the vector constructs described herein into the cells by techniques readily available to the person afordinary skill in the art. These include, but are not limited to, calcium phosphate transfection, DEAF-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, lipofection, and other techniques such as those found in Sambrook, ~l al.
(Molectzlcrr Cloning= ~
hczborcrloz~y t~lczzmcrl. Zntl, rcl , Cold Spz'ing Hcrz~boz~ l~crborcrtoz~~, Cold Spring I Iarbor Laboratory Press, Gold Spring I-larbor, NY, 1989.
I-lost cells can contain more than one vector. Thus, different nucleotide sequences can be introduced on different vectors ofthe same cell. Similarly, the Nucleic acid molecules can be introduced either alone or with other nucleic acid molecules that are not related to the nucleic acid molecules such as those providing traps-acting factors for expression vectors.
When more than one vector is introduced into a cell, the vectors can be introduced independently, co-introduced or joined to the nucleic acid molecule vector.
:-l~) In the case of bacteriophage and viral vectors. these can be introduced into cells as packaged or encapsulated virus by Standard procedures for infection and transduction.
Viral vectors can be replication-competent or replication-defective. In the: case in which viral replication is defective, repllcatlon Wlll L>CCllr 111 110St Ge115 pI~OVld111g 111I1Ct1()ll5 that Cplllplelnellt the deleCIS.
Vectors generally include selectable markers that enable the selection of the subpopulation of cells that contain the recombinant vector constructs. The marker can be contained in the same vector that contains the nucleic acid molecules described herein or may be on a separate vector.
Markers include tetracycline or ampicillin-resistance genes for prokaryotic host cells and dihydrofolate reductase or neomycin resistance for eukaryotic host cells.
However, any marker that provides selection for a phenotypic trait will be effective.
While the mature proteins can be produced in bacteria, yeast, mammalian cells, and other cells under the control of the appropriate regulatory sequences, cell- free transcription and translation systems can also be used to produce these proteins using RNA
derived from the DNA
constructs described herein.
Where secretion ofthe peptide is desired, which is difficult to achieve with multi-transmembrane domain containing proteins such as GPCRs, appropriate secretion signals are incorporated into the vector. The signal sequence can be endogenous to the peptides or heterologous to these peptides.
Where the peptide is not secreted into the medium, which is typically the case with GPGRs, the protein can be isolated from the host cell by standard disruption procedures, including freeze thaw, sonication, mechanical disruption, use of lysing agents and the like.
The peptide can then be recovered and purified by well-known purification methods including ammonium sulfate precipitation, acid extraction, anion or cationic exchange chromatography, phosphocellulose chromatography, hydrophobic-interaction chromatography, affinity chromatography, hydroxylapatite chromatography, lectin chromatography> or high performance liquid chromatography.
It is also understood that depending upon the host cell in recombinant production of the peptides described herein. the peptides can have various glycosylation patterns, depending upon the cell, or maybe non-glycosylated as when produced in bacteria. In addition, the peptides may include an initial modified methionine in some cases as a result of a host-mediated process.
Uses of vectors and host cells SO

fhe recombinant host calls expressing the peptides described herein have a variety ofrrses_ I~irst, the cells are useful For producing a GPCR protein or peptide that can be l-urther purit3ed to produce desired amounts of GP CR protein or (i~agments. "I~hus, host cells containing expression vectors are usefr,tl far- peptide pr~oductiot~.
Host cells are also useful for conducting cell-based assays involving the GPCR
protein or GPCR protein fragments, such as those described above as well as other formats known in the ar-t.
Thus, a recombinant host cell expressing a native GPCR protein is useful for assaying compounds that stimulate or inhibit GPCR protein function.
Host cells are also useful for identifying GPCR protein mutants in which these functions are affected. If the mutants naturally occur and give rise to a pathology, host cells containing the mutations are useful to assay compounds that have a desired effect an the mutant GPCR protein (for example, stimulating or inhibiting function) which may not be indicated by their effect on the native GPCR protein.
Genetically engineered host cells can be further used to produce non-human transgenic animals. A transgenic animal is preferably a mammal, for example a rodent, such as a rat or mouse, in which one or more of the cells of the animal include a transgene. A
transgene is exogenous DNA
which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal in one or more cell types or tissues of the transgenic animal. These animals are useful for studying the function of a GPCR protein and identifying and evaluating modulators of GPCR protein activity. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, and amphibians.
A transgenic animal can be produced by introducing 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. Any of the GPCR protein nucleotide sequences can be introduced as a transgene into the genome of a non-human animal, such as a mouse.
Any of the regulatory or other sequences useful in expression vectors can form part of the transgenic sequence. This includes intronic sequences and polyadenylation signals, if not already included. A tissue-specific regulatory sequences) can 6e operably linked to the transgene to direct expression of the GPCR 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 N'os. ~1,73G.8G6 and X1,870,009, both by Leder c1 crl., (.I'.S. Patent No.
X1,873,191 by Wagner cl ctl. and in I-Iogan, f3_. ~'I~Itrrliprtlcrtiry~l the ltlott.5r Fntbruu, (Cold spring ~l Harbor hahoratory Press, Cold Spring I-larbor, N.Y.. 1986). Similar methods are used for production of other transgenic animals, A transgenic lounder animal can be identified based upon the presence of the transgene in its gen ome and/or expression of transgenic mRNA in tissues or cells of the animals. A transgenic Founder animal can then be used to breed additional animals carrying the transgenc. Moreover, transgenic animals carrying a transgene can further be bred to ocher transgenic animals carrying other transgenes. A transgenic animal also includes animals in which the entire animal or tissues in the animal have been produced using the homologously recombinant host cells described herein.
In another embodiment, transgenic non-human animals can be produced which contain selected systems that allow for regulated expression of the transgene. One example of such a system is the orvlloxP recombinase system ofbacteriophage PI. For a description ofthe cf~elloxP
recombinase system, see, e.g., Lakso et al. PN~1S $9;6232-6236 ( 1992).
Another example of a recombinase system is the FLP recombinase system of S, cerevisiae (O'Gorman et al. Science 21:1351-1355 (1991). If a crelloxP recombinase system is used to regulate Expression of the transgene, animals containing transgenes encoding both the C're recombinase and a selected protein is required. Such animals can be provided through the consttltction 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 of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut, I. e1 al. lVatzn~e 385:810-813 (1997) and PCT
International Publication Nos. WO 97/07668 and WO 97107669. 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 G~ phase.
The quiescent cell can then be fused, e.g., tlwough the use of electrical pulses, to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated. The reconstructed oocyte is then cultured such that it develops to morula or blastocyst and then transferred to pseudopregnant female foster animal. The offspring born of this female foster animal will be a clone of the animal from which the cell, e.g.. the somatic cell, is isolated.
Transgenic animals containing recombinant cells that express the peptides described herein are useful to conduct the assays described herein in an ire uiuo context.
Accordingly, the various physiological factors that are present irmivo and that could effect ligand binding, GPCR protein activation, and signal transduction, may not be evident from in vulro cell-Ii-ee or cell-based assays.
Accordingly, it is useful to provide non-human tt°ans;~t~nic animals to assay irr uiv>U GPCR protein function, including ligand interaction, the effect oi~spocific mutant GfCR
proteins on GPCR protein Function and ligand interaction. and the effect of chimcric ~iPCR proteins. It is also possible to assess the ~I~fect of mill mutations. that is mutations that substantially or completely eliminate one or more Cif'C'R protein functions.
All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing From the scope and spirit of the invention. Although the invention has been described in connection with specil"ic preferred embodiments, it should be understood that tl~e invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described modes for carrying out the invention which are obvious to those skilled in the field of molecular biology or related fields are intended to be within the scope of the Following claims.
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<141> 2aa1-la-24 <15a> a~/~g~,8~1 <15~.> 2a0a-1a-24 <1~0> x91781,559 <1~7~> 2001-13-a~
<160> 6 <17a> FastSEQ ~o~ Wzndaws Vers9.on 4.a <21a> 1 <211> 2082 <212> I7N'A
<~G1~> I-~IkIT~ail <9aa> 1 taaataacag cgttaatgag cagcaattca tccctgctgg tggctgtgea gctgtgctae 6a gcgaacgtga atgggtcctg tgtgaaaatc cccttctcgc cgggatccrg ggtgattctg 22a tacatagtgt ttggctttgg ggctgtgctg gctgtgtttg gaaacctcct ggtgatgatt 18a tcaatcctcc atttcaagca gctgcactct ccgaccaatt ttctcgttgc etctctggcc 24a tgcgctgatt tcttggtggg tgtgactgtg atgcccttca gcatggtcag gacggtggag 3aa agctgctggt attttgggag gagtttttgt actt~ccaca cctgctgtga tgtggcattt 36a tgttactctt ctctctttca cttgtgcttc atctccatcg acaggtaeat tgcggttact X20 gaccccctgg tctatcctac caagttcacc gtatctgtgt caggaatttg catcagcgf':g 48a tcctggatcc tgcccctcat gtacagcggt gctgtgttct acacaggtgt ctatgacgat 54a gggctggagg aattatctga tgccctaaac tgtataggag gttgtcagac cgttgtaaat 6aa caaaactggg tgttgacaga ttttctatcc ttctttatac ctacctttat tatgataatt 66a ctgtatggta acatatttct tgtggctaga cgacaggcga aaaagataga aaatactggt °~2a agcaagacag aatcatccte agagagttac aaagccagag tggccaggag agagagaaaa ~8a gcagctaaaa ccctgggggt cacagtggta gcatttatga tttcatggtt accatatagc 840 attgattcat taattgatgc ctttatgggc tttataaccc ctgectgtat ttatgagatt 9aa tgctgttggt gtgcttatta taactcagcc atgaatcctt tgatttatgc tttattttac 960 ccatggttta ggaaagcaat aaaagttatt gtaactggtc aggttttaaa gaacagttca ~.a2Q
gcaaccatga atttgttttc tgaacatata taagcagttg gatagacgaa gttcaggata ~a8a cc 1x82 <~~~~>
<~11> 345 <~12> ~Ft"I' <213> F~~~nan ~ eI t~~ G?'~
Met Sex ~k=~ Asn SRr Sir Lxm~ ~~u vat. Ala Vat ~J! ~ ~~t~ ~y~ "I"yr Ana 1 5 1~ n 1 Asri VaJ! Asn fly Se ~ ~ys Va ~. Toys :T'~l~.e Lara ~k~e her F'r..~ G~.~~~ ~4r Arg V~~ ~ Ll~.t~ LF7tu "F'y~- :I: ll~ Va 1. ~"fux~ c~ l ~,,~ Fte ~~'1y ~~l~.a ~.j;~.1 L~e~,s AF-~ ~~a 1i. I~)~~

~.~y A:.:n t~eu L,eu VaJ Met. Ll.e S«r L1~ l'~~s ~fi~s Phe L~ys Gin 1."e~ H.a-s 50 rj"~ ~rJ
Ser ~~ro "T'~v~- Asrr T-'Efl~--~ L"e.e~ V".i~_ Ana 5er P~eta Al~.a wys Ala Asy L"hca ~~e:c.~
JL~ ~~5 80 Vd~. ~Ly V~.~L Tl-rr V~i~ M~~t- ~~rca I~he ;.~;ez~~ Met Va:~ Arg Thr Vxl~. GLu S~r-~ (1 Cys Trp Tyr Phe G1y Arty Ser ~~'~ie Cys "1'hr Phe His 'T'hr Sys ~ys Asp 1g0 1! 0'~ 110 Val A.La Phe Cys "L'yr Ser Ser Lieu Phe Hi.s ~eu Cys Phe I1e Ser :Ck.e Asp Arg Tyr Tle Ala Va1 'I"hr Asp Pro Lieu Val Tyr Pro 'Fhr Lys Phe Thr Va1 Spr Val Ser Gly I1e Cys Tle Ser val Ser Trp 11e Leu Pro Leu Met Tyr Ser Gly Ala Val Phe Tyr Thr fly Val Tyr Asp Asp fly 165 1.70 1~5 T~eu ~J! a flu Leu Ser Asp Ala Leu Asn ~'ys T1e fly ~1y ~'ys ~ln Thr Val Va1 Asn ~ln Asn Trp Val Leu Thr Asp Phe Leu Ser Phe Phe Ile Pro Thr Phe Tle Met Tle lle Leu Tyr fly Asn ale Phe Leu Val Ala 210' 215 220 Arg Arg ~1n Ala Lys Lays Tle g1u Asn Thr fly Ser Lys Thr Slu Ser 225 230 235 24'0 Ser Ser Glu Ser Tyr Lys Ala Arg Val Ala Arg Arg Glu Arg Lys Ala Ala Lys 'Fhr Leu ~1y Val Thr Val Val Ala Phe Met lle Ser Trp heu 260 265 2~g Pro Tyr Ser Lle Asp Ser Leu 11e Asp A1a Phe Met Gly Phe Tle Thr Pro Ala Cys 21e Tyr Glra lle Cys Cys Trp Cys Ala Tyr Tyr Asn Ser 29Q 295 30(~
Ala Met Asn Pro Leu lle Tyr A~.a T~eu Phe Tyr Pro Trp Phe Arg ~ys 3a5 .~~.~ ~1~ 32a Ala 11e ~ys Val lle Val Thr G1y Gln Va2 heu ~ys Asn Ser Ser Ala Thr Met Asn ~eu Phe Ser flu His Ile <210> 3 <211> 994 <212> ~1~1A
<213> Human <400> 3 ctt~aggaag acaataatat aataataaca atattttctt cactctgcag tgtctttaca 60 ttcoagggtt gggaacatta ctgaggattc l~Gtt~ccatt ttccagtttc otgtt~atta 1.2Q
tt~ttatttt tttgactgct tttagcatog ggagcaoaaa ggccagteac oaggaattgc X80 aaaoaaatgc gtagtcagag agagagggct cactgcc~cat ttgtc~atgtg gatgcagaca s40 cattg~agat gtgttcc~ag taa~.aatgto ttgagaagag gactggtctt tcca:~oagca 38L7 tctoagaaat gcGggtgtgt otaaaeagoa tgtcgttctt taatg~ttto atgcaatata 3~0 ttttat~caa~~ ctcaagtt~c ~ctcactat.g tattataata atttctgctt gttggtaao~ 420 aaL:g~agatg gaaaattgat tcttaacaga agagaaagag ocaagtattg atg~-tta~ta ~~80 tttaoa~~ct attgtat~-tt tgtaaoaaaa a~oogg9tgg ~-taag~tai:g attgggaa~a 540 agggaatggt t~:aagt~t~at gca~:taagga aaaaoaaato tttgg~,~taa aa~aataatg 6Q~0 ataata"~aa~ ttaatataga gtagagac~~t gttttgtaga ataactt~tr~~ i~agt~aatrsac~ 8~0 tgf~tgaaaat aatcatae L a gtt~a~acug "~~~oactarag ggatt"_~.:at~"
gar.~r~c~.atttt 'DLO
c~ccattr~aaf~ Ocattt~~-~ t ac~etaa~~agg a,~t'~.~at~tt taagr~~gg~:a at~~~ragg~tk.~~r 7~0~
gataaca~l'-~~~ G~~~a~~ag.~fi'_aa ~~~~~~caag ~~-gt~ ~:~ryt~ntcagwt e~~ataa~
~=ac~ ~at:t->-r-~~~E-afi-"_-a~,r"~~3~z~~k"~t'~t gcaragagaa ar:~tcaaaa":~'~ f~aaaaa~'~aaa atatgaaagq atatttaaaa ~~g t:"-aa.~a'aet~~a ttttatcaaa tt aagacfw.~~ _~~~"~acattta t~agti_".~aaa cast".~tccaa ~~Ct t'aat-"-t:t~y~t_g caatataatt ttt'4gt~rt_~ant- t t E-~nG-t~ttgt cat'-~aa"~aEk tgggatttaG- lfon.
aat ~:3,maat:q gaaacttgaa aa'~ti-at at t- r3"~r~ctat aata f_~.~tgal~~at<t tcctct<qgca 1~.~8r"}
t_"~~a~.s.ai.ttta~-+a tatgtgtttt ttLccc~rt~a"~"~ a'~t"~ac~L=gaaa ~rtt_"~a".~".~adC~a at~=ragtaf'_l_ lb4r~
i-t_t t. Yat:",t~rt~ a at":.aacaaqg a~~e3aa~~"-t t' ~- 1' ~~"°atot_gta a:~t'~aaC-actc_~a ttal_gagc~~r~ l2gt~
caat~"°a+~r~c ctgctggtgg ctgtgc=ag":f'= gt=qctacgcg aat°_gt~~a:~tc3 ggtcctgtgt ~
g,~a~tatLeccc ttctcgccgg qatcccqgg6_ gattctgtac atagtgtt tg gctttggggc L3; E~
I~"gtgrwtggct gtgtttggaa acctcci-gqH qai~gatttca atcctccatt tcaagcagct 1~38g gcact~:t'~ccg accaattttc tcgttgcci:v tctggcctgc gctgatttct tggtgggtgt 144E~
gactgtgatg cccttcagca tggtcaggac ggtggagagc tgctggtatt ttgggaggag 1500 tttttgtact ttccacacct gctgtgatgt gg"uattttgt tactcttctc tctttcactt 156Q
gtgcttcatc tccatcgaca ggtacattgc ggttactgac cccctggtct atcctaccaa 162a gttcaccgta tctgtgtcag gaatttgcat cagcgtgtcc tggatcctgc ccctcatgta 1680 cagcggtgct gtgttctaca caggtgtcta tgacgatggg ctggaggaat tatctgatgc 5740 cctaaactgt ataggaggtt gtcagaccgt tgtaaatcaa aactgggtgt tgacagattt 7~.8Q0 tctatccttc tttat~ccta cctttattat gataattctg tatggtaaca tatttcttgt 186Q
ggctagacga caggcgaaaa agatagaaaa tactggtagc aagacagaat catcctcaga 1920 gagttacaaa gccagagtgg ccaggagaga gagaaaagca gctaaaaccc tgggggtcac 1988 agtggtagca tttatgattt catggttacc atatagcatt gattcattaa ttgatgcctt 2040 tatgggcttt ataacccctg cctgtattta tgagatttgc tgttggtgtg cttattataa 21Ct0 ctcagccatg aatcctttga tttatgcttt attttaccea tggtttagga aagcaataaa 2160 agttattgta actggtcagg ttttaaagaa cagttcagca accatgaatt tgttttctga 2220 acatatataa gcagttgtat agacgaagtt caggatacct ttaaaattac caagcgaaat 2280 gagtttttaa aaatcaagta agactatgaa tgaatagcaa ataaattgct cttcaaatga 234(>
aaaacaaatc aatgtttttc agtcttgtta agatgtgcac tttcctgtcc cttctgcaaa 24Q0 agtatttact tggctaacaa atgttaaatt cctatttgtt aactgcttta gagctcagca 2460 tatcccactc cctgcagaca ctttttgtct tttaatccat tgactcttcc ctctgctctg 2520 gtatttttcc taaaaatatt tgtttttttt tttttattta ttccctttcc tcttttcttt 258a acaaagcttt ctactctttc ccagcctgcc aaaaatttca tttgtgaata gcctttatca 2640 aattattggt ttcttttgct ttggttattt taccacagga gtccttttag gtattaattt 27gE~
aatttattca atcttgggag agatctcagg gtgtatgggg caatttgcaa atgaagacat 2760 catcttgacc aggctgttgt aattgteaaa ccagttactg tcattcttgt aattatttcc 2820 tcccccaaag tgggaagcag aagccactgt acttcccaga atgatgttag gatgattatt 288a tggctgctgt tcttgctatt gcacaaaact gtttaaagag ttggtatgaa tagagccctg 2940 tgttacatta ttcagttcat acacattgaa tattacttgt tcctttaggg aggatatctt 3000 tcaagtgcag tctctagctt tcttttcttt ttttttctat attaaaactt aattacagca 3060 aggaatttgc aagattagaa gccatgtgga atatacatca atgagaagca ttcaggtatt 3120 catccatgtt ttttccattt accaaacatg aaacctgtgc ctatattgta tggaaatcag 318 tgctagatgc cttagacaca ggcataacat cccatttttg tctttaataa gctgtgactc 3?40 tggcaagaag caatgttggt cactgaaaat tttaaaaagg gcgaacacta gctcaaccag 33~g gtaattaagg ttcaatatca gtagtagtaa ggcatgtggc tattgtgtgt tcttgacatt 3368 atgtaatgag aatggctttt acctctatgt cctttcttcc ccaaatccat aacccccatg ~42p tagtcatgag aaaaatgtca gacgaaactt tatcgggaat attctgcata atatttgatc 248D
agtatttctc aaaactgtca gtcatcaaaa ataaagtatg agaaactgtc atgatctaca 8S4(?
ggaaactaag aagacatgac aactaaacgt agtatggcat cctaaatgga aagctagaaa 26aQ
gaaaagggac attaggggaa gtgaggaaat ccgaataatg aatggaaatt ttattgctat 366U
attgataaca atattggatc attagttatc acaaatgtac tatatgagtt taagatgtta ~72~J
atgagaaaet tcttgcaagg tatataggaa ttctcattac tatctttgca atttttcagt 3788 aattctacaa ctattctgaa attaaaagtt tattcaaaaa atatagagta cacaattcct 384f~
gcttgataaa gtttctagcc. tgtctatgtg aagacagcaa aqc_acttatc cttacagtca 39L7Q
ttcatttatt cattctgaat atatctttga agactgagtg tgtactagac tcttggttca 396p gtgtgatcag gaatagaaaa ccaggaactt agaatat'~ttt gtggcaaaac ccaaaatacc 4a2tj agtaattaag acttggaatg catgggaatt taagctataa aaqgctgtgt ttaaggaaca 4Q8a ~:aggagaaag gagaattcag acctggatgg aaaatqaagq agatgtatta aagaagtggc 41.4rf attCaagtag ggccttaaat tttaagaagg atttttgta~ ~a:~ggaaagg atgggaagcg 42f~g attttcaggc atgagcaaag aaactgagaa agtgcaaagt "-~tttdac~r.~aa tatgaaaata 4?6E~
aaacE~a~.~tafg gctggg=gtg gtgggtcacg Fwctgt"pat c"~ as"~"=a° tfi. 'tg ggaggccaaq 4.~20~
q.gggtggat cacaaggtca aaagatcgag as"~a:~~-"~tq~ -taa"_ae~r3"ai~ gaaa~cecgt 438t'~
cib"~tact~~aa aatacaaaaa i'-t-a~:ctc~s~at gtggi:-3q"'a"°
~.t4"er"_"°t"_~t ag t~c~caca~tt~= 444?
t~~.~g'~ta9~.tct qaggcagq"a:~ ~aat"ctctt'=~~u 'a"_~"~t~~-t:~r~'t~t t"rt;-~~~"r~t.c~~ actt~gag~=c"~.s 4"~t~rtf ~aat~Tatgcca ct<gt-~ic~t~c~ gcrtgggt~aa cxagagl-:~add cta~.t<atcada aaa~-~~3.:~rad~~ri ~156t~
~.ir'#dc3daagaa dcie4dd~lgaac'3 gs~-aa~aaciclh a~iaa~rlc~t~=te'3 7dr'=r3aCCaelC"C ac~gae~~_~C"ddg 4(~e_~1 ~aagt'dgteac gc~ttttaaac cctcctra~-~!~g f-tg~~'at~r-~c,a ar~~~c~tgcac cte"F~ir~tt~t: 4~a8rt "=t=tr~fgg~:r"~~c ~~tr c:at:tgt_g qtttggaqac: a~-f-~~t ~yr,cmr~~t r--1-c~C-wctcaa gt_Et_~-,-~~tc~'_c~ 4'14t1 --~at<~~attf~t t<G-+'a c:+_aagaa acattt:.~:C~e~d d~~fir~ti~~gaf~E t~ir~~3.~G-iac~°ig tc~r~+~t-"~m~iLr<~ 48Qt7 .aae-ii r3~tc~ca a'g"~adadd~d tttctgatG-r aE~ad~~t'_att.t_L Lr.f~ara~ict-gi'_t r.trv3t~,a-P r-Et= ~PB~at~
ttck~gttc°tt gctagadcact gl:atattagge"~ ai'att~~aga ~"+'~~:"tgagaag t~ccl-_~t~+:1~~ 49X1 ~+r"aaaaacct gLccaatttt gtttaadtgt gr~att~rtaa~3 cdttrtattaa accatgggta 4980 i:tcttttgat atgaggagct caataacaga g~~tattgtcc aagqaaaata tttdaggaac 5Q40 dccatctcta tcgttgattt ggttcttacc taatgt'_atta ttaatcttgt attggtcttt 5lpg tggtctttca catacaatac acttcattea atttgtattt ctaadaggta gaggtiggttt 5160 tccdacccat ggtataataa tgagtaagca ctcataacat gttttttttt gtcaattatg 522Q
atgtataaga taaattgtac agtatgtaaa atgggataaa ttatgtgact ttgaaggagg 5280 gactggatgc tgcagactga aaaatctcag aaggttccat ggagaactta gaatataata 5340 r_cagctaagc agtggaggat tggtttaaat agaaaaaaca atagtagaaa dggaatttct 540D
aggcagaata daaatctcat gaaaggtata aatttagtgg tgcatcaaat gcaattaagc 596a atacaaaaaa ggatagccaa taagatcagt cggcctaaag cacaccattt gggtaagaga 552(3 gtcacaatca atctatccat caatcaacat actattttaa gccctatatc tgcactgtcc 5580 agtatggtag ccatgagcaa catgtggtta ttgaggactt gaaatgtggc tagtctgagc 5648 tgagatdttc tataggtaga atttatatac cagatttcaa agacaaacaa aagaatgtaa 5700 gatgtctctt tttaaaatat tgattacatg ttgaaattat caattttggg atgtattggg 5760 ttaaataaaa atatgttatt tagattadtt tctcttttct ctttttactt tttaaaatgt 5820 ggctactaga aaaatgtaaa attatgtatg tagcttaaat tatatttcta ttggacagca 5888 ctactctaga gaaaacaaaa atgagccata ggacaattct agccctccag gacttaaaaa 5940 tcaagatggg gagagaatgc atgaacataa acaatgaagt aaatatata~ gaactatggt 6000 aaaagttaaa taatatttat ttagcaattg atgtttaaaa acagaggtac aaaactcata 6060 ttaatcttat agtctggtca ttttataaat gagaaaattg ggactcagag agadataaat 6120 taacttgcat gaaattatac agctagtagg ctaggtgcgg ttgctcacgc ctgtaatctc 6180 agcactttgg gaagctgaag tgggaggatc acttgagttc aggagtttga taccagcctg 6240 ggcaacatag tgagaccttg tctgtactaa aaatgadaaa gttagccagg cgtggtggtg 6300 adtgcctgta gtcccagcta ctcaggaggc tcaggtggga ggatcacttg tacacaggag 6360 tttgaggctg cagtgagcag tgatcgcatc tagadacaaa gtgagattct gtctcaaaaa 6420 ataaaaagtt atacagttag caaaaaatct ttgccatact ttgaacccaa attatttgaa 6480 ttctaagctc aatttttttt ctcgacatgg adatgagagt ttaggacaaa caataaggga 6540 tattacaaag aagtacagtt aaataaatgc tatccacaag aagatattta gcatttagag 6600 tttctctcat aagtcagtgg ttcttgataa tttttagdtc atagtcacat tgaaatatgc 6660 tgaaatctat gtattctttc tctagaaaaa tgcacacaat cacatataca caatatttag 6720 tgtactattc tggggg~tcc atadcctttg gggccadtta caggtcatgg atacactgtt 6780 cctaagttgg gcatagacta gacttggctg aagtgaaaac gacctcaact aactctgtgg 6840 cttdacacaa caaaggttta tttcctgctc atgtgaagtc cactatgagt ctgaggagat 6900 dtcagggcaa ttgtcctcaa catagtgcag gttgctttga tttcatggct ccatcttttc 6960 aacaagagac ttctatactt cagcctgaaa gaacacatga acacttaaat ctattagcca 7p20 gaaccaagca gacagtccac ctaatggctt gaaaaacaca ggacgataag cacaatgttt 7080 gctgcacaca aaggcctctt ccaaacactg ttttactgga tgggacatca cattgtgtga 7140 atgatgtatc tctcagggtt tcaatttggc agaaaatgtt ttaaactcat tagtgtttat X200 tttaatcaat attaagtcta agtgagatag ttgctggctt cggctggctg actcaagtat 7260 gttagagtca aggatgctgt acagattttc ctgatgcttg tctaatggtt ccaaagtgac 7320 tacaggagtt ccagccaaat cacacatgtt cctggaaata gggaggagga agacctagga X380 agdggggctc tctgaagccc tacttgacaa cttcagtt+.~c cccaagggag agaagattct 7440 gatgctatgg aggaaattaa cccattggtt gttaggtgag aaataagcat ttctgccaca '500 tgaagtaatc tggaaaatga aatccaacaa gtga~~aataa aactf~cttac caaattctct 7560 caca:~gtatt tgctgtataa atccataagg aagtgaactg agaadaagag aatggaaaaa 7620 ataacaggtt fmtctcaaaga tctctatacc atctttttct taaacttctc cttttgtatt 7680 atttdgtatt aatttttcag ccaatgaaca tgtactatat atgtgttaca tagtaaatgc ~~~0 ectgttgacd atgcagaagt gatgagdatg adtgagdctc aatcc~tttta gtagagtttt X800 caatatttcc caaaaggtag tggttacaag cdgagccaga caqccagtcc ttttatggga X860 cdaagatdtc ttgctdgagd tcacacagtg ggtcagcatc agggacagag tcceeagaat ~9<"-0 ttgcaatagc ctggttgtta ttgtcttgga greaatcdgta ga~~ar~atgta aaggcatatt X980 ttttaatt'tt a=~att*'caag gt~acatgtgr,~ aggatgtgCKa ggttt~f~ttdw ataggtaaa~:
80~f~a atgtc~c~atc~ gtgqtttg~-t gcacctatca a~~c"~dtE~dr:L tag~ta+-_taa g~:c~aq~~atg 810th ~ac'Statr',3C/',Et=dt ~t~WC~'W=~'Lac3t gr=trytC-~tt'.-jC=C:
!°~'_rac'r"c~fat"--.".'.S= rwGtr3tT~+~~~°da r=ai~~r'~~w".:'"C:ci~~7 ~~fr~l tgf''c~t ~,:~tgc- tcccttcctiet gtqtcr_~tt'~t attctcatt_g ttcdgc°_t'-c=ra actgaagaat~ ~?~~' ~,~~a~dt=r-,~t_~ aaaatggc=:a t_at t-=y°_e°r~aa a~caatttat agar=t"_w El~g ctafitctj"Maf s~,°3r~
tarid~~t-:~"~c~.~ ttgacattct t~car:agdat-t agaaaaaaaa gattt':aa~:i:aa ttcatat~g!~a ~ ~~lrt~' ~r'1'$'~3el.deldf~ ~l~Ct~gtelP~d t~(.~t'cl~~G.~!=taaa ~txCtaagf:_aa claei~r7ds:.l,7c'3 riC:as~c3r~r'~ag!~ ~~z~~'TCf rWl _t~~.ira~~r°~ct r'k~Caact'~f~c'c,1 dr~t~t_dti~drwt'a t,:aaggCtdr e'1 a.~t t'i~7r_'cw,,t.~,ac1 C:clCjr,dtP~v~l ri rrir~HEl r"t'".~r'~1_r3r~~acd at~r':ggr~a'lC.~r3t~ agaC".:ac~t:gC~ ~ldtuc7~aatag c3~d~3~lC~t_r'rdr~ct aat''r.~E3a~~dr~rl ~°_~.jfy tr an=ai "a ~~ga a6~c.a6=ctgc~t cttcaacaaa ctt~gacaaaa aaa~~aa~~at'-a t_t-gggaaagE ~3a~t;l at~ts~~~~t~r=art taataaatgg tgctggaaga actggctagc catat~acaaa aaaatt~gdaa 8t5=t0 ctggr~tfc~r=ct tccttacatc ctatacaaga attaactcaa gac~~gattaa agacttaaat: 8~~~g gcaa~~rmcad ttaatatttc actagtaggg ctgccctcta ccagat'.~ggct aatggagtca 8-160 cgatgcagag ctatgagatg gagggcaggg gatgctatgg ctaattttaa ccacagcagc 8820 aaacacagaa aaacccacta ggtcgtatag actcctgacc agccccagca tcagatcttg 8880 gtcactgtgg gctatggtga attcataatt caaaacccaa agcctaattt ctgttgtgtt 890' tggaataaaa ttctaactct gtgattctga tctatgaggc tttgcatagt cttagccccg 9000 cacaactctc tacccactac ctattctgct ctccctttac ttgttatgct ccaaatatat 8060 ttgtccaatc ccagcccctc ggctaccccc cacaggatgc ctcaggagct gtgcactggc 912D
tatcccatct gttgtactag gtcgctcagg agactcgcac agggtttttc atctgttgga 9.80 tcactcagtc ctcagatctt cacggagctg gttccttaca caggtctcag ctttaacatc 9240 gctccctcag aaaaatcaag atctcggcta atgttgttat ccatgtttta ttcttttact 930'0 caccttgttt ttatatcatt tccttcatgg tacatatcag aatgtgttac aatcttattc 9360' atctgt:ttat tttcttgtgt tttaactgtc tgtctcttaa tgacatgtaa gctgcaggag 940' gtcagatact tgctgaagta tcactatgag taaatcagac agtttgtact ggtgttttat 9480 ttctttgctc ctatgttgtt gttttacttt ggtcagttga gtaaataaat gagtgaataa 9590 ataaaataga aatagattcc ccagcattgg gaggatacat taaatgtatt cttttttttt 96D0 tttttaaagt tctggggtac atgtgctgga tgtgcagttt tcttacatag gtaaatgtgt 9660 gccatggtgg tttgctgcac ctatcaactc attacctagt tattaagcct ggaaaaatgc 9720 attcttttta taagcttttt tggggagggg cagggtcttg ctgtgaccta ggctggggta 980 cagtggrwatg atcatagctc aatgcagtct caaactcctg ggctcaggtg acactcccat 9840' gtagctgggg ctacaggcgt gtgcgaccat gcctggctaa tttttaaaaa aaatttattt 9900' ttgtagaaaa gggatctcat tttgttgccc aggttggtct caaacac 999'7 <z10'> 4 <2~.1> 107.
<~12> I~NA
<~13> Human <90'Q> 9 gaacagttca gcaaccatga atttgttttc tgaacatata taagcagttg katagacgaa 60 gttcaggata cctttaaaat taccaagcga aatgagtttt t 10'x.
<z10> 5 <2~~> 321 <~~,z> PRT
<2~3> Hurnan «0'0> 3 Cys "i"?/~ ~ln Val Asn Gly Ser Cys Prc Arg Thr Vat His Thr ~eu G.t.~
10' 15 ~1.~ ~ln T~~u Vat. Ile Tyr Leu Thr Cys AJ.a Ala fly Met ~eu Tle I.l.e zo 2~ 30 Val~ Leta ~~.y Asn Val Phe Va1 Ala Phe Ala Va L Ser "Fyr the .L'ys A.~a 3a ~0 93 ~e~' H.~s '~'~ar ~rQ "T'hr Asn the ~.ae~a Leu Lreu her Iaeu J~la Leu A?'a J~s~
'_~0' S5 60 ~Iet ~'he L~u ~~! y Leu l.~eu Val Leu ~rca Leu ~~r "I"hr T.~a Arg Ser Val 65 7D' ~S 80 ~~t~ See ~'~rs "~'r~a Phe ~'he ~~~!y Ash Phe Leu 0'"~s Jarg Leu fills '~h>~
"T'y~
R5 ~fi A
I~nn.~ asp "2"fns Pry"o~ Fhe C'ys I~e~a "z"hr per ~'tr~" ti'f~t-~ t~.~.s Leu ~'~Js the t.7!.~

~L~P 'Lr-~", l~.g :~er- T~'.e As~.~ Arg f-Ia.:~ ~'ys ALa T:1.F=~ r'y~: Asp Pra Iaeu Lreu "L"yr !'rte :~~~r L l r5 1;~'fi7 1~ ~
I~ys f"1're "1"hr. Va 1 Ar"I Va l ALa Lfi:t~ Arch 'i'y'~ L.l,e Laeu A.La Gl~ y 'I'r~~ r-rp y 131 ~ ~a l.tlg Va D I=rrr AT ~a II E ~r '1"yr "I~'hr Ser E,c-:u E'lm~ lieu "~yr "L""rm Asp Vci,L Va l rz M ur 125 Lag 15a I~s~r Thr Arg L,eu S"~r. ~Ln "1'rp I~eu C;~.u flu Met Pra Cys Va1 c:xly See ~y~;
.L6'~ 7_~p 1~a Gln T~eu ~~eu ~,eu Asn D,ys Phe Trp ~.Ly 'T'rp Leu Asn Phe Pro T~eu F'he 18~ 185 190 Phe Val Pro ~'ys Leu lle Met Ile Ser Leu Tyr Val L,ys l~.e Phe Va.L
~.~5 zoa za5 Val Ala 'Fhr Arg Gln Ala ~ln Gln Tle "~hr Thr ~,eu Ser Lys Ser Geu 211) 215 220 Ala Gly Ala Ala f~ys His ~1u Arg Lys Ala Ala Lys Thr ~eu G~.y 11.e Va1 Val GIy ~1e Tyr lieu Leu Cys 'Frp Leu Pro Phe Thr Tle Asp Thr 245 z50 255 Met Val Asp Ser Iseu Leu His Phe Ile Thr Pra Pro I~eu Val Phe Asp 260 z65 zip Ile Phe I.Le Trp Phe Ala '~yr Phe Asn Ser Ala Cys Asn Pro Ile 1.1.e 2?5 280 285 Tyr Val Phe Ser Tyr ~1n Trp Phe Arg Lys Ala ~eu Lys Leu 'Fhr I~eu 29(~ z~5 300 Ser ~ln I~ys Val Phe Sex Pra G~.n "Fhr Arg Thr Va1 Asp Leu Tyr ~1n 3a5 3~.0 37.5 321) Glu <210> 6 <z11> 296 <21.z> PRT
<z13> Hurnan <9Q0> 6 ~eu Phe ~~!y Asn )~eu Val 21e Met Val Ser lle Ser His Phe ~ys ~ln Leu His Ser Pra Thr Asn Phe Leu T1e Leu Ser Met Ala 1'hr '~'hr Asp Phe Leu Taeu G.1_y Phe Val Tle Met Pro 'Fyr Ser Ile Met Arg Ser Val 35 4(1 95 Glu Ser Cys "I'rp ~'yr Phe fly Asp fly Phe ~ys Lys Phe His 'I"hr See 50 55 6p Phe Asp Met Met Leu Arg Leu Thr Ser lle Phe His Leu ~ys Ser Tle Ala :Lle Asp Arg Phe ryr Ala Va.L ~ys T'yr Fra Leu His "T'yr Thr ~'hr 85 9D ~5 Lys Met "L'hr Asn Sex Thr Tle Lys ~ln ):~eu Iseu Ala Phe Cys Trp Ser ~.tJQ 1Q5 110 Val Pr<a A.La Lieu Phe Ser Phe ~.Ly T.~eu Va.~ beta Ser ~1u Ala Asp Vat.
115 12(~ 1z5 Ser ~ly Met ~a.ll n her Tyr ~~ys :~.~e Leu Val Ala Cys Ptse Asn Phe ~ys 13g 135 leg Ala ~pu "D'hr Fhe Asrr Lys Phe ~'rp ~.~y "P'hr :L~.e .~eu Phe "L°hr "~"hr ~'ys 195 15~? 1.55 1.6g Phe Phe "1"h~ Pr~.~ fly Ser :~l~e Met VaD ~l~.y .ale Tyr fly )!,ors '~~e L'tm 1~5 17fi~ ~.1~', :LLe Va.l Sr=~ ~.~v:~ c:~Ln His Ala Ar-:I Vat 1')Le 5er 1~Ir.s Vat. Pr~~ ~rlu Asrr ~ ~~' ~.8!w h uc~
C>

"~'h~ ~.ry5 1~1~~ X11 G+ V~1. ~rys ~~y:.: '' P -; ~.~=j": ~r~Y L~ya ~y5.s Lrys Ilsp Firs toys ~'r"~J s'~DO! ~~)~
T~~~ 1~~.~ I~ys "?''h r I~F~u ~"r.P y ~' 1~ ~~, ;Y,.a ~ N~Fe ~ I ~~ ~a l FhEr ~reu Al~a C'ys 'k'rF' _~~J ~l"', ~.W~
1.~~~u I'~o ~ys Fh~ I~."~e~ A7~rfi Vein ~~,an~~ I~r, n::~, ~-rr~ '~'~>r I~a~u Asia 'I'y~ °;F=r .' i' ~J rE-.~ C'.? .'..~ ~7 4' 'f qtr Fra I ~.e l~~e~ I ~a ,~Ct~ ~ls~~ D>>av h"r"ar V~ T "~"r~~ ~r~t~ T~rg 'I"yr ~'f~r~ hs,rr i .J V
~~r Thr Cys Asr~ Fca LreU I.l! ~~ I~ i s C~~ y l~h~ 1''h~ Asri Fra "~'rp F"he G~.r~
z~Q~ ~'_a 2~0 Lys A1a Fhe Lys "~y.r :~:Le V~.1. ~~r ~.l~y Lys :ale Fhe Se.r Ser l~lis Ser z75 2801' 285 Glu T'hr Ana Asr> L~eu ~'he ~'rc~ flu zoo z~~

Claims (23)

Claims That which is claimed is:

1. An isolated peptide consisting of an amino acid sequence selected from the group consisting of:
(a) an amino acid sequence shown in SEQ ID NO:2;
(b) an amino acid sequence of an allelic variant of an amino acid sequence shown in SEQ ID NO:2, wherein said allelic variant is encoded by a nucleic acid molecule that hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 (cDNA) or 3 (genomic);
(c) an amino acid sequence of an ortholog of an amino acid sequence shown in SEQ ID NO:2, wherein said ortholog is encoded by a nucleic acid molecule that hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 (cDNA) or 3 (genomic); and (d) a fragment of an amino acid sequence shown in SEQ ID NO:2, wherein said fragment comprises at least 10 contiguous amino acids.

2. An isolated peptide comprising an amino acid sequence selected from the group consisting of:
(a) an amino acid sequence shown in SEQ ID NO:2;
(b) an amino acid sequence of an allelic variant of an amino acid sequence shown in SEQ ID NO:2, wherein said allelic variant is encoded by a nucleic acid molecule that hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 (cDNA) or 3 (genomic);
(c) an amino acid sequence of an ortholog of an amino acid sequence shown in SEQ ID NO:2, wherein said ortholog is encoded by a nucleic acid molecule that hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 (cDNA) or 3 (genomic); and (d) a fragment of an amino acid sequence shown in SEQ ID NO:2, wherein said fragment comprises at least 10 contiguous amino acids.

3. An isolated antibody that selectively binds to a peptide of claim 2.

4. An isolated nucleic acid molecule consisting of a nucleotide sequence selected from the group consisting of:
(a) a nucleotide sequence that encodes an amino acid sequence shown in SEQ
ID NO:2;
(b) a nucleotide sequence that encodes of an allelic variant of an amino acid sequence shown in SEQ ID NO:2, wherein said nucleotide sequence hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID
NOS:1 (cDNA) or 3 (genomic);
(c) a nucleotide sequence that encodes an ortholog of an amino acid sequence shown in SEQ ID NO:2, wherein said nucleotide sequence hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 (cDNA) or 3 (genomic);
(d) a nucleotide sequence that encodes a fragment of an amino acid sequence shown in SEQ ID NO:2, wherein said fragment comprises at least 10 contiguous amino acids; and (e) a nucleotide sequence that is the complement of a nucleotide sequence of (a)-(d).

5. An isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of:
(a) a nucleotide sequence that encodes an amino acid sequence shown in SEQ
ID NO:2;
(b) a nucleotide sequence that encodes of an allelic variant of an amino acid sequence shown in SEQ ID NO:2, wherein said nucleotide sequence hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID
NOS:1 (cDNA) or 3 (genomic);
(c) a nucleotide sequence that encodes an ortholog of an amino acid sequence shown in SEQ ID NO:2, wherein said nucleotide sequence hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 (cDNA) or 3 (genomic);
(d) a nucleotide sequence that encodes a fragment of an amino acid sequence shown in SEQ ID NO:2, wherein said fragment comprises at least 10 contiguous amino acids, and (e) a nucleotide sequence that is the complement of a nucleotide sequence of (a)-(d).

6. A gene chip comprising a nucleic acid molecule of claim 5.

7. A transgenic non-human animal comprising a nucleic acid molecule of claim 5.

8. A nucleic acid vector comprising a nucleic acid molecule of claim 5.

9. A host cell containing the vector of claim 8.

10. A method for producing any of the peptides of claim 1 comprising introducing a nucleotide sequence encoding any of the amino acid sequences in (a)-(d) into a host cell, and culturing the host cell under conditions in which the peptides are expressed from the nucleotide sequence.

11. A method for producing any of the peptides of claim 2 comprising introducing a nucleotide sequence encoding any of the amino acid sequences in (a)-(d) into a host cell, and culturing the host cell under conditions in which the peptides are expressed from the nucleotide sequence.

12. A method for detecting the presence of any of the peptides of claim 2 in a sample, said method comprising contacting said sample with a detection agent that specifically allows detection of the presence of the peptide in the sample and then detecting the presence of the peptide.

13. A method for detecting the presence of a nucleic acid molecule of claim 5 in a sample, said method comprising contacting the sample with an oligonucleotide that hybridizes to said nucleic acid molecule under stringent conditions and determining whether the oligonucleotide binds to said nucleic acid molecule in the sample.

14. A method for identifying a modulator of a peptide of claim 2, said method comprising contacting said peptide with an agent and determining if said agent has modulated the function or activity of said peptide.

15. The method of claim 14, wherein said agent is administered to a host cell comprising an expression vector that expresses said peptide.

16. A method for identifying an agent that binds to any of the peptides of claim 2, said method comprising contacting the peptide with an agent and assaying the contacted mixture to determine whether a complex is formed with the agent bound to the peptide.

17. A pharmaceutical composition comprising an agent identified by the method of claim 16 and a pharmaceutically acceptable carrier therefor.

18. A method for treating a disease or condition mediated by a human proteases, said method comprising administering to a patient a pharmaceutically effective amount of an agent identified by the method of claim 16.

19. A method for identifying a modulator of the expression of a peptide of claim 2, said method comprising contacting a cell expressing said peptide with an agent, and determining if said agent has modulated the expression of said peptide.

20. An isolated human protease peptide having an amino acid sequence that shares at least 70% homology with an amino acid sequence shown in SEQ ID NO:2.

21. A peptide according to claim 20 that shares at least 90 percent homology with an amino acid sequence shown in SEQ ID NO:2.

22. An isolated nucleic acid molecule encoding a human protease peptide, said nucleic acid molecule sharing at least 80 percent homology with a nucleic acid molecule shown in SEQ ID
NOS:1 (cDNA) or 3 (genomic).

23. A nucleic acid molecule according to claim 22 that shares at least 90 percent homology with a nucleic acid molecule shown in SEQ ID NOS:1 (cDNA) or 3 (genomic).