AXOR39, A G-PROTEIN COUPLED RECEPTOR WITH 7-TM SEGMENTS

Abstract: AXOR39 polypeptides and polynucleotides and methods for producing such polypeptides by recombinant techniques are disclosed. Also disclosed are methods for utilizing AXOR39 polypeptides and polynucleotides in diagnostic assays.

Description: AXOR39, A G-PROTEIN COUPLED RECEPTOR WITH 7-TM SEGMENTS Field of the Invention This mvention relates to newly identified polypeptides and polynucleotides encoding such polypeptides, to their use in diagnosis and m identifying compounds that may be agonists, antagonists that are potentially useful in therapy, and to production of such polypeptides and polynucleotides Background of the Invention The drug discovery process is currently undergoing a fundamental revolution as it embraces "functional genomics", that is, high throughput genome- or gene-based biology. This approach as a means to identify genes and gene products as therapeutic targets is rapidly superseding earlier approaches based on "positional cloning". A phenotype, that is a biological function or genetic disease, would be identified and this would then be tracked back to the responsible gene, based on its genetic map position. Functional genomics relies heavily on high-throughput DNA sequencing technologies and the vaπous tools of bioinformatics to identify gene sequences of potential interest from the many molecular biology databases now available. There is a continuing need to identify and characterize further genes and their related polypeptides/protems, as targets for drug discovery. It is well established that many medically significant biological processes are mediated by protems participating in signal transduction pathways that involve G-proteins and/or second messengers, e.g., cAMP (Lefkowitz, Nature, 1991, 351:353-354). Herein these protems are referred to as protems participating m pathways with G-proteins or PPG protems. Some examples of these protems mclude the GPC receptors, such as those for adrenergic agents and dopamme (Kobilka, B.K., et al., Proc. Natl Acad. Sci., USA, 1987, 84:46-50; Kobilka, B.K., et al., Science, 1987, 238:650-656; Bunzow, J.R., et al., Nature, 1988, 336:783-787), G-proteins themselves, effector protems, e.g., phosphohpase C, adenyl cyclase, and phosphodiesterase, and actuator protems, e.g., protein kinase A and protein kinase C (Simon, M.I., et al., Science, 1991, 252:802-8) For example, in one form of signal transduction, the effect of hormone binding is activation of the enzyme, adenylate cyclase, inside the cell. Enzyme activation by hormones is dependent on the presence of the nucleotide GTP GTP also influences hormone binding. A G-protem connects the hormone receptor to adenylate cyclase. G-protem was shown to exchange GTP for bound GDP when activated by a hormone receptor. The GTP-carrying form then binds to activated adenylate cyclase Hydrolysis of GTP to GDP, catalyzed by the G-protem itself, returns the G-protem to its basal, inactive form. Thus, the G-protem serves a dual role, as an intermediate that relays the signal from receptor to effector, and as a clock that controls the duration of the signal. The membrane protein gene superfamily of G-protem coupled receptors has been characterized as having seven putative transmembrane domains. The domains are believed to represent transmembrane a-hehces connected by extracellular or cytoplasmic loops. G-protem coupled receptors include a wide range of biologically active receptors, such as hormone, viral, growth factor and neuroreceptors. G-protem coupled receptors (otherwise known as 7TM receptors) have been characterized as including these seven conserved hydrophobic stretches of about 20 to 30 ammo acids, connecting at least eight divergent hydrophilic loops. The G-protem family of coupled receptors mcludes dopam e receptors which bmd to neuroleptic drugs used for treating psychotic and neurological disorders. Other examples of members of this family include, but are not limited to, calcitomn, adrenergic, endothelm, cAMP, adenosine, muscarmic, acetylcholme, serotonm, histamine, thrombm, kimn, follicle stimulating hormone, opsms, endothehal differentiation gene-1, rhodopsms, odorant, and cytomegalovirus receptors. Most G-protem coupled receptors have s gle conserved cysteine residues m each of the first two extracellular loops which form disulfide bonds that are believed to stabilize functional protein structure. The 7 transmembrane regions are designated as TM1, TM2, TM3, TM4, TM5, TM6, and TM7. TM3 has been implicated in signal transduction. Phosphorylation and hpidation (palmitylation or farnesylation) of cysteine residues can influence signal transduction of some G-protem coupled receptors. Most G-protem coupled receptors contain potential phosphorylation sites within the third cytoplasmic loop and/or the carboxy terminus. For several G-protem coupled receptors, such as the b-adrenoreceptor, phosphorylation by protein kinase A and/or specific receptor kinases mediates receptor desensitization. For some receptors, the ligand binding sites of G-protem coupled receptors are believed to compπse hydrophilic sockets formed by several G-protem coupled receptor transmembrane domains, said socket being surrounded by hydrophobic residues of the G-protem coupled receptors. The hydrophilic side of each G-protem coupled receptor transmembrane helix is postulated to face mward and form polar ligand binding site. TM3 has been implicated m several G-protem coupled receptors as having a ligand binding site, such as the TM3 aspartate residue. TM5 sennes, a TM6 asparagine and TM6 or TM7 phenylalamnes or tyrosmes are also implicated m hgand binding. G-protem coupled receptors can be lntracellularly coupled by heterotπmeπc G-protems to vaπous lntracellular enzymes, ion channels and transporters (see, Johnson et al., Endoc. Rev., 1989, 10:317-331) Different G-protem a-subunits preferentially stimulate particular effectors to modulate vaπous biological functions m a cell Phosphorylation of cytoplasmic residues of G-protem coupled receptors have been identified as an important mechanism for the regulation of G-protem couplmg of some G-protem coupled receptors. G-protem coupled receptors are found in numerous sites within a mammalian host. Over the past 15 years, nearly 350 therapeutic agents targeting 7 transmembrane (7 TM) receptors have been successfully introduced onto the market. Summary of the Invention The present invention relates to AXOR39, m particular AXOR39 polypeptides and AXOR39 polynucleotides, recombmant mateπals and methods for their production. Such polypeptides and polynucleotides are of interest in relation to methods of treatment of certam diseases, including, but not limited to, infections such as bacteπal, fungal, protozoan and viral infections, particularly infections caused by HTV-1 or HIV-2; pain; cancers; diabetes, obesity; anorexia; bulimia; asthma; Parkinson's disease; acute heart failure; hypotension; hypertension; uπnary retention; osteoporosis; ang a pectoπs; myocardial infarction; stroke; ulcers; asthma; allergies; benign prostatic hypertrophy; migraine; vomiting; psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, depression, de πum, dementia, and severe mental retardation; and dyskmesias, such as Huntmgton's disease or Gilles dela Tourett's syndrome, hereinafter referred to as "diseases of the mvention". In a further aspect, the mvention relates to methods for identifying agonists and antagonists (e.g , inhibitors) using the materials provided by the invention, and treating conditions associated with AXOR39 imbalance with the identified compounds. In a still further aspect, the invention relates to diagnostic assays for detecting diseases associated with mappropπate AXOR39 activity or levels Description of the Invention In a first aspect, the present invention relates to AXOR39 polypeptides. Such polypeptides mclude: (a) an isolated polypeptide encoded by a polynucleotide compπsmg the sequence of SEQ ID NO 1 , (b) an isolated polypeptide compπsmg a polypeptide sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the polypeptide sequence of SEQ ID NO:2; (c) an isolated polypeptide compπsmg the polypeptide sequence of SEQ ID NO:2; (d) an isolated polypeptide having at least 95%, 96%, 97%, 98%, or 99% identity to the polypeptide sequence of SEQ ID NO:2, (e) the polypeptide sequence of SEQ ID NO:2; (f) an isolated polypeptide having or comprising a polypeptide sequence that has an Identity Index of 0.95, 0.96, 0.97, 0.98, or 0.99 compared to the polypeptide sequence of SEQ ID NO:2; and (g) fragments and vaπants of such polypeptides in (a) to (f). Polypeptides of the present invention are believed to be members of the extracellular calcium- sensmg receptor family of polypeptides. They are therefore of interest because G-protem coupled receptors are the basis of much of cell to cell communication m human bodies and because the transmembrane family more than any other gene family, are the targets of pharmaceutical intervention. As such they have been the basis of action of more pharmaceutical drugs than any other gene family The biological properties of the AXOR39 are hereinafter referred to as "biological activity of AXOR39" or "AXOR39 activity". Preferably, a polypeptide of the present mvention exhibits at least one biological activity of AXOR39. Polypeptides of the present invention also mclude vaπants of the aforementioned polypeptides, including all allelic forms and splice vaπants. Such polypeptides vary from the reference polypeptide by insertions, deletions, and substitutions that may be conservative or non-conservative, or any combination thereof. Particularly preferred vaπants are those m which several, for instance from 50 to 30, from 30 to 20, from 20 to 10, from 10 to 5, from 5 to 3, from 3 to 2, from 2 to 1 or 1 ammo acids are inserted, substituted, or deleted, in any combination. Preferred fragments of polypeptides of the present mvention mclude an isolated polypeptide compπsmg an ammo acid sequence having at least 30, 50 or 100 contiguous ammo acids from the ammo acid sequence of SEQ ID NO: 2, or an isolated polypeptide comprising an ammo acid sequence having at least 30, 50 or 100 contiguous ammo acids truncated or deleted from the amino acid sequence of SEQ ID NO. 2. Preferred fragments are biologically active fragments that mediate the biological activity of AXOR39, including those with a similar activity or an improved activity, or with a decreased undesirable activity. Also preferred are those fragments that are antigenic or lmmunogenic in an animal, especially m a human Fragments of the polypeptides of the mvention may be employed for producing the coπespondmg full-length polypeptide by peptide synthesis; therefore, these vaπants may be employed as intermediates for producing the full-length polypeptides of the invention. The polypeptides of the present mvention may be in the form of the "mature" protein or may be a part of a larger protein such as a precursor or a fusion protein. It is often advantageous to mclude an additional ammo acid sequence that contains secretory or leader sequences, pro-sequences, sequences that aid in purification, for instance multiple histidme residues, or an additional sequence for stability duπng recombinant production. Polypeptides of the present invention can be prepared in any suitable manner, for instance by isolation form naturally occurπng sources, from genetically engineered host cells compπsing expression systems (vide infra) or by chemical synthesis, using for instance automated peptide synthesizers, or a combination of such methods. Means for preparing such polypeptides are well understood in the art In a further aspect, the present invention relates to AXOR39 polynucleotides. Such polynucleotides mclude: (a) an isolated polynucleotide compπsing a polynucleotide sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the polynucleotide sequence of SEQ ID NO: 1 ; (b) an isolated polynucleotide compπsmg the polynucleotide of SEQ ID NO: 1 ; (c) an isolated polynucleotide having at least 95%, 96%, 97%, 98%, or 99% identity to the polynucleotide of SEQ ID NO: 1 ; (d) the isolated polynucleotide of SEQ ID NO: 1; (e) an isolated polynucleotide compπsmg a polynucleotide sequence encoding a polypeptide sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the polypeptide sequence of SEQ ID NO:2, (f) an isolated polynucleotide compπsmg a polynucleotide sequence encoding the polypeptide of SEQ K> NO:2; (g) an isolated polynucleotide having a polynucleotide sequence encoding a polypeptide sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the polypeptide sequence of SEQ ID NO:2; (h) an isolated polynucleotide encoding the polypeptide of SEQ ID NO:2; (I) an isolated polynucleotide having or compπsmg a polynucleotide sequence that has an Identity Index of 0.95, 0.96, 0.97, 0.98, or 0.99 compared to the polynucleotide sequence of SEQ ID NO:l; (j) an isolated polynucleotide having or compπsmg a polynucleotide sequence encodmg a polypeptide sequence that has an Identity Index of 0.95, 0.96, 0.97, 0.98, or 0.99 compared to the polypeptide sequence of SEQ ID NO:2; and (k) polynucleotides that are fragments and vaπants of the above mentioned polynucleotides or that are complementary to above mentioned polynucleotides, over the entire length thereof. Preferred fragments of polynucleotides of the present mvention include an isolated polynucleotide compπsmg an nucleotide sequence having at least 15, 30, 50 or 100 contiguous nucleotides from the sequence of SEQ ID NO: 1, or an isolated polynucleotide compπsing an sequence having at least 30, 50 or 100 contiguous nucleotides truncated or deleted from the sequence of SEQ ID NO: 1. Prefeπed vaπants of polynucleotides of the present mvention include splice vaπants, allehc vaπants, and polymorphisms, including polynucleotides having one or more single nucleotide polymorphisms (SNPs) Polynucleotides of the present mvention also include polynucleotides encoding polypeptide vaπants that compπse the ammo acid sequence of SEQ ID NO:2 and in which several, for instance from 50 to 30, from 30 to 20, from 20 to 10, from 10 to 5, from 5 to 3, from 3 to 2, from 2 to 1 or 1 ammo acid residues are substituted, deleted or added, in any combination In a further aspect, the present mvention provides polynucleotides that are RNA transcπpts of the DNA sequences of the present mvention. Accordingly, there is provided an RNA polynucleotide that: (a) comprises an RNA transcπpt of the DNA sequence encoding the polypeptide of SEQ ID NO:2; (b) is the RNA transcπpt of the DNA sequence encoding the polypeptide of SEQ ID NO:2, (c) comprises an RNA transcπpt of the DNA sequence of SEQ ID NO: 1 ; or (d) is the RNA transcπpt of the DNA sequence of SEQ ID NO: 1 ; and (e) RNA polynucleotides that are complementary thereto. The polynucleotide sequence of SEQ ID NO:l shows homology with RAIG1 (J. Biol. Chem. 273 (52) 35008-35015 (1998). The polynucleotide sequence of SEQ ID NO:l is a cDNA sequence that encodes the polypeptide of SEQ ID NO:2. The polynucleotide sequence encoding the polypeptide of SEQ ID NO:2 may be identical to the polypeptide encoding sequence of SEQ ID NO: 1 or it may be a sequence other than SEQ ID NO:l, which, as a result of the redundancy (degeneracy) of the genetic code, also encodes the polypeptide of SEQ ID NO:2. The polypeptide of the SEQ ID NO:2 is related to other protems of the extracellular calcium-sensmg receptor family, having homology and/or structural similarity with RAIGl (J. Biol. Chem. 273 (52) 35008-35015 (1998). Preferred polypeptides and polynucleotides of the present mvention are expected to have, inter aha, similar biological functions/properties to their homologous polypeptides and polynucleotides. Furthermore, preferred polypeptides and polynucleotides of the present mvention have at least one AXOR39 activity. Polynucleotides of the present mvention may be obtained using standard cloning and screening techniques from a cDNA library deπved from mRNA m cells of human prostate, (see for instance, Sambrook et al, Molecular Clonmg: A Laboratory Manual, 2nd Ed., Cold Spπng Harbor Laboratory Press, Cold Spπng Harbor, N.Y. (1989)). Polynucleotides of the invention can also be obtained from natural sources such as genomic DNA hbraπes or can be synthesized using well known and commercially available techniques. When polynucleotides of the present invention are used for the recombinant production of polypeptides of the present mvention, the polynucleotide may include the coding sequence for the mature polypeptide, by itself, or the coding sequence for the mature polypeptide m reading frame with other coding sequences, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protem sequence, or other fusion peptide portions. For example, a marker sequence that facilitates purification of the fused polypeptide can be encoded. In certam preferred embodiments of this aspect of the invention, the marker sequence is a hexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) and descπbed in Gentz et al, Proc Natl Acad Sci USA (1989) 86:821-824, or is an HA tag. The polynucleotide may also contain non-coding 5' and 3' sequences, such as transcπbed, non-translated sequences, splicing and polyadenylation signals, πbosome binding sites and sequences that stabilize mRNA. Polynucleotides that are identical, or have sufficient identity to a polynucleotide sequence of SEQ ID NO: 1, may be used as hybπdization probes for cDNA and genomic DNA or as pπmers for a nucleic acid amplification reaction (for instance, PCR). Such probes and pπmers may be used to isolate full-length cDNAs and genomic clones encodmg polypeptides of the present mvention and to isolate cDNA and genomic clones of other genes (including genes encodmg paralogs from human sources and orthologs and paralogs from species other than human) that have a high sequence similaπty to SEQ ID NO: 1 , typically at least 95% identity. Preferred probes and pπmers will generally compπse at least 15 nucleotides, preferably, at least 30 nucleotides and may have at least 50, if not at least 100 nucleotides. Particularly preferred probes will have between 30 and 50 nucleotides. Particularly preferred pπmers will have between 20 and 25 nucleotides. A polynucleotide encodmg a polypeptide of the present mvention, including homologs from species other than human, may be obtained by a process compπsing the steps of screenmg a library under stringent hybπdization conditions with a labeled probe having the sequence of SEQ ED NO: 1 or a fragment thereof, preferably of at least 15 nucleotides; and isolating full-length cDNA and genomic clones containing said polynucleotide sequence. Such hybπdization techniques are well known to the skilled artisan. Prefeπed stπngent hybπdization conditions include overnight incubation at 42°C in a solution compπsmg: 50% formamide, 5xSSC (150mM NaCl, 15mM tπsodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10 % dextran sulfate, and 20 microgram ml denatured, sheared salmon sperm DNA; followed by washing the filters m O.lx SSC at about 65°C. Thus the present mvention also includes isolated polynucleotides, preferably with a nucleotide sequence of at least 100, obtained by screenmg a library under stringent hybπdization conditions with a labeled probe having the sequence of SEQ ID NO: 1 or a fragment thereof, preferably of at least 15 nucleotides. The skilled artisan will appreciate that, many cases, an isolated cDNA sequence will be incomplete, in that the region coding for the polypeptide does not extend all the way through to the 5' terminus. This is a consequence of reverse transcπptase, an enzyme with inherently low "processivity" (a measure of the ability of the enzyme to remain attached to the template duπng the polymerization reaction), failing to complete a DNA copy of the mRNA template duπng first strand cDNA synthesis. There are several methods available and well known to those skilled in the art to obtain full- length cDNAs, or extend short cDNAs, for example those based on the method of Rapid Amplification of cDNA ends (RACE) (see, for example, Frohman et al., Proc Nat Acad Sci USA 85, 8998-9002, 1988). Recent modifications of the technique, exemplified by the Marathon (trade mark) technology (Clontech Laboratoπes Inc.) for example, have significantly simplified the search for longer cDNAs. In the Marathon (trade mark) technology, cDNAs have been prepared from mRNA extracted from a chosen tissue and an 'adaptor' sequence ligated onto each end. Nucleic acid amplification (PCR) is then earned out to amplify the "missing" 5' end of the cDNA using a combination of gene specific and adaptor specific ohgonucleotide pπmers. The PCR reaction is then repeated using 'nested' pπmers, that is, pπmers designed to anneal within the amplified product (typically an adapter specific pπmer that anneals further 3' in the adaptor sequence and a gene specific primer that anneals further 5' in the known gene sequence). The products of this reaction can then be analyzed by DNA sequencing and a full-length cDNA constructed either by joining the product directly to the existing cDNA to give a complete sequence, or carrying out a separate full- length PCR using the new sequence information for the design of the 5' pπmer. Recombmant polypeptides of the present invention may be prepared by processes well known m the art from genetically engineered host cells compπsing expression systems. Accordingly, in a further aspect, the present mvention relates to expression systems compπsmg a polynucleotide or polynucleotides of the present mvention, to host cells which are genetically engineered with such expression systems and to the production of polypeptides of the mvention by recombmant techniques. Cell-free translation systems can also be employed to produce such protems using RNAs deπved from the DNA constructs of the present mvention. For recombmant production, host cells can be genetically engineered to incorporate expression systems or portions thereof for polynucleotides of the present invention. Polynucleotides may be introduced to host cells by methods descπbed in many standard laboratory manuals, such as Davis et al., Basic Methods in Molecular Biology (1986) and Sambrook et al.(ιbιd). Prefeπed methods of introducing polynucleotides mto host cells mclude, for instance, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, micro-mjection, catiomc lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection Representative examples of appropπate hosts mclude bacteπal cells, such as Streptococci, Staphylococci, E coh, Streptomyces and Bacillus subtihs cells, fungal cells, such as yeast cells and Aspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells; and plant cells. A great vaπety of expression systems can be used, for instance, chromosomal, episomal and virus-deπved systems, e.g., vectors deπved from bacteπal plasmids, from bacteπophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccmia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors deπved from combinations thereof, such as those deπved from plasmid and bacteπophage genetic elements, such as cosmids and phagemids. The expression systems may contain control regions that regulate as well as engender expression. Generally, any system or vector that is able to maintain, propagate or express a polynucleotide to produce a polypeptide in a host may be used. The appropπate polynucleotide sequence may be mserted mto an expression system by any of a vaπety of well-known and routine techniques, such as, for example, those set forth m Sambrook et ah, (ibid). Appropπate secretion signals may be incorporated mto the desired polypeptide to allow secretion of the translated protein mto the lumen of the endoplasmic reticulum, the peπplasmic space or the extracellular environment. These signals may be endogenous to the polypeptide or they may be heterologous signals. If a polypeptide of the present mvention is to be expressed for use in screenmg assays, it is generally preferred that the polypeptide be produced at the surface of the cell. In this event, the cells may be harvested pπor to use in the screening assay. If the polypeptide is secreted mto the medium, the medium can be recovered in order to recover and puπfy the polypeptide. If produced mtracellularly, the cells must first be lysed before the polypeptide is recovered. Polypeptides of the present mvention can be recovered and puπfied from recombmant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography Most preferably, high performance liquid chromatography is employed for puπfication. Well known techniques for refolding protems may be employed to regenerate active conformation when the polypeptide is denatured duπng lntracellular synthesis, isolation and/or puπfication. Polynucleotides of the present mvention may be used as diagnostic reagents, through detecting mutations in the associated gene. Detection of a mutated form of the gene characteπzed by the polynucleotide of SEQ ID NO: 1 m the cDNA or genomic sequence and which is associated with a dysfunction will provide a diagnostic tool that can add to, or define, a diagnosis of a disease, or susceptibility to a disease, which results from under-expression, over-expression or altered spatial or temporal expression of the gene. Individuals carrying mutations m the gene may be detected at the DNA level by a vaπety of techniques well known m the art. Nucleic acids for diagnosis may be obtamed from a subject's cells, such as from blood, urine, saliva, tissue biopsy or autopsy mateπal. The genomic DNA may be used directly for detection or it may be amplified enzymatically by usmg PCR, preferably RT-PCR, or other amplification techniques pπor to analysis. RNA or cDNA may also be used in similar fashion. Deletions and insertions can be detected by a change m size of the amplified product m comparison to the normal genotype. Pomt mutations can be identified by hybndizing amplified DNA to labeled AXOR39 nucleotide sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences m melting temperatures. DNA sequence difference may also be detected by alterations in the electrophoretic mobility of DNA fragments m gels, with or without denatuπng agents, or by direct DNA sequencing (see, for instance, Myers et al, Science (1985) 230:1242). Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and SI protection or the chemical cleavage method (see Cotton et al, Proc Natl Acad Sci USA (1985) 85: 4397-4401). An array of oligonucleotides probes compπsmg AXOR39 polynucleotide sequence or fragments thereof can be constructed to conduct efficient screening of e g., genetic mutations. Such arrays are preferably high density arrays or gπds. Array technology methods are well known and have general applicability and can be used to address a vaπety of questions m molecular genetics including gene expression, genetic linkage, and genetic variability, see, for example, M. Chee et al., Science, 274, 610-613 (1996) and other references cited therein. Detection of abnormally decreased or increased levels of polypeptide or mRNA expression may also be used for diagnosing or determining susceptibility of a subject to a disease of the invention. Decreased or increased expression can be measured at the RNA level usmg any of the methods well known in the art for the quantitation of polynucleotides, such as, for example, nucleic acid amplification, for instance PCR, RT-PCR, RNase protection, Northern blotting and other hybπdization methods. Assay techniques that can be used to determine levels of a protein, such as a polypeptide of the present mvention, in a sample deπved from a host are well-known to those of skill in the art. Such assay methods mclude radio-immunoassays, competitive-binding assays, Western Blot analysis and ELISA assays Thus in another aspect, the present mvention relates to a diagnostic kit comprising- (a) a polynucleotide of the present invention, preferably the nucleotide sequence of SEQ ID NO. 1 , or a fragment or an RNA transcript thereof; (b) a nucleotide sequence complementary to that of (a); (c) a polypeptide of the present invention, preferably the polypeptide of SEQ ID NO:2 or a fragment thereof; or (d) an antibody to a polypeptide of the present invention, preferably to the polypeptide of SEQ ID NO:2. It will be appreciated that in any such kit, (a), (b), (c) or (d) may compπse a substantial component. Such a kit will be of use in diagnosing a disease or susceptibility to a disease, particularly diseases of the mvention, amongst others The polynucleotide sequences of the present mvention are valuable for chromosome localization studies. The sequence is specifically targeted to, and can hybπdize with, a particular location on an individual human chromosome. The mapping of relevant sequences to chromosomes according to the present mvention is an important first step m correlating those sequences with gene associated disease. Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found in, for example, V. McKusick, Mendehan Inheπtance m Man (available on-line through Johns Hopkins University Welch Medical Library). The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (co- mheπtance of physically adjacent genes). Precise human chromosomal localization's for a genomic sequence (gene fragment etc.) can be determined usmg Radiation Hybπd (RH) Mapping (Walter, M Spillett, D., Thomas, P., Weissenbach, J., and Goodfellow, P., (1994) A method for constructing radiation hybπd maps of whole genomes, Nature Genetics 7, 22-28). A number of RH panels are available from Research Genetics (Huntsville, AL, USA) e.g. the GeneBndge4 RH panel (Hum Mol Genet 1996 Mar;5(3):339-46 A radiation hybπd map of the human genome. Gyapay G, Schmitt K, Fizames C, Jones H, Vega-Czarny N, Spillett D, Muselet D, Prud'Homme JF, Dib C, Auffray C, Moπssette J, Weissenbach J, Goodfellow PN). To determine the chromosomal location of a gene usmg this panel, 93 PCRs are performed using pπmers designed from the gene of interest on RH DNAs. Each of these DNAs contains random human genomic fragments maintained in a hamster background (human / hamster hybrid cell lines). These PCRs result in 93 scores indicating the presence or absence of the PCR product of the gene of interest. These scores are compared with scores created usmg PCR products from genomic sequences of known location. This comparison is conducted at http://www.genome.wi.mit.edu . The gene of the present mvention maps to human chromosome 12pl2.3-pl3 The polynucleotide sequences of the present mvention are also valuable tools for tissue expression studies. Such studies allow the determination of expression patterns of polynucleotides of the present mvention which may give an indication as to the expression patterns of the encoded polypeptides m tissues, by detecting the mRNAs that encode them. The techniques used are well known in the art and include in situ hybπdization techniques to clones arrayed on a gπd, such as cDNA microaπay hybπdization (Schena et al, Science, 270, 467-470, 1995 and Shalon et al, Genome Res, 6, 639-645, 1996) and nucleotide amplification techniques such as PCR. A preferred method uses the TAQMAN (Trade mark) technology available from Perkm Elmer. Results from these studies can provide an indication of the normal function of the polypeptide m the organism. In addition, comparative studies of the normal expression pattern of mRNAs with that of mRNAs encoded by an alternative form of the same gene (for example, one having an alteration m polypeptide coding potential or a regulatory mutation) can provide valuable insights mto the role of the polypeptides of the present invention, or that of mappropπate expression thereof in disease. Such mappropπate expression may be of a temporal, spatial or simply quantitative nature. The polypeptides of the present mvention are expressed in testis, small intestine, lung, putuitary gland, spleen, pancreas, stomach with lower levels of expression m placenta and prostate. A further aspect of the present mvention relates to antibodies. The polypeptides of the invention or their fragments, or cells expressing them, can be used as lmmunogens to produce antibodies that are lmmunospecific for polypeptides of the present invention. The term "lmmunospecific" means that the antibodies have substantially greater affinity for the polypeptides of the invention than their affinity for other related polypeptides m the pπor art. Antibodies generated against polypeptides of the present mvention may be obtained by administering the polypeptides or epitope-beaπng fragments, or cells to an animal, preferably a non- human animal, using routine protocols. For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybπdoma technique (Kohler, G. and Milstein, C, Nature (1975) 256:495-497), the tπoma technique, the human B-cell hybπdoma technique (Kozbor et al, Immunology Today (1983) 4:72) and the EBV- hybπdoma technique (Cole et al, Monoclonal Antibodies and Cancer Therapy, 77-96, Alan R. Liss, Inc., 1985). Techmques for the production of single chain antibodies, such as those descπbed in U.S. Patent No 4,946,778, can also be adapted to produce smgle chain antibodies to polypeptides of this mvention Also, transgenic mice, or other organisms, including other mammals, may be used to express humanized antibodies The above-descπbed antibodies may be employed to isolate or to identify clones expressing the polypeptide or to punfy the polypeptides by affinity chromatography Antibodies against polypeptides of the present mvention may also be employed to treat diseases of the invention, amongst others Polypeptides and polynucleotides of the present invention may also be used as vaccines Accordingly, in a further aspect, the present invention relates to a method for inducing an immunological response m a mammal that comprises inoculating the mammal with a polypeptide of the present invention, adequate to produce antibody and/or T cell immune response, including, for example, cytokme-producmg T cells or cytotoxic T cells, to protect said animal from disease, whether that disease is already established with the individual or not. An immunological response in a mammal may also be induced by a method compπses dehveπng a polypeptide of the present invention via a vector directing expression of the polynucleotide and coding for the polypeptide in vivo in order to induce such an immunological response to produce antibody to protect said animal from diseases of the invention. One way of admmisteπng the vector is by accelerating it mto the desired cells as a coating on particles or otherwise Such nucleic acid vector may compπse DNA, RNA, a modified nucleic acid, or a DNA/RNA hybrid. For use a vaccine, a polypeptide or a nucleic acid vector will be normally provided as a vaccine formulation (composition). The formulation may further compπse a suitable earner. Since a polypeptide may be broken down in the stomach, it is preferably administered parenterally (for instance, subcutaneous, mtra-muscular, intravenous, or intra-dermal injection). Formulations suitable for parenteral administration include aqueous and non-aqueous steπle injection solutions that may contain anti-oxidants, buffers, bacteπostats and solutes that render the formulation lnstomc with the blood of the recipient; and aqueous and non- aqueous steπle suspensions that may mclude suspending agents or thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-dπed condition requiπng only the addition of the steπle liquid earner immediately pπor to use. The vaccine formulation may also mclude adjuvant systems for enhancing the lmmunogenicity of the formulation, such as oil-m water systems and other systems known in the art The dosage will depend on the specific activity of the vaccine and can be readily determined by routine expeπmentation. Polypeptides of the present mvention have one or more biological functions that are of relevance in one or more disease states, m particular the diseases of the mvention hereinbefore mentioned It is therefore useful to identify compounds that stimulate or inhibit the function or level of the polypeptide. Accordmgly, m a further aspect, the present invention provides for a method of screenmg compounds to identify those that stimulate or inhibit the function or level of the polypeptide Such methods identify agonists or antagonists that may be employed for therapeutic and prophylactic purposes for such diseases of the mvention as hereinbefore mentioned. Compounds may be identified from a vaπety of sources, for example, cells, cell-free preparations, chemical hbraπes, collections of chemical compounds, and natural product mixtures. Such agonists or antagonists so-identified may be natural or modified substrates, hgands, receptors, enzymes, etc., as the case may be, of the polypeptide, a structural or functional mimetic thereof (see Cohgan et al , Current Protocols in Immunology l(2):Chapter 5 (1991)) or a small molecule. Such small molecules preferably have a molecular weight below 2,000 daltons, more preferably between 300 and 1,000 daltons, and most preferably between 400 and 700 daltons. It is preferred that these small molecules are organic molecules The screenmg method may simply measure the binding of a candidate compound to the polypeptide, or to cells or membranes beaπng the polypeptide, or a fusion protein thereof, by means of a label directly or indirectly associated with the candidate compound. Alternatively, the screenmg method may involve measuπng or detecting (qualitatively or quantitatively) the competitive binding of a candidate compound to the polypeptide against a labeled competitor (e.g agonist or antagonist). Further, these screening methods may test whether the candidate compound results in a signal generated by activation or inhibition of the polypeptide, using detection systems appropπate to the cells beaπng the polypeptide. Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist by the presence of the candidate compound is observed. Further, the screenmg methods may simply compπse the steps of mixing a candidate compound with a solution containing a polypeptide of the present invention, to form a mixture, measuπng a AXOR39 activity m the mixture, and compaπng the AXOR39 activity of the mixture to a control mixture which contains no candidate compound. Polypeptides of the present invention may be employed m conventional low capacity screenmg methods and also m high-throughput screening (HTS) formats. Such HTS formats mclude not only the well-established use of 96- and, more recently, 384-well micotiter plates but also emerging methods such as the nanowell method descnbed by Schullek et al, Anal Biochem., 246, 20-29, (1997). Fusion protems, such as those made from Fc portion and AXOR39 polypeptide, as hereinbefore descnbed, can also be used for high-throughput screening assays to identify antagonists for the polypeptide of the present invention (see D. Bennett et al., J Mol Recognition, 8:52-58 (1995); and K. Johanson et al., J Biol Chem, 270(16):9459-9471 (1995)). One screenmg technique includes the use of cells which express the receptor of this mvention (for example, transfected CHO cells) in a system which measures extracellular pH or mtracellular calcium changes caused by receptor activation. In this technique, compounds may be contacted with cells expressmg the receptor polypeptide of the present mvention. A second messenger response, e.g , signal transduction, pH changes, or changes in calcium level, is then measured to determine whether the potential compound activates or inhibits the receptor Another method involves screenmg for receptor inhibitors by determining inhibition or stimulation of receptor-mediated cAMP and/or adenylate cyclase accumulation. Such a method involves transfecting a eukaryotic cell with the receptor of this mvention to express the receptor on the cell surface. The cell is then exposed to potential antagonists m the presence of the receptor of this mvention. The amount of cAMP accumulation is then measured If the potential antagonist binds the receptor, and thus inhibits receptor binding, the levels of receptor-mediated cAMP, or adenylate cyclase, activity will be reduced or mcreased. Another method for detecting agonists or antagonists for the receptor of the present mvention is the yeast based technology as descnbed in U.S. Patent No. 5,482,835. The polynucleotides, polypeptides and antibodies to the polypeptide of the present invention may also be used to configure screenmg methods for detecting the effect of added compounds on the production of mRNA and polypeptide in cells. For example, an ELISA assay may be constructed for measuπng secreted or cell associated levels of polypeptide using monoclonal and polyclonal antibodies by standard methods known in the art. This can be used to discover agents that may inhibit or enhance the production of polypeptide (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues. A polypeptide of the present invention may be used to identify membrane bound or soluble receptors, if any, through standard receptor binding techniques known in the art. These include, but are not limited to, ligand binding and crosslmking assays m which the polypeptide is labeled with a radioactive isotope (for instance, ^^1), chemically modified (for instance, biotmylated), or fused to a peptide sequence suitable for detection or punfϊcation, and incubated with a source of the putative receptor (cells, cell membranes, cell supernatants, tissue extracts, bodily fluids). Other methods include biophysical techniques such as surface plasmon resonance and spectroscopy. These screenmg methods may also be used to identify agonists and antagonists of the polypeptide that compete with the binding of the polypeptide to its receptors, if any. Standard methods for conducting such assays are well understood in the art. Examples of antagonists of polypeptides of the present mvention mclude antibodies or, in some cases, ohgonucleotides or protems that are closely related to the hgands, substrates, receptors, enzymes, etc., as the case may be, of the polypeptide, e g., a fragment of the hgands, substrates, receptors, enzymes, etc.; or a small molecule that bmd to the polypeptide of the present mvention but do not elicit a response, so that the activity of the polypeptide is prevented. Screenmg methods may also involve the use of transgemc technology and AXOR39 gene. The art of constructing transgemc animals is well established. For example, the AXOR39 gene may be introduced through micromjection mto the male pronucleus of fertilized oocytes, retroviral transfer mto pre- or post-implantation embryos, or injection of genetically modified, such as by electroporation, embryonic stem cells mto host blastocysts. Particularly useful transgemc animals are so-called "knock-m" animals in which an animal gene is replaced by the human equivalent within the genome of that animal. Knock-m transgemc animals are useful m the drug discovery process, for target validation, where the compound is specific for the human target. Other useful transgemc animals are so-called "knock-out" animals in which the expression of the animal ortholog of a polypeptide of the present invention and encoded by an endogenous DNA sequence m a cell is partially or completely annulled. The gene knock-out may be targeted to specific cells or tissues, may occur only in certam cells or tissues as a consequence of the limitations of the technology, or may occur in all, or substantially all, cells in the animal. Transgemc animal technology also offers a whole animal expression-clonmg system in which introduced genes are expressed to give large amounts of polypeptides of the present mvention Screening kits for use in the above descnbed methods form a further aspect of the present invention. Such screening kits comprise' (a) a polypeptide of the present mvention; (b) a recombmant cell expressing a polypeptide of the present mvention; (c) a cell membrane expressing a polypeptide of the present mvention; or (d) an antibody to a polypeptide of the present mvention; which polypeptide is preferably that of SEQ ID NO:2. It will be appreciated that in any such kit, (a), (b), (c) or (d) may compπse a substantial component. Glossary The following definitions are provided to facilitate understanding of certain terms used frequently hereinbefore "Antibodies" as used herein includes polyclonal and monoclonal antibodies, chimenc, single chain, and humanized antibodies, as well as Fab fragments, including the products of an Fab or other lmmunoglobuhn expression library "Isolated" means altered "by the hand of man" from its natural state, i e , if it occurs m nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or a polypeptide naturally present in a living organism is not "isolated," but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated", as the term is employed herem. Moreover, a polynucleotide or polypeptide that is introduced into an organism by transformation, genetic manipulation or by any other recombinant method is "isolated" even if it is still present in said organism, which organism may be living or non-living. "Polynucleotide" generally refers to any polynbonucleotide (RNA) or polydeoxπbonucleotide (DNA), which may be unmodified or modified RNA or DNA "Polynucleotides" mclude, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybπd molecules compnsmg DNA and RNA that may be smgle-stranded or, more typically, double-stranded or a mixture of single- and double- stranded regions. In addition, "polynucleotide" refers to tπple-stranded regions comprising RNA or DNA or both RNA and DNA The term "polynucleotide" also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons. "Modified" bases include, for example, tπtylated bases and unusual bases such as inosine A vanety of modifications may be made to DNA and RNA; thus, "polynucleotide" embraces chemically, enzymatically or metabohcally modified forms of polynucleotides as typically found m nature, as well as the chemical forms of DNA and RNA characteπstic of viruses and cells. "Polynucleotide" also embraces relatively short polynucleotides, often referred to as ohgonucleotides. "Polypeptide" refers to any polypeptide compπsmg two or more ammo acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres "Polypeptide" refers to both short chains, commonly refeπed to as peptides, oligopeptides or o gomers, and to longer chains, generally referred to as protems Polypeptides may contain ammo acids other than the 20 gene-encoded ammo acids "Polypeptides" mclude ammo acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques that are well known in the art. Such modifications are well descnbed m basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications may occur anywhere in a polypeptide, including the peptide backbone, the ammo acid side-chams and the ammo or carboxyl termini. It will be appreciated that the same type of modification may be present to the same or varying degrees at several sites m a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched and branched cyclic polypeptides may result from post-translation natural processes or may be made by synthetic methods. Modifications mclude acetylation, acylation, ADP-πbosylation, amidation, biotmylation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide denvative, covalent attachment of a hpid or hpid deπvative, covalent attachment of phosphotidylmositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma- carboxylation, glycosylation, GPI anchor formation, hydroxylation, lodmation, methylation, mynstoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of ammo acids to protems such as argmylation, and ubiquitination (see, for instance, Protems - Structure and Molecular Properties, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York, 1993; Wold, F., Post-translational Protein Modifications: Perspectives and Prospects, 1-12, in Post-translational Covalent Modification of Protems, B. C. Johnson, Ed., Academic Press, New York, 1983; Seifter et al, "Analysis for protein modifications and nonprotem cofactors", Meth Enzymol, 182, 626-646, 1990, and Rattan et al , "Protein Synthesis: Post-translational Modifications and Agmg", Ann NY Acad Sci, 663, 48-62, 1992). "Fragment" of a polypeptide sequence refers to a polypeptide sequence that is shorter than the reference sequence but that retains essentially the same biological function or activity as the reference polypeptide. "Fragment" of a polynucleotide sequence refers to a polynucleotide sequence that is shorter than the reference sequence of SEQ ID NO: 1. "Vanant" refers to a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, but retains the essential properties thereof. A typical vanant of a polynucleotide differs in nucleotide sequence from the reference polynucleotide. Changes m the nucleotide sequence of the vanant may or may not alter the ammo acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in ammo acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below. A typical vanant of a polypeptide differs in amino acid sequence from the reference polypeptide Generally, alterations are limited so that the sequences of the reference polypeptide and the vanant are closely similar overall and, in many regions, identical A vanant and reference polypeptide may differ in ammo acid sequence by one or more substitutions, insertions, deletions in any combination. A substituted or inserted ammo acid residue may or may not be one encoded by the genetic code. Typical conservative substitutions mclude Gly, Ala; Val, Le, Leu; Asp, Glu; Asn, Gin; Ser, Thr; Lys, Arg; and Phe and Tyr. A vanant of a polynucleotide or polypeptide may be naturally occumng such as an allele, or it may be a vanant that is not known to occur naturally. Non-naturally occurπng variants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis Also included as vaπants are polypeptides having one or more post-translational modifications, for instance glycosylation, phosphorylation, methylation, ADP πbosylation and the like. Embodiments include methylation of the N-termmal ammo acid, phosphorylations of sennes and threonmes and modification of C-termmal glycmes "Allele" refers to one of two or more alternative forms of a gene occurπng at a given locus in the genome "Polymorphism" refers to a vaπation m nucleotide sequence (and encoded polypeptide sequence, if relevant) at a given position in the genome within a population. "Single Nucleotide Polymorphism" (SNP) refers to the occurrence of nucleotide vaπability at a single nucleotide position m the genome, within a population. An SNP may occur within a gene or within mtergemc regions of the genome. SNPs can be assayed using Allele Specific Amplification (ASA). For the process at least 3 pπmers are required. A common pπmer is used m reverse complement to the polymorphism being assayed. This common pπmer can be between 50 and 1500 bps from the polymorphic base. The other two (or more) pπmers are identical to each other except that the final 3' base wobbles to match one of the two (or more) alleles that make up the polymorphism. Two (or more) PCR reactions are then conducted on sample DNA, each usmg the common pπmer and one of the Allele Specific Pπmers. "Splice Vanant" as used herein refers to cDNA molecules produced from RNA molecules initially transcπbed from the same genomic DNA sequence but which have undergone alternative RNA splicing. Alternative RNA splicing occurs when a primary RNA transcnpt undergoes splicing, generally for the removal of mtrons, which results in the production of more than one mRNA molecule each of that may encode different ammo acid sequences. The term splice vanant also refers to the protems encoded by the above cDNA molecules. "Identity" reflects a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, determined by compaπng the sequences. In general, identity refers to an exact nucleotide to nucleotide or ammo acid to amino acid correspondence of the two polynucleotide or two polypeptide sequences, respectively, over the length of the sequences being compared "% Identity" - For sequences where there is not an exact coπespondence, a "% identity" may be determined. In general, the two sequences to be compared are aligned to give a maximum correlation between the sequences. This may mclude inserting "gaps" in either one or both sequences, to enhance the degree of alignment. A % identity may be determined over the whole length of each of the sequences being compared (so-called global alignment), that is particularly suitable for sequences of the same or very similar length, or over shorter, defined lengths (so-called local alignment), that is more suitable for sequences of unequal length. "Similaπty" is a further, more sophisticated measure of the relationship between two polypeptide sequences. In general, "similaπty" means a compaπson between the ammo acids of two polypeptide chains, on a residue by residue basis, taking mto account not only exact coπespondences between a between pairs of residues, one from each of the sequences being compared (as for identity) but also, where there is not an exact correspondence, whether, on an evolutionary basis, one residue is a likely substitute for the other. This likelihood has an associated "score" from which the "% similaπty" of the two sequences can then be determined. Methods for companng the identity and similaπty of two or more sequences are well known in the art. Thus for instance, programs available in the Wisconsin Sequence Analysis Package, version 9.1 (Devereux J et al, Nucleic Acids Res, 12, 387-395, 1984, available from Genetics Computer Group, Madison, Wisconsin, USA), for example the programs BESTFIT and GAP, may be used to determine the % identity between two polynucleotides and the % identity and the % similaπty between two polypeptide sequences. BESTFΓT uses the "local homology" algonthm of Smith and Waterman (J Mol Biol, 147,195-197, 1981, Advances in Applied Mathematics, 2, 482- 489, 1981) and finds the best single region of similaπty between two sequences. BESTFIT is more suited to companng two polynucleotide or two polypeptide sequences that are dissimilar in length, the program assuming that the shorter sequence represents a portion of the longer. In companson, GAP aligns two sequences, finding a "maximum similaπty", according to the algonthm of Neddleman and Wunsch (J Mol Biol, 48, 443-453, 1970). GAP is more suited to companng sequences that are approximately the same length and an alignment is expected over the entire length. Preferably, the parameters "Gap Weight" and "Length Weight" used m each program are 50 and 3, for polynucleotide sequences and 12 and 4 for polypeptide sequences, respectively. Preferably, % identities and similaπties are determined when the two sequences being compared are optimally aligned Other programs for determining identity and/or similaπty between sequences are also known in the art, for instance the BLAST family of programs (Altschul S F et al, J Mol Biol, 215, 403-410, 1990, Altschul S F et al, Nucleic Acids Res., 25:389-3402, 1997, available from the National Center for Biotechnology Information (NCBI), Bethesda, Maryland, USA and accessible through the home page of the NCBI at www.ncbi.nlm.mh.gov) and FASTA (Pearson W R, Methods m Enzymology, 183, 63-99, 1990; Pearson W R and Lipman D J, Proc Nat Acad Sci USA, 85, 2444-2448,1988, available as part of the Wisconsin Sequence Analysis Package). Preferably, the BLOSUM62 ammo acid substitution matπx (Hemkoff S and Hemkoff J G, Proc. Nat. Acad Sci. USA, 89, 10915-10919, 1992) is used in polypeptide sequence compansons including where nucleotide sequences are first translated mto ammo acid sequences before compaπson Preferably, the program BESTFIT is used to determine the % identity of a query polynucleotide or a polypeptide sequence with respect to a reference polynucleotide or a polypeptide sequence, the query and the reference sequence being optimally aligned and the parameters of the program set at the default value, as hereinbefore descnbed. "Identity Index" is a measure of sequence relatedness which may be used to compare a candidate sequence (polynucleotide or polypeptide) and a reference sequence. Thus, for instance, a candidate polynucleotide sequence having, for example, an Identity Index of 0.95 compared to a reference polynucleotide sequence is identical to the reference sequence except that the candidate polynucleotide sequence may include on average up to five differences per each 100 nucleotides of the reference sequence. Such differences are selected from the group consistmg of at least one nucleotide deletion, substitution, including transition and transversion, or insertion. These differences may occur at the 5' or 3' terminal positions of the reference polynucleotide sequence or anywhere between these terminal positions, interspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups withm the reference sequence. In other words, to obtain a polynucleotide sequence having an Identity Index of 0.95 compared to a reference polynucleotide sequence, an average of up to 5 m every 100 of the nucleotides of the m the reference sequence may be deleted, substituted or inserted, or any combination thereof, as hereinbefore descnbed The same applies mutatis mutandis for other values of the Identity Index, for instance 0.96, 0.97, 0.98 and 0.99 Similarly, for a polypeptide, a candidate polypeptide sequence having, for example, an Identity Index of 0.95 compared to a reference polypeptide sequence is identical to the reference sequence except that the polypeptide sequence may mclude an average of up to five differences per each 100 am o acids of the reference sequence. Such differences are selected from the group consistmg of at least one ammo acid deletion, substitution, including conservative and non- conservative substitution, or insertion. These differences may occur at the ammo- or carboxy- termmal positions of the reference polypeptide sequence or anywhere between these terminal positions, interspersed either individually among the ammo acids in the reference sequence or in one or more contiguous groups within the reference sequence. In other words, to obtain a polypeptide sequence having an Identity Index of 0.95 compared to a reference polypeptide sequence, an average of up to 5 in every 100 of the ammo acids in the reference sequence may be deleted, substituted or mserted, or any combination thereof, as hereinbefore descnbed The same applies mutatis mutandis for other values of the Identity Index, for instance 0.96, 0.97, 0.98 and 0.99. The relationship between the number of nucleotide or ammo acid differences and the Identity Index may be expressed in the following equation n a < x a - (x a • I), m which: n a is the number of nucleotide or ammo acid differences, x a is the total number of nucleotides or ammo acids m SEQ ID NO:l or SEQ ID NO:2, respectively, I is the Identity Index, • is the symbol for the multiplication operator, and m which any non-mteger product of x a and I is rounded down to the nearest integer pπor to subtracting it from x a "Homolog" is a geneπc term used in the art to indicate a polynucleotide or polypeptide sequence possessing a high degree of sequence relatedness to a reference sequence. Such relatedness may be quantified by determining the degree of identity and/or similaπty between the two sequences as hereinbefore defined. Falling with this geneπc term are the terms "ortholog", and "paralog". "Ortholog" refers to a polynucleotide or polypeptide that is the functional equivalent of the polynucleotide or polypeptide in another species. "Paralog" refers to a polynucleotide or polypeptide that withm the same species which is functionally similar. "Fusion protein" refers to a protein encoded by two, often unrelated, fused genes or fragments thereof. In one example, EP-A-0 464 533-A discloses fusion proteins compπsing vaπous portions of constant region of immunoglobulin molecules together with another human protein or part thereof In many cases, employing an immunoglobulin Fc region as a part of a fusion protein is advantageous for use in therapy and diagnosis resulting m, for example, improved pharmacokmetic properties [see, e.g., EP-A 0232 262]. On the other hand, for some uses it would be desirable to be able to delete the Fc part after the fusion protein has been expressed, detected and puπfied. All publications and references, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference m their entirety as if each individual publication or reference were specifically and mdividually indicated to be incorporated by reference herein as being fully set forth Any patent application to which this application claims pnority is also incorporated by reference herein m its entirety in the manner descnbed above for publications and references. Examples Example 1 : Mammalian Cell Expression The receptors of the present mvention are expressed in either human embryonic kidney 293 (HEK293) cells or adherent dhfr CHO cells. To maximize receptor expression, typically all 5' and 3' untranslated regions (UTRs) are removed from the receptor cDNA pπor to insertion mto a pCDN or pCDNA3 vector. The cells are transfected with individual receptor cDNAs by hpofectin and selected in the presence of 400 mg/ml G418. After 3 weeks of selection, individual clones are picked and expanded for further analysis. HEK293 or CHO cells transfected with the vector alone serve as negative controls. To isolate cell lmes stably expressing the individual receptors, about 24 clones are typically selected and analyzed by Northern blot analysis. Receptor mRNAs are generally detectable in about 50% of the G418-resιstant clones analyzed Example 2 : Ligand bank for binding and functional assays. A bank of over 600 putative receptor hgands has been assembled for screenmg. The bank compπses: transmitters, hormones and chemok es known to act via a human seven transmembrane (7TM) receptor; naturally occurπng compounds which may be putative agonists for a human 7TM receptor, non-mammalian, biologically active peptides for which a mammalian counterpart has not yet been identified; and compounds not found in nature, but which activate 7TM receptors with unknown natural hgands. This bank is used to initially screen the receptor for known hgands, usmg both functional (i.e. . calcium, cAMP, microphysiometer, oocyte electrophysiology, etc, see below) as well as binding assays. Example 3 Ligand Binding Assays Ligand binding assays provide a direct method for ascertaining receptor pharmacology and are adaptable to a high throughput format. The puπfied ligand for a receptor is radiolabeled to high specific activity (50-2000 Ci/mmol) for binding studies. A determination is then made that the process of radiolabehng does not dimmish the activity of the ligand towards its receptor. Assay conditions for buffers, ions, pH and other modulators such as nucleotides are optimized to establish a workable signal to noise ratio for both membrane and whole cell receptor sources. For these assays, specific receptor binding is defined as total associated radioactivity minus the radioactivity measured m the presence of an excess of unlabeled competing ligand. Where possible, more than one competing ligand is used to define residual nonspecific binding. Example 4: Functional Assay in Xenopus Oocytes Capped RNA transcπpts from lmeaπzed plasmid templates encodmg the receptor cDNAs of the invention are synthesized in vitro with RNA polymerases in accordance with standard procedures In vitro transcπpts are suspended in water at a final concentration of 0.2 mg/ml. Ovaπan lobes are removed from adult female toads, Stage V defolliculated oocytes are obtained, and RNA transcπpts (10 ng/oocyte) are injected m a 50 nl bolus using a micromjection apparatus. Two electrode voltage clamps are used to measure the currents from individual Xenopus oocytes m response to agonist exposure. Recordings are made in Ca2+ free Barth's medium at room temperature. The Xenopus system can be used to screen known hgands and tissue/cell extracts for activating hgands. Example 5. Microphysiometπc Assays Activation of a wide vaπety of secondary messenger systems results m extrusion of small amounts of acid from a cell. The acid formed is largely as a result of the increased metabolic activity required to fuel the lntracellular signaling process. The pH changes m the media surrounding the cell are very small but are detectable by the CYTOSENSOR microphysiometer (Molecular Devices Ltd., Menlo Park, CA). The CYTOSENSOR is thus capable of detecting the activation of a receptor which is coupled to an energy utilizing tracellular signaling pathway such as the G-protem coupled receptor of the present invention. Example 6: Extract/Cell Supernatant Screenmg A large number of mammalian receptors exist for which there remains, as yet, no cognate activating ligand (agonist) Thus, active hgands for these receptors may not be included withm the ligand banks as identified to date Accordingly, the 7TM receptor of the invention is also functionally screened (usmg calcium, cAMP, microphysiometer, oocyte electrophysiology, etc., functional screens) against tissue extracts to identify natural hgands. Extracts that produce positive functional responses can be sequentially subfractionated until an activating ligand is isolated identified. Example 7- Calcium and cAMP Functional Assays 7TM receptors which are expressed in HEK 293 cells have been shown to be coupled functionally to activation of PLC and calcium mobilization and/or cAMP stimulation or inhibition Basal calcium levels m the HEK 293 cells in receptor-transfected or vector control cells were observed to be m the normal, 100 nM to 200 nM, range. HEK 293 cells expressmg recombmant receptors are loaded with fura 2 and in a smgle day > 150 selected hgands or tissue/cell extracts are evaluated for agomst induced calcium mobilization. Similarly, HEK 293 cells expressmg recombmant receptors are evaluated for the stimulation or inhibition of c AMP production usmg standard cAMP quantitation assays. Agonists presenting a calcium transient or cAMP fluctuation are tested m vector control cells to determine if the response is unique to the transfected cells expressing receptor.

Claims: What is claimed is: 1. An isolated polypeptide selected from the group consisting of: (a) an isolated polypeptide encoded by a polynucleotide compπsing the sequence of SEQ ID NO: 1 , (b) an isolated polypeptide compπsmg a polypeptide sequence having at least 95% identity to the polypeptide sequence of SEQ ID NO:2; (c) an isolated polypeptide compπsing the polypeptide sequence of SEQ ID NO:2; (d) an isolated polypeptide having at least 95%> identity to the polypeptide sequence of SEQ ID NO:2; (e) the polypeptide sequence of SEQ ID NO:2; and (f) fragments and vaπants of such polypeptides m (a) to (e) 2. An isolated polynucleotide selected from the group consistmg of: (a) an isolated polynucleotide compπsing a polynucleotide sequence having at least 95%) identity to the polynucleotide sequence of SEQ ID NO: 1 ; (b) an isolated polynucleotide compπsmg the polynucleotide of SEQ ID NO: 1; (c) an isolated polynucleotide having at least 95% identity to the polynucleotide of SEQ ID NO: 1 ; (d) the isolated polynucleotide of SEQ ID NO: 1 ; (e) an isolated polynucleotide compπsmg a polynucleotide sequence encodmg a polypeptide sequence having at least 95% identity to the polypeptide sequence of SEQ ID NO:2; (f) an isolated polynucleotide compπsmg a polynucleotide sequence encodmg the polypeptide of SEQ ID NO:2; (g) an isolated polynucleotide having a polynucleotide sequence encoding a polypeptide sequence having at least 95% identity to the polypeptide sequence of SEQ ID NO:2; (h) an isolated polynucleotide encodmg the polypeptide of SEQ ID NO:2; (l) an isolated polynucleotide with a nucleotide sequence of at least 100 nucleotides obtained by screenmg a library under stnngent hybπdization conditions with a labeled probe having the sequence of SEQ ID NO: 1 or a fragment thereof having at least 15 nucleotides; (j) a polynucleotide which is the RNA equivalent of a polynucleotide of (a) to (l); or a polynucleotide sequence complementary to said isolated polynucleotide and polynucleotides that are vaπants and fragments of the above mentioned polynucleotides or that are complementary to above mentioned polynucleotides, over the entire length thereof. 3. An antibody lmmunospecific for the polypeptide of claim 1. 4. An antibody as claimed in claim 3 which is a polyclonal antibody. 5. An expression vector compπsing a polynucleotide capable of producing a polypeptide of claim 1 when said expression vector is present in a compatible host cell. 6. A process for producing a recombmant host cell which compπses the step of introducing an expression vector compπsmg a polynucleotide capable of producing a polypeptide of claim 1 mto a cell such that the host cell, under appropriate culture conditions, produces said polypeptide. 7. A recombmant host cell produced by the process of claim 6. 8. A membrane of a recombmant host cell of claim 7 expressing said polypeptide. 9. A process for producing a polypeptide which compnses cultunng a host cell of claim 7 under conditions sufficient for the production of said polypeptide and recoveπng said polypeptide from the culture.

Inventors: ELSHOURBAGY NABIL, LANE PAMELA, MICHALOVICH DAVID, PANGALOS MENELAS, ROBBINS MELANIE, ELSHOURBAGY, NABIL, LANE, PAMELA, MICHALOVICH, DAVID, PANGALOS, MENELAS, ROBBINS, MELANIE
Applicants: SMITHKLINE BEECHAM CORP, SMITHKLINE BEECHAM PLC, SMITHKLINE BEECHAM CORPORATION
Application year: 2000
Main classification: C12N15
IPC codes: A61K39, C07K14, C12N15