WO2001019984A1

WO2001019984A1 - Rgs8like polypeptide and polynucleotides, a member of the regulators of g protein signalling family of polypeptides

Info

Publication number: WO2001019984A1
Application number: PCT/GB2000/003501
Authority: WO
Inventors: David Malcolm Duckworth
Original assignee: Smithkline Beecham P.L.C.
Priority date: 1999-09-14
Filing date: 2000-09-11
Publication date: 2001-03-22
Also published as: GB9921690D0; EP1131423A1

Abstract

RGS8LIKE polypeptides and polynucleotides member of the regulators of G protein signalling family of polypeptides and methods for producing such polypeptides by recombinant techniques are disclosed. Also disclosed are methods for utilizing RGS8LIKE polypeptides and polynucleotides in diagnostics assays.

Description

RGS8LIKE POLYPEPTIDE AND POLYNUCLEOTIDES, A MEMBER OF THE REGULATORS OF G PROTEIN SIGNALLING FAMILY OF

Field of the Invention POLYPEPTIDES

This invention relates to newly identified polypeptides and polynucleotides encoding such polypeptides, to their use in diagnosis and in identifying compounds that mav 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 genorrucs", 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 superceding 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 genorrucs relies heavily on high-throughput DNA sequencing technologies and the various 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 characterise further genes and their related polypeptides/proteins, as targets for drug discovery

Summary of the Invention

The present invention relates to RGS8LIKE, in particular RGS8LIKE polypeptides and RGS8LIKE polynucleotides, recombinant materials and methods for their production Such polypeptides and polynucleotides are of interest in relation to methods of treatment of certain diseases, including, but not limited to neurological conditions such as pain, hereinafter referred to as "diseases of the invention" In a further aspect, the invention relates to methods for identifying agonists and antagonists (e g , inhibitors) using the mateπals provided by the invention, and treating conditions associated with RGS8LIKE imbalance with the identified compounds In a still further aspect, the invention relates to diagnostic assays for detecting diseases associated with inappropriate RGS8LIKE activity or levels

Description of the Invention

In a first aspect, the present invention relates to RGS8LIKE polypeptides Such polypeptides include (a) an isolated polypeptide encoded by a polynucleotide comprising the sequence of SEQ ID NO 1 ,

(b) an isolated polypeptide comprising 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 comprising the polvpeptide sequence of SEQ ID NO 2 (d) an isolated polypeptide having at least 95% 967c, 977c 987c, or 997o identity to the polypeptide sequence of SEQ ID NO 2,

(e) the polypeptide sequence of SEQ ID NO 2, and

(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,

(g) fragments and variants of such polypeptides in (a) to (f)

Polypeptides of the present invention are believed to be members of the regulators of G protein signalling family of polypeptides They are therefore of interest because G proteins have an important function in cell signalling pathways These are GTP binding proteins and are heterotπmers consisting of alpha, beta and gamma subunits When G proteins are inactive, the alpha subumt is bound the beta and gamma subunits and GDP In contrast, when G proteins are active and the signalling pathway is on, the alpha subumt is dissociated form the beta-gamma complex and is bound to GTP Inactivation is accomplished through hydrolysis of the bound GTP to GDP and the rate of hydrolysis determines the time the G protein is active and the duration of the physiological response G proteins are GTPases but the process is accelerated by GTPase activating proteins (GAPs) Regulators of G protein signalling (RGS proteins) act as GAPs They are of interest because some negatively modulate G protein-coupled receptor neurotransrrutter responses in neurons or are involved in the activation and deactivation kinetics of potassium and calcium channels The biological properties of the RGS8LIKE are hereinafter referred to as "biological activity of RGS8LIKE" or "RGS8LIKE activity" Preferably, a polypeptide of the present invention exhibits at least one biological activity of RGS8LIKE

Polypeptides of the present invention also includes variants of the aforementioned polypeptides, including all allehc forms and splice variants 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 variants are those 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 amino acids are inserted, substituted, or deleted, in any combination

Preferred fragments of polypeptides of the present invention include an isolated polypeptide comprising an amino acid sequence having at least 30, 50 or 100 contiguous amino acids from the amino acid sequence of SEQ ID NO 2, or an isolated polypeptide comprising an amino acid sequence having at least 30 50 or 100 contiguous amino 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 RGS8LIKE, 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 in a human

Fragments of the polypeptides of the invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis, therefore, these variants may be

- 2 - employed as intermediates tor producing the full-length polypeptides of the invention The polypeptides of the present invention 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 include an additional amino acid sequence that contains secretory or leader sequences, pro-sequences, sequences that aid in purification, for instance multiple histidine residues, or an additional sequence tor stability during recombinant production

Polypeptides of the present invention can be prepared in any suitable manner, for instance by isolation form naturally occunng sources, from genetically engineered host cells compπsing expression systems (vide infra) or by chemical synthesis, using for instance automated peptide synthesisers, 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 RGS8LIKE polynucleotides Such polynucleotides include (a) an isolated polynucleotide compπsing a polynucleotide sequence having at least 957c, 967c, 977c, 987o, or 997o identity to the polynucleotide squence of SEQ ID NO 1 ,

(b) an isolated polynucleotide compπsing the polynucleotide of SEQ ID NO 1 ,

(c) an isolated polynucleotide having at least 957c, 967c, 977c, 987c, or 997c identity to the polynucleotide of SEQ TD NO 1 , (d) the isolated polynucleotide of SEQ ID NO 1 ,

(e) an isolated polynucleotide compπsing a polynucleotide sequence encoding a polypeptide sequence having at least 957o, 96%, 97%, 987c, or 997c identity to the polypeptide sequence of SEQ ID NO 2,

(f) an isolated polynucleotide compπsing a polynucleotide sequence encoding the polypeptide of SEQ ID NO 2,

(g) an isolated polynucleotide having a polynucleotide sequence encoding a polypeptide sequence having at least 957c, 967c, 977c, 987o, or 997o identity to the polypeptide sequence of SEQ ID NO 2,

(h) an isolated polynucleotide encoding the polypeptide of SEQ ID NO 2, (l) an isolated polynucleotide having or comprising 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 1

(j) an isolated polynucleotide having or comprising a polynucleotide sequence encoding 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 polynucleotides that are fragments and variants 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 invention include an isolated polynucleotide comprising an nucleotide sequence having at least 15 30, 50 or 100 contiguous nucleotides trom the sequence of SEQ ID NO 1 or an isolated polynucleotide comprising an sequence having at least 30, 50 or 100 contiguous nucleotides truncated or deleted from the sequence of SEQ ID NO 1

Preferred vaπants of polynucleotides of the present invention include splice vaπants, alle c vaπants, and polymorphisms, including polynucleotides having one or more single nucleotide polymorphisms (SNPs)

Polynucleotides of the present invention also include polynucleotides encoding polypeptide vaπants that compnse the amino 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 invention provides polynucleotides that are RNA transcπpts of the DNA sequences of the present invention Accordingly, there is provided an RNA polynucleotide that

(a) compπses 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 RNA polynucleotides that are complementary thereto

The polynucleotide sequence of SEQ ID NO 1 shows homology with rat RGS8 (O Saitoh et al , Nature 390 525-529, 1997) The polynucleotide sequence of SEQ ID NO 1 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 1, 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 proteins of the regulators of G protein signalling family, having homology and/or structural similaπty with rat RGS8 (O Saitoh et al , Nature 390 525-529, 1997)

Preferred polypeptides and polynucleotides of the present invention are expected to have inter alia, similar biological functions/properties to their homologous polypeptides and polynucleotides Furthermore, preferred polypeptides and polynucleotides of the present invention have at least one RGS8LIKE activity

Polynucleotides of the present invention may be obtained using standard cloning and screening techniques from a cDNA library deπved from mRNA in cells of human testes, (see for instance, Sambrook et al , Molecular Cloning A Laboratory Manual, 2nd Ed , Cold Spπng Harbor

- 4 - 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 invention, the polynucleotide may include the coding sequence for the mature polypeptide, by itself, or the coding sequence for the mature polypeptide in reading frame with other coding sequences, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protein sequence, or other fusion peptide portions For example, a marker sequence that facilitates puπfication of the fused polypeptide can be encoded. In certain 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 encoding polypeptides of the present invention and to isolate cDNA and genomic clones of other genes (including genes encoding paralogs from human sources and orthologs and paralogs from species other than human) that have a high sequence similaπty to SEQ ID NO. l, 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 encoding a polypeptide of the present invention, including homologs from species other than human, may be obtained by a process compπsing the steps of screening a library under stπngent hybπdization conditions with a labeled probe having the sequence of SEQ ID 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 Preferred stπngent hybπdization conditions include overnight incubation at 42°C in a solution compπsing 50% formamide, 5xSSC (150mM NaCl, 15mM tπsodium citrate), 50 mM sodium phosphate (pH7.6), 5x Denhardt's solution, 10 % dextran sulfate, and 20 microgram/ml denatured, sheared salmon sperm DNA, followed by washing the filters in O.lx SSC at about 65°C Thus the present invention also includes isolated polynucleotides, preferably with a nucleotide sequence of at least 100, obtained by screening a library under stπngent hybridization conditions with a labeled probe having the sequence of SEQ ED NO 1 or a fragment thereof, preferably of at least 15 nucleotides The skilled artisan will appreciate that, in 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 dunng the polymerisation reaction), tailing to complete a DNA copy of the mRNA template during 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 hgated 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 oligonucleotide pnmers The PCR reaction is then repeated using 'nested' pπmers, that is, pπmers designed to anneal within the amplified product (typically an adaptor specific primer 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 analysed 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

Recombinant polypeptides of the present invention may be prepared by processes well known in the art from genetically engineered host cells compπsing expression systems Accordingly, in a further aspect, the present invention relates to expression systems compπsing a polynucleotide or polynucleotides of the present invention, to host cells which are genetically engineered with such expression sytems and to the production of polypeptides of the invention by recombinant techniques Cell-free translation systems can also be employed to produce such proteins using RNAs deπved from the DNA constructs of the present invention

For recombinant production, host cells can be genetically engineered to incorporate expression systems or portions thereof for polynucleotides of the present invention Polynucleotides may be introduced into 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 (ibid) Preferred methods of introducing polynucleotides into host cells include, for instance, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, canonic lφid- mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection

- 6 - Representative examples of appropπate hosts include bacteπal cells, such as Streptococci.

E coll. Streptomvces and Bacillus subtilis cells, fungal cells, such as yeast cells and Aspergillus cells, insect cells such as Drosophda 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 ? , 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, vaccinia 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 appropnate polynucleotide sequence may be inserted into an expression system by any of a vaπety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al , (ibid) Appropnate secretion signals may be incorporated into the desired polypeptide to allow secretion of the translated protein into 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 invention is to be expressed for use in screening 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 into the medium, the medium can be recovered in order to recover and purify the polypeptide If produced intracellularly, the cells must first be lysed before the polypeptide is recovered

Polypeptides of the present invention can be recovered and puπfied from recombinant 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 lectm chromatography Most preferably, high performance liquid chromatography is employed for puπfication Well known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured dunng intracellular synthesis, isolation and/or puπfication

Polynucleotides of the present invention may be used as diagnostic reagents, through detecting mutations in the associated gene Detection of a mutated form of the gene characteπsed by the polynucleotide of SEQ ID NO 1 in 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 in the gene may be detected at the DNA level by a variety of techniques well known in the art Nucleic acids for diagnosis may be obtained from a subject s cells, such as from blood, uπne. saliva, tissue biopsy or autopsy mateπal The genomic DNA may be used directlv for detection or it may be amplified enzymatically by using 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 bv a change in size of the amplified product in compaπson to the normal genotype Point mutations can be identified by hybπdizing amplified DNA to labeled RGS8LIKE nucleotide sequences Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in melting temperatures DNA sequence difference may also be detected by alterations in the electrophoretic mobility of DNA fragments in 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 S 1 protection or the chemical cleavage method (see Cotton et al , Proc Natl Acad Scι USA (1985) 85 4397-4401)

An array of oligonucleotides probes compπsing RGS8LIKE 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 vanety of questions in molecular genetics including gene expression, genetic linkage, and genetic vaπabihty, 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 using 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 hybridization methods Assay techniques that can be used to determine levels of a protein, such as a polypeptide of the present invention, in a sample deπved from a host are well-known to those of skill in the art Such assay methods include radioimmunoassays, competitive-binding assays Western Blot analysis and ELISA assays

Thus in another aspect, the present invention relates to a diagonostic kit compπsing (a) a polynucleotide of the present invention, preferably the nucleotide sequence of SEQ ID NO 1 , or a fragment or an RNA transcπpt 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 comprise a substantial component Such a kit will be of use in diagnosing a disease or susceptibility to a disease, particularly diseases of the invention, amongst others

The polynucleotide sequences of the present invention are valuable for chromosome localisation studies The sequence is specifically targeted to and can hybndize with, a particular location on an individual human chromosome The mapping of relevant sequences to chromosomes according to the present invention is an important first step in 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 in 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-inhentance of physically adjacent genes) Precise human chromosomal localisations for a genomic sequence (gene fragment etc ) can be determined using Radiation Hybrid (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, PrudΗomme JF, Dib C, Auffray C, Moπssette J, Weissenbach J, Goodfellow PN) To determine the chromosomal location of a gene using 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 using PCR products from genomic sequences of known location This comparison is conducted at http //www genome wi mit edu/

The polynucleotide sequences of the present invention are also valuable tools for tissue expression studies Such studies allow the determination of expression patterns of polynucleotides of the present invention which may give an indication as to the expression patterns of the encoded polypeptides in tissues, by detecting the mRNAs that encode them The techniques used are well known in the art and include in situ hydπdisation techniques to clones arrayed on a grid, such as cDNA microarray hybπdisation (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 in the organism In addition, comparative studies of the normal expression pattern of mRNAs with that of mRNAs encoded bv an alternative form of the same gene (for example one having an alteration in polypeptide coding potential or a regulatory mutation) can provide valuable insights into the role of the polypeptides of the present invention, or that of inappropπate expression thereof in disease Such inappropπate expression may be of a temporal, spatial or simply quantitative nature The polynucleotides of the invention are expressed in brain and pituitary with small amounts detected in heart

A further aspect of the present invention relates to antibodies The polypeptides of the invention or their fragments, or cells expressing them, can be used as immunogens 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 in the pπor art

Antibodies generated against polypeptides of the present invention may be obtained by administeπng 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 hybndoma technique (Kohler, G and Milstein, C , Nature (1975) 256 495-497), the tπoma technique, the human B-cell hybndoma technique (Kozbor et al , Immunology Today (1983) 4 72) and the EBV-hybndoma technique (Cole et al , Monoclonal Antibodies and Cancer Therapy, 77-96, Alan R Liss, Inc , 1985) Techniques 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 single chain antibodies to polypeptides of this invention Also, transgenic mice, or other organisms, including other mammals, may be used to express humanized antibodies

The above-descnbed 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 invention 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 in 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, cytokine-producing T cells or cytotoxic T cells, to protect said animal from disease, whether that disease is already established within the individual or not An immunological response in a mammal mav also be induced by a method compπses delivering 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 wav of administering the vector is by

- 10 - accelerating it into the desired cells as a coating on particles or otherwise Such nucleic acid vector may comprise 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 comprise a suitable carrier Since a polypeptide may be broken down in the stomach, it is preferably administered parenterally (for instance, subcutaneous, intramuscular, intravenous, or intradermal 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 instomc with the blood of the recipient, and aqueous and non-aqueous steπle suspensions that may include 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 requiring only the addition of the steπle liquid earner immediately pπor to use The vaccine formulation may also include adjuvant systems for enhancing the immunogenicity of the formulation, such as oil-in 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 invention have one or more biological functions that are of relevance in one or more disease states, in particular the diseases of the invention which include neurological conditons, for example neuropathic pain, pain, chronic pain, postoperative pain, rheumatoid arthπtic pain, neuralgia, neuropathies, algesia, nerve injury, lschaemia, neurodegeneration, stroke, incontinence, inflammatory disorders, spasticity, myoclonus, epilepsy, head trauma, It is therefore useful to to identify compounds that stimulate or inhibit the function or level of the polypeptide Accordingly, in a further aspect, the present invention provides for a method of screening 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 invention as hereinbefore mentioned Compounds may be identified from a vaπety of sources, for example, cells, cell-free preparations, chemical branes collections of chemical compounds, and natural product mixtures Such agonists or antagonists so-identified may be natural or modified substrates, ligands, 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 1(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 screening 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 screening method may involve measuring 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 bv activation or inhibition of the polypeptide, using detection systems appropπate to the cells bearing the polypeptide Inhibitors of activation are generallv 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 screening methods may simply comprise the steps of mixing a candidate compound with a solution containing a polypeptide of the present invention, to form a mixture, measuring a RGS8LIKE activity in the mixture, and comparing the RGS8LIKE activity of the mixture to a control mixture which contains no candidate compound

Polypeptides of the present invention may be employed in conventional low capacity screening methods and also in high-throughput screening (HTS) formats Such HTS formats include 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 proteins, such as those made from Fc portion and RGS8LIKE polypeptide, as hereinbefore described, 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))

The polynucleotides, polypeptides and antibodies to the polypeptide of the present invention may also be used to configure screening 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 measuring 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, hgand binding and crosshnking assays in which the polypeptide is labeled with a radioactive isotope (for instance, ^~-^>\), chemically modified (for instance, biotinylated), or fused to a peptide sequence suitable for detection or punfication, 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 screening 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 invention include antibodies or, in some cases, o gonucleotides or proteins that are closely related to the ligands. substrates, receptors, enzymes, etc , as the case may be, of the polypeptide, e.g , a fragment of the ligands, substrates, receptors, enzymes, etc , or a small molecule that bind to the polypeptide of the present invention but do not elicit a response, so that the activity of the polypeptide is prevented

Screening methods may also involve the use of transgenic technology and RGS8LIKE gene The art of constructing transgenic animals is well established. For example, the RGS8LIKE gene may be introduced through microinjection into the male pronucleus of fertilized oocytes, retroviral transfer into pre- or post-implantation embryos, or injection of genetically modified, such as by electroporation, embryonic stem cells into host blastocysts Particularly useful transgenic animals are so-called "knock-in" animals in which an animal gene is replaced by the human equivalent within the genome of that animal. Knock-in transgenic animals are useful in the drug discovery process, for target validation, where the compound is specific for the human target. Other useful transgenic 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 in a cell is partially or completely annulled The gene knock-out may be targeted to specific cells or tissues, may occur only in certain cells or tissues as a consequence of the limitations of the technology, or may occur in all, or substantially all, cells in the animal Transgenic animal technology also offers a whole animal expression-cloning system in which introduced genes are expressed to give large amounts of polypeptides of the present invention

Screening kits for use in the above described methods form a further aspect of the present invention Such screening kits compπse

(a) a polypeptide of the present invention;

(b) a recombinant cell expressing a polypeptide of the present invention, (c) a cell membrane expressing a polypeptide of the present invention; or

(d) an antibody to a polypeptide of the present invention, 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 comprise 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, chimeπc, single chain, and humanized antibodies, as well as Fab fragments, including the products of an Fab or other immunoglobulin expression library

"Isolated" means altered "by the hand of man" from its natural state, : e , if it occurs in 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 herein 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" include, 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 compπsing DNA and RNA that may be single-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 vaπety of modifications may be made to DNA and RNA, thus, "polynucleotide" embraces chemically, enzymatically or metabohcally modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells "Polynucleotide" also embraces relatively short polynucleotides, often refened to as ohgonucleotides

"Polypeptide" refers to any polypeptide compπsing two or more amino acids joined to each other by peptide bonds or modified peptide bonds, I e , peptide isosteres "Polypeptide" refers to both short chains, commonly refened to as peptides, ohgopeptides or ohgomers, and to longer chains, generally refened to as proteins Polypeptides may contain amino acids other than the 20 gene-encoded amino acids "Polypeptides" include amino 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 described in 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 amino acid side-chains and the amino 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 in a given polypeptide Also, a given polypeptide may contain many types of modifications Polypeptides may be branched as a result of ubiquitmation, 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 include acetylation, acylation, ADP-πbosylation, amidation, biotmylation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide deπvative, covalent attachment of a hpid or pid derivative, covalent attachment of phosphotidvhnositol. cross-linking, cychzation, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation hydroxylation. lodmation, methylauon, myπstoylation, oxidation, proteolytic processing, phosphor) lation. prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination (see, for instance, Proteins - 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 Proteins, B C Johnson, Ed , Academic Press, New York, 1983, Seifter et al , "Analysis for protein modifications and nonprotein cofactors", Meth Enzymol. 182, 626-646, 1990, and Rattan et al , "Protein Synthesis Post-translational Modifications and Aging", 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 polynucloetide 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 in the nucleotide sequence of the vanant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide Nucleotide changes may result in am o 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 variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, insertions, deletions in any combination A substituted or inserted amino acid residue may or may not be one encoded by the genetic code Typical conservative substitutions include Gly, Ala, Val, lie, Leu, Asp, Glu, Asn, Gin, Ser, Thr, Lys, Arg, and Phe and Tyr A variant of a polynucleotide or polypeptide may be naturally occurnng such as an allele, or it may be a variant that is not known to occur naturallv Non-naturally occurnng variants of polynucleotides and polvpeptides may be made by mutagenesis techniques or by direct synthesis Also included as variants are polypeptides having one or more post-translational modifications, for instance glycosylation, phosphorylation, methylauon, ADP πbosylation and the like Embodiments include methylauon of the N-terminal amino acid, phosphorylations of serines and threonines and modification of C-terminal glycines

"Allele" refers to one of two or more alternative forms of a gene occuπng at a given locus in the genome

- 15 - "Polymoφhism refers to a variation in nucleotide sequence (and encoded polypeptide sequence, if relevant) at a given position in the genome within a population

"Single Nucleotide Polymoφhism ' (SNP) refers to the occurence of nucleotide variability at a single nucleotide position in the genome, within a population. An SNP may occur within a gene or within intergenic regions of the genome SNPs can be assayed using Allele

Specific Amplification (ASA) For the process at least 3 pπmers are required A common primer is used in reverse complement to the polymoφhism being assayed This common pπmer can be between 50 and 1500 bps from the polymoφhic 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 polymoφhism. Two (or more) PCR reactions are then conducted on sample DNA, each using the common primer and one of the Allele Specific Pπmers

"Splice Vanant" as used herein refers to cDNA molecules produced from RNA molecules initially transcribed 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 introns, which results in the production of more than one mRNA molecule each of that may encode different amino acid sequences The term splice variant also refers to the proteins encoded by the above cDNA molecules.

"Identity" reflects a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, determined by comparing the sequences. In general, identity refers to an exact nucleotide to nucleotide or amino acid to amino acid conespondence 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 conespondence, 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 include inserting "gaps" in either one or both sequences, to enhance the degree of alignment. A 7σ 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 "Similarity ' is a further, more sophisticated measure of the relationship between two polypeptide sequences In general, "similarity" means a comparison between the amino acids of two polypeptide chains, on a residue by residue basis, taking into account not only exact conespondences between a between pairs of residues, one from each of the sequences being compared (as tor 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 "% similarity" of the two sequences can then be determined

Methods for comparing the identity and similarity of two or more sequences are well known in the ait Thus for instance, programs available in the Wisconsin Sequence Analysis

- 16 - 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 % similarity between two polypeptide sequences BESTFTT uses the "local homology ' algoπthm 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 similarity between two sequences BESTFIT is more suited to comparing 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 comparison, GAP aligns two sequences, finding a "maximum similaπty", according to the algoπthm of Neddleman and Wunsch (J Mol Biol. 48, 443-453,

1970) GAP is more suited to comparing 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 in each program are 50 and 3, for polynucleotide sequences and 12 and 4 for polypeptide sequences, respectively. Preferably, % identities and similarities are determined when the two sequences being compared are optimally aligned

Other programs for determining identity and/or similarity 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.nih.gov) and FASTA (Pearson W R,

Methods in 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 amino acid substitution matrix (Henikoff S and Henikoff J G, Proc. Nat. Acad Sci. USA, 89, 10915-10919, 1992) is used in polypeptide sequence comparisons including where nucleotide sequences are first translated into amino 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 consisting 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 within 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 in every 100 of the nucleotides of the in the reference sequence may be deleted, substituted or inserted, or any combination thereof, as hereinbefore described 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 include an average of up to five differences per each 100 ammo acids of the reference sequence Such differences are selected from the group consisting of at least one amino acid deletion, substitution, including conservative and non- conservative substitution, or insertion These differences may occur at the amino- or carboxy- terminal 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 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.

The relationship between the number of nucleotide or amino acid differences and the Identity Index may be expressed in the following equation n_a < x_a - (χ_a • I), in which n_a is the number of nucleotide or amino acid differences, χ_a is the total number of nucleotides or am o acids in SEQ ID NO 1 or SEQ ID NO 2, respectively, I is the Identity Index , • is the symbol for the multiplication operator, and in which any non-integer product of x_a and I is rounded down to the nearest integer prior to subtracting it from x_a

"Homolog" is a generic 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 similanty between the two sequences as hereinbefore defined Falling within this genenc 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 polynucleotideor polypeptide that within 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 comprising 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 in, for example, improved pharmacokinetic 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 incoφorated by reference in their entirety as if each individual publication or reference were specifically and individually indicated to be incoφorated by reference herein as being fully set forth Any patent application to which this application claims priority is also incoφorated by reference herein in its entirety in the manner described above for publications and references

- 19 - Examples Example 1: TaqMan Analysis of mRNA distribution

The expression pattern of RGS8LIKE was investigated using Syber-Green fluorescent PCR (Perkin Elmer) and human cDNAs prepared from vaπous brain areas and peripheral tissues All Syber-Green analysis was earned out according to the manufacturers instructions using the following ohgonucleotides

Forward pπmer 5'- CTG CAT TCC GTG CCT TCT TG-3' Reverse pnmer 5'- TTT ACC TCC CGT GGA GCC TG-3'

20 tissues examined in the Syber-Green experiments were Brain*, pituitary, heart, lung, liver, foetal liver kidney, skeletal muscle, stomach, small/large intestine, spleen, lymphocytes (PBMC), macrophages, adipose, pancreas, prostate (4 males), placenta, cartilage, bone (l male, 3 females) and bone marrow

(* Brain = an equal-part mix of the 18 most distinct brain regions representing 75% of sample and 25% of sample is spinal cord This approach was designed to maximise the chance of detecting genes expressed speftcally in small brain sub-regions)

(key for relative expression levels 0 - no detectable expression, + low expression, medium expression, +++ high expression)

Brain +++

Pituitary ++

Heart +

Lung 0

Liver 0

Foetal liver 0

Kidney 0

Skeletal muscle 0

Stomach 0

Small/large intestine 0

Spleen 0

Lymphocytes (PBMC) 0

Macrophages 0

Adipose 0

Pancreas 0 Prostate 0

Placenta 0

Cartilage 0

Bone 0 Bone Marow 0

SEQUENCE INFORMATION

SEQ ID NO :1

>

ATGTGGAACACCTTAACCCGAAGCCTCTCTGACCATCCAGTTGGCAAAGACCCTCAGGCC ATGAGGACTGGCCAAAGACAGAACAAAGGGATGAGGACTCGACTGGGATGCCTGTCTCAC AAGTCAGACTCGTGTAGTGATTTCACAGCTATTCTTCCAGACAAACCCAACCGCGCTCTC AAGAGATTATCGACAGAAGAAGCTACGAGGTGGGCAGATTCCTTTGATGTGCTTCTCTCT CATAAGTATGGGGTGGCTGCATTCCGTGCCTTCTTGAAGACGGAGTTCAGTGAGGAGAAC CTGGAATTCTGGTTGGCCTGTGAGGAGTTCAAGAAGACCAGGTCAACTGCAAAACTGGTC TCTAAGGCCCATAGGATCTTTGAGGAGTTTGTGGATGTGCAGGCTCCACGGGAGGTAAAC ATTGACTTCCAGACCCGAGAAGCCACGAGGAAGAACCTGCAGGAGCCATCCCTGACTTGC TTTGACCAAGCCCAAGGAAAAGTACACAGCCTCATGGAGAAAGACTCTTACCCCAGGTTC CTGAGGTCCAAAATGTACTTAGATCTGCTGTCCCAAAGCCAGAGGAGGCTCAGTTAG

SEQIDNO:2 >

M TLTRSLSDHPVGKDPQAMRTGQRQNKGMRTRLGCLSHKSDSCSDFTAILPDKPNRAL KR STEEATRWADSFDVLLSHKYGVAAFRAFLKTEFSEEN EF LACEEFKKTRSTAKLV SKAHRIFEEFVDVQAPREVNIDFQTREATRKNLQEPSLTCFDQAQGKVHSLMEKDSYPRF LRSKMY DLLSQSQRRLS

Claims

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 comprising a polypeptide sequence having at least 957o identity to the polypeptide sequence of SEQ ID NO 2,

(c) an isolated polypeptide having at least 957c identity to the polypeptide sequence of SEQ ID NO 2, and (d) fragments and variants of such polypeptides in (a) to (e)

2 The isolated polypeptide as claimed in claim 1 compπsing the polypeptide sequence of SEQ ID NO 2

3 The isolated polypeptide as claimed in claim 1 which is the polypeptide sequence of SEQ ID NO 2

4 An isolated polynucleotide selected from the group consisting 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 having at least 95% identity to the polynucleotide of SEQ ID NO 1,

(c) an isolated polynucleotide compπsing a polynucleotide sequence encoding a polypeptide sequence having at least 95% identity to the polypeptide sequence of SEQ ED NO 2,

(d) 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,

(e) an isolated polynucleotide with a nucleotide sequence of at least 100 nucleotides obtained by screening a library under stnngent hybndization conditions with a labeled probe having the sequence of SEQ ED NO 1 or a fragment thereof having at least 15 nucleotides,

(f) a polynucleotide which is the RNA equivalent of a polynucleotide of (a) to (e), 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

5 An isolated polynucleotide as claimed in claim 4 selected from the group consisting of (a) an isolated polynucleotide compnsmg the polynucleotide of SEQ ED NO 1,

(b) the isolated polynucleotide of SEQ ID NO 1,

(c) an isolated polynucleotide compnsmg a polynucleotide sequence encoding the polypeptide of SEQ ID NO 2, and

- 23 - (d) an isolated polynucleotide encoding the polypeptide of SEQ ED NO:2.

6. An expression system comprising a polynucleotide capable of producing a polypeptide of claim 1 when said expression vector is present in a compatible host cell.

7. A recombinant host cell comprising the expression vector of claim 6 or a membrane thereof expressing the polypeptide of claim 1.

8. A process for producing a polypeptide of claim 1 comprising the step of culturing a host cell as defined in claim 7 under conditions sufficient for the production of said polypeptide and recovering the polypeptide from the culture medium.

9. An antibody immunospecific for the polypeptide of any one of claims 1 to 3.

10. A method for screening to identify compounds that stimulate or inhibit the function or level of the polypeptide of claim 1 comprising a method selected from the group consisting of: (a) measuring or, detecting, quantitatively or qualitatively, the binding of a candidate compound to the polypeptide (or to the cells or membranes expressing the polypeptide) or a fusion protein thereof by means of a label directly or indirectly associated with the candidate compound; (b) measuring the competition of binding of a candidate compound to the polypeptide (or to the cells or membranes expressing the polypeptide) or a fusion protein thereof in the presence of a labeled competitior;

(c) testing whether the candidate compound results in a signal generated by activation or inhibition of the polypeptide, using detection systems appropriate to the cells or cell membranes expressing the polypeptide;

(d) mixing a candidate compound with a solution containing a polypeptide of claim 1, to form a mixture, measuring activity of the polypeptide in the mixture, and comparing the activity of the mixture to a control mixture which contains no candidate compound; or

(e) detecting the effect of a candidate compound on the production of mRNA encoding said polypeptide or said polypeptide in cells, using for instance, an ELISA assay.