GB2364308A - AXOR64, a G-protein coupled receptor polypeptide and its encoding polynucleotide sequence - Google Patents

Abstract

An isolated polynucleotide of SEQ ID NO: 1, variants and fragments thereof are claimed encoding a polypeptide of SEQ ID NO: 2, variants and fragments thereof. The polypeptide encoded is a G protein-coupled receptor (AXOR64) that is related to a histamine receptor. Also claimed are antibodies to said polypeptide, expression vectors and recombinant host cells and preferably their membranes expressing the said polypeptide.

Description

GP-70684-1 2364308 Molecular Cloning of a 7TM Receptor (AXOR64)

Field of the Invention

5 This invention relates to newly identified polypeptides and polynucleotides encoding such polypeptides, to their use in diagnosis and in 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

10 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 11positional 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.

15 Functional genomics 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 characterize further genes and their related polypeptides/proteins, as targets for drug discovery.

It is well established that many medically significant biological processes are mediated by proteins 20 participating in signal transduction pathways that involve G-proteins and/or second messengers, e.g., cANP (Lefkowitz, Nature, 1991, 351:353-354). Herein these proteins are referred to as proteins participating in pathways with G-proteins or PPG proteins. Some examples of these proteins include the GPC receptors, such as those for adrenergic agents and dopamine (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, JR., et al., Nature, 1988, 336:783 25 787), G-proteins themselves, effector proteins, e.g., phospholipase C, adenyl cyclase, and phosphodiesterase, and actuator proteins, 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 30 the nucleotide, GTP. GTP also influences hormone binding. A G-protein connects the hormone receptor to adenylate cyclase. G-protein 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-protein itself, returns the G-protein to its basal, inactive form. Thus, the G-protein serves a dual role, as an intermediate that relays the signal from receptor to effector, and as a clock that controls the 35 duration of the signal.

The membrane protein gene superfamily of G-protein coupled receptors has been characterized as having seven putative transmembrane domains. The domains are believed to represent transmembranea- GP-70684-1 2 helices connected by extracellular or cytoplasmic loops. G-protein coupled receptors include a wide range of biologically active receptors, such as hormone, viral, growth factor and neuroreceptors.

G-protein coupled receptors (otherwise known as 7TM receptors) have been characterized as including these seven conserved hydrophobic stretches of about 20 to 30 amino acids, connecting at least 5 eight divergent hydrophilic loops. The G-protein family of coupled receptors includes dopamine receptors which bind to neuroleptic drugs used for tredting psychotic and neurological disorders. Other examples of members of this family include, but are not limited to, calcitonin, adrenergic, endothelin, cAMP, adenosine, muscarinic, acetylcholine, serotonin, histamine, thrombin, kinin, follicle stimulating hormone, opsins, endothelial differentiation gene-1, rhodopsins, odorant, and cytornegalovirus receptors. In particular, 10 histamine is a mediator of inflammation and allergy which is released by mast cells. It is also released by parietal cells of the gastric mucosa and is distributed widely in both neuronal and non-neuronal cells in the brain and spinal cord. Histamine has been implicated in CNS functions such as arousal, sexual function, and analgesia.

15 Most G-protein coupled receptors have single conserved cysteine residues in 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 TM 1, TM2, TM3, TM4, TM5, TM6, and TM7. TM3 has been implicated in signal transduction.

Phosphorylation and lipidation (palinitylation or farnesylation) of cysteine residues can influence 20 signal transduction of some G-protein coupled receptors. Most G- protein coupled receptors contain potential phosphorylation sites within the third cytoplasmic loop and/or the carboxy terminus. For several G- protein coupled receptors, such as the P-adrenoreceptor, phosphorylation by protein kinase A and/or specific receptor kinases mediates receptor desensitization, For some receptors, the ligand binding sites of G-protein coupled receptors are believed to comprise 25 hydrophilic sockets formed by several G-protein coupled receptor transmembrane domains, said sockets being surrounded by hydrophobic residues of the G-protein coupled receptors. The hydrophilic side of each G-protein coupled receptor transmembrane helix is postulated to face inward and form a polar ligand binding site. TM3 has been implicated in several G-protein coupled receptors as having a ligand binding site, such as the TM3 aspartate residue. TM5 serines, a TM6 asparagine and TM6 or TM7 phenylalanines or tyrosines are 30 also implicated in ligand binding.

G-protein coupled receptors can be intracellularly coupled by heterotrimeric G-proteins to various intracellular enzymes, ion channels and transporters (see, Johnson et al., Endoc. Rev., 1989, 10:3 17-3 3 1). Different G-protein (x-subunits preferentially stimulate particular effectors to modulate various biological functions in a cell. Phosphorylation of cytoplasmic residues of G-protein coupled receptors has been 35 identified as an important mechanism for the regulation of G-protein coupling of some G-protein coupled receptors. G-protein coupled receptors are found in numerous sites within a mammalian host.

Over the past IS years, nearly 3 50 therapeutic agents targeting 7 transmembrane (7 TM) receptors have been successfully introduced onto the market.

GP-70684-1 3 Summary of the Invention

The present invention relates to AXOR64, in particular AXOR64 polypeptides and AXOR64 polynucleotides, recombinant materials and methods for their production. Axor 64 shows relation to a 5 histamine receptor. Such polypeptides and polynucleotides are of interest in relation to methods of treatment of certain diseases, including, but not limited to, infections such as bacterial, fungal, protozoan and viral infections, particularly infections caused by HIV- I or FHV-2; pain; cancers; diabetes, obesity; anorexia; bulimia; asthma; Parkinson's disease; acute heart failure; hypotension; hypertension; urinary retention; osteoporosis; angina pectoris; myocardial infarction; stroke; ulcers; asthma; allergies; benign prostatic 10 hypertrophy; migraine; vomiting; psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, depression, delirium, dementia, and severe mental retardation; and dyskinesias, such as Huntington's disease, progressive supranuclear palsy (PSP) or Gilles dela Tourett's syndrome, hereinafter referred to as "diseases of the invention". Preferably, the disease of the invention is a neurological disorder, and particularly preferred is when it is Huntington's disease or progressive supranuclear palsy 15 (PSP). In a further aspect, the invention relates to methods for identifying agonists and antagonists (e.g., inhibitors) using the materials provided by the invention, and treating conditions associated with AXOR64 imbalance with the identified compounds In a still further aspect, the invention relates to diagnostic assays for detecting diseases associated with inappropriateAXOR64 activity or levels, in particular for the diagnosis of neurological diseases such as Huntingtons disease or PSP.

Description of the Invention

In a first aspect, the present invention relates to AXOR64 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 25 99% identity to the polypeptide sequence of SEQ ID NO:2; (c) an isolated polypeptide comprising 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; and 30 (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).

The term "variant" refers to a polypeptide which has the same essential character or basic biological functionality as AXOR64. The essential character of AXOR64 can be defined as follows:

35 AXOR64 is a histamine receptor-like receptor. Preferably a variant polypeptide is one which binds to the same ligand as AXOR64. Preferably the polypeptide is capable of binding histamine or a structurally related molecule. A polypeptide having the same essential character as AXOR64 may be identified by monitoring for a function of the histamine receptor-like polypeptide such as effects on immune function GP-70684-1 4 and inflammation. A full length variant polypeptide is preferably one which includes a seven transmembrane region. Preferably, a full length variant polypeptide may couple to G-protein to mediate intracellular responses.

5 Polypeptides of the present invention are believed to be members of theG-protein Coupled (7TM) Receptor family of polypeptides. They are therefore of interest because G- Protein Coupled Receptors, more than any other gene family, are the objects of pharmaceutical intervention The biological properties of the AXOR64 are hereinafter referred to as "biological activity of AXOR64" or "AXOR64 activity."

Preferably, a polypeptide of the present invention exhibits at least one biological activity of AXOR64.

10 Polypeptides of the present invention also include variants of the aforementioned polypeptides, including all allelic forms and splice variants. Such polypeptides vary from the reference polypeptide by insertions, deletions, and substitutions that may be conservative or nonconservative, 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 I amino acids are inserted, substituted, or 15 deleted, in any combination.

Amino acid substitutions may be made, for example from 1, 2 or 3 to 10, 20 or 30 substitutions.

The modified polypeptide generally retains activity as a histamine receptor-like polypeptide.

Conservative substitutions may be made, for example according to the following Table. Amino acids in the same block in the second column and preferably in the same line in the third column may be 20 substituted for each other.

ALIPHATIC Non-polar GAP ILV Polar-uncharged CSTM N Q Polar-charged D E K R AROMATIC HFWY Preferred fragments of polypeptides of the present invention include &I isolated polypeptide comprising an amino acid sequence having at least 30, 50 or 100 contiguous amino acids from the amino 25 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 mediatethe biological activity of GP-70684-1 5 AXOR64, 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 immunogenic in an animal, especially in a human. In particular, but not exclusively, this aspect of the invention encompasses the situation when the protein is a fragment of the complete protein sequence and may represent a ligand-binding region (N 5 terminal extracellular domain) or an effector binding region (C- terminal intracellular domain). Such fragments can be used to construct chimeric receptors preferably with another 7-transmembrane receptor, more preferably with another member of the family of histamine receptor- like polypeptides. Such fragments of AXOR64 or a variant thereof can also be used to raise anti- AXOR64 antibodies. In this embodiment the fragment may comprise an epitope of the AXOR64 polypeptide and may otherwise not 10 demonstrate the ligand binding or other properties of AXOR64.

Fragments of the polypeptides of the invention may also be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, these variants may be employed as intermediates for producing the full-length polypeptides of the invention. Thepolypeptides 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 15 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 for stability during recombinant production.

Polypeptides of the present invention can be prepared in any suitable manner, for instance by isolation form naturally occurring sources, from genetically engineered host cells comprising expression 20 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 ftirther aspect, the present invention relates to AXOR64 polynucleotides. Such polynucleotides include:

(a) an isolated polynucleotide comprising a polynucleotide sequence having at least 95%, 96%, 97%, 25 98%, or 99% identity to the polynucleotide sequence of SEQ ID NO: 1; (b) an isolated polynucleotide comprising 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; 30 (e) an isolated polynucleotide comprising a polynucleotide sequence encoding a polypeptide sequence having at least 95%, 96%, 97%, 98%, or 99% identity to thepolypeptide sequence of SEQ ID NO:2; (f) an isolated polynucleotide comprising 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 35 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 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:I;.

GP-70684-1 6 0) 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 5 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 from 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.

10 Preferred variants of polynucleotides of the present invention include splice variants, allelic variants, and polymorphisms, including polynucleotides having one or more single nucleotide polymorphisms (SNPs).

Polynucleotides of the present invention also include polynucleotides encoding polypeptide variants that comprise the amino acid sequence of SEQ ID NO:2 and in which several, for instance from 50 to 30, 15 from 30 to 20, from 20 to 10, from 10 to 5, from 5 to 3, from 3 to 2, from 2 to I or I amino acid residues are substituted, deleted or added, in any combination. The polynucleotide of SEQ ID NO: I may alternatively or additonally be modified by one or more insertions and/or deletions and/or by an extension at either or both ends. A polynucleotide may include one or more introns, for example may comprise genomic DNA.

Typically a polynucleotide of the invention comprises a contiguous sequence of nucleotides 20 which is capable of hybridizing under selective conditions to the coding sequence or the complement of the coding sequence of SEQ ID NO: 1.

A polynucleotide of the invention can hybridize to the coding sequence or the complement of the coding sequence of SEQ ID NO: I at a level significantly above background. Background hybridization may occur, for example, because of other cDNAs present in a cDNA library. The signal level generated

25 by the interaction between a polynucleotide of the invention and the coding sequence or complement of the coding sequence of SEQ ID NO: I is typically at least 10 fold, preferably at least 100 fold, as intense as interactions between other polynucleotides and the coding sequence of SEQ ID NO: 1. The intensity of interaction may be measured, for example, by radiolabelling the probe, e.g. with 32p. Selective hybridisation may typically be achieved using conditions of medium to high stringency. However, such 30 hybridisation may be carried out under any suitable conditions known in the art (see Sambrook et al, 1989. For example, if high stringency is required suitable conditions include from 0. 1 to 0.2 x SSC at 60 OC up to 65'C. If lower stringency is required suitable conditions include 2 x SSC at 60'C.

In a further aspect, the present invention provides polynucleotides that are RNA transcripts of the 35 DNA sequences of the present invention. Accordingly, there is provided an RNA polynucleotide that:

(a) comprises an RNA transcript of the DNA sequence encoding the polypeptide of SEQ ID NO:2; (b) is the RNA transcript of the DNA sequence encoding the polypeptide of SEQ ID NO:2; GP-70684-1 7 (c) comprises an RNA transcript of the DNA sequence of SEQ ID NO: 1; or (d) is the RNA transcript of the DNA sequence of SEQ ID NO: 1; and RNA polynucleotides that are complementary thereto.

The polynucleotide sequence of SEQ ID NO: I shows homology with GPRXORLYA [A. Yosouka, 5 et.al., Biochem. Biophys. Acta 1235 (2), 467-469 (1995)]. The polynucleotide sequence of SEQ ID NO: I 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: I 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 SEQ ID NO:2 is 10 related to other proteins of the G-protein Coupled family, having homology and/or structural similarity with GPRX-ORLYA [A. Yasuoka, et. al., Biochim, Biophys. Acta 1235 (2), 467-469 (1995)].

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 oneAXOR64 activity.

15 Polynucleotides of the present invention may be obtained using standard cloning and screening techniques from a cDNA library derived from mRNA in cells of the human brain, (see for instance, Sambrook etal., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)). Polynucleotides of the invention can also be obtained from natural sources such as genomic DNA libraries or can be synthesized using well known and commercially 20 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, 25 or other fusion peptide portions. For example, a marker sequence that facilitates purification 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 described in Gentzet al., Proc Natl Acad Sci USA (1989) 86:821-824, or is an HA tag. The polynucleotide may also contain noncoding 5' and 3' sequences, such as transcribed, non-translated sequences, splicing and polyadenylation 30 signals, ribosome 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 hybridization probes for cDNA and genomic DNA or as primers for a nucleic acid amplification reaction (for instance, PCR). Such probes and primers may be used to isolate full-length cDNAs and genomic clones encoding polypeptides of the present invention and to isolate cDNA and 35 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 similarity to SEQ ID NO: 1, typically at least 95% identity. Preferred probes and primers will generally comprise at least 15 nucleotides, preferably, at least 30 nucleotides and may have at least 50, if not at least 100 nucleotides. Particularly preferred probes GP-70684-1 8 will have between 30 and 50 nucleotides. Particularly preferred primers 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 comprising the steps of screening a library under stringent 5 hybridization conditions with a labeled probe having the sequence of SEQ ID NO: I or a ftagment thereof, preferably of at least 15 nucleotides; and isolating full-length cDNA and genomic clones containing said polynucleotide sequence. Such hybridization techniques are well known to the skilled artisan. Preferred stringent hybridization conditions include overnight incubation at 42DC in a solution comprising: 50% formamide, 5xSSC (1 50mM NaC1, 15mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x 10 Denhardt's solution, 10 % dextran sulfate, and 20 microgram/m I denatured, sheared salmon sperm DNA; followed by washing the filters in O.lx SSC at about 650C. Thus the present invention also includes isolated polynucleotides, preferably with a nucleotide sequence of at least 100, obtained by screening a library under stringent hybridization conditions with a labeled probe having the sequence of SEQ ID NO: I or a fragment thereof, preferably of at least 15 nucleotides.

15 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 transcriptase, an enzyme with inherently low "processivity" (a measure of the ability of the enzyme to remain attached to the template during the polymerization reaction), failing to complete a DNA copy of the mRNA template during first strand cDNA synthesis.

20 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 Laboratories Inc.) for example, have significantly simplified the search for longer cDNAs. In the 25 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 carried out to amplify the "missing" 5' end of the cDNA using a combination of gene specific and adaptor specific oligonucleotide primers. The PCR reaction is then repeated using'nested' primers, that is, primers designed to anneal within the amplified product (typically an adapter specific primer that anneals 30 ftirther 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' primer.

35 The present invention also includes expression vectors that comprise nucleotide sequences encoding the proteins or variants thereof of the invention. Such expression vectors are routinely constructed in the art of molecular biology and may for example involve the use of plasmid DNA and appropriate initiators, promoters, enhancers and other elements, such as for example polyadenylation signals which may be GP-70684-1 9 necessary, and which are positioned in the correct orientation, in order to allow for protein expression.

Other suitable vectors would be apparent to persons skilled in the art. By way of further example in this regard we refer to Sambrook et al. 1989.

Polynucleotides according to the invention may also be inserted into the vectors described above 5 in an antisense orientation in order to provide for the production of antisense RNA. Antisense RNA or other antisense polynucleotides may also be produced by synthetic means. Such antisense polynucleotides may be used as test compounds in the assays of the invention or may be useful in a method of treatment of the human or animal body by therapy.

Preferably, a polynucleotide of the invention or for use in the invention in a vector is operably 10 linked to a control sequence which is capable of providing for the expression of the coding sequence by the host cell, i.e. the vector is an expression vector. The term "operably linked" refers to ajuxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A regulatory sequence, such as a promoter, "operably linked" to a coding sequence is positioned in such a way that expression of the coding sequence is achieved under conditions compatible with the 15 regulatory sequence.

The vectors may be for example, plasmid, virus or phage vectors provided with an origin of replication, optionally a promoter for the expression of the said polynucleotide and optionally a regulator of the promoter. The vectors may contain one or more selectable marker genes, for example an ampicillin resistence gene in the case of a bacterial plasmid or a resistance gene for a fungal vector. Vectors may be 20 used in vitro, for example for the production of DNA or RNA or used to transfect or transform a host cell, for example, a mammalian host cell. The vectors may also beadapted to be used in vivo, for example in a method of gene therapy.

Promoters and other expression regulation signals may be selected to be compatible with the host cell for which expression is designed. For example, yeast promoters include S. cerevisiae GAL4 and 25 ADH promoters, S. pombe nmtl and adh promoter. Mammalian promoters include the metallothionein promoter which can be induced in response to heavy metals such as cadmium. Viral promoters such as the SV40 large T antigen promoter or adenovirus promoters may also be used. All these promoters are readily available in the art.

Mammalian promoters, such as P-actin promoters, may be used. Tissuespecific promoters are 30 especially preferred. Viral promoters may also be used, for example the Moloney murine leukaemia virus long terminal repeat (MMLV LTR), the rous sarcoma virus (RSV) LTR promoter, the SV40 promoter, the human cytomegalovirus (CMV) IE promoter, adenovirus, HSV promoters (such as the HSV IE promoters), or HPV promoters, particularly the HPV upstream regulatory region (URR). Viral promoters are readily available in the art.

35 The vector may further include sequences flanking the polynucleotide giving rise to polynucleotides which comprise sequences homologous to eukaryotic genomic sequences, preferably mammalian genomic sequences, or viral genomic sequences. This will allow the introduction of the polynucleotides of the invention into the genome of eukaryotic cells or viruses by homologous GP-70684-1 10 recombination. In particular, a plasmid vector comprising the expression cassette flanked by viral sequences can be used to prepare a viral vector suitable for delivering the polynucleotides of the invention to a mammalian cell. Other examples of suitable viral vectors include herpes simplex viral vectors and retroviruses, including lentiviruses, adenoviruses, adeno-associated viruses and HPV viruses. Gene 5 transfer techniques using these viruses are known to those skilled in the art. Retrovirus vectors for example may be used to stably integrate the polynucleotide giving rise to the polynucleotide into the host genome. Replication-defective adenovirus vectors by contrast remain episomal and therefore allow transient expression.

10 Recombinant polypeptides of the present invention may be prepared by processes well known in the art from genetically engineered host cells comprising expression systems. Accordingly, ina further aspect, the present invention relates to expression systems comprising a polynucleotide or polynucleotides of the present invention, to host cells which are genetically engineered with such expression systems and to the production of polypeptides of the invention by recombinant techniques. Cell-free translation systems can 15 also be employed to produce such proteins using RNAs derived 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 described in many standard laboratory manuals, such as Davis et al, Basic 20 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, micro-injection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection.

Representative examples of appropriate hosts include bacterial cells, such asSireptococci, 25 Staphylococci, E. coli, Streptomyces and Bacillus subtilis 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, C 127, 3T3, BHK, HEK 293 and Bowes melanoma cells; and plant cells.

A great variety of expression systems can be used, for instance, chromosomal, episomal and virus derived systems, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from 30 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 derived from combinations thereof, such as those derived from plasmid and bacteriophage 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 35 maintain, propagate or express a polynucleotide to produce a polypeptide in a host may be used. The appropriate polynucleotide sequence may be inserted into an expression system by any of a variety of well known and routine techniques, such as, for example, those set forth in Sambrook el al., (ibid). Appropriate secretion signals may be incorporated into the desired polypeptide to allow secretion of the translated protein GP-70684-1 I I into the lumen of the endoplasmic reticulum, the periplasmic 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 5 harvested prior 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 purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation 10 exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography is employed for purification. Well known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured during intracellular synthesis, isolation and/or purification.

15 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 characterized by the polynucleotide of SEQ ID NO: I in the cDNA or genornic 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 20 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, urine, saliva, tissue biopsy or autopsy material. The genomic DNA may be used directly for detection or it may be amplified enzymatically by using PCR, preferably RT-PCR, or other amplification techniques prior to 25 analysis. RNA or cDNA may also be used in similar fashion. Deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to labeledAXOR64 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 30 of DNA fragments in gels, with or without denaturing 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 I protection or the chemical cleavage method (see Cottonet aL, Proc Natl Acad Sci USA (1985) 85: 4397-4401).

An array of oligonucleotides probes comprising AXOR64 polynucleotide sequence or fragments 35 thereof can be constructed to conduct efficient screening ofe.g., genetic mutations. Such arrays are preferably high density arrays or grids. Array technology methods are well known and have general applicability and can be used to address a variety of questions in molecular genetics including gene

GP-70684-1 12 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 toa disease of the invention. Decreased or 5 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 derived from a host are well-known to those of skill in the art. Such assay methods include radio-immunoassays, 10 competitive-binding assays, Western Blot analysis and ELISA assays.

Thus in another aspect, the present invention 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); 15 (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 20 diseases of the invention, amongst others.

The polynucleotide sequences of the present invention are valuable for chromosome localization studies. The sequence is specifically targeted to, and can hybridize 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 25 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, Mendelian Inheritance 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-inheritance of physically adjacent genes).AXOR64 has been 30 mapped to I p 13.

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 35 hybridization techniques to clones arrayed on a grid, such as cDNA microarray hybridization (Schenaet 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 Perkin Elmer. Results from these studies can provide an indication of the normal function of the polypeptide GP-70684-1 13 in 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 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 inappropriate expression thereof in disease. Such 5 inappropriate expression may be of a temporal, spatial or simply quantitative nature. TAQMAN has been carried out on this receptor, and the results can be seen in Example 1.

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 10 immunospecific for polypeptides of the present invention. The term "immunosp6cific" means that the antibodies have substantially greater affinity for the polypeptides of the invention than their affinity for other related polypeptides in the prior art.

Antibodies generated against polypeptides of the present invention may be obtained by administering the polypeptides or epitope-bearing fragments, or cells to an animal, preferably a non-human animal, using 15 routine protocols. For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler, G. and Milstein, C., Nature (1975) 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., Immunology Today (1983) 4:72) and the EBV- hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, 77-96, Alan R. Liss, Inc., 1985) .

20 Techniques for the production of single chain antibodies, such as those described 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. Antibodies of the invention may be antibodies to human polypeptides or fragments thereof.

rD For the purposes of this invention, the term "antibody", unless specified to the contrary, includes 25 fragments which bind a polypeptide of the invention. Such fragments include Fv, F(ab') and F(ab')2 fragments, as well as single chain antibodies. Furthermore, the antibodies and fragment thereof may be chimeric antibodies, CDR-grafted antibodies or humanised antibodies.

The above-described antibodies may be employed to isolate or to identify clones expressing the 30 polypeptide or to purify 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 polymicleotides 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 35 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 may also be induced by a method comprises delivering a polypeptide of the present invention via a vector directing GP-70684-1 14 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 administering the vector is by 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 5 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, intra-muscular, intravenous, or intra-dermal injection). Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions that may contain 10 anti-oxidants, buffers, bacteriostats and solutes that render the formulation instonic with the blood of the recipient; and aqueous and non-aqueous sterile 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-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use. The vaccine formulation may also include adjuvant 15 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 experimentation.

Polypeptides of the present invention have one or more biological functions that are of relevance in 20 one or more disease states, in particular the diseases of the invention hereinbefore mentioned. It is therefore useful 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 25 hereinbefore mentioned. Compounds may be identified from a variety of sources, for example, cells, cell free preparations, chemical libraries, 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 Coliganet al., Current Protocols in Immunology 1(2):Chapter 5 (1991)) or a small molecule. Such small molecules preferably 30 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. Candidate products can be biomolecules including, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. Known pharmacological 35 agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs.

Test substances may be used in an initial screen of, for example, 10 substances per reaction, and the substances of these batches which show inhibition or activation tested individually. Test substances GP-70684-1 15 may be used at a concentration of from I nM to I OOOpLM, preferably from I gM to I OOgM, more preferably from I gM to I OiM. Preferably, the activity of a test substance is compared to the activity shown by a known activator or inhibitor. A test substance which acts as an inhibitor may produce a 50% inhibition of activity of the receptor. Alternatively a test substance which acts as an activator may 5 produce 50% of the maximal activity produced using a known activator.

The screenina method may simply measure the binding of a candidate compound to the polypeptide, or to cells or membranes bearing the polypeptide, or a fusion protein thereof, by means of a label directly or indirectly associated with the candidate compound. The binding of a test substance to a polypeptide of the invention can be determined directly. For example, a radiolabelled test substance can 10 be incubated with the polypeptide of the invention and binding of the test substance to the polypeptide can be monitored. Typically, the radiolabelled test substance can be incubated with cell membranes containing the polypeptide until equilibrium is reached. The membranes can then be separated from a non-bound test substance and dissolved in scintillation fluid to allow the radioactive content to be determined by scintillation counting. Non-specific binding of the test substance may also be determined 15 by repeating the experiment in the presence of a saturating concentration of a non-radioactive ligand.

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 by activation or inhibition of the polypeptide, using detection 20 systems appropriate to the cells bearing 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 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 AXOR64 activity in the mixture, and comparing the AXOR64 activity of the 25 mixture to a control mixture which contains no candidate compound.

Assays may be carried out using cells expressing AXOR64, and incubating such cells with the test substance optionally in the presence of AXOR64 ligand. Alternatively an antibody may be used to complex AXOR64 and thus mediate AXOR64 activity. Test substances may then be added to assess the effect on such activity. Cells expressing AXOR64 constitutively may be provided for use in assays for 30 AXOR64 function. Such constitutively expressed AXOR64 may demonstrate AXOR64 activity in the absence of ligand binding. Additional test substances may be introduced in any assay to look for inhibitors of ligand binding or inhibitors of AXOR64-mediated activity.

In preferred aspects, a host cell is provided expressing the polypeptide and containing a G-protein coupled pathway responsive reporter construct. The host cell is treated with a substance under test for a 35 defined time. The expression of the reporter gene, such as SP alkaline phosphatase or luciferase is assayed. The assay enables determination of whether the compound modulates the induction of the G protein coupled pathway by AXOR64 in target cells.

GP-70684-1 16 Assays may also be carried out to identify modulators of receptor- shedding. A polypeptide of the invention can be cleaved from the cell surface. Shedding the receptor would act to down regulate receptor signalling. Thus, cell-based assays may be used to screen for compounds which promote or inhibit receptor-shedding.

5 Other assays which can be used to monitor the effect of a test substance on AXOR64 expression include using a reporter crene construct driven by the AXOR64 regulatory sequences as the promoter sequence and monitoring for expression of the reporter polypeptide. Further possible assays could utilise membrane fractions from overexpression of AXOR64 polypeptide either in X laevis oocytes or cell lines such as HEK293, CHO, COS7 and HeLa cells and assessment of displacement of a radiolabelled ligand.

10 Additional control experiments may be carried out. Assays may also be carried out using known ligands of other histamine receptor-like polypeptides to identify ligands which are specific for polypeptides of the invention. Preferably, the assays of the invention are carried out under conditions which would result in G-protein coupled pathway mediated activity in the absence of the test substance, to identify inhibitors or activators of histamine receptor-like polypeptide mediated activity, or agents 15 which inhibit ligand-induced histamine receptor-like polypeptide activity. An assay of the invention may be carried out using a known histamine receptor-like polypeptide agonist or histamine receptor-like polypeptide antagonist to provide a comparison with a compound under test.

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 20 well-established use of 96- and, more recently, 384-well micotiter plates but also emerging methods such as the nanowell method described by Schullek et al, Anal Biochem., 246, 20-29, (1997).

Fusion proteins, such as those made from Fe portion and AXOR64 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.

25 Johanson et al., J Biol Chem, 270(16):9459-9471 (1995)).

One screening technique includes the use of cells which express the receptor of this invention (for example, transfected CHO cells) in a system which measures extracellular pH or intracelfular calcium changes caused by receptor activation. In this technique, compounds may be contacted with cells expressing the receptor polypeptide of the present invention. A second messenger response, e.g., signal transduction, pH 30 changes, or changes in calcium level, is then measured to determine whether the potential compound activates or inhibits the receptor.

Another method involves screening 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 invention to express the receptor on the cell surface. The cell is then 35 exposed to potential antagonists in the presence of the receptor of this invention. 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 increased.

GP-70684-1 17 Another method for detecting agonists or antagonists for the receptor of the present invention is the yeast based technology as described in U.S. Patent No. 5,482,83 5.

The polynucleotides, polypeptides and antibodies to the polypeptide of the present invention may 5 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 10 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 crosslinking assays in which the polypeptide islabeled with a radioactive isotope (for instance, 125J), chemically modified (for instance, biotinylated), or fused to a peptide sequence suitable for detection or purification, and incubated with a source of the putative 15 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. Standardmethods for conducting such assays are well understood in the art.

20 Examples of antagonists of polypeptides of the present invention include antibodies or, in some cases, oligonucleotides 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.

25 Screening methods may also involve the use of transgenic technology and AXOR64 gene. The art of constructing transgenic animals is well established. For example, the AXOR64 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 30 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 35 out may be targeted to specific cells or tissues, may occur only in certain cells or tissues as a consequence o f 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 express ion-clon ing system in which introduced genes are expressed to give large amounts of polypeptides of the present invention M GP-70684-1 18 Screening kits for use in the above described methods form a further aspect of the present invention. Such screening kits comprise: (a) a polypeptide of the present invention; (b) a recombinant cell expressing a polypeptide of the present invention; 5 (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.

10 Another aspect of the present invention is the use of the substances that have been identified by screening techniques referred to above in the treatment of disease states, which are responsive to regulation of histamine receptor-like polypeptide activity. The treatment may be therapeutic or prophylactic. The condition of a patient suffering from such a disease state can thus be improved.

In particular, such substances may be used in the treatment of the diseases of the invention 15 hereinbefore described. Substances identified according to the screening methods outlined above may be formulated with standard pharmaceutically acceptable carriers and/or excipients as is routine in the pharmaceutical art. For example, a suitable substance may be dissolved in physiological saline or water for injections. The exact nature of a formulation will depend upon several factors including the particular substance to be administered and the desired route of administration. Suitable types of formulation are 20 fully described in Remington's Pharmaceutical Sciences, Mack Publishing Company, Eastern.

Pennsylvania, 17 1h Ed. 1985, the disclosure of which is included herein of its entirety by way of reference.

The substances may be administered by enteral or parenteral routes such as via oral, buccal, anal, pulmonary, intravenous, intra-arterial, intramuscular, intraperitoneal, topical or other appropriate administration routes' 25 A therapeutically effective amount of a modulator is administered to a patient. The dose of a modulator may be determined according to various parameters, especially according to the substance used; the age, weight and condition of the patient to be treated; the route of administration; and the required regimen. A physician will be able to determine the required route of administration and dosage for any particular patient. A typical daily dose is from about 0.1 to 50 mg per kg of body weight, 30 according to the activity of the specific modulator, the age, weight and conditions of the subject to be treated, the type and severity of the degeneration and the frequency and route of administration.

Preferably, daily dosage levels are from 5 mg to 2 g.

Nucleic acid encoding AXOR64 or a variant thereof which inhibits AXOR64 activity may be administered to the mammal. Nucleic acid, such as RNA or DNA, and preferably, DNA, is provided in 35 the form of a vector, such as the polymicleotides described above, which may be expressed in the cells of the mammal.

Nucleic acid encoding the polypeptide may be administered by any available technique. For example, the nucleic acid may be introduced by needle injection, preferably intradermally, GP-70684-1 19 subcutaneously or intramuscularly. Alternatively, the nucleic acid may be delivered directly across the skin using a nucleic acid delivery device such as particle-mediated gene delivery. The nucleic acid may be administered topically to the skin, or to mucosal surfaces for example by intranasal, oral, intravaginal or intrarectal administration.

5 Uptake of nucleic acid constructs may be enhanced by several known transfection techniques, for example those including the use of transfection agents. Examples of these agents includes cationic -agents, for example, calcium phosphate and DEAE-Dextran and lipofectants, for example, lipofectam and transfectam. The dosage of the nucleic acid to be administered can be altered. Typically the nucleic acid is administered in the range of lpg to Inig, preferably to Ipg to I Ogg nucleic acid for particle mediated 10 gene delivery and I Ogg to I mg for other routes.

Glossary The following definitions are provided to facilitate understanding of certain terms used frequently hereinbefore.

15 "Antibodies" as used herein includes polyclonal and monoclonal antibodies, chimeric, 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, Le., if it occurs in nature, it has been changed or removed from its original environment, or both. For example, a 20 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 25 non-living.

"Polynucleotide" generally refers to any polyribonucleotide (RNA) or polydeoxribonucleotide (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 30 regions, hybrid molecules comprising 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 triple-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, tritylated bases and 35 unusual bases such as inosine. A variety of modifications may be made to DNA and RNA; thus, "polynucleotide" embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and GP-70684-1 20 cells. "Polynucleotide" also embraces relatively short polynucteotides, often referred to as oligonucleotides.

"Polypeptide" refers to any polypeptide comprising 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 5 chains, commonly referred to as peptides, oligopeptides or oligomers, and to longer chains, generally referred 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 10 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 ubiquitination, and they may be cyclic, with or without branching. Cyclic, 15 branched and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, arnidation, biotinylation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, 20 formation of covalent cross-links, formation of cystine, formation of pyroglutarnate, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racernization, 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., 25 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).

30 "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.

"Variant" refers to a polynucleotide or polypeptide that differs from a reference polynucleotide or 35 polypeptide, but retains the essential properties thereof A typical variant of a polynucleotide differs in nucleotide sequence from the reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide.

Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in GP-70684-1 21 the polypeptide encoded by the reference sequence, as discussed below. A typical variant 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 variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one 5 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, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe and Tyr. A variant of a polynucleotide or polypeptide may be naturally occurring such as an allele, or it may be a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides may 10 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, methylation, ADP ribosylation and the like. Embodiments include methylation 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 occurring at a given locus in the 15 genome.

"Polymorphism" 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 Polymorphism" (SNP) refers to the occurrence of nucleotide variability at a single nucleotide'position in the genome, within a population. An SNP may occur within a gene or within 20 intergenic regions of the genome. SNPs can be assayed using Allele Specific Amplification (ASA). For the process at least 3 primers are required. A common primer is used in reverse complement to the polymorphism being assayed. This common primer can be between 50 and 1500 bps from the polymorphic base. The other two (or more) primers 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) 25 PCR reactions are then conducted on sample DNA, each using the common primer and one of the Allele Specific Primers.

"Splice Variant" 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 transcript undergoes splicing, generally 30 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 polyriucleotide sequences, determined by comparing the sequences. In general, identity refers to an exact 35 nucleotide to nucleotide or amino 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 correspondence, a "% identity" may be determined. In general, the two sequences to be compared are aligned to give a maximum correlation GP-70684-1 22 between the sequences. This may include 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 (socalled local alignment), that is more suitable for 5 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 correspondences between a between pairs of residues, one from each of the sequences being compared (as for identity) but also, 10 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 art. Thus for instance, programs available in the Wisconsin Sequence Analysis Package, version 9.1 15 (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. BESTFIT uses the "local homology" algorithm of Smith and Watennan (J Mol Biol, 147,195 197, 198 1, Advances in Applied Mathematics, 2, 482-489, 198 1) and finds the best single region of 20 similarity between two sequences. BESTFIT is more suited to comparing two po lynucleotide 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 similarity", according to the algorithm 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 25 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 30 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 35 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 comparison.

GP-70684-1 23 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 described.

5 "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 10 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 15 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 20 Index of 0.95 compared to a reference polypeptide sequence is identical to thereference sequence except that the po lypeptide sequence may include an average of up to five differences per each 100 amino 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 25 or anywhere between these terminal positions, interspersed either individually among the amino 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 amino acids in the reference sequence may be deleted, substituted or inserted, or any combination thereof, as hereinbefore described. The same 30 applies mutatis mutandis for other values of the Identit 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:

na: Xa - (Xa in which:

35 na is the number of nucleotide or amino acid differences, Xa is the total number of nucleotides or amino acids in SEQ ID NO: I or SEQ ID NO:2, respectively, I is the Identity Index, GP-70684-1 24 is the symbol for the multiplication operator, and in which any non-integer product Of xa and I is rounded down to the nearest integer prior to subtracting it from xa.

" Homolog" is a generic term used in the art to indicate a polynucleotide or polypeptide sequence 5 possessing a high degree of sequence relatedness to a reference sequence. Such relatedness may be quantified by determining the degree of identity and/or similarity between the two sequences as hereiribefore defined. Failing within this generic term are the terms "ortholog", and "paralog".

"Ortholog" refers to a polynucleotide or polypeptide that is the functional equivalent of the poiynucleotide or polypeptide in another species. "Paralog" refers to a polynucleotide or polypeptide that within the 10 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 various 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 15 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 purified.

All publications and references, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference in their entirety as if each individual publication or

20 reference were specifically and individually indicated to be incorporated by reference herein as being fully set forth. Any patent application to which this application claims priority is also incorporated by reference herein in its entirety in the manner described above for publications and references.

Examples

Example 1: Characterisation of the sequence. A histamine receptor-like polypeptide, designated as AXOR64 has been identified. The nucleotide and amino acid sequences o f the polypeptide have been determined. These are set out below in SEQ ID NOs: 1 and 2. Suitable primers and probes were designed and used tp analyse tissue expression. AXOR64 was 30 found to be primarily expressed in the central nervous system: cerebellum and whole brain, Lower expression was observed in hypothalamus and cerebral cortex as well as in thymus, thyroid, tonsil, lung, skin, ovary, omentum, rectum and jejunum (Figure 1). The polypeptide was not expressed in resting immune cells; however it was upregulated in growth factor-stimulated bone marrow cells, osteoarthritic knee cartilage and in Hepatitis BVirus and Herpes Simplex Virus-infected cells. The polypeptide 35 appeared to be more highly expressed in frontal cortex from Huntingdon's and progressive supranuclear palsy. Cerebellar expression in amyotrophic lateral sclerosis and Huntingdon's appeared to be downregulated compared to normal (Figure 2); however, the as in frontal cor-tex expression, the polypeptide is GP-70684-1 25 highly expressed in PSP cerebellum.

The chromosomal localization was also mapped. Human AXOR64has been mapped to I p 13.

Example 2: Mammalian Cell Expression and Screening for substances which exhibit protein modulating 5 activity.

The receptors of the present invention 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 prior to insertion into a pCDN or pCDNA3 vector. The cells are transfected with individual receptor cDNAs by lipofectin and selected in the 10 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 lines 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 resistant clones analyzed.

15 A brief screening assay protocol is as follows:

Mammalian cells stably over-expressing a polypeptide of the invention are cultured in black wall, clear bottom, tissue culture-coated, 96 or 3 84 well plates with a volume of I 00gl cell culture medium in each well 3 days before use in a FLIPR (Fluorescence Imaging Plate Reader - Molecular Devices). Cells are incubated with 4gM FLUO-3AM at 300C in 5%CO2 for 90 mins and then washed once in Tyrodes 20 buffer containing 3mM probenecid. Basal fluorescence is determined prior to addition of test substances.

The polypeptide is activated upon the addition of a known agonist. Activation results in an increase in intracellular calcium which can be measured directly in the FLIPR. For antagonist studies, substances are preincubated with the cells for 4 minutes following dye loading and washing and fluorescence measured for 4 minutes. Agonists are then added and cell fluorescence measured for a further I minute.

25 Assays may also be carried out as follows:

Gs-coupled receptors are expressed and assayed in mammalian cells which express the 6xCRE luciferase reporter gene such as CHO cells. Gq-coupled and Gi-coupled receptors are expressed and assayed in mammalian cells which express the Gal4/Elk-l chimeric protein and 5xUAS-luciferase reporter gene. Cells are propagated in either in suspension or adherent cultures.

30 For adherent culture, cells are propagated in T225 flasks in DMEM/F12 containing 5% fetal bovine serum and I mM glutamine. Forty-eight hoursprior to assay, cells are harvested with 2 ml of 0.05% trypsin, washed with complete medium and plated at a concentration of 4,000 cells/well in complete medium. Sixteen hours prior to the assay, the medium is removed from the cells and replaced with 90 gl/well of serum-free DMEM/F 12. At the time of the assay, test substances are added to the 35 wells at a final concentration of 10 gM and the plates are incubated for four hours at 370C in a cell culture incubator. The medium is aspirated by vacuum followed by the addition of 50 gI of a 1: 1 mixture of LucLite Tm and dPBS/l mM CaCl,/l MM MgC12- Plates are sealed and subjected to dark adaptation at GP-70684-1 26 room temperature for 10 minutes before luciferase activity is quantitated on a TopCountT1 microplate scintillation counter (Packard) using 3 seconds/well count time.

For suspension cultures, cells are propagated in Excel 301 medium containing 5% FBS and 2 mM glutamine at a minimum of Ix 105 cells/ml for one week. Sixteen hours prior to an assay, cells are 5 removed from suspension by centrifugation and resuspended in serum-free Excel 301 at a concentration Of IX106 cells/mi. At the time of assay, the cells are resuspended in serum-free DMEMIF12 at a concentration of 50,000 cells/mi. 100 Ll/well or5O pd/well of this suspension is pipetted into black 96well or 384-well plates, respectively. The 96-well and 384-well plate contained I ul or 0.5 fl of agonist compounds in 100% DMSO at a final concentration of 10 pi.M. A Multidrop S20 cell dispenser is used to 10 dispense cells into either 96- or 384-well plates. The reminder of the assay is the same as described for adherent culture above.

Example 3 Ligand bank for binding and functional assays.

15 A bank of over 600 putative receptor ligands has been assembled for screening. The bank comprises: transmitters, hormones and chernokines known to act via a human seven transmembrane (7TM) receptor; naturally occurring 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 ligands. This bank 20 is used to initially screen the receptor for known ligands, using both functional (i.e. calcium, cAMP, microphysiometer, oocyte electrophysiology, etc, see below) as well as binding assays.

Example 4: Ligand Binding Assays Ligand binding assays provide a direct method for ascertaining receptor pharmacology and are 25 adaptable to a high throughput format. The purified 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 radiolabeling does not diminish 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 30 total associated radioactivity minus the radioactivity measured in the presence of an excess of unlabeled competing ligand. Where possible, more than one competing ligand is used to define residual nonspecific binding.

Example 5: Functional Assay in Xenopus Oocytes 35 Capped RNA transcripts from linearized plasmid templates encoding the receptor cDNAs of the invention are synthesized in vitro with RNA polymerases in accordance with standard procedures. In vitro transcripts are suspended in water at a final concentration of 0.2 mg/ml. Ovarian lobes are removed from adult female toads, Stage V defolliculated oocytes are obtained, and RNA transcripts (10 ng/oocyte) are GP-70684-1 27 injected in a 50 nt bolus using a microinjection apparatus. Two electrode voltage clamps are used to measure the currents from individual Xenopus oocytes in 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 ligands and tissue/cell extracts for activating ligands.

Example 6: Microphysiometric Assays Activation of a wide variety of secondary messenger systems results in 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 intracellular signaling process. The pH changes in the media surrounding the cell are very small but are 10 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 intracellular signaling pathway such as the G-protein coupled receptor of the present invention.

Example 7: Melanophore Screens 15 Modified or unmodified receptors are expressed in melanophores using appropriate vector constructs including pJG3.6. The expressed receptors are then screened for Gs, Gq, Gi or Go activity.

When a ligand binds to a Gs-coupled receptor, it activates adenylyl cyclase that in turn activates protein kinase A. This results in the initiation of phosphorylation events that cause the melanosomes to disperse.

When a Gi-coupled receptor is activated, it inhibits adenylyl cyclase which in turn reverses the pigment 20 dispersion process to result in aggregation. When a Gq-COUPled receptor is activated, it activates phospholipase C, which in turn activates protein kinase C. This results in the initiation of phosphorylation events to cause melanosome dispersion. The expressed receptors can be screened in agonist, antagonist or constitutive modes using bead-based lawn format or 96-well, 384-well or 1536-well formats.

25 Melanophores are grown in conditioned fibroblast medium (CFM) at room temperature. After harvesting the cells with trypsin/EDTA, approximately 6 to 10 million cells are electroporated with relevant receptor- expression vectors at 475 V, 425 IAFd, 720 ohms. The transfected cells are then plated into T225 flasks and are incubated for 24 hours. Cells are then harvested and plated into assay plates and incubated for24hours. Test substances are added to wells at 10 uM final concentration and 30minutes later 30 the dispersion or aggregation is measured using an SLT Spectra plate reader. For dispersion assays, cells are first treated with 2 nM melatonin in assay buffer (0.7X LI 510.1% BSA) for 60 minutes before addition of test compounds. For aggregation assays, CFM is replaced with the assay buffer and cells are incubated for 60 minutes before addition of test compounds.

35 Example 8: Extract/Cell Supernatant Screening A large number of mammalian receptors exist for which there remains, as yet, no cognate activating ligand (agonist). Thus, active ligands for these receptors may not be included within the ligand banks as identified to date. Accordingly, the 7TM receptor of the invention is also functionally screened (using GP-70684-1 28 calcium, cAMP, microphysiometer, oocyte electrophysiology, etc., functional screens) against tissue extracts to identify natural ligands. Extracts that produce positive functional responses can be sequentially subfractionated until an activating ligand is isolated and identified.

5 Example 9: 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 in the HEK 293 cells in receptor-transfected or vector control cells were observed to be in the normal, 100 nM to 200 nM, range. HEK 293 cells expressing recombinant receptors are loaded with fara 2 and in a single day 10 > 150 selected ligands or tissue/cell extracts are evaluated for agonist induced calcium mobilization.

Similarly, HEK 293 cells expressing recombinant receptors are evaluated for the stimulation or inhibition of cANIP production using standard cANIP quantitation assays. Agonists presenting a calcium transient or cANW fluctuation are tested in vector control cells to determine if the response is unique to the transfected cells expressing receptor.

GP-70684-1 29 SEQUENCE INFORMATION SEQ ED NO: I ATGGAGTCCTCACCCATCCCCCAGTCATCAGGGAACTCTTCCACTTTGGGGAGGGTCCCT CAAACCCCAGGTCCCTCTACTGCCAGTGGGGTCCCGGAGGTGGGGCTACGGGATGTTGCT 5 TCGGAATCTGTGGCCCTCTTCTTCATGCTCCTGCTGGACTTGACTGCTGTGGCTGGCAAT GCCGCT G T GAT GGCCGTGATCGCCAAGACGCCT GCCCTCCGAAAATT TGTCT TCGT C T TC CACCTCTGCCTGGTGGACCTGCTGGCTGCCCTGACCCTCATGCCCCTGGCCATGCTCTCC AGCTCTGCCCTCTTTGACCACGCCCTCTTTGGGGAGGTGGCCTGCCGCCTCTACTTGTTT CTGAGCGTGTGCTTTGTCAGCCTGGCCATCCTCTCGGTGTCAGCCATCAATGTGGAGCGC 10 TACTATTACGTAGTCCACCCCATGCGCTACGAGGTGCGCATGACGCTGGGGCTGGTGGCC TCTGTGCTGGTGGGTGTGTGGGTGAAGGCCTTGGCCATGGCTTCTGTGCCAGTGTTGGGA AGGGTCTCCTGGGAGGAAGGAGCTCCCAGTGTCCCCCCAGGCTGTTCACTCCAGTGGAGC CACAGTGCCTACTGCCAGCTTTTTGTGGTGGTCTTTGCTGTCCTTTACTTTCTGTTGCCC CTGCTCCTCATACTTGTGGTCTACTGCAGCATGTTCCGAGTGGCCCGCGTGGCTGCCATG 15 CAGCACGGGCCGCTGCCCACGTGGATGGAGACACCCCGGCAACGCTCCGAATCTCTCAGC AGCCGCTCCACGATGGTCACCAGCTCGGGGGCCCCCCAGACCACCCCACACCGGACGTTT GGGGGAGGGAAAGCAGCAGTGGTTCTCCTGGCTGTGGGGGGACAGTTCCTGCTCTGTTGG TTGCCCTACTTCTCTTTCCACCTCTATGTTGCCCTGAGTGCTCAGCCCATTTCAACTGGG CAGGTGGAGAGTGTGGTCACCTGGATTGGCTACTTTTGCTTCACTTCCAACCCTTTCTTC 20 TATGGATGTCTCAACCGGCAGATCCGGGGGGAGCTCAGCAAGCAGTTTGTCTGCTTCTTC AAGCCAGCTCCAGAGGAGGAGCTGAGGCTGCCTAGCCGGGAGGGCTCCATTGAGGAGAAC TTCCTGCAGTTCCTTCAGGGGACTGGCTGTCCTTCTGAGTCCTGGGTTTCCCGACCCCTA CCCAGCCCCAAGCAGGAGCCACCTGCTGTTGACTTTCGAATCCCAGGCCAGATAGCTGAG GAGACCTCTGAGTTCCTGGAGCAGCAACTCACCAGCGACATCATCATGTCAGACAGCTAC 25 CTCCGTCCTGCCGCCTCACCCCGGCTGGAGTCATGA SEQ 11D NO:2 MESSPIPQSSGNSSTLGRVPQTPGPSTASGVPEVGLRDVASESVALFFMLLLDLTAVAGN AAVMAVIAKTPALRKFVFVFHLCLVDLLAALTLMPLAMLSSSALFDIIALFGEVACRLYLF LSVCFVSLAILSVSAINVERYYYVVHPMRYEVRMTLGLVASVLVGVWVKALAMASVPVLG 30 RVSWEEGAPSVPPGCSLQWSHSAYCQLFVVVFAVLYFLLPLLLILVVYCSMFRVARVAAM QHGPLPTWMETPRQRSESLSSRSTMVTSSGAPQTTPHRTFGGGKAAVVLLAVGGQFLLCW LPYFSFHLYVALSAQPISTGQVESVVTWIGYFCFTSNPFFYGCLNRQIRGELSKQFVCFF KPAPEEELRLPSREGSIEENFLQFLQGTGCPSESWVSRPLPSPKQEPPAVDFRIPGQIAE ETSEFLE. QQLTSDIIMSDSYLRPAASPRLES GP-70684-1 30

Claims (9)

What is claimed is:

1. An isolated polypeptide selected from the group consisting of:

(a) an isolated polypeptide encoded by a polynucleotide comprising the sequence of SEQ ID NO: 1; 5 (b) an isolated polypeptide comprising a polypeptide, sequence having at least 95% identity to the polypeptide sequence of SEQ ID NO:2; (c) an isolated polypeptide comprising 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 10 (f) fragments and variants of such polypeptides in (a) to (e).

2. An isolated polynucleotide selected from the group consisting of: (a) an isolated polynucleotide comprising a polynucleotide sequence having at least 95% identity to the polynucleotide sequence of SEQ ID NO: 1; 15 (b) an isolated polynucleotide comprising 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 comprising a polynucleotide sequence encoding a polypeptide sequence having at least 95% identity to the polypeptide sequence of SEQ ID NO:2; 20 ( an isolated polynucleotide comprising 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 95% identity to the polypeptide sequence of SEQ ID NO:2; (h) an isolated polynucleotide encoding the polypeptide of SEQ ID NO:2; 25 (i) an isolated polynucleotide with a nucleotide sequence of at least 100 nucleotidesobtained by screening a library under stringent hybridization conditions with a labeled probe having the sequence of SEQ ID NO: I or a fragment thereof having at least 15 nucleotides; and 0) a polynucleotide which is the RNA equivalent of a polynucleotide of (a) to (i); or a polynucleotide sequence complementary to said isolated polynucleotide, 30 and polynucleotides that are variants and fragments of the above mentioned polynucleotides or that are complementary to above mentioned polynucleotides, over the entire length thereof.

3 ). An antibody immunospecific for the polypeptide, of claim 1.

35

4. An antibody as claimed in claim 3 which is a polyclonal antibody.

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

GP-70684-1 31

6. A process for producing a recombinant host cell which comprises the step of introducing an expression vector comprising a polynucleotide capable of producing a polypeptide of claim I into a cell such that the host cell, under appropriate culture conditions, produces said polypeptide.

7. A recombinant host cell produced by the process of claim 6.

8. A membrane of a recombinant host cell of claim 7 expressing said polypeptide.

10

9. A process for producing a polypeptide which comprises culturing a host cell of claim 7 under conditions sufficient for the production of said polypeptide and recovering said polypeptide from the culture.