WO2001004139A2 - Human axor29 receptor - Google Patents

Human axor29 receptor Download PDF

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Publication number
WO2001004139A2
WO2001004139A2 PCT/US2000/019001 US0019001W WO0104139A2 WO 2001004139 A2 WO2001004139 A2 WO 2001004139A2 US 0019001 W US0019001 W US 0019001W WO 0104139 A2 WO0104139 A2 WO 0104139A2
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Prior art keywords
polypeptide
sequence
polynucleotide
seq
receptor
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PCT/US2000/019001
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French (fr)
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WO2001004139A3 (en
Inventor
Robert S. Ames
Nabil Elshourbagy
James J. Foley
David Michalovich
Henry M. Sarau
Randall_F. Smith
Ping Tsui
Lisa Vawter
Pankaj Agarwal
Pamela A. Lane
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Smithkline Beecham Corporation
Smithkline Beecham Plc
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Priority claimed from GBGB9916417.0A external-priority patent/GB9916417D0/en
Application filed by Smithkline Beecham Corporation, Smithkline Beecham Plc filed Critical Smithkline Beecham Corporation
Publication of WO2001004139A2 publication Critical patent/WO2001004139A2/en
Publication of WO2001004139A3 publication Critical patent/WO2001004139A3/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to newly identified polypeptides and polynucleotides encoded by them and to the use of such polynucleotides and polypeptides, and to their production. More particularly, the polynucleotides and polypeptides of the present invention relate to the G-protein coupled receptors, hereinafter referred to as human AXOR29 receptor.
  • This invention also relates to methods for discove ⁇ ng agonists and antagonists of the interaction between sphmgosme 1- phosphate (hereinafter referred to as "S-l-P") and di-hydro sphmgosme- 1 -phosphate (hereinafter referred to as "di -hydro S-l-P”) and their cellular receptor, human AXOR29 receptor.
  • the invention also relates to the use of human AXOR29 receptor polynucleotides and polypeptides m therapy and in identifying compounds which may be agonists, antagonists and /or inhibitors which are potentially useful in therapy, and to production of such polypeptides and polynucleotides
  • the drug discovery process is currently undergoing a fundamental revolution as it embraces "functional genomics", that is, high throughput genome- or gene-based biology. This approach as a means to identify genes and gene products as therapeutic targets is rapidly superseding earlier approaches based on “positional cloning”. A phenotype, that is a biological function or genetic disease, would be identified and this would then be tracked back to the responsible gene, based on its genetic map position.
  • proteins participating in signal transduction pathways that involve G-protems and/or second messengers, e.g., cAMP (Lefkowitz, Nature, 1991, 351:353-354).
  • these proteins are referred to as protems participating in pathways with G-protems 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.
  • the effect of hormone binding is activation of the enzyme, adenylate cyclase, mside the cell.
  • Enzyme activation by hormones is dependent on the presence of the nucleotide GTP.
  • GTP also influences hormone binding.
  • a G-protem connects the hormone receptor to adenylate cyclase.
  • G-protem was shown to exchange GTP for bound GDP when activated by a hormone receptor.
  • the GTP-carrying form then binds to activated adenylate cyclase.
  • Hydrolysis of GTP to GDP catalyzed by the G-protem itself, returns the G-protem to its basal, mactive form.
  • the G-protem serves a dual role, as an intermediate that relays the signal from receptor to effector, and as a clock that controls the duration of the signal.
  • G-protem coupled receptors include a wide range of biologically active receptors, such as hormone, viral, growth factor and neuroreceptors .
  • G-protem coupled receptors (otherwise known as 7TM receptors) have been characte ⁇ zed as including these seven conserved hydrophobic stretches of about 20 to 30 ammo acids, connecting at least eight divergent hydrophilic loops.
  • the G-protem family of coupled receptors includes dopamme receptors which bmd to neuroleptic drugs used for treating psychotic and neurological disorders.
  • members of this family mclude, but are not limited to, calcitomn, adrenergic, endothelm, cAMP, adenosme, musca ⁇ nic, acetylcho ne, serotonin, histamme, thrombm, kmm, follicle stimulating hormone, opsms, endothehal differentiation gene-1, rhodopsms, odorant, and cytomegalovirus receptors.
  • G-protem coupled receptors have single conserved cysteme residues in each of the first two extracellular loops which form disulfide bonds that are believed to stabilize functional protein structure.
  • TM1, TM2, TM3, TM4, TM5, TM6, and TM7 The 7 transmembrane regions are designated as TM1, TM2, TM3, TM4, TM5, TM6, and TM7.
  • TM3 has been implicated in signal transduction.
  • G-protem coupled receptors Phosphorylation and hpidation (palmitylation or farnesylation) of cysteme residues can influence signal transduction of some G-protem coupled receptors.
  • Most G-protem coupled receptors contain potential phosphorylation sites within the third cytoplasmic loop and or the carboxy terminus.
  • G-protem coupled receptors such as the b-adrenoreceptor
  • phosphorylation by protem kmase A and/or specific receptor kinases mediates receptor desensitization.
  • the ligand binding sites of G-protein coupled receptors are believed to comp ⁇ se hydrophilic sockets formed by several G-protem coupled receptor transmembrane domains, said socket being surrounded by hydrophobic residues of the G-protem coupled receptors.
  • the hydrophilic side of each G-protem coupled receptor transmembrane helix is postulated to face mward and form polar ligand binding site.
  • TM3 has been implicated m several G-protein coupled receptors as having a ligand binding site, such as the TM3 aspartate residue TM5 se ⁇ nes, a TM6 asparagrne and TM6 or TM7 phenylalanmes or tyrosmes are also implicated m ligand binding.
  • G-protem coupled receptors can be mtracellularly coupled by heterot ⁇ me ⁇ c G-protems to va ⁇ ous mtracellular enzymes, ion channels and transporters (see, Johnson, et al, Endoc. Rev., 1989, 10:317-331)
  • Different G-protem a-subunits preferentially stimulate particular effectors to modulate va ⁇ ous biological functions in a cell.
  • Phosphorylation of cytoplasmic residues of G-protem coupled receptors have been identified as an important mechanism for the regulation of G-protem couplmg of some G-protem coupled receptors.
  • G-protein coupled receptors are found in numerous sites withm a mammalian host. Over the past 15 years, nearly 350 therapeutic agents targeting 7 transmembrane (7 TM) receptors have been successfully introduced onto the market.
  • the present invention relates to human AXOR29 receptor, m particular human AXOR29 receptor polypeptides and human AXOR29 receptor polynucleotides, recombinant mate ⁇ als and methods for their production.
  • Such polypeptides and polynucleotides are of interest m relation to methods of treatment of certain diseases, including, but not limited to infections such as bacte ⁇ al, fungal, protozoan and viral infections, particularly infections caused by HTV-1 or HIV-2; pa ; cancers; diabetes, obesity; anorexia; bulimia; asthma; Parkinson's disease; acute heart failure; hypotension; hypertension; u ⁇ nary retention; osteoporosis; angina pecto ⁇ s; myocardial infarction; stroke; ulcers; asthma; allergies; benign prostatic hypertrophy; migraine; vomiting; psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, depression, delirium, dementia, and severe mental retardation, hereinafter referred to as "diseases of the invention.
  • the preferred method for identifying agonist or antagonist of a human AXOR29 receptor polypeptide comp ⁇ ses: (a) contacting a cell expressing on the surface thereof the polypeptide, said polypeptide being associated with a second component capable of providmg a detectable signal m response to the bmdmg of a compound to said polypeptide, with a compound to be screened under conditions to permit bmdmg to the polypeptide; and (b) determining whether the compound binds to and activates or inhibits the polypeptide by measu ⁇ ng the level of a signal generated from the interaction of the compound with the polypeptide.
  • the method further comp ⁇ ses conductmg the identification of agonist or antagonist in the presence of labeled or unlabeled S-l-P or di-hydro S-l-P.
  • the method for identifying agonist or antagonist of a human AXOR29 receptor polypeptide comp ⁇ ses: determining the inhibition of bmdmg of a ligand to cells which have the polypeptide on the surface thereof, or to cell membranes containing the polypeptide, m the presence of a candidate compound under conditions to permit binding to the polypeptide, and determining the amount of ligand bound to the polypeptide, such that a compound capable of causing reduction of bmdmg of a ligand is an agonist or antagonist.
  • the ligand is S-l-P or di-hydro S-l-P. Yet more preferably, S-l-P or di-hydro S-l-P is labeled.
  • the present invention relates to treating conditions associated with human AXOR29 recpeptor imbalance with the identified compounds.
  • Yet another aspect of the mvention relates to diagnostic assays for detecting diseases associated with mapprop ⁇ ate human AXOR29 receptor activity or levels.
  • Figure 1 shows the nucleotide sequence of the human AXOR29 receptor (SEQ ID NO:l).
  • Figure 2 shows the deduced ammo acid sequence of the human AXOR29 receptor (SEQ ID NO:2).
  • Figure 3 shows concentration-response curves for S-l-P with 3 clones of the human AXOR29 receptor stably expressed in RBL 2H3 cells and also for RBL 2H3 cells and RBL 2H3 vector control cells.
  • Figure 4 shows concentration-response curves for lysophosphatidic acid (LPA) with 3 clones of the AXOR29 receptor stably expressed in RBL 2H3 cells and also for RBL 2H3 cells and RBL 2H3 vector control cells.
  • the present invention relates to AXOR29 polypeptides
  • Such polypeptides include:
  • Polypeptides of the present mvention are believed to be members of the 7 transmembrane G- protem coupled receptor family of polypeptides.
  • the biological properties of the human AXOR29 receptor are hereinafter referred to as "biological activity of human AXOR29 receptor” or "human AXOR29 receptor activity”.
  • a polypeptide of the present invention exhibits at least one biological activity of human AXOR29 receptor.
  • Polypeptides of the present invention also include va ⁇ ants of the aforementioned polypeptides, including all allehc forms and splice va ⁇ ants. Such polypeptides vary from the reference polypeptide by insertions, deletions, and substitutions that may be conservative or non-conservative, or any combination thereof. Particularly preferred va ⁇ ants are those m which several, for instance from 50 to 30, from 30 to 20, from 20 to 10, from 10 to 5, from 5 to 3, from 3 to 2, from 2 to 1 or 1 ammo acids are inserted, substituted, or deleted, in any combination.
  • Preferred fragments of polypeptides of the present invention include an isolated polypeptide comp ⁇ sing an ammo acid sequence having at least 30, 50 or 100 contiguous ammo acids from the ammo acid sequence of SEQ ID NO: 2, or an isolated polypeptide comp ⁇ sing an ammo acid sequence having at least 30, 50 or 100 contiguous ammo acids truncated or deleted from the ammo acid sequence of SEQ ID NO: 2.
  • Preferred fragments are biologically active fragments that mediate the biological activity of human AXOR29 receptor, including those with a similar activity or an improved activity, or with a decreased undesirable activity. Also preferred are those fragments that are antigenic or lmmunogenic m an animal, especially m a human.
  • polypeptides of the invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, these va ⁇ ants may be employed as intermediates for producing the full-length polypeptides of the mvention
  • the polypeptides of the present invention may be in the form of the "mature" protem or may be a part of a larger protem such as a precursor or a fusion protem. It is often advantageous to include an additional ammo acid sequence that contains secretory or leader sequences, pro-sequences, sequences that aid in pu ⁇ f ⁇ cation, for instance multiple histidme residues, or an additional sequence for stability du ⁇ ng recombinant production.
  • Polypeptides of the present invention can be prepared in any suitable manner, for instance by isolation form naturally occur ⁇ ng sources, from genetically engmeered host cells comp ⁇ sing expression systems (vide infra) or by chemical synthesis, using for instance automated peptide synthesisers, or a combination of such methods.
  • Means for preparing such polypeptides are well understood m the art
  • the present mvention relates to human AXOR29 receptor polynucleotides.
  • Such polynucleotides include: (a) an isolated polynucleotide comp ⁇ smg a polynucleotide sequence having at least 95%, 96%, 97%,
  • Preferred fragments of polynucleotides of the present mvention include an isolated polynucleotide comp ⁇ sing 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 comp ⁇ sing an sequence having at least 30, 50 or 100 contiguous nucleotides truncated or deleted from the sequence of SEQ ID NO: 1.
  • Preferred va ⁇ ants of polynucleotides of the present invention include splice va ⁇ ants, allehc va ⁇ ants, and polymorphisms, including polynucleotides having one or more smgle nucleotide polymorphisms (SNPs).
  • SNPs smgle nucleotide polymorphisms
  • Polynucleotides of the present mvention also include polynucleotides encodmg polypeptide va ⁇ ants that comp ⁇ se the ammo acid sequence of SEQ ID NO:2 and m which several, for instance from 50 to 30, from 30 to 20, from 20 to 10, from 10 to 5, from 5 to 3, from 3 to 2, from 2 to 1 or 1 ammo acid residues are substituted, deleted or added, m any combination.
  • the present invention provides polynucleotides that are RNA transc ⁇ pts of the DNA sequences of the present mvention. Accordingly, there is provided an RNA polynucleotide that:
  • (b) is the RNA transc ⁇ pt of the DNA sequence encoding the polypeptide of SEQ ID NO:2;
  • RNA transc ⁇ pt of the DNA sequence of SEQ ID NO: 1; or (d) is the RNA transc ⁇ pt of the DNA sequence of SEQ ID NO: 1 ; and RNA polynucleotides that are complementary thereto.
  • the polynucleotide sequence of SEQ ID NO: 1 shows homology with Mouse edg-1 G-protem coupled receptor (CH. Liu and T. Hla, Genomics 43 : 15-24, 1997).
  • the polynucleotide sequence of SEQ ID NO: 1 is a cDNA sequence that encodes the polypeptide of SEQ ID NO:2.
  • the polynucleotide sequence encoding the polypeptide of SEQ ID NO:2 may be identical to the polypeptide encodmg sequence of SEQ ID NO: 1 or it may be a sequence other than SEQ ED NO: 1 , which, as a result of the redundancy (degeneracy) of the genetic code, also encodes the polypeptide of SEQ ED NO.2
  • the polypeptide of the SEQ ED NO:2 is related to other protems of the 7 transmembrane G-protem coupled receptor family, having homology and/or structural simila ⁇ ty with Mouse edg-1 G-protem coupled receptor (CH. Liu and T. Hla, Genomics 43:15-24, 1997).
  • AXOR29 was identified to be an EDG receptor.
  • EDG receptors comprise a small, closely-related group of GPCRs.
  • Known EDG receptors are
  • EDG-1, EDG-2, EDG-3, EDG-4, EDG-5, EDG-6 and EDG-7 See Moolenaar, et al, J Curr Opin. Cell Biol. 9(2): 168-173 (1997); MacLennan, et al, J Cell. Neurosci. 5(3) 201- 209 (1994); Graler, et al , J Genomics 53(2): 164-169 (1998); Bandoh, Acid J Bwl Chem.274 27776-27785 (1999).
  • EDG-2, -4 and-7 are more closely-related to each other than any of them is to the S-l-P - responding EDG receptor group.
  • the human AXOR29 receptor shares 42% ammo acid identity with EDG-1 and shares the same de ⁇ ved, fixed mutations that we identified in the S-1-P-respondmg group of EDG receptors (EDG- 1, -3 and -5). We we have confirmed through expe ⁇ mentation that S-l-P is the ligand for AXOR29.
  • Preferred polypeptides and polynucleotides of the present mvention are expected to have, inter alia, similar biological functions/properties to their homologous polypeptides and polynucleotides. Furthermore, preferred polypeptides and polynucleotides of the present mvention have at least one human AXOR29 receptor activity.
  • Polynucleotides of the present mvention may be obtained using standard cloning and screenmg techmques from a cDNA library de ⁇ ved from mRNA in cells of human lymph, prostate, pancreas and multiple sclerosis lesions, (see for instance, Sambrook, et al., MOLECULAR CLONTNG: A LABORATORY MANUAL, 2nd Ed., Cold Sp ⁇ ng Harbor Laboratory Press, Cold Sp ⁇ ng Harbor, N.Y (1989)).
  • Polynucleotides of the invention can also be obtained from natural sources such as genomic DNA hbra ⁇ es or can be synthesized using well known and commercially available techniques
  • 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 codmg sequences, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protem sequence, or other fusion peptide portions.
  • a marker sequence that facilitates pu ⁇ f ⁇ cation of the fused polypeptide can be encoded.
  • the marker sequence is a hexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) and descnbed in Gentz et al , Proc NatlAcad Sci USA (1989) 86-821-824, or is an HA tag
  • the polynucleotide may also contain non-codmg 5' and 3' sequences, such as transcnbed, non-translated sequences, splicing and polyadenylation signals, ⁇ bosome bmdmg sites and sequences that stabilize mRNA.
  • Polynucleotides that are identical, or have sufficient identity to a polynucleotide sequence of SEQ ED NO: 1 may be used as hyb ⁇ dization probes for cDNA and genomic DNA or as p ⁇ mers for a nucleic acid amplification reaction (for instance, PCR). Such probes and p ⁇ mers may be used to isolate full-length cDNAs and genomic clones encoding polypeptides of the present mvention and to isolate cDNA and genomic clones of other genes (including genes encodmg paralogs from human sources and orthologs and paralogs from species other than human) that have a high sequence simila ⁇ ty to SEQ ED NO: 1 , typically at least 95% identity.
  • Preferred probes and p ⁇ mers will generally comp ⁇ se at least 15 nucleotides, preferably, at least 30 nucleotides and may have at least 50, if not at least 100 nucleotides Particularly preferred probes will have between 30 and 50 nucleotides. Particularly preferred p ⁇ mers will have between 20 and 25 nucleotides.
  • a polynucleotide encoding a polypeptide of the present mvention, mcludmg homologs from species other than human, may be obtained by a process comp ⁇ smg the steps of screening a library under stringent hyb ⁇ dization conditions with a labeled probe having the sequence of SEQ ED NO 1 or a fragment thereof, preferably of at least 15 nucleotides; and isolating full-length cDNA and genomic clones containing said polynucleotide sequence.
  • hyb ⁇ dization techniques are well known to the skilled artisan.
  • Preferred stringent hyb ⁇ dization conditions mclude overnight incubation at 42°C in a solution comp ⁇ smg: 50% formamide, 5xSSC (150mM NaCl, 15mM t ⁇ sodium citrate), 50 mM sodium phosphate (pH7.6), 5x Denhardt's solution, 10 % dextran sulfate, and 20 microgram/ml denatured, sheared salmon sperm DNA; followed by washing the filters in 0.
  • the present mvention also includes isolated polynucleotides, preferably with a nucleotide sequence of at least 100, obtained by screenmg a library under st ⁇ ngent hyb ⁇ dization conditions with a labeled probe having the sequence of SEQ ED NO: 1 or a fragment thereof, preferably of at least 15 nucleotides.
  • processing (a measure of the ability of the enzyme to remain attached to the template du ⁇ ng the polymerisation reaction), failing to complete a DNA copy of the mRNA template du ⁇ ng first strand cDNA synthesis.
  • the PCR reaction is then repeated using 'nested' p ⁇ mers, that is, p ⁇ mers designed to anneal withm the amplified product (typically an adaptor specific p ⁇ mer that anneals further 3' m the adaptor sequence and a gene specific p ⁇ mer that anneals further 5' in the known gene sequence).
  • the products of this reaction can then be analysed by DNA sequencing and a full-length cDNA constructed either by joining the product directly to the existing cDNA to give a complete sequence, or carrying out a separate full- length PCR using the new sequence information for the design of the 5' p ⁇ mer.
  • Recombmant polypeptides of the present invention may be prepared by processes well known in the art from genetically engmeered host cells comp ⁇ sing expression systems. Accordingly, m a further aspect, the present invention relates to expression systems comp ⁇ sing a polynucleotide or polynucleotides of the present mvention, to host cells which are genetically engmeered with such expression systems and to the production of polypeptides of the invention by recombinant techmques. Cell-free translation systems can also be employed to produce such protems using RNAs de ⁇ ved from the DNA constructs of the present mvention.
  • host cells can be genetically engineered to incorporate expression systems or portions thereof for polynucleotides of the present mvention.
  • Polynucleotides may be mtroduced mto host cells by methods desc ⁇ bed m many standard laboratory manuals, such as Davis, et al, BASIC METHODS IN MOLECULAR BIOLOGY (1986) and Sambrook, et al.(ibid).
  • Preferred methods of introducing polynucleotides mto host cells mclude, for instance, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, micromjection, cationic hpid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection.
  • approp ⁇ ate hosts include bacte ⁇ al cells, such as Streptococci, Staphylococci, E. coli, Streptomyces and Bacillus subtihs cells; fungal cells, such as yeast cells and
  • Aspergillus cells insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells; and plant cells.
  • a great va ⁇ ety of expression systems can be used, for instance, chromosomal, episomal and virus-de ⁇ ved systems, e g , vectors de ⁇ ved from bacte ⁇ al plasrmds, from bacte ⁇ ophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccmia viruses, adenovrruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors de ⁇ ved from combinations thereof, such as those de ⁇ ved from plasrmd and bacte ⁇ ophage genetic elements, such as cosmids and phagemids
  • the expression systems may contain control regions that regulate as well as engender expression.
  • any system or vector that is able to maintain, propagate or express a polynucleotide to produce a polypeptide m a host may be used.
  • the approp ⁇ ate polynucleotide sequence may be mserted into an expression system by any of a va ⁇ ety of well-known and routine techniques, such as, for example, those set forth m Sambrook et al., (ibid).
  • Approp ⁇ ate secretion signals may be incorporated mto the desired polypeptide to allow secretion of the translated protem into the lumen of the endoplasmic reticulum, the penplasmic space or the extracellular environment. These signals may be endogenous to the polypeptide or they may be heterologous signals.
  • a polypeptide of the present mvention is to be expressed for use m screenmg assays, it is generally preferred that the polypeptide be produced at the surface of the cell.
  • the cells may be harvested p ⁇ or to use in the screening assay. If the polypeptide is secreted mto the medium, the medium can be recovered m order to recover and pu ⁇ fy the polypeptide. If produced mtracellularly, the cells must first be lysed before the polypeptide is recovered.
  • Polypeptides of the present invention can be recovered and pu ⁇ fied from recombmant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, amon or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography
  • polypeptides of the present invention may be used as diagnostic reagents, through detecting mutations m the associated gene.
  • Detection of a mutated form of the gene characte ⁇ sed by the polynucleotide of SEQ ED NO: 1 m the cDNA or genomic sequence and which is associated with a dysfunction will provide a diagnostic tool that can add to, or define, a diagnosis of a disease, or susceptibility to a disease, which results from under-expression, over-expression or altered spatial or temporal expression of the gene.
  • Individuals carrying mutations m the gene may be detected at the DNA level by a va ⁇ ety of techniques well known m the art.
  • Nucleic acids for diagnosis may be obtamed from a subject's cells, such as from blood, urine, saliva, tissue biopsy or autopsy mate ⁇ al.
  • the genomic DNA may be used directly for detection or it may be amplified enzymatically by usmg PCR, preferably RT-PCR, or other amplification techniques p ⁇ or to analysis.
  • RNA or cDNA may also be used m similar fashion. Deletions and insertions can be detected by a change in size of the amplified product m comparison to the normal genotype. Pomt mutations can be identified by hyb ⁇ dizmg amplified DNA to labeled human AXOR29 receptor nucleotide sequences.
  • DNA sequence difference may also be detected by alterations m the electrophoretic mobility of DNA fragments in gels, with or without denatu ⁇ ng agents, or by direct DNA sequencing (see, for instance, Myers et al, Science (1985) 230: 1242). Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and S 1 protection or the chemical cleavage method (see Cotton, et al , Proc Natl Acad Sci USA (1985) 85: 4397-4401).
  • An array of oligonucleotides probes comp ⁇ sing human AXOR29 receptor polynucleotide sequence or fragments thereof can be constructed to conduct efficient screening of e.g , genetic mutations.
  • Such arrays are preferably high density arrays or gnds.
  • Array technology methods are well known and have general applicability and can be used to address a va ⁇ ety of questions m molecular genetics including gene expression, genetic linkage, and genetic va ⁇ abihty, see, for example, M.Chee, et al, Science, 21 A, 610-613 (1996) and other references cited therem.
  • Detection of abnormally decreased or increased levels of polypeptide or mRNA expression may also be used for diagnosing or determining susceptibility of a subject to a disease of the invention. Decreased or increased expression can be measured at the RNA level using any of the methods well known in the art for the quantitation of polynucleotides, such as, for example, nucleic acid amplification, for instance PCR, RT-PCR, RNase protection, Northern blotting and other hyb ⁇ dization methods.
  • Assay techniques that can be used to determine levels of a protem, such as a polypeptide of the present invention, in a sample de ⁇ ved from a host are well-known to those of skill in the art. Such assay methods mclude radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays.
  • the present invention relates to a diagnostic kit comp ⁇ sing
  • a polynucleotide of the present mvention preferably the nucleotide sequence of SEQ ED NO 1 , or a fragment or an RNA transc ⁇ pt thereof;
  • polypeptide of the present invention preferably the polypeptide of SEQ ED NO:2 or a fragment thereof; or
  • kits may comp ⁇ se a substantial component.
  • Such a kit will be of use diagnosing a disease or susceptibility to a disease, particularly diseases of the invention, amongst others.
  • the polynucleotide sequences of the present mvention are valuable for chromosome localisation studies.
  • the sequence is specifically targeted to, and can hyb ⁇ dize with, a particular location on an individual human chromosome.
  • the mapping of relevant sequences to chromosomes accordmg to the present mvention is an important first step in correlating those sequences with gene associated disease. Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found m, for example, V. McKusick, Mendelian Inhe ⁇ tance m Man (available on-lme through Johns
  • PCRs result m 93 scores indicating the presence or absence of the PCR product of the gene of interest. These scores are compared with scores created using PCR products from genomic sequences of known location. This compa ⁇ son is conducted at http://www.genome.wi.mit.edu/.
  • the gene of the present invention maps to human chromosome 19pl3.2.
  • the polynucleotide sequences of the present mvention are also valuable tools for tissue expression studies. Such studies allow the determination of expression patterns of polynucleotides of the present mvention which may give an indication as to the expression patterns of the encoded polypeptides in tissues, by detecting the mRNAs that encode them.
  • results from these studies can provide an indication of the normal function of the polypeptide in the organism.
  • comparative studies of the normal expression pattern of mRNAs with that of mRNAs encoded by an alternative form of the same gene can provide valuable msights mto the role of the polypeptides of the present invention, or that of inappropnate expression thereof in disease.
  • mapprop ⁇ ate expression may be of a temporal, spatial or simply quantitative nature.
  • a further aspect of the present mvention relates to antibodies.
  • the polypeptides of the mvention or their fragments, or cells expressmg them, can be used as immunogens to produce antibodies that are lmmunospecific for polypeptides of the present mvention.
  • the term "lmmunospecific" means that the antibodies have substantially greater affinity for the polypeptides of the mvention than their affinity for other related polypeptides in the p ⁇ or art.
  • Antibodies generated against polypeptides of the present mvention may be obtained by administering the polypeptides or epitope-bea ⁇ ng fragments, or cells to an animal, preferably a non- human animal, using routine protocols.
  • any technique which provides antibodies produced by continuous cell lme cultures can be used. Examples include the hyb ⁇ doma technique (Kohler, G. and Milstein, C, Nature (1975) 256:495-497), the t ⁇ oma technique, the human B-cell hyb ⁇ doma technique (Kozbor et al, Immunology Today (1983) 4:72) and the EBV- hybridoma technique (Cole, et al. , MONOCLONAL ANTIBODIES AND CANCER THERAPY, 77-
  • smgle chain antibodies such as those desc ⁇ bed in U.S. Patent No. 4,946,778, can also be adapted to produce smgle chain antibodies to polypeptides of this mvention.
  • transgenic mice, or other orgamsms, including other mammals may be used to express humanized antibodies.
  • the above-desc ⁇ bed antibodies may be employed to isolate or to identify clones expressing the polypeptide or to pu ⁇ fy the polypeptides by affinity chromatography.
  • Antibodies against polypeptides of the present invention may also be employed to treat diseases of the invention, amongst others
  • polypeptides and polynucleotides of the present invention may also be used as vaccines. Accordingly, in a further aspect, the present invention relates to a method for inducing an immunological response in a mammal that comprises inoculating the mammal with a polypeptide of the present invention, adequate to produce antibody and or T cell immune response, including, for example, cytokme-producmg T cells or cytotoxic T cells, to protect said animal from disease, whether that disease is already established withm the individual or not.
  • An immunological response in a mammal may also be induced by a method comprises dehve ⁇ ng a polypeptide of the present invention via a vector directing expression of the polynucleotide and coding for the polypeptide in vivo m order to induce such an immunological response to produce antibody to protect said animal from diseases of the invention.
  • One way of admmiste ⁇ ng the vector is by accelerating it mto the desired cells as a coating on particles or otherwise.
  • Such nucleic acid vector may comp ⁇ se DNA, RNA, a modified nucleic acid, or a DNA/RNA hyb ⁇ d.
  • a polypeptide or a nucleic acid vector will be normally provided as a vaccine formulation (composition).
  • the formulation may further comp ⁇ se a suitable earner. Since a polypeptide may be broken down m the stomach, it is preferably administered parenterally (for instance, subcutaneous, intramuscular, intravenous, or mtradermal injection).
  • parenterally for instance, subcutaneous, intramuscular, intravenous, or mtradermal injection.
  • Formulations suitable for parenteral administration include aqueous and non- aqueous ste ⁇ le injection solutions that may contain anti-oxidants, buffers, bacte ⁇ ostats and solutes that render the formulation lsotomc with the blood of the recipient; and aqueous and non-aqueous ste ⁇ le suspensions that may include suspending agents or thickening agents.
  • the formulations may be presented m unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-d ⁇ ed condition requi ⁇ ng only the addition of the ste ⁇ le liquid earner immediately pnor to use.
  • the vaccine formulation may also include adjuvant systems for enhancing the lmmunogemcity of the formulation, such as oil-m water systems and other systems known in the art. The dosage will depend on the specific activity of the vaccine and can be readily determined by routine expe ⁇ mentation.
  • the human AXOR29 receptor polypeptide of the present invention may be employed m a process for screening for compounds that bmd to and activate the human AXOR29 receptor polypeptides of the present invention (called agonists), or inhibit the interaction of the human AXOR29 receptor polypeptides with receptor ligands (called antagonists).
  • polypeptides of the mvention may also be used to assess the binding of small molecule substrates and ligands m, for example, cells, cell-free preparations, chemical bra ⁇ es, and natural product mixtures.
  • substrates and ligands may be natural substrates and ligands or may be structural or functional mimetics. See Cohgan, et al, CURRENT PROTOCOLS IN IMMUNOLOGY l(2):Chapter 5 (1991).
  • Human AXOR29 receptor prote s are responsible for many biological functions, including many pathologies. Provided by the invention are screening methods to identify compounds and drugs that stimulate human AXOR29 receptor or that inhibit the function or level of the polypeptide.
  • agonists are employed for therapeutic and prophylactic purposes for such conditions as infections such as diseases of the mvention, among others.
  • Antagonists may be employed for a va ⁇ ety of therapeutic and prophylactic purposes for such conditions as diseases of the mvention.
  • such screening procedures involve providing approp ⁇ ate cells that express the receptor polypeptide of the present invention on the surface thereof.
  • Such cells mclude cells from mammals, yeast, Drosophila or E coli.
  • a polynucleotide encoding the receptor of the present mvention is employed to transfect cells to thereby express the human AXOR29 receptor polypeptide.
  • the expressed receptor is then contacted with a test compound to observe bmdmg, stimulation or inhibition of a functional response.
  • One such screenmg procedure involves the use of melanophores that are transfected to express the human AXOR29 receptor polypeptide of the present invention.
  • a screening technique is desc ⁇ bed m PCT WO 92/01810, published February 6, 1992.
  • Such an assay may be employed to screen for a compound which inhibits activation of the receptor polypeptide of the present invention by contacting the melanophore cells that encode the receptor with both a receptor ligand, such as S-l-P, and a compound to be screened. Inhibition of the signal generated by the ligand indicates that a compound is a potential antagonist for the receptor, i e., inhibits activation of the receptor.
  • the technique may also be employed for screening of compounds that activate the receptor by contacting such cells with compounds to be screened and determining whether such compound generates a signal, i.e., activates the receptor.
  • screening techmques include the use of cells which express the human AXOR29 receptor polypeptide (for example, transfected CHO cells) in a system that measures extracellular pH changes caused by receptor activation.
  • compounds may be contacted with cells expressing the receptor polypeptide of the present invention.
  • a second messenger response e.g., signal transduction or pH changes, is then measured to determine whether the potential compound activates or inhibits the receptor.
  • Another screening technique involves expressing the human AXOR29 receptor polypeptide m which the receptor is linked to phosphohpase C or D.
  • Such cells include, but are not limited to: endothehal cells, smooth muscle cells, and embryonic kidney cells
  • the screening may be accomplished as hereinabove descnbed by detecting activation of the receptor or inhibition of activation of the receptor from the phosphohpase second signal.
  • Another method involves screenmg for compounds that are antagonists, and thus inhibit activation of the receptor polypeptide of the present invention by determining inhibition of binding of labeled ligand, such as S-l-P or di-hydro S-l-P, to cells expressing the receptor on their surface, or cell membranes containing the receptor.
  • labeled ligand such as S-l-P or di-hydro S-l-P
  • Such a method involves transfectmg a eukaryotic cell with DNA encoding the human AXOR29 receptor polypeptide, such that the cell expresses the receptor on its surface.
  • the cell is then contacted with a potential antagonist in the presence of a labeled form of a ligand, such as S-l-P or di-hydro S-l-P.
  • the ligand can be labeled, e g., by radioactivity.
  • the amount of labeled ligand bound to the receptors is measured, e g., by measu ⁇ ng radioactivity associated with transfected cells or membrane from these cells. If the compound binds to the receptor, the binding of labeled ligand to the receptor is inhibited as determined by a reduction of labeled ligand which binds to the receptors. This method is called bmdmg assay Naturally, this same technique can be used to look for an agonist.
  • the screening method may simply measure the bmdmg of a candidate compound to the polypeptide, or to cells or membranes bea ⁇ ng the polypeptide, or a fusion protem thereof by means of a label directly or indirectly associated with the candidate compound
  • the screenmg method may involve measu ⁇ ng or, qualitatively or quantitatively, detecting the competition of binding of a candidate compound to the polypeptide with a labeled competitor (e g agonist or antagonist).
  • these screening methods may test whether the candidate compound results in a signal generated by activation or inhibition of the polypeptide, using detection systems approp ⁇ ate to the cells bea ⁇ ng the polypeptide.
  • Inhibitors of activation are generally assayed m 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 comp ⁇ se the steps of mixing a candidate compound with a solution containing a polypeptide of the present invention, to form a mixture, measunng human AXOR29 receptor activity in the mixture, and compa ⁇ ng the AXOR29 activity of the mixture to a control mixture which contains no candidate compound
  • Another screening procedure involves the use of mammalian cells (CHO, HEK 293, Xenopus Oocytes, RBL-2H3, etc that are transfected to express human AXOR29 receptor.
  • the cells are loaded with an indicator dye that produces a fluorescent signal when bound to calcium, and the cells are contacted with a test substance and a receptor agonist, such as S-l-P. Any change m fluorescent signal is measured over a defined pe ⁇ od of time using, for example, a fluorescence spectrophotometer or a fluorescence imaging plate reader.
  • a change in the fluorescence signal pattern generated by the ligand indicates that a compound is a potential antagonist or agonist for the receptor.
  • Another screening procedure involves use of mammalian cells (CHO, HEK293, Xenopus
  • Oocytes, RBL-2H3, etc. that are transfected to express the receptor of interest, and that are also transfected with a reporter gene construct that is coupled to activation of the receptor (for example, luciferase or beta-galactosidase behind an appropriate promoter).
  • the cells are contacted with a test substance and the receptor agonist (ligand), such as S-l-P or di-hydro S-l-P, and the signal produced by the reporter gene is measured after a defined period of time
  • the signal can be measured using a luminometer, spectrophotometer, fluo ⁇ meter, or other such instrument approp ⁇ ate for the specific reporter construct used
  • Inhibition of the signal generated by the ligand indicates that a compound is a potential antagonist for the receptor
  • Another screening technique for antagonists or agonists involves introducing RNA encodmg the AXOR29 polypeptide into Xenopus oocytes (or CHO, HEK 293 , RBL-2H3. etc to transiently or stably express the receptor.
  • the receptor oocytes are then contacted with the receptor ligand, such as S-l-P or di-hydro S-l-P, and a compound to be screened. Inhibition or activation of the receptor is then determined by detection of a signal, such as, cAMP, calcium, proton, or other ions
  • Another method involves screening for AXOR29 polypeptide inhibitors by determining inhibition or stimulation of AXOR29 polypeptide-mediated cAMP and/or adenylate cyclase accumulation or dimumtion.
  • Such a method involves transiently or stably transfectmg a eukaryotic cell with AXOR29 polypeptide receptor to express the receptor on the cell surface. The cell is then exposed to potential antagonists m the presence of AXOR29 polypeptide ligand, such as S-l-P or di- hydro S-l-P.
  • Changes m levels of cAMP is then measured over a defined pe ⁇ od of time, for example, by radio-immuno or protem binding assays (for example using Flashplates or a scintillation proximity assay). Changes m cAMP levels can also be determined by directly measuring the activity of the enzyme, adenylyl cyclase, m broken cell preparations.
  • Another screening method for agonists and antagonists relies on the endogenous pheromone response pathway in the yeast, Saccharomyces cerevisiae. Heterothallic strains of yeast can exist in two mitotically stable haploid mating types, MATa and MATa.
  • Each cell type secretes a small peptide hormone that binds to a G-protem coupled receptor on opposite mating-type cells which tnggers a MAP kinase cascade leadmg to Gl arrest as a prelude to cell fusion.
  • Genetic alteration of certain genes in the pheromone response pathway can alter the normal response to pheromone, and heterologous expression and coupling of human G-protein coupled receptors and humanized G- protein subunits in yeast cells devoid of endogenous pheromone receptors can be linked to downstream signalling pathways and reporter genes (e.g., U.S. Patents 5,063,154; 5,482,835; 5,691,188).
  • Such genetic alterations include, but are not limited to: (i) deletion of the STE2 or
  • STE3 gene encoding the endogenous G-protein coupled pheromone receptors; (ii) deletion of the FAR1 gene encoding a protein that normally associates with cyclin-dependent kinases leading to cell cycle arrest; and (iii) construction of reporter genes fused to the FUS1 gene promoter (where FUS1 encodes a membrane-anchored glycoprotein required for cell fusion).
  • Downstream reporter genes can permit either a positive growth selection (e.g., histidine prototrophy using the FUS1-HIS3 reporter), or a colorimetric, fluorimetric or spectrophotometric readout, depending on the specific reporter construct used (e.g., b-galactosidase induction using a FUSl-LacZ reporter).
  • the yeast cells can be further engineered to express and secrete small peptides from random peptide libraries, some of which can permit autocrine activation of heterologously expressed human (or mammalian) G-protein coupled receptors (Broach, et al, Nature 384: 14-16, 1996; Manfredi, et al, Mol. Cell. Biol 16: 4700-4709, 1996).
  • This provides a rapid direct growth selection (e.g., using the FUS1-HIS3 reporter) for surrogate peptide agonists that activate characterized or orphan receptors.
  • yeast cells that functionally express human (or mammalian) G-protein coupled receptors linked to a reporter gene readout can be used as a platform for high-throughput screening of known ligands, fractions of biological extracts and libraries of chemical compounds for either natural or surrogate ligands.
  • Functional agonists of sufficient potency can be used as screening tools in yeast cell-based assays for identifying G-protein coupled receptor antagonists. For example, agonists will promote growth of a cell with FUS-HIS3 reporter or give positive readout for a cell with FUSl-LacZ.
  • yeast system offers advantages over mammalian expression systems due to its ease of utility and null receptor background (lack of endogenous G-protein coupled receptors), which often interferes with the ability to identify agonists or antagonists.
  • the present invention also provides a method for identifying new ligands not known to be capable of binding to a human AXOR29 receptor polypeptide.
  • the screening assays described above for identifying agonists may be used to identify new ligands.
  • the present invention also contemplates agonists and antagonists obtained from the above described screening methods.
  • potential human AXOR29 polypeptide receptor antagonists include peptidomimetics, synthetic organic molecules, natural products, antibodies, etc., that bmd to the receptor but do not elicit a second messenger response, such that the activity of the receptor is prevented.
  • Potential antagonists also include protems which are closely related to the ligand of the
  • AXOR29 polypeptide receptor i e , a fragment of the ligand, which have lost biological function, and when they bmd to human AXOR29 polypeptide receptor, elicit no response.
  • the present invention relates to a screening kit for identifying agonists, antagonists, and ligands for human AXOR29 polypeptides, comp ⁇ smg: (a) a human AXOR29 polypeptide, preferably that of SEQ ED NO.2; and further preferably comp ⁇ ses labeled or unlabeled S-l-P or di-hydro S-l-P,
  • a recombinant cell expressing a human AXOR29 polypeptide, preferably that of SEQ ED NO:2; and further preferably comp ⁇ ses labeled or unlabeled S-l-P or di-hydro S-l-P; or
  • a potential antagonist is a small molecule that binds to the human AXOR29 polypeptide, making it inaccessible to ligands such that normal biological activity is prevented.
  • small molecules include, but are not limited to, small peptides or peptide-hke molecules.
  • Potential antagonists also include soluble forms of a human AXOR29 polypeptide receptor, e g , fragments of the receptor, which bmd to the ligand and prevent the ligand from interacting with membrane bound human AXOR29 polypeptide.
  • the screening method may simply measure the bmdmg of a candidate compound to the polypeptide, or to cells or membranes bea ⁇ ng the polypeptide, or a fusion protem thereof by means of a label directly or indirectly associated with the candidate compound.
  • the screenmg method may involve competition with a labeled competitor.
  • these screening methods may test whether the candidate compound results in a signal generated by activation or inhibition of the polypeptide, using detection systems approp ⁇ ate to the cells bea ⁇ ng the polypeptide.
  • Inhibitors of activation are generally assayed m the presence of a known agonist and the effect on activation by the agonist by the presence of the candidate compound is observed.
  • Constituitively active polypeptides may be employed in screenmg methods for inverse agonists or inhibitors, in the absence of an agonist or inhibitor, by testing whether the candidate compound results m inhibition of activation of the polypeptide Further, the screening methods may simply comp ⁇ se the steps of mixing a candidate compound with a solution containing a polypeptide of the present invention, to form a mixture, measuring human AXOR29 receptor activity m the mixture, and comparmg the human AXOR29 receptor activity of the mixture to a standard.
  • Fusion protems such as those made from Fc portion and AXOR29 polypeptide, as hereinbefore desc ⁇ bed, can also be used for high-throughput screening assays to identify antagonists for the polypeptide of the present invention (see D. Bennett et al, J Mol Recognition, 8:52-58 (1995); and K. Johanson et al , J. Bwl Chem., 270(16):9459-9471 (1995).
  • Polypeptides of the present invention may be employed in conventional low capacity screening methods and also in high-throughput screenmg (HTS) formats.
  • HTS formats include not only the well-established use of 96- and, more recently, 384-well microtiter plates but also emerging methods such as the nanowell method desc ⁇ bed by Schullek, et al , Anal Biochem., 246 20-29 (1997).
  • S-l-P sphmgosme- 1 -phosphate
  • Di -hydro S-l-P (di-hvdro sphmgosme- 1 -phosphate)" refers to the sphmgohpid metabolite having the structure-
  • Antibodies as used herein includes polyclonal and monoclonal antibodies, chime ⁇ c, single chain, and humanized antibodies, as well as Fab fragments, including the products of an
  • Isolated means altered “by the hand of man” from its natural state, i.e., if it occurs in nature, it has been changed or removed from its ongmal environment, or both.
  • a polynucleotide or a polypeptide naturally present m a living organism is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting matenals of its natural state is “isolated”, as the term is employed herein.
  • a polynucleotide or polypeptide that is introduced mto an organism by transformation, genetic manipulation or by any other recombmant method is "isolated” even if it is still present m said organism, which organism may be living or non-living.
  • Polynucleotide generally refers to any polynbonucleotide (RNA) or polydeox ⁇ bonucleotide (DNA), which may be unmodified or modified RNA or DNA.
  • Polynucleotides include, without limitation, smgle- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hyb ⁇ d molecules comprising DNA and RNA that may be smgle-stranded or, more typically, double-stranded or a mixture of single- and double- stranded regions.
  • polynucleotide refers to tnple-stranded regions comp ⁇ sing RNA or DNA or both RNA and DNA.
  • 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 mclude, for example, tntylated bases and unusual bases such as inosine.
  • polynucleotide embraces chemically, enzymatically or metabohcally modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells.
  • Polynucleotide also embraces relatively short polynucleotides, often referred to as ohgonucleotides.
  • Polypeptide refers to any polypeptide comp ⁇ sing two or more ammo acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres "Polypeptide” refers to both short chains, commonly refe ⁇ ed to as peptides, ohgopeptides or ohgomers, and to longer chains, generally referred to as protems. Polypeptides may contain ammo acids other than the 20 gene-encoded ammo acids. "Polypeptides” include ammo acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques that are well known in the art.
  • Modifications may occur anywhere m a polypeptide, including the peptide backbone, the ammo acid side-chams and the ammo or carboxyl termini. It will be appreciated that the same type of modification may be present to the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched as a result of ubiquitmation, and they may be cyclic, with or without branching. Cyclic, branched and branched cyclic polypeptides may result from post-translation natural processes or may be made by synthetic methods.
  • Modifications include acetylation, acylation, ADP- ⁇ bosylation, amidation, biotmylation, covalent attachment of flavm, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide de ⁇ vative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylmositol, cross-linking, cychzation, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteme, formation of pyroglutamate, formylation, gamma- carboxylation, glycosylation, GPI anchor formation, hydroxylation, lodmation, methylation, my ⁇ stoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of ammo acids to protems such as argmylation, and ubiquit
  • “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 ED NO: 1..
  • Va ⁇ ant refers to a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, but retains the essential properties thereof.
  • a typical va ⁇ ant of a polynucleotide differs m nucleotide sequence from the reference polynucleotide. Changes m the nucleotide sequence of the va ⁇ ant may or may not alter the ammo acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in ammo acid substitutions, additions, deletions, fusions and truncations m the polypeptide encoded by the reference sequence, as discussed below.
  • a typical vanant of a polypeptide differs m ammo acid sequence from the reference polypeptide. Generally, alterations are limited so that the sequences of the reference polypeptide and the va ⁇ ant are closely similar overall and, m many regions, identical
  • a va ⁇ ant and reference polypeptide may differ in ammo acid sequence by one or more substitutions, insertions, deletions m any combination.
  • a substituted or inserted ammo acid residue may or may not be one encoded by the genetic code. Typical conservative substitutions include Gly, Ala; Val, Be, Leu; Asp, Glu; Asn, Gin; Ser, Thr; Lys, Arg; and Phe and Tyr.
  • a va ⁇ ant of a polynucleotide or polypeptide may be naturally occurnng such as an allele, or it may be a variant that is not known to occur naturally.
  • Non-naturally occur ⁇ ng va ⁇ ants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis.
  • polypeptides having one or more post-translational modifications for instance glycosylation, phosphorylation, methylation, ADP nbosylation and the like.
  • Embodiments include methylation of the N-termmal ammo acid, phosphorylations of se ⁇ nes and threonmes and modification of C-termmal glycmes
  • Allele refers to one of two or more alternative forms of a gene occur ⁇ ng at a given locus in the genome.
  • Polymorphism refers to a variation in nucleotide sequence (and encoded polypeptide sequence, if relevant) at a given position in the genome withm a population.
  • Single Nucleotide Polymorphism refers to the occurrence of nucleotide vanabihty at a single nucleotide position m the genome, withm a population.
  • An SNP may occur withm a gene or within lntergemc regions of the genome.
  • SNPs can be assayed using Allele Specific Amplification (ASA). For the process at least 3 p ⁇ mers are required. A common p ⁇ mer is used m reverse complement to the polymorphism being assayed.
  • ASA Allele Specific Amplification
  • This common p ⁇ mer can be between 50 and 1500 bps from the polymorphic base.
  • the other two (or more) p ⁇ mers are identical to each other except that the final 3' base wobbles to match one of the two (or more) alleles that make up the polymorphism.
  • Two (or more) PCR reactions are then conducted on sample DNA, each using the common p ⁇ mer and one of the Allele Specific P ⁇ mers.
  • RNA molecules produced from RNA molecules initially transc ⁇ bed from the same genomic DNA sequence but which have undergone alternative
  • RNA splicing occurs when a p ⁇ mary RNA transc ⁇ pt undergoes splicing, generally for the removal of mtrons, which results in the production of more than one mRNA molecule each of that may encode different ammo acid sequences
  • the term splice va ⁇ ant also refers to the protems encoded by the above cDNA molecules "Identity" reflects a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, determined by compa ⁇ ng the sequences.
  • identity refers to an exact nucleotide to nucleotide or ammo acid to ammo 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.
  • the two sequences to be compared are aligned to give a maximum correlation between the sequences. This may include inserting "gaps" in either one or both sequences, to enhance the degree of alignment.
  • a % identity may be determined over the whole length of each of the sequences being compared (so-called global alignment), that is particularly suitable for sequences of the same or very similar length, or over shorter, defined lengths (so-called local alignment), that is more suitable for sequences of unequal length.
  • Similarity is a further, more sophisticated measure of the relationship between two polypeptide sequences.
  • 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, 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.
  • programs available in the Wisconsin Sequence Analysis Package, version 9.1 may be used to determine the % identity between two polynucleotides and the % identity and the % similarity between two polypeptide sequences.
  • BESTFLT uses the "local homology" algorithm of
  • BESTFLT is more suited to comparing two polynucleotide or two polypeptide sequences that are dissimilar in length, the program assuming that the shorter sequence represents a portion of the longer.
  • GAP aligns two sequences, finding a "maximum similarity", according to the algorithm of
  • GAP is more suited to comparing sequences that are approximately the same length and an alignment is expected over the entire length.
  • 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.
  • % identities and similarities are determined when the two sequences being compared are optimally aligned.
  • the program BESTFLT 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 desc ⁇ bed.
  • Identity Index is a measure of sequence relatedness which may be used to compare a candidate sequence (polynucleotide or polypeptide) and a reference sequence.
  • 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 mclude on average up to five differences per each 100 nucleotides of the reference sequence. Such differences are selected from the group consistmg of at least one nucleotide deletion, substitution, including transition and transversion, or insertion.
  • a candidate polypeptide sequence having, for example, an Identity Index of 0.95 compared to a reference polypeptide sequence is identical to the reference sequence except that the polypeptide sequence may include an average of up to five differences per each 100 amino acids of the reference sequence. Such differences are selected from the group consisting of at least one ammo acid deletion, substitution, including conservative and non- conservative substitution, or insertion. These differences may occur at the ammo- or carboxy- terminal positions of the reference polypeptide sequence or anywhere between these terminal positions, interspersed either individually among the ammo acids m the reference sequence or m one or more contiguous groups withm the reference sequence.
  • an average of up to 5 m every 100 of the ammo acids in the reference sequence may be deleted, substituted or inserted, or any combination thereof, as hereinbefore desc ⁇ bed.
  • n a is the number of nucleotide or ammo acid differences
  • x a is the total number of nucleotides or ammo acids m SEQ ED NO.1 or SEQ ED NO:2, respectively,
  • I is the Identity Index
  • • is the symbol for the multiplication operator, and in which any non-mteger product of x a and I is rounded down to the nearest integer p ⁇ or to subtracting it from x a .
  • “Homolog” is a gene ⁇ c term used m the art to indicate a polynucleotide or polypeptide sequence possessing a high degree of sequence relatedness to a reference sequence. Such relatedness may be quantified by determining the degree of identity and/or simila ⁇ ty between the two sequences as hereinbefore defined. Falling withm this gene ⁇ c term are the terms “ortholog”, and “paralog”. "Ortholog” refers to a polynucleotide or polypeptide that is the functional equivalent of the polynucleotide or polypeptide in another species. "Paralog” refers to a polynucleotide or polypeptide that withm the same species which is functionally similar.
  • Fusion protem refers to a protein encoded by two, often unrelated, fused genes or fragments thereof.
  • EP-A-0 464 533 (Canadian counterpart 2045869) discloses fusion proteins compnsmg va ⁇ ous portions of constant region of immunoglobulm molecules together with another human protein or part thereof.
  • employing an immunoglobulm Fc region as a part of a fusion protem is advantageous for use in therapy and diagnosis resulting m, for example, improved pharmacokmetic properties [see, e g , EP-A 0232 262].
  • Example 1 Mammalian Cell Expression
  • the receptors of the present mvention are expressed m either human embryonic kidney 293 (HEK293) cells or adherent dhfr CHO cells.
  • HEK293 human embryonic kidney 293
  • adherent dhfr CHO cells typically all 5' and 3' untranslated regions (UTRs) are removed from the receptor cDNA p ⁇ or to insertion mto a pCDN or pCDNA3 vector.
  • the cells are transfected with individual receptor cDNAs by hpofectin and selected in the presence of 400 mg/ml G418. After 3 weeks of selection, individual clones are picked and expanded for further analysis.
  • HEK293 or CHO cells transfected with the vector alone serve as negative controls.
  • Receptor mRNAs are generally detectable in about 50% of the G418-res ⁇ stant clones analyzed.
  • a bank of over 600 putative receptor ligands has been assembled for screening.
  • the bank comp ⁇ ses transmitters, hormones and chemokmes known to act via a human seven transmembrane
  • (7TM) receptor naturally occur ⁇ ng compounds which may be putative agomsts for a human 7TM receptor, non-mammalian, biologically active peptides for which a mammalian counterpart has not yet been identified; and compounds not found m nature, but which activate 7TM receptors with unknown natural ligands.
  • This bank is used to initially screen the receptor for known ligands, usmg both functional (i.e . calcium, cAMP, microphysiometer, oocyte electrophysiology, etc, see below) as well as bmdmg assays.
  • Ligand bmdmg assays provide a direct method for ascertaining receptor pharmacology and are adaptable to a high throughput format.
  • the punfied ligand for a receptor is radiolabeled to high specific activity (50-2000 Ci/mmol) for bmdmg studies.
  • a determination is then made that the process of radiolabeling does not dimmish the activity of the ligand towards its receptor.
  • Assay conditions for buffers, ions, pH and other modulators such as nucleotides are optimized to establish a workable signal to noise ratio for both membrane and whole cell receptor sources.
  • specific receptor bmdmg is defined as total associated radioactivity minus the radioactivity measured m the presence of an excess of unlabeled competing ligand. Where possible, more than one competing ligand is used to define residual nonspecific bmdmg.
  • RNA transc ⁇ pts from lmeanzed plasmid templates encodmg the receptor cDNAs of the invention are synthesized in vitro with RNA polymerases m accordance with standard procedures In vitro transc ⁇ pts are suspended m water at a final concentration of 0.2 mg/ml. Ova ⁇ an lobes are removed from adult female toads, Stage V defolliculated oocytes are obtained, and RNA transc ⁇ pts (10 ng/oocyte) are injected in a 50 nl bolus usmg a micromjection apparatus. Two electrode voltage clamps are used to measure the currents from individual Xenopus oocytes m response to agonist exposure
  • Activation of a wide va ⁇ ety of secondary messenger systems results m extrusion of small amounts of acid from a cell.
  • the acid formed is largely as a result of the increased metabolic activity required to fuel the lntracellular signaling process.
  • the pH changes m the media surrounding the cell are very small but are detectable by the CYTOSENSOR microphysiometer (Molecular Devices Ltd., Menlo Park, CA).
  • the CYTOSENSOR is thus capable of detecting the activation of a receptor which is coupled to an energy utilizing lntracellular signaling pathway such as the G-protem coupled receptor of the present mvention.
  • the 7TM receptor of the mvention is also functionally screened (usmg calcium, cAMP, microphysiometer, oocyte electrophysiology, etc., functional screens) agamst tissue extracts to identify natural ligands. Extracts that produce positive functional responses can be sequencially subfractionated until an activating ligand is isolated identified.
  • HEK 293 cells m receptor-transfected or vector control cells were observed to be m the normal, 100 nM to 200 nM, range.
  • HEK 293 cells expressing recombmant receptors are loaded with fura 2 and in a smgle day > 150 selected ligands or tissue/cell extracts are evaluated for agomst induced calcium mobilization.
  • HEK 293 cells expressing recombmant receptors are evaluated for the stimulation or inhibition of cAMP production usmg standard cAMP quantitation assays. Agonists presenting a calcium transient or cAMP flucuation are tested in vector control cells to determine if the response is unique to the transfected cells expressing receptor.
  • Example 9 Calcium Mobilization Assay for Human AXOR29

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Abstract

Human AXOR29 polypeptides and polynucleotides and methods for producing such polypeptides by recombinant techniques are disclosed. Human AXOR29 is identified as a selective receptor for sphingosine-1-phosphate ('S-1-P') and di-hydro sphingosine-1-phosphate ('di-hydro S-1-P'). Also disclosed are methods for discovering agonists and antagonists of the interaction between S-1-P and di-hydro S-1-P and their cellular receptor, human AXOR29, which may have utility in the treatment of several human diseases and disorders, including, but not limited to the treatment of infections such as bacterial, fungal, protozoan and viral infections, particularly infections caused by HIV-1 or HIV-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; allergie; benign prostatic hypertrophy; migraine; vomiting; psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, depression, delirium, dementia, and severe mental retardation.

Description

POLYNUCLEOTTDE AND POLYPEPTIDE SEQUENCES OF HUMAN AXOR29
RECEPTOR AND METHODS OF SCREENING FOR AGONISTS AND
ANTAGONISTS OF THE INTERACTION BETWEEN HUMAN
AXOR29 RECEPTOR AND ITS LIGANDS
FLELD OF THE INVENTION
This invention relates to newly identified polypeptides and polynucleotides encoded by them and to the use of such polynucleotides and polypeptides, and to their production. More particularly, the polynucleotides and polypeptides of the present invention relate to the G-protein coupled receptors, hereinafter referred to as human AXOR29 receptor This invention also relates to methods for discoveπng agonists and antagonists of the interaction between sphmgosme 1- phosphate (hereinafter referred to as "S-l-P") and di-hydro sphmgosme- 1 -phosphate (hereinafter referred to as "di -hydro S-l-P") and their cellular receptor, human AXOR29 receptor. The invention also relates to the use of human AXOR29 receptor polynucleotides and polypeptides m therapy and in identifying compounds which may be agonists, antagonists and /or inhibitors which are potentially useful in therapy, and to production of such polypeptides and polynucleotides
BACKGROUND OF THE INVENTION
The drug discovery process is currently undergoing a fundamental revolution as it embraces "functional genomics", that is, high throughput genome- or gene-based biology. This approach as a means to identify genes and gene products as therapeutic targets is rapidly superseding earlier approaches based on "positional cloning". A phenotype, that is a biological function or genetic disease, would be identified and this would then be tracked back to the responsible gene, based on its genetic map position.
Functional genomics relies heavily on high-throughput DNA sequencing technologies and the vaπous tools of bioinformatics to identify gene sequences of potential interest from the many molecular biology databases now available. There is a continuing need to identify and characterise further genes and their related polypeptides/protems, as targets for drug discovery.
It is well established that many medically significant biological processes are mediated by proteins participating in signal transduction pathways that involve G-protems and/or second messengers, e.g., cAMP (Lefkowitz, Nature, 1991, 351:353-354). Herein these proteins are referred to as protems participating in pathways with G-protems 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. NatlAcad Set , USA, 1987, 8446-50, Kobilka, B.K., et al, Science, 1987, 238:650-656; Bunzow, J.R., et al , Nature, 1988, 336:783-787), G-proteins themselves, effector protems, eg, phosphohpase C, adenyl cyclase, and phosphodiesterase, and actuator protems, e g., protein kinase A and protem kmase 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, mside the cell. Enzyme activation by hormones is dependent on the presence of the nucleotide GTP. GTP also influences hormone binding. A G-protem connects the hormone receptor to adenylate cyclase. G-protem was shown to exchange GTP for bound GDP when activated by a hormone receptor. The GTP-carrying form then binds to activated adenylate cyclase. Hydrolysis of GTP to GDP, catalyzed by the G-protem itself, returns the G-protem to its basal, mactive form. Thus, the G-protem serves a dual role, as an intermediate that relays the signal from receptor to effector, and as a clock that controls the duration of the signal.
The membrane protein gene superfamily of G-protein coupled receptors has been characteπzed as having seven putative transmembrane domains. The domains are believed to represent transmembrane a-helices connected by extracellular or cytoplasmic loops. G-protem coupled receptors include a wide range of biologically active receptors, such as hormone, viral, growth factor and neuroreceptors .
G-protem coupled receptors (otherwise known as 7TM receptors) have been characteπzed as including these seven conserved hydrophobic stretches of about 20 to 30 ammo acids, connecting at least eight divergent hydrophilic loops. The G-protem family of coupled receptors includes dopamme receptors which bmd to neuroleptic drugs used for treating psychotic and neurological disorders. Other examples of members of this family mclude, but are not limited to, calcitomn, adrenergic, endothelm, cAMP, adenosme, muscaπnic, acetylcho ne, serotonin, histamme, thrombm, kmm, follicle stimulating hormone, opsms, endothehal differentiation gene-1, rhodopsms, odorant, and cytomegalovirus receptors.
Most G-protem coupled receptors have single conserved cysteme 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 TM1, TM2, TM3, TM4, TM5, TM6, and TM7. TM3 has been implicated in signal transduction.
Phosphorylation and hpidation (palmitylation or farnesylation) of cysteme residues can influence signal transduction of some G-protem coupled receptors. Most G-protem coupled receptors contain potential phosphorylation sites within the third cytoplasmic loop and or the carboxy terminus.
For several G-protem coupled receptors, such as the b-adrenoreceptor, phosphorylation by protem kmase A and/or specific receptor kinases mediates receptor desensitization. For some receptors, the ligand binding sites of G-protein coupled receptors are believed to compπse hydrophilic sockets formed by several G-protem coupled receptor transmembrane domains, said socket being surrounded by hydrophobic residues of the G-protem coupled receptors. The hydrophilic side of each G-protem coupled receptor transmembrane helix is postulated to face mward and form polar ligand binding site. TM3 has been implicated m several G-protein coupled receptors as having a ligand binding site, such as the TM3 aspartate residue TM5 seπnes, a TM6 asparagrne and TM6 or TM7 phenylalanmes or tyrosmes are also implicated m ligand binding.
G-protem coupled receptors can be mtracellularly coupled by heterotπmeπc G-protems to vaπous mtracellular enzymes, ion channels and transporters (see, Johnson, et al, Endoc. Rev., 1989, 10:317-331) Different G-protem a-subunits preferentially stimulate particular effectors to modulate vaπous biological functions in a cell. Phosphorylation of cytoplasmic residues of G-protem coupled receptors have been identified as an important mechanism for the regulation of G-protem couplmg of some G-protem coupled receptors. G-protein coupled receptors are found in numerous sites withm a mammalian host. Over the past 15 years, nearly 350 therapeutic agents targeting 7 transmembrane (7 TM) receptors have been successfully introduced onto the market.
SUMMARY OF THE INVENTION
The present invention relates to human AXOR29 receptor, m particular human AXOR29 receptor polypeptides and human AXOR29 receptor polynucleotides, recombinant mateπals and methods for their production. Such polypeptides and polynucleotides are of interest m relation to methods of treatment of certain diseases, including, but not limited to infections such as bacteπal, fungal, protozoan and viral infections, particularly infections caused by HTV-1 or HIV-2; pa ; cancers; diabetes, obesity; anorexia; bulimia; asthma; Parkinson's disease; acute heart failure; hypotension; hypertension; uπnary retention; osteoporosis; angina pectoπs; myocardial infarction; stroke; ulcers; asthma; allergies; benign prostatic hypertrophy; migraine; vomiting; psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, depression, delirium, dementia, and severe mental retardation, hereinafter referred to as "diseases of the invention".
In accordance with another aspect of the present invention there are provided methods of screenmg for compounds which bmd to and activate (agonist) or inhibit activation (antagonist) of human AXOR29 receptor polypeptides, and for their hgands.
In particular, the preferred method for identifying agonist or antagonist of a human AXOR29 receptor polypeptide compπses: (a) contacting a cell expressing on the surface thereof the polypeptide, said polypeptide being associated with a second component capable of providmg a detectable signal m response to the bmdmg of a compound to said polypeptide, with a compound to be screened under conditions to permit bmdmg to the polypeptide; and (b) determining whether the compound binds to and activates or inhibits the polypeptide by measuπng the level of a signal generated from the interaction of the compound with the polypeptide.
In a further preferred embodiment, the method further compπses conductmg the identification of agonist or antagonist in the presence of labeled or unlabeled S-l-P or di-hydro S-l-P.
In another embodiment, the method for identifying agonist or antagonist of a human AXOR29 receptor polypeptide compπses: determining the inhibition of bmdmg of a ligand to cells which have the polypeptide on the surface thereof, or to cell membranes containing the polypeptide, m the presence of a candidate compound under conditions to permit binding to the polypeptide, and determining the amount of ligand bound to the polypeptide, such that a compound capable of causing reduction of bmdmg of a ligand is an agonist or antagonist. Preferably, the ligand is S-l-P or di-hydro S-l-P. Yet more preferably, S-l-P or di-hydro S-l-P is labeled.
Furthermore, the present invention relates to treating conditions associated with human AXOR29 recpeptor imbalance with the identified compounds. Yet another aspect of the mvention relates to diagnostic assays for detecting diseases associated with mappropπate human AXOR29 receptor activity or levels. BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows the nucleotide sequence of the human AXOR29 receptor (SEQ ID NO:l).
Figure 2 shows the deduced ammo acid sequence of the human AXOR29 receptor (SEQ ID NO:2).
Figure 3 shows concentration-response curves for S-l-P with 3 clones of the human AXOR29 receptor stably expressed in RBL 2H3 cells and also for RBL 2H3 cells and RBL 2H3 vector control cells.
Figure 4 shows concentration-response curves for lysophosphatidic acid (LPA) with 3 clones of the AXOR29 receptor stably expressed in RBL 2H3 cells and also for RBL 2H3 cells and RBL 2H3 vector control cells. DESCRIPTION OF THE INVENTION
In a first aspect, the present invention relates to AXOR29 polypeptides Such polypeptides include:
(a) an isolated polypeptide encoded by a polynucleotide comprising the sequence of SEQ ID NO.1 ; (b) an isolated polypeptide compπsing a polypeptide sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the polypeptide sequence of SEQ ID NO:2;
(c) an isolated polypeptide comprising the 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
(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 vaπants of such polypeptides m (a) to (f)
Polypeptides of the present mvention are believed to be members of the 7 transmembrane G- protem coupled receptor family of polypeptides. The biological properties of the human AXOR29 receptor are hereinafter referred to as "biological activity of human AXOR29 receptor" or "human AXOR29 receptor activity". Preferably, a polypeptide of the present invention exhibits at least one biological activity of human AXOR29 receptor.
Polypeptides of the present invention also include vaπants of the aforementioned polypeptides, including all allehc forms and splice vaπants. Such polypeptides vary from the reference polypeptide by insertions, deletions, and substitutions that may be conservative or non-conservative, or any combination thereof. Particularly preferred vaπants are those m which several, for instance from 50 to 30, from 30 to 20, from 20 to 10, from 10 to 5, from 5 to 3, from 3 to 2, from 2 to 1 or 1 ammo acids are inserted, substituted, or deleted, in any combination. Preferred fragments of polypeptides of the present invention include an isolated polypeptide compπsing an ammo acid sequence having at least 30, 50 or 100 contiguous ammo acids from the ammo acid sequence of SEQ ID NO: 2, or an isolated polypeptide compπsing an ammo acid sequence having at least 30, 50 or 100 contiguous ammo acids truncated or deleted from the ammo acid sequence of SEQ ID NO: 2. Preferred fragments are biologically active fragments that mediate the biological activity of human AXOR29 receptor, including those with a similar activity or an improved activity, or with a decreased undesirable activity. Also preferred are those fragments that are antigenic or lmmunogenic m an animal, especially m a human. Fragments of the polypeptides of the invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, these vaπants may be employed as intermediates for producing the full-length polypeptides of the mvention The polypeptides of the present invention may be in the form of the "mature" protem or may be a part of a larger protem such as a precursor or a fusion protem. It is often advantageous to include an additional ammo acid sequence that contains secretory or leader sequences, pro-sequences, sequences that aid in puπfϊcation, for instance multiple histidme residues, or an additional sequence for stability duπng recombinant production.
Polypeptides of the present invention can be prepared in any suitable manner, for instance by isolation form naturally occurπng sources, from genetically engmeered host cells compπsing expression systems (vide infra) or by chemical synthesis, using for instance automated peptide synthesisers, or a combination of such methods.. Means for preparing such polypeptides are well understood m the art
In a further aspect, the present mvention relates to human AXOR29 receptor polynucleotides. Such polynucleotides include: (a) an isolated polynucleotide compπsmg a polynucleotide sequence having at least 95%, 96%, 97%,
98%, or 99% identity to the polynucleotide sequence of SEQ ID NO: 1 ;
(b) an isolated polynucleotide compπsmg the polynucleotide of SEQ ID NO.1 ;
(c) an isolated polynucleotide having at least 95%, 96%, 97%, 98%, or 99% identity to the polynucleotide of SEQ ID NO: 1 ; (d) the isolated polynucleotide of SEQ ID NO: 1 ;
(e) an isolated polynucleotide compπsmg a polynucleotide sequence encodmg a polypeptide sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the polypeptide sequence of SEQ ID NO:2,
(f) an isolated polynucleotide compπsing a polynucleotide sequence encodmg the polypeptide of SEQ ID NO:2; (g) an isolated polynucleotide having a polynucleotide sequence encodmg a polypeptide sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the polypeptide sequence of SEQ ID NO.2,
(h) an isolated polynucleotide encodmg the polypeptide of SEQ ID NO:2;
(l) an isolated polynucleotide having or compπsing a polynucleotide sequence that has an Identity Index of 0.95, 0.96, 0.97, 0.98, or 0.99 compared to the polynucleotide sequence of SEQ ID NO:l, (j) an isolated polynucleotide having or comprising a polynucleotide sequence encodmg a polypeptide sequence that has an Identity Index of 0.95, 0.96, 0.97, 0.98, or 0.99 compared to the polypeptide sequence of SEQ ID NO:2; and polynucleotides that are fragments and vaπants of the above mentioned polynucleotides or that are complementary to above mentioned polynucleotides, over the entire length thereof.
Preferred fragments of polynucleotides of the present mvention include an isolated polynucleotide compπsing an nucleotide sequence having at least 15, 30, 50 or 100 contiguous nucleotides from the sequence of SEQ ID NO: 1, or an isolated polynucleotide compπsing an sequence having at least 30, 50 or 100 contiguous nucleotides truncated or deleted from the sequence of SEQ ID NO: 1.
Preferred vaπants of polynucleotides of the present invention include splice vaπants, allehc vaπants, and polymorphisms, including polynucleotides having one or more smgle nucleotide polymorphisms (SNPs).
Polynucleotides of the present mvention also include polynucleotides encodmg polypeptide vaπants that compπse the ammo acid sequence of SEQ ID NO:2 and m which several, for instance from 50 to 30, from 30 to 20, from 20 to 10, from 10 to 5, from 5 to 3, from 3 to 2, from 2 to 1 or 1 ammo acid residues are substituted, deleted or added, m any combination.
In a further aspect, the present invention provides polynucleotides that are RNA transcπpts of the DNA sequences of the present mvention. Accordingly, there is provided an RNA polynucleotide that:
(a) compπses an RNA transcπpt of the DNA sequence encoding the polypeptide of SEQ ED NO:2;
(b) is the RNA transcπpt of the DNA sequence encoding the polypeptide of SEQ ID NO:2;
(c) compπses an RNA transcπpt of the DNA sequence of SEQ ID NO: 1; or (d) is the RNA transcπpt of the DNA sequence of SEQ ID NO: 1 ; and RNA polynucleotides that are complementary thereto.
The polynucleotide sequence of SEQ ID NO: 1 shows homology with Mouse edg-1 G-protem coupled receptor (CH. Liu and T. Hla, Genomics 43 : 15-24, 1997). The polynucleotide sequence of SEQ ID NO: 1 is a cDNA sequence that encodes the polypeptide of SEQ ID NO:2. The polynucleotide sequence encoding the polypeptide of SEQ ID NO:2 may be identical to the polypeptide encodmg sequence of SEQ ID NO: 1 or it may be a sequence other than SEQ ED NO: 1 , which, as a result of the redundancy (degeneracy) of the genetic code, also encodes the polypeptide of SEQ ED NO.2 The polypeptide of the SEQ ED NO:2 is related to other protems of the 7 transmembrane G-protem coupled receptor family, having homology and/or structural similaπty with Mouse edg-1 G-protem coupled receptor (CH. Liu and T. Hla, Genomics 43:15-24, 1997).
Through phylogenetic analysis, AXOR29 was identified to be an EDG receptor. EDG receptors comprise a small, closely-related group of GPCRs. Known EDG receptors are
EDG-1, EDG-2, EDG-3, EDG-4, EDG-5, EDG-6 and EDG-7. See Moolenaar, et al, J Curr Opin. Cell Biol. 9(2): 168-173 (1997); MacLennan, et al, J Cell. Neurosci. 5(3) 201- 209 (1994); Graler, et al , J Genomics 53(2): 164-169 (1998); Bandoh, Acid J Bwl Chem.274 27776-27785 (1999). Reports on the ligand paiπng of the EDG receptors indicate that the EDG-1, -3 and -5 receptors respond to S-l-P, whereas the EDG-2, -4 and -7 receptors use LPA as their ligand. See Lee, et al , Science 279: 1552-1555 (1998); Moolenaar, et al , supra; Bandoh, et al., supra; Okamoto, et ah, Biotech. Biophys. Res. Comm. 260:203 (1999); Ancellm, et al, J Bio Chem 274: 18997 (1999). The ligand for the EDG-6 receptor has yet to be identified. We have found the ligand usage pattern of the EDG receptors to reflect the phylogenetic relationships among these genes, identified by shared, deπved mutations among the hgand-usage groups. These relationships are evidenced by the presence of shared, derived fixed mutations that are individual to each of the groups. Swofford, et al, (1996) Chap. 11, PHYLOGENETIC INFERENCE IN MOLECULAR SYSTEMATICS (Eds. Hilhs, D.M., C Moπtz, B.K. Mable, Sinauer Assoc, Sunderland Mass.), 655. In short, EDG-1, -3 and -5 receptors are closest relatives.
Likewise, EDG-2, -4 and-7 are more closely-related to each other than any of them is to the S-l-P - responding EDG receptor group.
The human AXOR29 receptor shares 42% ammo acid identity with EDG-1 and shares the same deπved, fixed mutations that we identified in the S-1-P-respondmg group of EDG receptors (EDG- 1, -3 and -5). We we have confirmed through expeπmentation that S-l-P is the ligand for AXOR29.
See Example 9 and Figures 3 and 4.
Preferred polypeptides and polynucleotides of the present mvention are expected to have, inter alia, similar biological functions/properties to their homologous polypeptides and polynucleotides. Furthermore, preferred polypeptides and polynucleotides of the present mvention have at least one human AXOR29 receptor activity.
Polynucleotides of the present mvention may be obtained using standard cloning and screenmg techmques from a cDNA library deπved from mRNA in cells of human lymph, prostate, pancreas and multiple sclerosis lesions, (see for instance, Sambrook, et al., MOLECULAR CLONTNG: A LABORATORY MANUAL, 2nd Ed., Cold Spπng Harbor Laboratory Press, Cold Spπng Harbor, N.Y (1989)). Polynucleotides of the invention can also be obtained from natural sources such as genomic DNA hbraπes or can be synthesized using well known and commercially available techniques
When polynucleotides of the present invention are used for the recombinant production of polypeptides of the present 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 codmg sequences, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protem sequence, or other fusion peptide portions. For example, a marker sequence that facilitates puπfϊcation of the fused polypeptide can be encoded. In certam preferred embodiments of this aspect of the invention, the marker sequence is a hexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) and descnbed in Gentz et al , Proc NatlAcad Sci USA (1989) 86-821-824, or is an HA tag The polynucleotide may also contain non-codmg 5' and 3' sequences, such as transcnbed, non-translated sequences, splicing and polyadenylation signals, πbosome bmdmg sites and sequences that stabilize mRNA.
Polynucleotides that are identical, or have sufficient identity to a polynucleotide sequence of SEQ ED NO: 1 , may be used as hybπdization probes for cDNA and genomic DNA or as pπmers for a nucleic acid amplification reaction (for instance, PCR). Such probes and pπmers may be used to isolate full-length cDNAs and genomic clones encoding polypeptides of the present mvention and to isolate cDNA and genomic clones of other genes (including genes encodmg paralogs from human sources and orthologs and paralogs from species other than human) that have a high sequence similaπty to SEQ ED NO: 1 , typically at least 95% identity. Preferred probes and pπmers will generally compπse at least 15 nucleotides, preferably, at least 30 nucleotides and may have at least 50, if not at least 100 nucleotides Particularly preferred probes will have between 30 and 50 nucleotides. Particularly preferred pπmers will have between 20 and 25 nucleotides.
A polynucleotide encoding a polypeptide of the present mvention, mcludmg homologs from species other than human, may be obtained by a process compπsmg the steps of screening a library under stringent hybπdization conditions with a labeled probe having the sequence of SEQ ED NO 1 or a fragment thereof, preferably of at least 15 nucleotides; and isolating full-length cDNA and genomic clones containing said polynucleotide sequence. Such hybπdization techniques are well known to the skilled artisan. Preferred stringent hybπdization conditions mclude overnight incubation at 42°C in a solution compπsmg: 50% formamide, 5xSSC (150mM NaCl, 15mM tπsodium citrate), 50 mM sodium phosphate (pH7.6), 5x Denhardt's solution, 10 % dextran sulfate, and 20 microgram/ml denatured, sheared salmon sperm DNA; followed by washing the filters in 0. lx SSC at about 65°C Thus the present mvention also includes isolated polynucleotides, preferably with a nucleotide sequence of at least 100, obtained by screenmg a library under stπngent hybπdization conditions with a labeled probe having the sequence of SEQ ED NO: 1 or a fragment thereof, preferably of at least 15 nucleotides.
The skilled artisan will appreciate that, m many cases, an isolated cDNA sequence will be incomplete, m that the region coding for the polypeptide does not extend all the way through to the 5' terminus. This is a consequence of reverse transcπptase, an enzyme with inherently low
"processivity" (a measure of the ability of the enzyme to remain attached to the template duπng the polymerisation reaction), failing to complete a DNA copy of the mRNA template duπng first strand cDNA synthesis.
There are several methods available and well known to those skilled m the art to obtain full- length cDNAs, or extend short cDNAs, for example those based on the method of Rapid
Amplification of cDNA ends (RACE) (see, for example, Frohman, et al , Proc Nat Acad Sci USA 85, 8998-9002, 1988). Recent modifications of the technique, exemplified by the Marathon (trade mark) technology (Clontech Laboratoπes Inc.) for example, have significantly simplified the search for longer cDNAs. In the Marathon (trade mark) technology, cDNAs have been prepared from mRNA extracted from a chosen tissue and an 'adaptor' sequence ligated onto each end. Nucleic acid amplification (PCR) is then carried out to amplify the "missing" 5' end of the cDNA using a combination of gene specific and adaptor specific ohgonucleotide pπmers. The PCR reaction is then repeated using 'nested' pπmers, that is, pπmers designed to anneal withm the amplified product (typically an adaptor specific pπmer that anneals further 3' m the adaptor sequence and a gene specific pπmer that anneals further 5' in the known gene sequence). The products of this reaction can then be analysed by DNA sequencing and a full-length cDNA constructed either by joining the product directly to the existing cDNA to give a complete sequence, or carrying out a separate full- length PCR using the new sequence information for the design of the 5' pπmer.
Recombmant polypeptides of the present invention may be prepared by processes well known in the art from genetically engmeered host cells compπsing expression systems. Accordingly, m a further aspect, the present invention relates to expression systems compπsing a polynucleotide or polynucleotides of the present mvention, to host cells which are genetically engmeered with such expression systems and to the production of polypeptides of the invention by recombinant techmques. Cell-free translation systems can also be employed to produce such protems using RNAs deπved from the DNA constructs of the present mvention.
For recombmant production, host cells can be genetically engineered to incorporate expression systems or portions thereof for polynucleotides of the present mvention. Polynucleotides may be mtroduced mto host cells by methods descπbed m many standard laboratory manuals, such as Davis, et al, BASIC METHODS IN MOLECULAR BIOLOGY (1986) and Sambrook, et al.(ibid). Preferred methods of introducing polynucleotides mto host cells mclude, for instance, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, micromjection, cationic hpid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection.
Representative examples of appropπate hosts mclude bacteπal cells, such as Streptococci, Staphylococci, E. coli, Streptomyces and Bacillus subtihs cells; fungal cells, such as yeast cells and
Aspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells; and plant cells.
A great vaπety of expression systems can be used, for instance, chromosomal, episomal and virus-deπved systems, e g , vectors deπved from bacteπal plasrmds, from bacteπophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccmia viruses, adenovrruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors deπved from combinations thereof, such as those deπved from plasrmd and bacteπophage genetic elements, such as cosmids and phagemids The expression systems may contain control regions that regulate as well as engender expression. Generally, any system or vector that is able to maintain, propagate or express a polynucleotide to produce a polypeptide m a host may be used. The appropπate polynucleotide sequence may be mserted into an expression system by any of a vaπety of well-known and routine techniques, such as, for example, those set forth m Sambrook et al., (ibid). Appropπate secretion signals may be incorporated mto the desired polypeptide to allow secretion of the translated protem into the lumen of the endoplasmic reticulum, the penplasmic space or the extracellular environment. These signals may be endogenous to the polypeptide or they may be heterologous signals.
If a polypeptide of the present mvention is to be expressed for use m screenmg assays, it is generally preferred that the polypeptide be produced at the surface of the cell. In this event, the cells may be harvested pπor to use in the screening assay. If the polypeptide is secreted mto the medium, the medium can be recovered m order to recover and puπfy the polypeptide. If produced mtracellularly, the cells must first be lysed before the polypeptide is recovered.
Polypeptides of the present invention can be recovered and puπfied from recombmant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, amon or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography
Most preferably, high performance liquid chromatography is employed for puπfication. Well known techniques for refoldmg protems may be employed to regenerate active conformation when the polypeptide is denatured duπng intracellular synthesis, isolation and/or puπfication. Polynucleotides of the present invention may be used as diagnostic reagents, through detecting mutations m the associated gene. Detection of a mutated form of the gene characteπsed by the polynucleotide of SEQ ED NO: 1 m the cDNA or genomic sequence and which is associated with a dysfunction will provide a diagnostic tool that can add to, or define, a diagnosis of a disease, or susceptibility to a disease, which results from under-expression, over-expression or altered spatial or temporal expression of the gene. Individuals carrying mutations m the gene may be detected at the DNA level by a vaπety of techniques well known m the art.
Nucleic acids for diagnosis may be obtamed from a subject's cells, such as from blood, urine, saliva, tissue biopsy or autopsy mateπal. The genomic DNA may be used directly for detection or it may be amplified enzymatically by usmg PCR, preferably RT-PCR, or other amplification techniques pπor to analysis. RNA or cDNA may also be used m similar fashion. Deletions and insertions can be detected by a change in size of the amplified product m comparison to the normal genotype. Pomt mutations can be identified by hybπdizmg amplified DNA to labeled human AXOR29 receptor nucleotide sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences m melting temperatures. DNA sequence difference may also be detected by alterations m the electrophoretic mobility of DNA fragments in gels, with or without denatuπng agents, or by direct DNA sequencing (see, for instance, Myers et al, Science (1985) 230: 1242). Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and S 1 protection or the chemical cleavage method (see Cotton, et al , Proc Natl Acad Sci USA (1985) 85: 4397-4401).
An array of oligonucleotides probes compπsing human AXOR29 receptor polynucleotide sequence or fragments thereof can be constructed to conduct efficient screening of e.g , genetic mutations. Such arrays are preferably high density arrays or gnds. Array technology methods are well known and have general applicability and can be used to address a vaπety of questions m molecular genetics including gene expression, genetic linkage, and genetic vaπabihty, see, for example, M.Chee, et al, Science, 21 A, 610-613 (1996) and other references cited therem.
Detection of abnormally decreased or increased levels of polypeptide or mRNA expression may also be used for diagnosing or determining susceptibility of a subject to a disease of the invention. Decreased or increased expression can be measured at the RNA level using any of the methods well known in the art for the quantitation of polynucleotides, such as, for example, nucleic acid amplification, for instance PCR, RT-PCR, RNase protection, Northern blotting and other hybπdization methods. Assay techniques that can be used to determine levels of a protem, such as a polypeptide of the present invention, in a sample deπved from a host are well-known to those of skill in the art. Such assay methods mclude radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays.
Thus in another aspect, the present invention relates to a diagnostic kit compπsing
(a) a polynucleotide of the present mvention, preferably the nucleotide sequence of SEQ ED NO 1 , or a fragment or an RNA transcπpt thereof;
(b) a nucleotide sequence complementary to that of (a);
(c) a polypeptide of the present invention, preferably the polypeptide of SEQ ED NO:2 or a fragment thereof; or
(d) an antibody to a polypeptide of the present invention, preferably to the polypeptide of SEQ ED NO:2.
It will be appreciated that in any such kit, (a), (b), (c) or (d) may compπse a substantial component. Such a kit will be of use diagnosing a disease or susceptibility to a disease, particularly diseases of the invention, amongst others.
The polynucleotide sequences of the present mvention are valuable for chromosome localisation studies. The sequence is specifically targeted to, and can hybπdize with, a particular location on an individual human chromosome. The mapping of relevant sequences to chromosomes accordmg to the present mvention is an important first step in correlating those sequences with gene associated disease. Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found m, for example, V. McKusick, Mendelian Inheπtance m Man (available on-lme 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- lnhentance of physically adjacent genes). Precise human chromosomal localisations for a genomic sequence (gene fragment etc.) can be determined using Radiation Hybrid (RH) Mapping (Walter, M Spillett, D., Thomas, P., Weissenbach, J., and Goodfellow, P., (1994) A method for constructing radiation hybπd maps of whole genomes, Nature Genetics 7, 22-28). A number of RH panels are available from Research Genetics (Huntsville, AL, USA) e.g. the GeneBπdge4 RH panel (Hum Mol Genet 1996 Mar;5(3):339-46 A radiation hybπd map of the human genome. Gyapay G, Schmitt K, Fizames C, Jones H, Vega-Czarny N, Spillett D, Muselet D, Prud'Homme JF, Dib C, Auffray C, Moπssette J, Weissenbach J, Goodfellow PN). To determine the chromosomal location of a gene using this panel, 93 PCRs are performed using pπmers designed from the gene of interest on RH DNAs. Each of these DNAs contains random human genomic fragments maintained m a hamster background (human / hamster hybπd cell lines). These PCRs result m 93 scores indicating the presence or absence of the PCR product of the gene of interest. These scores are compared with scores created using PCR products from genomic sequences of known location. This compaπson is conducted at http://www.genome.wi.mit.edu/. The gene of the present invention maps to human chromosome 19pl3.2. The polynucleotide sequences of the present mvention are also valuable tools for tissue expression studies. Such studies allow the determination of expression patterns of polynucleotides of the present mvention which may give an indication as to the expression patterns of the encoded polypeptides in tissues, by detecting the mRNAs that encode them. The techniques used are well known in the art and include in situ hydπdisation techmques to clones arrayed on a gπd, such as cDNA microarray hybπdisation (Schena et al, Science, 270, 467-470, 1995 and Shalon et al, Genome Res, 6,
639-645, 1996) and nucleotide amplification techniques such as PCR. A preferred method uses the TAQMAN (Trade mark) technology available from Perkin Elmer. Results from these studies can provide an indication of the normal function of the polypeptide in the organism. In addition, comparative studies of the normal expression pattern of mRNAs with that of mRNAs encoded by an alternative form of the same gene (for example, one having an alteration m polypeptide coding potential or a regulatory mutation) can provide valuable msights mto the role of the polypeptides of the present invention, or that of inappropnate expression thereof in disease. Such mappropπate expression may be of a temporal, spatial or simply quantitative nature.
A further aspect of the present mvention relates to antibodies. The polypeptides of the mvention or their fragments, or cells expressmg them, can be used as immunogens to produce antibodies that are lmmunospecific for polypeptides of the present mvention. The term "lmmunospecific" means that the antibodies have substantially greater affinity for the polypeptides of the mvention than their affinity for other related polypeptides in the pπor art.
Antibodies generated against polypeptides of the present mvention may be obtained by administering the polypeptides or epitope-beaπng fragments, or cells to an animal, preferably a non- human animal, using routine protocols. For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell lme cultures can be used. Examples include the hybπdoma technique (Kohler, G. and Milstein, C, Nature (1975) 256:495-497), the tπoma technique, the human B-cell hybπdoma technique (Kozbor et al, Immunology Today (1983) 4:72) and the EBV- hybridoma technique (Cole, et al. , MONOCLONAL ANTIBODIES AND CANCER THERAPY, 77-
96, Alan R. Liss, Inc., 1985).
Techniques for the production of smgle chain antibodies, such as those descπbed in U.S. Patent No. 4,946,778, can also be adapted to produce smgle chain antibodies to polypeptides of this mvention. Also, transgenic mice, or other orgamsms, including other mammals, may be used to express humanized antibodies.
The above-descπbed antibodies may be employed to isolate or to identify clones expressing the polypeptide or to puπfy the polypeptides by affinity chromatography. Antibodies against polypeptides of the present invention may also be employed to treat diseases of the invention, amongst others
Polypeptides and polynucleotides of the present invention may also be used as vaccines. Accordingly, in a further aspect, the present invention relates to a method for inducing an immunological response in a mammal that comprises inoculating the mammal with a polypeptide of the present invention, adequate to produce antibody and or T cell immune response, including, for example, cytokme-producmg T cells or cytotoxic T cells, to protect said animal from disease, whether that disease is already established withm the individual or not. An immunological response in a mammal may also be induced by a method comprises dehveπng a polypeptide of the present invention via a vector directing expression of the polynucleotide and coding for the polypeptide in vivo m order to induce such an immunological response to produce antibody to protect said animal from diseases of the invention. One way of admmisteπng the vector is by accelerating it mto the desired cells as a coating on particles or otherwise. Such nucleic acid vector may compπse DNA, RNA, a modified nucleic acid, or a DNA/RNA hybπd. For use a vaccine, a polypeptide or a nucleic acid vector will be normally provided as a vaccine formulation (composition). The formulation may further compπse a suitable earner. Since a polypeptide may be broken down m the stomach, it is preferably administered parenterally (for instance, subcutaneous, intramuscular, intravenous, or mtradermal injection). Formulations suitable for parenteral administration include aqueous and non- aqueous steπle injection solutions that may contain anti-oxidants, buffers, bacteπostats and solutes that render the formulation lsotomc with the blood of the recipient; and aqueous and non-aqueous steπle suspensions that may include suspending agents or thickening agents. The formulations may be presented m unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-dπed condition requiπng only the addition of the steπle liquid earner immediately pnor to use. The vaccine formulation may also include adjuvant systems for enhancing the lmmunogemcity of the formulation, such as oil-m water systems and other systems known in the art. The dosage will depend on the specific activity of the vaccine and can be readily determined by routine expeπmentation.
The human AXOR29 receptor polypeptide of the present invention may be employed m a process for screening for compounds that bmd to and activate the human AXOR29 receptor polypeptides of the present invention (called agonists), or inhibit the interaction of the human AXOR29 receptor polypeptides with receptor ligands (called antagonists). Thus, polypeptides of the mvention may also be used to assess the binding of small molecule substrates and ligands m, for example, cells, cell-free preparations, chemical braπes, and natural product mixtures. These substrates and ligands may be natural substrates and ligands or may be structural or functional mimetics. See Cohgan, et al, CURRENT PROTOCOLS IN IMMUNOLOGY l(2):Chapter 5 (1991).
Human AXOR29 receptor prote s are responsible for many biological functions, including many pathologies. Provided by the invention are screening methods to identify compounds and drugs that stimulate human AXOR29 receptor or that inhibit the function or level of the polypeptide. In general, agonists are employed for therapeutic and prophylactic purposes for such conditions as infections such as diseases of the mvention, among others. Antagonists may be employed for a vaπety of therapeutic and prophylactic purposes for such conditions as diseases of the mvention.
In general, such screening procedures involve providing appropπate cells that express the receptor polypeptide of the present invention on the surface thereof. Such cells mclude cells from mammals, yeast, Drosophila or E coli. In particular, a polynucleotide encoding the receptor of the present mvention is employed to transfect cells to thereby express the human AXOR29 receptor polypeptide. The expressed receptor is then contacted with a test compound to observe bmdmg, stimulation or inhibition of a functional response.
One such screenmg procedure involves the use of melanophores that are transfected to express the human AXOR29 receptor polypeptide of the present invention. Such a screening technique is descπbed m PCT WO 92/01810, published February 6, 1992. Such an assay may be employed to screen for a compound which inhibits activation of the receptor polypeptide of the present invention by contacting the melanophore cells that encode the receptor with both a receptor ligand, such as S-l-P, and a compound to be screened. Inhibition of the signal generated by the ligand indicates that a compound is a potential antagonist for the receptor, i e., inhibits activation of the receptor.
The technique may also be employed for screening of compounds that activate the receptor by contacting such cells with compounds to be screened and determining whether such compound generates a signal, i.e., activates the receptor.
Other screening techmques include the use of cells which express the human AXOR29 receptor polypeptide (for example, transfected CHO cells) in a system that measures extracellular pH 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 or pH changes, is then measured to determine whether the potential compound activates or inhibits the receptor. Another screening technique involves expressing the human AXOR29 receptor polypeptide m which the receptor is linked to phosphohpase C or D. Representative examples of such cells include, but are not limited to: endothehal cells, smooth muscle cells, and embryonic kidney cells The screening may be accomplished as hereinabove descnbed by detecting activation of the receptor or inhibition of activation of the receptor from the phosphohpase second signal.
Another method involves screenmg for compounds that are antagonists, and thus inhibit activation of the receptor polypeptide of the present invention by determining inhibition of binding of labeled ligand, such as S-l-P or di-hydro S-l-P, to cells expressing the receptor on their surface, or cell membranes containing the receptor. Such a method involves transfectmg a eukaryotic cell with DNA encoding the human AXOR29 receptor polypeptide, such that the cell expresses the receptor on its surface. The cell is then contacted with a potential antagonist in the presence of a labeled form of a ligand, such as S-l-P or di-hydro S-l-P. The ligand can be labeled, e g., by radioactivity. The amount of labeled ligand bound to the receptors is measured, e g., by measuπng radioactivity associated with transfected cells or membrane from these cells. If the compound binds to the receptor, the binding of labeled ligand to the receptor is inhibited as determined by a reduction of labeled ligand which binds to the receptors. This method is called bmdmg assay Naturally, this same technique can be used to look for an agonist.
The screening method may simply measure the bmdmg of a candidate compound to the polypeptide, or to cells or membranes beaπng the polypeptide, or a fusion protem thereof by means of a label directly or indirectly associated with the candidate compound Alternatively, the screenmg method may involve measuπng or, qualitatively or quantitatively, detecting the competition of binding of a candidate compound to the polypeptide with a labeled competitor (e g agonist or antagonist). Further, these screening methods may test whether the candidate compound results in a signal generated by activation or inhibition of the polypeptide, using detection systems appropπate to the cells beaπng the polypeptide. Inhibitors of activation are generally assayed m 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 compπse the steps of mixing a candidate compound with a solution containing a polypeptide of the present invention, to form a mixture, measunng human AXOR29 receptor activity in the mixture, and compaπng the AXOR29 activity of the mixture to a control mixture which contains no candidate compound
Another screening procedure involves the use of mammalian cells (CHO, HEK 293, Xenopus Oocytes, RBL-2H3, etc that are transfected to express human AXOR29 receptor. The cells are loaded with an indicator dye that produces a fluorescent signal when bound to calcium, and the cells are contacted with a test substance and a receptor agonist, such as S-l-P. Any change m fluorescent signal is measured over a defined peπod of time using, for example, a fluorescence spectrophotometer or a fluorescence imaging plate reader. A change in the fluorescence signal pattern generated by the ligand indicates that a compound is a potential antagonist or agonist for the receptor. Another screening procedure involves use of mammalian cells (CHO, HEK293, Xenopus
Oocytes, RBL-2H3, etc.) that are transfected to express the receptor of interest, and that are also transfected with a reporter gene construct that is coupled to activation of the receptor (for example, luciferase or beta-galactosidase behind an appropriate promoter). The cells are contacted with a test substance and the receptor agonist (ligand), such as S-l-P or di-hydro S-l-P, and the signal produced by the reporter gene is measured after a defined period of time The signal can be measured using a luminometer, spectrophotometer, fluoπmeter, or other such instrument appropπate for the specific reporter construct used Inhibition of the signal generated by the ligand indicates that a compound is a potential antagonist for the receptor
Another screening technique for antagonists or agonists involves introducing RNA encodmg the AXOR29 polypeptide into Xenopus oocytes (or CHO, HEK 293 , RBL-2H3. etc to transiently or stably express the receptor. The receptor oocytes are then contacted with the receptor ligand, such as S-l-P or di-hydro S-l-P, and a compound to be screened. Inhibition or activation of the receptor is then determined by detection of a signal, such as, cAMP, calcium, proton, or other ions
Another method involves screening for AXOR29 polypeptide inhibitors by determining inhibition or stimulation of AXOR29 polypeptide-mediated cAMP and/or adenylate cyclase accumulation or dimumtion. Such a method involves transiently or stably transfectmg a eukaryotic cell with AXOR29 polypeptide receptor to express the receptor on the cell surface. The cell is then exposed to potential antagonists m the presence of AXOR29 polypeptide ligand, such as S-l-P or di- hydro S-l-P. The changes m levels of cAMP is then measured over a defined peπod of time, for example, by radio-immuno or protem binding assays (for example using Flashplates or a scintillation proximity assay). Changes m cAMP levels can also be determined by directly measuring the activity of the enzyme, adenylyl cyclase, m broken cell preparations. If the potential antagonist binds the receptor, and thus inhibits AXOR29 polypeptide-hgand bmdmg, the levels of AXOR29 polypeptide-mediated cAMP, or adenylate cyclase activity, will be reduced or increased Another screening method for agonists and antagonists relies on the endogenous pheromone response pathway in the yeast, Saccharomyces cerevisiae. Heterothallic strains of yeast can exist in two mitotically stable haploid mating types, MATa and MATa. Each cell type secretes a small peptide hormone that binds to a G-protem coupled receptor on opposite mating-type cells which tnggers a MAP kinase cascade leadmg to Gl arrest as a prelude to cell fusion. Genetic alteration of certain genes in the pheromone response pathway can alter the normal response to pheromone, and heterologous expression and coupling of human G-protein coupled receptors and humanized G- protein subunits in yeast cells devoid of endogenous pheromone receptors can be linked to downstream signalling pathways and reporter genes (e.g., U.S. Patents 5,063,154; 5,482,835; 5,691,188). Such genetic alterations include, but are not limited to: (i) deletion of the STE2 or
STE3 gene encoding the endogenous G-protein coupled pheromone receptors; (ii) deletion of the FAR1 gene encoding a protein that normally associates with cyclin-dependent kinases leading to cell cycle arrest; and (iii) construction of reporter genes fused to the FUS1 gene promoter (where FUS1 encodes a membrane-anchored glycoprotein required for cell fusion). Downstream reporter genes can permit either a positive growth selection (e.g., histidine prototrophy using the FUS1-HIS3 reporter), or a colorimetric, fluorimetric or spectrophotometric readout, depending on the specific reporter construct used (e.g., b-galactosidase induction using a FUSl-LacZ reporter).
The yeast cells can be further engineered to express and secrete small peptides from random peptide libraries, some of which can permit autocrine activation of heterologously expressed human (or mammalian) G-protein coupled receptors (Broach, et al, Nature 384: 14-16, 1996; Manfredi, et al, Mol. Cell. Biol 16: 4700-4709, 1996). This provides a rapid direct growth selection (e.g., using the FUS1-HIS3 reporter) for surrogate peptide agonists that activate characterized or orphan receptors. Alternatively, yeast cells that functionally express human (or mammalian) G-protein coupled receptors linked to a reporter gene readout (e.g., FUSl-LacZ) can be used as a platform for high-throughput screening of known ligands, fractions of biological extracts and libraries of chemical compounds for either natural or surrogate ligands. Functional agonists of sufficient potency (whether natural or surrogate) can be used as screening tools in yeast cell-based assays for identifying G-protein coupled receptor antagonists. For example, agonists will promote growth of a cell with FUS-HIS3 reporter or give positive readout for a cell with FUSl-LacZ. However, a candidate compound that inhibits growth or negates the positive readout induced by an agonist is an antagonist. For this purpose, the yeast system offers advantages over mammalian expression systems due to its ease of utility and null receptor background (lack of endogenous G-protein coupled receptors), which often interferes with the ability to identify agonists or antagonists.
The present invention also provides a method for identifying new ligands not known to be capable of binding to a human AXOR29 receptor polypeptide. The screening assays described above for identifying agonists may be used to identify new ligands.
The present invention also contemplates agonists and antagonists obtained from the above described screening methods. Examples of potential human AXOR29 polypeptide receptor antagonists include peptidomimetics, synthetic organic molecules, natural products, antibodies, etc., that bmd to the receptor but do not elicit a second messenger response, such that the activity of the receptor is prevented. Potential antagonists also include protems which are closely related to the ligand of the
AXOR29 polypeptide receptor, i e , a fragment of the ligand, which have lost biological function, and when they bmd to human AXOR29 polypeptide receptor, elicit no response.
Thus m another aspect, the present invention relates to a screening kit for identifying agonists, antagonists, and ligands for human AXOR29 polypeptides, compπsmg: (a) a human AXOR29 polypeptide, preferably that of SEQ ED NO.2; and further preferably compπses labeled or unlabeled S-l-P or di-hydro S-l-P,
(b) a recombinant cell expressing a human AXOR29 polypeptide, preferably that of SEQ ED NO:2; and further preferably compπses labeled or unlabeled S-l-P or di-hydro S-l-P; or
(c) a cell membrane expressing a human AXOR29 polypeptide; preferably that of SEQ ED NO: 2; and further preferably compπses labeled or unlabled S-l-P or di-hydro S-l-P.
It will be appreciated that in any such kit, (a), (b), or (c) may compnse a substantial component.
As noted above, a potential antagonist is a small molecule that binds to the human AXOR29 polypeptide, making it inaccessible to ligands such that normal biological activity is prevented. Examples of small molecules include, but are not limited to, small peptides or peptide-hke molecules.
Potential antagonists also include soluble forms of a human AXOR29 polypeptide receptor, e g , fragments of the receptor, which bmd to the ligand and prevent the ligand from interacting with membrane bound human AXOR29 polypeptide. The screening method may simply measure the bmdmg of a candidate compound to the polypeptide, or to cells or membranes beaπng the polypeptide, or a fusion protem thereof by means of a label directly or indirectly associated with the candidate compound. Alternatively, the screenmg method may involve competition with a labeled competitor. Further, these screening methods may test whether the candidate compound results in a signal generated by activation or inhibition of the polypeptide, using detection systems appropπate to the cells beaπng the polypeptide. Inhibitors of activation are generally assayed m the presence of a known agonist and the effect on activation by the agonist by the presence of the candidate compound is observed. Constituitively active polypeptides may be employed in screenmg methods for inverse agonists or inhibitors, in the absence of an agonist or inhibitor, by testing whether the candidate compound results m inhibition of activation of the polypeptide Further, the screening methods may simply compπse the steps of mixing a candidate compound with a solution containing a polypeptide of the present invention, to form a mixture, measuring human AXOR29 receptor activity m the mixture, and comparmg the human AXOR29 receptor activity of the mixture to a standard. Fusion protems, such as those made from Fc portion and AXOR29 polypeptide, as hereinbefore descπbed, can also be used for high-throughput screening assays to identify antagonists for the polypeptide of the present invention (see D. Bennett et al, J Mol Recognition, 8:52-58 (1995); and K. Johanson et al , J. Bwl Chem., 270(16):9459-9471 (1995).
Polypeptides of the present invention may be employed in conventional low capacity screening methods and also in high-throughput screenmg (HTS) formats. Such HTS formats include not only the well-established use of 96- and, more recently, 384-well microtiter plates but also emerging methods such as the nanowell method descπbed by Schullek, et al , Anal Biochem., 246 20-29 (1997).
Glossary
The following definitions are provided to facilitate understanding of certam terms used frequently hereinbefore.
"S-l-P (sphmgosme- 1 -phosphate) " refers to the sphmgohpid metabolite having the structure:
CH
Figure imgf000023_0001
"Di -hydro S-l-P" (di-hvdro sphmgosme- 1 -phosphate)" refers to the sphmgohpid metabolite having the structure-
Figure imgf000023_0002
"Antibodies" as used herein includes polyclonal and monoclonal antibodies, chimeπc, single chain, and humanized antibodies, as well as Fab fragments, including the products of an
Fab or other lmmunoglobulm expression library.
"Isolated" means altered "by the hand of man" from its natural state, i.e., if it occurs in nature, it has been changed or removed from its ongmal environment, or both. For example, a polynucleotide or a polypeptide naturally present m a living organism is not "isolated," but the same polynucleotide or polypeptide separated from the coexisting matenals of its natural state is "isolated", as the term is employed herein. Moreover, a polynucleotide or polypeptide that is introduced mto an organism by transformation, genetic manipulation or by any other recombmant method is "isolated" even if it is still present m said organism, which organism may be living or non-living.
"Polynucleotide" generally refers to any polynbonucleotide (RNA) or polydeoxπbonucleotide (DNA), which may be unmodified or modified RNA or DNA. "Polynucleotides" include, without limitation, smgle- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybπd molecules comprising DNA and RNA that may be smgle-stranded or, more typically, double-stranded or a mixture of single- and double- stranded regions. In addition, "polynucleotide" refers to tnple-stranded regions compπsing 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 mclude, for example, tntylated bases and unusual bases such as inosine. A vaπety of modifications may be made to DNA and RNA; thus, "polynucleotide" embraces chemically, enzymatically or metabohcally modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. "Polynucleotide" also embraces relatively short polynucleotides, often referred to as ohgonucleotides.
"Polypeptide" refers to any polypeptide compπsing two or more ammo acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres "Polypeptide" refers to both short chains, commonly refeπed to as peptides, ohgopeptides or ohgomers, and to longer chains, generally referred to as protems. Polypeptides may contain ammo acids other than the 20 gene-encoded ammo acids. "Polypeptides" include ammo acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques that are well known in the art. Such modifications are well descπbed in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications may occur anywhere m a polypeptide, including the peptide backbone, the ammo acid side-chams and the ammo or carboxyl termini. It will be appreciated that the same type of modification may be present to the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched as a result of ubiquitmation, and they may be cyclic, with or without branching. Cyclic, branched and branched cyclic polypeptides may result from post-translation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-πbosylation, amidation, biotmylation, covalent attachment of flavm, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide deπvative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylmositol, cross-linking, cychzation, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteme, formation of pyroglutamate, formylation, gamma- carboxylation, glycosylation, GPI anchor formation, hydroxylation, lodmation, methylation, myπstoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of ammo acids to protems such as argmylation, and ubiquitmation (see, for instance, Protems - Structure and Molecular Properties, 2nd
Ed., T. E. Creighton, W. H. Freeman and Company, New York, 1993, Wold, F., Post-translational Protem Modifications: Perspectives and Prospects, 1-12, in Post-translational Covalent Modification of Protems, B. C. Johnson, Ed., Academic Press, New York, 1983; Seifter et al , "Analysis for protem modifications and nonprotem cofactors", Meth Enzymol, 182, 626-646, 1990, and Rattan et al , "Protem Synthesis: Post-translational Modifications and Agmg", Ann NY Acad Sci, 663, 48-62,
1992).
"Fragment" of a polypeptide sequence refers to a polypeptide sequence that is shorter than the reference sequence but that retains essentially the same biological function or activity as the reference polypeptide. "Fragment" of a polynucleotide sequence refers to a polynucleotide sequence that is shorter than the reference sequence of SEQ ED NO: 1..
"Vaπant" refers to a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, but retains the essential properties thereof. A typical vaπant of a polynucleotide differs m nucleotide sequence from the reference polynucleotide. Changes m the nucleotide sequence of the vaπant may or may not alter the ammo acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in ammo acid substitutions, additions, deletions, fusions and truncations m the polypeptide encoded by the reference sequence, as discussed below. A typical vanant of a polypeptide differs m ammo acid sequence from the reference polypeptide. Generally, alterations are limited so that the sequences of the reference polypeptide and the vaπant are closely similar overall and, m many regions, identical A vaπant and reference polypeptide may differ in ammo acid sequence by one or more substitutions, insertions, deletions m any combination. A substituted or inserted ammo acid residue may or may not be one encoded by the genetic code. Typical conservative substitutions include Gly, Ala; Val, Be, Leu; Asp, Glu; Asn, Gin; Ser, Thr; Lys, Arg; and Phe and Tyr. A vaπant of a polynucleotide or polypeptide may be naturally occurnng such as an allele, or it may be a variant that is not known to occur naturally. Non-naturally occurπng vaπants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis. Also included as variants are polypeptides having one or more post-translational modifications, for instance glycosylation, phosphorylation, methylation, ADP nbosylation and the like. Embodiments include methylation of the N-termmal ammo acid, phosphorylations of seπnes and threonmes and modification of C-termmal glycmes
"Allele" refers to one of two or more alternative forms of a gene occurπng at a given locus in the genome.
"Polymorphism" refers to a variation in nucleotide sequence (and encoded polypeptide sequence, if relevant) at a given position in the genome withm a population. "Single Nucleotide Polymorphism" (SNP) refers to the occurrence of nucleotide vanabihty at a single nucleotide position m the genome, withm a population. An SNP may occur withm a gene or within lntergemc regions of the genome. SNPs can be assayed using Allele Specific Amplification (ASA). For the process at least 3 pπmers are required. A common pπmer is used m reverse complement to the polymorphism being assayed. This common pπmer can be between 50 and 1500 bps from the polymorphic base. The other two (or more) pπmers are identical to each other except that the final 3' base wobbles to match one of the two (or more) alleles that make up the polymorphism. Two (or more) PCR reactions are then conducted on sample DNA, each using the common pπmer and one of the Allele Specific Pπmers.
"Splice Vaπant" as used herein refers to cDNA molecules produced from RNA molecules initially transcπbed from the same genomic DNA sequence but which have undergone alternative
RNA splicing. Alternative RNA splicing occurs when a pπmary RNA transcπpt undergoes splicing, generally for the removal of mtrons, which results in the production of more than one mRNA molecule each of that may encode different ammo acid sequences The term splice vaπant also refers to the protems encoded by the above cDNA molecules "Identity" reflects a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, determined by compaπng the sequences. In general, identity refers to an exact nucleotide to nucleotide or ammo acid to ammo 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 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 (so-called local alignment), that is more suitable for sequences of unequal length.
"Similarity" is a further, more sophisticated measure of the relationship between two polypeptide sequences. In general, "similarity" means a comparison between the amino acids of two polypeptide chains, on a residue by residue basis, taking into account not only exact correspondences between a between pairs of residues, one from each of the sequences being compared (as for identity) but also, where there is not an exact correspondence, whether, on an evolutionary basis, one residue is a likely substitute for the other. This likelihood has an associated "score" from which the "% 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 (Devereux J, et al, Nucleic Acids Res, 12, 387-395, 1984, available from Genetics Computer Group, Madison, Wisconsin, USA), for example the programs BESTFLT and GAP, may be used to determine the % identity between two polynucleotides and the % identity and the % similarity between two polypeptide sequences. BESTFLT uses the "local homology" algorithm of
Smith and Waterman (JMol Biol, 147,195-197, 1981, Advances in Applied Mathematics, 2, 482- 489, 1981) and finds the best single region of similarity between two sequences. BESTFLT is more suited to comparing two polynucleotide or two polypeptide sequences that are dissimilar in length, the program assuming that the shorter sequence represents a portion of the longer. In comparison, GAP aligns two sequences, finding a "maximum similarity", according to the algorithm of
Neddleman and Wunsch (JMolBiol, 48, 443-453, 1970). GAP is more suited to comparing sequences that are approximately the same length and an alignment is expected over the entire length. Preferably, the parameters "Gap Weight" and "Length Weight" used in each program are 50 and 3, for polynucleotide sequences and 12 and 4 for polypeptide sequences, respectively. Preferably, % identities and similarities are determined when the two sequences being compared are optimally aligned.
Other programs for determining identity and/or similarity between sequences are also known in the art, for instance the BLAST family of programs (Altschul S F, et al, J MolBiol, 215, 403-410, 1990, Altschul S F, et al, Nucleic Acids Res., 25:389-3402, 1997, available from the National Center for Biotechnology Information (NCBI), Bethesda, Maryland, USA and accessible through the home page of the NCBI at www.ncbi.nlm.nih.gov) and FASTA (Pearson W R, Methods in Enzymology, 183, 63-99, 1990; Pearson W R and Lipman D J, Proc Nat Acad Sci USA, 85, 2444- 2448,1988, available as part of the Wisconsin Sequence Analysis Package). Preferably, the BLOSUM62 ammo acid substitution matπx (Henikoff S and Hemkoff J G,
Proc. Nat. Acad Sci. USA, 89, 10915-10919, 1992) is used m polypeptide sequence comparisons including where nucleotide sequences are first translated mto ammo acid sequences before companson.
Preferably, the program BESTFLT 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 descπbed.
"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 mclude on average up to five differences per each 100 nucleotides of the reference sequence. Such differences are selected from the group consistmg of at least one nucleotide deletion, substitution, including transition and transversion, or insertion. These differences may occur at the 5' or 3' terminal positions of the reference polynucleotide sequence or anywhere between these terminal positions, interspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups withm the reference sequence. In other words, to obtain a polynucleotide sequence having an Identity Index of 0.95 compared to a reference polynucleotide sequence, an average of up to 5 in every 100 of the nucleotides of the in the reference sequence may be deleted, substituted or mserted, or any combination thereof, as hereinbefore descπbed. The same applies mutatis mutandis for other values of the Identity Index, for instance 0.96, 0.97, 0.98 and 0.99.
Similarly, for a polypeptide, a candidate polypeptide sequence having, for example, an Identity Index of 0.95 compared to a reference polypeptide sequence is identical to the reference sequence except that the polypeptide sequence may include an average of up to five differences per each 100 amino acids of the reference sequence. Such differences are selected from the group consisting of at least one ammo acid deletion, substitution, including conservative and non- conservative substitution, or insertion. These differences may occur at the ammo- or carboxy- terminal positions of the reference polypeptide sequence or anywhere between these terminal positions, interspersed either individually among the ammo acids m the reference sequence or m one or more contiguous groups withm 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 m every 100 of the ammo acids in the reference sequence may be deleted, substituted or inserted, or any combination thereof, as hereinbefore descπbed. The same applies mutatis mutandis for other values of the Identity Index, for instance 0.96, 0.97, 0.98 and 0.99.
The relationship between the number of nucleotide or ammo acid differences and the Identity Index may be expressed in the following equation na < xa - (xa • I), in which: na is the number of nucleotide or ammo acid differences, xa is the total number of nucleotides or ammo acids m SEQ ED NO.1 or SEQ ED NO:2, respectively,
I is the Identity Index ,
• is the symbol for the multiplication operator, and in which any non-mteger product of xa and I is rounded down to the nearest integer pπor to subtracting it from xa.
"Homolog" is a geneπc term used m the art to indicate a polynucleotide or polypeptide sequence possessing a high degree of sequence relatedness to a reference sequence. Such relatedness may be quantified by determining the degree of identity and/or similaπty between the two sequences as hereinbefore defined. Falling withm this geneπc term are the terms "ortholog", and "paralog". "Ortholog" refers to a polynucleotide or polypeptide that is the functional equivalent of the polynucleotide or polypeptide in another species. "Paralog" refers to a polynucleotide or polypeptide that withm the same species which is functionally similar.
"Fusion protem" refers to a protein encoded by two, often unrelated, fused genes or fragments thereof. In one example, EP-A-0 464 533 (Canadian counterpart 2045869) discloses fusion proteins compnsmg vaπous portions of constant region of immunoglobulm molecules together with another human protein or part thereof. In many cases, employing an immunoglobulm Fc region as a part of a fusion protem is advantageous for use in therapy and diagnosis resulting m, for example, improved pharmacokmetic properties [see, e g , EP-A 0232 262]. On the other hand, for some uses it would be desirable to be able to delete the Fc part after the fusion protein has been expressed, detected and puπfied. All publications and references, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference m their entirety as if each individual publication or reference were specifically and individually indicated to be incorporated by reference herein as being fully set forth. Any patent application to which this application claims pnonty is also incorporated by reference herein m its entirety in the manner descπbed above for publications and references.
Examples
Example 1 : Mammalian Cell Expression
The receptors of the present mvention are expressed m either human embryonic kidney 293 (HEK293) cells or adherent dhfr CHO cells. To maximize receptor expression, typically all 5' and 3' untranslated regions (UTRs) are removed from the receptor cDNA pπor to insertion mto a pCDN or pCDNA3 vector. The cells are transfected with individual receptor cDNAs by hpofectin and selected in the presence of 400 mg/ml G418. After 3 weeks of selection, individual clones are picked and expanded for further analysis. HEK293 or CHO cells transfected with the vector alone serve as negative controls. To isolate cell lines stably expressmg the individual receptors, about 24 clones are typically selected and analyzed by Northern blot analysis. Receptor mRNAs are generally detectable in about 50% of the G418-resιstant clones analyzed.
Example 2 Ligand bank for bmdmg and functional assays
A bank of over 600 putative receptor ligands has been assembled for screening. The bank compπses: transmitters, hormones and chemokmes known to act via a human seven transmembrane
(7TM) receptor; naturally occurπng compounds which may be putative agomsts for a human 7TM receptor, non-mammalian, biologically active peptides for which a mammalian counterpart has not yet been identified; and compounds not found m nature, but which activate 7TM receptors with unknown natural ligands. This bank is used to initially screen the receptor for known ligands, usmg both functional (i.e . calcium, cAMP, microphysiometer, oocyte electrophysiology, etc, see below) as well as bmdmg assays.
Example 3 : Ligand Binding Assays
Ligand bmdmg assays provide a direct method for ascertaining receptor pharmacology and are adaptable to a high throughput format. The punfied ligand for a receptor is radiolabeled to high specific activity (50-2000 Ci/mmol) for bmdmg studies. A determination is then made that the process of radiolabeling does not dimmish the activity of the ligand towards its receptor. Assay conditions for buffers, ions, pH and other modulators such as nucleotides are optimized to establish a workable signal to noise ratio for both membrane and whole cell receptor sources. For these assays, specific receptor bmdmg is defined as total associated radioactivity minus the radioactivity measured m the presence of an excess of unlabeled competing ligand. Where possible, more than one competing ligand is used to define residual nonspecific bmdmg.
Example 4- Functional Assay in Xenopus Oocytes Capped RNA transcπpts from lmeanzed plasmid templates encodmg the receptor cDNAs of the invention are synthesized in vitro with RNA polymerases m accordance with standard procedures In vitro transcπpts are suspended m water at a final concentration of 0.2 mg/ml. Ovaπan lobes are removed from adult female toads, Stage V defolliculated oocytes are obtained, and RNA transcπpts (10 ng/oocyte) are injected in a 50 nl bolus usmg a micromjection apparatus. Two electrode voltage clamps are used to measure the currents from individual Xenopus oocytes m response to agonist exposure
Recordings are made in Ca2+ free Barth's medium at room temperature. The Xenopus system can be used to screen known ligands and tissue/cell extracts for activating ligands.
Example 5: Microphysiometnc Assays
Activation of a wide vaπety of secondary messenger systems results m extrusion of small amounts of acid from a cell. The acid formed is largely as a result of the increased metabolic activity required to fuel the lntracellular signaling process. The pH changes m the media surrounding the cell are very small but are detectable by the CYTOSENSOR microphysiometer (Molecular Devices Ltd., Menlo Park, CA). The CYTOSENSOR is thus capable of detecting the activation of a receptor which is coupled to an energy utilizing lntracellular signaling pathway such as the G-protem coupled receptor of the present mvention.
Example 6: Extract/Cell Supernatant Screenmg
A large number of mammalian receptors exist for which there remains, as yet, no cognate activating ligand (agonist). Thus, active ligands for these receptors may not be mcluded within the ligands banks as identified to date. Accordmgly, the 7TM receptor of the mvention is also functionally screened (usmg calcium, cAMP, microphysiometer, oocyte electrophysiology, etc., functional screens) agamst tissue extracts to identify natural ligands. Extracts that produce positive functional responses can be sequencially subfractionated until an activating ligand is isolated identified.
Example 8: Calcium and cAMP Functional Assays
7TM receptors which are expressed m HEK 293 cells have been shown to be coupled functionally to activation of PLC and calcium mobilization and/or cAMP stimuation or inhibition.
Basal calcium levels m the HEK 293 cells m receptor-transfected or vector control cells were observed to be m the normal, 100 nM to 200 nM, range. HEK 293 cells expressing recombmant receptors are loaded with fura 2 and in a smgle day > 150 selected ligands or tissue/cell extracts are evaluated for agomst induced calcium mobilization. Similarly, HEK 293 cells expressing recombmant receptors are evaluated for the stimulation or inhibition of cAMP production usmg standard cAMP quantitation assays. Agonists presenting a calcium transient or cAMP flucuation are tested in vector control cells to determine if the response is unique to the transfected cells expressing receptor. Example 9: Calcium Mobilization Assay for Human AXOR29
Based on the results generated in Figure 3, it was shown that an S-l-P exhibited an endogenous response in RBL 2H3 cells only at very high concentrations. Stable cell lines of the human AXOR29 receptor were prepared in the RBL 2H3 cell line. The expression of functionally active clones were followed using Fluorescent Imaging Plate Reader (FLEPR). The concentration- dependent responses for three clones to S-l-P is presented in Figure 3. As can be seen from this figure, the best clones respond in a concentration-dependent manner with EC50S of about 120 nM. The best clones were charactenzed further, and that is shown m Figure 4. The cells responded with high potency through the human AXOR29 receptor to S-l-P. The cells also responded to dihydro-S- 1-P. The cells did not respond to LPA, the other EDG family receptor ligand.

Claims

Claims
1. An isolated polypeptide selected from the group consisting of:
(a) an isolated polypeptide encoded by a polynucleotide compnsmg the sequence of SEQ ED NO- 1,
(b) an isolated polypeptide comprising a polypeptide sequence having at least 95% identity to the polypeptide sequence of SEQ ED NO:2;
(c) an isolated polypeptide having at least 95% identity to the polypeptide sequence of SEQ ED NO:2; and
(d) fragments and vaπants of such polypeptides in (a) to (e).
2. The isolated polypeptide as claimed in Claim 1 comprising the polypeptide sequence of SEQ ED NO:2.
3. The isolated polypeptide as claimed m Claim 1 which is the polypeptide sequence of SEQ ED NO:2.
4. An isolated polynucleotide selected from the group consistmg of:
(a) an isolated polynucleotide compπsmg a polynucleotide sequence having at least 95% identity to the polynucleotide sequence of SEQ ED NO: 1 ; (b) an isolated polynucleotide having at least 95% identity to the polynucleotide of SEQ ED NO: 1 ,
(c) an isolated polynucleotide compπsmg a polynucleotide sequence encodmg a polypeptide sequence having at least 95% identity to the polypeptide sequence of SEQ ED NO:2;
(d) an isolated polynucleotide having a polynucleotide sequence encoding a polypeptide sequence having at least 95% identity to the polypeptide sequence of SEQ ED NO:2; (e) an isolated polynucleotide with a nucleotide sequence of at least 100 nucleotides obtamed by screenmg a library under stπngent hybπdization conditions with a labeled probe having the sequence of SEQ ED NO: 1 or a fragment thereof having at least 15 nucleotides;
(f) a polynucleotide which is the RNA equivalent of a polynucleotide of (a) to (e); or a polynucleotide sequence complementary to said isolated polynucleotide and polynucleotides that are variants and fragments of the above mentioned polynucleotides or that are complementary to above mentioned polynucleotides, over the entire length thereof.
5. An isolated polynucleotide as claimed in claim 4 selected from the group consisting of:
(a) an isolated polynucleotide comprising the polynucleotide of SEQ ED NO: 1;
(b) the isolated polynucleotide of SEQ ED NO: 1 ;
(c) an isolated polynucleotide comprising a polynucleotide sequence encoding the polypeptide of SEQ ED NO:2; and
(d) an isolated polynucleotide encoding the polypeptide of SEQ ED NO:2.
6. An expression system comprising a polynucleotide capable of producing a polypeptide of claim 1 when said expression vector is present in a compatible host cell.
7. A recombinant host cell comprising the expression vector of claim 6 or a membrane thereof expressing the polypeptide of claim 1.
8. A process for producing a polypeptide of claim 1 comprising the step of culturing a host cell as defined in claim 7 under conditions sufficient for the production of said polypeptide and recovering the polypeptide from the culture medium.
9. An antibody immunospecific for the polypeptide of any one of claims 1 to 3.
10. A method for identifying agonist or antagonist of the polypeptide as claimed in Claim 2 comprising the steps of: (a) contacting a cell expressing on the surface thereof the polypeptide, said polypeptide being associated with a second component capable of providing a detectable signal in response to the binding of a compound to said polypeptide, with a compound to be screened under conditions to permit binding to the polypeptide; and (b) determining whether the compound binds to and activates or inhibits the polypeptide by measuπng the level of a signal generated from the interaction of the compound with the polypeptide
11. The method as claimed in Claim 10, wherein the ligand is labeled or unlabeled sphmgosme- 1- phosphate (S-l-P) or di-hydro sphmgosme- 1 -phosphate (dι-hydro-S-1-P).
12. A method for identifying an agonist or antagonist of the polypeptide as claimed Claim 2 compπsing the steps of: determining the inhibition of binding of a ligand to cells expressing the polypeptide on the surface thereof, or to cell membranes containing the polypeptide, in the presence of a candidate compound under conditions to permit bmdmg to the polypeptide, and determining the amount of ligand bound to the polypeptide, such that a compound capable of causing reduction of bmdmg of a ligand is an agonist or antagonist.
13. The method as claimed in Claim 12, wherein the ligand is labeled or unlabeled S-l-P or di- hydro S-l-P.
14. A method for screening to identify compounds that stimulate or that inhibit a function or level of the polypeptide as claimed m Claim 2, compπsing a method selected from the group consisting of (a) measuπng or, quantitatively or qualitatively, detecting the bmdmg of a candidate compound to the polypeptide (or to the cells or membranes beaπng the polypeptide) or a fusion protem thereof by means of a label directly or indirectly associated with the candidate compound;
(b) measuπng the competition of the bmdmg of a candidate compound to the polypeptide (or to the cells or membranes beaπng the polypeptide) or a fusion protem thereof m the presence of a labeled competitor, preferably S-l-P or di-hydro S-l-P;
(c) testing whether the candidate compound results m a signal generated by activation or inhibition of the polypeptide, using detection systems appropπate to the cells or cell membranes beaπng the polypeptide; (d) mixing a candidate compound with a solution comprising said polypeptide to form a mixture, measuring activity of the polypeptide in the mixture, and comparing the activity of the mixture to a to a control mixture which contains no candidate compound; or
(e) detecting the effect of a candidate compound on the production of mRNA encoding said polypeptide and said polypeptide in cells.
15. An antagonist identified by the method Claims 14.
16. An agonist identified by the method of Claim 14.
PCT/US2000/019001 1999-07-13 2000-07-13 Human axor29 receptor WO2001004139A2 (en)

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WO2001036473A3 (en) * 1999-11-16 2002-07-11 Upjohn Co Human g protein-coupled receptors
WO2001081573A1 (en) * 2000-04-26 2001-11-01 Aventis Pharma Deutschland Gmbh Edg8 receptor, its preparation and use
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US7901682B2 (en) 2000-12-22 2011-03-08 Lpath, Inc. Compositions and methods for the treatment and prevention of cancer, angiogenesis, and inflammation
US6858383B2 (en) 2000-12-22 2005-02-22 Medlyte, Inc. Compositions and methods for the treatment and prevention of cardiovascular diseases and disorders, and for identifying agents therapeutic therefor
US6881546B2 (en) 2000-12-22 2005-04-19 Medlyte, Inc., Sdsu Heart Institute Compositions and methods for the treatment and prevention of cardiovascular diseases and disorders, and for identifying agents therapeutic therefor
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WO2004058149A2 (en) 2002-12-20 2004-07-15 Merck & Co., Inc. 1-(amino)indanes and (1,2-dihydro-3-amino)-benzofurans, benzothiophenes and indoles
EP1845971A4 (en) * 2005-01-21 2009-12-30 Medvet Science Pty Ltd A method of treating cellular damage
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US9274130B2 (en) 2006-05-31 2016-03-01 Lpath, Inc. Prevention and treatment of pain using antibodies to lysophosphatidic acid
US9274129B2 (en) 2006-05-31 2016-03-01 Lpath, Inc. Methods and reagents for detecting bioactive lipids
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US8067549B2 (en) 2006-10-27 2011-11-29 Lpath, Inc. Humanized antibodies and compositions for binding sphingosine-1-phosphate
US8026342B2 (en) 2006-10-27 2011-09-27 Lpath, Inc. Compositions and methods for binding sphingosine-1-phosphate
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