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WO2000078809A1 - Axor16, récepteur couplé à une protéine g - Google Patents

Axor16, récepteur couplé à une protéine g Download PDF

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Publication number
WO2000078809A1
WO2000078809A1 PCT/US2000/016869 US0016869W WO0078809A1 WO 2000078809 A1 WO2000078809 A1 WO 2000078809A1 US 0016869 W US0016869 W US 0016869W WO 0078809 A1 WO0078809 A1 WO 0078809A1
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Prior art keywords
polypeptide
polynucleotide
sequence
seq
isolated
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PCT/US2000/016869
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English (en)
Inventor
Nabil Elshourbagy
Usman Shabon
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Smithkline Beecham Corporation
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Priority to EP00942952A priority Critical patent/EP1189944A4/fr
Publication of WO2000078809A1 publication Critical patent/WO2000078809A1/fr

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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

Definitions

  • This invention relates to newly identified polypeptides and polynucleotides encoding such polypeptides, to their use in diagnosis and in identifying compounds that may be agonists, antagonists that are potentially useful m 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 supercedmg 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 proteins participating in pathways with G-proteins or PPG proteins.
  • Some examples of these proteins include the GPC receptors, such as those for adrenergic agents and doparmne (Kobilka, B.K., et al., Proc. Natl Acad.
  • G-proteins themselves, effector proteins, e.g., phospholipase C, adenyl cyclase, and phosphodiesterase, and actuator proteins, e.g., protein kmase A and protein kmase C (Simon, M.I., et al., Science, 1991, 252:802-8).
  • effector proteins e.g., phospholipase C, adenyl cyclase, and phosphodiesterase
  • actuator proteins e.g., protein kmase A and protein kmase C (Simon, M.I., et al., Science, 1991, 252:802-8).
  • the effect of hormone binding is activation of the enzyme, adenylate cyclase, inside the cell.
  • Enzyme activation by hormones is dependent on the presence of the nucleotide GTP.
  • GTP also influences hormone binding.
  • a G-protem connects the hormone receptor to adenylate cyclase.
  • G-protem was shown to exchange GTP for bound GDP when activated by a hormone receptor.
  • the GTP-carrying form then binds to activated adenylate cyclase Hydrolysis of GTP to GDP, catalyzed by the G-protem itself, returns the G-protem to its basal, inactive form.
  • 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 hydrophihc 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 include, but are not limited to, calcitonm, adrenergic, endothelm, cAMP, adenosme, muscanmc, acetylcholine, serotonin, histamme, thrombm, kmm, follicle stimulating hormone, opsins, endothelial differentiation gene-1, rhodopsins, odorant, and cytomegalovirus receptors.
  • G-protem coupled receptors have smgle conserved cysteine residues in each of the first two extracellular loops which form disulfide bonds that are believed to stabilize functional protem structure.
  • the 7 transmembrane regions are designated as TM1, TM2, TM3, TM4, TM5, TM6, and TM7.
  • TM3 has been implicated in signal transduction.
  • Phosphorylation and hpidation (palmitylation or farnesylation) of cysteine residues can influence signal transduction of some G-protem coupled receptors.
  • Most G-protem coupled receptors contain potential phosphorylation sites within the third cytoplasmic loop and/or the carboxy terminus.
  • G-protem coupled receptors such as the ⁇ -adrenoreceptor
  • phosphorylation by protein kmase A and/or specific receptor kinases mediates receptor desensitization.
  • the hgand bmdmg sites of G-protein coupled receptors are believed to comp ⁇ se hydrophihc sockets formed by several G-protem coupled receptor transmembrane domains, said socket being surrounded by hydrophobic residues of the G-protem coupled receptors.
  • the hydrophihc side of each G-protem coupled receptor transmembrane helix is postulated to face inward and form polar hgand bindmg site.
  • TM3 has been implicated m several G-protem coupled receptors as having a hgand bmding site, such as the TM3 aspartate residue.
  • TM6 or TM7 phenylalanmes or tyrosmes are also imphcated in hgand bmdmg.
  • G-protem coupled receptors can be lntracellularly coupled by heterotnme ⁇ c G-protems to vanous mtracellular enzymes, ion channels and transporters (see, Johnson et al., Endoc. Rev., 1989, 10:317-331)
  • Different G-protem ⁇ -subunits preferentially stimulate particular effectors to modulate vanous 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-protem coupled receptors are found in numerous sites within a mammalian host.
  • the present invention relates to AXOR16, in particular AXOR16 polypeptides and AXOR16 polynucleotides, recombmant matenals and methods for their production.
  • Such polypeptides and polynucleotides are of interest in relation to methods of treatment of certain diseases, including, but not limited to, infections such as bactenal, fungal, protozoan and viral infections, particularly infections caused by H ⁇ V-1 or HIV-2; pain; cancers; diabetes, obesity; anorexia; bulimia; asthma; Parkinson's disease; acute heart failure; hypotension; hypertension; u ⁇ nary retention; osteoporosis; ang a pecto ⁇ s; myocardial infarction; stroke; ulcers; asthma; allergies; benign prostatic hypertrophy; migraine; vomiting; psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, depression, deh ⁇ um, dementia, and severe mental retardation; and dyskinesias, such as Huntmgton's disease or Gilles dela
  • the invention relates to methods for identifying agonists and antagonists (e.g , inhibitors) using the mate ⁇ als provided by the invention, and treating conditions associated with AXOR16 imbalance with the identified compounds.
  • the invention relates to diagnostic assays for detecting diseases associated with mapprop ⁇ ate AXOR16 activity or levels. Description of the Invention
  • the present invention relates to AXOR16 polypeptides.
  • Such polypeptides include: (a) an isolated polypeptide encoded by a polynucleotide comprising the sequence of SEQ ID NO: 1 ; (b) an isolated polypeptide comprising a polypeptide sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the polypeptide sequence of SEQ ID NO:2;
  • AXOR16 The biological properties of the AXOR16 are hereinafter referred to as "biological activity of AXOR16" or "AXOR16 activity”.
  • a polypeptide of the present invention exhibits at least one biological activity of AXOR16.
  • Polypeptides of the present invention also mcludes va ⁇ ants of the aforementioned polypeptides, including all allelic 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 in which several, for instance from 50 to 30, from 30 to 20, from 20 to 10, from 10 to 5, from 5 to 3, from 3 to 2, from 2 to 1 or 1 ammo acids are inserted, substituted, or deleted, in any combination.
  • Preferred fragments of polypeptides of the present invention include an isolated polypeptide comprising an ammo acid sequence having at least 30, 50 or 100 contiguous ammo acids from the ammo acid sequence of SEQ ID NO: 2, or an isolated polypeptide comprising an ammo acid sequence having at least 30, 50 or 100 contiguous ammo acids truncated or deleted from the ammo acid sequence of SEQ ID NO. 2.
  • Preferred fragments are biologically active fragments that mediate the biological activity of AXOR16, 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 in a human.
  • Fragments of the polypeptides of the invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, these vanants 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" protein or may be a part of a larger protein such as a precursor or a fusion protein. It is often advantageous to include an additional ammo acid sequence that contains secretory or leader sequences, pro-sequences, sequences that aid in purification, for instance multiple histidme residues, or an additional sequence for stability during recombinant production.
  • Polypeptides of the present invention can be prepared in any suitable manner, for instance by isolation form naturally occurmg sources, from genetically engineered host cells comp ⁇ smg expression systems (vide infra) or by chemical synthesis, using for instance automated peptide synthesisers, or a combination of such methods. Means for preparing such polypeptides are well understood in the art.
  • the present invention relates to AXOR16 polynucleotides.
  • Such polynucleotides include:
  • an isolated polynucleotide comp ⁇ smg a polynucleotide sequence encoding a polypeptide sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the polypeptide sequence of SEQ ID NO:2;
  • Preferred fragments of polynucleotides of the present invention include an isolated polynucleotide comprising an nucleotide sequence having at least 15, 30, 50 or 100 contiguous nucleotides from the sequence of SEQ ED NO: 1 , or an isolated polynucleotide compnsing an sequence having at least 30, 50 or 100 contiguous nucleotides truncated or deleted from the sequence of SEQ ED NO: 1.
  • Preferred va ⁇ ants of polynucleotides of the present invention include splice va ⁇ ants, allelic variants, and polymorphisms, including polynucleotides having one or more single nucleotide polymorphisms (SNPs).
  • SNPs single nucleotide polymorphisms
  • Polynucleotides of the present invention also include polynucleotides encoding polypeptide vanants that comp ⁇ se the ammo acid sequence of SEQ ED NO:2 and in which several, for instance from 50 to 30, from 30 to 20, from 20 to 10, from 10 to 5, from 5 to 3, from 3 to 2, from 2 to 1 or 1 ammo acid residues are substituted, deleted or added, in any combination.
  • the present invention provides polynucleotides that are RNA transcnpts of the DNA sequences of the present invention. Accordingly, there is provided an RNA polynucleotide that:
  • (a) comprises an RNA transcnpt of the DNA sequence encoding the polypeptide of SEQ ID NO:2;
  • (b) is the RNA transcnpt of the DNA sequence encoding the polypeptide of SEQ ED NO:2,
  • (c) comprises an RNA transcnpt of the DNA sequence of SEQ ED NO: 1 ;
  • (d) is the RNA transcnpt of the DNA sequence of SEQ ED NO: 1 ; and RNA polynucleotides that are complementary thereto.
  • the polynucleotide sequence of SEQ D NO: 1 shows homology with Gadus morhua neuropeptide (NPYRB) F.
  • the polynucleotide sequence of SEQ ED NO: 1 is a cDNA sequence that encodes the polypeptide of SEQ ED NO:2.
  • the polynucleotide sequence encoding the polypeptide of SEQ ED NO:2 may be identical to the polypeptide encoding sequence of SEQ ED NO: 1 or it may be a sequence other than SEQ ED NO:l, which, as a result of the redundancy (degeneracy) of the genetic code, also encodes the polypeptide of SEQ ED NO:2.
  • polypeptide of the SEQ ED NO:2 is related to other protems of the G protein coupled receptor 7TM family, having homology and/or structural simila ⁇ ty with Danio reno neuropeptide Y (NPYRYA) [ P. Starback et. al. DNA Cell Biology 16(11), 1357-1363, 1997]
  • NPYRYA Danio reno neuropeptide Y
  • P. Starback et. al. DNA Cell Biology 16(11), 1357-1363, 1997 P. Starback et. al. DNA Cell Biology 16(11), 1357-1363, 1997
  • Preferred polypeptides and polynucleotides of the present invention are expected to have, inter aha, similar biological functions/properties to their homologous polypeptides and polynucleotides.
  • preferred polypeptides and polynucleotides of the present invention have at least one AXOR16 activity.
  • Polynucleotides of the present invention may be obtained using standard cloning and screening techniques from a cDNA library de ⁇ ved from mRNA in cells of human kidney and testis, (see for instance, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spnng 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 readmg frame with other coding sequences, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protein sequence, or other fusion peptide portions.
  • a marker sequence that facilitates punfication 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 Natl Acad Sci USA (1989) 86:821-824, or is an HA tag.
  • the polynucleotide may also contain non-codmg 5' and 3' sequences, such as transc ⁇ bed, 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 hybndization 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 invention and to isolate cDNA and genomic clones of other genes (including genes encoding paralogs from human sources and orthologs and paralogs from species other than human) that have a high sequence 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 pnmers will have between 20 and 25 nucleotides.
  • a polynucleotide encoding a polypeptide of the present invention, mcludmg homologs from species other than human, may be obtained by a process comp ⁇ smg the steps of screening a library under st ⁇ ngent hybndization 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 containmg said polynucleotide sequence
  • hybndization techniques are well known to the skilled artisan.
  • Preferred stnngent hybndization conditions include overnight incubation at 42°C in a solution compnsing: 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 O.lx SSC at about 65°C.
  • the present invention also includes isolated polynucleotides, preferably with a nucleotide sequence of at least 100, obtained by screenmg a library under stnngent hybndization conditions with a labeled probe having the sequence of SEQ ED NO:l or a fragment thereof, preferably of at least 15 nucleotides
  • a labeled probe having the sequence of SEQ ED NO:l or a fragment thereof, preferably of at least 15 nucleotides
  • PCR Nucleic acid amplification
  • Recombinant polypeptides of the present invention may be prepared by processes well known in the art from genetically engineered host cells compnsing expression systems. Accordingly, in a further aspect, the present invention relates to expression systems compnsing a polynucleotide or polynucleotides of the present invention, to host cells which are genetically engineered with such expression sytems and to the production of polypeptides of the invention by recombinant techniques. Cell-free translation systems can also be employed to produce such proteins using RNAs de ⁇ ved from the DNA constructs of the present invention.
  • host cells can be genetically engineered to incorporate expression systems or portions thereof for polynucleotides of the present invention.
  • Polynucleotides may be introduced into host cells by methods descnbed in many standard laboratory manuals, such as Davis et al , Basic Methods m Molecular Biology (1986) and Sambrook et al.( ⁇ b ⁇ d).
  • Preferred methods of introducing polynucleotides mto host cells include, for instance, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, micromjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection.
  • approp ⁇ ate hosts include bactenal cells, such as Streptococci, Staphylococci, E coh, Streptomyces and Bacillus subtihs cells; fungal cells, such as yeast cells and Aspergillus cells; insect cells such as Drosoph ⁇ a S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells; and plant cells.
  • bactenal cells such as Streptococci, Staphylococci, E coh, Streptomyces and Bacillus subtihs cells
  • fungal cells such as yeast cells and Aspergillus cells
  • insect cells such as Drosoph ⁇ a S2 and Spodoptera Sf9 cells
  • animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells
  • a great vanety of expression systems can be used, for instance, chromosomal, episomal and virus-de ⁇ ved systems, e g , vectors denved from bactenal plasmids, from bactenophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccmia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors de ⁇ ved from combinations thereof, such as those denved from plasmid and bactenophage 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 appropnate polynucleotide sequence may be inserted into an expression system by any of a vanety 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 into the desired polypeptide to allow secretion of the translated protem into the lumen of the endoplasmic reticulum, the pe ⁇ plasmic space or the extracellular environment. These signals may be endogenous to the polypeptide or they may be heterologous signals.
  • a polypeptide of the present invention 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 pnor to use in the screening assay If the polypeptide is secreted mto the medium, the medium can be recovered in order to recover and purify 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 punfied from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography Most preferably, high performance liquid chromatography is employed for punfication.
  • Well known techniques for refoldmg protems may be employed to regenerate active conformation when the polypeptide is denatured dunng mtracellular synthesis, isolation and/or punfication.
  • Polynucleotides of the present invention may be used as diagnostic reagents, through detecting mutations in the associated gene. Detection of a mutated form of the gene charactensed by the polynucleotide of SEQ ED NO.1 in the cDNA or genomic sequence and which is associated with a dysfunction will provide a diagnostic tool that can add to, or define, a diagnosis of a disease, or susceptibility to a disease, which results from under-expression, over-expression or altered spatial or temporal expression of the gene. Individuals carrying mutations in the gene may be detected at the DNA level by a vanety of techniques well known in the art.
  • Nucleic acids for diagnosis may be obtained from a subject's cells, such as from blood, urine, saliva, tissue biopsy or autopsy mate ⁇ al.
  • the genomic DNA may be used directly for detection or it may be amplified enzymatically by using PCR, preferably RT-PCR, or other amplification techniques pnor to analysis.
  • RNA or cDNA may also be used in similar fashion. Deletions and insertions can be detected by a change in size of the amplified product m compa ⁇ son to the normal genotype. Pomt mutations can be identified by hybndizing amplified DNA to labeled AXOR16 nucleotide sequences.
  • DNA sequence difference may also be detected by alterations m the electrophoretic mobility of DNA fragments m 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 SI protection or the chemical cleavage method (see Cotton et al, Proc Natl Acad Sci USA (1985) 85: 4397-4401).
  • An array of oligonucleotides probes compnsing AXOR16 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 g ⁇ ds.
  • Array technology methods are well known and have general applicability and can be used to address a vanety of questions m molecular genetics including gene expression, genetic linkage, and genetic vanabihty, see, for example, M.Chee et al., Science, 274, 610-613 (1996) and other references cited therein.
  • Detection of abnormally decreased or mcreased levels of polypeptide or mRNA expression may also be used for diagnosing or determining susceptibility of a subject to a disease of the invention.
  • Decreased or increased expression can be measured at the RNA level using any of the methods well known in the art for the quantitation of polynucleotides, such as, for example, nucleic acid amplification, for instance PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods.
  • Assay techniques that can be used to determine levels of a protein, such as a polypeptide of the present invention, a sample denved from a host are well-known to those of skill in the art.
  • Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays.
  • the present invention relates to a diagonostic kit comprising:
  • a polynucleotide of the present invention preferably the nucleotide sequence of SEQ ED NO: 1, or a fragment or an RNA transcnpt thereof;
  • polypeptide of the present invention preferably the polypeptide of SEQ ED NO:2 or a fragment thereof; or
  • any such kit may comprise a substantial component.
  • Such a kit will be of use in diagnosing a disease or susceptibility to a disease, particularly diseases of the invention, amongst others.
  • the polynucleotide sequences of the present invention are valuable for chromosome localisation studies.
  • the sequence is specifically targeted to, and can hybndize with, a particular location on an individual human chromosome.
  • the mappmg of relevant sequences to chromosomes according to the present invention is an important first step m correlating those sequences with gene associated disease. Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found in, for example, V. McKusick, Mendehan Inhe ⁇ tance m Man (available on-line through Johns
  • RH Mappmg Radiation Hybrid Mappmg (Walter, M. Spillett, D., Thomas, P., Weissenbach, J., and Goodfellow, P., (1994) A method for constructing radiation hybnd maps of whole genomes, Nature Genetics 7, 22-28). A number of RH panels are available from Research Genetics (Huntsville, AL, USA) e.g.
  • 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 comparison is conducted at http://www.genome.wi.mit.edu/.
  • the gene of the present invention maps to human chromosome l lql2.2.
  • the polynucleotide sequences of the present invention are also valuable tools for tissue expression studies. Such studies allow the determination of expression patterns of polynucleotides of the present invention which may give an indication as to the expression patterns of the encoded polypeptides m tissues, by detecting the mRNAs that encode them.
  • the polypeptides of the present invention are expressed m kidney and testis.
  • a fiirther aspect of the present invention relates to antibodies.
  • the polypeptides of the invention or their fragments, or cells expressing them, can be used as l munogens to produce antibodies that are lmmunospecific for polypeptides of the present invention.
  • the term "lmmunospecific" means that the antibodies have substantially greater affinity for the polypeptides of the invention than their affinity for other related polypeptides m the pnor art.
  • Antibodies generated against polypeptides of the present invention may be obtained by administering the polypeptides or epitope-beanng fragments, or cells to an animal, preferably a non- human animal, usmg routine protocols.
  • any technique which provides antibodies produced by continuous cell line cultures can be used.
  • Examples m include the hyb ⁇ doma technique (Kohler, G. and Milstein, C, Nature (1975) 256:495-497), the t ⁇ oma technique, - 13 -
  • transgemc mice or other organisms, including other mammals, may be used to express humanized antibodies.
  • antibodies may be employed to isolate or to identify clones expressing the polypeptide or to punfy the polypeptides by affinity chromatography.
  • Antibodies against polypeptides of the present invention may also be employed to treat diseases of the invention, amongst others
  • the present invention relates to a method for inducing an immunological response in a mammal that comprises inoculating the mammal with a polypeptide of the present invention, adequate to produce antibody and or T cell immune response, including, for example, cytokine-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 m 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 in order to induce such an immunological response to produce antibody to protect said animal from diseases of the invention
  • 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 in order to induce such an immunological response to produce antibody to protect said animal from diseases of the invention
  • One way of admmistenng the vector is by accelerating it mto the desired cells as a coating on particles or otherwise.
  • Such nucleic acid vector may comprise DNA, RNA, a modified nucleic acid, or a DNA/RNA hybnd.
  • a polypeptide or a nucleic acid vector will be normally provided as a vaccine formulation (composition).
  • the formulation may further compnse a suitable earner. Since a polypeptide may be broken down m the stomach, it is preferably administered parenterally (for instance, subcutaneous, intramuscular, intravenous, or intradermal injection).
  • parenterally for instance, subcutaneous, intramuscular, intravenous, or intradermal injection.
  • Formulations suitable for parenteral administration include aqueous and non- aqueous sterile injection solutions that may contain anti-oxidants, buffers, bactenostats and solutes that render the formulation mstonic with the blood of the recipient; and aqueous and non-aqueous sterile suspensions that may include suspending agents or thickening agents.
  • the formulations may be presented m unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-dned condition requinng only the addition of the stenle liquid earner immediately pnor to use.
  • the vaccine formulation may also include adjuvant systems for enhancing the immunogemcity 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.
  • Polypeptides of the present invention have one or more biological functions that are of relevance m one or more disease states, in particular the diseases of the mvention hereinbefore mentioned. It is therefore useful to to identify compounds that stimulate or inhibit the function or level of the polypeptide. Accordingly, m a further aspect, the present invention provides for a method of screening compounds to identify those that stimulate or inhibit the function or level of the polypeptide. Such methods identify agonists or antagonists that may be employed for therapeutic and prophylactic purposes for such diseases of the invention as hereinbefore mentioned. Compounds may be identified from a vanety of sources, for example, cells, cell-free preparations, chemical hbranes, collections of chemical compounds, and natural product mixtures.
  • Such agonists or antagonists so-identified may be natural or modified substrates, hgands, receptors, enzymes, etc., as the case may be, of the polypeptide; a structural or functional mimetic thereof (see Cohgan et al , Current Protocols in Immunology l(2):Chapter 5 (1991)) or a small molecule.
  • Such small molecules preferably have a molecular weight below 2,000 daltons, more preferably between 300 and 1,000 daltons, and most preferably between 400 and 700 daltons. It is preferred that these small molecules are organic molecules
  • the screening method may simply measure the bmdmg of a candidate compound to the polypeptide, or to cells or membranes beanng the polypeptide, or a fusion protein thereof, by means of a label directly or indirectly associated with the candidate compound.
  • the screening method may involve measuring or detecting (qualitatively or quantitatively) the competitive binding of a candidate compound to the polypeptide against a labeled competitor (e.g agonist or antagonist). Further, these screening methods may test whether the candidate compound results in a signal generated by activation or inhibition of the polypeptide, using detection systems appropnate to the cells bearing the polypeptide.
  • Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist by the presence of the candidate compound is observed.
  • the screening methods may simply comprise the steps of mixing a candidate compound with a solution containing a polypeptide of the present invention, to form a mixture, measuring a AXOR16 activity in the mixture, and comparing the AXOR16 activity of the mixture to a control mixture which contains no candidate compound.
  • Polypeptides of the present invention may be employed in conventional low capacity screening methods and also in high-throughput screening (HTS) formats.
  • HTS formats include not only the well-established use of 96- and, more recently, 384-well micotiter plates but also emerging methods such as the nanowell method described by Schullek et al, Anal Biochem., 246, 20-29, (1997). - 15 -
  • Fusion proteins such as those made from Fc portion and AXOR16 polypeptide, as hereinbefore described, can also be used for high-throughput screening assays to identify antagonists for the polypeptide of the present invention (see D. Bennett et al., J Mol Recognition, 8:52-58 (1995); and K. Johanson et al, J Biol Chem, 270(16):9459-9471 (1995)).
  • One screening technique includes the use of cells which express receptors of this invention (for example, transfected CHO cells) m a system which measures extracellular pH or mtracellular calcium changes caused by receptor activation.
  • compounds may be contacted with cells expressing a receptor polypeptide of the present invention.
  • a second messenger response e.g., signal transduction, pH changes, or changes in calcium level, is then measured to determine whether the potential compound activates or inhibits the receptor.
  • Another method mvolves screening for receptor inhibitors by determining inhibition or stimulation of receptor-mediated cAMP and/or adenylate cyclase accumulation.
  • Such a method involves transfecting a eukaryotic cell with the receptor of this invention to express the receptor on the cell surface. The cell is then exposed to potential antagonists in the presence of the receptor of this invention. The amount ofcAMP accumulation is then measured. If the potential antagonist binds the receptor, and thus inhibits receptor binding, the levels of receptor-mediated cAMP, or adenylate cyclase, activity will be reduced or mcreased.
  • polypeptides or antibodies to the polypeptide of the present invention may also be used to configure screening methods for detecting the effect of added compounds on the production of mRNA and polypeptide m cells.
  • an ELISA assay may be constructed for measunng secreted or cell associated levels of polypeptide usmg monoclonal and polyclonal antibodies by standard methods known m the art. This can be used to discover agents that may inhibit or enhance the production of polypeptide (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues.
  • a polypeptide of the present invention may be used to identify membrane bound or soluble receptors, if any, through standard receptor binding techniques known in the art. These include, but are not limited to, hgand bmdmg and crosslmking assays in which the polypeptide is labeled with a radioactive isotope (for instance, 1 ⁇ 1), chemically modified (for instance, biotmylated), or fused to a peptide sequence suitable for detection or purification, and incubated with a source of the putative receptor (cells, cell membranes, cell supernatants, tissue extracts, bodily fluids). Other methods include biophysical techniques such as surface plasmon resonance and spectroscopy. These screening methods may also be used to identify agonists and antagonists of the polypeptide that compete with the binding of the polypeptide to its receptors, if any. Standard methods for conducting such assays are well understood in the art.
  • antagonists of polypeptides of the present invention include antibodies or, in some cases, oligonucleotides or proteins that are closely related to the hgands, substrates, receptors, enzymes, etc., as the case may be, of the polypeptide, eg , a fragment of the hgands, substrates, receptors, enzymes, etc.; or a small molecule that bind to the polypeptide of the present invention but do not elicit a response, so that the activity of the polypeptide is prevented.
  • transgemc technology Screening methods may also involve the use of transgemc technology and AXOR16 gene.
  • the art of constructing transgemc animals is well established.
  • the AXOR16 gene may be introduced through micromjection mto the male pronucleus of fertilized oocytes, retro viral transfer mto pre- or post-implantation embryos, or injection of genetically modified, such as by electroporation, embryonic stem cells into host blastocysts.
  • Particularly useful transgemc animals are so-called "knock-m” animals m which an animal gene is replaced by the human equivalent withm the genome of that animal. Knock-in transgemc animals are useful in the drug discovery process, for target validation, where the compound is specific for the human target.
  • transgemc animals are so-called "knock-out" animals in which the expression of the animal ortholog of a polypeptide of the present invention and encoded by an endogenous DNA sequence in a cell is partially or completely annulled.
  • the gene knock-out may be targeted to specific cells or tissues, may occur only in certain cells or tissues as a consequence of the limitations of the technology, or may occur in all, or substantially all, cells in the animal.
  • Transgemc animal technology also offers a whole animal expression-cloning system in which introduced genes are expressed to give large amounts of polypeptides of the present invention
  • Screening kits for use in the above described methods form a further aspect of the present invention.
  • Such screening kits compnse: (a) a polypeptide of the present invention
  • 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 original environment, or both
  • a polynucleotide or a polypeptide naturally present in a living organism is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is “isolated”, as the term is employed herein
  • a polynucleotide or polypeptide that is introduced mto an organism by transformation, genetic manipulation or by any other recombinant method is "isolated” even if it is still present in said organism, which organism may be living or non-hvmg.
  • Polynucleotide generally refers to any polynbonucleotide (RNA) or polydeoxnbonucleotide (DNA), which may be unmodified or modified RNA or DNA.
  • Polynucleotides include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid 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 triple-stranded regions comprising RNA or
  • polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons.
  • Modified bases include, for example, t ⁇ tylated bases and unusual bases such as lnosme A vanety of modifications may be made to DNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically or metabohcally modified forms of polynucleotides as typically found m nature, as well as the chemical forms of DNA and RNA charactenstic of viruses and cells "Polynucleotide” also embraces relatively short polynucleotides, often referred to as ohgonucleotides.
  • Polypeptide refers to any polypeptide comprising 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 proteins. Polypeptides may contain ammo acids other than the 20 gene-encoded ammo acids. "Polypeptides” include ammo acid sequences modified either by natural - 18 -
  • Modifications may occur anywhere in a polypeptide, including the peptide backbone, the ammo acid side-chams and the ammo or carboxyl termini. It will be appreciated that the same type of modification may be present to the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications.
  • Polypeptides may be branched as a result of ubiquitination, and they may be cyclic, with or without branching Cyclic, 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 flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a hpid or hpid derivative, covalent attachment of phosphotidyhnositol, cross-linking, cychzation, disulfide bond formation, demethylation, formation of covalent cross-lmks, formation of cystine, 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 proteins such as argmylation, and ubiquitination (
  • “Fragment” of a polypeptide sequence refers to a polypeptide sequence that is shorter than the reference sequence but that retains essentially the same biological function or activity as the reference polypeptide
  • “Fragment” of a polynucleotide sequence refers to a polynucloetide sequence that is shorter than the reference sequence of SEQ ID NO: 1.
  • Vanant refers to a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, but retains the essential properties thereof.
  • a typical vanant of a polynucleotide differs m nucleotide sequence from the reference polynucleotide. Changes in the nucleotide sequence of the vanant may or may not alter the ammo acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in ammo acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below.
  • a typical variant of a polypeptide differs in ammo acid sequence from the reference polypeptide. Generally, alterations are limited so that the sequences of the reference polypeptide and the vanant are closely similar overall and, in many regions, identical.
  • a variant and reference polypeptide may differ m ammo acid sequence by one or more substitutions, insertions, deletions in any combination.
  • a substituted or inserted ammo acid residue may or may not be one encoded by the genetic code. Typical conservative substitutions include Gly, Ala; Val, He, Leu; Asp, Glu; Asn, Gin; Ser, Thr; Lys, Arg; and Phe and Tyr.
  • a variant of a polynucleotide or polypeptide may be naturally occurnng such as an allele, or it may be a vanant that is not known to occur naturally.
  • Non-naturally occurring vanants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis.
  • va ⁇ ants are polypeptides having one or more post-translational modifications, for instance glycosylation, phosphorylation, methylation, ADP ⁇ bosylation and the like.
  • Embodiments include methylation of the N-termmal ammo acid, phosphorylations of sennes and threonmes and modification of C-termmal glycmes.
  • Allele refers to one of two or more alternative forms of a gene occunng at a given locus in the genome.
  • Polymorphism refers to a vanation in nucleotide sequence (and encoded polypeptide sequence, if relevant) at a given position in the genome withm a population.
  • SNP Single Nucleotide Polymorphism
  • ASA ASA
  • a common pnmer is used in reverse complement to the polymorphism being assayed.
  • This common primer can be between 50 and 1500 bps from the polymorphic base.
  • the other two (or more) pnmers 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 pnmer and one of the Allele Specific P ⁇ mers.
  • RNA molecules produced from RNA molecules initially transcnbed from the same genomic DNA sequence but which have undergone alternative RNA splicing.
  • Alternative RNA splicing occurs when a primary RNA transcnpt undergoes splicing, generally for the removal of mtrons, which results m the production of more than one mRNA molecule each of that may encode different ammo acid sequences.
  • the term splice vanant also refers to the proteins encoded by the above cDNA molecules.
  • Identity reflects a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, determined by compa ⁇ ng 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.
  • the two sequences to be compared are aligned to give a maximum correlation between the sequences. This may include inserting "gaps" m 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.
  • “similanty” means a comparison between the ammo 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 "% similanty" of the two sequences can then be determined.
  • BESTFLT and GAP may be used to determine the % identity between two polynucleotides and the % identity and the % similanty between two polypeptide sequences.
  • BESTFrf uses the "local homology" algorithm of Smith and Waterman (J Mol Biol, 147,195-197, 1981, Advances in Applied Mathematics, 2, 482- 489, 1981) and finds the best single region of similarity between two sequences.
  • BESTFIT is more suited to companng two polynucleotide or two polypeptide sequences that are dissimilar in length, the program assuming that the shorter sequence represents a portion of the longer.
  • GAP aligns two sequences, finding a "maximum similarity", according to the algonthm of Neddleman and Wunsch (J Mol Biol, 48, 443-453, 1970). GAP is more suited to companng sequences that are approximately the same length and an alignment is expected over the entire length.
  • 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 similanties are determined when the two sequences being compared are optimally aligned.
  • the BLOSUM62 ammo acid substitution matrix (Hemkoff S and Hemkoff J G, Proc. Nat. Acad Sci. USA, 89, 10915-10919, 1992) is used in polypeptide sequence comparisons including where nucleotide sequences are first translated into ammo acid sequences before comparison.
  • the program BESTFIT is used to determine the % identity of a query polynucleotide or a polypeptide sequence with respect to a reference polynucleotide or a polypeptide sequence, the query and the reference sequence being optimally aligned and the parameters of the program set at the default value, as hereinbefore 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 include on average up to five differences per each 100 nucleotides of the reference sequence. Such differences are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion, or insertion.
  • a candidate polypeptide sequence having, for example, an Identity Index of 0.95 compared to a reference polypeptide sequence is identical to the reference sequence except that the polypeptide sequence may include an average of up to five differences per each 100 ammo acids of the reference sequence. Such differences are selected from the group consistmg of at least one ammo acid deletion, substitution, including conservative and non- conservative substitution, or insertion. These differences may occur at the ammo- or carboxy- 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 in one or more contiguous groups withm the reference sequence.
  • an average of up to 5 in every 100 of the ammo acids in the reference sequence may be deleted, substituted or inserted, or any combination thereof, as hereinbefore desc ⁇ bed.
  • n a is the number of nucleotide or ammo acid differences
  • x a is the total number of nucleotides or ammo acids in SEQ ED NO: 1 or SEQ ED NO:2, respectively,
  • I is the Identity Index
  • Homolog is a generic term used in the art to indicate a polynucleotide or polypeptide sequence possessing a high degree of sequence relatedness to a reference sequence. Such relatedness may be quantified by determining the degree of identity and/or similanty between the two sequences as hereinbefore defined. Falling withm this genenc term are the terms "ortholog", and "paralog”.
  • “Ortholog” refers to a polynucleotide or polypeptide that is the functional equivalent of the polynucleotide or polypeptide in another species.
  • “Paralog” refers to a polynucleotideor polypeptide that within the same species which is functionally similar.
  • Fusion protein refers to a protein encoded by two, often unrelated, fused genes or fragments thereof.
  • EP-A-0 464 533-A discloses fusion proteins comprising vanous 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 protein is advantageous for use m therapy and diagnosis resulting m, for example, improved pharmacokmetic - 23 -
  • Example 1 Mammalian Cell Expression
  • the receptors of the present mvention are expressed in either human embryonic kidney 293 (HEK293) cells or adherent dhfr CHO cells.
  • HEK293 human embryonic kidney 293
  • adherent dhfr CHO cells typically all 5' and 3' untranslated regions (UTRs) are removed from the receptor cDNA pnor to insertion mto a pCDN or pCDNA3 vector.
  • the cells are transfected with individual receptor cDNAs by lipofectm 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.
  • Example 2 Ligand bank for bindmg and functional assays.
  • a bank of over 600 putative receptor hgands has been assembled for screening.
  • the bank compnses transmitters, hormones and chemokmes known to act via a human seven transmembrane
  • (7TM) receptor naturally occurring compounds which may be putative agonists for a human 7TM receptor, non-mammalian, biologically active peptides for which a mammalian counterpart has not yet been identified; and compounds not found m nature, but which activate 7TM receptors with unknown natural hgands.
  • This bank is used to initially screen the receptor for known hgands, usmg both functional (i.e . calcium, cAMP, microphysiometer, oocyte electrophysiology, etc, see below) as well as bmdmg assays.
  • Example 3 Ligand Bindmg 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 radiolabelmg 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 bindmg is defined as total associated radioactivity minus the radioactivity measured in the presence of an excess of unlabeled competing ligand. Where possible, more than one competing ligand is used to define residual nonspecific bmdmg.
  • RNA transcnpts from lineanzed plasmid templates encoding the receptor cDNAs of the invention are synthesized in vitro with RNA polymerases m accordance with standard procedures. In vitro transcnpts are suspended in water at a final concentration of 0.2 mg/ml. Ovanan lobes are removed from adult female toads, Stage V defolliculated oocytes are obtained, and RNA transcnpts (10 ng/oocyte) are injected in a 50 nl bolus using a micromjection apparatus. Two electrode voltage clamps are used to measure the currents from individual Xenopus oocytes in response to agonist exposure. Recordings are made in Ca2+ free Barth's medium at room temperature. The Xenopus system can be used to screen known hgands and tissue/cell extracts for activating hgands.
  • Activation of a wide vanety of secondary messenger systems results in extrusion of small amounts of acid from a cell.
  • the acid formed is largely as a result of the increased metabolic activity required to fuel the mtracellular signaling process.
  • the pH changes in 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 mtracellular signaling pathway such as the G-protem coupled receptor of the present invention. - 25 -
  • the 7TM receptor of the invention is also functionally screened (usmg calcium, cAMP, microphysiometer, oocyte electrophysiology, etc., functional screens) agamst tissue extracts to identify natural hgands. Extracts that produce positive functional responses can be sequencially subfractionated until an activating ligand is isolated identified.
  • Example 7 Calcium and cAMP Functional Assays 7TM receptors which are expressed in HEK 293 cells have been shown to be coupled functionally to activation of PLC and calcium mobilization and or cAMP stimulation or inhibition. Basal calcium levels m the HEK 293 cells in receptor-transfected or vector control cells were observed to be in the normal, 100 nM to 200 nM, range. HEK 293 cells expressing recombinant receptors are loaded with fura 2 and m a single day > 150 selected hgands or tissue/cell extracts are evaluated for agonist induced calcium mobilization.
  • HEK 293 cells expressing recombinant 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.

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Abstract

Cette invention, qui a trait à des polypeptides et à des polynucléotides d'AXOR16, concerne également des procédés de production de ces polypeptides par le biais de techniques de recombinaison. Elle concerne également des méthodes d'utilisation des polypeptides et des polynucléotides d'AXOR16 dans des doses diagnostiques.
PCT/US2000/016869 1999-06-21 2000-06-19 Axor16, récepteur couplé à une protéine g WO2000078809A1 (fr)

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WO2002050265A1 (fr) * 2000-12-19 2002-06-27 Takeda Chemical Industries, Ltd. Nouvelles proteines transmembranaires du type recepteur et adn correspondants
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Cited By (20)

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Publication number Priority date Publication date Assignee Title
EP1207201A1 (fr) * 1999-08-27 2002-05-22 Takeda Chemical Industries, Ltd. Proteine recepteur couplee a une proteine g et adn correspondant
EP1207201A4 (fr) * 1999-08-27 2003-03-12 Takeda Chemical Industries Ltd Proteine recepteur couplee a une proteine g et adn correspondant
WO2001087930A3 (fr) * 2000-05-18 2002-08-29 Bayer Ag Regulation du recepteur couple a la proteine g analogue au recepteur de la galanine humaine
WO2001087930A2 (fr) * 2000-05-18 2001-11-22 Bayer Aktiengesellschaft Regulation du recepteur couple a la proteine g analogue au recepteur de la galanine humaine
US6927041B2 (en) 2000-07-06 2005-08-09 Bayer Corporation Human neuropeptide Y-like G protein-coupled receptor
WO2002004518A2 (fr) * 2000-07-06 2002-01-17 Bayer Corporation Recepteur humain couple aux proteines g de type neuropeptide y
WO2002004518A3 (fr) * 2000-07-06 2003-01-09 Bayer Ag Recepteur humain couple aux proteines g de type neuropeptide y
WO2002050265A1 (fr) * 2000-12-19 2002-06-27 Takeda Chemical Industries, Ltd. Nouvelles proteines transmembranaires du type recepteur et adn correspondants
WO2003033536A2 (fr) * 2001-10-19 2003-04-24 Paradigm Therapeutics Limited Recepteur
WO2003033536A3 (fr) * 2001-10-19 2003-07-03 Paradigm Therapeutics Ltd Recepteur
EP1532446A4 (fr) * 2002-04-12 2006-06-07 Schering Corp Ligands des recepteurs couples aux proteine g et methodes
EP1532446A2 (fr) * 2002-04-12 2005-05-25 Schering Corporation Ligands des recepteurs couples aux proteine g et methodes
JP2005528894A (ja) * 2002-04-12 2005-09-29 シェーリング　コーポレイション Ｇタンパク質共役レセプターリガンドおよび方法
WO2003087134A2 (fr) 2002-04-12 2003-10-23 Schering Corporation Ligands des recepteurs couples aux proteine g et methodes
JP2006254916A (ja) * 2002-04-12 2006-09-28 Schering Plough Corp Ｇタンパク質共役レセプターリガンドおよび方法
US7338772B2 (en) 2002-04-12 2008-03-04 Schering Corporation G-protein coupled receptor ligands and methods
US7807390B2 (en) 2002-04-12 2010-10-05 Schering Corporation G-protein coupled receptor ligands and methods
JP2010279373A (ja) * 2002-04-12 2010-12-16 Schering Corp Ｇタンパク質共役レセプターリガンドおよび方法
WO2004022086A1 (fr) * 2002-09-02 2004-03-18 Takeda Pharmaceutical Company Limited Regulateur de secretion d'hormones adrenocorticales
WO2006039256A1 (fr) * 2004-09-29 2006-04-13 Glaxo Group Limited Utilisation d'agonistes du gpr103 pour moduler le comportement alimentaire

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