WO2000060056A1 - Gene edg5 de la souris - Google Patents

Gene edg5 de la souris Download PDF

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Publication number
WO2000060056A1
WO2000060056A1 PCT/US2000/008965 US0008965W WO0060056A1 WO 2000060056 A1 WO2000060056 A1 WO 2000060056A1 US 0008965 W US0008965 W US 0008965W WO 0060056 A1 WO0060056 A1 WO 0060056A1
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polypeptide
identity
seq
subject
sequence
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PCT/US2000/008965
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English (en)
Inventor
Ping Tsui
Nabil Elshourbagy
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Smithkline Beecham Corporation
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Publication of WO2000060056A1 publication Critical patent/WO2000060056A1/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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • This invention relates to newly identified polypeptides and polynucleotides encoding such polypeptides, to their use 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 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 proteins participating m pathways with G-protems or PPG proteins.
  • Some examples of these proteins mclude the GPC receptors, such as those for adrenergic agents and dopamme (Kobilka, B.K., et al., Proc. Natl Acad. Sci, USA, 1987, 84:46-50; Kobilka, B.K, et al.
  • G-protems themselves, effector proteins, e.g., phosphohpase C, adenyl cyclase, and phosphodiesterase, and actuator proteins, e.g., protein kmase A and protein kinase C (Simon, M.I, et al. Science, 1991, 252:802-8).
  • effector proteins e.g., phosphohpase C, adenyl cyclase, and phosphodiesterase
  • actuator proteins e.g., protein kmase A and protein kinase C (Simon, M.I, et al. Science, 1991, 252:802-8).
  • 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
  • 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 The membrane protein gene superfamily of G-protem coupled receptors has been characte ⁇ zed as having seven putative transmembrane domains. The domains are believed to represent transmembrane ⁇ -hehces connected by extracellular or cytoplasmic loops. G-protem coupled receptors mclude 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 dopamine receptors which bind to neuroleptic drugs used for treating psychotic and neurological disorders.
  • members of this family include, but are not limited to, calcitomn, adrenergic, endothelm, cAMP, adenosme, musca ⁇ mc, acetylchohne, serotonm, histamme, thrombm, kin , follicle stimulating hormone, opsins, endothehal differentiation gene-1, rhodopsms, odorant, and cytomegalovirus receptors
  • G-protem coupled receptors have smgle conserved cysteme residues m each of the first two extracellular loops which form disulfide bonds that are believed to stabilize functional protein structure.
  • the 7 transmembrane regions are designated as TM1, TM2, TM3, TM4, TM5, TM6, and TM7.
  • TM3 has been implicated in signal transduction Phosphorylation and hpidation (palmitylation or farnesylation) of 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 ⁇ -adrenoreceptor, phosphorylation by protein kmase A and/or specific receptor kmases mediates receptor desensitization.
  • the ligand binding sites of G-protem coupled receptors are believed to comp ⁇ se hydrophihc sockets formed by several G-protem coupled receptor transmembrane domains, said sockets 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 a polar ligand binding site.
  • TM3 has been implicated in several G-protem coupled receptors as having a ligand binding site, such as the TM3 aspartate residue TM5 se ⁇ nes, a TM6 asparagme and
  • TM6 or TM7 phenylalanmes or tyrosines are also implicated m ligand binding.
  • G-protein coupled receptors can be lntracellularly coupled by heterot ⁇ me ⁇ c G-protems to va ⁇ ous intracellular enzymes, ion channels and transporters (see, Johnson et al, Endoc. Rev, 1989, 10:317- 331)
  • Different G-protein ⁇ -subumts preferentially stimulate particular effectors to modulate vanous biological functions m a cell.
  • Phosphorylation of cytoplasmic residues of G-protem coupled receptors has been identified as an important mechanism for the regulation of G-protein coupling of some G- protem coupled receptors.
  • G-protein coupled receptors are found m numerous sites withm a mammalian host. Over the past 15 years, nearly 350 therapeutic agents targeting 7 transmembrane (7
  • TM TM receptors
  • the present invention relates to mouse EDG5, in particular mouse EDG5 polypeptides and mouse EDG5 polynucleotides, recombinant mate ⁇ als and methods for their production.
  • the invention relates to methods for using such polypeptides and polynucleotides, including the treatment of infections such as bacte ⁇ al, 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; u ⁇ nary retention; osteoporosis; angma pectons; myocardial infarction; stroke; ulcers; asthma; allergies; benign prostatic hypertrophy; migraine; vomiting; psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, depression, de ⁇ um, dementia, and severe mental retardation; and dyskmesias, such as Huntington's disease or Gilles dela Tourett's syndrome, hereinafter
  • the invention relates to methods for identifying agonists and antagonists/ inhibitors using the mate ⁇ als provided by the invention, and treating conditions associated with mouse EDG5 imbalance with the identified compounds.
  • the invention relates to diagnostic assays for detecting diseases associated with inappropriate mouse EDG5 activity or levels.
  • the present invention relates to mouse EDG5 polypeptides.
  • Such peptides mclude isolated polypeptides comprising an amino acid sequence which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least
  • polypeptides include those comprising the ammo acid of SEQ ID NO:2.
  • peptides of the present invention include isolated polypeptides in which the amino acid sequence has at least 70% identity, preferably at least 80% identity, more preferably at least
  • polypeptides include the polypeptide of SEQ ED NO.2
  • polypeptides of the present invention include isolated polypeptides encoded by a polynucleotide comprising the sequence contained in SEQ ID NO. l.
  • Polypeptides of the present invention are believed to be members of the EDG family of polypeptides They are therefore of interest because understanding of the biological activities of EDG5 m mouse would help to understand the biological function of its human counter part, human
  • mouse EDG5 activity or “mouse EDG5 polypeptide activity” or “biological activity of mouse EDG5”
  • antigemc and lmmunogenic activities of said mouse EDG5 polypeptides in particular the antigemc and lmmunogenic activities of the polypeptide of SEQ ED NO:2.
  • a polypeptide of the present invention exhibits at least one biological activity of mouse EDG5
  • 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 fusion protein. It is often advantageous to include an additional ammo acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in pu ⁇ fication such as multiple histidme residues, or an additional sequence for stability du ⁇ ng recombinant production.
  • the present invention also includes include variants of the aforementioned polypeptides, that is polypeptides that vary from the referents by conservative ammo acid substitutions, whereby a residue is substituted by another with like characte ⁇ stics.
  • Typical such substitutions are among Ala, Val, Leu and Be; among Ser and Thr; among the acidic residues Asp and Glu; among Asn and Gin; and among the basic residues Lys and Arg; or aromatic residues Phe and Tyr
  • va ⁇ ants m which several, 5-10, 1-5, 1-3, 1-2 or 1 ammo acids are substituted, deleted, or added in any combination
  • Polypeptides of the present invention can be prepared m any suitable manner. Such polypeptides include isolated naturally occumng polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for prepa ⁇ ng such polypeptides are well understood m the art.
  • the present invention relates to mouse EDG5 polynucleotides
  • Such polynucleotides include isolated polynucleotides comprising a nucleotide sequence encodmg a polypeptide which has at least 70% identity, preferably at least 80% identity, more preferably at least
  • polypeptides which have at least 97% identity are highly preferred, whilst those with at least 98-99% identity are more highly preferred, and those with at least 99% identity are most highly preferred
  • polynucleotides include a polynucleotide comp ⁇ sing the nucleotide sequence contained in SEQ ED NO.l encoding the polypeptide of SEQ ED NO:2.
  • polynucleotides of the present invention include isolated polynucleotides comp ⁇ smg a nucleotide sequence that has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, to a nucleotide sequence encoding a polypeptide of SEQ ED NO:2, over the entire coding region.
  • polynucleotides which have at least 97% identity are highly preferred, whilst those with at least 98-99% identity are more highly preferred, and those with at least 99% identity are most highly preferred.
  • polynucleotides of the present invention mclude isolated polynucleotides comprising a nucleotide sequence which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, to SEQ ED NO.1 over the entire length of SEQ ED NO: 1.
  • polynucleotides which have at least 97% identity are highly preferred, whilst those with at least 98-99% identify are more highly preferred, and those with at least 99% identity are most highly preferred.
  • Such polynucleotides include a polynucleotide comp ⁇ sing the polynucleotide of SEQ ED NO: 1 as well as the polynucleotide of SEQ ED NO: 1.
  • the invention also provides polynucleotides which are complementary to all the above described polynucleotides.
  • the nucleotide sequence of SEQ ED NO: 1 shows homology with rat pH218 gene (U 10699 MacLennan A.J. et al, Mol. Cell. Neurosci. 5. 201-209 (1994).
  • the nucleotide sequence of SEQ ED NO: 1 is a cDNA sequence and comp ⁇ ses a polypeptide encoding sequence (nucleotide 1 to 1056) encoding a polypeptide of 352 ammo acids, the polypeptide of SEQ ED NO.2
  • the nucleotide sequence encoding the polypeptide of SEQ ED NO:2 may be identical to the polypeptide encoding sequence contained in SEQ ED NO:l or it may be a sequence other than the one contained m SEQ ID 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 SEQ ED NO:2 is structurally related to other proteins of the EDG family, having homology and/or structural similanty with rat pH218 (U 10699 MacLennan A. J. et al, Mol. Cell. Neurosci. 5, 201-209 (1994))
  • Preferred polypeptides and polynucleotides of the present invention are expected to have, inter aha, similar biological functions/properties to their homologous polypeptides and polynucleotides Furthermore, preferred polypeptides and polynucleotides of the present invention have at least one mouse EDG5 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 mouse brain and testis, using the expressed sequence tag (EST) analysis (Adams, M.D, et al Science (1991) 252: 1651-1656: Adams, M.D. et al , Nature, (1992) 355:632-634, Adams, M.D, et al , Nature (1995) 377 Supp:3-174) 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.
  • EST expressed sequence tag
  • the polynucleotide may include the coding sequence for the mature polypeptide, by itself; or the coding sequence for the mature polypeptide in reading frame with other coding sequences, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protem sequence, or other fusion peptide portions.
  • a marker sequence which facilitates pu ⁇ fication of the fused polypeptide can be encoded.
  • the marker sequence is a hexa-histidme peptide, as provided in the pQE vector (Qiagen, Inc.) and desc ⁇ bed 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 transc ⁇ bed, non-translated sequences, splicing and polyadenylation signals, ⁇ bosome binding sites and sequences that stabilize mRNA.
  • polypeptide va ⁇ ants which comp ⁇ se the ammo acid sequence of SEQ ED NO:2 and m which several, for instance from 5 to 10, 1 to 5, 1 to 3, 1 to 2 or 1, ammo acid residues are substituted, deleted or added, in any combination.
  • Polynucleotides which are identical or sufficiently identical to a nucleotide sequence contained m 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 (PCR) reaction, 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 homologs and orthologs from species other than mouse) that have a high sequence simila ⁇ ty to SEQ ED NO.1.
  • these nucleotide sequences are 70% identical, preferably 80% identical, more preferably 90% identical, most preferably 95% identical to that of the referent.
  • the probes or p ⁇ mers will generally comp ⁇ se at least 15 nucleotides, preferably, at least 30 nucleotides and may have at least 50 nucleotides. Particularly preferred probes will have between 30 and 50 nucleotides.
  • a polynucleotide encodmg a polypeptide of the present invention, including homologs and orthologs from species other than mouse, may be obtained by a process which comp ⁇ ses the steps of screening an approp ⁇ ate library under st ⁇ ngent hyb ⁇ dization conditions with a labeled probe having the sequence of SEQ ED NO- 1 or a fragment thereof; and isolating full-length cDNA and genomic clones containing said polynucleotide sequence.
  • st ⁇ ngent hyb ⁇ dization conditions include overnight incubation at 42°C m a solution comp ⁇ sing: 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 polynucleotides obtainable by screening an approp ⁇ ate library under st ⁇ ngent hybridization conditions with a labeled probe having the sequence of SEQ ED NO:l or a fragment thereof.
  • the PCR reaction is then repeated using 'nested' primers, that is, p ⁇ mers designed to anneal within the amplified product (typically an adaptor specific p ⁇ mer that anneals further 3' in the adaptor sequence and a gene specific p ⁇ mer that anneals further 5' m the known gene sequence).
  • the products of this reaction can then be analyzed by DNA sequencing and a full-length cDNA constructed either by joining the product directly to the existing cDNA to give a complete sequence, or carrying out a separate full-length PCR using the new sequence information for the design of the 5' primer
  • Recombinant polypeptides of the present invention may be prepared by processes well known in the art from genetically engineered host cells comp ⁇ sing expression systems. Accordingly, m a further aspect, the present invention relates to expression systems which comp ⁇ se a polynucleotide or polynucleotides of the present invention, to host cells which are genetically engineered with such expression systems and to the production of polypeptides of the invention by recombinant techniques Cell-free translation systems can also be employed to produce such proteins using RNAs de ⁇ ved from the DNA constructs of the present invention For recombinant production, host cells can be genetically engineered to incorporate expression systems or portions thereof for polynucleotides of the present invention.
  • Preferred such methods mclude, for instance, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic hpid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection.
  • approp ⁇ ate hosts include bacte ⁇ al cells, such as streptococci, staphylococct, 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
  • 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 plasmids, 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, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors de ⁇ ved from combinations thereof, such as those de ⁇ ved from plasmid 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 which is able to maintain, propagate or express a polynucleotide to produce a polypeptide in a host may be used.
  • the approp ⁇ ate nucleotide sequence may be inserted into an expression system by any of a va ⁇ ety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al, MOLECULAR CLONING, A LABORATORY MANUAL (supra).
  • Approp ⁇ ate secretion signals may be incorporated into the desired polypeptide to allow secretion of the translated protein 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 in screening assays, it is generally preferred that the polypeptide be produced at the surface of the cell. In this event, the cells may be harvested prior to use in the screening assay If the polypeptide is secreted into the medium, the medium can be recovered m 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 pu ⁇ fied from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, a on 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 pu ⁇ fication Well known techniques for refoldmg proteins may be employed to regenerate active conformation when the polypeptide is denatured du ⁇ ng isolation and or pu ⁇ fication.
  • This mvention also relates to the use of polynucleotides of the present invention as diagnostic reagents Detection of a mutated form of the gene characte ⁇ zed by the polynucleotide of SEQ ED NO.1 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 expression of the gene. Individuals carrying mutations in the gene may be detected at the DNA level by a va ⁇ ety of techniques
  • Nucleic acids for diagnosis may be obtained from a subject's cells, such as from blood, u ⁇ ne, saliva, tissue biopsy or autopsy mate ⁇ al.
  • the genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR or other amplification techniques p ⁇ or to analysis.
  • RNA or cDNA may also be used m similar fashion. Deletions and msertions can be detected by a change in size of the amplified product in compa ⁇ son to the normal genotype. Point mutations can be identified by hyb ⁇ dizmg amplified DNA to labeled mouse EDG5 nucleotide sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in melting temperatures. DNA sequence differences may also be detected by alterations in electrophoretic mobility of DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencmg
  • an array of ohgonucleotides probes comp ⁇ sing mouse EDG5 nucleotide sequence or fragments thereof can be constructed to conduct efficient screening of e.g, genetic mutations.
  • the diagnostic assays offer a process for diagnosing or determining a susceptibility to the Diseases through detection of mutation in the mouse EDG5 gene by the methods desc ⁇ bed
  • diseases may be diagnosed by methods comprising determining from a sample derived from a sub j ect an abnormally decreased or increased level of polypeptide or mRNA.
  • 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 mstance 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, m a sample denved 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 diagonostic kit which comp ⁇ ses:
  • a polynucleotide of the present invention preferably the nucleotide sequence of SEQ ID NO: 1, or a fragment thereof ;
  • any such kit, (a), (b), (c) or (d) may comprise a substantial component.
  • a kit will be of use in diagnosing a disease or susceptibility to a disease, particularly infections such as bacte ⁇ al, fungal, protozoan and viral infections, particularly infections caused by HFV-1 or HEV-2; pam; cancers; diabetes, obesity; anorexia; bulimia; asthma; Parkinson's disease: acute heart failure, hypotension; hypertension; u ⁇ nary retention; osteoporosis; angina pectons, 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 dyskmesias, such as Huntmgton's disease or Gilles dela Tourett's syndrome, amongst others.
  • the nucleotide sequences of the present invention are also valuable for chromosome identification.
  • the sequence is specifically targeted to, and can hybridize with, a particular location on an individual human chromosome.
  • the mapping of relevant sequences to chromosomes according to the present invention is an important first step in correlating those sequences with gene associated disease. Once 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, Mendehan Inhe ⁇ tance in Man (available on-line through Johns Hopkins University Welch Medical Library).
  • the relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (comhe ⁇ tance of physically adjacent genes).
  • linkage analysis corhe ⁇ tance of physically adjacent genes.
  • the differences in the cDNA or genomic sequence between affected and unaffected individuals can also be determined. If a mutation is observed in some or all of the affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent of the disease.
  • the polypeptides of the invention or their fragments or analogs thereof, or cells expressing them can also be used as immunogens to produce antibodies lmmunospecific for polypeptides of the present invention.
  • the term "lmmunospecific" means that the antibodies have substantially greater affinity for the polypeptides of the invention than their affinity for other related polypeptides in the p ⁇ or art.
  • Antibodies generated against polypeptides of the present invention may be obtained by administering the polypeptides or epitope-beanng fragments, analogs or cells to an animal, preferably a non-human animal, using routine protocols.
  • an animal preferably a non-human animal
  • any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybndoma technique (Kohler, G.
  • the above-desc ⁇ bed 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 the Diseases, amongst others.
  • the present invention relates to genetically engineered soluble fusion proteins comprising a polypeptide of the present invention, or a fragment thereof, and various portions of the constant regions of heavy or light chains of mrmunoglobulms of various subclasses (IgG, IgM, IgA, IgE) Preferred as an immunoglobulin is the constant part of the heavy chain of human IgG, particularly IgGl , where fusion takes place at the hinge region
  • the Fc part can be removed simply by incorporation of a cleavage sequence which can be cleaved with blood clotting factor Xa
  • this invention relates to processes for the preparation of these fusion proteins by genetic engineering, and to the use thereof for drug screenmg, diagnosis and therapy
  • a further aspect of the invention also relates to polynucleotides encoding such fusion proteins Examples of fusion protein technology can be found in International Patent Application Nos. W094/29458 and W094/22914
  • Another aspect of the invention relates to a method for inducing an immunological response m a mammal which comprises inoculating the mammal with a polypeptide of the present invention, adequate to produce antibody and/or T cell immune response to protect said animal from the Diseases hereinbefore mentioned, amongst others
  • Yet another aspect of the invention relates to a method of inducing immunological response m a mammal which comprises, delivering a polypeptide of the present invention via a vector directing expression of the polynucleotide and coding for the polypeptide in vivo in order to induce such an immunological response to produce antibody to protect said animal from diseases
  • a further aspect of the invention relates to an lmmunological/vaccme formulation (composition) which, when introduced into a mammalian host, induces an immunological response in that mammal to a polypeptide of the present invention wherein the composition comprises a polypeptide or polynucleotide of the present invention
  • the vaccine formulation may further comprise a suitable carrier. Since a polypeptide may be broken down in the stomach, it is preferably administered parenterally (for instance, subcutaneous, intramuscular, intravenous, or mtradermal injection).
  • Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacte ⁇ ostats and solutes which render the formulation lsotomc with the blood of the recipient; and aqueous and non-aqueous sterile suspensions which 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 sterile liquid carrier immediately prior 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 m the art
  • the dosage will depend on the specific activity of the vaccine and can be readily determined by routine expenmentation.
  • Polypeptides of the present invention are responsible for many biological functions, including many disease states, m particular the Diseases hereinbefore mentioned It is therefore desirous to devise screening methods to identify compounds which stimulate or which inhibit the function of the polypeptide Accordingly, m a further aspect, the present invention provides for a method of screenmg compounds to identify those which stimulate or which inhibit the function of the polypeptide.
  • agonists or antagonists may be employed for therapeutic and prophylactic purposes for such Diseases as hereinbefore mentioned
  • Compounds may be identified from a va ⁇ ety of sources, for example, cells, cell-free preparations, chemical hbra ⁇ es. and natural product mixtures.
  • Such agonists, antagonists or inhibitors so-identified may be natural or modified substrates, ligands, receptors, enzymes, etc, as the case may be, of the polypeptide; or may be structural or functional mimetics thereof (see Cohgan et al , Current Protocols in Immunology l(2):Chapter 5 (1991)).
  • the screening method may simply measure the binding of a candidate compound to the polypeptide, or to cells or membranes bearing the polypeptide, or a fusion protein thereof by means of a label directly or indirectly associated with the candidate compound. Alternatively, the screening 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 appropriate to the cells bea ⁇ ng 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.
  • Constitutively active polypeptides may be employed m screening methods for inverse agonists or inhibitors, m the absence of an agonist or inhibitor, by testing whether the candidate compound results in inhibition of activation of the polypeptide.
  • 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 mouse EDG5 activity in the mixture, and comparing the mouse EDG5 activity of the mixture to a standard.
  • Fusion proteins such as those made from Fc portion and mouse EDG5 polypeptide, as hereinbefore descnbed, can also be used for high-throughput screening assays to identify antagonists for the polypeptide of the present invention
  • 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 intracellular calcium changes caused by receptor activation.
  • compounds may be contacted with cells expressing receptor polypeptides 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 involves screening for receptor inhibitors by determining inhibition or stimulation of receptor-mediated cAMP and/or adenylate cyclase accumulation.
  • Such a method involves transfectmg a eukaryotic cell with a 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 of cAMP accumulation is then measured. If the potential antagonist binds the receptor, and thus inhibits receptor binding, the levels of receptor-mediated cAMP, or adenylate cyclase, activity will be reduced or increased
  • Another method for detecting agonists or antagonists for the receptor of the present invention is the yeast based technology as descnbed m U.S. Patent No. 5,482,835
  • polypeptides and antibodies to the polypeptide of the present invention may also be used to configure screening methods for detecting the effect of added compounds on the production of mRNA and polypeptide m cells.
  • an ELISA assay may be constructed for measu ⁇ ng secreted or cell associated levels of polypeptide using monoclonal and polyclonal antibodies by standard methods known in the art. This can be used to discover agents which may inhibit or enhance the production of polypeptide (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues.
  • the polypeptide may be used to identify membrane bound or soluble receptors, if any, through standard receptor binding techniques known in the art.
  • ligand binding and crosshnkmg assays which the polypeptide is labeled with a radioactive isotope (for instance, 1 ⁇ 1), chemically modified (for instance, biotinylated), 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).
  • 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 which compete with the binding of the polypeptide to its receptors, if any Standard methods for conducting such assays are well understood m the art.
  • polypeptide antagonists examples include antibodies or, in some cases, ohgonucleotides or proteins which are closely related to the ligands, substrates, receptors, enzymes, etc , as the case may be, of the polypeptide, e g, a fragment of the ligands, substrates, receptors, enzymes, etc , or small molecules which bind to the polypeptide of the present invention but do not elicit a response, so that the activity of the polypeptide is prevented.
  • the present invention relates to a screening kit for identifying agonists, antagonists, ligands, receptors, substrates, enzymes, etc. for polypeptides of the present invention; or compounds which decrease or enhance the production of such polypeptides, which comprises.
  • kits may comprise a substantial component.
  • polypeptide of the present invention may also be used in a method for the structure-based design of an agonist, antagonist or inhibitor of the polypeptide, by:
  • the present invention provides methods of treating abnormal conditions such as, for instance, infections such as bactenal, fungal, protozoan and viral infections, particularly infections caused by HEV-1 or HEV-2; pam; cancers; diabetes, obesity; anorexia; bulimia; asthma;
  • infections such as bactenal, fungal, protozoan and viral infections, particularly infections caused by HEV-1 or HEV-2; pam; cancers; diabetes, obesity; anorexia; bulimia; asthma;
  • Parkinson's disease acute heart failure; hypotension; hypertension; u ⁇ nary retention; osteoporosis; ang a pectons; 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; and dyskmesias, such as
  • One approach compnses admmistenng to a subject in need thereof an inhibitor compound (antagonist) as heremabove descnbed, optionally m combmation with a pharmaceutically acceptable earner, in an amount effective to inhibit the function of the polypeptide, such as, for example, by blocking the binding of ligands, substrates, receptors, enzymes, etc, or by inhibiting a second signal, and thereby alleviating the abnormal condition.
  • soluble forms of the polypeptides still capable of binding the ligand, substrate, enzymes, receptors, etc. m competition with endogenous polypeptide may be administered.
  • Typical examples of such competitors include fragments of the mouse EDG5 polypeptide
  • expression of the gene encodmg endogenous mouse EDG5 polypeptide can be inhibited using expression blocking techniques.
  • Known such techniques involve the use of antisense sequences, either internally generated or separately administered (see, for example, O'Connor, J Neurochem (1991) 56:560 in Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)).
  • ohgonucleotides which form triple helices with the gene can be supplied (see, for example, Lee et al, Nucleic Acids Res (1979) 3:173; Cooney et al, Science (1988) 241 :456; Dervan et al, Science (1991) 251:1360). These ohgomers can be administered per se or the relevant ohgomers can be expressed in vivo.
  • a polynucleotide of the invention may be engineered for expression in a replication defective retroviral vector, as discussed above.
  • the retroviral expression construct may then be isolated and introduced into a packaging cell transduced with a retroviral plasmid vector containing RNA encoding a polypeptide of the present invention such that the packaging cell now produces infectious viral particles containing the gene of interest.
  • These producer cells may be administered to a subject for engineering cells in vivo and expression of the polypeptide in vivo.
  • Another approach is to administer a therapeutic amount of a polypeptide of the present invention in combmation with a suitable pharmaceutical earner.
  • the present invention provides for pharmaceutical compositions comp ⁇ sing a therapeutically effective amount of a polypeptide, such as the soluble form of a polypeptide of the present invention, agonist/antagonist peptide or small molecule compound, in combination with a pharmaceutically acceptable earner or excipient.
  • a pharmaceutically acceptable earner or excipient include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the invention further relates to pharmaceutical packs and kits comp ⁇ sing one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention Polypeptides and other compounds of the present invention may be employed alone or m conjunction with other compounds, such as therapeutic compounds.
  • composition will be adapted to the route of administration, for instance by a systemic or an oral route.
  • Preferred forms of systemic administration include injection, typically by intravenous injection.
  • Other injection routes such as subcutaneous, intramuscular, or mtrape ⁇ toneal, can be used
  • Alternative means for systemic administration include transmucosal and transdermal administration using penetrants such as bile salts or fusidic acids or other detergents.
  • penetrants such as bile salts or fusidic acids or other detergents.
  • oral administration may also be possible. Administration of these compounds may also be topical and or localized, m the form of salves, pastes, gels, and the like.
  • the dosage range required depends on the choice of peptide or other compounds of the present invention, the route of administration, the nature of the formulation, the nature of the subject's condition, and the judgment of the attending practitioner. Suitable dosages, however, are in the range of 0 1-100 ⁇ g/kg of subject. Wide vanations in the needed dosage, however, are to be expected m view of the vanety of compounds available and the diffenng efficiencies of va ⁇ ous routes of administration For example, oral administration would be expected to require higher dosages than administration by intravenous injection. Vanations m these dosage levels can be adjusted using standard empi ⁇ cal routines for optimization, as is well understood in the art.
  • Polypeptides used in treatment can also be generated endogenously in the subject, m treatment modalities often referred to as "gene therapy" as descnbed above.
  • cells from a subject may be engineered with a polynucleotide, such as a DNA or RNA, to encode a polypeptide ex vivo, and for example, by the use of a retroviral plasmid vector. The cells are then introduced into the subject.
  • polypeptide sequences form a valuable information resource with which to identify further sequences of similar homology. This is most easily facilitated by sto ⁇ ng the sequence in a computer readable medium and then using the stored data to search a sequence database usmg well known searchmg tools, such as GCC.
  • searchmg tools such as GCC.
  • the present invention provides for a computer readable medium having stored thereon a polynucleotide comprising the sequence of SEQ D NO' 1 and/or a polypeptide sequence encoded thereby
  • 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 Fab or other immunoglobulin expression library "Isolated” means altered “by the hand of man” from the natural state. If an “isolated” composition or substance occurs in nature, it has been changed or removed from its original environment, or both.
  • a polynucleotide or a polypeptide naturally present m a living animal 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.
  • Polynucleotide generally refers to any poly ⁇ bonucleotide or polydeox ⁇ bonucleotide, which may be unmodified RNA or DNA 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 t ⁇ ple-stranded regions comprising 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 include, for example, t ⁇ tylated bases and unusual bases such as mosme. A variety 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 characte ⁇ stic of viruses and cells.
  • Polynucleotide also embraces relatively short polynucleotides, often referred to as ohgonucleotides.
  • Polypeptide refers to any peptide or protein 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 referred to as peptides, ohgopeptides or ohgomers, and to longer chains, generally refe ⁇ ed 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 processes, such as post-translational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and m more detailed monographs, as well as in a voluminous research literature. Modifications may occur anywhere in a polypeptide, including the peptide backbone, the ammo acid side-chams and the ammo or carboxyl termini It will be appreciated that the same type of modification may be present to the same or varying degrees at several sites 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, covalent attachment of flavm, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylmositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-lmks, formation of cystme, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, lodmation, methylation, my ⁇ stoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation
  • Changes in the nucleotide sequence of the variant 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 va ⁇ ant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the va ⁇ ant are closely similar overall and, in many regions, identical.
  • a va ⁇ ant and reference polypeptide may differ in ammo acid sequence by one or more substitutions, additions, deletions in any combination.
  • a substituted or inserted amino acid residue may or may not be one encoded by the genetic code.
  • a variant of a polynucleotide or polypeptide may be a naturally occurring such as an allelic va ⁇ ant, or it may be a variant that is not known to occur naturally.
  • Non- naturally occumng variants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis. "Identity,” as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences.
  • identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences.
  • Identity and similarity can be readily calculated by known methods, including but not limited to those descnbed in (Computational Molecular Biology, Lesk, A.M., ed, Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W, ed.
  • Prefe ⁇ ed methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified m publicly available computer programs. Prefe ⁇ ed computer program methods to determine identity and simila ⁇ ty between two sequences include, but are not limited to, the GCG program package (Devereux, J, et al. Nucleic Acids Research 12(1). 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S.F. et al, J Molec. Biol 215 403-410 (1990).
  • the BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S, et al, NCBI NLM NIH Bethesda, MD 20894; Altschul, S, et al , J Mol. Biol 215: 403-410 (1990).
  • the well known Smith Waterman algo ⁇ thm may also be used to determine identity.
  • Preferred parameters for polypeptide sequence comparison include the following:
  • a program useful with these parameters is publicly available as the "gap" program from Genetics Computer Group, Madison WI.
  • the aforementioned parameters are the default parameters for peptide comparisons (along with no penalty for end gaps).
  • Preferred parameters for polynucleotide comparison include the following:
  • Gap Length Penalty 3 Available as: The "gap" program from Genetics Computer Group, Madison WI These are the default parameters for nucleic acid comparisons.
  • a polynucleotide sequence of the present invention may be identical to the reference sequence of SEQ ED NO: 1, that is be 100% identical, or it may include up to a certain integer number of nucleotide alterations as compared to the reference sequence.
  • Such alterations are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion, or insertion, and wherein said alterations may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleotides in the reference sequence or m one or more contiguous groups withm the reference sequence.
  • the number of nucleotide alterations is determined by multiplying the total number of nucleotides m SEQ ED NO: 1 by the numerical percent of the respective percent ⁇ dent ⁇ ty(d ⁇ v ⁇ ded by 100) and subtracting that product from said total number of nucleotides in SEQ ED NO.1, or n n ⁇ x n - (x n • y), wherein n n is the number of nucleotide alterations, x n is the total number of nucleotides in SEQ ED NO: l, and y is, for instance, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%,etc, and wherein any non-mteger product of x n and y is rounded down to the nearest integer prior to subtracting it from x n Alterations of a polynucleotide sequence encoding the polypeptide of SEQ ED NO.2 may create nonsense, missense
  • a polypeptide sequence of the present invention may be identical to the reference sequence of SEQ ED NO:2, that is be 100% identical, or it may mclude up to a certain integer number of ammo acid alterations as compared to the reference sequence such that the % identity is less than 100%.
  • Such alterations are selected from the group consisting of at least one ammo acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the amino- or carboxy-termmal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the ammo acids in the reference sequence or in one or more contiguous groups within the reference sequence
  • the number of amino acid alterations for a given % identity is determined by multiplying the total number of ammo acids m SEQ ID NO.2 by the numerical percent of the respective percent ⁇ dent ⁇ ty(d ⁇ v ⁇ ded by 100) and then subtracting that product from said total number of ammo acids m SEQ ED NO:2, or
  • n a is the number of ammo acid alterations
  • x a is the total number of ammo acids in SEQ ID NO.2
  • y is, for instance 0.70 for 70%, 0 80 for 80%, 0 85 for 85% etc, and wherein any non- mteger product of x a and y is rounded down to the nearest integer prior to subtracting it from x a .
  • Fusion protein refers to a protein encoded by two, often unrelated, fused genes or fragments thereof.
  • EP-A-0 464 discloses fusion proteins comprising various portions of constant region of immunoglobulin molecules together with another human protein or part thereof.
  • employing an immunoglobulin Fc region as a part of a fusion protein is advantageous for use in therapy and diagnosis resulting in, for example, improved pharmacokmetic properties [see, e.g, EP-A 0232 262].
  • 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 p ⁇ or to insertion into a pCDN or pCDNA3 vector.
  • the cells are transfected with individual receptor cDNAs by lipofectin 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.
  • a bank of over 200 putative receptor ligands has been assembled for screening
  • the bank compnses: transmitters, hormones and chemokmes known to act via a human seven transmembrane (7TM) receptor; naturally occumng 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 ligands.
  • This bank is used to initially screen the receptor for known ligands, using both functional (i.e calcium, cAMP, microphysiometer, oocyte electrophysiology, etc, see below) as well as binding assays
  • Example 3 Ligand Binding Assays
  • Ligand binding assays provide a direct method for ascertaining receptor pharmacology and are adaptable to a high throughput format.
  • the pu ⁇ fied ligand for a receptor is radiolabeled to high specific activity (50-2000 Ci/mmol) for binding studies.
  • a determination is then made that the process of radiolabe ng 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 binding 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 binding.
  • RNA transc ⁇ pts from lmea ⁇ zed plasmid templates encoding the receptor cDNAs of the invention are synthesized in vitro with RNA polymerases in accordance with standard procedures.
  • In vitro transcnpts are suspended in water at a final concentration of 0.2 mg/ml. Ova ⁇ an lobes are removed from adult female toads, Stage V defolhculated oocytes are obtained, and RNA transcnpts (10 ng/oocyte) are injected in a 50 nl bolus using a microinjection 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 Microphysiomet ⁇ c Assays
  • Activation of a wide va ⁇ ety of secondary messenger systems results in extrusion of small amounts of acid from a cell.
  • the acid formed is largely as a result of the increased metabolic activity required to fuel the intracellular signaling process.
  • the pH changes in the media surrounding the cell are very small but are detectable by the CYTOSENSOR microphysiometer (Molecular Devices Ltd, Menlo Park, CA).
  • the CYTOSENSOR is thus capable of detecting the activation of a receptor which is coupled to an energy utilizing intracellular signaling pathway such as the G-protem coupled receptor of the present invention.
  • the 7TM receptor of the invention is also functionally screened (using calcium, cAMP, microphysiometer, oocyte electrophysiology, etc, functional screens) agamst tissue extracts to identify natural ligands Extracts that produce positive functional responses can be sequentially subfractionated until an activating ligand is isolated and identified
  • Basal calcium levels in 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 recombinant receptors are loaded with fura 2 and m a single day more than 150 selected ligands or tissue/cell extracts are evaluated for agonist induced calcium mobilization Similarly, HEK 293 cells expressing recombinant receptors are evaluated for the stimulation or inhibition of cAMP production using standard cAMP quantitation assays Agonists presenting a calcium transient or cAMP fluctuation are tested in vector control cells to determine if the response is unique to the transfected cells expressing receptor

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Abstract

L'invention concerne les polypeptides et polynucléotides EDG5 de la souris, et des procédés utilisant des techniques recombinantes pour produire lesdits polypeptides. L'invention concerne en outre des procédés mettant en oeuvre les polypeptides et polynucléotides EDG5 de la souris dans des thérapies, et des méthodes diagnostiques aux fins desdites thérapies.
PCT/US2000/008965 1999-04-05 2000-04-05 Gene edg5 de la souris WO2000060056A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1071708A1 (fr) * 1998-04-23 2001-01-31 Smithkline Beecham Corporation Gene de la famille edg, polypeptides humains h218
WO2003051395A2 (fr) * 2001-11-30 2003-06-26 Solvay Pharmaceuticals Gmbh Utilisation d'agonistes de recepteur edg pour le traitement de l'hypertension
WO2004058149A2 (fr) 2002-12-20 2004-07-15 Merck & Co., Inc. 1-(amino)indanes et (1,2-dihydro-3-amino)-benzofuranes, benzothiophenes et indoles utilises en tant qu'agonistes du recepteur edg

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5585476A (en) * 1994-02-15 1996-12-17 Maclennan; Alexander J. Molecular cloning and expression of G-protein coupled receptors

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5585476A (en) * 1994-02-15 1996-12-17 Maclennan; Alexander J. Molecular cloning and expression of G-protein coupled receptors

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
OKAZAKI ET. AL.: "Molecular cloning of a novel putative G protein coupled receptor expressed in the cardiovascular system", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATION, vol. 190, no. 3, 15 February 1993 (1993-02-15), pages 1104 - 1109, XP002929595 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1071708A1 (fr) * 1998-04-23 2001-01-31 Smithkline Beecham Corporation Gene de la famille edg, polypeptides humains h218
EP1071708A4 (fr) * 1998-04-23 2005-01-19 Smithkline Beecham Corp Gene de la famille edg, polypeptides humains h218
WO2003051395A2 (fr) * 2001-11-30 2003-06-26 Solvay Pharmaceuticals Gmbh Utilisation d'agonistes de recepteur edg pour le traitement de l'hypertension
WO2003051395A3 (fr) * 2001-11-30 2004-03-11 Solvay Pharm Gmbh Utilisation d'agonistes de recepteur edg pour le traitement de l'hypertension
WO2004058149A2 (fr) 2002-12-20 2004-07-15 Merck & Co., Inc. 1-(amino)indanes et (1,2-dihydro-3-amino)-benzofuranes, benzothiophenes et indoles utilises en tant qu'agonistes du recepteur edg

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