WO2003078466A1 - Recepteurs couples a la proteine g et genes correspondants - Google Patents

Recepteurs couples a la proteine g et genes correspondants Download PDF

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Publication number
WO2003078466A1
WO2003078466A1 PCT/JP2003/003042 JP0303042W WO03078466A1 WO 2003078466 A1 WO2003078466 A1 WO 2003078466A1 JP 0303042 W JP0303042 W JP 0303042W WO 03078466 A1 WO03078466 A1 WO 03078466A1
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Prior art keywords
polypeptide
protein
ligand
activity
nucleic acid
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PCT/JP2003/003042
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English (en)
Japanese (ja)
Inventor
Tetsuo Onuki
Yutaka Koguchi
Emiko Hosoi
Aiko Chikada
Takeshi Hosoi
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Tanabe Seiyaku Co., Ltd.
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Priority to AU2003220913A priority Critical patent/AU2003220913A1/en
Priority to JP2003576470A priority patent/JP3949111B2/ja
Publication of WO2003078466A1 publication Critical patent/WO2003078466A1/fr
Priority to US10/943,848 priority patent/US7527935B2/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4719—G-proteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
    • G01N2333/726—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the present invention relates to a novel receptor (eicosanoid receptor) protein and a gene thereof.
  • the present invention also relates to a method for screening drug candidate compounds using the same. Background art
  • G protein-binding protein a trimeric GTP-binding protein present in cells, and transmit information into cells through activation. It has been known. For this reason, these receptors are collectively referred to as G protein-coupled receptors. It is known that all G protein-coupled receptors have a common structure containing seven transmembrane regions.
  • G protein is a trimer composed of ⁇ ,] 3 and ⁇ subunits, and exists in an inactive form (GDP-bound form) in which these subunits are associated in the basic state.
  • An inactive (GDP-bound) G protein is converted into an active (GTP-bound) G protein-coupled receptor stimulated by a ligand, and the ⁇ subunit (Gc and J3 / Y It dissociates into the subunit complex (G] 3o) and controls signal effectors such as GTP-bound Ga (and in some cases G / 3 ⁇ /), adenylate cyclase, and phospholipase C Convey the file.
  • G proteins are diverse and control different effectors depending on the type of Ga.
  • G protein-coupled receptors recognize specific types of G proteins. Activates and becomes more specific Transmit information into cells.
  • G protein-coupled receptors include hitherto and / 3-adrenergic receptor, muscarinic acetylcholine receptor, adenosine receptor, angiotensin receptor, endothelin receptor, gonadotropin releasing factor receptor, HI — And H 2 —histamine receptor, dopamine receptor, metabotropic glutamate receptor, somatostatin receptor, and the like are known.
  • G protein-coupled receptors are attracting attention as targets for drug development. Finding new G protein-coupled receptors, identifying their ligands, and finding ways to screen or identify their agonists and antagonists are all about screening new drug candidates. Is strongly desired.
  • Eicosanoides include prostaglandins, prostacyclin, thromboxanthin, leukotriene, and eicosatetraenoic acids such as 5-oxo-ETE and 5-H ETE.
  • Receptors with eicosanoide as a ligand so far include prostaglandins EP1, EP2, EP3, EP4, F2 receptor, prostacyclin PI2 receptor, thromboxanthin TA2 receptor, leukotriene B4 receptor which are all G protein-coupled receptors (Narumiya et al., Physiol. Rev., Vol. 79, pp.
  • An object of the present invention is to provide a novel G protein-coupled receptor and its gene. It is to be. More specifically, it is to provide a novel eicosanoid receptor and its gene.
  • Another object of the present invention is to provide a method for screening, identifying, and characterizing a ligand and an agonist (agonist or antagonist) for the receptor protein.
  • the present inventors have isolated a full-length cDNA encoding a novel G protein-coupled receptor from humans. We have also succeeded in expressing this receptor protein in cells by genetic recombination technology. Further, a ligand for this receptor was identified, and this receptor was found to be an eicosanoide receptor, and the present invention was completed.
  • the present invention relates to a polypeptide selected from the following (A), (B) and (C), which has a function or activity as an eicosanoid receptor.
  • (C) A polypeptide encoded by a nucleic acid that can hybridize under stringent conditions with a nucleic acid consisting of the nucleotide sequence represented by SEQ ID NO: 1 or 20, or a complement thereof.
  • the present invention also relates to a nucleic acid encoding the polypeptide.
  • the present invention also relates to a recombinant vector containing the nucleic acid and a host cell.
  • the present invention also relates to a method for detecting the function or activity of the polypeptide using the polypeptide. Further, the present invention provides a method for using the It also relates to a method of modulating (enhancing or suppressing) the activity or activity.
  • the present invention also relates to a method for screening or identifying a ligand or an agonist (agonist or antagonist) of the polypeptide using the same.
  • FIG. 1 is a diagram showing the amino acid sequence and base sequence of TG 1019 and a putative seven transmembrane region (underlined).
  • FIG. 2 is a diagram showing the distribution of the expression of the TG1019 gene in each human tissue or cell (the results of the dot blot).
  • FIG. 3 is a schematic diagram showing the outline of the structure of a fusion protein of the TG1019 protein and various G proteins.
  • Figure 4 A is a variety of test substances on specific binding to the membrane fraction and GTP y S containing fusion proteins with TG 101 9 protein and various G proteins when using 5 Okiso one ETE as test compound It is a figure showing the influence.
  • the specific binding amount is expressed as a relative value ( 0 /. Of the control) with respect to the binding amount at the time when the test substance was not added with the control.
  • “ ⁇ Gial (351Cys ⁇ lie)” shows the test results for the membrane fraction containing the fusion protein of TG1019 protein and Gial (351Cys ⁇ Ile).
  • ⁇ G qa indicates the test result in the membrane fraction containing the fusion protein of TGI019 protein and G qa .
  • “Mouth G saL indicates the test result in the membrane fraction containing the fusion protein of TG 1019 protein and G saL .
  • FIG. 4B shows the effect of various test substances on the specific binding of GTP ⁇ S to the membrane fraction containing the fusion protein of TG 1019 protein and various G proteins when 5-HP ETE was used as the test compound.
  • FIG. The specific binding amount is expressed as a relative value ( 0 /. Of the control) relative to the binding amount at the time when no test substance was added as a control.
  • “ ⁇ Gial (351Cys ⁇ lie)” is a fusion protein of TG1019 protein and Gial (351Cys ⁇ Ile).
  • 3 shows the test results for the membrane fraction containing the protein.
  • “ ⁇ G qa ” indicates the test result in the membrane fraction containing the fusion protein of TGI019 protein and G qa .
  • “Mouth G saL indicates the test result in the membrane fraction containing the fusion protein of TG 1019 protein and G saL .
  • Figure 4C shows the effect of various test substances on specific binding to the membrane fraction and the GTP gamma S containing fusion proteins with TG 1019 protein and various G proteins when using Arakidon acid as a test compound
  • the specific binding amount is expressed as a relative value ( 0 in the control ) relative to the binding amount at the time when no test substance was added as a control.
  • ⁇ Gial (351Cys ⁇ lie) shows the test results for the membrane fraction containing the fusion protein of TG1019 protein and Gial (351Cys ⁇ Ile).
  • ⁇ G qa indicates the test result in the membrane fraction containing the fusion protein of TGI019 protein and G qa .
  • “Mouth G saL indicates the test result in the membrane fraction containing the fusion protein of TG 1019 protein and G saL .
  • FIG. 4D is specific to the membrane fraction and GTP gamma S containing fusion proteins with TG 1019 protein and various G proteins when using eicosa an 5 Z- 8 Z-1 1 Z- Toryen acid as a test compound
  • FIG. 4 is a diagram showing the effect of various test substances on the amount of binding.
  • the specific binding amount is expressed as a relative value ( 0 /. Of the control) relative to the binding amount at the time when no test substance was added as a control.
  • “ ⁇ Gial (351Cys ⁇ Ile)” indicates the test results for the membrane fraction containing the fusion protein of TG11019 protein and G; a (351Cys ⁇ Ile).
  • ⁇ G qa indicates the test result in the membrane fraction containing the fusion protein of TGI 019 protein and G qa .
  • “Mouth GsaL ” indicates the test results for the membrane fraction containing the fusion protein of TGI019 protein and GsaL .
  • Figure 4E the various test substances on specific binding to the membrane fraction and the GTP gamma S containing fusion proteins with TG 1019 protein and various G proteins when using the 5-HET r E as a test compound It is a figure showing the influence.
  • the specific binding amount is defined as the control when no test substance is added, Expressed as a relative value ( 0 /. Of control).
  • ⁇ Gial (351Cys ⁇ lie) shows the test results for the membrane fraction containing the fusion protein of TG1019 protein and Gial (351Cys ⁇ Ile).
  • ⁇ G qa indicates the test result in the membrane fraction containing the fusion protein of TG1019 protein and G qa .
  • Mcouth G saL indicates the test result in the membrane fraction containing the fusion protein of TG 1019 protein and G saL .
  • FIG. 3 is a diagram showing the influence of a test substance.
  • the specific binding amount is expressed as a relative value (% of control) with respect to the binding amount at the time when no test substance was added as a control.
  • ⁇ Gial (351Cys ⁇ Ile) indicates the test results for the membrane fraction containing the fusion protein of TG1019 protein and Gial (351Cys ⁇ Ile).
  • ⁇ G qa indicates the test result in the membrane fraction containing the fusion protein of TG 1019 protein and G qa .
  • Mcouth GsaL indicates the test result in the membrane fraction containing the fusion protein of TG1019 protein and GsaL .
  • Figure 4G shows the effect of various test substances on the specific binding of GTP ⁇ S to the membrane fraction containing the fusion protein of TG 1019 protein and various G proteins when 5-HTE was used as the test compound.
  • FIG. The specific binding amount is expressed as a relative value ( 0 /. Of the control) relative to the binding amount at the time when no test substance was added as a control.
  • " ⁇ G i al (351Cys ⁇ lie)" shows the test results in the membrane fraction containing the fusion protein with TG 1019 protein G ial (351Cys ⁇ Ile).
  • ⁇ G qa indicates the test result in the membrane fraction containing the fusion protein of TG1019 protein and G qa .
  • “Mouth GsaL ” indicates the test result in the membrane fraction containing the fusion protein of TGI019 protein and GsaL .
  • FIG. 4H shows the fusion protein of TG 1019 protein and various G proteins when 5, 8, 11-eicosatriic acid (ETI) was used as a test compound.
  • FIG. 4 is a graph showing the effect of various test substances on the specific binding amount between a membrane fraction containing a protein and GTP ⁇ S. The specific binding amount is expressed as a relative value ( 0 /. Of the control) relative to the binding amount at the time when no test substance was added as a control.
  • ⁇ G i al (351Cys ⁇ Ile) shows the test results in the membrane fraction containing the fusion protein with TG 1 0 1 9 protein G, a j (351Cys ⁇ Ile ).
  • SEQ ID NO: 1 in the sequence listing below is a human-derived cDNA (including the entire translation region) of the receptor protein (referred to as TG1019 protein) gene (referred to as TG1019 gene) isolated by the inventors.
  • TG1019 protein a human-derived cDNA (including the entire translation region) of the receptor protein (referred to as TG1019 protein) gene (referred to as TG1019 gene) isolated by the inventors.
  • Represents the base sequence of SEQ ID NO: 2 represents an amino acid sequence of a receptor protein (TG1019 protein) encoded by the cDNA.
  • the receptor protein of the present invention is a G protein-coupled receptor protein, and has a function (biological activity) as an eicosanoid receptor. Therefore, the receptor protein of the present invention specifically binds to eicosanoid. The specific binding of eicosanoide to the receptor protein of the present invention stimulates the receptor protein and induces intracellular signal transduction.
  • Eicosanoid which is a ligand of the receptor protein of the present invention, refers to a substance derived from a fatty acid having 20 carbon atoms, such as eicosanoic acid, and is preferably an unsaturated fatty acid at the 8-position.
  • 5-oxo-6E, 8Z, 11Z, 14Z-eicosatetraenoic acid (5-oxo-6E, 8Z, llZ, 14Z-eicosatetraenoic acid, 5-abbreviated as 5-oxo-ETE)
  • 5—Hydroperoxyeicosa-6E, 8Z, 11Z, 14Z—Tetraenoic acid (5-hydroperoxyeicosa-6E, 8Z, 11Z, 14Z-tetraenoic acid, 5—HPETE)
  • Arachidonic acid arachidonic acid or eicosa-5Z, 8Z, llZ, 14Z-tetraenoic acid
  • eicosa-5Z, 8 Z, 11Z-trienoic acid eicosa-5Z, 8Z, 11Z-trienoic acid
  • 5-hydroxyeicosa 6E, 8Z, 11Z-trienoic acid (5-hydroxyeicosa-eE'SZ'llZ-te
  • 5-oxo-ETE is synthesized from arachidonic acid via 5-HETE and is known to be a potent chemoattractant for eosinophils and neutrophils (Schwenk et al., J. Biol. Chem., Vol. 270, pp. 15029-15036, 1995, Guilbert et al., Am. J. Respir. Cell Mol. Biol., Vol. 21, pp. 97-104, 1999). Also, 5-oxo-ETE is known to induce eosinophil migration at the animal level (Stamatiou et al., J. Clin. Invest., Vol. 102, pp. 2165-2172, 1998. ).
  • the antagonist of the novel G protein-coupled receptor of the present invention may be useful as a therapeutic agent for allergy and inflammation such as asthma in which antioxidant is involved.
  • Arachidonic acid is known to be an intermediate in the biosynthesis of prostaglandins and leukotrienes
  • 5-HPETE is known to be an intermediate in the biosynthesis of arachidonic acid to leukotriene.
  • the antagonist of the novel G protein-coupled receptor of the present invention may be an anticancer agent against the above-mentioned cancer.
  • the receptor protein of the present invention activates a G protein belonging to the Gi subfamily when stimulated by a ligand (agonist). alpha Focusing on Sabuyu knit, receptor proteins of the present invention, the ligand ( ⁇ Gore two strike) by (alpha subunit of G proteins belonging to the G i subfamily one, for example, G ial) G ia when stimulated activity To a modified form (with GTP binding ability). Then, a signal can be transmitted in a cell through activation of the G protein.
  • the receptor protein (polypeptide) of the present invention has the following functions (i) to (iii).
  • G protein ⁇ subunit of G protein belonging to Gi subfamily
  • ligand ligand acting as agonist
  • the ligands include, for example, 5-oxo-, 5--, arachidonic acid, eicosa-5-, 8-, 11-triene, 5- Eicosanoids such as HET r ⁇ , eicosa-5 ⁇ , 8 ⁇ -genic acid, 5- ⁇ , and 5,8,11-eicosatriic acid, among which, among others, 5-oxo-one A preferred example is given.
  • ligand refers to a compound that can specifically bind to a receptor protein.
  • Ligands include both naturally occurring and artificially synthesized compounds.
  • agonist means a compound capable of inducing intracellular signal transduction by stimulating the receptor protein by specifically binding to the receptor protein.
  • antagonist means a compound capable of suppressing the action of a compound capable of inducing intracellular signal transduction by stimulating a receptor protein.
  • the protein or polypeptide of the present invention includes a protein or polypeptide comprising an amino acid sequence represented by SEQ ID NO: 2.
  • an amino acid sequence in which one or more amino acids are deleted, substituted, or added may be used. Deletion, substitution or addition of amino acids can function as an eicosanoid receptor (biological activity; biological activity). As long as activity is not lost, it is usually 1 to about 80, preferably 1 to about 60, more preferably 1 to about 45, and still more preferably 1 to about 30, Still more preferably, from 1 to about 15.
  • polypeptide of the present invention in addition to the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2, as well as one or more polypeptides comprising the amino acid sequence represented by SEQ ID NO: 2 Polypeptides with conservative amino acid substitutions.
  • Such proteins or polypeptides include mutant proteins or polypeptides found in nature, artificially modified mutant proteins or polypeptides, proteins or polypeptides derived from heterologous organisms, etc. It is.
  • proteins or polypeptides include conservative substitution variants of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 and naturally occurring mutants (naturally occurmg allelic variants). )
  • Such a protein or polypeptide is usually composed of about 75% or more, preferably about 80% or more, more preferably about 85% or more, more preferably about 9% or more, of the amino acid sequence represented by SEQ ID NO: 2. It has a homology of 0% or more, even more preferably about 95% or more of amino acids.
  • nucleic acid (DNA or RNA) of the present invention examples include those containing a nucleic acid having a base sequence represented by SEQ ID NO: 1. Further, a nucleic acid consisting of the base sequence represented by SEQ ID NO: 1 and a nucleic acid capable of hybridizing under stringent conditions (more preferably, under high stringent conditions) or a complement thereof (ie, a nucleic acid having a complementary sequence) are used. Including. Such a hybridizable nucleic acid may be any as long as it encodes a protein or a polypeptide having a function (biological activity) as an eicosanoid receptor.
  • Such a nucleic acid has a nucleotide sequence represented by SEQ ID NO: 1, usually about 70% or more, It preferably has a homology of about 80% or more, more preferably about 85% or more, still more preferably 90% or more, and even more preferably 95% or more.
  • genes or nucleic acids include mutant genes found in nature, artificially modified mutant genes, and homologous genes (orthologs) derived from heterologous organisms.
  • the hybridization under stringent conditions is usually a temperature of 50 to 60 ° C. in a hybridization solution having a salt concentration of 6 XSSC or equivalent. Under conditions, hybridize for about 16 hours, pre-wash as necessary with 6 X SSC or a solution with equivalent salt concentration, and in a solution with 1 X SSC or equivalent salt concentration. Washing.
  • the condition having a higher stringency means that the washing is performed in a solution having a concentration of 0.1 X SSC or a salt concentration equivalent thereto.
  • Sequence homology can be analyzed using a conventional method such as the blast method (Blast Method, Altschul et al., J. Mol. Biol., No. 215, pp. 403-410, 1990).
  • the nucleic acid of the present invention can be isolated and obtained by performing screening using mammalian tissues or cells as a gene source.
  • Mammals include non-human animals such as dogs, lions, pomas, goats, sheep, monkeys, pigs, puppies, rats and mice, as well as humans. Of these, human-derived drugs are desirable for use in research and development of human therapeutics.
  • the nucleic acid of the present invention can be obtained by using the sequence information disclosed in the present specification (SEQ ID NO: 1 in the sequence listing below).
  • primers and probes are designed based on the disclosed nucleotide sequence information, and the PCR (polymerase chain reaction) method, colony hybridization method, and plaque hybridization method using these primers are appropriately combined. You can select and obtain from a DNA library.
  • a cDNA fragment is obtained by the PCR method.
  • a cDNA library is screened by a colony hybridization method or a plaque hybridization method, and a full-length cDNA can be obtained.
  • genomic genes can be isolated by screening a genomic DNA library. Further, by screening a DNA library of another mammal, a homologous gene (ortholog) derived from a heterologous organism can be isolated.
  • DNA libraries such as cDNA libraries and genomic DNA libraries are, for example, L: "Molecular Cloning J (Sambrook, J., Fritsch, EF and Maniatis, T., Cold Spring Harbor Laboratory Press, 1989 Can be prepared by the method described in (1), or a commercially available library may be used.
  • the translation region encoding the protein of the gene product can be determined, and the amino acid sequence of this protein can be obtained.
  • the protein or polypeptide of the present invention can be produced by overexpression by a conventional gene recombination technique. It can also be expressed and produced in the form of a fusion protein with other proteins or peptides.
  • Cells overexpressing the protein or polypeptide of the present invention can be obtained, for example, as follows. First, an expression vector is constructed by inserting a DNA encoding the protein or polypeptide of the present invention into a vector in a form ligated downstream of an appropriate promoter. Next, the obtained expression vector is introduced into a host cell.
  • Examples of the expression system include expression systems for bacteria, yeast, insect cells, and mammalian cells.
  • insect cells Spodoptera frugiperda SF9, SF21, etc.
  • mammalian cells Monkey COS-7 cells, Chinese hamster CH) O cells, human HeLa cells, etc.
  • promoters for expressing the protein or polypeptide of the present invention include SV40 promoter, LTR promoter, elongation ⁇ ⁇ ⁇ promoter, etc. in mammalian cell systems, and polyhedrin promoter in insect cell systems. One or the like can be used.
  • the vector one used in this expression system in the case of mammalian cell lines, retroviral Uinoresu based vectors, Nono 0 pyromellitic one Maui Roh-less vector, vaccinia ⁇ Ino-less base click terpolymers, SV 4 0 based vectors and the like, insect cell systems
  • a paculovirus vector or the like can be used.
  • a cDNA for example, one consisting of the base sequence shown in SEQ ID NO: 1
  • a DNA corresponding to the amino acid sequence of the target protein 1 to 6 types of codons each encode one amino acid are known, and the codon to be used may be selected arbitrarily.For example, the expression efficiency may be further increased in consideration of the codon usage of the host used for expression. Sequence can be designed.
  • DNA having the designed nucleotide sequence can be obtained by chemical synthesis of DNA, fragmentation and binding of the above-mentioned cDNA, partial modification of the nucleotide sequence, and the like. Partial alterations and mutations in artificial base sequences can be performed by PCR using primers consisting of synthetic oligonucleotides encoding the desired alterations or site-specific mutagenesis (Mark et al., Proceedings ⁇ National Academy ⁇ Sciences, Vol. 81, pp. 5662-5666, 1984).
  • the protein or polypeptide of the present invention can be purified from a culture of cells into which an expression vector has been introduced by a known purification method (salting out with inorganic salts, fractionation and precipitation with an organic solvent, ion-exchange resin column chromatography, affinity chromatography). Separation and purification can be achieved by appropriate combination of single column chromatography, gel filtration, etc.).
  • Overexpression of a protein or polypeptide of the invention This can enhance (enhance) the function or activity of the protein or polypeptide in cells.
  • Nucleic acids that hybridize with the nucleic acids of the present invention under stringent conditions or their complements can be used as probes for detecting the genes of the present invention.
  • this nucleic acid or its complement can be used, for example, as an antisense oligonucleotide, ribozyme, or decoy to modulate (eg, suppress) gene expression.
  • Examples of such a nucleic acid include, for example, a nucleotide having a continuous partial sequence of at least 14 bases or a complementary sequence thereof of a nucleic acid (sense strand or antisense strand) consisting of the nucleotide sequence shown in SEQ ID NO: 1. Can be used.
  • the protein or polypeptide of the present invention is used as an antigen by using the protein or polypeptide of the present invention or a protein or peptide having immunological equivalence to the protein or polypeptide (eg, a synthetic peptide having a protein fragment or a partial sequence).
  • An antibody that recognizes the peptide can be obtained.
  • Having immunological equivalence means, for example, that a cross-reactivity occurs with an antibody against the protein or polypeptide of the present invention.
  • the polyclonal antibody can be prepared by a conventional method of inoculating a host animal (for example, rat or egret) with an antigen and collecting immune serum.
  • Monoclonal antibodies can be prepared by a conventional technique such as the hybridoma method.
  • a humanized monoclonal antibody or the like can be prepared by modifying the gene of the monoclonal antibody.
  • expression of the protein or polypeptide of the present invention in cells or tissues can be detected by a conventional immunochemical method (such as immunochemical assay).
  • the protein or polypeptide of the present invention can be purified by affinity chromatography using an antibody.
  • the function or activity of the protein or polypeptide of the present invention is modulated (eg, suppressed) using a neutralizing antibody. Can be.
  • the receptor protein or polypeptide of the present invention has a function or activity (biological activity) as an eicosanoid receptor.
  • functions or activities include, for example, the following.
  • G protein more specifically, G ia (a G protein subunit belonging to the Gi subfamily, such as Gial )
  • a ligand a ligand that acts as an agonist
  • the ligands include eicosanoides (eg, 5_oxo-ETE, 5-HPETE, arachidonic acid, eicosa-1 5Z, 8Z, 11Z-trienic acid) , 5-HET r E, eicosa-5Z, 8Z-genic acid, 5-HETE, and 5,8,11-eicosatriic acid).
  • Such a function or activity of the receptor protein or polypeptide of the present invention can be detected, for example, as follows.
  • the receptor protein or polypeptide of the present invention (in the form of a membrane fraction containing the same, or in the form of a cell expressing the same on the cell surface, etc.) is used as a ligand (eicosanoid, for example, 5—oxo ETE, 5—HPETE, Rachidonic acid, eicosa 5 Z, 8 Z, 11 Z—trienoic acid, 5—HET r E, eicosa 5 Z, 8 Z—genic acid, 5_HETE, and 5 , 8, 11-eicosatriic acid] to detect the specific binding of both.
  • a ligand with a label eg, RI label, fluorescent label, etc.
  • Specific binding can be detected by a conventional competitive assay using a mixture of a labeled ligand and an unlabeled ligand.
  • a cell expressing the receptor protein or polypeptide of the present invention on the cell surface is treated with a ligand [eicosanoide (eg, 5-oxo-ETE, 5-HP ETE, arachidonic acid, eicosa-5Z, 8Z, 1 1 Z-trienoic acid, 5-HET r E, eicosa-5 Z, 8 Z-genic acid, 5_HETE, and 5,8,11-eicosatriic acid, etc., which act as agonists)
  • a ligand eicosanoide (eg, 5-oxo-ETE, 5-HP ETE, arachidonic acid, eicosa-5Z, 8Z, 1 1 Z-trienoic acid, 5-HET r E, eicosa-5 Z, 8 Z-genic acid, 5_HETE, and 5,8,11-eicosatriic acid, etc., which act as agonists)
  • induced intracellular signaling eg, changes in Ca 2+ concentration, changes
  • Cells that express (or do not express) the receptor protein or polypeptide of the present invention at lower levels are used as controls and compared to the level of intracellular signaling in such control cells. If the level of intracellular signal transduction is higher than that, it is recognized that it has the function or activity (ii) depending on the degree.
  • Receptor protein or polypeptide and G ia (alpha subunit of G proteins belonging to the Gi subfamily one, for example, G ial) of the present invention the fusion protein from cells that are expressed on the cell membrane, the membrane fraction prepared I do.
  • This membrane fraction was purified with a ligand [eicosanoide (eg, 5-oxo ETE, 5-HPETE, Chidonic acid, eicosa 5 Z, 8 Z, 11 Z-trienic acid, 5-HETR E, dicosa 5 Z, 8 Z-genic acid, 5-HETE, and 5, 8, 11-eicosatriic acid, etc.
  • a ligand eicosanoide (eg, 5-oxo ETE, 5-HPETE, Chidonic acid, eicosa 5 Z, 8 Z, 11 Z-trienic acid, 5-HETR E, dicosa 5 Z, 8 Z-genic acid, 5-HETE, and 5, 8, 11-eicosatriic acid, etc.
  • GTP analogs such as labeled GTP or analogs thereof (eg, GTPyS (guanosine 5'-O- (3-thiotriphosphate)), in the presence and absence of ]. Thereafter, the binding of the labeled GTP or its analog to the membrane fraction is detected. If the level of binding in the presence of the ligand is higher than the level of binding in the absence of the ligand, the function or activity (iii) described above is recognized to have a corresponding degree.
  • labeled GTP or analogs thereof eg, GTPyS (guanosine 5'-O- (3-thiotriphosphate)
  • G ia (a Sabuyuni' of G proteins belonging to the Gi subfamily Bok, for example, G ial) and for the gene already known amino acid sequence and the base sequence [human G ial (351Cys ⁇ Ile) ZBahia Biochemistry, Vol. 37, pp. 11555-11562, 1998: Human Gial / Genbank / EMBL accession no. AF055013, PIR / SWISS-PROT accession no. P04898: etc.].
  • DNA encoding Gia can be prepared by utilizing the known sequence information disclosed, such as the PCR (polymerase chain reaction) method, the coagulation hybridization method, the plaque hybridization method, or a combination thereof. You can select and obtain from the DNA library.
  • DNA encoding G ia attached to Downstream of the DNA of the receptor protein or Poribe peptide co Solo de of the present invention, incorporate it into a vector one containing a suitable promoter, a fusion protein A vector for expression can be obtained. Then, the expression vector of the fusion protein can be introduced into cells to express the fusion protein.
  • the receptor protein or polypeptide of the present invention can be used for screening or identifying a ligand or agonist (agonist or antagonist) thereto.
  • Method for screening or identifying a ligand or agonist (agonist or antagonist) for the receptor protein or polypeptide of the present invention Contacting the receptor protein or polypeptide of the present invention (in the form of a membrane fraction containing the same or in the form of a cell expressing the same on the cell surface) with a test compound; and Detecting (1) specific binding of the receptor protein or polypeptide to a test compound, (2) intracellular signal transduction, or (3) activation of G protein (G ia ) in the presence of the test compound It can be carried out using a method including a step.
  • the method of screening or identifying a ligand, an agonist and an antagonist can be respectively carried out as follows.
  • the detection of such specific binding is carried out, for example, by a conventional competitive assay using a known ligand labeled (for example, RI label, fluorescent label, etc.) mixed with an unlabeled test compound.
  • a known ligand labeled for example, RI label, fluorescent label, etc.
  • test compound (ligand) with specific binding capacity is likely to be an agonist (agonist or antagonist).
  • the receptor protein or polypeptide of the present invention (in the form of a membrane fraction containing the same or in the form of a cell expressing the same on the cell surface, etc.) is brought into contact with a test compound, and (2) ) Detecting intracellular signal transmission or activation of G protein (G ia ) in the presence of the test compound, and (3) detecting the presence of the test compound based on the stimulation of the receptor protein or polypeptide. It can be carried out by determining whether or not it has the ability to induce intracellular signal transmission or activation of G protein, or determining the strength of the ability.
  • Detection of intracellular signaling eg, changes in Ca 2+ or c AMP concentration, activation of phospholipase C, changes in pH, changes in K + concentration, etc.
  • detection of G protein (G ia ) Activation can be detected using the same method as described above.
  • test compound and a ligand preferably, a receptor protein or a polypeptide of the present invention (in the form of a membrane fraction containing the same or in the form of a cell expressing the same on the cell surface)
  • a ligand preferably, a receptor protein or a polypeptide of the present invention (in the form of a membrane fraction containing the same or in the form of a cell expressing the same on the cell surface)
  • detecting the function or activity of the receptor protein or polypeptide of the present invention detecting the test compound with the receptor protein or polypeptide of the present invention. It can be carried out by determining whether or not it has the ability to suppress the function or activity of, or the strength of the ability.
  • the ligands include, for example, 5-oxo-ETE, 5-HPETE, arachidonic acid, eicosa-1 5Z, 8Z, 11Z-trienic acid, 5-HETR E, eicosa-5Z, 8Z-genic acid, 5-HETE, and eicosanoids such as 5,8,11_eicosatriic acid.
  • 5-oxo-ETE is particularly preferred. It is mentioned as.
  • the function or activity of the protein or polypeptide of the present invention includes (i) (i i) or (i i i), ie,
  • Detection of these functions or activities can be carried out, for example, by adding or not adding a test compound together with a ligand (preferably, a ligand acting as an agonist), or by applying the same method as described above.
  • a ligand preferably, a ligand acting as an agonist
  • a compound selected or identified as an antagonist by the above method or the like is brought into contact with a cell expressing the polypeptide of the present invention, whereby the function of the polypeptide (eicosanoid receptor) in the cell is determined. Or activity can be suppressed.
  • an antagonist to the polypeptide (eicosanoide receptor) of the present invention can be used as an active ingredient of a medicine. Therefore, a compound selected or identified as an antagonist for the polypeptide (eicosanoid receptor) of the present invention by the above-mentioned method or the like can be used for the production of a medicament.
  • a pharmaceutical composition can be produced by mixing with a conventional carrier, and such a composition can be sold as a medicament.
  • the administration method is not particularly limited, and general oral or parenteral methods (oral, intravenous, intramuscular, subcutaneous, etc.) may be applied. Further, if necessary, it may be formulated as a conventional pharmaceutical preparation (tablets, granules, capsules, powders, injections, inhalants, etc.) together with an inert carrier according to the administration method.
  • the compound can be used in a usual manner by formulating a compound together with an activator or diluent such as a binder, disintegrant, bulking agent, filler, lubricant, etc. which are acceptable in general pharmaceuticals. .
  • the dosage varies depending on the administration method, age, weight, and condition of the patient, but general dosages, for example, 1 to 300 mg Z kg per day for oral administration, parenteral administration Is set in the range of 0.01 to 5 O mg Z kg.
  • the receptor protein or polypeptide used for screening or identification of the ligand or agonist may be in the form of a membrane fraction containing the receptor protein or polypeptide, or a receptor protein or polypeptide.
  • the cells It can be used in the form of cells expressing on the surface.
  • Cells expressing the receptor protein or polypeptide on the cell surface include cells overexpressing the receptor protein or polypeptide or the like.
  • cells into which a recombinant vector containing a nucleic acid encoding the receptor protein or polypeptide has been introduced can be used.
  • a cell that can express the foreign receptor protein on the cell membrane without impairing its function
  • a cell that does not express the target receptor protein or polypeptide or expresses only a low level of the target receptor protein before introducing the recombinant vector.
  • the membrane fraction containing the receptor protein or polypeptide is subjected to centrifugal force such as fractionation centrifugation or density gradient centrifugation after disrupting cells expressing the receptor protein or polypeptide. It can be prepared using the fractionation method used. For example, the cell lysate is centrifuged at a low speed (approximately 500-300 rpm) for a short period of time (usually approximately 1-10 minutes), and the supernatant is centrifuged at a higher speed (approximately 150-000 rpm). (Centrifugation at ⁇ 300 rpm) for about 30 to 120 minutes, and the obtained precipitate fraction is used as a membrane fraction.
  • centrifugal force such as fractionation centrifugation or density gradient centrifugation after disrupting cells expressing the receptor protein or polypeptide. It can be prepared using the fractionation method used. For example, the cell lysate is centrifuged at a low speed (approximately 500-300 rpm) for a
  • Example 1 Isolation of human TG 10 19
  • the amino acid sequences of 62 G protein-coupled receptors that use peptides as ligands were aligned, and the amino acid sequence (SEQ ID NO: 3) that was well conserved among these G protein-coupled receptors was determined.
  • This sequence (SEQ ID NO: 3) was used as a query sequence and NCBI was used as a homology search method by using tb1astn (Altschul SF et al., J. Mol. Biol., 215: 403-410, 1990). (The National Center for Biotecnnology information CO High-Throughput Genomic Sequences database was searched.
  • SEQ ID NO: 4 a sequence with the highest homology (SEQ ID NO: 4) was found in clone A CO 13 396.3.
  • the amino acid sequence of the polypeptide encoded by this open reading frame was determined by using HMMT0P (membrane protein identification and membrane). Prediction of transmembrane domains using a transmembrane helix prediction system (Tusnady GE et al., J. Mol. Biol., Vol. 283, pp. 489-506, 1998) and known G protein-coupled receptor With the body Homology analysis was performed, etc. As a result, it was inferred that the nucleotide sequence shown in SEQ ID NO: 5 encodes the full length of the novel G protein-coupled receptor.
  • a primer was designed based on the nucleotide sequence of AC013396.3 in (1) above, and a DNA containing SEQ ID NO: 5 was obtained by PCR.
  • the first PCR was carried out using a human cDNA library (trade name: “Hmnan Universal QUICK-Clone cDNAj”; manufactured by Clontech Co., Ltd.).
  • a nucleotide a primer corresponding to the region including bases 97735 to 97760 of AC013396.3
  • an antisense primer was a synthetic oligonucleotide consisting of the base sequence shown in SEQ ID NO: 7 (96299 to 299299 of AC013396.3).
  • a PCR reaction solution 25 ⁇ ⁇
  • type III cDNA (trade name “Human Universal QUICK-Clone cDNA”) was used.
  • TG1019 protein The amino acid sequence (423 amino acid residues) of the encoded protein (referred to as TG1019 protein) was as shown in SEQ ID NO: 2.
  • SEQ ID NO: 2 the transmembrane region was predicted by HMMTOP, and homology analysis with a known G protein-coupled receptor was performed.
  • the TG1019 protein was presumed to be a novel G protein-coupled receptor, and the cDNA (1462 bp) obtained above contained the entire translation region of the TG1019 protein gene (referred to as TG1019 gene). It was considered to be cDNA containing.
  • FIG. 1 shows the transmembrane region (underlined) together with the base sequence and the amino acid sequence.
  • PCR (independently twice) was performed using human cDNA library 1 (trade name: Marathon Ready cDNA library, human fetal spleen; manufactured by Clonetech) as a type II, and the base containing the above differences was found.
  • human cDNA library 1 (trade name: Marathon Ready cDNA library, human fetal spleen; manufactured by Clonetech) as a type II, and the base containing the above differences was found.
  • the Ap a I fragment was obtained, and the nucleotide sequences of multiple clones were analyzed.
  • the difference (CA) at nucleotide 1022 in SEQ ID NO: 1 was considered to be due to nucleotide polymorphism, and was thought to be derived from naturally occurring allelic variants. . This difference in nucleotide sequence does not cause a difference in the amino acid sequence of the encoded polypeptide.
  • the 487th, 771st, 1022th, and 1038th bases in SEQ ID NO: 1 are “A” (adenine).
  • the 150th, 245th, and 334th amino acid residues in SEQ ID NO: 2 are Asp, Lys, and Thr, respectively. It was considered.
  • SEQ ID NO: 20 The nucleotide sequence after the PCR error correction is shown in SEQ ID NO: 20 in the sequence listing below, and the amino acid sequence of the polypeptide encoded thereby is shown in SEQ ID NO: 21.
  • TG1019 gene expression in various human tissues and cells was examined by dot plot analysis.
  • Dot blots show mRNA from human tissues and human cultured cells (dotted on a nylon membrane) (Multiple Tissue Expression Array, Clontech) and RI-labeled probe.
  • the membrane has dots (adsorption-fixed) of mRNA [poly (A) RNA] derived from various human tissues and human cultured cells shown in Fig. 2.
  • the RI-labeled probe used was prepared as follows. That is, after treating the plasmid containing the cDNA fragment obtained in the section (2) of Example 1 with the restriction enzyme NotI, a DNA fragment of about 1500 bp (a full length translation region of the TG1019 gene) was subjected to agarose gel electrophoresis. The purified cDNA fragment was obtained. This cDNA fragment was designated as type I and labeled with a random primer (random hexadeoxyribonucleotides), a nucleotide mixture (dATP, ⁇ 0 and 01 TTP) and DNA polymerase I (Large (Klenow) Fragment). use kit (Prime-a- Gene labeling system, Promega) and [ ⁇ - 32 P] after labeling with d CTP, and purified by gel filtration to prepare an RI-labeled probe.
  • the dot blot hybridization was performed as follows. After treating the mRNA-immobilized membrane with 160 ⁇ l of Solution I (Salraon Testes DNA (9.4 ⁇ g / ⁇ l: Sigma)) at 97 ° C for 5 minutes, cool it on ice and mix it. Prepared by adding 15 ml of ExpressHyb hybridization Solution (manufactured by Clontech) at 60 ° C] for 30 minutes at 68 ° C. Then, the membrane, the labeled probe containing Haiburidize one Chillon solution [the RI-labeled probe 20 1, human COT- 1 DNA (1 ⁇ g / ⁇ 1: Roche Co.) 30 mu 1, salmon testes DNA (9.
  • TG1019 protein was presumed to be a G protein-coupled receptor as in Example 1 above.
  • GTPyS guanosine 5'_O— (3_thiotriphosphate)
  • FIG. 3 shows a schematic diagram of the fusion protein.
  • G ial (351Cys ⁇ Ile) is that it means a mutant G ial protein converts 351 th cis Tin residues of G ial protein Isoroishin residues.
  • PCR was performed using the plasmid containing the TG1019 gene cDNA (including the entire translation region) obtained in section (2) of Example 1 as type III.
  • the sense primer primer TG 1019-1
  • the antisense primer primer TG 1019-2
  • synthetic oligonucleotides having the nucleotide sequences shown in SEQ ID NO: 8 and SEQ ID NO: 9 were used, respectively.
  • These primers were designed based on the cDNA base sequence of the TG1019 gene (SEQ ID NO: 1), and contained a cDNA encoding the entire length of the TG1019 protein (excluding the stop codon) as a PCR product. It is designed to obtain a DNA fragment with a restriction enzyme recognition site (Bg1II site at the N-terminus and Cp0I and Hindill sites at the C-terminus).
  • the PCR product thus obtained was ligated to Vector Plasmid (a vector system for cloning PCR products) (pGEM-T Easy Vector, manufactured by Promega), and the obtained plasmid was ligated with restriction enzymes Not I and Cp. After treatment with oI, the resulting DNA fragment of about 1300 bp was recovered.
  • Vector Plasmid a vector system for cloning PCR products
  • PCR was performed using human brain-derived cDNA (Marathon-Ready cDNA Brain, Clontech) as type II. At that time, as the sense primer (primer Gial -1) and the antisense primer (primer Gial -2), synthetic oligonucleotides having the nucleotide sequences shown in SEQ ID NO: 10 and SEQ ID NO: 11, respectively, were used. .
  • Gial (351Cys ⁇ Ile) (0 ⁇ ! It contains cDNA encoding the full length of a mutant Gial protein in which the cysteine residue at position 351 of the protein has been replaced with an isoleucine residue, and has restriction enzyme recognition sites ( CpOI site and B a It is designed to obtain a DNA fragment to which an mHI site has been added.
  • the obtained PCR product was ligated to Vector-plasmid (pGEM-T Easy Vector, manufactured by Promega).
  • This plasmid was treated with restriction enzymes NotI and BamHI, and the resulting DNA fragment of about 1100 bp was digested with the NotIZBamHI site of baculovirus vector plasmid PVL1392 (Pharmingen).
  • pVL1392ZGial 351Cys ⁇ Ile.
  • the DNA fragment obtained in the above (a) was inserted into the restriction enzyme Not I / Cpo I site of this plasmid, and the plasmid PVL 1392 / TG 1019-G iQl (351 Cys ⁇ Ile) for fusion protein expression was inserted. Obtained.
  • G qa G protein
  • G qa already known nucleotide sequence of the c DNA encoding (Genbank / EMBL accession no. U43083 ) all SANYO designed based on, c DNA is obtained that encodes the full length of G qa as P CR product It is designed to be used.
  • a PCR product comprising the c DNA encoding the full-length G qa, restriction enzyme recognition sites on both ends (C p 0 I site and B AMH I site) is appended DN a fragment obtained It is designed to be
  • the obtained PCR product was ligated to Vector Plasmid (pGEM-T Easy Vector, manufactured by Promega).
  • the obtained plasmid was treated with the restriction enzymes NotI and BamHI, and the resulting DNA fragment of about 1100 bp was digested with the NotI / BamHI site of the baculovirus vector plasmid pVL1392. It was inserted into, to give the plasmid p VL 1392ZG qa.
  • primers were designed based on the known nucleotide sequence of cDNA encoding GsaL (Genbank / EMBL accession no.X04408), and contained cDNA encoding the full-length GsaL as a PCR product. It is designed so as to obtain a DNA fragment having restriction enzyme recognition sites (Cp0I site and XbaI site) at both ends.
  • the obtained PCR product was ligated to Vector Plasmid (pGEM-T Easy Vector, manufactured by Promega).
  • the resulting plasmid was treated with restriction enzymes NotI and XbaI, and the resulting DNA fragment of about 1200 bp was digested with NotI DNA of baculovirus vector plasmid pVL1392 (Pharmingen).
  • Plasmid P VL 1392Z G saL was obtained by insertion into the Xba I site.
  • the DNA fragment obtained in (1) was inserted into the restriction enzyme NotI / CpoI site of this plasmid to obtain a plasmid pVL1392TG1019-GsaL for fusion protein expression.
  • TG1019 protein full length
  • G protein G
  • ial 351Cys ⁇ Ile
  • GsaL is a plasmid that expresses a fusion protein having a linked structure.
  • 92 TG101 9- GsaL was expressed in insect cells as follows, and a membrane fraction containing the fusion protein was prepared.
  • a culture medium [10%] was prepared by dissolving insect cells Sf9 (Spodoptera frugiperda SF9) (manufactured by Pharmingen Co., Ltd.) in a collagen-coated 3 cm Petri dish so that about 60% confluent. The cells were incubated at 27 ° C. for 15 minutes in Grace's Insect Cell Culture Medium (pH 6.2: manufactured by Lifetech Oriental) containing fetal serum, 0.1 mg Zml streptomycin and 10 OU / ml penicillin.
  • Transfection Buffer A Transfection Buffer A, manufactured by Pharmingen
  • DNA solution prepared for fusion protein expression 1 ⁇ g, Linearized BaculoGold Baculovilus DNA (Pharmingen) 0.1 Mix 25 ⁇ g in 25 ⁇ l of sterile water, incubate at 25 ° C for 15 minutes, and transfer 375 ⁇ l of Transfection Buffer B (Pharmingen). 400 ⁇ 1 was added dropwise. After culturing at 27 ° C for 4 hours, the culture solution was removed, 1.2 ml of medium was added, and the cells were cultured at 27 ° C for 5 days. The obtained culture was centrifuged (1,000 Xg, 5 minutes), and the supernatant was recovered as virus I.
  • Transfection Buffer A Transfection Buffer A, manufactured by Pharmingen
  • Sf9 cells are seeded on a collagen-coated 3 cm Petri dish at about 30% confluence, and the virus solution I ( ⁇ ⁇ ⁇ ⁇ ) obtained above and 1.2 ml of medium are added and the mixture is incubated at 27 ° C. Cultured for 4 days. The obtained culture solution was centrifuged (1,000 xg, 5 minutes), and the supernatant was recovered as virus solution II. Sf9 cells are seeded on a collagen-coated 10 cm Petri dish at about 70% confluency, and the virus solution II (500 ⁇ ) obtained above and 12 ml of medium are added, and the mixture is added at 27 ° C for 4 days. Cultured.
  • the culture solution thus obtained was centrifuged (1,000 X g, 5 minutes), and the supernatant was recovered as a virus solution.
  • Sf9 cells were seeded on a collagen-coated treated 1 Ocm Petri dish at about 70% confluence, and the virus solution III ( ⁇ ⁇ ⁇ ) obtained above and 12 ml of medium were added, and the mixture was incubated at 27 ° C. For 4 days.
  • the cells thus obtained were washed with cold PBS (phosphate-buffered saline, pH 7.4), and then cooled with a lysis buffer [20 mM Tris-HCl, pH 7.5, lmMEDTA, 0.2 mM fluorinated phenol.
  • the cell lysate was centrifuged (600 ⁇ g, 10 minutes), and the obtained supernatant was further centrifuged (50,000 ⁇ g, 20 minutes).
  • the resulting precipitate was suspended in a cooled reaction buffer [20 mM ris—HC1, pH 7.5, 5 OmM sodium chloride, 1 OmM magnesium chloride] 450 ⁇ using a Teflon homogenizer to suspend the fusion protein.
  • the expressed membrane fraction was obtained.
  • the amount of [ 35 S] GTPyS on the filter was measured by a liquid scintillation assay.
  • the specific binding of S] GTP yS was calculated by subtracting the non-specific binding (the binding measured in the presence of l O MGTP S) from the [35 S] GTP Y S binding thus determined. The amount was determined.
  • TG 1019 protein was considered to be G protein-coupled receptor of the type conjugated with G ial or G qa protein.
  • 5-oxo-ETE, 5-HPETE, arachidonic acid, eicosa-5Z, 8Z, 11Z-trienic acid, 5-HETRE, eicosa-5Z, 8Z-genic acid, 5-HETE And 5, 8, 11-eicosatriic acid were found to act as their ligands (agonists).
  • TG 1019 protein is composed of 5_oxo-ETE, 5-HPET E, arachidonic acid, eicosa-5Z, 8Z, 11Z-trienoic acid, 5-HET rE, eicosa-5,8-genic acid , 5-HETE, and 5,8,11-eicosatriic acid as ligands (agonists).
  • G protein activation was detected in the same manner as in Example 3, (3) using the membrane fraction containing the fusion protein of TG1019 protein and Gia1 and labeled GT PyS. did.
  • the reaction was started by adding 20 ⁇ l of [ 35 S] GTPyS thereto, and after incubating at 30 ° C. for 1 hour, the reaction was stopped by filtration through a glass filter.
  • Table 2 shows those compounds identified as acting as antagonists of the TG01019 protein (G protein-coupled receptor) and their IC50 values.
  • the TG101 protein expression vector is transiently introduced into CHO cells to express the recombinant TG109 protein as follows, and the TG109 protein is expressed using the cells.
  • the function of the 0.19 protein as an eicosanoid receptor was confirmed.
  • Example 1 (1) the DNA encoding the full-length TG01019 protein obtained in Example 1 (1) (a) was used for NotI recognition of vector plasmid pcDNA3.1 (expression vector, manufactured by invitrogen). Subcloning was performed at the site to obtain a plasmid vector pcDNA3.1-TG1019 for expression of the TG109 protein.
  • CHO cells (lxl O 6 cells) were cultured at 37 ° C. for 20 hours in DMEMZF-12 medium containing 10% fetal calf serum. Using these cells, plasmid DNA (pcDNA3.1-TG1019 or, as a control, vector pcDNA3.1) (5 ⁇ g) and a reagent for transfection (LipofectAMINE, Invitrogen) And transfection was carried out, followed by culturing for 24 hours. In the case of pretreatment with pertussis toxin, pertussis toxin (100 ng) was added after culturing for 20 hours, followed by culturing for 4 hours.
  • plasmid DNA pcDNA3.1-TG1019 or, as a control, vector pcDNA3.1
  • LipofectAMINE LipofectAMINE, Invitrogen
  • the cells after culture are collected, washed, suspended in KRH buffer (Krebs-Ringer Hepes buffer, pH 7.4) containing rolipram (25 mM), and incubated at 37 ° C for 30 minutes. Inoculate 6-well plate (5-7.5 xlO 4 cells / 90 ⁇ 1 / well) was.
  • KRH buffer Krebs-Ringer Hepes buffer, pH 7.4
  • rolipram 25 mM
  • the cAMP production was measured using a cAMP Enzym 'Imnoassy' system manufactured by Amersham Biosciences.
  • TG1019 protein is an eicosanoid receptor that uses 5-oxo-ETE as a ligand (a ligand that acts as an agonist), and Gi (an adenylate cyclase-inhibiting G protein). It was confirmed to be a G-protein-coupled receptor that couples with G.
  • the receptor protein of the present invention and its gene are useful for studying the mechanism of intracellular signal transduction. It can also be a target molecule for therapeutic drugs for new diseases.
  • the method for screening, identifying and characterizing an agonist (agonist or antagonist) using the receptor protein and its gene of the present invention is useful for research and development of new drugs.

Abstract

L'invention concerne des polypeptides appartenant à l'un des trois types définis ci-après, qui ont une fonction ou une activité de récepteur éicosanoïde: (A) polypeptide comprenant une séquence d'acides aminés représentée par SEQ ID N0:2 ou 21: (B) polypeptide comprenant une séquence d'acides aminés représentée par SEQ ID N0:2 ou 21, par suppression, substitution ou adjonction d'un ou plusieurs acides aminés; et (C) polypeptide codé par un acide nucléique hybridable avec un acide nucléique comprenant une séquence de base représentée par SEQ ID N0:1 ou 20, dans des conditions rigoureuses, ou son complémentaire. L'invention concerne également: acides nucléiques codant ces polypeptides; vecteurs de recombinaison et cellules hôtes renfermant les acides nucléiques en question; procédé de détection de fonction ou d'activité du polypeptide considéré, à travers ces éléments; et procédé de criblage ou d'identification d'un ligand, d'un agoniste ou d'un antagoniste vis-à-vis de ce polypeptide, à travers lesdits éléments.
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