WO2003087364A1 - Novel g protein-coupled receptor and its gene - Google Patents

Novel g protein-coupled receptor and its gene Download PDF

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Publication number
WO2003087364A1
WO2003087364A1 PCT/JP2003/004759 JP0304759W WO03087364A1 WO 2003087364 A1 WO2003087364 A1 WO 2003087364A1 JP 0304759 W JP0304759 W JP 0304759W WO 03087364 A1 WO03087364 A1 WO 03087364A1
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Prior art keywords
protein
ligand
nucleic acid
cell
reaction product
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PCT/JP2003/004759
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French (fr)
Japanese (ja)
Inventor
Tetsuo Onuki
Koji Ogawa
Yutaka Koguchi
Emiko Hosoi
Aiko Chikada
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Tanabe Seiyaku Co., Ltd.
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Priority to AU2003227499A priority Critical patent/AU2003227499A1/en
Priority to JP2003584306A priority patent/JPWO2003087364A1/en
Publication of WO2003087364A1 publication Critical patent/WO2003087364A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • 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
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • 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

Definitions

  • the present invention relates to a novel G protein-coupled receptor protein having a lipid peroxidation reaction product as a ligand and a gene thereof.
  • the present invention also relates to a method for identifying a drug candidate compound and a pharmaceutical composition using the same.
  • G protein-binding protein a trimeric GTP-binding protein present in cells, and are known to transmit information into cells through their activation. ing. 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.
  • the G protein is a trimer composed of spike and ⁇ subunits, and exists in an inactive form (GDP binding form) in which these subunits are associated in the ground state.
  • Inactive (GDP-bound) G protein is converted to active (GTP-bound) by ligand-stimulated G protein-coupled receptor, and ⁇ submit (Ga) bound to GT ⁇ and Dissociates with the / 3 / ⁇ subunit complex (Gj3 y).
  • Gee coupled with GTP (and in some cases, G] 3 ⁇ ) controls signals such as adenylate cyclase and phospholipase C to transmit signals.
  • G proteins are diverse in G ⁇ and control different effectors depending on the type of G ⁇ .
  • a G protein-coupled receptor activates a specific type of G protein, and transmits specific information into a cell by the G protein.
  • G protein-coupled receptors there have been one and the following: adrenergic receptor, muscarinic acetylcholine receptor, adenosine receptor, angiotensin Receptors, endothelin receptors, gonadotropin-releasing factor receptors, are known: ⁇ 1- and 12-histamine receptors, dopamine receptors, metabotropic glutamate receptors, somatostatin receptors, purine receptors, etc. .
  • Each of the above receptors plays an important role in vivo as a target of a physiologically active substance. Furthermore, the fact that many of the drugs known to date were also ligands targeting G protein-coupled receptors, or agonists-antagonists, is also very significant.
  • G protein-coupled receptors are attracting attention as targets for drug development. Therefore, finding new G protein-coupled receptors, identifying their ligands, and finding ways to identify their agonists and antagonists will lead to the identification of new drug candidates. Such identification and development are strongly desired.
  • arteriosclerosis Pal inski, W., et al., Proc. Natl. Acad. Sci.
  • the present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, isolated a full-length cDNA encoding a novel G protein-coupled receptor from human. We have also succeeded in expressing this receptor protein in cells by genetic recombination technology. Further, the ligand was identified, and it was found that this receptor was a G protein-coupled receptor using a lipid peroxidation reaction product as a ligand, thereby completing the present invention.
  • the present invention provides the following (1) to (37).
  • G protein conjugate having an amino acid sequence represented by SEQ ID NO: 2 in which one or several amino acids are deleted, substituted or added, and a lipid peroxidation reaction product as a ligand A protein having a function as a type receptor or a biological activity.
  • the function or biological activity as a G protein-coupled receptor having a lipid peroxidation reaction product as a ligand is selected from the following (i), (ii) and (iii): The protein according to (2) or (3), which is as described above.
  • the protein according to any one of (1) to (6) which is a mammalian protein.
  • a G protein-conjugated type that hybridizes under stringent conditions with a nucleic acid having a nucleotide sequence complementary to the nucleic acid having the nucleotide sequence represented by SEQ ID NO: 1 and uses a lipid peroxidation reaction product as a ligand
  • a nucleic acid of SEQ ID NO: 1 having at least 75% homology over the entire length of its translated region, and a function or organism as a G protein-coupled receptor having a lipid peroxidation reaction product as a ligand An isolated nucleic acid encoding a protein having biological activity.
  • the function or biological activity as a G protein-coupled receptor having a lipid peroxidation reaction product as a ligand is one or more selected from the following (i), (ii) and (iii): (10) or the nucleic acid according to (11).
  • nucleic acid according to any one of (9) to (13), which is a DNA is a DNA.
  • nucleic acid according to any one of (9) to (14), which is a mammalian nucleic acid.
  • a recombinant vector comprising the nucleic acid according to any one of (9) to (16).
  • (21) A method for enhancing the function or biological activity in a cell of a G protein-coupled receptor using a lipid peroxidation reaction product as a ligand, using the recombinant vector according to (17).
  • a method for producing a protein having a function as a G-protein-coupled receptor or a biological activity using a lipid peroxidation reaction product as a ligand comprising the following steps:
  • An antisense nucleic acid having a nucleotide sequence complementary to the nucleic acid according to any one of (9) to (11) and capable of suppressing the expression of the nucleic acid.
  • (C) a step of determining the presence or absence or the strength of specific binding between the protein and a test compound.
  • (C) a step of determining whether or not the test compound has the ability to induce or suppress intracellular signal transduction or activation of G protein.
  • (C) a step of determining whether or not the test compound has the ability to induce or suppress intracellular signal transduction by measuring the change.
  • a membrane fraction is prepared from a cell in which a fusion protein of the protein according to any one of (1) to (8) and a G protein belonging to the Gi subfamily is expressed on a cell membrane.
  • (31) A method for identifying a gonist or antagonist to the protein according to any one of (1) to (8), comprising the following steps.
  • a membrane fraction is prepared from cells in which a fusion protein of the protein according to any one of (1) to (8) with a G protein subunit of the Gi subfamily is expressed on the cell membrane.
  • the protein according to any one of (1) to (8) is in the form of a membrane fraction containing the protein, or in the form of a cell in which the protein is expressed on the cell surface.
  • a cell in which the protein is expressed on the cell surface is a cell in which a nucleic acid encoding the protein or an expression vector containing the same has been introduced to express the protein.
  • a method for producing a pharmaceutical composition comprising: identifying an agonist or antagoust by the method according to any one of (29) to (31); and mixing the agonist or antagonist with a carrier. .
  • FIG. 1 is a diagram showing the amino acid sequence and base sequence of the TG0039 protein and the putative seven transmembrane regions (underlined).
  • FIG. 2 is a diagram showing the distribution of TG0039 gene expression (results of dot blot) in human tissues or cells. Circles indicate tissues or cells in which gene expression was observed at the mRNA level.
  • FIG. 3 is a schematic diagram showing an outline of the structure of a fusion protein of the TG 0039 protein and various G proteins.
  • FIG. 4 is a view showing the effect of 4-HNE on the specific binding amount between GTP P ⁇ S and a membrane fraction containing a fusion protein of TGO039 protein and various G proteins.
  • the specific binding amount is expressed as a relative value (% of control) relative to the binding amount at the time when no test substance was added as a control.
  • ⁇ Gial (351Cys ⁇ Ile) j represents the test results in the membrane fraction containing the fusion protein with TG 00 3 9 protein and G i a l (351Cy S ⁇ Ile)
  • ⁇ ⁇ Gqa '' indicates the test results for the membrane fraction containing the fusion protein of TG 0039 protein and Gqa
  • ⁇ Mouth GsH L '' indicates the test results for the membrane fraction containing the fusion protein of TG 003 9 protein and GsccL.
  • the present invention provides the following proteins.
  • a protein having the amino acid sequence represented by SEQ ID NO: 2 is provided.
  • Such proteins include those having the amino acid sequence of a receptor protein (hereinafter, referred to as “TG0039 protein”) encoded by the cDNA shown in SEQ ID NO: 1.
  • the second protein has an amino acid sequence represented by SEQ ID NO: 2 in which one or several amino acids have been deleted, substituted or added, and Provided is a protein having a function or a biological activity as a G protein-coupled receptor using a lipid peroxidation reaction product as a ligand.
  • Examples of such a protein include a protein having an amino acid sequence represented by SEQ ID NO: 2 in which one or several amino acids have been deleted, substituted or added.
  • the deletion, substitution or addition of amino acids may be performed to such an extent that the function or biological activity as a G protein-coupled receptor using a lipid peroxidation reaction product as a ligand is not lost.
  • 1 to about 80 preferably 1 to about 60, more preferably 1 to about 45, still more preferably 1 to about 30, and still more preferably 1 to about 15 is there.
  • specific examples of the protein of the present invention include one or more conservative amino acid substituting amino acids compared to the protein consisting of the amino acid sequence represented by SEQ ID NO: 2. ).
  • Such proteins include mutant proteins found in nature, artificially modified mutant proteins, proteins derived from heterologous organisms, and the like. Therefore, such proteins include conservative substitution variants and naturally occurring variants of the protein consisting of the amino acid sequence shown in SEQ ID NO: 2. Naturally occurring allelic variants; 0 3 rare.
  • a protein consisting of the amino acid sequence of SEQ ID NO: 2 and 75% or more, preferably 80% or more, more preferably about 85% or more, and still more preferably about 90% over its entire length
  • proteins are even more preferably proteins having homology of about 95% or more amino acid sequences, and have a function or biological activity as a G protein-coupled receptor having a lipid peroxidation reaction product as a ligand.
  • Homology is a relationship between two or more proteins (or polynucleotides), as known in the art, determined by comparing the sequences, Means the degree of sequence correlation between proteins (or polynucleotide sequences) as determined by the match between the sequences. "Homology” can be readily determined by methods known in the art. For example, it can be determined using the BLAST (Basic Local Alignment Search Tool) program by Altschul et al.
  • the above various proteins is a kind of G protein-coupled receptors, in c herein having a function or biological activity of lipid peroxidation products as G protein coupled receptors to ligands, lipid peroxidation
  • the reaction product is, for example, a decomposition product of lipid peroxides, and includes 4-hydroxy-2-nonenal (also known as 4-hydroxynonenone or 4-hydroxynone-2-enal). , Hereinafter referred to as 4-HNE).
  • the protein of the present invention specifically binds to a lipid peroxidation reaction product (eg, 4-HNE). Then, by these specific bindings, the receptor protein is stimulated to induce intracellular signal transduction.
  • the receptor protein of the present invention activates G ; a G protein belonging to subfamily 1 when stimulated by the above-mentioned substance that specifically binds to the protein.
  • G a G protein belonging to subfamily 1 when stimulated by the above-mentioned substance that specifically binds to the protein.
  • G singular bind substances
  • G G j Sabufuamiri one belonging G protein ⁇ Sapuyunitto, for example G i ft l
  • an activated form a state having GTP binding ability
  • the protein of the present invention is a receptor for a lipid peroxidation reaction product, and has one or more functions or biological activities (i) to (iii) summarized below. This is as described in the examples of the present specification.
  • examples of ligands include 4HNE.
  • the protein of the present invention may be derived from a mammal, for example, a non-human animal or a human derived animal such as a dog, a magpie, a poma, a goat, a hidge, a sal, a pig, a penguin, a rat and a mouse. It may be a tide or a synthetic protein.
  • the protein can be synthesized by a conventional method known in the technical field.
  • the present invention provides a nucleic acid (DNA or RNA) encoding the protein. More specifically, the present invention provides an isolated nucleic acid having the following nucleotide sequence of (a) or (b).
  • a function or biological function as a G protein-coupled receptor that hybridizes under stringent conditions with a nucleic acid consisting of the nucleotide sequence represented by SEQ ID NO: 1 and uses a lipid peroxidation reaction product as a ligand An isolated nucleic acid encoding a protein having activity.
  • SEQ ID NO: 1 represents the nucleotide sequence of human-derived cDNA (including the entire translation region) of the gene of TG0039 protein (hereinafter, referred to as “TG0039 gene”).
  • “can hybridize under stringent conditions” generally means that in a 6 X SSC or a hybridization solution having a salt concentration equivalent thereto, a temperature of 50 to 60 ° C and a temperature of about 1 Perform hybridization for 6 hours, perform pre-washing as necessary with 6 XSSC or a solution with a salt concentration equivalent to this, and then wash in a solution with 1 XSSC or a salt concentration equivalent to this. Means that hybridization can be performed.
  • “high stringent conditions (conditions having higher stringency)” means that washing can be performed in 0.1 XSSC or a solution having a salt concentration equivalent thereto in the above. means.
  • the nucleic acid that hybridizes under stringent conditions with the TG0039 gene represented by SEQ ID NO: 1 has a function or biological activity as a G protein-coupled receptor using a lipid peroxidation reaction product as a ligand. It only needs to encode a protein having
  • nucleic acid of the present invention has a nucleotide sequence represented by SEQ ID NO: 1 and usually about 70% or more, preferably about 80% or more, more preferably about 85% or more, more preferably about the entire length of the translation region thereof. Nucleic acids having 90% or more, more preferably 95% or more homology are also included.
  • genes or nucleic acids include mutant genes found in nature or artificially modified, homologous genes derived from heterologous organisms, and the like.
  • the nucleic acid of the present invention can be isolated and obtained by, for example, performing identification using mammalian tissues or cells as a gene source. Mammals include non-human animals such as dogs, magpies, magpies, goats, sheep, monkeys, pigs, magpies, rats and mice, as well as humans. Of these, it is desirable to use human-derived drugs for research and development of therapeutic drugs for humans.
  • the nucleic acid of the present invention can also be obtained by utilizing the sequence information of the gene represented by SEQ ID NO: 1.
  • a primer profile is designed based on the sequence information of SEQ ID NO: 1, and a PCR (polymerase chain reaction) method, a colony hybridization method, and a plaque hybridization method using these primers are appropriately combined. You can select and obtain from a DNA library.
  • cDNA is synthesized from mRNA prepared from mammalian cells and tissues, and this is used as a type II to obtain a cDNA fragment by the PCR method.
  • a full-length cDNA can be obtained by identifying a cDNA library by colony hybridization or plaque hybridization.
  • genomic genes can be isolated by identifying a genomic DNA library. By isolating a DNA library of another mammal, a homologous gene (ortholog) derived from a heterologous organism can be isolated.
  • DNA libraries such as DNA libraries and genomic DNA libraries are described in, for example, "Molecular Cloning J (Sambrook, J., Fritsch, EF and Maniatis, T., Cold Spring Harbor Laboratory Press, 1989) Can be prepared by the method described in (2) Alternatively, if there is a commercially available library, this may be used.
  • the translation region encoding the protein of the gene product can be determined, and the amino acid sequence of the obtained protein can be obtained.
  • the nucleic acid of the present invention obtained as described above encodes a G protein-coupled receptor protein having a lipid peroxidation reaction product as a ligand, and a G protein-coupled receptor having a lipid peroxidation reaction product as a ligand. Having a bodily function or biological activity As described in Examples of the present specification.
  • the present invention provides, in a third aspect, a method for producing the above protein.
  • the method for producing is to prepare a recombinant vector containing the nucleic acid of the present invention, obtain a cell transformed with the nucleic acid of the present invention or the recombinant vector, culture the cell, and recover the protein from the culture. Can be achieved by:
  • the above-mentioned method includes a method of expressing and producing the protein of the present invention by a conventional gene recombination technique. Expression of the protein in the form of a fusion protein with another protein peptide is performed. Production methods are also included.
  • Cells expressing the protein of the present invention can be obtained, for example, as follows. First, DNA encoding the protein of the present invention is inserted into a vector in a form ligated downstream of an appropriate promoter to construct an expression vector. Next, the obtained expression vector is introduced into a host cell.
  • Examples of the expression system include, for example, bacterial, yeast, insect cell and mammalian cell expression systems. Among them, insect cells (Spodoptera frugiperda SF9, SF21, etc.) and mammalian cells (monkey COS-7 cells, Chinese hamster CH ⁇ cells, human HeLa) Cells) are preferably used as hosts.
  • insect cells Spodoptera frugiperda SF9, SF21, etc.
  • mammalian cells monkey COS-7 cells, Chinese hamster CH ⁇ cells, human HeLa Cells
  • an SV40 promoter As a promoter for expressing the protein of the present invention, an SV40 promoter, an LTR promoter, an elongation 1 ⁇ promoter and the like in a mammalian cell system, and a polyhedrin promoter and the like in an insect cell system can be used. it can.
  • a retrovirus-based vector in the case of a mammalian cell line, a retrovirus-based vector, a virion-winores-better, a vaccinia winores-better, an SV40-based vector, and the like, and in an insect cell line, a baculovirus vector can be used. .
  • cDNA corresponding to mRNA existing in nature for example, one having the nucleotide sequence shown in SEQ ID NO: 1
  • a DNA corresponding to the amino acid sequence of the protein of the present invention can be designed and used.
  • one to six codons each encoding one amino acid are known.
  • the selection of codons may be arbitrarily selected.
  • a sequence having higher expression efficiency can be designed in consideration of the frequency of codon usage of a host used for expression.
  • DNA having the designed base sequence can be obtained by chemical synthesis of DNA, fragmentation and binding of the cDNA, partial modification of the base sequence, and the like. Partial alterations and mutations in artificial nucleotide sequences can be performed by PCR using primers consisting of synthetic oligonucleotides that encode the desired alterations, or by site-specific mutagenesis (Mark et al., Proceedings of Nations ⁇ Academy of Sciences, Vol. 81, 5 pp. 2-5666, 1984).
  • the protein of the present invention is produced in a culture of cells into which the above-described expression vector has been introduced, and is purified by known purification methods (salting out with inorganic salts, fractional precipitation with an organic solvent, ion exchange resin column chromatography). Separation and purification can be carried out by appropriately combining chromatography, affinity column chromatography, gel filtration, etc.).
  • the present invention provides a method for enhancing the function or biological activity of the protein of the present invention in a cell.
  • a method for enhancing the expression it can be achieved by increasing the expression of the protein of the present invention in cells. Specifically, it can be carried out by introducing the recombinant expression vector obtained in the third embodiment into a cell. Further, a nucleic acid having a base sequence encoding the protein of the present invention may be directly introduced into cells using a technique known in the art.
  • an antisense nucleic acid having a base sequence complementary to the nucleic acid of the present invention and capable of suppressing the expression of the nucleic acid, and a protein of the present invention using the same.
  • an antisense nucleic acid having a nucleotide sequence complementary to the nucleic acid according to any one of claims 9 to 11 of the present invention and capable of suppressing the expression of the nucleic acid is used as a lipozyme or decoy. You can also. In this case, for example, it is usually preferable to use 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 represented by SEQ ID NO: 1. preferable.
  • the antisense nucleic acid is introduced into a cell, and the expression of a gene encoding the protein is expressed. Modulation can alter (eg, inhibit) the function or biological activity of the receptor protein of the present invention.
  • an antisense nucleic acid can be added to a cell containing a target nucleic acid sequence by a DNA transfection method such as a calcium phosphate method, a lipofection method, an electoral poration method, a microinjection method, or a gene transfer vector such as a virus.
  • the function or biological activity of the protein of the present invention can be modulated (for example, suppressed) by introduction using a method known in the art such as a gene introduction method including use.
  • a vector that expresses the oligonucleotide is prepared using an appropriate retroviral vector, and then the expression vector may be introduced into a cell containing the target nucleic acid sequence by contacting the cell with the cell in vivo or exvivo. .
  • the gene of the present invention can also be detected by using the oligonucleotide or its complement as a probe. .
  • the present invention provides, in the sixth aspect, an antibody that specifically binds to the protein of the present invention and neutralizes the function or biological activity of the protein, and the function or the function of the protein of the present invention using the antibody. Methods for inhibiting biological activity are provided.
  • An antibody that recognizes and specifically binds to the protein of the present invention includes the protein of the present invention, or a protein or peptide having immunological equivalence thereto, for example, a synthetic peptide having a protein fragment or partial sequence. And the like as an antigen.
  • having immunological equivalence means, for example, that a cross-reactivity occurs with an antibody against the protein of the present invention.
  • the antibody may be a polyclonal antibody or a monoclonal antibody.
  • the polyclonal antibody can be produced by a usual method of inoculating a host animal (for example, rat or egret) with an antigen and collecting immune serum.
  • Monoclonal antibodies can be produced by techniques such as the conventional hybridoma method.
  • a humanized monoclonal antibody or the like can be prepared by modifying the gene of the monoclonal antibody.
  • the expression of the protein of the present invention in cells or tissues can be detected by a usual immunochemical method (such as immunochemical assay). Alternatively, affinity chromatography using antibodies Thus, the protein of the present invention can be purified.
  • the function or biological activity of the protein of the present invention can be suppressed by contacting the neutralizing antibody of the present invention with a cell.
  • the neutralizing antibody of the present invention is combined with an inactive ingredient such as an immunogenic adjuvant or a further active ingredient for therapeutic use together with a stabilizer and an excipient, sterilized by filtration, and then lyophilized, or It can be obtained as a stock in a stabilized aqueous preparation.
  • an inactive ingredient such as an immunogenic adjuvant or a further active ingredient for therapeutic use together with a stabilizer and an excipient, sterilized by filtration, and then lyophilized, or It can be obtained as a stock in a stabilized aqueous preparation.
  • a method known in the art such as subcutaneous injection, intraarterial injection, or intravenous injection.
  • the dose can be appropriately selected by those skilled in the art according to the patient and the administration method.
  • the protein of the present invention has a function or a biological activity selected from the following (i), (ii) and (iiii) as described above.
  • ligand ⁇ Gore second ligand that acts as a strike
  • 4-HNE is included, the intracellular signaling, C a 2 + concentration change, change in concentration of c AM P, activity of phosphorylase Horipaze C , Changes in pH, changes in K + concentration, and the like.
  • the protein (receptor) of the present invention specifically binds to a lipid peroxidation reaction product, and the binding stimulates the protein, activates the G protein, and transduces a signal into a cell through the activation. Is induced.
  • Activating a G protein and activating it means that the G protein is converted into an active form (GTP type) and the ⁇ ,
  • G a include ⁇ subunits of G proteins belonging to G i subfamily, for example, G i ⁇ 1 .
  • the method for detecting the function or biological activity of (i) can be achieved, for example, by performing the following steps.
  • (B) a step of detecting the presence or absence or the strength of specific binding between the protein and a ligand.
  • binding can be detected, for example, by using a ligand to which a detectable label (for example, RI label, fluorescent label, etc.) is attached, and measuring the label.
  • a detectable label for example, RI label, fluorescent label, etc.
  • specific binding may be detected by a general competitive assay using a mixture of a labeled ligand and an unlabeled ligand.
  • the method for detecting the function or biological activity of (ii) can be achieved, for example, by performing the following steps.
  • (A) a step of bringing the protein of the present invention into contact with a ligand (a ligand that acts as an agonist);
  • (B) C a 2 + concentration change within the cell, changes in the concentration of c AM P, activity of phospholipase C, measuring the change or K + concentration change of p H.
  • Cells which express (or do not express) the protein 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 is high, it is deemed to have (ii) a function or biological activity according to the degree.
  • the method for detecting the function or biological activity of (iii) may be, for example, as follows. It can be carried out.
  • (A) a step of preparing a membrane fraction from cells in which a fusion protein of the protein of the present invention and an ⁇ -subunit of a G protein belonging to the Gi subfamily is expressed on a cell membrane;
  • the stimulation by ligand By comparing the presence or absence or the strength of the binding in the presence or absence of the ligand (ligand acting as agonist), the stimulation by ligand (ligand acting as agonist) can be performed. Determining activation of the G protein based on the activation. For the labeled GTP or its analog, for example, a GTP analog that is hardly decomposed, such as GTPyS (guanosine 5, -0- (3-thiotriphosphate)), can be used. If the binding level in the presence of the lipid peroxidation reaction product is higher than the binding level in the absence of the lipid peroxidation reaction product, the function or biological function of (iii) depends on the degree. Recognized as active.
  • the amino acid sequence and base sequence of G i and its gene are already known (human G i ⁇ 1 (351 Cys ⁇ Ile) / Bahia et al., Biochemistry, Vol. 37, pp. 11555-11562, 1998: Human Gi ⁇ 1 / Genbank / EMBL accession no. AF055013, PIR / SWISS-PROT accession no. P04898: etc.). Therefore, the DNA encoding Gia can be obtained by utilizing the disclosed known sequence information, such as the PCR (polymerase chain reaction) method, colony neutralization method, and plaque hybridization method. Alternatively, these can be appropriately combined and selected from a DNA library.
  • a DNA encoding Gi ⁇ can be ligated downstream of the DNA encoding the polypeptide of the present invention and inserted into a vector containing an appropriate promoter to obtain a vector for expressing the fusion protein.
  • the expression vector of the fusion protein can be introduced into cells to express the fusion protein.
  • the present invention in a seventh aspect, identifies a ligand for the protein of the present invention. Provide a way to:
  • ligand means a compound that specifically binds to a receptor protein.
  • Ligands include both naturally occurring and artificially synthesized compounds.
  • the ligand may include the following agonist or antagonist.
  • the protein of the present invention used in the present method also includes a protein in the form of a membrane fraction containing the protein or a cell in which the protein is expressed on the cell surface.
  • the identification of the ligand can be specifically performed, for example, as follows.
  • (C) a step of determining the presence or absence or the strength of the binding ability between the protein and a test compound.
  • Such specific binding can be detected, for example, by a conventional competitive assay method using a known ligand compound with a detectable label (eg, RI label, fluorescent label, etc.) mixed with an unlabeled test compound.
  • a detectable label eg, RI label, fluorescent label, etc.
  • the test compound is not particularly limited, and includes a low-molecular compound, a peptide, and the like.
  • the test compound may be artificially synthesized or naturally occurring.
  • a test compound (ligand) having specific binding ability is likely to be an agonist (agonist or antagonist).
  • the present invention provides, in an eighth aspect, a method for identifying an agonist or an antagonist for the protein of the present invention.
  • agonist means a compound capable of inducing intracellular signal transduction by stimulating the receptor protein by specifically binding to the receptor protein.
  • antagonist refers to a compound having the ability to suppress the action of a compound capable of inducing intracellular signal transmission by stimulating a receptor protein.
  • the identification of the agonist or antagonist can be performed, for example, as follows.
  • (A) a step of bringing the protein of the present invention into contact with a test compound and, if necessary, a ligand (preferably a ligand acting as agonist);
  • Intracellular signaling e.g., changes in the concentration of C a 2 tau, changes in the concentration of cAMP, phosphorylase Activation of Horipaze C, change in p H, ⁇ Pi, or to detect changes in the concentration, etc.
  • K + Intracellular signaling (e.g., changes in the concentration of C a 2 tau, changes in the concentration of cAMP, phosphorylase Activation of Horipaze C, change in p H, ⁇ Pi, or to detect changes in the concentration, etc.) of K +, or, the step of detecting the activation of G proteins (subunit of G protein belonging to the G t family, etc.) , as well as
  • (C) a step of determining whether or not the test compound has the ability to induce or suppress intracellular signal transduction or activation of G protein.
  • (A) a step of bringing the protein of the present invention into contact with a test compound and, if necessary, a ligand (preferably a ligand acting as agonist);
  • (C) a step of determining whether or not the test compound has the ability to induce or suppress intracellular signal transduction by measuring the change.
  • (A) a step of preparing a membrane fraction from cells in which a fusion protein of the protein of the present invention and an ⁇ -subunit of a G protein belonging to a Gi subfamily is expressed on a cell membrane;
  • test compound (D) a step of determining whether or not the test compound has the ability to induce or suppress the activation of G protein by detecting the binding.
  • control In determining the ability of a test compound, it is advisable to take appropriate controls. Examples of such a control include detection in the absence of a test compound, detection using a cell which does not express the receptor protein of the present invention, or which has a lower expression level of the receptor protein. Tests are given. More than one of these controls may be combined for more accurate determination.
  • Compounds identified as antagonists by the above method or the like include (the protein of the present invention) By contacting the cells with cells expressing white matter), the biological function or activity of the receptor for the lipid peroxidation reaction product in the cells can be suppressed.
  • the antagonist against the protein substance (receptor of lipid peroxidation reaction product) of the present invention can be used as an active ingredient of a medicine. Therefore, a compound identified as an antagonist to the protein (receptor of lipid peroxidation reaction product) 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 it 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.
  • it may be formulated as a conventional pharmaceutical preparation (tablets, granules, capsules, powders, injections, inhalants, etc.) together with an inert carrier according to the method of administration.
  • a compound may be used together with activators or diluents such as binders, disintegrants, bulking agents, fillers, lubricants, and the like, which are acceptable in general pharmaceuticals, and may be used in the form of a preparation by ordinary methods. it can.
  • the dosage varies depending on the method of administration, the age of the patient, body weight, and condition, but general dosages, for example, l-300 mg / kg per day for oral administration, parenteral administration Is set in the range of 0.01 to 5 O mg Z kg.
  • the protein of the present invention used for identifying a ligand, agonist or antagonist can be used in the form of a membrane fraction containing the protein or in the form of a cell expressing the protein on the cell surface. it can.
  • a cell or the like in which the receptor protein or the protein is overexpressed can be used.
  • cells into which a recombinant vector containing the desired nucleic acid has been introduced can be used.
  • a cell capable of expressing the foreign receptor protein on the cell membrane without impairing its function
  • the cell itself before the introduction of the recombinant vector does not express the target receptor protein or protein or has only a low level. It is desirable to use expressing cells.
  • the membrane fraction containing the protein is obtained by disrupting the protein or cells expressing the protein, and then using a fractionation method utilizing centrifugal force such as fractionation centrifugation or density gradient centrifugation.
  • a fractionation method utilizing centrifugal force such as fractionation centrifugation or density gradient centrifugation.
  • the cell lysate is centrifuged at a low speed (about 500 to 300 rpm) for a short time (usually about 1 to 10 minutes).
  • the suspension is usually centrifuged at about 3000 rpm for about 30 to 120 minutes, and the obtained precipitate fraction is used as a membrane fraction.
  • the ligand, agonist and antagonist compounds of the present invention have an action of inducing or suppressing the activation of a G protein-coupled receptor using a lipid peroxidation reaction product as a ligand, a G protein using a lipid peroxidation reaction product as a ligand It can be an effective component of a pharmaceutical composition for treating or preventing a disease associated with the function or biological activity of a conjugated receptor.
  • the present invention provides a pharmaceutical composition for treating or preventing a disease associated with the function or biological activity of a G protein-coupled receptor having a lipid peroxidation reaction product as a ligand. .
  • a pharmaceutical composition can be prepared by the following conventional method. For example, an effective amount of a compound identified as a ligand, agonist or antagonist, or a pharmaceutically acceptable salt thereof, is mixed with a pharmaceutically acceptable carrier. Next, the dosage form is adjusted to the dosage form.
  • the dosage form of the pharmaceutical composition used in the present invention includes oral administration, intravenous administration, intramuscular administration, intraarterial administration, intramedullary administration, intrathecal administration, intraventricular administration, transdermal administration, subcutaneous administration, intraperitoneal administration Examples include, but are not limited to, internal, nasal mucosal, enteral, topical, sublingual, or rectal administration.
  • compositions for oral administration include solid forms such as tablets, granules, capsules, pills, and powders, and liquid forms such as solutions, syrups, elixirs, and suspensions. It is. When used for parenteral administration, dosage forms such as sterile solutions, suspensions, and emulsions are included.
  • the carrier include sugars, starches, fatty acids, talc, vegetable oils, gums, glycols, physiological saline, buffered saline, and the like. I can do it.
  • 4-HNE which is an agonist of the protein (receptor) of the present invention, is produced in vivo as a lipid peroxidation reaction product when the organism is subjected to oxidative stress (Esterbauer, H , et al., Free Radical Biology & Medicine, 11: 81-128, 1991).
  • HNE and other lipid peroxidation products include, for example, the following diseases, aging (Stadtman, ER, Science, 257: 1220-1224, 1992), arterial sclerosis (Palinski, W., et al.) Natl. Acad. Sci. USA, 86: 1372-1376, 1989), Parkinson's disease (Yoritaka, A., et al., Proc. Natl. Acad. Sci. 93: 2696-2701, 1996), Kanoman, et al. (Mark, RJ, et al., J. Neurochem., 68: 255-264, 1997; Kruman,
  • the pharmaceutical composition containing the protein (receptor) agonist, antagonist, antibody or the like of the present invention as an active ingredient, which is obtained by the above identification method, can be used for aging, atherosclerosis, Parkinson's disease, Alzheimer's disease, etc. It is expected to be effective in treating or preventing various diseases.
  • Human genomic DNA (trade name “Human Genomic DNAJ; manufactured by Kukuntec Co., Ltd.) was used as the type II of the PCR, and an oligonucleotide having the base sequence shown in SEQ ID NO: 3 was used as a sense primer. Oligonucleotides having the nucleotide sequence shown in SEQ ID NO: 4 were used as sense primers. These primers were obtained by adding restriction enzyme recognition sites (Kpnl site and Hindlll site) to both ends of the PCR product. It is designed to obtain fragments.
  • PCR buffer (Advantage 2 PCR Buffer, Clontech) 5 ⁇ 1 Deoxynucleotide solution (dATP, dCTP, dGTP and dTTP, 10 mM each) 1 ⁇ 1 Polymerase solution (Advantage 2 Polymerase Mix, manufactured by Kukuntec) 1 ⁇ 1 Sense primer (10 ⁇ ) 1 l ⁇ 1
  • PCR buffer Advantage 2 PCR Buffer, Clontech
  • Deoxynucleotide solution dATP, dCTP, dGTP and dTTP, 10 mM each
  • Polymerase solution (Advantage 2 Polymerase Mix, manufactured by Kukuntec) 1 ⁇ 1 Sense primer (10 ⁇ ) 1 l ⁇ 1
  • the obtained PCR product is subjected to agarose gel electrophoresis, and a band is cut out to cut out about 1,000 bp.
  • the cDNA fragment was purified and obtained.
  • nucleotide sequence of the cloned TG003 gene was compared with the nucleotide sequence of GenBank / EMBL accession no. AF000545 in the NCBI human genome database, a difference of 1 It was considered due to a genetic polymorphism or due to a PCR error.
  • the amino acid sequence (339 amino acid residues) corresponding to the nucleotide sequence of the TG03039 gene represented by SEQ ID NO: 1 thus obtained was as shown in SEQ ID NO: 2.
  • the transmembrane region was predicted by S0SUI, and as a result, a seven-transmembrane region characteristic of a G protein-coupled receptor was estimated.
  • FIG. 1 shows the base sequence of the TG03039 gene, the amino acid sequence corresponding to the sequence, and the transmembrane region (underlined).
  • mRNA poly (A) RNA
  • mRNA derived from various human tissues and human cultured cells shown in FIG. 2 is adsorbed and fixed.
  • the RI-labeled probe used was prepared as follows. That is, the implementation The plasmid containing the cDNA fragment obtained in section (2) of Example 1 was subjected to PCR using the plasmid as type III. At that time, as the sense primer and the antisense primer, synthetic oligonucleotides having the nucleotide sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively, were used. The obtained PCR product was purified and obtained by agarose gel electrophoresis to purify a DNA fragment of about 560 bp (cDNA fragment containing the translation region of the TG03039 gene).
  • This cDNA fragment was used as a type III primer kit containing random hexadeoxyribonucleotides, a mixture of nucleotides (dATP, dGTP and dTTP), and DNA polymerase I (Large (Klenow) Fragment). After labeling using a-Gene Labeling System ⁇ (Flomega) and [hi-32P] dCTP, purification was performed by gel filtration to prepare an RI-labeled probe.
  • the hybridization of the dot blot was performed as follows.
  • the membrane with the immobilized mRNA was treated with 160 ⁇ l of Solution I [Salmon Testes DNA (9.4 g / ⁇ 1: SIGMA) at 97 ° C for 5 minutes, cooled on ice, and then cooled to 60 ° C.
  • ExpressHyb hybridization Solution (prepared by adding 15 ml of Clontech)] at 68 ° C for 30 minutes. Then, this membrane was added to a hybridization solution containing a labeled probe [the RI-labeled probe 20 ⁇ 1, human
  • COT-1 DNA (1 / zg / l: Roche) 30 ⁇ 1, Salmon Testes DNA (9.4 ⁇ g // x1: SIGMA) 16 ⁇ 1, 20XSSC (3M sodium chloride, 300 mM sodium citrate) , PH7.0)
  • Figure 2 shows the results of detecting the signal of hybridization using an image analyzer (Bio-imaging Analysis System 2000, manufactured by Fuji Huinolem).
  • image analyzer Bio-imaging Analysis System 2000, manufactured by Fuji Huinolem.
  • FIG. 2 strong signals were observed in mRNAs from lymph nodes, peripheral blood leukocytes, spleen, appendix, ileum, thymus, lung and Daudi cells. From these results, The TGOO39 gene was considered to be relatively highly expressed in lymph nodes, peripheral blood leukocytes, spleen, appendix, ileum, thymus, lung and Daudi cells.
  • GTP T / S (guanosine 5'-O- (3-thiotriphosphate) was used as follows by using a membrane fraction containing a fusion protein of TG003 protein and G protein. Method for Detecting Ligand-Dependent Binding of Cwenzel-Seifert et al., Mol. Pharmacol., Vol. 58, 954-966, 2000; Bahia et al., Biochemistry, 37, 11555-11562, 1998 ], The ligand was identified.
  • (I) ⁇ a manner as (two), TG 0 0 3 9 protein and G protein plasmid for expressing the [G id (351Cys ⁇ Ile), G qa, or G S fd] fusion proteins with was prepared.
  • a schematic diagram of the fusion protein is shown in FIG.
  • G id (351Cys ⁇ Ile) refers to mutation type G i protein obtained by converting the 351 th cysteine residues in G i protein with an isoleucine residue.
  • PCR was performed using the plasmid containing the TG03039 gene cDNA (including the entire length of the translation region) obtained in section (2) of Example 1 as type III.
  • the sense primer and the antisense primer synthetic oligonucleotides having the nucleotide sequences shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively, were used.
  • These primers were designed based on the cDNA base sequence of the TG03039 gene (SEQ ID NO: 1), and as a PCR product, cDNA encoding the full-length TG003 protein (with a stop codon). This is designed to obtain a DNA fragment with restriction enzyme recognition sites at both ends (Xhol site at the N-terminus and Cpol and Hindlll sites at the C-terminus).
  • the PCR product thus obtained was ligated to Vector Plasmid (a vector system for cloning the PCR product) (pGEM-T Easy Vector, manufactured by Promega), and the resulting plasmid was ligated with restriction enzymes NotI and ⁇ The DNA fragment of about 1,000 bp was recovered by treatment with CpoI.
  • Vector Plasmid a vector system for cloning the PCR product
  • pGEM-T Easy Vector manufactured by Promega
  • PCR was performed using human brain-derived cDNA (Marathon-Ready cDNA Brain, manufactured by Clontech) as type III. At that time, as the sense primer (primer G iffH ) and the antisense primer (primer G id — 2 ), synthetic oligonucleotides having the base sequences shown in SEQ ID NO: 9 and SEQ ID NO: 10 were used, respectively.
  • primers are known nucleotide sequence of the c DN A encoding a G i protein
  • Genbank / EMBL accession no. AF055013 are those designed based on, that is designed to c DN A encoding the full-length G id protein as PCR products can be obtained.
  • Mutant Gid protein in which the 351st cysteine residue has been replaced with an isoleucine residue including cDNA encoding the full length, and restriction enzyme recognition sites (CpoI and BamHI sites) at both ends It is designed to obtain a DNA fragment to which is added.
  • the obtained PCR product was ligated to Vectorplasmid (pGEM-T Easy Vector, manufactured by Promega).
  • the resulting plasmid was treated with restriction enzymes NotI and BamHI, and the resulting DNA fragment of about 1,100 bp was purified from the baculovirus vector plasmid pVL1392 (Pharmingen). It was inserted into the N ot I / B a mH I site to give the plasmid pVL1392 / G id (351Cys ⁇ Ile ).
  • a second PCR was performed using the product obtained by the PCR as a ⁇ type.
  • the sense primer primer Gqf 3
  • the antisense primer primer Gq ( )
  • synthetic oligonucleotides having the nucleotide sequences shown in SEQ ID NOS: 15 and 16, respectively, were used.
  • the obtained PCR product was ligated to Vector Plasmid (pGEM-T Easy Vector, manufactured by Promega).
  • the obtained plasmid was treated with restriction enzymes Not I and BamHI, and the resulting DNA fragment of about 1,100 bp was digested with the Not I / B of baculovirus vector plasmid pVL1392. Insertion into the amHI site yielded the plasmid pVL13392 / Gq.
  • PCR was performed using human bone marrow-derived cDNA (Marathon-Ready cDNA Bone marrow ⁇ Clontech) as type II. At that time, as the sense primer (primer aH) and antisense primer (primer G sa), respectively, using synthetic oligonucleotides having the nucleotide sequences shown in SEQ ID NO 1 7 ⁇ Pi SEQ ID NO: 1 8.
  • These primers have a known nucleotide sequence of cDNA encoding GsaI /.
  • the obtained PCR product was ligated to Vectorplasmid (pGEM-T Easy Vector, manufactured by Promega).
  • the resulting plasmid was treated with the restriction enzymes NotI and XbaI, and the resulting DNA fragment of about 1,200 bp was digested with the Bacchus Winores vector plasmid p.
  • Plasmid PVL1392ZGS (d was obtained by inserting into NotI / XbaI site of VL1392 (manufactured by Farmingzin).
  • the DNA fragment obtained in (1) above was inserted into the restriction enzyme NotiZCpoI site of this plasmid, and the plasmid for expression of a fusion protein, pVL1392 / TG0039—
  • the three expression plasmids obtained in the above (1) (mouth) and (2) are linker sequences added to the C-terminal of the TG0039 protein. through (an G ly -P ro-), TG00 3 9 protein (full length) and G protein [Gi d (351Cys ⁇ Ile), G qa, or G S (tL] is expressed fusion protein having a structure linked It is a plus.
  • Plasmids for expression of these fusion proteins p VLl 39 2 / TG 0 39 -G 1
  • TG0039 -G SttIj was expressed in insect cells as follows, and a membrane fraction containing the fusion protein was prepared.
  • a culture medium (10% fetal bovine serum, 0.1 mg / l) was prepared by dissolving insect cells Sf9 (Spodoptera frugiperda SF9) (Pharmingen) in a collagen-coated 3 cm Petri dish so that about 60% confluent was obtained.
  • Sf9 Spodoptera frugiperda SF9
  • the mixture was incubated at 27 ° C for 15 minutes in Grace's Insect Cell Culture Medium (pH 6.2: manufactured by Lifetech Oriental) containing ml streptomycin and 100 units / ml penicillin.
  • the culture solution was removed, and 1.2 ml of medium was added, followed by culturing at 27 ° C for 5 days.
  • the obtained culture solution was centrifuged (1, 000 Xg, 5 minutes), and the supernatant was recovered as virus solution I.
  • Sf9 cells were seeded in a collagen-coated 3 cm Petri dish at a confluence of about 30%, and the virus solution I (100 ⁇ ) obtained above and 1.2 ml of medium were added and cultured at 27 ° C for 4 days. .
  • the obtained culture solution was centrifuged (1, 000 Xg, 5 minutes), and the supernatant was collected as a virus solution.
  • Sf9 cells were seeded on a collagen-coated 10 cm Petri dish so as to have a confluence of about 70%, the virus solution II (500 ⁇ 1) obtained above and 12 ml of medium were added, and the cells were cultured at 27 ° C for 4 days. The culture solution thus obtained was centrifuged (1, 000 Xg, 5 minutes), and the supernatant was recovered as virus solution III.
  • Sf9 cells are seeded in a collagen-coated 10 cm Petri dish so as to have about 70% confluence, and the virus solution ⁇ (100 ⁇ ) obtained above and 12 ml of medium are added, and the mixture is incubated at 27 ° C for 4 days. Cultured.
  • the cells thus obtained were washed with cooled PBS (Phosphate buffered saline, pH 7.4), and then cooled with a lysis buffer (20 mM Tris-HCl, pH 7.5, lmMEDTA, 0.2 raM phenylmethylsulfonyl fluoride, 10 ⁇ g / ml of pepstatin, 10 ⁇ g / ml of leptin, and 2 ⁇ g / ml of aprotin], and the cells were disrupted using a Teflon homogenizer. This cell lysate was centrifuged (600 ⁇ g, 10 minutes), and the obtained supernatant was further centrifuged (50,000 ⁇ g, 20 minutes).
  • PBS Phosphate buffered saline, pH 7.4
  • a lysis buffer 20 mM Tris-HCl, pH 7.5, lmMEDTA, 0.2 raM phenylmethylsulfony
  • the resulting precipitate was suspended in 450 ⁇ l of a cooled reaction buffer (20 mM Tris-HCl, pH 7.5, 50 mM sodium chloride, 10 mM magnesium chloride) using a Teflon homogenizer, and the fusion protein was suspended.
  • the expressed membrane fraction was obtained.
  • the membrane fraction (450 ⁇ ) obtained in the above (2) was suspended in a reaction buffer (7.54 ml), and 10 ⁇ GDP of GDP (10 mM) was added thereto.
  • Fig. 4 shows the results.
  • a membrane fraction containing the TG 0 0 3 9 fusion protein between the protein and Gi ftl (351Cys ⁇ Ile) by the child added 4-HNE, a concentration-dependent manner [35 S] of GTP y S The amount of specific binding increased.
  • the TG03039 protein was a G protein-coupled receptor of the type that couples to the Gi ⁇ 1 protein.
  • 4- ⁇ ⁇ ⁇ acts as the agonist (ligand). That is, the TG003 protein was considered to be a receptor for a peroxidation reaction product of lipid having 4- ⁇ as an agonist.
  • the G protein-coupled receptor protein having the lipid peroxidation reaction product of the present invention as a ligand and its gene are useful for studying the mechanism of intracellular signal transduction.
  • it can be a target molecule of a therapeutic drug for a disease relating to a G protein-coupled receptor using a lipid peroxidation reaction product as a ligand.
  • SEQ ID NO: 3 Synthetic DNA
  • SEQ ID NO: 5 synthetic DNA
  • SEQ ID NO: 6 synthetic DNA
  • SEQ ID NO: 9 synthetic DNA
  • SEQ ID NO: 10 synthetic DNA
  • SEQ ID NO: 11 synthetic DNA
  • SEQ ID NO: 12 synthetic DNA
  • SEQ ID NO: 14 Synthetic DNA
  • SEQ ID NO: 15 Synthetic DNA
  • SEQ ID NO: 16 Synthetic DNA
  • SEQ ID NO: 17 Synthetic DNA
  • SEQ ID NO: 18 Synthetic DNA

Abstract

It is intended to provide a novel G protein-coupled receptor protein with the use of a lipid peroxidation reaction product as a ligand and its gene. It is also intended to provide a method of identifying a novel ligand, an agonist or an antagonist and medicinal compositions comprising as the active ingredient a ligand, an agonist, an antagonist, etc. More specifically speaking, a purified protein having the amino acid sequence represented by SEQ ID NO:2; and a purified protein having an amino acid sequence derived from the amino acid sequence represented by SEQ ID NO:2 by deletion, substitution or addition of one to several amino acids and having a function or a biological activity as a G protein-coupled receptor protein with the use of a lipid peroxidation product as a ligand.

Description

明 細 書 新規 G蛋白質共役型受容体及びその遺伝子 技術分野  Description Novel G protein-coupled receptor and its gene
本発明は、 脂質過酸化反応産物をリガンドとする新規な G蛋白質共役型受容体 蛋白質及びその遺伝子に関する。 また、 これらを用いた医薬品候補化合物の同定 方法及び医薬組成物に関する。 背景技術  The present invention relates to a novel G protein-coupled receptor protein having a lipid peroxidation reaction product as a ligand and a gene thereof. The present invention also relates to a method for identifying a drug candidate compound and a pharmaceutical composition using the same. Background art
多くの神経伝達物質、 ホルモン、 ォータコイドなどの生理活性物質は、 細胞表 面に存在する特異的な受容体蛋白質を介して、 生体の機能を調節している。 この ような受容体の多くは、 細胞内に存在する三量体の GTP結合蛋白質 （以下、 G 蛋白質と称する） と共役しており、 その活性化を通じて情報を細胞内に伝達する ことが知られている。 このことから、 これら受容体は G蛋白質共役型受容体と総 称される。 G蛋白質共役型受容体は、 いずれも 7個の膜貫通領域を含む共通の構 造を有していることが知られている。  Many biologically active substances such as neurotransmitters, hormones, and otacoids regulate the functions of living organisms through specific receptor proteins present on the cell surface. Many of these receptors are coupled to a trimeric GTP-binding protein (hereinafter referred to as G protein) present in cells, and are known to transmit information into cells through their activation. ing. 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蛋白質は、 ひ、 及び γサブユニットで構成される三量体であり、 基底状態 ではこれらサブュ,ニットが会合した不活性型 （GDP結合型） として存在する。 リガンド刺激を受けた G蛋白質共役型受容体により、 不活性型 （GDP結合型） G蛋白質が活性型 （GTP結合型） へと変換され、 GT Ρと結合した αサブュ- ット （Ga) と /3/γサブユニット複合体 （Gj3 y) とに解離する。 そして、 G TPと結合した Gee (また場合によっては G ]3 γ) 力 アデ二ル酸シクラーゼ、 ホスホリパーゼ Cなどの効果器を制御してシグナルを伝達する。 The G protein is a trimer composed of spike and γ subunits, and exists in an inactive form (GDP binding form) in which these subunits are associated in the ground state. Inactive (GDP-bound) G protein is converted to active (GTP-bound) by ligand-stimulated G protein-coupled receptor, and α submit (Ga) bound to GTΡ and Dissociates with the / 3 / γ subunit complex (Gj3 y). Gee coupled with GTP (and in some cases, G] 3γ), controls signals such as adenylate cyclase and phospholipase C to transmit signals.
G蛋白質は G αに多様性があり、 G αの種類によって異なる効果器を制御する。 一般に、 G蛋白質共役型受容体は特定の種類の G蛋白質を活性化し、 その G蛋 白質により特定の情報を細胞内に伝える。  G proteins are diverse in G α and control different effectors depending on the type of G α. Generally, a G protein-coupled receptor activates a specific type of G protein, and transmits specific information into a cell by the G protein.
G蛋白質共役型受容体としては、 これまでに、 一及び —アドレナリン受容 体、 ムスカリン性ァセチルコリン受容体、 アデノシン受容体、 アンジォテンシン 受容体、 エンドセリン受容体、 ゴナドトロピン放出因子受容体、 ：《1—及び1 2 一ヒスタミン受容体、 ドーパミン受容体、 代謝型グルタミン酸受容体、 ソマ トス タチン受容体、 プリン受容体などが知られている。 As G protein-coupled receptors, there have been one and the following: adrenergic receptor, muscarinic acetylcholine receptor, adenosine receptor, angiotensin Receptors, endothelin receptors, gonadotropin-releasing factor receptors, are known: << 1- and 12-histamine receptors, dopamine receptors, metabotropic glutamate receptors, somatostatin receptors, purine receptors, etc. .
上記のような受容体は、 いずれも生理活性物質の標的として、 生体内での重要 な役割を担っている。 さらに、 現在までに知られている医薬品の多くが、 やはり G蛋白質共役型受容体を標的とするリガンド、 あるいはァゴニストゃアンタゴニ ストであったという事実も非常に意義深い。  Each of the above receptors plays an important role in vivo as a target of a physiologically active substance. Furthermore, the fact that many of the drugs known to date were also ligands targeting G protein-coupled receptors, or agonists-antagonists, is also very significant.
これらのことから、 G蛋白質共役型受容体は、 医薬品開発の標的として注目さ れている。 従って、 新たな G蛋白質共役型受容体を見出すこと、 そのリガンドを 同定すること、 及び、 それらのァゴ-ストやアンタゴニストを同定する方法を見 出すことは、 新たな医薬品候補化合物の同定につながり、 そのような同定や開発 が強く望まれている。  For these reasons, G protein-coupled receptors are attracting attention as targets for drug development. Therefore, finding new G protein-coupled receptors, identifying their ligands, and finding ways to identify their agonists and antagonists will lead to the identification of new drug candidates. Such identification and development are strongly desired.
しかしながら、 これまでにすべての G蛋白質共役型受容体が確認されているわ けではなく、 未だ、 未知の受容体 （ォーファン受容体） が数多く存在していると 考えられる。 このため、 それらのォーファン受容体の特定とそれに対応するリガ ンドの探索を行い、 その機能解明と利用が切望されている。  However, not all G protein-coupled receptors have been confirmed so far, and it is thought that there are still many unknown receptors (orphan receptors). For this reason, identification of these orphan receptors and search for the corresponding ligands are required, and elucidation of their functions and utilization are eagerly awaited.
近年、 生体内で発現している c D N Aの配列をランダムに解析し、 得られた c D N Aの断片配列が Expressed Sequence Tag (EST) としてデータベースに登録 され、 公開されてきているが、 E S T情報の多くは配列情報のみが開示されてい るにすぎず、 G蛋白質共役型受容体蛋白質の探索の分野においても、 E S T配列 から遺伝子の活性や機能を推定することは容易ではない。  In recent years, the sequence of cDNA expressed in vivo has been randomly analyzed, and the obtained cDNA fragment sequence has been registered in a database as an Expressed Sequence Tag (EST) and published. In many cases, only sequence information is disclosed, and it is not easy to estimate the activity or function of a gene from an EST sequence in the field of searching for a G protein-coupled receptor protein.
一方、 生体が酸化ス トレスを受けた場合、 脂質膜の過酸化反応産物 （脂質の過 酸化物の分解物） として 4 -ヒ ドロキシ- 2-ノネナール （4 -ヒ ドロキシノネナール または 4 -ヒドロキシノン- 2 -ェナールとも称する) 等のアルデヒ ドが生体内で産 生されること力 s失口られてレヽる (Esterbauer, H. , et al. , Free Radical Biology &On the other hand, when an organism undergoes oxidative stress, 4-hydroxy-2-nonenal (4-hydroxynonenal or 4-hydroxynone) is produced as a peroxidation reaction product (decomposed product of lipid peroxide) of the lipid membrane. - 2 -. Enaru also referred to) and the like aldehydes can be that force s lost opening produced in vivo in Rereru (Esterbauer, H., et al, Free Radical Biology &
Medicine, 11 : 81-128， 1991)。 これらの脂質過酸化反応産物は、 生成後にタンパ ク質と結合することが報告されており、例えば、老化 （Stadtman, E. R.， Science,Medicine, 11: 81-128, 1991). These lipid peroxidation products have been reported to bind to proteins after formation, and are described, for example, in aging (Stadtman, ER, Science,
257 : 1220-1224， 1992)、動脈硬化 (Pal inski , W. , et al.， Proc. Natl. Acad. Sci.257: 1220-1224, 1992), arteriosclerosis (Pal inski, W., et al., Proc. Natl. Acad. Sci.
USA, 86 : 1372-1376, 1989)、 パーキンソン病 （Yoritaka, A.， et al. , Proc. Natl. Acad. Sci. USA, 93 : 2696—2701， 1996)、 ァノレツハイマー病 （Mark, R. J. , et al. , J. Neurochem. , 68： 255-264, 1997； Kruman, I. , et al. , J. Neurosci. , 17 : 5089-5100， 1997) 等の疾患と関係していることが報告されている。 しかしな がら、 脂質過酸化反応産物をリガンドとする G蛋白質共役型受容体にっレ、てはこ れまで報告されていない。 発明の開示 USA, 86: 1372-1376, 1989), Parkinson's disease (Yoritaka, A., et al., Proc. Natl. Acad. Sci. USA, 93: 2696-2701, 1996), Panorezheimer's disease (Mark, RJ, et al., J. Neurochem., 68: 255-264, 1997; Kruman, I., et al., J.). Neurosci., 17: 5089-5100, 1997). However, no G protein-coupled receptor using a lipid peroxidation reaction product as a ligand has been reported. Disclosure of the invention
本発明の目的は、 脂質過酸化反応産物をリガンドとする新規な G蛋白質共役型 受容体及びその遺伝子を提供することにある。 また、 当該受容体蛋白質を用いた リガンド及ぴ作用薬 （ァゴ二スト又はアンタゴニスト） の同定方法及ぴ医薬組成 物を'提供することにある。 さらには、 上記以外の目的については以下の記載より 明らかである。  An object of the present invention is to provide a novel G protein-coupled receptor using a lipid peroxidation reaction product as a ligand and a gene thereof. Another object of the present invention is to provide a method for identifying a ligand and an agonist (agonist or antagonist) using the receptor protein and a pharmaceutical composition. Further, the purpose other than the above is clear from the following description.
本発明者等は、 上記課題を解決するため鋭意研究を行った結果、 新しい G蛋白 質共役型受容体をコードする全長 c D N Aをヒ トから単離した。 また、 この受容 体蛋白質を遺伝子組換え技術により細胞中で発現させることに成功した。 さらに リガンドの同定を行い、 この受容体が脂質過酸化反応産物をリガンドとする G蛋 白質共役型受容体であることを見出し、 本発明を完成するに到った。  The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, isolated a full-length cDNA encoding a novel G protein-coupled receptor from human. We have also succeeded in expressing this receptor protein in cells by genetic recombination technology. Further, the ligand was identified, and it was found that this receptor was a G protein-coupled receptor using a lipid peroxidation reaction product as a ligand, thereby completing the present invention.
すなわち、 本発明は、 以下の （1 ) 〜 （3 7 ) を提供する。  That is, the present invention provides the following (1) to (37).
( 1 ) 配列番号 2で表されるアミノ酸配列を有する蛋白質。  (1) A protein having the amino acid sequence represented by SEQ ID NO: 2.
( 2 ) 配列番号 2で表されるアミノ酸配列において、 1若しくは数個のァミノ 酸が欠失、 置換若しくは付加されたアミノ酸配列を有し、 かつ、 脂質過酸化反応 産物をリガンドとする G蛋白質共役型受容体としての機能又は生物学的活性を有 する蛋白質。  (2) G protein conjugate having an amino acid sequence represented by SEQ ID NO: 2 in which one or several amino acids are deleted, substituted or added, and a lipid peroxidation reaction product as a ligand A protein having a function as a type receptor or a biological activity.
( 3 ) 配列番号 2のアミノ酸配列を有する蛋白質とその全長にわたり 7 5 %以 上のアミノ酸配列の相同性を有する蛋白質であり、 かつ、 脂質過酸化反応産物を リガンドとする G蛋白質共役型受容体としての機能又は生物学的活性を有する蛋 白質。  (3) a protein having homology of 75% or more amino acid sequence over its entire length with a protein having the amino acid sequence of SEQ ID NO: 2 and a G protein-coupled receptor having a lipid peroxidation reaction product as a ligand Having a function or biological activity as a protein.
( 4 ) 脂質過酸化反応産物をリガンドとする G蛋白質共役型受容体としての機 能又は生物学的活性が、 以下の （ i )、 ( i i ) 及ぴ （ i i i ) から選択される 1 以上のものである、 （2) 又は （3) に記載の蛋白質。 (4) The function or biological activity as a G protein-coupled receptor having a lipid peroxidation reaction product as a ligand is selected from the following (i), (ii) and (iii): The protein according to (2) or (3), which is as described above.
( i ) リガンドとの特異的結合。  (i) Specific binding with ligand.
( i i ) リガンドによる刺激に基づく細胞内シグナル伝達の誘導。  (ii) Induction of intracellular signaling upon stimulation by ligand.
( i i i ) リガンドによる刺激に基づく G蛋白質の活性化。  (iii) Activation of G protein upon stimulation by ligand.
(5) 脂質過酸化反応産物が 4 -ヒ ドロキシ -2-ノネナールである、 （2) 〜 （4) のいずれかに記載の蛋白質。  (5) The protein according to any one of (2) to (4), wherein the lipid peroxidation reaction product is 4-hydroxy-2-nonenal.
(6) 組換え蛋白質である、 （1) 〜 （5) のいずれかに記載の蛋白質。  (6) The protein according to any one of (1) to (5), which is a recombinant protein.
(7) 哺乳動物の蛋白質である、 （1) 〜 （6) のいずれかに記載の蛋白質。 (7) The protein according to any one of (1) to (6), which is a mammalian protein.
(8) 哺乳動物がヒトである、 （7) に記載の蛋白質。 (8) The protein according to (7), wherein the mammal is a human.
(9) (1) 〜 （3) のいずれかに記載の蛋白質をコードする塩基配列を有す る単離された核酸。  (9) An isolated nucleic acid having a base sequence encoding the protein according to any one of (1) to (3).
(1 0) 以下の （a) 又は （b) の塩基配列を有する単離された核酸。  (10) An isolated nucleic acid having the following nucleotide sequence of (a) or (b).
( a ) 配列番号 1で表される塩基配列。  (a) The nucleotide sequence represented by SEQ ID NO: 1.
( b ) 配列番号 1で表される塩基配列を有する核酸と相補的な塩基配列を有する 核酸とストリンジェントな条件下でハイブリダィズし、 かつ、 脂質過酸化反応産 物をリガンドとする G蛋白質共役型受容体としての機能又は生物学的活性を有す る蛋白質をコードする単離された核酸。  (b) a G protein-conjugated type that hybridizes under stringent conditions with a nucleic acid having a nucleotide sequence complementary to the nucleic acid having the nucleotide sequence represented by SEQ ID NO: 1 and uses a lipid peroxidation reaction product as a ligand An isolated nucleic acid encoding a protein having a function as a receptor or a biological activity.
(1 1) 配列番号 1の核酸と、 その翻訳領域の全長にわたり少なくとも 75% 以上の相同性を有し、 つ、 脂質過酸化反応産物をリガンドとする G蛋白質共役 型受容体としての機能又は生物学的活性を有する蛋白質をコードする単離された 核酸。  (11) a nucleic acid of SEQ ID NO: 1 having at least 75% homology over the entire length of its translated region, and a function or organism as a G protein-coupled receptor having a lipid peroxidation reaction product as a ligand An isolated nucleic acid encoding a protein having biological activity.
(1 2) 脂質過酸化反応産物をリガンドとする G蛋白質共役型受容体としての 機能又は生物学的活性が、 以下の （ i )、 （ i i ) 及ぴ （ i i i ) から選択される 1以上のものである、 （1 0) 又は （1 1) に記載の核酸。  (1 2) The function or biological activity as a G protein-coupled receptor having a lipid peroxidation reaction product as a ligand is one or more selected from the following (i), (ii) and (iii): (10) or the nucleic acid according to (11).
( i ) リガンドとの特異的結合。  (i) Specific binding with ligand.
( i i ) リガンドによる刺激に基づく細胞内シグナル伝達の誘導。  (ii) Induction of intracellular signaling upon stimulation by ligand.
( i i i ) リガンドによる刺激に基づく G蛋白質の活性化。  (iii) Activation of G protein upon stimulation by ligand.
(1 3) 脂寳過酸化反応産物が 4-ヒ ドロキシ- 2 -ノネナールである、 （1 0) 〜 (1 2) のいずれかに記載の核酸。 (14) DNAである、 （9) 〜 （1 3) のいずれかに記載の核酸。 (13) The nucleic acid according to any one of (10) to (12), wherein the fatty acid peroxidation reaction product is 4-hydroxy-2-nonenal. (14) The nucleic acid according to any one of (9) to (13), which is a DNA.
(1 5) 哺乳動物の核酸である、 （9) 〜 （1 4) のいずれかに記載の核酸。 (15) The nucleic acid according to any one of (9) to (14), which is a mammalian nucleic acid.
(1 6) 哺乳動物がヒ トである、 （1 5) に記載の核酸。 (16) The nucleic acid according to (15), wherein the mammal is a human.
(1 7) (9) 〜 （1 6) のいずれかに記載の核酸を含む組換えベクター。 (17) A recombinant vector comprising the nucleic acid according to any one of (9) to (16).
(1 8) (9) 〜 （1 6) のいずれかに記載の核酸、 又は、 （1 7) に記載の組 換えべクタ一で形質転換された細胞。 (18) A cell transformed with the nucleic acid according to any one of (9) to (16) or the recombinant vector according to (17).
(1 9) (6) に記載の組換え蛋白質を細胞表面上に発現している、 （1 8) に 記載の細胞。  (19) The cell according to (18), which expresses the recombinant protein according to (6) on a cell surface.
(20) (1 0) 〜 （1 2) のいずれかに記載の核酸によってコードされるポ リぺプチド。  (20) A polypeptide encoded by the nucleic acid according to any one of (10) to (12).
(2 1) (1 7) に記載の組換えベクターを用いて、 脂質過酸化反応産物をリ ガンドとする G蛋白質共役型受容体の細胞における機能又は生物学的活性を増強 する方法。  (21) A method for enhancing the function or biological activity in a cell of a G protein-coupled receptor using a lipid peroxidation reaction product as a ligand, using the recombinant vector according to (17).
(22) 以下の工程を含む、 脂質過酸化反応産物をリガンドとする G蛋白質共 役型受容体としての機能又は生物学的活性を有する蛋白質の製造方法。  (22) A method for producing a protein having a function as a G-protein-coupled receptor or a biological activity using a lipid peroxidation reaction product as a ligand, comprising the following steps:
(A) (1 8) 又は （1 9) に記載の細胞を培養する工程、  (A) culturing the cells according to (18) or (19),
(B) 培養物から蛋白質を回収する工程。  (B) a step of recovering the protein from the culture.
(23) (9) 〜（1 1) のいずれかに記載の核酸に相補的な塩基配列を有し、 かつ、 該核酸の発現を抑制し得るアンチセンス核酸。  (23) An antisense nucleic acid having a nucleotide sequence complementary to the nucleic acid according to any one of (9) to (11) and capable of suppressing the expression of the nucleic acid.
(24) (23) に記載のアンチセンス核酸を細胞に導入することからなる、 (1) 〜 （3) のいずれかに記載の蛋白質の機能又は生物学的活性を抑制する方 法。  (24) A method for suppressing the function or biological activity of the protein according to any one of (1) to (3), which comprises introducing the antisense nucleic acid according to (23) into a cell.
(25) (1) 〜 （8) のいずれかに記載の蛋白質に特異的に結合し、 該蛋白 質の機能又は生物学的活性を中和する抗体。  (25) An antibody that specifically binds to the protein according to any one of (1) to (8) and neutralizes the function or biological activity of the protein.
(26) (25) に記載の抗体と細胞とを接触させることを含んでなる、 （1) 〜 （8) のいずれかに記載の蛋白質の機能又は生物学的活性を抑制する方法。  (26) A method for inhibiting the function or biological activity of the protein according to any one of (1) to (8), which comprises contacting the antibody according to (25) with a cell.
(27) (1) 〜 （8) のいずれかに記載の蛋白質を用いて、 （1) 〜 （8) の いずれかに記載の蛋白質に対するリガンド、 ァゴニスト又はアンタゴニストを同 定する方法。 (28) 下記の工程を含む、 （1) 〜 （8) のいずれかに記載の蛋白質に対する リガンドを同定する方法。 (27) A method for identifying a ligand, agonist or antagonist for the protein according to any one of (1) to (8) using the protein according to any one of (1) to (8). (28) A method for identifying a ligand for the protein according to any one of (1) to (8), comprising the following steps.
(A) 該蛋白質を試験化合物と接触させる工程、  (A) contacting the protein with a test compound,
(B) 該蛋白質と該試験化合物との特異的結合を検出する工程、  (B) a step of detecting specific binding between the protein and the test compound,
(C) 該蛋白質と試験化合物との特異的結合の有無又は強度を判定する工程。 (29) 下記の工程を含む、 （1) 〜 （8) のいずれかに記載の蛋白質に対する ァゴニスト又はアンタゴニストを同定する方法。  (C) a step of determining the presence or absence or the strength of specific binding between the protein and a test compound. (29) A method for identifying an agonist or an antagonist to the protein according to any one of (1) to (8), comprising the following steps.
(A) 該蛋白質を試験化合物及ぴ所望によりリガンドと接触せしめる工程、 (A) contacting the protein with a test compound and optionally a ligand,
(B) 細胞内シグナル伝達又は G蛋白質の活性化を検出する工程、 及ぴ (B) a step of detecting intracellular signal transduction or activation of a G protein; and
(C) 該試験化合物が、 細胞内シグナル伝達又は G蛋白質の活性化を誘導又は抑 制する能力の有無又は強度を判定する工程。  (C) a step of determining whether or not the test compound has the ability to induce or suppress intracellular signal transduction or activation of G protein.
(30) 下記の工程を含む、 （1) 〜 （8) のいずれかに記載の蛋白質に対する ァゴニスト又はアンタゴニストを同定する方法。  (30) A method for identifying an agonist or antagonist for the protein according to any one of (1) to (8), comprising the following steps.
(A) 該蛋白質を試験化合物及ぴ所望によりリガンドと接触させる工程、 (A) contacting the protein with a test compound and optionally a ligand,
(B) 細胞内の C a の濃度変化、 c AMPの濃度変化、 ホスホリパーゼ Cの活 性化、 p Hの変化又は Κτの濃度変化を測定する工程、 (B) a step of measuring change in the concentration of C a in the cell, changes in the concentration of c AMP, activity of phospholipase C, the change in concentration of a change in p H or kappa tau,
(C) 該変化の測定により、 該試験化合物が、 細胞内シグナル伝達を誘導又は抑 制する能力の有無又は強度を判定する工程。  (C) a step of determining whether or not the test compound has the ability to induce or suppress intracellular signal transduction by measuring the change.
(30) 下記の工程を含む、 （1) 〜 （8) のいずれかに記載の蛋白質に対する ァゴニスト又はアンタゴニストを同定する方法。  (30) A method for identifying an agonist or antagonist for the protein according to any one of (1) to (8), comprising the following steps.
(A) (1) 〜 （8) のいずれかに記載の蛋白質と G iサブファミリーに属する G 蛋白質のひサブュ-ットとの融合蛋白質を細胞膜上に発現させた細胞から膜画分 を調製する工程、  (A) A membrane fraction is prepared from a cell in which a fusion protein of the protein according to any one of (1) to (8) and a G protein belonging to the Gi subfamily is expressed on a cell membrane. Process,
(B) 試験化合物及び所望によりリガンドの存在下で、 該膜画分を検出可能に標 識された G TP又はそのアナ口グと接触させる工程、  (B) contacting the membrane fraction with a detectably labeled GTP or its analog in the presence of a test compound and optionally a ligand,
(C) 検出可能な標識の測定により、 該膜画分と GTP又はそのアナログとの結 合を検出する工程、  (C) detecting the binding of the membrane fraction to GTP or an analog thereof by measuring a detectable label;
(D) 該結合を検出することにより、 該試験化合物が G蛋白質の活性化を誘導又 は抑制する能力の有無又は強度を判定する工程。 (3 1) 下記の工程を含む、 （1) 〜 （8) のいずれかに記載の蛋白質に対する 了ゴニスト又はアンタゴニストを同定する方法。 (D) a step of determining the presence or absence or the strength of the test compound by inducing or suppressing the activation of G protein by detecting the binding. (31) A method for identifying a gonist or antagonist to the protein according to any one of (1) to (8), comprising the following steps.
(A) (1) 〜 （8) のいずれかに記載の蛋白質と G iサブファミリーに属す る G蛋白質のひサブュニッ トとの融合蛋白質を細胞膜上に発現させた細胞から膜 画分を調製する工程、  (A) A membrane fraction is prepared from cells in which a fusion protein of the protein according to any one of (1) to (8) with a G protein subunit of the Gi subfamily is expressed on the cell membrane. Process,
(B) 試験化合物及び所望により リガンドの存在下で、 該膜画分を検出可能に標 識された G T P又はそのアナログと接触させる工程、  (B) contacting the membrane fraction with a detectably labeled GTP or an analog thereof in the presence of a test compound and optionally a ligand;
(C) 検出可能な標識の測定により、 該膜画分と GTP又はそのアナログとの結 合を検出する工程、  (C) detecting the binding of the membrane fraction to GTP or an analog thereof by measuring a detectable label;
(D) 該結合を検出することにより、 該試験化合物が G蛋白質の活性化を誘導又 は抑制する能力の有無又は強度を判定する工程。- (D) a step of determining the presence or absence or the strength of the test compound by inducing or suppressing the activation of G protein by detecting the binding. -
(3 2) (1) 〜 （8) のいずれかに記載の蛋白質が、 該蛋白質を含む膜画分 の形態のもの、 又は、 該蛋白質が細胞表面上に発現している細胞の形態のもので ある （27) 〜 （3 1) に記載の方法。 (32) The protein according to any one of (1) to (8) is in the form of a membrane fraction containing the protein, or in the form of a cell in which the protein is expressed on the cell surface. The method according to any one of (27) to (31).
(33) 該蛋白質が細胞表面上に発現している細胞が、 該蛋白質をコードする 核酸又はこれを含む発現ベクターを導入して該蛋白質を発現させた細胞である (33) A cell in which the protein is expressed on the cell surface is a cell in which a nucleic acid encoding the protein or an expression vector containing the same has been introduced to express the protein.
(3 2) に記載の方法。 The method described in (32).
(34) 医薬の選別、 同定もしくは特徴づけのために使用されるものである、 (2 7) 〜 （3 1) のいずれか記載の方法。  (34) The method according to any one of (27) to (31), which is used for selecting, identifying or characterizing a medicine.
(3 5) リガンドが、 4 -ヒ ドロキシ -2 -ノネナールである、 (29) 〜 (3 1) のいずれか記載の方法。  (35) The method according to any one of (29) to (31), wherein the ligand is 4-hydroxy-2-nonenal.
(36) (29) 〜 （3 1) のいずれか記載の方法により、 ァゴニスト又はァ ンタゴュストを同定し、 当該ァゴニスト又はアンタゴ-ストを、 担体とともに混 合することからなる、 医薬組成物の製造方法。  (36) A method for producing a pharmaceutical composition, comprising: identifying an agonist or antagoust by the method according to any one of (29) to (31); and mixing the agonist or antagonist with a carrier. .
(3 7) (29) - (3 1) のいずれか記載の方法により同定されたアンタゴ 二ストを、 細胞と接触せしめて、 該細胞における脂質過酸化反応産物の受容体の 機能又は生物学的活性を抑制する方法。 本明細書は本願の優先権の基礎である日本国特許出願 2002- 1 1 3 8 29 号の明細書および/または図面に記載される内容を包含する。 図面の簡単な説明 (37) The antagonist identified by the method according to any one of (29) to (31) is contacted with a cell, and the function of the receptor for lipid peroxidation reaction product in the cell or the biological function How to suppress activity. This description is based on Japanese Patent Application No. 2002-1 1 3 8 29 which is the basis of priority of the present application. This includes the contents described in the specification and / or drawings of the issue. BRIEF DESCRIPTION OF THE FIGURES
図 1は、 TG 003 9蛋白質のアミノ酸配列、 塩基配列及び推測される 7回膜 貫通領域 （下線部） を示した図である。  FIG. 1 is a diagram showing the amino acid sequence and base sequence of the TG0039 protein and the putative seven transmembrane regions (underlined).
図 2は、 ヒ トの各組織又は細胞における T G 00 3 9遺伝子の発現分布 （ドッ トブロットの結果） を示した図である。 丸印は、 mRN Aレベルで遺伝子の発現 が認められた組織又は細胞を示す。  FIG. 2 is a diagram showing the distribution of TG0039 gene expression (results of dot blot) in human tissues or cells. Circles indicate tissues or cells in which gene expression was observed at the mRNA level.
図 3は、 TG 0039蛋白質と各種 G蛋白質との融合蛋白質の構造の概略を示 した模式図である。  FIG. 3 is a schematic diagram showing an outline of the structure of a fusion protein of the TG 0039 protein and various G proteins.
図 4は、 TGO 03 9蛋白質と各種 G蛋白質との融合蛋白質を含む膜画分と G T P γ Sとの特異的結合量に対する 4- HNEの影響を示した図である。 特異的結合 量は、 試験物質を添加しない場合をコントロールとし、 その時の結合量に対する 相対値 （％ of control) で表している。  FIG. 4 is a view showing the effect of 4-HNE on the specific binding amount between GTP PγS and a membrane fraction containing a fusion protein of TGO039 protein and various G proteins. The specific binding amount is expressed as a relative value (% of control) relative to the binding amount at the time when no test substance was added as a control.
また、 図 4中、 「 〇 Gial(351Cys→Ile) j は、 T G 00 3 9蛋白質と G i al(351CyS→Ile)との融合蛋白質を含む膜画分における試験結果を示し、 「 △ Gqa 」 は、 TG 0039蛋白質と Gqaとの融合蛋白質を含む膜画分における 試験結果を示し、 「 口 Gsひ L 」 は、 TG 003 9蛋白質と GsccL との融合 蛋白質を含む膜画分における試験結果を示す。 発明を実施するための形態 Further, in FIG. 4, "〇 Gial (351Cys → Ile) j represents the test results in the membrane fraction containing the fusion protein with TG 00 3 9 protein and G i a l (351Cy S → Ile), " △ `` Gqa '' indicates the test results for the membrane fraction containing the fusion protein of TG 0039 protein and Gqa, and `` Mouth GsH L '' indicates the test results for the membrane fraction containing the fusion protein of TG 003 9 protein and GsccL. Is shown. BEST MODE FOR CARRYING OUT THE INVENTION
以下、 本発明を詳細に説明する。  Hereinafter, the present invention will be described in detail.
本発明は、 第 1の態様において、 以下の蛋白質を提供する。  In a first aspect, the present invention provides the following proteins.
第 1の蛋白質としては、 配列番号 2で表されるアミノ酸配列を有する蛋白質を 提供する。 このような蛋白質としては、 配列番号 1で示される当該 c DNAにコ 一ドされる受容体蛋白質 （以下、 「TG 0039蛋白質」 と称する) のアミノ酸配 列を有するものが挙げられる。  As the first protein, a protein having the amino acid sequence represented by SEQ ID NO: 2 is provided. Such proteins include those having the amino acid sequence of a receptor protein (hereinafter, referred to as “TG0039 protein”) encoded by the cDNA shown in SEQ ID NO: 1.
第 2の蛋白質としては、 配列番号 2で表されるアミノ酸配列において、 1若し くは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列を有し、かつ、 脂質過酸化反応産物をリガンドとする G蛋白質共役型受容体としての機能又は生 物学的活性を有する蛋白質を提供する。 The second protein has an amino acid sequence represented by SEQ ID NO: 2 in which one or several amino acids have been deleted, substituted or added, and Provided is a protein having a function or a biological activity as a G protein-coupled receptor using a lipid peroxidation reaction product as a ligand.
このような蛋白質としては、 配列番号 2で示されるアミノ酸配列において、 1 若しくは数個のアミノ酸が欠失、 置換若しくは付加されたアミノ酸配列からなる ものが挙げられる。 ここで、 アミノ酸の欠失、 置換若しくは付加は、 脂質過酸化 反応産物をリガンドとする G蛋白質共役型受容体としての機能又は生物学的活性 (biological activity) が失われない程度であればよく、 通常 1〜約 8 0個、 好 ましくは 1〜約 6 0個、 より好ましくは 1〜約 4 5個、 さらに好ましくは 1〜約 3 0個、 さらに一層好ましくは 1〜約 1 5個である。 また、 本発明の蛋白質の具 体例としては、 配列番号 2で示されるアミノ酸配列からなる蛋白質と比較して 1 若し く はそれ以上の保存的ア ミ ノ酸置換 （ conservative amino aci d subst itut ions) を有する蛋白質が挙げられる。  Examples of such a protein include a protein having an amino acid sequence represented by SEQ ID NO: 2 in which one or several amino acids have been deleted, substituted or added. Here, the deletion, substitution or addition of amino acids may be performed to such an extent that the function or biological activity as a G protein-coupled receptor using a lipid peroxidation reaction product as a ligand is not lost. Usually 1 to about 80, preferably 1 to about 60, more preferably 1 to about 45, still more preferably 1 to about 30, and still more preferably 1 to about 15 is there. In addition, specific examples of the protein of the present invention include one or more conservative amino acid substituting amino acids compared to the protein consisting of the amino acid sequence represented by SEQ ID NO: 2. ).
このような蛋白質には、 自然界で発見される変異型蛋白質のほか、 人為的に改 変した変異型蛋白質、 異種生物由来の蛋白質等が含まれる。 従って、 このような 蛋白質には、 配列番号 2で示されるアミノ酸配列からなる蛋白質の保存的置換変 異体 (conservative substitution variants)及び自然 生？ 立変異体 (naturally occuring allelic variants) ；0 3まれ 。  Such proteins include mutant proteins found in nature, artificially modified mutant proteins, proteins derived from heterologous organisms, and the like. Therefore, such proteins include conservative substitution variants and naturally occurring variants of the protein consisting of the amino acid sequence shown in SEQ ID NO: 2. Naturally occurring allelic variants; 0 3 rare.
第 3の蛋白質としては、 配列番号 2のアミノ酸配列からなる蛋白質とその全長 にわたり 7 5 %以上、 好ましくは 8 0 %以上、 より好ましくは約 8 5 %以上、 さ らに好ましくは約 9 0 %以上、 さらに一層好ましくは約 9 5 %以上のアミノ酸配 列の相同性を有する蛋白質であり、 かつ、 脂質過酸化反応産物をリガンドとする G蛋白質共役型受容体としての機能又は生物学的活性を有する蛋白質を提供する。  As the third protein, a protein consisting of the amino acid sequence of SEQ ID NO: 2 and 75% or more, preferably 80% or more, more preferably about 85% or more, and still more preferably about 90% over its entire length These proteins are even more preferably proteins having homology of about 95% or more amino acid sequences, and have a function or biological activity as a G protein-coupled receptor having a lipid peroxidation reaction product as a ligand. A protein having the same.
「相同性」 とは、 当該技術分野で知られているとおり、 配列を比較することに より決定される、 2以上の蛋白質（又はポリヌクレオチド）の間の関係であって、 蛋白質 （又はポリヌクレオチド配列） 間の適合によって決定されるような、 蛋白 質（又はポリヌクレオチド配列）間の配列の相関の程度を意味する。「相同性」は、 当該技術分野において公知の方法により容易に決定できる。 例えば、 Altschul ら による BLAST (Basic Local Al ignment Search Tool) プログラムを用いて決定す ることができる。 上記の各種蛋白質は G蛋白質共役型受容体の一種であり、 脂質過酸化反応産物 をリガンドとする G蛋白質共役型受容体としての機能又は生物学的活性を有する c 本明細書において、 脂質過酸化反応産物とは脂質の過酸化物の分解物などを意味 し、 これには、 4 -ヒ ドロキシ -2-ノネナ一ル (別称： 4 -ヒ ドロキシノネナールまた は 4-ヒドロキシノン- 2-ェナール、 以下、 4- HNEと称する） が含まれる。 本発明の 蛋白質は、 脂質過酸化反応産物 （4 - HNE等） と特異的に結合する。 そして、 これ らの特異的結合により、 受容体蛋白質は刺激されて細胞内シグナル伝達が誘導さ れる。 "Homology" is a relationship between two or more proteins (or polynucleotides), as known in the art, determined by comparing the sequences, Means the degree of sequence correlation between proteins (or polynucleotide sequences) as determined by the match between the sequences. "Homology" can be readily determined by methods known in the art. For example, it can be determined using the BLAST (Basic Local Alignment Search Tool) program by Altschul et al. The above various proteins is a kind of G protein-coupled receptors, in c herein having a function or biological activity of lipid peroxidation products as G protein coupled receptors to ligands, lipid peroxidation The reaction product is, for example, a decomposition product of lipid peroxides, and includes 4-hydroxy-2-nonenal (also known as 4-hydroxynonenone or 4-hydroxynone-2-enal). , Hereinafter referred to as 4-HNE). The protein of the present invention specifically binds to a lipid peroxidation reaction product (eg, 4-HNE). Then, by these specific bindings, the receptor protein is stimulated to induce intracellular signal transduction.
また、 本発明の受容体蛋白質は、 該蛋白質に特異的に結合する上記のような物 質により刺激を受けると、 G ;サブフアミリ一に属する G蛋白質を活性化する。 例えば、 G蛋白質の αサブユニットに着目すると、 本発明の受容体蛋白質は、 特 異的に結合する物質による刺激を受けると G ( G jサブフアミリ一に属する G 蛋白質の αサプュニット、 例えば G i ft l ) を活性化型 （G T P結合能を有する状 態） に変換する。 そして、 かかる G蛋白質の活性化を介して細胞内でシグナルを 伝える。 In addition, the receptor protein of the present invention activates G ; a G protein belonging to subfamily 1 when stimulated by the above-mentioned substance that specifically binds to the protein. For example, focusing on the α subunit of the G protein, receptor proteins of the present invention, the Upon stimulation by singular bind substances G (G j Sabufuamiri one belonging G protein α Sapuyunitto, for example G i ft l ) is converted to an activated form (a state having GTP binding ability). Then, it transmits a signal inside the cell through activation of the G protein.
従って、 本発明の蛋白質は、 脂質過酸化反応産物の受容体であり、 以下にまと めた （ i ) 〜 （ i i i ) の 1以上の機能又は生物学的活性を有する。 そして、 こ のことは、 本願明細書実施例に記載の通りである。  Therefore, the protein of the present invention is a receptor for a lipid peroxidation reaction product, and has one or more functions or biological activities (i) to (iii) summarized below. This is as described in the examples of the present specification.
( i ) リガンドとの特異的結合。  (i) Specific binding with ligand.
( i i ) リガンド （ァゴ二ストとして作用するリガンド） による刺激に基づく細 胞内シグナル伝達の誘導。  (ii) Induction of intracellular signaling upon stimulation by a ligand (a ligand that acts as an agonist).
( i i i ) リガンド （ァゴ二ストとして作用するリガンド） による刺激に基づく G蛋白質の活性化。  (iii) Activation of G proteins upon stimulation by ligands (ligands acting as agonists).
ここで、 リガンド （ァゴ二ストとして作用するリガンド） としては、 4 HNE な どが挙げられる。  Here, examples of ligands (ligands acting as agonists) include 4HNE.
本発明の蛋白質は、 哺乳動物、 例えば、 ィヌ、 ゥシ、 ゥマ、 ャギ、 ヒッジ、 サ ル、 ブタ、 ゥサギ、 ラット及ぴマウスなどの非ヒ ト動物又はヒ トに由来するぺプ チドであってよく、 また、 合成蛋白質であってもよい。 蛋白質の合成は、 当該技 術分野で公知の常法によって実施することができる。 本発明は、 第 2の態様において、 前記蛋白質をコードする核酸 （DNA又は R NA) を提供する。 さらに詳しくは、 以下の （a) 又は （b) のいずれかの塩基 配列を包含する単離された核酸を提供する。 The protein of the present invention may be derived from a mammal, for example, a non-human animal or a human derived animal such as a dog, a magpie, a poma, a goat, a hidge, a sal, a pig, a penguin, a rat and a mouse. It may be a tide or a synthetic protein. The protein can be synthesized by a conventional method known in the technical field. In a second aspect, the present invention provides a nucleic acid (DNA or RNA) encoding the protein. More specifically, the present invention provides an isolated nucleic acid having the following nucleotide sequence of (a) or (b).
(a) 配列番号 1で表される塩基配列。  (a) The base sequence represented by SEQ ID NO: 1.
(b) 配列番号 1で表される塩基配列からなる核酸とストリンジェントな条件下 でハイプリダイズし、 かつ、 脂質過酸化反応産物をリガンドとする G蛋白質共役 型受容体としての機能又は生物学的活性を有する蛋白質をコードする単離された 核酸。  (b) a function or biological function as a G protein-coupled receptor that hybridizes under stringent conditions with a nucleic acid consisting of the nucleotide sequence represented by SEQ ID NO: 1 and uses a lipid peroxidation reaction product as a ligand An isolated nucleic acid encoding a protein having activity.
なお、 配列番号 1は、 TG 003 9蛋白質の遺伝子 （以下、 「TG 00 39遺伝 子」 と称する） のヒ ト由来 c DNA (翻訳領域全長を含む） の塩基配列を表すも のである。  SEQ ID NO: 1 represents the nucleotide sequence of human-derived cDNA (including the entire translation region) of the gene of TG0039 protein (hereinafter, referred to as “TG0039 gene”).
ここで、 「ストリンジェントな条件下でハイプリダイズし得る」 とは、 通常、 6 X S S C又はこれと同等の塩濃度のハイプリダイゼーション溶液中、 5 0〜6 0°Cの温度条件下、 約 1 6時間ハイプリダイゼーションを行い、 6 X S S C又は これと同等の塩濃度の溶液等で必要に応じて予備洗浄を行った後、 1 X S S C又 はこれと同等の塩濃度の溶液中で洗浄を行うことによりハイプリダイゼーション を実施できることを意味する。 また、 「ハイストリンジェントな条件 (より高いス トリンジエンシーを有する条件）」 とは、 前記において、 洗浄を 0. 1 X S S C又 はこれと同等の塩濃度の溶液中で行うことにより実施できることを意味する。 な お、 配列番号 1で示される TG00 3 9遺伝子とストリンジヱントな条件下でハ イブリダィズする核酸は、 脂質過酸化反応産物をリガンドとする G蛋白質共役型 受容体受容体としての機能又は生物学的活性を有する蛋白質をコードするもので あればよい。  Here, "can hybridize under stringent conditions" generally means that in a 6 X SSC or a hybridization solution having a salt concentration equivalent thereto, a temperature of 50 to 60 ° C and a temperature of about 1 Perform hybridization for 6 hours, perform pre-washing as necessary with 6 XSSC or a solution with a salt concentration equivalent to this, and then wash in a solution with 1 XSSC or a salt concentration equivalent to this. Means that hybridization can be performed. In addition, “high stringent conditions (conditions having higher stringency)” means that washing can be performed in 0.1 XSSC or a solution having a salt concentration equivalent thereto in the above. means. The nucleic acid that hybridizes under stringent conditions with the TG0039 gene represented by SEQ ID NO: 1 has a function or biological activity as a G protein-coupled receptor using a lipid peroxidation reaction product as a ligand. It only needs to encode a protein having
また、 本発明の核酸には、 配列番号 1で示される塩基配列と、 その翻訳領域の 全長にわたり、 通常約 70 %以上、 好ましくは約 80 %以上、 より好ましくは約 85%以上、 さらに好ましくは 90%以上、 さらに一層好ましくは 95%以上の 相同性を有する核酸をも含まれる。  Further, the nucleic acid of the present invention has a nucleotide sequence represented by SEQ ID NO: 1 and usually about 70% or more, preferably about 80% or more, more preferably about 85% or more, more preferably about the entire length of the translation region thereof. Nucleic acids having 90% or more, more preferably 95% or more homology are also included.
以上のような遺伝子又は核酸には、 自然界で発見されるか、 又は、 人為的に改 変された変異型遺伝子、 異種生物由来の相同遺伝子等も含まれる。 本発明の核酸は、 例えば、 哺乳動物の組織や細胞を遺伝子源として同定を行う ことにより単離取得できる。 哺乳動物としては、 ィヌ、 ゥシ、 ゥマ、 ャギ、 ヒッ ジ、 サル、 ブタ、 ゥサギ、 ラット及びマウスなどの非ヒ ト動物のほか、 ヒ トが挙 げられる。 これらのうち、 ヒ トの治療薬の研究開発に利用する上では、 ヒ ト由来 のものを用いることが望ましい。 The above-mentioned genes or nucleic acids include mutant genes found in nature or artificially modified, homologous genes derived from heterologous organisms, and the like. The nucleic acid of the present invention can be isolated and obtained by, for example, performing identification using mammalian tissues or cells as a gene source. Mammals include non-human animals such as dogs, magpies, magpies, goats, sheep, monkeys, pigs, magpies, rats and mice, as well as humans. Of these, it is desirable to use human-derived drugs for research and development of therapeutic drugs for humans.
また、 本発明の核酸は、 配列番号 1で表される遺伝子の配列情報を利用して取 得することもできる。 例えば、 配列番号 1の配列情報に基づいてプライマーゃプ ローフを設計し、 これらを用いる P CR (polymerase chain reaction) 法、 コロ ニーハイプリダイゼーション法、 プラークハイブリダィゼーション法を適宜組み 合わせて、 DNAライブラリーから選択 ·取得することができる。  Further, the nucleic acid of the present invention can also be obtained by utilizing the sequence information of the gene represented by SEQ ID NO: 1. For example, a primer profile is designed based on the sequence information of SEQ ID NO: 1, and a PCR (polymerase chain reaction) method, a colony hybridization method, and a plaque hybridization method using these primers are appropriately combined. You can select and obtain from a DNA library.
具体的には、 哺乳動物の細胞や組織から調製した mRNAから c DNAを合成 し、 これを铸型として、 P CR法により c DNA断片を得る。 得られた c DNA をプローブとして用い、 コロニーハイプリダイゼーション法又はプラークハイブ リダイゼーション法により c DNAライブラリ一を同定して、 全長 c DNAを取 得できる。 また、 ゲノミック DNAライブラリーを同定することにより、 ゲノム 遺伝子を単離することができる。 また、 他の哺乳動物の DNAライブラリーを同 定することにより、 異種生物由来の相同遺伝子 （オルソローグ） を単離すること ができる。  Specifically, cDNA is synthesized from mRNA prepared from mammalian cells and tissues, and this is used as a type II to obtain a cDNA fragment by the PCR method. Using the obtained cDNA as a probe, a full-length cDNA can be obtained by identifying a cDNA library by colony hybridization or plaque hybridization. In addition, genomic genes can be isolated by identifying a genomic DNA library. By isolating a DNA library of another mammal, a homologous gene (ortholog) derived from a heterologous organism can be isolated.
c DNAライブラリー及ぴゲノミック DN Aライブラリー等の DNAライブラ リ' "は、例えば、「Molecular CloningJ (Sambrook, J. , Fritsch, E. F.及ひ Maniatis, T.著、 Cold Spring Harbor Laboratory Pressより 1989年に発刊） に記載の方法 により調製することができる。 あるいは、 市販のライブラリーがある場合はこれ を用いてもよい。  c) DNA libraries such as DNA libraries and genomic DNA libraries are described in, for example, "Molecular Cloning J (Sambrook, J., Fritsch, EF and Maniatis, T., Cold Spring Harbor Laboratory Press, 1989) Can be prepared by the method described in (2) Alternatively, if there is a commercially available library, this may be used.
得られた c DN Aの塩基配列を決定することにより、 遺伝子産物の蛋白質をコ 一ドする翻訳領域を決定でき、 取得した蛋白質のアミノ酸配列を得ることができ る。  By determining the nucleotide sequence of the obtained cDNA, the translation region encoding the protein of the gene product can be determined, and the amino acid sequence of the obtained protein can be obtained.
上記のようにして得られる本発明の核酸は、 脂質過酸化反応産物をリガンドと する G蛋白質共役型受容体蛋白質をコードするものであり、 脂質過酸化反応産物 をリガンドとする G蛋白質共役型受容体の機能又は生物学的活性を有することは、 本願明細書実施例に記載の通りである。 The nucleic acid of the present invention obtained as described above encodes a G protein-coupled receptor protein having a lipid peroxidation reaction product as a ligand, and a G protein-coupled receptor having a lipid peroxidation reaction product as a ligand. Having a bodily function or biological activity As described in Examples of the present specification.
本発明は、 第 3の態様において、 上記蛋白質を産生する方法を提供する。 産生 する方法は、 本発明の核酸を含む組換えベクターを調製し、 本発明の核酸又は該 組換えベクターで形質転換した細胞を取得し、 該細胞を培養し、 培養物から蛋白 質を回収することによって達成することができる。  The present invention provides, in a third aspect, a method for producing the above protein. The method for producing is to prepare a recombinant vector containing the nucleic acid of the present invention, obtain a cell transformed with the nucleic acid of the present invention or the recombinant vector, culture the cell, and recover the protein from the culture. Can be achieved by:
上記方法は、 具体的には、 通常の遺伝子組換え技術により本発明の蛋白質を発 現させ生産する方法が挙げられる力 他の蛋白質ゃぺプチドとの融合蛋白（fusion protein) の形で発現させ生産する方法も含まれる。  Specifically, the above-mentioned method includes a method of expressing and producing the protein of the present invention by a conventional gene recombination technique. Expression of the protein in the form of a fusion protein with another protein peptide is performed. Production methods are also included.
本発明の蛋白質を発現する細胞は、 例えば、 以下のようにして得ることができ る。 まず、 本発明の蛋白質をコードする D N Aを、 適当なプロモーターの下流に 連結される形でベクターに揷入し、 発現ベクターを構築する。 ついで得られた発 現ベクターを宿主細胞に導入する。  Cells expressing the protein of the present invention can be obtained, for example, as follows. First, DNA encoding the protein of the present invention is inserted into a vector in a form ligated downstream of an appropriate promoter to construct an expression vector. Next, the obtained expression vector is introduced into a host cell.
発現系 （宿主一ベクター系） としては、 例えば、 細菌、 酵母、 昆虫細胞及び哺 乳動物細胞の発現系などが挙げられる。 このうち、 機能がよく保存された蛋白質 を得るためには、 昆虫細胞 （Spodoptera frugiperda SF9、 SF21 等） 及び哺乳動 物細胞 （サル C O S— 7細胞、 チャイニーズハムスター C H〇細胞、 ヒ ト H e L a細胞等） を宿主として用いることが好ましい。  Examples of the expression system (host-vector system) include, for example, bacterial, yeast, insect cell and mammalian cell expression systems. Among them, insect cells (Spodoptera frugiperda SF9, SF21, etc.) and mammalian cells (monkey COS-7 cells, Chinese hamster CH〇 cells, human HeLa) Cells) are preferably used as hosts.
本発明の蛋白質を発現させるためのプロモーターとしては、 哺乳動物細胞系の 場合、 S V 4 0プロモーター、 L T Rプロモーター、 ェロンゲーシヨン 1 αプロ モーター等、 昆虫細胞系の場合、 ポリヘドリンプロモーター等を用いることがで きる。  As a promoter for expressing the protein of the present invention, an SV40 promoter, an LTR promoter, an elongation 1α promoter and the like in a mammalian cell system, and a polyhedrin promoter and the like in an insect cell system can be used. it can.
ベクターとしては、 哺乳動物細胞系の場合、 レトロウイルス系ベクター、 ノ ピ ローマウイノレスベタター、ワクシニアウイノレスベタター、 S V 4 0系ベクター等、 昆虫細胞系の場合、 バキュロウィルスベクター等を用いることができる。  As a vector, in the case of a mammalian cell line, a retrovirus-based vector, a virion-winores-better, a vaccinia winores-better, an SV40-based vector, and the like, and in an insect cell line, a baculovirus vector can be used. .
上記発現系で用いる本発明の蛋白質をコードする D N Aとしては、 自然界に存 在する m R N Aに対応する c D N A (例えば、 配列番号 1に示される塩基配列か らなるもの） を用いることができるが、 これに限定されない。 例えば、 本発明の 蛋白質のアミノ酸配列に対応する D N Aを設計して用いることもできる。 この場 合、 ひとつのアミノ酸をコードするコドンは各々 1〜6種類知られており、 用い るコドンの選択は任意でよいが、 例えば発現に利用する宿主のコ ドン使用頻度を 考慮して、 より発現効率の高い配列を設計することができる。 設計した塩基配列 を持つ D N Aは、 D N Aの化学合成、 前記 c D N Aの断片化と結合、 塩基配列の 一部改変等によって取得できる。 人為的な塩基配列の一部改変、 変異導入は、 所 望の改変をコードする合成オリゴヌクレオチドからなるプライマーを利用した P C R法や部位特異的変異導入法 ( site specific mutagenesi s ) ( Mark ら、 Proceedings of Nations丄 Academy of Sciences、 第 81卷、 5りり 2〜5666頁、 1984 年） 等によって実施できる。 As the DNA encoding the protein of the present invention used in the above-mentioned expression system, cDNA corresponding to mRNA existing in nature (for example, one having the nucleotide sequence shown in SEQ ID NO: 1) can be used. However, it is not limited to this. For example, a DNA corresponding to the amino acid sequence of the protein of the present invention can be designed and used. In this case, one to six codons each encoding one amino acid are known. The selection of codons may be arbitrarily selected. For example, a sequence having higher expression efficiency can be designed in consideration of the frequency of codon usage of a host used for expression. DNA having the designed base sequence can be obtained by chemical synthesis of DNA, fragmentation and binding of the cDNA, partial modification of the base sequence, and the like. Partial alterations and mutations in artificial nucleotide sequences can be performed by PCR using primers consisting of synthetic oligonucleotides that encode the desired alterations, or by site-specific mutagenesis (Mark et al., Proceedings of Nations 丄 Academy of Sciences, Vol. 81, 5 pp. 2-5666, 1984).
本発明の蛋白質は、 上記のような発現ベクターを導入した細胞の培養物中に産 生され、 公知の精製方法 （無機塩類による塩析、 有機溶媒による分画沈殿、 ィォ ン交換樹脂カラムクロマトグラフィー、 ァフィ二ティーカラムクロマトグラフィ 一、 ゲルろ過法など） を適宜組合せることによって、 分離精製できる。  The protein of the present invention is produced in a culture of cells into which the above-described expression vector has been introduced, and is purified by known purification methods (salting out with inorganic salts, fractional precipitation with an organic solvent, ion exchange resin column chromatography). Separation and purification can be carried out by appropriately combining chromatography, affinity column chromatography, gel filtration, etc.).
本発明は、 第 4の態様において、 細胞における本発明の蛋白質の機能又は生物 学的活性を増強する方法を提供する。  In a fourth aspect, the present invention provides a method for enhancing the function or biological activity of the protein of the present invention in a cell.
増強する方法としては、 細胞における本発明の蛋白質の発現を高めることによ つて達成できる。 具体的には、 上記第 3の態様において得られた、 組換え発現べ クタ一を細胞に導入することによって行うことができる。 また、 本発明の蛋白質 をコードする塩基配列を有する核酸を当技術分野において公知の技術を用いて細 胞に直接導入してもよい。  As a method for enhancing the expression, it can be achieved by increasing the expression of the protein of the present invention in cells. Specifically, it can be carried out by introducing the recombinant expression vector obtained in the third embodiment into a cell. Further, a nucleic acid having a base sequence encoding the protein of the present invention may be directly introduced into cells using a technique known in the art.
本発明は、 第 5の態様において、 本発明の核酸に相補的な塩基配列を有し、 か つ、 該核酸の発現を抑制し得るアンチセンス核酸と、 それを用いた本発明の蛋白 質としての機能又は生物学的活性を抑制する方法を提供する。  According to a fifth aspect of the present invention, there is provided an antisense nucleic acid having a base sequence complementary to the nucleic acid of the present invention and capable of suppressing the expression of the nucleic acid, and a protein of the present invention using the same. Methods for inhibiting the function or biological activity of
例えば、 本発明の請求項 9〜1 1のいずれか 1項に記載の核酸に相補的な塩基 配列を有し、かつ、該核酸の発現を抑制し得るアンチセンス核酸は、リポザィム、 デコイとして使用することもできる。 この場合、 例えば、 配列番号 1で示される 塩基配列からなる核酸 （センス鎖又はアンチセンス鎖） の、 通常、 連続する 1 4 塩基以上の部分配列若しくはその相補的な配列を有するヌクレオチドを用いるの が好ましい。  For example, an antisense nucleic acid having a nucleotide sequence complementary to the nucleic acid according to any one of claims 9 to 11 of the present invention and capable of suppressing the expression of the nucleic acid is used as a lipozyme or decoy. You can also. In this case, for example, it is usually preferable to use 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 represented by SEQ ID NO: 1. preferable.
該アンチセンス核酸は、 細胞に導入し、 該蛋白質をコードする遺伝子の発現を 変調させることによって、 本発明の受容体蛋白質としての機能又は生物学的活性 を変化 （例えば、 抑制） することができる。 The antisense nucleic acid is introduced into a cell, and the expression of a gene encoding the protein is expressed. Modulation can alter (eg, inhibit) the function or biological activity of the receptor protein of the present invention.
例えば、 アンチセンス核酸は、 標的核酸配列を含む細胞に、 リン酸カルシウム 法、 リポフエクシヨン法、 エレク ト口ポレーシヨン法、 マイクロインジェクショ ン法などの DNA トランスフエクシヨン法、 又はウィルスなどの遺伝子導入べクタ 一の使用を含む遺伝子導入法などの当該技術分野で公知の方法を用いて導入する ことによって、 本発明の蛋白質としての機能又は生物学的活性を変調 （例えば、 抑制） することができる。 例えば、 適切なレトロウイルスベクターを用いて該ォ リゴヌクレオチドを発現するベクターを調製し、 その後、 該発現ベクターを細胞 と i n v i v o又は e x v i v oで接触させることにより、 標的核酸配列を 含む細胞に導入すればよい。  For example, an antisense nucleic acid can be added to a cell containing a target nucleic acid sequence by a DNA transfection method such as a calcium phosphate method, a lipofection method, an electoral poration method, a microinjection method, or a gene transfer vector such as a virus. The function or biological activity of the protein of the present invention can be modulated (for example, suppressed) by introduction using a method known in the art such as a gene introduction method including use. For example, a vector that expresses the oligonucleotide is prepared using an appropriate retroviral vector, and then the expression vector may be introduced into a cell containing the target nucleic acid sequence by contacting the cell with the cell in vivo or exvivo. .
また、 該オリゴヌクレオチド又はその相補物をプローブとして用いることによ つて、 本発明の遺伝子を検出することもできる。 .  The gene of the present invention can also be detected by using the oligonucleotide or its complement as a probe. .
本発明は、 第 6の態様において、 本発明の蛋白質に特異的に結合し、 該蛋白質 の機能又は生物学的活性を中和する抗体、 及び、 該抗体を用いて本発明の蛋白質 の機能又は生物学的活性を抑制する方法を提供する。  The present invention provides, in the sixth aspect, an antibody that specifically binds to the protein of the present invention and neutralizes the function or biological activity of the protein, and the function or the function of the protein of the present invention using the antibody. Methods for inhibiting biological activity are provided.
本発明の蛋白質を認識し、 特異的に結合する抗体は、 本発明の蛋白質、 又はこ れと免疫学的同等性を有する蛋白質若しくはペプチド、 例えば、 蛋白質の断片又 は部分配列を有する合成ぺプチド等を抗原として用いて取得することができる。 ここで、 免役学的同等性を有するとは、 例えば、 本発明の蛋白質に対する抗体と 交差反応を生じるということを意味する。 また、 抗体はポリクローナル抗体であ つてもモノクローナル抗体であってもよい。  An antibody that recognizes and specifically binds to the protein of the present invention includes the protein of the present invention, or a protein or peptide having immunological equivalence thereto, for example, a synthetic peptide having a protein fragment or partial sequence. And the like as an antigen. Here, having immunological equivalence means, for example, that a cross-reactivity occurs with an antibody against the protein of the present invention. Further, the antibody may be a polyclonal antibody or a monoclonal antibody.
ポリクローナル抗体は、 宿主動物 (例えば、 ラットやゥサギ等） に抗原を接種 し、 免疫血清を回収する通常の方法により製造することができる。 モノクローナ ル抗体は、 通常のハイプリ ドーマ法などの技術により製造できる。 また、 モノク ローナル抗体の遺伝子を改変してヒ ト化モノクローナル抗体等を作製できる。 上記で得られた抗体を用いて、 通常の免疫化学的方法 （immunochemical assay 法など） により、 本発明の蛋白質の細胞中又は組織中などにおける発現を検出す ることができる。 あるいは、 抗体を用いるァフィ二ティクロマトグラフィーによ り本発明の蛋白質の精製を実施することができる。 The polyclonal antibody can be produced by a usual method of inoculating a host animal (for example, rat or egret) with an antigen and collecting immune serum. Monoclonal antibodies can be produced by techniques such as the conventional hybridoma method. In addition, a humanized monoclonal antibody or the like can be prepared by modifying the gene of the monoclonal antibody. Using the antibody obtained above, the expression of the protein of the present invention in cells or tissues can be detected by a usual immunochemical method (such as immunochemical assay). Alternatively, affinity chromatography using antibodies Thus, the protein of the present invention can be purified.
また、 本発明の中和抗体と細胞とを接触させることによって、 本発明の蛋白質 の機能又は生物学的活性を抑制することができる。 例えば、 本発明の中和抗体を 免疫原性アジュバント等の不活性成分又は治療的使用のためにさらなる有効成分 と、 安定化剤及ぴ賦形剤とともに組み合わせ、 濾過滅菌後、 凍結乾燥物、 又は安 定化水性調製物中の貯蔵物として得ることができる。被験者へ投与する場合には、 例えば、 皮下注射、 動脈内注射、 静脈内注射などの当該技術分野において公知で ある方法により行い得る。 投与量は、 患者、 投与方法に応じて、 当業者であれば 適当な投与量を適宜選択し得る。  In addition, the function or biological activity of the protein of the present invention can be suppressed by contacting the neutralizing antibody of the present invention with a cell. For example, the neutralizing antibody of the present invention is combined with an inactive ingredient such as an immunogenic adjuvant or a further active ingredient for therapeutic use together with a stabilizer and an excipient, sterilized by filtration, and then lyophilized, or It can be obtained as a stock in a stabilized aqueous preparation. When administered to a subject, it can be performed by a method known in the art, such as subcutaneous injection, intraarterial injection, or intravenous injection. The dose can be appropriately selected by those skilled in the art according to the patient and the administration method.
本発明の蛋白質は、 前記のように以下の （ i )、 （ i i ) 及び （ i i i ) から選 択される機能又は生物学的活性を有する。  The protein of the present invention has a function or a biological activity selected from the following (i), (ii) and (iiii) as described above.
( i ) リガンドとの特異的結合。  (i) Specific binding with ligand.
( i i ) リガンド （ァゴ二ストとして作用するリガンド） による刺激に基づく細 胞内シグナル伝達の誘導。  (ii) Induction of intracellular signaling upon stimulation by a ligand (a ligand that acts as an agonist).
( i i i ) リガンド （ァゴ二ストとして作用するリガンド） による刺激に基づく G蛋白質の活性化。  (iii) Activation of G proteins upon stimulation by ligands (ligands acting as agonists).
ここで、 リガンド （ァゴ二ストとして作用するリガンド） には 4— H N Eが含 まれ、 細胞内シグナル伝達には、 C a 2 +の濃度変化、 c AM Pの濃度変化、 ホス ホリパーゼ Cの活性化、 p Hの変化及ぴ K +の濃度変化等が含まれる。 Here, ligand (§ Gore second ligand that acts as a strike) is 4-HNE is included, the intracellular signaling, C a 2 + concentration change, change in concentration of c AM P, activity of phosphorylase Horipaze C , Changes in pH, changes in K + concentration, and the like.
本発明の蛋白質 （受容体） は、 脂質過酸化反応産物と特異的に結合し、 その結 合により蛋白質が刺激され、 G蛋白質を活性ィ匕し、 その活性化を介して細胞内に シグナル伝達が誘導される。  The protein (receptor) of the present invention specifically binds to a lipid peroxidation reaction product, and the binding stimulates the protein, activates the G protein, and transduces a signal into a cell through the activation. Is induced.
「G蛋白質を活性化し、 その活性化を介して」 とは、 G蛋白質を活性型 （G T P型） へと変換し、 G蛋白質を構成する α、 |3及び τ /サブユニッ トをひサブュニ ット （以下、 G aと称する) と β Ζ γサブュニット複合体 (以下、 G /3 γと称す る） とに解離し、 G がアデ-ル酸シクラーゼ、 ホスホリパーゼ Cなどの効果器 を制御してシグナルを細胞内に伝達することを意味する。 また、 G aとしては、 G iサブフアミリーに属する G蛋白質の αサブュニット、 例えば、 G i α 1が挙 げられる。 ( i ) の機能又は生物学的活性の検出方法は、 例えば、 次の工程を行うことに よって達成できる。 “Activating a G protein and activating it” means that the G protein is converted into an active form (GTP type) and the α, | 3 and τ / subunits that constitute the G protein are subunits. (Hereinafter, referred to as Ga) and βΖγ subunit complex (hereinafter, referred to as G / 3γ), and G controls signal effectors such as adenylate cyclase and phospholipase C to signal. Is transmitted to cells. Examples of G a include α subunits of G proteins belonging to G i subfamily, for example, G i α1 . The method for detecting the function or biological activity of (i) can be achieved, for example, by performing the following steps.
(A) 本発明の蛋白質と検出可能に標識されたリガンドとを接触させる工程、 (A) contacting the protein of the present invention with a detectably labeled ligand,
( B ) 該蛋白質とリガンドとの特異的結合の有無又は強度を検出する工程。 (B) a step of detecting the presence or absence or the strength of specific binding between the protein and a ligand.
かかる結合の検出には、 例えば、 検出可能な標識 （例えば、 R I標識、 蛍光標 識など） を付したリガンドを用い、 この標識を測定することにより実施できる。 また、 その際、 標識を付したリガンドとともに、 非標識のリガンドを混合して用 いる通常の競合ァッセィにより特異的結合を検出してもよい。  Such binding can be detected, for example, by using a ligand to which a detectable label (for example, RI label, fluorescent label, etc.) is attached, and measuring the label. At that time, specific binding may be detected by a general competitive assay using a mixture of a labeled ligand and an unlabeled ligand.
( i i ) の機能又は生物学的活性の検出方法は、 例えば、 次の工程を行うことに よって達成できる。  The method for detecting the function or biological activity of (ii) can be achieved, for example, by performing the following steps.
(A) 本発明の蛋白質とリガンド （ァゴ二ストとして作用するリガンド） とを接 触させる工程、  (A) a step of bringing the protein of the present invention into contact with a ligand (a ligand that acts as an agonist);
( B ) 細胞内の C a 2 +の濃度変化、 c AM Pの濃度変化、 ホスホリパーゼ Cの活 性化、 p Hの変化又は K +の濃度変化を測定する工程。 (B) C a 2 + concentration change within the cell, changes in the concentration of c AM P, activity of phospholipase C, measuring the change or K + concentration change of p H.
本発明の蛋白質をより低いレベルで発現している （又は発現していない） 細胞 を対照として用い、 このような対照細胞における細胞内シグナル伝達のレベルと 比較して、 それよりも細胞内シグナル伝達のレベルが高ければ、 その程度に応じ ( i i ) の機能又は生物学的活性を有すると認められる。  Cells which express (or do not express) the protein 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 is high, it is deemed to have (ii) a function or biological activity according to the degree.
細胞内シグナル伝達の検出の具体的な方法については、 例えば、 文献 〔Chenら の方法、 Analytical Biochemistry, 第 226卷、 第 349-354頁、 1995年 ( C a 2 十の濃度変化、 c AM Pの濃度変化） ； Graminski らの方法、 J. Biol. Chem.、 第 268卷、 第 5957- 5964頁、 1993年 （ホスホリパーゼ Cの活性化） ； Sakurai らの 方法、 Cell、第 92巻、第 573-585頁、 I"8年（C a 2 +の濃度変化）； Hollopeter らの方法、 Nature、第 409卷、第 202— 207頁、 2001年（K +の濃度変化）； Tatemoto らの方法、 Biochem. Biophys. Res. Commun.、 第 251巻、 第 471- 476頁、 1998年 ( pFor the specific method of detecting intracellular signal transduction, see, for example, the literature [Chen et al., Analytical Biochemistry, Vol. 226, pp. 349-354, 1995 (Ca20 concentration change, cAMP). Grainski et al., J. Biol. Chem., Vol. 268, pp. 5957-5964, 1993 (Activation of phospholipase C); Sakurai et al., Cell, Vol. 92, 573. -585, I " 8 years (C 2+ concentration change); Hollopeter et al., Nature, Vol. 409, pages 202-207, 2001 (K + concentration change); Tatemoto et al., Biochem. Biophys. Res. Commun., 251: 471-476, 1998 (p.
Hの変化） ； Hinuma らの方法、 Nature、 第 393 卷、 第 272-273 頁、 1998 年 rachidonic acid metabolite releasing) ；特開平 9-268 公報なと」 に記載 された方法に準じて行うことができる。 H change); Hinuma et al., Nature, Vol. 393, pp. 272-273, rachidonic acid metabolite releasing, 1998; Japanese Unexamined Patent Publication No. 9-268. it can.
( i i i ) の機能又は生物学的活性の検出方法は、 例えば、 以下のようにして 行うことができる。 The method for detecting the function or biological activity of (iii) may be, for example, as follows. It can be carried out.
(A) 本発明の蛋白質と G iサブファミリ一に属する G蛋白質の αサブュニット との融合蛋白質を細胞膜上に発現させた細胞から膜画分を調製する工程、  (A) a step of preparing a membrane fraction from cells in which a fusion protein of the protein of the present invention and an α-subunit of a G protein belonging to the Gi subfamily is expressed on a cell membrane;
(Β) リガンド（ァゴ二ストとして作用するリガンド）の存在下又は非存在下で、 該膜画分を検出可能に標識された G TP又はそのアナログと接触させる工程、 (Ii) contacting the membrane fraction with a detectably labeled GTP or an analog thereof in the presence or absence of a ligand (a ligand that acts as an agonist);
(C) 検出可能な標識を測定する工程、 (C) measuring a detectable label,
(D) 該検出可能な標識の測定により、 該膜画分と GTP又はそのアナログとの 結合の存在又はレベルを検出する工程、  (D) detecting the presence or level of binding between the membrane fraction and GTP or an analog thereof by measuring the detectable label;
(E) リガンド （ァゴ二ストとして作用するリガンド） の存在下又は非存在下に おける該結合の有無又は強度を比較することにより、 リガンド （ァゴ二ストとし て作用するリガンド） による刺激に基づく G蛋白質の活性化を判定する工程。 標識した GT P又はそのアナログには、 例えば、 GTP y S (グアノシン 5， -0- (3—チォ三リン酸））などの分解されにくい GTPアナログを利用するこ とができる。 脂質過酸化反応産物の非存在下での結合レベルと比較して、 脂質過 酸化反応産物の存在下での結合レベルの方がより高ければ、 その程度に応じ （ i i i ) の機能又は生物学的活性があるものと認められる。  (E) By comparing the presence or absence or the strength of the binding in the presence or absence of the ligand (ligand acting as agonist), the stimulation by ligand (ligand acting as agonist) can be performed. Determining activation of the G protein based on the activation. For the labeled GTP or its analog, for example, a GTP analog that is hardly decomposed, such as GTPyS (guanosine 5, -0- (3-thiotriphosphate)), can be used. If the binding level in the presence of the lipid peroxidation reaction product is higher than the binding level in the absence of the lipid peroxidation reaction product, the function or biological function of (iii) depends on the degree. Recognized as active.
ここで、 G i 及びその遺伝子については、 既にアミノ酸配列及び塩基配列が 知られている 〔ヒ ト G i α 1 (351Cys→Ile) /Bahia ら、 Biochemistry、 第 37 卷、 第 11555-11562 頁、 1998 年： ヒ 卜 G i α 1 /Genbank/EMBL accession no. AF055013、 PIR/SWISS-PROT accession no. P04898 ： 等〕。 従って、 G i αをコ ードする DNAは、 開示されている前記の既知配列情報を利用して、 P CR (polymerase chain reaction) 法、 コロニーノヽイブジダイゼーシヨン法、 プラー クハイプリダイゼーション法又はこれらを適宜組合わせて、 DNAライブラリ一 から選択 '取得できる。 G i αをコードする DNAを、 本発明のポリペプチドを コードする DNAの下流に結合し、 それを適当なプロモータを含むベクターに組 込んで、 融合蛋白質を発現させるためのベクターを得ることができる。 その融合 蛋白質の発現ベクターを、 細胞に導入して、 融合蛋白質を発現させることができ る。  Here, the amino acid sequence and base sequence of G i and its gene are already known (human G i α1 (351 Cys → Ile) / Bahia et al., Biochemistry, Vol. 37, pp. 11555-11562, 1998: Human Giα1 / Genbank / EMBL accession no. AF055013, PIR / SWISS-PROT accession no. P04898: etc.). Therefore, the DNA encoding Gia can be obtained by utilizing the disclosed known sequence information, such as the PCR (polymerase chain reaction) method, colony neutralization method, and plaque hybridization method. Alternatively, these can be appropriately combined and selected from a DNA library. A DNA encoding Giα can be ligated downstream of the DNA encoding the polypeptide of the present invention and inserted into a vector containing an appropriate promoter to obtain a vector for expressing the fusion protein. . The expression vector of the fusion protein can be introduced into cells to express the fusion protein.
本発明は、 第 7の態様において、 本発明の蛋白質に対するリガンドを同定する ための方法を提供する。 The present invention, in a seventh aspect, identifies a ligand for the protein of the present invention. Provide a way to:
ここで、「リガンド」とは、受容体蛋白質に特異的に結合する化合物を意味する。 リガンドには、 天然の化合物及び人工的に合成された化合物の両方が含まれる。 なお、リガンドには下記のァゴニスト又はアンタゴニストが含まれ得る。また、 本方法で用いる本発明の蛋白質には、該蛋白質を含む膜画分の形態のもの、又は、 該蛋白質が細胞表面上に発現している細胞の形態のものをも含む。  Here, "ligand" means a compound that specifically binds to a receptor protein. Ligands include both naturally occurring and artificially synthesized compounds. The ligand may include the following agonist or antagonist. The protein of the present invention used in the present method also includes a protein in the form of a membrane fraction containing the protein or a cell in which the protein is expressed on the cell surface.
リガンドの同定は、具体的には、例えば、以下のようにして行うことができる。 The identification of the ligand can be specifically performed, for example, as follows.
(A) 本発明の蛋白質を試験化合物と接触させる工程、 (A) contacting the protein of the present invention with a test compound,
( B ) 該蛋白質と該試験化合物との特異的結合を検出する工程、  (B) a step of detecting specific binding between the protein and the test compound,
( C ) 該蛋白質と試験化合物との結合能力の有無又は強度を判定する工程。 かかる特異的結合の検出は、 例えば、 検出可能な標識 （例えば、 R I標識、 蛍 光標識など） を付した既知リガンド化合物を、 非標識の試験化合物とともに混合 して用いる通常の競合アツセィ法などにより実施できる。 試験化合物には特に制 限はなく、 低分子化合物、 ペプチドなどが挙げられ、 また、 人工的に合成したも のであっても、 天然に存在するものであっても良い。 特異的結合能を有する試験 化合物 （リガンド） は、 作用薬 （ァゴ二スト又はアンタゴニスト） である可能性 が高い。  (C) a step of determining the presence or absence or the strength of the binding ability between the protein and a test compound. Such specific binding can be detected, for example, by a conventional competitive assay method using a known ligand compound with a detectable label (eg, RI label, fluorescent label, etc.) mixed with an unlabeled test compound. Can be implemented. The test compound is not particularly limited, and includes a low-molecular compound, a peptide, and the like. The test compound may be artificially synthesized or naturally occurring. A test compound (ligand) having specific binding ability is likely to be an agonist (agonist or antagonist).
本発明は、 第 8の態様において、 本発明の蛋白質に対するァゴニス ト又はアン タゴニストを同定するための方法を提供する。  The present invention provides, in an eighth aspect, a method for identifying an agonist or an antagonist for the protein of the present invention.
ここで、 「ァゴ二スト」 とは、 受容体蛋白質と特異的に結合することなどにより 該受容体蛋白質を刺激して細胞内シグナル伝達を誘導する能力を有する化合物を 意味する。 また、 「アンタゴニス ト」 とは、 受容体蛋白質を刺激して細胞内シグナ ル伝達を誘導する能力を有する化合物の作用に対して、 これを抑制する能力を有 する化合物を意味する。  Here, “agonist” means a compound capable of inducing intracellular signal transduction by stimulating the receptor protein by specifically binding to the receptor protein. The term “antagonist” refers to a compound having the ability to suppress the action of a compound capable of inducing intracellular signal transmission by stimulating a receptor protein.
ァゴ-ス ト又はアンタゴニス トの同定は、 例えば、 以下のようにして実施でき る。  The identification of the agonist or antagonist can be performed, for example, as follows.
(A) 本発明の蛋白質を試験化合物および必要に応じリガンド （好ましくはァゴ 二ストとして作用するリガンド） と接触せしめる工程、  (A) a step of bringing the protein of the present invention into contact with a test compound and, if necessary, a ligand (preferably a ligand acting as agonist);
( B ) 細胞内シグナル伝達 （例えば、 C a 2 τの濃度変化、 cAMPの濃度変化、 ホス ホリパーゼ Cの活性化、 p Hの変化、 及ぴ、 K+の濃度変化等） を検出するか、 又 は、 G蛋白質 （G tファミリーに属する G蛋白質の サブユニット等） の活性化 を検出する工程、 及び (B) Intracellular signaling (e.g., changes in the concentration of C a 2 tau, changes in the concentration of cAMP, phosphorylase Activation of Horipaze C, change in p H,及Pi, or to detect changes in the concentration, etc.) of K +, or, the step of detecting the activation of G proteins (subunit of G protein belonging to the G t family, etc.) , as well as
( C ) 該試験化合物が、 細胞内シグナル伝達又は G蛋白質の活性化を誘導又は抑 制する能力の有無又は強度を判定する工程。  (C) a step of determining whether or not the test compound has the ability to induce or suppress intracellular signal transduction or activation of G protein.
具体的には、 次のような方法を実施することができる。  Specifically, the following method can be implemented.
1 . 細胞内シグナル伝達の検出による同定  1. Identification by detecting intracellular signal transduction
(A) 本発明の蛋白質を試験化合物および必要に応じリガンド （好ましくはァゴ 二ス トとして作用するリガンド） と接触させる工程、  (A) a step of bringing the protein of the present invention into contact with a test compound and, if necessary, a ligand (preferably a ligand acting as agonist);
( B ) 細胞内の C a 2 +の濃度変化、 c AM Pの濃度変化、 ホスホリパーゼ Cの活 性化、 p Hの変化又は K +の濃度変化を測定する工程、 (B) a step of measuring C a 2 + concentration change within the cell, changes in the concentration of c AM P, activity of phospholipase C, a change or K + concentration change of p H,
( C ) 該変化の測定により、 該試験化合物が、 細胞内シグナル伝達を誘導又は抑 制する能力の有無又は強度を判定する工程。  (C) a step of determining whether or not the test compound has the ability to induce or suppress intracellular signal transduction by measuring the change.
2 . G蛋白質の活性化の検出による同定  2. Identification of G protein activation by detection
(A) 本発明の蛋白質と G iサブフアミリーに属する G蛋白質の αサブュニット との融合蛋白質を細胞膜上に発現させた細胞から膜画分を調製する工程、  (A) a step of preparing a membrane fraction from cells in which a fusion protein of the protein of the present invention and an α-subunit of a G protein belonging to a Gi subfamily is expressed on a cell membrane;
( Β ) 試験化合物および必要に応じリガンド （好ましくはァゴニス トとして作用 するリガンド） の存在下で、 該膜画分を検出可能に標識された G T P又はそのァ ナログと接触させる工程、  (Ii) contacting the membrane fraction with a detectably labeled GTP or its analog in the presence of a test compound and, if necessary, a ligand (preferably a ligand acting as an agonist);
( C ) 検出可能な標識の測定により、 該膜画分と G T P又はそのアナログとの結 合を検出する工程、  (C) detecting the binding of the membrane fraction to GTP or its analog by measuring a detectable label;
( D ) 該結合を検出することにより、 該試験化合物が G蛋白質の活性化を誘導又 は抑制する能力の有無又は強度を判定する工程。  (D) a step of determining whether or not the test compound has the ability to induce or suppress the activation of G protein by detecting the binding.
試験化合物の能力を決定するにあたっては、適切な対照をとることが望ましい。 このような対照としては、 試験化合物の非存在下での検出 ·試験、 本発明の受容 体蛋白質を発現していないか、 あるいは同受容体蛋白質の発現レベルがより少な い細胞を用いた検出 ·試験があげられる。 さらに正確な決定のためにこれら対照 を複数組合せてもよい。  In determining the ability of a test compound, it is advisable to take appropriate controls. Examples of such a control include detection in the absence of a test compound, detection using a cell which does not express the receptor protein of the present invention, or which has a lower expression level of the receptor protein. Tests are given. More than one of these controls may be combined for more accurate determination.
前記方法などにより、アンタゴニストとして同定された化合物は、 （本発明の蛋 白質を発現している細胞） と接触せしめることにより、 該細胞における脂質過酸 化反応産物の受容体の生物学的機能又は活性を抑制することができる。 Compounds identified as antagonists by the above method or the like include (the protein of the present invention) By contacting the cells with cells expressing white matter), the biological function or activity of the receptor for the lipid peroxidation reaction product in the cells can be suppressed.
また、 本発明の蛋白實 （脂質過酸化反応産物の受容体） に対するアンタゴニス トは、 医薬の有効成分として使用できることが期待される。 従って、 前記方法な どにより、 本発明の蛋白質 （脂質過酸化反応産物の受容体） に対するアンタゴニ ストとして同定された化合物は、 医薬の製造のために使用できる。 医薬の製造に おいては、 慣用の担体とともに混合して医薬組成物を製造することができ、 かか る組成物を医薬として販売することができる。  Further, it is expected that the antagonist against the protein substance (receptor of lipid peroxidation reaction product) of the present invention can be used as an active ingredient of a medicine. Therefore, a compound identified as an antagonist to the protein (receptor of lipid peroxidation reaction product) of the present invention by the above-mentioned method or the like can be used for the production of a medicament. In the manufacture of a medicament, a pharmaceutical composition can be produced by mixing it with a conventional carrier, and such a composition can be sold as a medicament.
医薬として用いる際. 投与方法は特に限定されず、 一般的な経口もしくは非経 口的な方法 （経口、 静脈内、 筋肉内、 皮下など） を適用すればよい。 また， 必要 に応じては、 投与方法に応じた不活性な担体と共に、 慣用の医薬製剤 （錠剤、 顆 粒剤、 カプセル剤、 散剤、 注射剤、 吸入剤等） として製剤化して用いればよい。 例えば、 一般的な医薬において許容される結合剤、 崩壌剤、 増量剤， 充填剤、 滑 沢剤などの賦活剤あるいは希釈剤とともに化合物を用い、 通常の方法により、 製 剤化して用いることができる。  When used as a medicament. The administration method is not particularly limited, and general oral or parenteral methods (oral, intravenous, intramuscular, subcutaneous, etc.) may be applied. In addition, 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 method of administration. For example, a compound may be used together with activators or diluents such as binders, disintegrants, bulking agents, fillers, lubricants, and the like, which are acceptable in general pharmaceuticals, and may be used in the form of a preparation by ordinary methods. it can.
投与量は、 投与方法、 患者の年令、 体重、 状態によっても異なるが、 一般的な 投与量、 例えば 1日当たり、 経口投与の場合は、 l〜3 0 0 m g / k g、 非経口 投与の場合には、 0 . 0 1〜5 O m g Z k gの範囲で設定される。  The dosage varies depending on the method of administration, the age of the patient, body weight, and condition, but general dosages, for example, l-300 mg / kg per day for oral administration, parenteral administration Is set in the range of 0.01 to 5 O mg Z kg.
リガンド、 ァゴニスト又はアンタゴニストの同定のために使用する本発明の蛋 白質は、 該蛋白質を含む膜画分の形態、 あるいは、 該蛋白質を細胞表面上に発現 している細胞の形態等で用いることができる。  The protein of the present invention used for identifying a ligand, agonist or antagonist can be used in the form of a membrane fraction containing the protein or in the form of a cell expressing the protein on the cell surface. it can.
受容体蛋白質若しくは蛋白質を細胞表面上に発現している細胞としては、 該受 容体蛋白質若しくは蛋白質を過剰発現 （overexpression) させた細胞などを用い ることができ、 例えば、 該受容体蛋白質若しくは蛋白質コードする核酸を含む組 換えベクターを導入した細胞などを用いることができる。  As the cell that expresses the receptor protein or the protein on the cell surface, a cell or the like in which the receptor protein or the protein is overexpressed can be used. For example, cells into which a recombinant vector containing the desired nucleic acid has been introduced can be used.
組換えベクターを導入する宿主細胞としては、 外来受容体蛋白質を、 その機能 を損なうことなく細胞膜上に発現し得る細胞 （哺乳類の細胞、 昆虫細胞など） を 使用できる。 また、 宿主細胞としては、 組換えベクターを導入する前の細胞それ 自体は、 対象の受容体蛋白質又は蛋白質を発現していないか、 低いレベルでのみ 発現している細胞を用いることが望ましい。 As the host cell into which the recombinant vector is introduced, a cell (mammalian cell, insect cell, etc.) capable of expressing the foreign receptor protein on the cell membrane without impairing its function can be used. In addition, as a host cell, the cell itself before the introduction of the recombinant vector does not express the target receptor protein or protein or has only a low level. It is desirable to use expressing cells.
該蛋白質を含む膜画分は、 該蛋白質又は該蛋白質を発現している細胞を破砕し た後、 分画遠心分離法や密度勾配遠心分離法などの遠心力を利用した分画法を用 いて、 調製できる。 例えば、 細胞破砕液を、 低速 （約 5 0 0〜3 0 0 0 r p m) で短時間 （通常、 約 1〜1 0分） 遠心し、 上清について、 さらに高速 （約 1 5 0 0 0〜3 0 0 0 0 r p m) で通常約 3 0〜 1 2 0分程度遠心し、 得られた沈殿画 分を、 膜画分として用いる。  The membrane fraction containing the protein is obtained by disrupting the protein or cells expressing the protein, and then using a fractionation method utilizing centrifugal force such as fractionation centrifugation or density gradient centrifugation. Can be prepared. For example, the cell lysate is centrifuged at a low speed (about 500 to 300 rpm) for a short time (usually about 1 to 10 minutes). The suspension is usually centrifuged at about 3000 rpm for about 30 to 120 minutes, and the obtained precipitate fraction is used as a membrane fraction.
本発明のリガンド、 ァゴニスト及びアンタゴニストの化合物は脂質過酸化反応 産物をリガンドとする G蛋白質共役型受容体の活性化を誘導又は抑制する作用を 有するので、 脂質過酸化反応産物をリガンドとする G蛋白質共役型受容体の機能 又は生物学的活性が関連する疾患を治療又は予防するための医薬組成物の有効成 分となり得る。  Since the ligand, agonist and antagonist compounds of the present invention have an action of inducing or suppressing the activation of a G protein-coupled receptor using a lipid peroxidation reaction product as a ligand, a G protein using a lipid peroxidation reaction product as a ligand It can be an effective component of a pharmaceutical composition for treating or preventing a disease associated with the function or biological activity of a conjugated receptor.
本発明は、 第 9の態様において、 脂質過酸化反応産物をリガンドとする G蛋白 質共役型受容体の機能又は生物学的活性が関連する疾患を治療又は予防するため の医薬組成物を提供する。  In a ninth aspect, the present invention provides a pharmaceutical composition for treating or preventing a disease associated with the function or biological activity of a G protein-coupled receptor having a lipid peroxidation reaction product as a ligand. .
第 8の態様によって得られる化合物を有効成分として含有する医薬組成物を得 るための工程としては、 以下のような常法により製剤化することができる。 例え ば、 有効量のリガンド、 ァゴニスト又はアンタゴニストとして確認された化合物 又はその薬理的に許容される塩と、 薬理学的に許容され得る担体とを混合する。 次いで、 投与形態に応じた剤型とする。 本発明で用いられる医薬品組成物の投与 形態には、 経口投与、 静脈内投与、 筋肉内投与、 動脈内投与、 髄内投与、 くも膜 下内投与、 脳室内投与、 経皮投与、 皮下投与、 腹腔内投与、 経鼻粘膜投与、 経腸 投与、 局所投与、 舌下投与、 或いは経直腸投与が挙げられるが、 これに限定され るものではない。  As a step for obtaining a pharmaceutical composition containing the compound obtained according to the eighth embodiment as an active ingredient, a pharmaceutical composition can be prepared by the following conventional method. For example, an effective amount of a compound identified as a ligand, agonist or antagonist, or a pharmaceutically acceptable salt thereof, is mixed with a pharmaceutically acceptable carrier. Next, the dosage form is adjusted to the dosage form. The dosage form of the pharmaceutical composition used in the present invention includes oral administration, intravenous administration, intramuscular administration, intraarterial administration, intramedullary administration, intrathecal administration, intraventricular administration, transdermal administration, subcutaneous administration, intraperitoneal administration Examples include, but are not limited to, internal, nasal mucosal, enteral, topical, sublingual, or rectal administration.
経口投与に用いる組成物としては、 錠剤、 顆粒剤、 カプセル剤、 丸剤、 及び散 剤などの固体剤型、 溶液剤、 シロップ剤、 エリキシル剤、 及び懸濁液剤などの液 体剤型が含まれる。 非経口投与に用いる場合には、 無菌溶液剤、 懸濁液剤、 乳剤 等の剤型が含まれる。 また、 上記担体としては、 例えば、 糖類、 デンプン類、 脂 肪酸、 タルク、 植物油、 ガム、 グリコール類、 生理食塩水、 緩衝食塩水、 等が挙 げられる。 Compositions for oral administration include solid forms such as tablets, granules, capsules, pills, and powders, and liquid forms such as solutions, syrups, elixirs, and suspensions. It is. When used for parenteral administration, dosage forms such as sterile solutions, suspensions, and emulsions are included. Examples of the carrier include sugars, starches, fatty acids, talc, vegetable oils, gums, glycols, physiological saline, buffered saline, and the like. I can do it.
以下に、 本発明の上記の同定方法により確認された、 本発明の脂質過酸化反応 産物をリガンドとする G蛋白質共役型受容体蛋白質のリガンド （ァゴニスト）、 4-HNE (4-ヒドロキシ- 2 -ノネナール） について説明する。  The ligand (agonist) of the G protein-coupled receptor protein having the lipid peroxidation reaction product of the present invention as a ligand, confirmed by the above-described identification method of the present invention, 4-HNE (4-hydroxy-2-) Nonenal) will be described.
本発明の蛋白質 （受容体） のァゴニス トである 4- HNEは、 生体が酸化ス トレス を受けた場合、 脂質過酸化反応産物として生体内で産生されることが知られてい る (Esterbauer, H. , et al. , Free Radical Biology & Medicine, 11 : 81 - 128， 1991)。  It is known that 4-HNE, which is an agonist of the protein (receptor) of the present invention, is produced in vivo as a lipid peroxidation reaction product when the organism is subjected to oxidative stress (Esterbauer, H , et al., Free Radical Biology & Medicine, 11: 81-128, 1991).
そして、 4一 HNEやその他の脂質過酸化反応産物は、 例えば、 以下の疾患、 老化 (Stadtman, E. R.， Science, 257 : 1220-1224, 1992)、 動脈硬ィ匕 （Palinski , W. , et al. , Proc. Natl. Acad. Sci. USA, 86 : 1372-1376, 1989)、 パーキンソン病 (Yoritaka, A. , et al. , Proc. Natl. Acad. Sci . 93 : 2696-2701， 1996)、 ァノレ ッノヽイマ一病 (Mark, R. J. , et al. , J. Neurochem. , 68 : 255-264, 1997； Kruman, And 41 HNE and other lipid peroxidation products include, for example, the following diseases, aging (Stadtman, ER, Science, 257: 1220-1224, 1992), arterial sclerosis (Palinski, W., et al.) Natl. Acad. Sci. USA, 86: 1372-1376, 1989), Parkinson's disease (Yoritaka, A., et al., Proc. Natl. Acad. Sci. 93: 2696-2701, 1996), Kanoman, et al. (Mark, RJ, et al., J. Neurochem., 68: 255-264, 1997; Kruman,
I.， et al. , J. Neurosci. , 17 : 5089-5100, 1997) 等と関係していることが報告 されている。 I., et al., J. Neurosci., 17: 5089-5100, 1997).
従って、 上記同定方法によって得られた、 本発明の蛋白質 （受容体） のァゴニ ス ト、 アンタゴニス ト、 抗体等を有効成分として含有する医薬組成物は、 老化、 動脈硬化、 パーキンソン病又はアルツハイマー病等の疾患の治療又は予防に有効 であると期待される。  Therefore, the pharmaceutical composition containing the protein (receptor) agonist, antagonist, antibody or the like of the present invention as an active ingredient, which is obtained by the above identification method, can be used for aging, atherosclerosis, Parkinson's disease, Alzheimer's disease, etc. It is expected to be effective in treating or preventing various diseases.
以下、 実施例をもって本発明をさらに詳しく説明するが、 これらの実施例は本 発明を制限するものではない。  Hereinafter, the present invention will be described in more detail with reference to Examples, but these Examples do not limit the present invention.
なお、 下記実施例において、各操作は特に明示がない限り、 「モレキュラークロ 一二ング (Molecular Cloning)」 (Sambrook, J. , Fritsch, E. F.及び Maniat is, T. 著、 Cold Spring Harbor Laboratory Pressより 1989年に発刊） に記載の方法に より行うか、 又は、 市販の試薬やキットを用いる場合には市販品の指示書に従つ て使用した。  In the following examples, unless otherwise specified, each operation is referred to as "Molecular Cloning" (Sambrook, J., Fritsch, EF and Maniat is, T., Cold Spring Harbor Laboratory Press). (Published in 1989)), or in the case of using a commercially available reagent or kit, following the instructions for the commercial product.
[実施例 1 ] ヒ ト T G 0 0 3 9遺伝子の cDNAの単離  [Example 1] Isolation of cDNA for human TG003 gene
( 1 ) ヒ トプリン受容体 P2Y5 (PIR/SWISS-PR0T accession no. P43657)、 P2Y7 (1) Human purine receptor P2Y5 (PIR / SWISS-PR0T accession no.P43657), P2Y7
(PIR/SWISS-PR0T accession no. Q15722) 及ぴ P2Y9 (PIR/SWISS-PR0T accession no. Q99677) のアミノ酸配列をクエリー配列とし、 ホモロジ一検索法として tblastn (Altschul SF ら、 J. ol. Biol.、 第 215卷、 第 403- 410頁、 1990年） を用いて、 NCBI (National Center for Biotechnology Information) のヒ ト EST データベースに対する検索を行った。 その結果、 ホモロジ一の高い ESTクローン として 「IMAGE ; 682942」 (GenBank/EMBL accession no. AA210739) を見出した。 この ESTクローンの塩基配列中には一つの大きなオープンリーディングフレーム が存在すると推定された。 (PIR / SWISS-PR0T accession no.Q15722) and P2Y9 (PIR / SWISS-PR0T accession no. No. Q99677) was used as a query sequence, and using tblastn (Altschul SF et al., J. ol. Biol., Vol. 215, pp. 403-410, 1990) as a homology search method, NCBI (National A search was performed against the human EST database of the Center for Biotechnology Information). As a result, "IMAGE;682942" (GenBank / EMBL accession no. AA210739) was found as an EST clone with the highest homology. It was presumed that one large open reading frame was present in the nucleotide sequence of this EST clone.
次に、 NCBIのヒ トゲノムデータベースに対して、 前記 AA210739配列で blastn 検索 （Altschul SF ら、 J. Mol. Biol.、 第 215巻、 第 403-410頁、 1990年） を 行った結果、 この ESTは、 その塩基配列からヒ ト G蛋白質共役型受容体遺伝子が コードされていることが推定されている配列 （GenBank/EMBL accession no. AF000545) の一部であることが判明した。  Next, a blastn search (Altschul SF et al., J. Mol. Biol., 215, 403-410, 1990) was performed on the NCA human genome database with the AA210739 sequence. Was found to be a part of a sequence (GenBank / EMBL accession no. AF000545), whose nucleotide sequence is predicted to encode a human G protein-coupled receptor gene.
( 2 ) 上記 （1 ) で得られた AF000545の塩基配列情報をもとにプライマーを設計 し、 polymerase chain reaction (PGR) により、 翻訳領域全長を含む cDNAを取得 した。 PCRは、 以下のように行った。  (2) Primers were designed based on the base sequence information of AF000545 obtained in (1) above, and cDNA containing the entire translation region was obtained by polymerase chain reaction (PGR). PCR was performed as follows.
PCRの铸型としては、 ヒ トゲノム DNA (商品名 「Human Genomic DNAJ ； ク口ンテ ック社製） を用いた。 センスプライマーとしては、 配列番号 3に示す塩基配列を 有するオリゴヌクレオチドを用い、 アンチセンスプライマーとしては、 配列番号 4に示す塩基配列を有するオリゴヌクレオチドを用いた。 なお、 これらのプライ マーは、 PCR産物の両端に制限酵素認識部位 （Kpnl部位及び Hindlll部位） が付 加された DNA断片が得られるように設計されている。  Human genomic DNA (trade name “Human Genomic DNAJ; manufactured by Kukuntec Co., Ltd.) was used as the type II of the PCR, and an oligonucleotide having the base sequence shown in SEQ ID NO: 3 was used as a sense primer. Oligonucleotides having the nucleotide sequence shown in SEQ ID NO: 4 were used as sense primers.These primers were obtained by adding restriction enzyme recognition sites (Kpnl site and Hindlll site) to both ends of the PCR product. It is designed to obtain fragments.
これらのプライマー及び鎵型 DNAを含む以下の組成の PCR反応液（50 1) を調 製し、 PCR 〔条件： 95°C 1分、 （95°C 30秒→66°C 3分） X 35回、 66°C 3分〕 を 実施した。  Prepare a PCR reaction solution (501) containing these primers and type I DNA with the following composition, and perform PCR [Condition: 95 ° C for 1 minute, (95 ° C for 30 seconds → 66 ° C for 3 minutes) X 35 Times at 66 ° C for 3 minutes].
PCR反応液組成：  PCR reaction solution composition:
鎳型画 A 1 μ 1  鎳 Model A 1 μ 1
滅菌水 40 μ 1  Sterile water 40 μ 1
PCR用緩衝液 (Advantage 2 PCR Buffer, クロンテック社製） 5 μ 1 デォキシヌクレオチド溶液 （dATP、 dCTP、 dGTP及ぴ dTTP、 各 10 mM) 1 μ 1 ポリメラーゼ溶液 (Advantage 2 Polymerase Mix、 ク口ンテック社製） 1 μ 1 センスプライマー (10 μ Μ) 1 ιχ 1 得られた PCR産物をァガロースゲル電気泳動に供し、 パンドを切り出して、 約 1, 000 bpの cDNA断片を精製取得した。これを、ベクタープラスミ ド（pGEM- T Easy、 プロメガ社製） に違結し、 得られたプラスミ ドを用いて、 cDNA断片の塩基配列を 決定した。 塩基配列は、 自動 DNAシークェンサ一 （Li- COR LIC-4200L (S) -2 DNA アナリシスシステム、 ァロカ社製） を用い、 ダイデォキシ法 (Thermo Sequenase Cycle Sequencing Kit, USB 社製） により決定した。 その結果、 配列番号 1に示 した塩基配列 （1， 038 bp) を取得した。 この塩基配列がコードしていると推定さ れる G蛋白質共役型受容体の遺伝子を T G 0 0 3 9遺伝子と称することにした。 このクローン化された T G 0 0 3 9遺伝子の塩基配列を NCBI のヒ トゲノムデー タベース中の GenBank/EMBL accession no. AF000545の塩基配列と比較すると、 1 塩基の相違が見られたが、 これは、 遺伝的多型性のため、 若しくは PCRの誤りの ためと考えられた。 PCR buffer (Advantage 2 PCR Buffer, Clontech) 5 μ 1 Deoxynucleotide solution (dATP, dCTP, dGTP and dTTP, 10 mM each) 1 μ 1 Polymerase solution (Advantage 2 Polymerase Mix, manufactured by Kukuntec) 1 μ 1 Sense primer (10 μΜ) 1 l μ 1 The obtained PCR product is subjected to agarose gel electrophoresis, and a band is cut out to cut out about 1,000 bp. The cDNA fragment was purified and obtained. This was ligated to vector plasmid (pGEM-T Easy, manufactured by Promega), and the nucleotide sequence of the cDNA fragment was determined using the obtained plasmid. The nucleotide sequence was determined by the dideoxy method (Thermo Sequenase Cycle Sequencing Kit, USB) using an automatic DNA sequencer (Li-CORLIC-4200L (S) -2 DNA analysis system, Aroka). As a result, the nucleotide sequence shown in SEQ ID NO: 1 (1,038 bp) was obtained. The G protein-coupled receptor gene presumed to be encoded by this nucleotide sequence was referred to as the TG03039 gene. When the nucleotide sequence of the cloned TG003 gene was compared with the nucleotide sequence of GenBank / EMBL accession no. AF000545 in the NCBI human genome database, a difference of 1 It was considered due to a genetic polymorphism or due to a PCR error.
また、 かくして得られた配列番号 1で表される T G 0 0 3 9遺伝子の塩基配列 に対応するアミノ酸配列 （339 アミノ酸残基） は配列番号 2に示した通りであつ た。 配列番号 2で表されるアミノ酸について、 S0SUI による膜貫通領域の予測を 行った結果、 G蛋白質共役型受容体に特徴的である 7回膜貫通領域が推定された。 図 1に、 前記 T G 0 0 3 9遺伝子の塩基配列及ぴ該配列に対応するアミノ酸配 列とともに、 その膜貫通領域 （下線部） を示す。  The amino acid sequence (339 amino acid residues) corresponding to the nucleotide sequence of the TG03039 gene represented by SEQ ID NO: 1 thus obtained was as shown in SEQ ID NO: 2. For the amino acid represented by SEQ ID NO: 2, the transmembrane region was predicted by S0SUI, and as a result, a seven-transmembrane region characteristic of a G protein-coupled receptor was estimated. FIG. 1 shows the base sequence of the TG03039 gene, the amino acid sequence corresponding to the sequence, and the transmembrane region (underlined).
[実施例 2 ] T G 0 0 3 9遺伝子の発現分布  [Example 2] Expression distribution of TG003 gene
ヒ トの各種組織 ·細胞における T G 0 0 3 9遺伝子の発現分布を、 ドットブ口 ッ ト解析により調べた。 ドットプロットは、 ヒ ト組織由来及びヒ ト培養細胞由来 の mRNA (ナイロンメンプレンにドットされたもの） （Multiple Tissue Expression The distribution of the expression of the TG03039 gene in various human tissues and cells was examined by dot-bit analysis. Dot plots show mRNA from human tissue and human cultured cells (dotted on nylon membrane) (Multiple Tissue Expression
Array, クロンテック社製） 及ぴ RI標識プローブを用いて行った。 メンプレンに は、 図 2に示した各種ヒ ト組織由来及ぴヒ ト培養細胞由来の mRNA 〔ポリ（A) RNA〕 が吸着固定されている。 Array, Clontech) and RI-labeled probe. On the membrane, mRNA [poly (A) RNA] derived from various human tissues and human cultured cells shown in FIG. 2 is adsorbed and fixed.
RI標識プローブは、 以下のように調製したものを用いた。 すなわち、 前記実施 例 1の （2) 項で得た cDNA断片を含むプラスミ ドを铸型にして PCRを行った。 そ の際、 センスプライマー及びアンチセンスプライマーとしては、 各々、 配列番号 5及び配列番号 6に示す塩基配列を有する合成オリゴヌクレオチドを用いた。 得 られた PCR産物をァガロースゲル電気泳動により約 560 bpの DNA断片（TG 0 0 3 9遺伝子の翻訳領域を含む cDNA断片） を精製 '取得した。 この cDNA断片を铸 型とし、 ランタムフフづマー (random hexadeoxyribonucleotides)、 ヌクレ才チ ド混液 （dATP, dGTP 及ぴ dTTP) 及び DNAポリメラーゼ I (Large (Klenow) Fragment) を含むフぺリンク用キット (Prime - a - Gene Labeling system^ フ—ロメ ガ社製） と [ひ- 32P]dCTP を用いてラベリングを行った後、 ゲルろ過で精製し、 RI 標識プローブを調製した。 The RI-labeled probe used was prepared as follows. That is, the implementation The plasmid containing the cDNA fragment obtained in section (2) of Example 1 was subjected to PCR using the plasmid as type III. At that time, as the sense primer and the antisense primer, synthetic oligonucleotides having the nucleotide sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively, were used. The obtained PCR product was purified and obtained by agarose gel electrophoresis to purify a DNA fragment of about 560 bp (cDNA fragment containing the translation region of the TG03039 gene). This cDNA fragment was used as a type III primer kit containing random hexadeoxyribonucleotides, a mixture of nucleotides (dATP, dGTP and dTTP), and DNA polymerase I (Large (Klenow) Fragment). After labeling using a-Gene Labeling System ^ (Flomega) and [hi-32P] dCTP, purification was performed by gel filtration to prepare an RI-labeled probe.
ドッ トブロッ トのハイブリダィゼーシヨンは、 以下のようにして行った。 mRNA の固定されたメンブレンを、 溶液 I [Salmon Testes DNA (9.4 g/ μ 1： SIGMA 社製） 160 μ 1を 97°Cで 5分間処理後、 氷中で冷却し、 これに 60°Cの ExpressHyb hybridization Solution (クロンテック社製） 15 ml を加えて調製したもの〕 中 で、 68°C、 30分間プレインキュペートした。 ついで、 このメンブレンを、 標識プ ローブ含有ハイブリダィゼーシヨン溶液 〔前記 RI 標識プローブ 20^ 1、 human The hybridization of the dot blot was performed as follows. The membrane with the immobilized mRNA was treated with 160 µl of Solution I [Salmon Testes DNA (9.4 g / µ1: SIGMA) at 97 ° C for 5 minutes, cooled on ice, and then cooled to 60 ° C. ExpressHyb hybridization Solution (prepared by adding 15 ml of Clontech)] at 68 ° C for 30 minutes. Then, this membrane was added to a hybridization solution containing a labeled probe [the RI-labeled probe 20 ^ 1, human
COT- 1 DNA (1 /z g/ l: Roche社製） 30 μ 1、 Salmon Testes DNA (9.4 μ g//x 1： SIGMA 社製） 16 μ 1、 20XSSC (3M 塩化ナトリゥム、 300 mMクェン酸ナトリゥム、 pH7.0)COT-1 DNA (1 / zg / l: Roche) 30 μ1, Salmon Testes DNA (9.4 μg // x1: SIGMA) 16 μ1, 20XSSC (3M sodium chloride, 300 mM sodium citrate) , PH7.0)
50 μ 1、 及び Nuclease Free Η20 (プロメガ社製） 84 を加えて、 200 1の溶 液を調製し、 これを 97°Cで 5分間処理後、 68°Cで 30分間インキュベートし、 さ らに前記溶液 I 5 mlを加えて調製したもの〕 中で、 65°C、 12時間ハイブリダィ ズさせた。 ついで、 メンブレンを、 1% SDSを含む 2XSSC (300 mM 塩化ナトリゥ ム、 30raM クェン酸ナトリウム、 pH7.0) にて、 65°C、 20分間、 5回、 プレ洗浄し た後、 さらに、 0.5% SDSを含む 0.1XSSC (15 mM 塩化ナトリウム、 1.5 mM タエ ン酸ナトリウム、 PH7.0) にて 55°C、 20分間、 2回洗浄した。 Add 50 μl and Nuclease Free Η20 (Promega) 84 to prepare a 200 1 solution, treat it at 97 ° C for 5 minutes, incubate at 68 ° C for 30 minutes, and further Prepared by adding 5 ml of the above solution I] at 65 ° C for 12 hours. Next, the membrane was pre-washed 5 times at 65 ° C for 20 minutes in 2XSSC (300 mM sodium chloride, 30 raM sodium citrate, pH 7.0) containing 1% SDS, and then 0.5% The plate was washed twice with SDS-containing 0.1X SSC (15 mM sodium chloride, 1.5 mM sodium catenate, PH 7.0) at 55 ° C for 20 minutes.
イメージアナライザー (Bio-imaging Analysis System 2000, フジフイノレム社 製） を用いてハイプリダイゼーションのシグナルを検出した結果を図 2に示す。 図 2から明らかなように、 リンパ節、 末梢血白血球、 脾臓、 虫垂、 回盲腸、 胸腺、 肺及び Daudi細胞由来の mRNAで強いシグナルが見られた。 これらの結果から、 TG O 0 3 9遺伝子は、 リンパ節、 末梢血白血球、 脾臓、 虫垂、 回盲腸、 胸腺、 肺及び Daudi細胞において比較的高発現していると考えられた。 Figure 2 shows the results of detecting the signal of hybridization using an image analyzer (Bio-imaging Analysis System 2000, manufactured by Fuji Huinolem). As is clear from FIG. 2, strong signals were observed in mRNAs from lymph nodes, peripheral blood leukocytes, spleen, appendix, ileum, thymus, lung and Daudi cells. From these results, The TGOO39 gene was considered to be relatively highly expressed in lymph nodes, peripheral blood leukocytes, spleen, appendix, ileum, thymus, lung and Daudi cells.
[実施例 3] TG 0 0 3 9蛋白質のリガンド同定  [Example 3] Ligand identification of TG003 protein
TG 0 0 3 9蛋白質は、 前記実施例 1の結果から、 G蛋白質共役型受容体であ ると推定された。 そこで、 TG 0 0 3 9蛋白質と G蛋白質の融合蛋白質を含む膜 画分を用い、 以下のようにして、 GTP T/ S (グアノシン 5 ' — O— （3—チォ三リ ン酸）） とのリガンド依存的な結合を検出する方法 CWenzel- Seifert ら、 Mol. Pharmacol.、 第 58卷、 第 954 - 966頁、 2000年； Bahia ら、 Biochemistry, 第 37卷、 第 11555- 11562頁、 1998年〕 により、 リガンドの同定を行った。  From the results of Example 1 above, it was estimated that the TG03039 protein was a G protein-coupled receptor. Therefore, GTP T / S (guanosine 5'-O- (3-thiotriphosphate)) was used as follows by using a membrane fraction containing a fusion protein of TG003 protein and G protein. Method for Detecting Ligand-Dependent Binding of Cwenzel-Seifert et al., Mol. Pharmacol., Vol. 58, 954-966, 2000; Bahia et al., Biochemistry, 37, 11555-11562, 1998 ], The ligand was identified.
( 1 ) 融合蛋白質を発現させるためのプラスミ ドの調製  (1) Preparation of plasmid for expressing fusion protein
以下、（ィ）〜（二）のようにして、 TG 0 0 3 9蛋白質と G蛋白質〔Gid (351Cys →Ile) 、 Gq a 、 又は GS fd 〕 との融合蛋白質を発現させるためのプラスミ ド を調製した。 融合蛋白質の概略図は図 3に示した。 ここで、 Gid (351Cys→Ile) は、 Gi 蛋白質の 351 番目のシスティン残基をイソロイシン残基に変換した変 異型 Gi 蛋白質を意味する。 Hereinafter, (I) ~ a manner as (two), TG 0 0 3 9 protein and G protein plasmid for expressing the [G id (351Cys → Ile), G qa, or G S fd] fusion proteins with Was prepared. A schematic diagram of the fusion protein is shown in FIG. Here, G id (351Cys → Ile) refers to mutation type G i protein obtained by converting the 351 th cysteine residues in G i protein with an isoleucine residue.
(ィ） TG 0 ◦ 3 9遺伝子 c DNAの調製  (B) Preparation of cDNA for TG0◦39 gene
前記実施例 1の （2) 項で得られた TG 0 0 3 9遺伝子 c DNA (翻訳領域全 長を含む） を含むプラスミ ドを铸型として用い、 P C Rを行った。 その際、 セン スプライマー及びアンチセンスプライマーとしては、 各々、 配列番号 7及ぴ配列 番号 8に示す塩基配列を有する合成オリゴヌクレオチドを用いた。 これらプライ マーは TG 0 0 3 9遺伝子の c DNA塩基配列 （配列番号 1 ) を元に設計したも のであり、 P C R産物として、 TG 0 0 3 9蛋白質の全長をコードする c DNA (終止コドンを除く） を含み、 その両端に制限酵素認識部位 （N末端に X h o l 部位、 C末端に C p o l及び H i n dlll部位）が付加された D N A断片が得られ るよう設計されている。  PCR was performed using the plasmid containing the TG03039 gene cDNA (including the entire length of the translation region) obtained in section (2) of Example 1 as type III. At that time, as the sense primer and the antisense primer, synthetic oligonucleotides having the nucleotide sequences shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively, were used. These primers were designed based on the cDNA base sequence of the TG03039 gene (SEQ ID NO: 1), and as a PCR product, cDNA encoding the full-length TG003 protein (with a stop codon). This is designed to obtain a DNA fragment with restriction enzyme recognition sites at both ends (Xhol site at the N-terminus and Cpol and Hindlll sites at the C-terminus).
かくして得られた P CR産物を、 ベクタープラスミ ド （P CR産物クローニン グ用ベクターシステム） （pGEM - T Easy Vector, プロメガ社製） に連結し、 得られ たプラスミ ドを、 制限酵素 N o t I及ぴ C p o Iで処理して、 生じた約 1, 000 bp の DNA断片を回収した。 (口） G蛋白質 （Gid(351CyS→Ile)) との融合蛋白質発現用プラスミ ドの調 製 The PCR product thus obtained was ligated to Vector Plasmid (a vector system for cloning the PCR product) (pGEM-T Easy Vector, manufactured by Promega), and the resulting plasmid was ligated with restriction enzymes NotI and処理 The DNA fragment of about 1,000 bp was recovered by treatment with CpoI. (Mouth) G protein (G id (351Cy S → Ile )) and fusion protein expression plasmid made tone of
ヒ ト脳由来 c DNA (Marathon-Ready cDNA Brain, クロンテック社製） を鎳型 として用い、 P CRを行った。 その際、 センスプライマー （プライマー GiffH) 及びアンチセンスプライマー （プライマー Gid_2) としては、 各々、 配列番号 9 及び配列番号 1 0に示す塩基配列を有する合成オリゴヌクレオチドを用いた。 PCR was performed using human brain-derived cDNA (Marathon-Ready cDNA Brain, manufactured by Clontech) as type III. At that time, as the sense primer (primer G iffH ) and the antisense primer (primer G id — 2 ), synthetic oligonucleotides having the base sequences shown in SEQ ID NO: 9 and SEQ ID NO: 10 were used, respectively.
これらプライマーは、 Gi 蛋白質をコードする c DN Aの既知塩基配列These primers are known nucleotide sequence of the c DN A encoding a G i protein
(Genbank/EMBL accession no. AF055013) を元に設計したものであり、 PCR産 物として Gid蛋白質の全長をコードする c DN Aが得られるよう設計されてい る。 (Genbank / EMBL accession no. AF055013 ) are those designed based on, that is designed to c DN A encoding the full-length G id protein as PCR products can be obtained.
ついで、 前記 P CRで得られた産物を錶型として、 2回目の PCRを行った。 その際、 センスプライマー （プライマー Gid_3) 及ぴアンチセンスプライマー （プ ライマー Gid_ としては、 各々、 配列番号 11及ぴ配列番号 12 に示す塩基配列 を有する合成オリゴヌクレオチドを用いた。 Next, a second PCR was performed using the product obtained by the PCR as a type II. At that time, the sense primer as _ (primer G id _ 3)及Pi antisense primer (primer G id, respectively, using synthetic oligonucleotides having the nucleotide sequences shown in SEQ ID NO: 11及Pi SEQ ID NO: 12.
これらプライマーは、 P CR産物として、 Gid(351CyS→Ile) (Gid蛋白質のThese primers, as P CR product, G id (351Cy S → Ile ) ( of the G id protein
351番目のシスティン残基がイソロイシン残基に置換された変異型 Gid蛋白質） の全長をコードする c DNAを含み、 その両端に制限酵素認識部位 （C p o I部 位及び B a mH I部位） が付加された D N A断片が得られるように設計されてい る。 Mutant Gid protein in which the 351st cysteine residue has been replaced with an isoleucine residue), including cDNA encoding the full length, and restriction enzyme recognition sites (CpoI and BamHI sites) at both ends It is designed to obtain a DNA fragment to which is added.
得られた PCR産物を、 ベクタープラスミ ド （pGEM- T Easy Vector, プロメガ 社製） に連結した。 得られたプラスミ ドを制限酵素 N o t I及ぴ B amH Iで処 理し、 生じた約 1， 100 bpの DNA断片を、 バキュロウィルスベクタープラスミ ド p V L 1 3 92 (ファーミンジェン社製） の N o t I /B a mH I部位に挿入し て、 プラスミ ド pVL1392/ Gid(351Cys→Ile)を得た。 The obtained PCR product was ligated to Vectorplasmid (pGEM-T Easy Vector, manufactured by Promega). The resulting plasmid was treated with restriction enzymes NotI and BamHI, and the resulting DNA fragment of about 1,100 bp was purified from the baculovirus vector plasmid pVL1392 (Pharmingen). It was inserted into the N ot I / B a mH I site to give the plasmid pVL1392 / G id (351Cys → Ile ).
このプラスミ ドの制限酵素 No t I /C p o I部位に、 前記 （ィ） で得た DN A断片を挿入し、 融合蛋白質発現用プラスミ ド PVL1392/TG0039- Gi (351Cys→ lie)を得た。 Restriction enzyme No t I / C po I site of this plasmid, the insert a DN A fragment obtained in (I), to obtain a fusion protein expression plasmid PVL1392 / TG0039- G i (351Cys → lie) .
(ハ） G蛋白質 （Gqa) との融合蛋白質発現用プラスミ ドの調製 (C) Preparation of plasmid for expression of fusion protein with G protein ( Gqa )
ヒ ト前立腺由来 c DNA (Marathon-Ready cDNA Prostate, クロンテック社製） を鎳型として用い、 P CRを行った。 その際、 センスプライマー （プライマー G q(rl) 及びアンチセンスプライマー （プライマー Gqft_2) としては、 各々、 配列 番号 1 3及び配列番号 1 4に示す塩基配列を有する合成オリゴヌクレオチドを用 いた。 これらプライマーは、 G q ft をコー ドする c D N Aの既知塩基配列Human prostate cDNA (Marathon-Ready cDNA Prostate, Clontech) Was used as type III and PCR was performed. At that time, as the sense primer (primer G q (rl) and antisense primer (primer G qft _ 2), respectively, had use a synthetic oligonucleotide having the nucleotide sequence shown in SEQ ID NO: 1: 3 and SEQ ID NO: 1 4. these primers are known nucleotide sequence of code sul c DNA to G q ft
(Genbank/EMBL accession no. U43083) を元に設計したものであり、 P CR産物 として Gq(l の全長をコードする c DNAが得られるよう設計されている。 (Genbank / EMBL accession no. U43083), and is designed to obtain a cDNA encoding the full length of Gq (l ) as a PCR product.
ついで、 前記 P CRで得られた産物を錄型として、 2回目の P CRを行った。 その際、 センスプライマー （プライマー Gqf 3) 及びアンチセンスプライマー （プ ライマー Gq( ) としては、 各々、 配列番号 1 5及び配列番号 16に示す塩基配列 を有する合成オリゴヌクレオチドを用いた。 これらプライマーは、 P CR産物と して、 Gqa の全長をコードする c DNAを含み、 その両端に制限酵素認識部位Next, a second PCR was performed using the product obtained by the PCR as a 錄 type. At that time, as the sense primer (primer Gqf 3 ) and the antisense primer (primer Gq ( )), synthetic oligonucleotides having the nucleotide sequences shown in SEQ ID NOS: 15 and 16, respectively, were used. Contains a cDNA encoding the entire length of Gqa as a PCR product, and a restriction enzyme recognition site at each end.
(C p o I部位及び B a mH I部位） が付加された D N A断片が得られるように 設計されている。 (CpoI site and BamHI site) are designed to obtain a DNA fragment.
得られた P CR産物を、 ベクタープラスミ ド (pGEM-T Easy Vector, プロメガ 社製） に連結した。 得られたプラスミ ドを制限酵素 N o t I及び B a mH Iで処 理し、 生じた約 1, 100 bpの DNA断片を、 バキュロウィルスベクタープラスミ ド p VL 1 3 9 2の N o t I /B a mH I部位に挿入して、 プラスミ ド p VL 1 3 9 2/G q を得た。  The obtained PCR product was ligated to Vector Plasmid (pGEM-T Easy Vector, manufactured by Promega). The obtained plasmid was treated with restriction enzymes Not I and BamHI, and the resulting DNA fragment of about 1,100 bp was digested with the Not I / B of baculovirus vector plasmid pVL1392. Insertion into the amHI site yielded the plasmid pVL13392 / Gq.
このプラ'スミ ドの制限酵素 N o t I /C p o I部位に、 前記 （ィ） で得た DN The restriction enzyme NotI / CpoI site of this plasmid was replaced with the DN obtained in (i) above.
A断片を揷入し、 融合蛋白質発現用プラスミ ド p VL 1 3 9 2/TG 0 0 3 9 - Gqaを得た。 . And揷入the A fragment, plasmid for fusion protein expression de p VL 1 3 9 2 / TG 0 0 3 9 - to obtain a G qa. .
(二） G蛋白質 （GS (^ ) との融合蛋白質発現用プラスミ ドの調製 (2) Preparation of plasmid for expression of fusion protein with G protein (GS ( ^))
ヒ ト骨髄由来 c DNA (Marathon-Ready cDNA Bone marrow^ クロンテック社製) を铸型として用い、 P CRを行った。 その際、 センスプライマー （プライマー aH) 及びアンチセンスプライマー （プライマー Gsa ) としては、 各々、 配列番 号 1 7及ぴ配列番号 1 8に示す塩基配列を有する合成オリゴヌクレオチドを用い た。 PCR was performed using human bone marrow-derived cDNA (Marathon-Ready cDNA Bone marrow ^ Clontech) as type II. At that time, as the sense primer (primer aH) and antisense primer (primer G sa), respectively, using synthetic oligonucleotides having the nucleotide sequences shown in SEQ ID NO 1 7及Pi SEQ ID NO: 1 8.
これらプライマーは、 G s a I/ をコー ドする c D N Aの既知塩基配列These primers have a known nucleotide sequence of cDNA encoding GsaI /.
(Genbank/EMBL accession no. X04408) を元に設計されたものであり、 P CR産 物として の全長をコードする c DNAを含み、 その両端に制限酵素認識部 位 （C p o I部位及び Xb a I部位） が付加された DNA断片が得られるように 設計されている。 (Genbank / EMBL accession no.X04408) It is designed to obtain a DNA fragment containing cDNA encoding the full length of the product, and having restriction enzyme recognition sites (CpoI site and XbaI site) added to both ends.
得られた PCR産物を、 ベクタープラスミ ド （pGEM-T Easy Vector, プロメガ 社製） に連結した。 得られたプラスミ ドを制限酵素 N o t I及び Xb a Iで処理 し、 生じた約 1，200 bpの DNA断片を、 バキュ口ウイノレスベクタープラスミ ド p The obtained PCR product was ligated to Vectorplasmid (pGEM-T Easy Vector, manufactured by Promega). The resulting plasmid was treated with the restriction enzymes NotI and XbaI, and the resulting DNA fragment of about 1,200 bp was digested with the Bacchus Winores vector plasmid p.
VL 1 3 92 (ファーミンジヱン社製） の N o t I /X b a I部位に揷入して、 プラスミ ド PVL 1 3 9 2ZGS(dを得た。 Plasmid PVL1392ZGS (d was obtained by inserting into NotI / XbaI site of VL1392 (manufactured by Farmingzin).
このプラスミ ドの制限酵素 No t iZC p o I部位に、 前記 （ィ） で得た DN A断片を挿入し、 融合蛋白質発現用プラスミ ド pVL 1 3 92/TG00 3 9— The DNA fragment obtained in (1) above was inserted into the restriction enzyme NotiZCpoI site of this plasmid, and the plasmid for expression of a fusion protein, pVL1392 / TG0039—
G S«Lを得た。 G S «L.
(2) 融合蛋白質を含む膜画分の調製  (2) Preparation of membrane fraction containing fusion protein
前記 （1) の （口） 〜 （二） で得られた 3種の発現用プラスミ ドは、 図 3 (概 略図） で示したように、 TG 0039蛋白質の C末端に付加されたリンカ一配列 (一 G l y -P r o—） を介して、 TG00 3 9蛋白質 （全長） と G蛋白質 〔Gi d (351Cys→Ile) 、 Gqa 、 又は GS(tL 〕 が連結した構造の融合蛋白質を発現 するプラスミ ドである。 As shown in FIG. 3 (schematic diagram), the three expression plasmids obtained in the above (1) (mouth) and (2) are linker sequences added to the C-terminal of the TG0039 protein. through (an G ly -P ro-), TG00 3 9 protein (full length) and G protein [Gi d (351Cys → Ile), G qa, or G S (tL] is expressed fusion protein having a structure linked It is a plus.
これら融合蛋白質発現用プラスミ ド p VL l 3 9 2/TG 0 0 3 9 -G 1 Plasmids for expression of these fusion proteins p VLl 39 2 / TG 0 39 -G 1
(351Cys→Ile)、 p V L 1 3 92/TG 0 0 3 9— Gqa、 又は pVL 1 3 9 2/(351Cys → Ile), p VL 1 3 92 / TG 0 0 3 9—G qa , or pVL 1 3 9 2 /
TG 00 3 9— GSttIjを、 以下のようにして、 昆虫細胞内で発現させ、 融合蛋白 質を含む膜画分を調製した。 TG0039 -G SttIj was expressed in insect cells as follows, and a membrane fraction containing the fusion protein was prepared.
まず、 昆虫細胞 Sf9 (Spodoptera frugiperda SF9) (ファーミンジェン社製） をコラーゲンコート処理済みの 3 cmシャーレに約 60%コンフノレェントとなるよ うに、 1.5 mlの培地 〔10%牛胎仔血清、 0.1 mg/ml ストレプトマイシン、 及ぴ 100 units/ml ペニシリンを含む Grace' s Insect Cell Culture Medium (pH6.2： ライ フテックオリエンタル社製）〕 中、 27°C、 15分間インキュベートした。  First, 1.5 ml of a culture medium (10% fetal bovine serum, 0.1 mg / l) was prepared by dissolving insect cells Sf9 (Spodoptera frugiperda SF9) (Pharmingen) in a collagen-coated 3 cm Petri dish so that about 60% confluent was obtained. The mixture was incubated at 27 ° C for 15 minutes in Grace's Insect Cell Culture Medium (pH 6.2: manufactured by Lifetech Oriental) containing ml streptomycin and 100 units / ml penicillin.
培地を取り除き、 トランスフエクシヨン用緩衝液 （Transfection Buffer A、 フ ァーミンジヱン社製） 375 Ail を加えた後、 予め調製しておいた DNA溶液 〔融 合蛋白質発現用プラスミ ド 1 μ g、 Linearized BaculoGold Baculovilus DNA (フ ァーミンジェン社製） 0.125 μ g、滅菌水 25 μ 1中で混和し、 25°Cで 15分間ィン キュペートした後、 Transfection Buffer B (ファーミンジェン社製） 375 1を 加えたもの〕 400 /z lを滴下した。 Remove the medium, add 375 Ail of Transfection Buffer (Transfection Buffer A, Farming Ind.), And prepare a DNA solution [prepared for fusion protein expression 1 μg, Linearized BaculoGold Baculovilus DNA (F Mix 0.125 μg in 25 μl of sterile water, incubate at 25 ° C for 15 minutes, and add Transfection Buffer B (Pharmingen) 375 1] 400 / zl Was added dropwise.
これを 27°Cで 4時間培養した後、 培養液を除き、 培地 1.2 mlを加えて 27°Cで 5 日間培養した。 得られた培養液を遠心分離 （l, 000Xg、 5分間） し、 上清をウイ ルス液 Iとして回収した。  After culturing at 27 ° C for 4 hours, the culture solution was removed, and 1.2 ml of medium was added, followed by culturing at 27 ° C for 5 days. The obtained culture solution was centrifuged (1, 000 Xg, 5 minutes), and the supernatant was recovered as virus solution I.
Sf9細胞をコラーゲンコート処理済みの 3 cmシャーレに約 30%コンフルェン トとなるように播き、 前記で得たウィルス液 I (100 μ ΐ) と培地 1.2 mlを加え て 27°Cで 4日間培養した。 得られた培養液を遠心分離 （l, 000Xg、 5分間） し、 その上清をウィルス液 Πとして回収した。  Sf9 cells were seeded in a collagen-coated 3 cm Petri dish at a confluence of about 30%, and the virus solution I (100 μΐ) obtained above and 1.2 ml of medium were added and cultured at 27 ° C for 4 days. . The obtained culture solution was centrifuged (1, 000 Xg, 5 minutes), and the supernatant was collected as a virus solution.
Sf9細胞をコラーゲンコート処理済みの 10 cmシャーレに約 70%コンフルェン トとなるように播き、前記で得たウィルス液 II (500 ^ 1)と培地 12mlを加え、 27°Cで 4日間培養した。 こうして得られた培養液を遠心分離 （l， 000Xg、 5分間） し、 その上清をウィルス液 IIIとして回収した。  Sf9 cells were seeded on a collagen-coated 10 cm Petri dish so as to have a confluence of about 70%, the virus solution II (500 ^ 1) obtained above and 12 ml of medium were added, and the cells were cultured at 27 ° C for 4 days. The culture solution thus obtained was centrifuged (1, 000 Xg, 5 minutes), and the supernatant was recovered as virus solution III.
Sf9細胞をコラーゲンコー ト処理済みの 10 cmシャーレに約 70%コンフノレェン トとなるように播き、 前記で得たウィルス液 ΠΙ (100 μ ΐ) と培地 12 mlを加 え、 27°Cで 4 日間培養した。 こうして得られた細胞を冷却した PBS (Phosphate buffered saline, pH7.4)で洗浄後、冷却した溶解緩衝液〔20 mM Tris-HCl, pH7.5、 l mMEDTA、 0.2 raM フッ化フエニルメチルスルホニル、 10 μ g/ml ぺプスタチン、 10 μ g/ml ロイぺプチン、 及ぴ 2 μ g/ml ァプロチュン〕 3.6 mlに懸濁し、 テフ ロンホモジナイザーを用いて細胞を破砕した。 この細胞破砕液を遠心分離 （600 Xg、 10分間） し、 得られた上清をさらに遠心分離 （50, 000Xg、 20分間） した。 得られた沈殿物を冷却した反応緩衝液 〔20 mM Tris-HCl, pH7.5、 50 mM塩化ナト リウム、 10 mM塩化マグネシウム〕 450 μ 1 に、 テフロンホモジナイザーを用い て懸濁して、 融合蛋白質を発現した膜画分を得た。  Sf9 cells are seeded in a collagen-coated 10 cm Petri dish so as to have about 70% confluence, and the virus solution ΠΙ (100 μΐ) obtained above and 12 ml of medium are added, and the mixture is incubated at 27 ° C for 4 days. Cultured. The cells thus obtained were washed with cooled PBS (Phosphate buffered saline, pH 7.4), and then cooled with a lysis buffer (20 mM Tris-HCl, pH 7.5, lmMEDTA, 0.2 raM phenylmethylsulfonyl fluoride, 10 μg / ml of pepstatin, 10 μg / ml of leptin, and 2 μg / ml of aprotin], and the cells were disrupted using a Teflon homogenizer. This 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 450 μl of a cooled reaction buffer (20 mM Tris-HCl, pH 7.5, 50 mM sodium chloride, 10 mM magnesium chloride) using a Teflon homogenizer, and the fusion protein was suspended. The expressed membrane fraction was obtained.
( 3 ) 融合蛋白質を含む膜画分と GTPT/ Sとの結合試験  (3) Binding test between membrane fraction containing fusion protein and GTPT / S
前記 （2) で得た膜画分 （450 ΐ) を反応緩衝液 （7.54 ml) に懸濁し、 これ に GDP (10 mM) 10 μ ΐを加えた。 この溶液 160 μ 1に試験検体 20 μ ΐを加え、The membrane fraction (450ΐ) obtained in the above (2) was suspended in a reaction buffer (7.54 ml), and 10 µ GDP of GDP (10 mM) was added thereto. The solution 160 mu 1 to test specimens 2 0 μ ΐ added,
30°Cで 10分間インキュベーションした後、 [35S]GTPyS (5 nM, 5 nCi/μ Ι: アマ シャム フアルマシア バイオテク社製） 20 μ ΐをカ卩え、 反応を開始した。 30°C で 1時間インキュベーションした後、 ガラスフィルター （UniFilter_96 GF/B、 パ ッカード社製） を用いてろ過することで反応を停止した。 フィルターを、 冷却し た反応緩衝液 200 i lで 3回洗浄した後、 フィルター上の [35S]GTPy S量 （膜画 分との結合量） を液体シンチレーシヨン測定法によって測定した。 こうして測定 した [35S]GTPy S結合量から、 非特異的結合量 （10 M GTPy S存在下で測定した 結合量） を差し引き、 [35S]GTP T/ Sの特異的結合量を求めた。 After incubation at 30 ° C for 10 minutes, [ 35 S] GTPyS (5 nM, 5 nCi / μ Ι: Siam Pharmacia Biotech Co., Ltd.) (20 μL) was added to initiate the reaction. After incubation at 30 ° C for 1 hour, the reaction was stopped by filtration using a glass filter (UniFilter_96 GF / B, manufactured by Packard). The filters were washed three times with cold reaction buffer 200 il, [35 S] GTPy S amount on the filter (the amount of binding between the membrane fraction) was measured by liquid scintillation measurement. Thus from the measured [35 S] GTPy S binding amount, subtracted the nonspecific binding amount (10 M GTPy S binding amount measured in the presence), was determined specific binding of [35 S] GTP T / S .
核酸、 アミノ酸、 ペプチド関連化合物、 組織抽出物及び脂質関連化合物約 370 種を試験検体として検定した。 その結果を図 4に示す。 TG 0 0 3 9蛋白質と Gi ftl(351Cys→Ile)との融合蛋白質を含む膜画分を用いた場合、 4- HNEを添加するこ とによって、 濃度依存的に [35S]GTPy Sの特異的結合量が増加した。 Approximately 370 nucleic acids, amino acids, peptide-related compounds, tissue extracts and lipid-related compounds were assayed as test samples. Fig. 4 shows the results. When using a membrane fraction containing the TG 0 0 3 9 fusion protein between the protein and Gi ftl (351Cys → Ile), by the child added 4-HNE, a concentration-dependent manner [35 S] of GTP y S The amount of specific binding increased.
一方、 TG 0 0 3 9蛋白質と Gq との融合蛋白質を含む膜画分を用いた場合、 及び、 TG 0 0 3 9蛋白質と GsaLとの融合蛋白質を含む膜画分を用いた場合に は、 これら化合物による特異的結合量の増加は、 認められなかった。  On the other hand, when a membrane fraction containing a fusion protein of TG003 protein and Gq was used, and when a membrane fraction containing a fusion protein of TG003 protein and GsaL was used, No increase in the amount of specific binding by these compounds was observed.
これらの結果から、 TG 0 0 3 9蛋白質は、 G i α 1蛋白質と共役するタイプ の G蛋白質共役型受容体であると考えられた。 また、 4- ΗΝΕがそのァゴニスト （リ ガンド） として作用することが明らかとなった。 すなわち、 TG 0 0 3 9蛋白質 は、 4-ΗΝΕ をァゴニストとする脂質の過酸化反応産物の受容体であると考えられ た。 産業上の利用の可能性  From these results, it was considered that the TG03039 protein was a G protein-coupled receptor of the type that couples to the Giα1 protein. In addition, it was found that 4- と し て acts as the agonist (ligand). That is, the TG003 protein was considered to be a receptor for a peroxidation reaction product of lipid having 4-ΗΝΕ as an agonist. Industrial applicability
本発明の脂質過酸化反応産物をリガンドとする G蛋白質共役型受容体蛋白質及 びその遺伝子は、 細胞内情報伝達のメカニズム研究のために有用である。 また、 脂質過酸化反応産物をリガンドとする G蛋白質共役型受容体に関する疾患に対す る治療薬の標的分子となり得る。  The G protein-coupled receptor protein having the lipid peroxidation reaction product of the present invention as a ligand and its gene are useful for studying the mechanism of intracellular signal transduction. In addition, it can be a target molecule of a therapeutic drug for a disease relating to a G protein-coupled receptor using a lipid peroxidation reaction product as a ligand.
さらに、 本発明の脂質過酸化反応産物をリガンドとする G蛋白質共役型受容体 蛋白質及びその遺伝子を利用した作用薬 (ァゴニスト又はアンタゴニスト) の同 定、 同定及び特徴付けの方法は、 新しい医薬の研究開発のために有用である。 本明細書で引用した全ての刊行物、 特許おょぴ特許出願をそのまま参考として 本明細書にとり入れるものとする。 配列表フリーテキスト Furthermore, the method of identifying, identifying and characterizing an agonist (agonist or antagonist) using a G protein-coupled receptor protein and its gene using the lipid peroxidation reaction product of the present invention as a ligand is a novel drug research. Useful for development. All publications, patents and patent applications cited in this specification are incorporated herein by reference in their entirety. Sequence listing free text
配列番号 3 ：合成 DNA  SEQ ID NO: 3: Synthetic DNA
配列番号 4 ：合成 DNA  Sequence number 4: Synthetic DNA
配列番号 5 ：合成 DNA  SEQ ID NO: 5: synthetic DNA
配列番号 6 ：合成 DNA  SEQ ID NO: 6: synthetic DNA
配列番号 9 ：合成 DNA  SEQ ID NO: 9: synthetic DNA
配列番号 1 0 ：合成 DNA  SEQ ID NO: 10: synthetic DNA
配列番号 1 1 ：合成 DNA  SEQ ID NO: 11: synthetic DNA
配列番号 1 2 ：合成 DNA  SEQ ID NO: 12: synthetic DNA
配列番号 1 3 ：合成 DNA  SEQ ID NO: 13: Synthetic DNA
配列番号 1 4 ：合成 DNA  SEQ ID NO: 14: Synthetic DNA
配列番号 1 5 ：合成 DNA  SEQ ID NO: 15: Synthetic DNA
配列番号 1 6 ：合成 DNA  SEQ ID NO: 16: Synthetic DNA
配列番号 1 7 ：合成 DNA  SEQ ID NO: 17: Synthetic DNA
配列番号 1 8 ：合成 DNA  SEQ ID NO: 18: Synthetic DNA

Claims

請 求 の 範 囲 The scope of the claims
1 . 配列番号 2で表されるアミノ酸配列を有する蛋白質。 1. A protein having the amino acid sequence represented by SEQ ID NO: 2.
2 . 配列番号 2で表されるアミノ酸配列において、 1若しくは数個のァミノ 酸が欠失、 置換若しくは付加されたアミノ酸配列を有し、 かつ、 脂質過酸化反応 産物をリガンドとする G蛋白質共役型受容体としての機能又は生物学的活性を有 する蛋白質。 2. In the amino acid sequence represented by SEQ ID NO: 2, the amino acid sequence has one or several amino acids deleted, substituted or added, and has a G protein-conjugated type having a lipid peroxidation reaction product as a ligand. A protein having a function as a receptor or a biological activity.
3 . 配列番号 2のアミノ酸配列を有する蛋白質とその全長にわたり 7 5 %以 上のアミノ酸配列の相同性を有する蛋白質であり、 かつ、 脂質過酸化反応産物を リガンドとする G蛋白質共役型受容体としての機能又は生物学的活性を有する蛋 白質。 3. A protein having the amino acid sequence homology of 75% or more over its entire length with the protein having the amino acid sequence of SEQ ID NO: 2, and as a G protein-coupled receptor having a lipid peroxidation reaction product as a ligand. A protein having the function or biological activity of
4 . 脂質過酸化反応産物をリガンドとする G蛋白質共役型受容体としての機 能又は生物学的活性が、 以下の （ i )、 （ i i ) 及び （ i i i ) から選択される 1 以上のものである、 請求項 2又は 3に記載の蛋白質。 4. The function or biological activity as a G protein-coupled receptor having a lipid peroxidation reaction product as a ligand is one or more selected from the following (i), (ii) and (iii): The protein according to claim 2, wherein the protein is present.
( i ) リガンドとの特異的結合。  (i) Specific binding with ligand.
( i i ) リガンドによる刺激に基づく細胞内シグナル伝達の誘導。  (ii) Induction of intracellular signaling upon stimulation by ligand.
( i i i ) リガンドによる刺激に基づく G蛋白質の活性化。  (iii) Activation of G protein upon stimulation by ligand.
5 . 脂質過酸化反応産物が 4 -ヒ ドロキシ- 2-ノネナールである、 請求項 2〜 4のいずれか 1項に記載の蛋白質。 5. The protein according to any one of claims 2 to 4, wherein the lipid peroxidation reaction product is 4-hydroxy-2-nonenal.
6 . 組換え蛋白質である、 請求項 1〜 5のいずれか 1項に記載の蛋白質。 6. The protein according to any one of claims 1 to 5, which is a recombinant protein.
7 . 哺乳動物の蛋白質である、請求項 1〜 6のいずれか 1項に記載の蛋白質。 7. The protein according to any one of claims 1 to 6, which is a mammalian protein.
8 . 哺乳動物がヒ トである、 請求項 7記載の蛋白質 c 8. The protein c according to claim 7, wherein the mammal is human.
9 . 請求項 1〜3のいずれか 1項に記載の蛋白質をコードする塩基配列を有 する単離された核酸。 9. An isolated nucleic acid having a base sequence encoding the protein according to any one of claims 1 to 3.
1 0 . 以下の （a ) 又は （b ) の塩基配列を有する単離された核酸。 10. An isolated nucleic acid having the following base sequence (a) or (b):
( a ) 配列番号 1で表される塩基配列。  (a) The nucleotide sequence represented by SEQ ID NO: 1.
( b ) 配列番号 1で表される塩基配列を有する核酸と相補的な塩基配列を有する 核酸とストリンジェントな条件下でハイブリダィズし、 かつ、 脂質過酸化反応産 物をリガンドとする G蛋白質共役型受容体としての機能又は生物学的活性を有す る蛋白質をコードする単離された核酸。  (b) a G protein-conjugated type that hybridizes under stringent conditions with a nucleic acid having a nucleotide sequence complementary to the nucleic acid having the nucleotide sequence represented by SEQ ID NO: 1 and uses a lipid peroxidation reaction product as a ligand An isolated nucleic acid encoding a protein having a function as a receptor or a biological activity.
1 1 . 配列番号 1の核酸と、 その翻訳領域の全長にわたり少なくとも 7 5 % 以上の相同性を有し、 かつ、 脂質過酸化反応産物をリガンドとする G蛋白質共役 型受容体としての機能又は生物学的活性を有する蛋白質をコードする単離された 核酸。 11. A function or organism as a G protein-coupled receptor that has at least 75% or more homology with the nucleic acid of SEQ ID NO: 1 over the entire length of its translation region and has a lipid peroxidation reaction product as a ligand An isolated nucleic acid encoding a protein having biological activity.
1 2 . 脂質過酸化反応産物をリガンドとする G蛋白質共役型受容体としての 機能又は生物学的活性が、 以下の （ i )、 （ i i ) 及び （ i i i ) から選択される 1以上のものである、 請求項 1 0又は 1 1に記載の核酸。 12. The function or biological activity as a G protein-coupled receptor using a lipid peroxidation reaction product as a ligand is one or more selected from the following (i), (ii) and (iii): The nucleic acid according to claim 10 or 11, which is present.
( i ) リガンドとの特異的結合。  (i) Specific binding with ligand.
( i i ) リガンドによる刺激に基づく細胞内シグナル伝達の誘導。  (ii) Induction of intracellular signaling upon stimulation by ligand.
( i i i ) リガンドによる刺激に基づく G蛋白質の活性化。  (iii) Activation of G protein upon stimulation by ligand.
1 3 . 脂質過酸化反応産物が 4 -ヒ ドロキシ- 2-ノネナールである、 請求項 1 0〜 1 2のいずれか 1項に記載の核酸。 13. The nucleic acid according to any one of claims 10 to 12, wherein the lipid peroxidation reaction product is 4-hydroxy-2-nonenal.
1 4 . D N Aである、 請求項 9〜 1 3のいずれか 1項に記載の核酸。 14. The nucleic acid according to any one of claims 9 to 13, which is a DNA.
1 5 . 哺乳動物の核酸である、請求項 9〜 1 4のいずれか 1項に記載の核酸。 15. The nucleic acid according to any one of claims 9 to 14, which is a mammalian nucleic acid.
1 6 . 哺乳動物がヒ トである、 請求項 1 5に記載の核酸。 16. The nucleic acid according to claim 15, wherein the mammal is a human.
1 7 . 請求項 9〜1 6のいずれか 1項に記載の核酸を含む組換えベクター。 17. A recombinant vector comprising the nucleic acid according to any one of claims 9 to 16.
1 8 . 請求項 9〜 1 6のいずれか 1項に記載の核酸、 又は、 請求項 1 7に記 載の組換えべクタ一で形質転換された細胞。 18. A cell transformed with the nucleic acid according to any one of claims 9 to 16 or the recombinant vector according to claim 17.
1 9 . 請求項 6に記載の組換え蛋白質を細胞表面上に発現している、 請求項 1 8に記載の細胞。 19. The cell according to claim 18, wherein the recombinant protein according to claim 6 is expressed on the cell surface.
2 0 . 請求項 1 0〜1 2のいずれか 1項に記載の核酸によってコードされる ポリぺプチド。 20. A polypeptide encoded by the nucleic acid according to any one of claims 10 to 12.
2 1 . 請求項 1 7に記載の組換えベクターを用いて、 脂質過酸化反応産物を リガンドとする G蛋白質共役型受容体の細胞における機能又は生物学的活性を増 強する方法。 21. A method for enhancing the function or biological activity in a cell of a G protein-coupled receptor using a lipid peroxidation reaction product as a ligand, using the recombinant vector according to claim 17.
2 2 . 以下の工程を含む、 脂質過酸化反応産物をリガンドとする G蛋白質共 役型受容体としての機能又は生物学的活性を有する蛋白質の製造方法。 22. A method for producing a protein having a function as a G protein-combined receptor or a biological activity using a lipid peroxidation reaction product as a ligand, comprising the following steps.
(A) 請求項 1 8又は 1 9に記載の細胞を培養する工程、  (A) culturing the cell according to claim 18 or 19,
( B ) 培養物から蛋白質を回収する工程。  (B) a step of recovering the protein from the culture.
2 3 . 請求項 9〜1 1のいずれか 1項に記載の核酸に相補的な塩基配列を有 し、 かつ、 該核酸の発現を抑制し得るアンチセンス核酸。 23. An antisense nucleic acid having a base sequence complementary to the nucleic acid according to any one of claims 9 to 11, and capable of suppressing expression of the nucleic acid.
2 4 . 請求項 2 3に記載のアンチセンス核酸を細胞に導入することからなる、 請求項 1〜 3のいずれか 1項に記載の蛋白質の機能又は生物学的活性を抑制する 方法。 24. A method for suppressing the function or biological activity of the protein according to any one of claims 1 to 3, comprising introducing the antisense nucleic acid according to claim 23 into a cell.
25. 請求項 1〜8のいずれか 1項に記載の蛋白質に特異的に結合し、 該蛋 白質の機能又は生物学的活性を中和する抗体。 25. An antibody that specifically binds to the protein according to any one of claims 1 to 8 and neutralizes the function or biological activity of the protein.
26. 請求項 25に記載の抗体と細胞とを接触させることを含んでなる、 請 求項 1〜 8のいずれか 1項に記載の蛋白質の機能又は生物学的活性を抑制する方 法。 26. A method for suppressing the function or biological activity of the protein according to any one of claims 1 to 8, comprising contacting a cell with the antibody according to claim 25.
27. 請求項 1〜8のいずれか 1項に記載の蛋白質を用いて、 請求項 1〜8 のいずれか 1項に記載の蛋白質に対するリガンド、 ァゴニスト又はアンタゴニス トを同定する方法。 27. A method for identifying a ligand, agonist or antagonist for the protein according to any one of claims 1 to 8, using the protein according to any one of claims 1 to 8.
28. 下記の工程を含む、 請求項 1〜 8のいずれか 1項に記載の蛋白質に対 するリガンドを同定する方法。 28. A method for identifying a ligand for the protein according to any one of claims 1 to 8, comprising the following steps.
(A) 該蛋白質を試験化合物と接触させる工程、  (A) contacting the protein with a test compound,
(B) 該蛋白質と該試験化合物との特異的結合を検出する工程、  (B) a step of detecting specific binding between the protein and the test compound,
(C) 該蛋白質と試験化合物との特異的結合の有無又は強度を判定する工程。  (C) a step of determining the presence or absence or the strength of specific binding between the protein and a test compound.
29. 下記の工程を含む、 請求項 1〜8のいずれか 1項に記載の蛋白質に対 するァゴニスト又はアンタゴニストを同定する方法。 29. A method for identifying an agonist or antagonist for the protein according to any one of claims 1 to 8, comprising the following steps.
(A) 該蛋白質を試験化合物及び所望によりリガンドと接触せしめる工程、 (A) contacting the protein with a test compound and optionally a ligand,
(B) 細胞内シグナル伝達又は G蛋白質の活性化を検出する工程、 及ぴ (B) a step of detecting intracellular signal transduction or activation of a G protein; and
(C) 該試験化合物が、 細胞内シグナル伝達又は G蛋白質の活性化を誘導又は抑 制する能力の有無又は強度を判定する工程。  (C) a step of determining whether or not the test compound has the ability to induce or suppress intracellular signal transduction or activation of G protein.
30. 下記の工程を含む、 請求項 1〜 8のいずれか 1項に記載の蛋白質に対 するァゴニス ト又はアンタゴニストを同定する方法。 30. A method for identifying an agonist or antagonist for the protein according to any one of claims 1 to 8, comprising the following steps.
(A) 該蛋白質を試験化合物及ぴ所望によりリガンドと接触させる工程、 (A) contacting the protein with a test compound and optionally a ligand,
(B) 細胞内の C a 2τの濃度変化、 c AMPの濃度変化、 ホスホリパーゼ Cの活 性化、 p Hの変化又は K +の濃度変化を測定する工程、 (B) Changes in intracellular C a 2τ concentration, c AMP concentration, and phospholipase C activity Measuring the change in pH, the change in pH or the change in K + concentration,
(C) 該変化の測定により、 該試験化合物が、 細胞内シグナル伝達を誘導又は抑 制する能力の有無又は強度を判定する工程。  (C) a step of determining whether or not the test compound has the ability to induce or suppress intracellular signal transduction by measuring the change.
31. 下記の工程を含む、 請求項 1〜8のいずれか 1項に記載の蛋白質に対 するァゴニス ト又はアンタゴニストを同定する方法。 31. A method for identifying an agonist or antagonist for the protein according to any one of claims 1 to 8, comprising the following steps.
(A) 請求項 1〜 8のいずれか 1項に記載の蛋白質と G iサブフアミリーに属す る G蛋白質の αサブュニットとの融合蛋白質を細胞膜上に発現させた細胞から膜 画分を調製する工程、  (A) a step of preparing a membrane fraction from cells in which a fusion protein of the protein according to any one of claims 1 to 8 and an α-subunit of a G protein belonging to the Gi subfamily is expressed on a cell membrane; ,
(Β) 試験化合物及び所望によりリガンドの存在下で、 該膜画分を検出可能に標 識された G TP又はそのアナログと接触させる工程、  (Ii) contacting the membrane fraction with a detectably labeled GTP or an analog thereof in the presence of a test compound and optionally a ligand;
(C) 検出可能な標識の測定により、 該膜画分と GTP又はそのアナログとの結 合を検出する工程、  (C) detecting the binding of the membrane fraction to GTP or an analog thereof by measuring a detectable label;
(D) 該結合を検出することにより、 該試験化合物が G蛋白質の活性化を誘導又 は抑制する能力の有無又は強度を判定する工程。  (D) a step of determining the presence or absence or the strength of the test compound by inducing or suppressing the activation of G protein by detecting the binding.
32. 請求項 1〜 8のいずれか 1項に記載の蛋白質が、 該蛋白質を含む膜画 分の形態のもの、 又は、 該蛋白質が細胞表面上に発現している細胞の形態のもの である請求項 27〜3 1のいずれか 1項に記載の方法。 32. The protein according to any one of claims 1 to 8, which is in the form of a membrane fraction containing the protein, or in the form of a cell in which the protein is expressed on the cell surface. The method according to any one of claims 27 to 31.
33. 該蛋白質が細胞表面上に発現している細胞が、 該蛋白質をコードする 核酸又はこれを含む発現ベクターを導入して該蛋白質を発現させた細胞である請 求項 32に記載の方法。 33. The method according to claim 32, wherein the cell in which the protein is expressed on a cell surface is a cell in which a nucleic acid encoding the protein or an expression vector containing the protein has been introduced to express the protein.
34. 医薬の選別、 同定もしくは特徴づけのために使用されるものである、 請求項 27〜 3 1のいずれか 1項記載の方法。 34. The method according to any one of claims 27 to 31 for use in the selection, identification or characterization of a medicament.
35. リガンドが、 4-ヒ ドロキシ- 2-ノネナールである、請求項 29〜 3 1の いずれか 1項記載の方法。 35. The method according to any one of claims 29 to 31, wherein the ligand is 4-hydroxy-2-nonenal.
3 6 . 請求項 2 9〜3 1のいずれか 1項記載の方法により、 ァゴニス ト又は アンタゴニストを同定し、 当該ァゴ-スト又はアンタゴニストを、 担体とともに 混合することからなる、 医薬組成物の製造方法。 36. A method for producing a pharmaceutical composition comprising: identifying an agonist or an antagonist by the method according to any one of claims 29 to 31; and mixing the agonist or the antagonist with a carrier. Method.
3 7 . 請求項 2 9〜 3 1のいずれか 1項記載の方法により同定されたアンタ ゴニス トを、 細胞と接触せしめて、 該細胞における脂質過酸化反応産物の受容体 の機能又は生物学的活性を抑制する方法。 37. Contacting the antagonist identified by the method according to any one of claims 29 to 31 with a cell, the function of a receptor for a lipid peroxidation reaction product in the cell, or the biological function of the receptor. How to suppress activity.
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