WO2003087364A1 - Nouveau recepteur couple a la proteine g et son gene - Google Patents

Nouveau recepteur couple a la proteine g et son gene Download PDF

Info

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
Authority
WO
WIPO (PCT)
Prior art keywords
protein
ligand
nucleic acid
cell
reaction product
Prior art date
Application number
PCT/JP2003/004759
Other languages
English (en)
Japanese (ja)
Inventor
Tetsuo Onuki
Koji Ogawa
Yutaka Koguchi
Emiko Hosoi
Aiko Chikada
Original Assignee
Tanabe Seiyaku Co., Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Tanabe Seiyaku Co., Ltd. filed Critical Tanabe Seiyaku Co., Ltd.
Priority to JP2003584306A priority Critical patent/JPWO2003087364A1/ja
Priority to AU2003227499A priority patent/AU2003227499A1/en
Publication of WO2003087364A1 publication Critical patent/WO2003087364A1/fr

Links

Classifications

    • 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

L'invention concerne la production d'une nouvelle protéine réceptrice couplée à la protéine G au moyen d'un produit de réaction de peroxydation lipidique comme ligand et son gène. Elle concerne également un procédé permettant d'identifier un nouveau ligand, un agoniste ou antagoniste et des compositions médicales comprenant comme ingrédient actif un ligand, un agoniste, un antagoniste, etc. Plus précisément, l'invention concerne une protéine purifiée possédant une séquence d'acides aminés représentée par SEQ ID NO:2; et une protéine purifiée possédant une séquence d'acides aminés dérivée de la séquence d'acides aminés représentée par SEQ ID NO:2 par délétion, substitution ou addition d'au moins un acide aminé et possédant une fonction ou une activité biologique comme protéine réceptrice couplée à la protéine G au moyen d'un produit de peroxydation de lipidique servant de ligand.
PCT/JP2003/004759 2002-04-16 2003-04-15 Nouveau recepteur couple a la proteine g et son gene WO2003087364A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
JP2003584306A JPWO2003087364A1 (ja) 2002-04-16 2003-04-15 新規ｇ蛋白質共役型受容体及びその遺伝子
AU2003227499A AU2003227499A1 (en) 2002-04-16 2003-04-15 Novel g protein-coupled receptor and its gene

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2002113829 2002-04-16
JP2002-113829 2002-04-16

Publications (1)

Publication Number Publication Date
WO2003087364A1 true WO2003087364A1 (fr) 2003-10-23

Family

ID=29243369

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2003/004759 WO2003087364A1 (fr) 2002-04-16 2003-04-15 Nouveau recepteur couple a la proteine g et son gene

Country Status (3)

Country Link
JP (1) JPWO2003087364A1 (fr)
AU (1) AU2003227499A1 (fr)
WO (1) WO2003087364A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005121356A1 (fr) * 2004-06-10 2005-12-22 Astellas Pharma Inc. Nouveau procédé de recherche par criblage

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0835933A2 (fr) * 1996-10-08 1998-04-15 Smithkline Beecham Corporation Un récepteur couplé à une protéine G - HLTEX11
WO2000077256A1 (fr) * 1999-06-11 2000-12-21 Human Genome Sciences, Inc. 48 proteines secretees humaines
WO2001098351A2 (fr) * 2000-06-16 2001-12-27 Incyte Genomics, Inc. Recepteurs couples a la proteine g
WO2002061087A2 (fr) * 2000-12-19 2002-08-08 Lifespan Biosciences, Inc. Peptides antigeniques destines a des recepteurs couples a la proteine g (gpcr), anticorps s'y rapportant, et systeme d'identification desdits peptides antigeniques

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0835933A2 (fr) * 1996-10-08 1998-04-15 Smithkline Beecham Corporation Un récepteur couplé à une protéine G - HLTEX11
WO2000077256A1 (fr) * 1999-06-11 2000-12-21 Human Genome Sciences, Inc. 48 proteines secretees humaines
WO2001098351A2 (fr) * 2000-06-16 2001-12-27 Incyte Genomics, Inc. Recepteurs couples a la proteine g
WO2002061087A2 (fr) * 2000-12-19 2002-08-08 Lifespan Biosciences, Inc. Peptides antigeniques destines a des recepteurs couples a la proteine g (gpcr), anticorps s'y rapportant, et systeme d'identification desdits peptides antigeniques

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HOSOI T. ET AL.: "Identification of a novel human eicosanoid receptor coupled to G(i/o)", J. BIOL. CHEM., vol. 277, no. 35, August 2002 (2002-08-01), pages 31459 - 31465, XP002970211 *
WENZEL-SEIFERT K. ET AL.: "Molecular analysis of beta(2)-adrenoceptor coupling to G(s)-, G(i)- and G(q)-proteins", MOL. PHARMACOL., vol. 58, no. 5, 2000, pages 954 - 966, XP002970210 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005121356A1 (fr) * 2004-06-10 2005-12-22 Astellas Pharma Inc. Nouveau procédé de recherche par criblage

Also Published As

Publication number Publication date
JPWO2003087364A1 (ja) 2005-08-18
AU2003227499A1 (en) 2003-10-27

Similar Documents

Publication Publication Date Title
WO1998039448A2 (fr) 186 nouvelles proteines secretees
WO1999007738A2 (fr) Nouveau recepteur orphelin
JP2001245666A (ja) 新規ポリペプチド
US7527935B2 (en) G-protein coupled receptor having eicosanoid as ligand and gene thereof
US20040014169A1 (en) Novel G protein-coupled receptors
CN109563516A (zh) Gpr156变体及其用途
JPH08193099A (ja) 新規ｇ蛋白質共役型レセプター蛋白質、その製造法および用途
JP2003534765A (ja) 単離されたｖｓｈｋ−１レセプターポリペプチドおよびそれを使用する方法
WO2003087364A1 (fr) Nouveau recepteur couple a la proteine g et son gene
JP2001500389A (ja) 選択的ａｔｐ親和性を示すｇ―結合レセプター
KR100971270B1 (ko) 이피에프 수용체 에세이, 화합물 및 치료학적 조성물
WO2001046415A1 (fr) Polypeptides de type tachykinine et utilisation associee
JP3949111B2 (ja) 新規ｇタンパク質共役型受容体およびその遺伝子
EP1219638A2 (fr) Récepteurs couplés aux protéines-G et ayant de l'homologie avec le P2Y purinorécepteur 1 (P2Y1)
US20030059878A1 (en) Novel purinoceptor and gene thereof
WO2002101002A2 (fr) Nouveaux polynucleotides et polypeptides du gene hgh-v
JP2003038184A (ja) 新規プリン受容体およびその遺伝子
US20030073162A1 (en) Signal peptide-containing proteins
EP1445315B1 (fr) Polypeptides de survie neuronale derives de cellules souches mammiferes et humaines
WO2001046414A1 (fr) Nouvelle proteine receptrice couplee a la proteine de fixation de guanosine triphosphate, bg26
WO2002044212A2 (fr) Recepteur couple aux proteines g humaines et ses utilisations
US20040137574A1 (en) Novel polypeptide
JP2003245082A (ja) 糸球体硬化症の疾患マーカーおよびその利用
EP1215214A1 (fr) Nouveau polypeptide
JP2005102623A (ja) 統合失調症関連タンパク質及びそれをコードする遺伝子

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NI NO NZ OM PH PL PT RO RU SC SD SE SG SK SL TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 2003584306

Country of ref document: JP

122 Ep: pct application non-entry in european phase