JP2004180668A - Method for determining ligand - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for determining a ligand of an orphan receptor. <P>SOLUTION: This method for determining the ligand to a receptor protein comprises using a fusion protein between the receptor protein the ligand of which is not determined and a fluorescent protein. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

【図面の簡単な説明】
【図１】ＴＧＲ５発現ベクターを導入したＨＥＫ２９３細胞（Ａ）および元のベクターのみを導入したＨＥＫ２９３細胞（Ｂ）における、コレステロール代謝関連物質によるレポーター活性化の検出（ｎ＝２）の結果を示す。
【図２】ＨＥＫ２９３細胞におけるリガンド刺激に対するレポーター遺伝子の発現誘導の結果を示す。
【図３】Ｇｉ共存下でのＴＧＲ５のリトコール酸に対する反応の結果を示す。
【図４】リガンド非存在下における、ＣＨＯ細胞に発現させたＴＧＲ５−ＧＦＰ融合タンパク質の局在を示す。図中の白線は４μｍを示す。
【図５】ＴＧＲ５−ＧＦＰを発現させたＣＨＯ細胞にＴＬＣＡを添加して３０分後の融合タンパク質の局在を示す。図中の白線は４μｍを示す。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a method for determining a ligand for a receptor protein present on a cell membrane, and more particularly to a method for determining a ligand for a so-called orphan receptor whose ligand is completely unknown.
[0002]
[Prior art]
Many physiologically active substances such as hormones and neurotransmitters regulate the functions of living organisms through specific receptor proteins present in cell membranes. Many of these receptor proteins perform intracellular signal transduction through activation of a conjugated guanine nucleotide-binding protein (hereinafter abbreviated as G protein), and have a common transmembrane region having seven transmembrane regions. Because of its structure, it is collectively referred to as a G protein-coupled receptor protein or a seven-transmembrane receptor protein (7TMR).
G protein-coupled receptor proteins are present on the surface of various functional cells of living cells and organs, and are physiologically targeted as molecules that regulate the functions of those cells and organs, such as hormones, neurotransmitters and bioactive substances. Plays an important role. The receptor transmits a signal into a cell through binding to a physiologically active substance, and this signal causes various reactions such as activation and suppression of the cell.
To clarify the relationship between substances that regulate the functions of cells and organs of various living organisms and their specific receptor proteins, especially G protein-coupled receptor proteins, elucidation of the functions of cells and organs of various living organisms, It will provide a very important means for drug development closely related to those functions.
[0003]
Conventionally, substances that inhibit the binding of a G protein-coupled receptor protein to a physiologically active substance (that is, a ligand) or substances that bind to cause the same signal transduction as a physiologically active substance (that is, a ligand) have been used for these receptors. It has been utilized as a specific antagonist or agonist as a drug for regulating biological functions.
However, even at this time, there are many so-called orphan receptors for which no corresponding ligand has been identified, and determination of the ligand and elucidation of its function are eagerly awaited.
It is useful to search for a new physiologically active substance (that is, a ligand) and to search for an agonist or antagonist for the G protein-coupled receptor using the signal transduction action of the receptor as an index. These ligands, agonists or antagonists to the receptor can be expected to be used as preventive and / or therapeutic or diagnostic agents for diseases associated with dysfunction of G protein-coupled receptors.
GFP (Green Fluorescent Protein) is a kind of photoprotein derived from the Mesozoan O jellyfish (Patent Documents 1 to 5).
There has been reported a method for determining a Gi, Gs or Gq-coupled orphan receptor ligand by combining a Multiple response element (MRE) and a cAMP response element (CRE) with a reporter activity (Non-Patent Document 1).
A method for screening a molecule that interacts with a Gs- or Gq-coupled orphan receptor by combining a cAMP response element (CRE) and a reporter activity has been reported (Non-Patent Document 2).
There has been reported a method of examining the characteristics of Gi or Gs-coupled receptors whose ligands are known by combining a cAMP response element (CRE) with a reporter activity (Non-Patent Document 3).
A method has been reported for detecting a reaction of a G protein-coupled receptor using cells expressing a chimeric G protein α subunit or chimeric G protein in which the ambiguity of the coupling relationship with the G protein-coupled receptor has been increased. (Patent Document 6).
A method for identifying a ligand for an orphan G protein-coupled receptor using a recombinant yeast expression library has been reported (Patent Document 7).
There has been reported a method for identifying a receptor activity modulator of a cell consisting of a cell containing both a receptor and a test peptide and useful for identifying or screening for an orphan receptor (Patent Document 8).
An expression system using Saccharomyces sexual reproductive system for identifying orphan receptor ligands has been reported (Patent Document 9).
[Patent Document 1]
WO 96/23810
[Patent Document 2]
WO 96/27675
[Patent Document 3]
WO97 / 26333
[Patent Document 4]
WO97 / 28261
[Patent Document 5]
WO97 / 42320
[Patent Document 6]
WO 01/36481
[Patent Document 7]
USP 6,255,059
[Patent Document 8]
US2001 / 0026926
[Patent Document 9]
WO 00/031261
[Non-patent document 1]
Analytical Biochemistry, 275, 54-61, 1999.
[Non-patent document 2]
Analytical Biochemistry, 226, 349-354, 1995
[Non-Patent Document 3]
Current Opinion in Biotechnology, 1995, 6: 574-581.
[0004]
[Problems to be solved by the invention]
In a conventional ligand search / determination method, for example, when screening ligands using eukaryotic cells, a so-called stable cell line that stably expresses the receptor must be established. Rather, the establishment of this cell line required special cells. In addition, screening requires a combination of a plurality of measurements, and when there are a plurality of test compounds, it is difficult to carry out the test because it takes time. In other words, conventional methods for searching and determining ligands and the like include (1) limited cell lines that can be used, (2) time-consuming establishment of the cell lines, and (3) a combination of a plurality of measurement methods. However, when the number of specimens is increased, there is a problem that the implementation becomes difficult. In order to solve these problems, a method for determining a ligand that can be used in various cell lines and can be performed in a short time is desired.
[0005]
[Means for Solving the Problems]
The present inventors have conducted intensive studies and as a result, by producing a cell in which a fusion protein of a receptor protein whose ligand has not been determined and a fluorescent protein such as GFP has been stably or transiently expressed, Using an immunostaining method using a fluorescent protein such as GFP or a fluorescent protein antibody such as a GFP antibody or a Western blot method,
(1) It can be confirmed that the receptor protein is expressed at the protein level,
(2) it can be confirmed that the receptor protein is expressed on the cell membrane,
(3) the amount of receptor protein expression can be estimated;
(4) selecting cells that highly express the receptor protein, and
(5) It has been found that a specific reaction of a receptor caused by a ligand can be detected as internalization of a fusion protein of a receptor and a fluorescent protein into cells, and the ligand is not determined by utilizing these characteristics. It has been found that a ligand of a receptor protein (hereinafter sometimes abbreviated as an orphan receptor) can be efficiently determined. The present inventors have further studied based on these findings, and have completed the present invention.
[0006]
That is, the present invention
[1] A method for determining a ligand for a receptor protein, wherein a fusion protein of a receptor protein and a fluorescent protein for which a ligand has not been determined is used,
[2] The method for determining a ligand according to the above [1], wherein a fusion protein of a receptor protein and a GFP whose ligand has not been determined is used.
[3] The determination of the ligand of the above-mentioned [1], wherein the test compound is brought into contact with a cell or a cell membrane fraction thereof expressing a fusion protein of a receptor protein and a GFP whose ligand has not been determined. Method,
[4] (1) Arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ Activity to promote or suppress release, intracellular cAMP production, intracellular cGMP production, inositol phosphate production, fluctuation of cell membrane potential, phosphorylation of intracellular protein, c-fos activation or pH decrease, (2) MAP Kinase activation, (3) transcription factor activation, (4) diacylglycerol production, (5) opening and closing of ion channels on cell membrane, (6) induction of apoptosis, (7) morphological change, (8) the fusion The ligand according to the above [1], which is characterized by measuring protein transfer from the cell membrane to the cytoplasm, (9) activation of a low-molecular-weight G protein, (10) cell division promoting activity or (11) DNA synthesis promoting activity. How to determine
[5] The method for determining a ligand according to the above [1], wherein the translocation of the fusion protein from the cell membrane to the cytoplasm is measured.
[6] the method for determining a ligand according to the above [5], wherein the translocation of the fusion protein from the cell membrane to the cytoplasm is measured by observing GFP fluorescence;
[7] A cell containing a plasmid expressing a fusion protein of a receptor protein and a fluorescent protein for which a ligand has not been determined and having a DNA encoding a reporter protein downstream of the cAMP response element / promoter, and a test compound. And determining the activity of the reporter protein by contacting
[8] (1) A plasmid containing a DNA encoding a fusion protein of a receptor protein whose ligand has not been determined and a fluorescent protein, and (2) a DNA encoding a reporter protein was ligated downstream of a cAMP response element / promoter. The method according to the above [7], wherein the cells containing the plasmid are cultured, and the activity of the reporter protein is measured by bringing the cells into contact with a test compound.
[9] A test compound and a cell containing a plasmid which expresses a fusion protein of GFP and a receptor protein whose ligand has not been determined, and contains a plasmid in which DNA encoding a reporter protein is linked downstream of a cAMP response element / promoter. Contacting and measuring the activity of the reporter protein, wherein the method for determining a ligand according to the above [2],
[10] (1) a plasmid containing a DNA encoding a fusion protein of a receptor protein whose ligand has not been determined and GFP, and (2) a plasmid having a DNA encoding a reporter protein downstream of a cAMP response element / promoter. The method according to the above-mentioned [9], wherein the cell containing the is cultured, and the activity of the reporter protein is measured by bringing the cell into contact with a test compound.
[11] the method of the above-mentioned [1], wherein the receptor protein is a G protein-coupled receptor protein;
[12] The above-mentioned [1], wherein GFP is a protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7. the method of,
[13] The method of the above-mentioned [7], wherein the promoter is a TATA-like sequence,
[14] the method of the above-mentioned [7], wherein the reporter protein is luciferase;
[15] the method of the above-mentioned [7], wherein the plasmid is obtained by ligating a gene encoding a TATA-like promoter and a reporter protein downstream of the cAMP response element;
[16] The method of the above-mentioned [7], wherein the cell expresses two or more types of receptor proteins whose ligands have not been determined,
[17] The method of the above-mentioned [7], wherein the cell further comprises a plasmid containing a gene encoding an inhibitory G protein α subunit Gi.
[18] The method of the above-mentioned [7], further comprising adding forskolin,
[19] the method of the above-mentioned [16], wherein two or more receptor proteins have similar characteristics;
[20] the method of the above-mentioned [19], wherein the similar characteristics are the basal expression level of the reporter protein and / or the expression level of the reporter protein upon addition of forskolin;
[21] The basal expression level of the reporter protein when two or more receptor proteins are independently expressed in advance and / or the expression level of the reporter protein when forskolin is added are measured in advance, and the expression level of the reporter protein is determined to be the same. The method of the above-mentioned [16], wherein two or more types of receptor proteins are expressed in combination.
[22] a fusion protein of a receptor protein whose ligand has not been determined and a fluorescent protein or a salt thereof,
[23] the fusion protein of the above-mentioned [22], wherein the fluorescent protein is GFP, or a salt thereof;
[24] a DNA containing the DNA encoding the fusion protein of the above-mentioned [22],
[25] a recombinant vector containing the DNA of the above-mentioned [22],
[26] a transformant transformed with the recombinant vector according to the above [25],
[27] use of a fluorescent protein to determine a ligand for a receptor protein whose ligand has not been determined,
[28] Use of GFP for determining a ligand for a receptor protein for which a ligand has not been determined.
[0007]
BEST MODE FOR CARRYING OUT THE INVENTION
The ligand determination method of the present invention is a method characterized by using (1) a fusion protein of a receptor protein whose ligand has not been determined and a fluorescent protein, and specifically, a receptor protein whose ligand has not been determined. A method comprising the steps of: bringing a test compound into contact with a cell or a cell membrane fraction thereof expressing a fusion protein of E. coli and a fluorescent protein (ligand determination method A).
Furthermore, the method for determining a ligand of the present invention comprises the steps of (1) expressing a fusion protein of a receptor protein whose ligand has not been determined and a fluorescent protein, and ligating a DNA encoding a reporter protein downstream of an enhancer / promoter. A method for measuring the activity of a reporter protein whose expression is induced by contacting a cell containing a plasmid with a test compound, (2) (i) encoding a fusion protein of a receptor protein and a fluorescent protein whose ligand has not been determined; A cell containing the plasmid containing the DNA to be expressed and (ii) a plasmid having a DNA encoding the reporter protein downstream of the enhancer / promoter are cultured and brought into contact with a test compound, and the expression of the reporter protein is induced. Those who measure It is (or, the ligand determination method B).
[0008]
As the orphan receptor used in the present invention, for example, a G protein-coupled receptor protein or the like is used. Specific examples include a G protein-coupled receptor protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 9, WO96 / 05302, EP-A-711831, EP-A-789076. EP-A-1103563, EP-A-1103562, JP-A-8-154682, JP-A-8-283295, JP-A-8-196278, JP-A-8-245697, and JP-A-8-266280. And G protein-coupled receptor proteins described in JP-A-9-51795, JP-A-9-121865, JP-A-9-2388686, and JP-A-10-146192.
[0009]
The fluorescent protein is not particularly limited as long as it is a protein that can be visually recognized. For example, GFP (Green Fluorescent Protein), Tag sequence, EGFP (enhanced green fluorescent protein), ECFP (enhanced cyan fluorescent protein, FP) (Enhanced yellow fluorescent protein), DsRED (Discosoma sp. Red Fluorescent Protein), EBFP (Enhanced blue fluorescent protein) and the like are used.
GFP is a kind of photoprotein derived from Mesozoan woolly jellyfish, and is identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7. Such proteins include amino acid sequences.
Examples of the amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7 include, for example, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: An amino acid sequence having about 70% or more, preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more homology with the amino acid sequence represented by SEQ ID NO: 5 or SEQ ID NO: 7. .
Examples of a protein having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7 include, for example, SEQ ID NO: 1, sequence Having substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7, and having SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7 A protein having substantially the same activity as the protein consisting of the amino acid sequence represented by
Examples of the activity of GFP include light emission by excitation light irradiation. Substantially the same activity indicates that those activities are the same in nature. Therefore, it is preferable that their activities are equivalent (eg, about 0.01 to 100 times, preferably about 0.5 to 20 times, more preferably about 0.5 to 2 times). Quantitative factors such as the molecular weight of the protein may be different.
The activity of GFP, such as light emission by excitation light irradiation, can be measured according to a known method.
[0010]
As GFP, (1) one or two or more (preferably, about 1 to 30) of the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7; More preferably, about 1 to 10, more preferably several (1 to 5) amino acids are deleted, (2) SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: : 1 or 2 or more (preferably about 1 to 30, more preferably about 1 to 10, and still more preferably several (1 to 5)) amino acids are added or inserted into the amino acid sequence represented by 7 (3) 1 or 2 or more (preferably about 1 to 30, more preferably 1) in the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7. Is about 1-10 And more preferably, a protein containing an amino acid sequence in which several (1 to 5) amino acids are substituted with another amino acid, or (4) an amino acid sequence obtained by combining deletion, addition and substitution thereof. And (ii) an amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7, wherein the N-terminal methionine residue is deleted, or (ii) A protein containing an amino acid sequence in which the methionine residue at the N-terminus of the amino acid sequence represented by SEQ ID NO: 1 has been deleted and the next alanine residue has been substituted with a threonine residue or a serine residue is preferably used. .
Specifically, for example, (i) GFP consisting of the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7, (ii) SEQ ID NO: 1, SEQ ID NO: 3 GFP comprising an amino acid sequence in which the N-terminal methionine residue of the amino acid sequence represented by SEQ ID NO: 5 or SEQ ID NO: 7 is deleted, (iii) N-terminal of the amino acid sequence represented by SEQ ID NO: 1 For example, GFP comprising an amino acid sequence in which a methionine residue is deleted and the next alanine residue is replaced with a threonine residue or a serine residue is used.
[0011]
For example, the following known sequences are used as the Tag sequence.
(1) His-tag (PCDNA3.1 / His A)
His His His His His (SEQ ID NO: 17)
(2) V5-tag (PCDNA3.1 / V5-His A)
Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr (SEQ ID NO: 18)
(3) myc-tag (pCDNA3.1 / myc-His A)
Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu (SEQ ID NO: 19)
(4) Xpress-tag (PCDNA3.1 / His A)
Asp Leu Tyr Asp Asp Asp Asp Lys (SEQ ID NO: 20)
(5) HA-tag (PCluzHA Expression vector)
Met Gly Ser Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser Leu Glu Phe (SEQ ID NO: 21)
[0012]
As EGFP, a protein consisting of the amino acid sequence represented by SEQ ID NO: 7 is used.
As ECFP, a protein consisting of the amino acid sequence represented by SEQ ID NO: 22 is used.
As EYFP, a protein consisting of the amino acid sequence represented by SEQ ID NO: 24 is used.
As DsRED, a protein consisting of the amino acid sequence represented by SEQ ID NO: 26 is used.
As EBFP, a protein consisting of the amino acid sequence represented by SEQ ID NO: 28 is used.
As long as these EGFP, ECFP, EYFP, DsRED, and EBFP have activities such as emission by excitation light irradiation, (1) one or more (preferably about 1 to 30, more preferably about 1 to 30) amino acids in the amino acid sequence described above. Is an amino acid sequence in which about 1 to 10, more preferably several (1 to 5) amino acids have been deleted, (2) one or two or more (preferably about 1 to 30) More preferably about 1 to 10, more preferably several (1 to 5) amino acids added or inserted; (3) one or two or more amino acids in the above amino acid sequence (preferably Is an amino acid sequence in which about 1 to 30, more preferably about 1 to 10, and still more preferably several (1 to 5) amino acids have been substituted with another amino acid, or ( ) May be a protein having an amino acid sequence that combines their deletion, addition and substitution.
[0013]
As the DNA encoding GFP, specifically, for example, a DNA containing the base sequence represented by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8, or SEQ ID NO: 2 SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8, which has a base sequence that hybridizes under high stringent conditions with a complementary base sequence to the base sequence represented by SEQ ID NO: 6, and SEQ ID NO: 1, SEQ ID NO: : 3, any DNA that encodes a protein having substantially the same activity as GFP consisting of the amino acid sequence represented by SEQ ID NO: 5 or SEQ ID NO: 7 (eg, emission by irradiation with excitation light). It may be something.
Examples of the DNA that can hybridize with the base sequence represented by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8 include, for example, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or DNA containing a nucleotide sequence having a homology of about 70% or more, preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more with the nucleotide sequence represented by No. 8 is used. Can be
[0014]
Hybridization can be performed according to a known method or a method analogous thereto, for example, a method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). When a commercially available hybridization reagent is used, the hybridization can be performed according to the method described in the attached instruction manual. More preferably, it can be carried out under high stringent conditions.
The high stringent conditions are, for example, conditions in which the sodium concentration is about 19 to 40 mM, preferably about 19 to 20 mM, and the temperature is about 50 to 70 ° C, preferably about 60 to 65 ° C. In particular, the case where the sodium concentration is about 19 mM and the temperature is about 65 ° C. is most preferable.
More specifically, the DNA encoding GFP consisting of the amino acid sequence represented by SEQ ID NO: 1 is a DNA containing the nucleotide sequence represented by SEQ ID NO: 2 (WO97 / 42320), and the like; DNA encoding the GFP consisting of the amino acid sequence represented by SEQ ID NO: 4 is a DNA containing the base sequence represented by SEQ ID NO: 4 (WO96 / 23810), etc., and GFP comprising the amino acid sequence represented by SEQ ID NO: 5 (GFPuv) The DNA encoding the GFP (EGFP) consisting of the amino acid sequence represented by SEQ ID NO: 7 is, for example, a DNA containing the base sequence represented by SEQ ID NO: 6 (WO97 / 26333). DNA containing the nucleotide sequence represented by SEQ ID NO: 8 (NCBI Accession No. AB0572), and the like.
[0015]
As the DNA encoding EGFP consisting of the amino acid sequence represented by SEQ ID NO: 7, a DNA consisting of the base sequence represented by SEQ ID NO: 8 or the like is used.
As the DNA encoding ECFP consisting of the amino acid sequence represented by SEQ ID NO: 22, a DNA comprising the base sequence represented by SEQ ID NO: 23 or the like is used.
As the DNA encoding EYFP having the amino acid sequence represented by SEQ ID NO: 24, a DNA comprising the base sequence represented by SEQ ID NO: 25 is used.
As the DNA encoding DsRED consisting of the amino acid sequence represented by SEQ ID NO: 26, a DNA comprising the base sequence represented by SEQ ID NO: 27 or the like is used.
As the DNA encoding EBFP consisting of the amino acid sequence represented by SEQ ID NO: 28, DNA consisting of the base sequence represented by SEQ ID NO: 29 and the like are used.
[0016]
A fusion protein of an orphan receptor and a fluorescent protein can be produced using a DNA obtained by linking a DNA encoding an orphan receptor protein and a DNA encoding GFP.
The ligated DNA is constructed by ligating the 5 'end of the DNA encoding GFP in-frame to the 3' end of the nucleotide sequence of the DNA encoding the orphan receptor.
In addition, a DNA encoding an amino acid sequence called about 1 to 5 linkers selected from amino acid residues having a small molecular weight such as Ala, Gly, and Ser may be inserted between both DNAs.
An expression vector for a fusion protein of an orphan receptor and a fluorescent protein (hereinafter sometimes abbreviated as a fusion protein) may be prepared by, for example, (1) preparing a DNA fragment encoding the fusion protein, and (2) preparing the DNA fragment. It can be produced by ligating downstream of a promoter in an appropriate expression vector.
As a fusion protein of a receptor protein and a fluorescent protein for which a ligand has not been determined, for example, a ligand comprising an amino acid sequence represented by any one of SEQ ID NOs: 30 to 131 has not been determined. A fusion protein of 102 types of each receptor protein and GFP having the amino acid sequence represented by SEQ ID NO: 1 is preferably used. In the amino acid sequence represented by any one of SEQ ID NOs: 30 to 131, the N-terminal methionine residue of the amino acid sequence represented by SEQ ID NO: 1 is deleted. In some cases, the next alanine residue is further substituted with a threonine residue or a serine residue.
The DNA encoding this fusion protein is a DNA encoding GFP consisting of the amino acid sequence represented by SEQ ID NO: 1 downstream of the portion encoding each of the 102 types of receptor proteins for which a ligand has not been determined (SEQ ID NO: The DNA can be prepared by ligating 2), and specifically, a DNA consisting of the base sequence represented by any one of SEQ ID NOs: 132 to 233 is used. In the base sequence represented by any one of SEQ ID NOs: 132 to 233, a methionine residue codon at the 5 ′ end of the base sequence represented by SEQ ID NO: 2 is deleted. In some cases, the next alanine residue codon has been replaced with a threonine residue codon or a serine residue codon.
[0017]
Examples of expression vectors include Escherichia coli-derived plasmids (eg, pCR4, pCR2.1, pBR322, pBR325, pUC12, pUC13), Bacillus subtilis-derived plasmids (eg, pUB110, pTP5, pC194), yeast-derived plasmids (eg, pSH19, In addition to pSH15), bacteriophages such as λ phage, animal viruses such as retrovirus, vaccinia virus, and baculovirus, pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo, and the like are used.
The promoter used in the vector may be any suitable promoter that functions in the host used for gene expression. For example, when animal cells are used as a host, SRα promoter, SV40 promoter, LTR promoter, CMV promoter, HSV-TK promoter and the like are used. Of these, CMV promoter, SRα promoter and the like are preferable.
When expressed in various tissues, insulin II promoter (pancreas), Glycoprotein-α subunit promoter (pituitary gland), transthyretin promoter (liver), renin promoter (kidney), PSE promoter (prostate) ), CD2 promoter (T cell), IgG-heavy chain promoter (B cell), scavenger receptor A promoter (macrophage) and the like.
When the host is Escherichia, trp promoter, lac promoter, recA promoter, λP _L When the host is a bacterium belonging to the genus Bacillus, an SPO1 promoter, an SPO2 promoter, a penP promoter, and the like are preferable. When the host is a yeast, a PHO5 promoter, a PGK promoter, a GAP promoter, an ADH promoter, and the like are preferable. When the host is an insect cell, a polyhedrin promoter, a P10 promoter and the like are preferable.
[0018]
The expression vector may contain, in addition to the above, an enhancer, a splicing signal, a polyA addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV40 ori), and the like, if desired. Examples of selectable markers include a dihydrofolate reductase (hereinafter sometimes abbreviated as dhfr) gene [methotrexate (MTX) resistance] and an ampicillin resistance gene (hereinafter Amp). ^r Neomycin resistance gene (hereinafter Neo). ^r G418 resistance). In particular, CHO (dhfr ⁻ ) When the dhfr gene is used as a selection marker using cells, the target gene can be selected using a thymidine-free medium.
If necessary, a signal sequence suitable for the host is added to the N-terminal side of the receptor protein of the present invention. When the host is a genus Escherichia, a PhoA signal sequence, an OmpA signal sequence, or the like is used. In some cases, MFα signal sequence, SUC2 signal sequence, etc., and when the host is an animal cell, insulin signal sequence, α-interferon signal sequence, antibody molecule, signal sequence, etc. can be used.
Using the expression vector containing the DNA encoding the fusion protein of the receptor protein and the fluorescent protein thus constructed, a transformant can be produced.
[0019]
As the host, for example, Escherichia, Bacillus, yeast, insect cells, insects, animal cells and the like are used.
Specific examples of the genus Escherichia include Escherichia coli K12 DH1 (Proc. Natl. Acad. Sci. USA, 60, 160 (1968)), JM103 (Nucleic Acids Research, 19, 9) ), JA221 (Journal of Molecular Biology, 120, 517 (1978)), HB101 (Journal of Molecular Biology, 41, 459 (1969)), C600 (Genetics, 39, 540, 440, 540, 440). , Nojima, H. and Okayayama, H., Gene, 96, 23-28 (1990)), DH10B (Proc. Nat). . Acad. Sci. USA, 87, 4645-4649 (1990)) and the like can be used.
As the Bacillus bacterium, for example, Bacillus subtilis MI114 (Gene, 24, 255 (1983)), 207-21 (Journal of Biochemistry, 95, 87 (1984)) and the like are used.
Examples of yeast include Saccharomyces cerevisiae AH22 and AH22R. ⁻ , NA87-11A, DKD-5D, 20B-12, Schizosaccharomyces pombe NCYC1913, NCYC2036, Pichia pastoris and the like.
[0020]
When the virus is AcNPV, for example, when the virus is AcNPV, a cell line derived from the larva of Spodoptera litura (Spodoptera frugiperda cell; Sf cell), MG1 cell derived from the midgut of Trichoplusia ni, and Hig derived from egg of Trichoplusia ni ni. ^TM Cells, cells derived from Mamestra brassicae, cells derived from Estigmena acrea, and the like are used. When the virus is BmNPV, a silkworm-derived cell line (Bombyx mori N; BmN cell) or the like is used. As the Sf cells, for example, Sf9 cells (ATCC CRL 1711), Sf21 cells (Vaughn, JL, et al., In Vivo, 13, 213-217 (1977)) and the like are used.
As an insect, for example, a silkworm larva is used [Maeda et al., Nature, 315, 592 (1985)].
Examples of animal cells include monkey cells COS-7, Vero, Chinese hamster cells CHO (hereinafter abbreviated as CHO cells), and dhfr gene-deficient Chinese hamster cells CHO (hereinafter CHO (dhfr). ⁻ ) Cells), mouse L cells, mouse AtT-20, mouse myeloma cells, rat GH3, human FL cells, pancreatic cells (RINm5F, HIT-T15, etc.), pituitary cells (GH3, GH1, RC4 / BC) ), Placenta-derived cells (BeWo, JAR, JEG-3, etc.), liver-derived cells (HepG2, etc.), kidney-derived cells (ACHN, etc.), blood cell-derived cells (H9, THP-1, U-937, etc.), HeLa cells and the like are used.
[0021]
For transformation of Escherichia, for example, Proc. Natl. Acad. Sci. USA, 69, 2110 (1972), and Gene, 17, 107 (1982).
Transformation of Bacillus sp. Can be performed, for example, according to the method described in Molecular & General Genetics, 168, 111 (1979).
To transform yeast, see, for example, Methods in Enzymology, 194, 182-187 (1991), Proc. Natl. Acad. Sci. USA, 75, 1929 (1978).
Insect cells or insects can be transformed, for example, according to the method described in Bio / Technology, 6, 47-55 (1988).
To transform animal cells, for example, see Cell Engineering Separate Volume 8, New Cell Engineering Experiment Protocol. 263-267 (1995) (published by Shujunsha) and Virology, 52, 456 (1973).
Thus, a transformant transformed with the expression vector containing the DNA encoding the fusion protein is obtained.
[0022]
When culturing a transformant whose host is a genus Escherichia or Bacillus, a liquid medium is suitable as a medium used for the culturing, and among them, a carbon source necessary for growth of the transformant, Nitrogen sources, inorganic substances, etc. are contained. As a carbon source, for example, glucose, dextrin, soluble starch, sucrose, etc.As a nitrogen source, for example, ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extract, soybean meal, potato extract and the like Examples of the inorganic or organic substance and the inorganic substance include calcium chloride, sodium dihydrogen phosphate, and magnesium chloride. In addition, yeast extract, vitamins, growth promoting factors and the like may be added. The pH of the medium is preferably about 5 to 8.
[0023]
As a medium for culturing the genus Escherichia, for example, an M9 medium containing glucose and casamino acids [Miller, Journal of Experiments in Molecular Genetics, 431-433, Cold Spring Harbor Laboratory, N.K., N.K. Is preferred. If necessary, an agent such as 3β-indolylacrylic acid can be added in order to make the promoter work efficiently.
When the host is a bacterium belonging to the genus Escherichia, the cultivation is usually performed at about 15 to 43 ° C. for about 3 to 24 hours, and if necessary, aeration and stirring can be added.
When the host is a bacterium belonging to the genus Bacillus, the cultivation is usually performed at about 30 to 40 ° C. for about 6 to 24 hours, and if necessary, aeration and stirring may be added.
When culturing a transformant in which the host is yeast, for example, Burkholder's minimum medium [Bostian, K. et al. L. Proc. Natl. Acad. Sci. USA, 77, 4505 (1980)] or an SD medium containing 0.5% casamino acid [Bitter, G. et al. A. Proc. Natl. Acad. Sci. USA, 81, 5330 (1984)]. Preferably, the pH of the medium is adjusted to about 5-8. The cultivation is usually performed at about 20 ° C. to 35 ° C. for about 24 to 72 hours, and if necessary, aeration or stirring is added.
[0024]
When culturing a transformant in which the host is an insect cell or an insect, the medium used is 10% inactivated by Grace's Insect Medium (Grace, TCC, Nature, 195, 788 (1962)). Those to which additives such as bovine serum are appropriately added are used. The pH of the medium is preferably adjusted to about 6.2 to 6.4. The cultivation is usually performed at about 27 ° C. for about 3 to 5 days, and if necessary, aeration and / or agitation are added.
When culturing a transformant whose animal host is an animal cell, examples of the medium include a MEM medium containing about 5 to 20% fetal bovine serum [Science, 122, 501 (1952)], and a DMEM medium [Virology, 8]. , 396 (1959)], an RPMI 1640 medium [The Journal of the American Medical Association, 199, 519 (1967)], and a 199 medium [Proceeding of the Society in Biotechnology, Inc., 1973. Preferably, the pH is between about 6 and 8. The cultivation is usually performed at about 30 ° C. to 40 ° C. for about 15 to 60 hours, and if necessary, aeration and stirring are added.
Suitable media and culturing conditions for various hosts are known (in particular, WO 00/14227, page 24, line 24 to page 26, line 8, EP1111047A2, paragraphs [0090] to [0096]. ).
As described above, the fusion protein can be expressed in the cell membrane of the transformant.
[0025]
The separation and purification of the fusion protein from the above culture can be performed, for example, by the following method.
When the fusion protein is extracted from the cultured cells or cells, the cells or cells are collected by a known method after culture, suspended in an appropriate buffer, and sonicated, lysozyme and / or freeze-thawed. After destroying the body or cells, a method of obtaining a crude extract of the fusion protein by centrifugation or filtration is used as appropriate. In a buffer, a protein denaturant such as urea or guanidine hydrochloride, or Triton X-100 is used. ^TM And the like. When the receptor protein is secreted into the culture solution, after the culture is completed, the supernatant is separated from the cells or cells by a known method, and the supernatant is collected.
Purification of the fusion protein contained in the thus obtained culture supernatant or extract can be performed by appropriately combining known separation and purification methods. These known separation and purification methods include methods utilizing solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis, mainly molecular weight. Using differences in charge, methods using charge differences such as ion exchange chromatography, methods using specific affinity such as affinity chromatography, and using differences in hydrophobicity such as reversed-phase high-performance liquid chromatography A method utilizing a difference between isoelectric points, such as an isoelectric focusing method, is used.
When the thus obtained fusion protein is obtained in a free form, it can be converted into a salt by a known method or a method analogous thereto, and conversely, when the fusion protein is obtained as a salt, a known method or a method analogous thereto , Free form or other salts.
The fusion protein produced by the recombinant can be arbitrarily modified or the polypeptide can be partially removed by applying an appropriate protein modifying enzyme before or after purification. As the protein modifying enzyme, for example, trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like are used.
The activity or presence of the fusion protein or a salt thereof thus produced can be measured by a binding experiment with a labeled ligand, an enzyme immunoassay using a specific antibody, or the like.
[0026]
As the test compound used in the method for determining the ligand of the present invention, known ligands (eg, angiotensin, bombesin, cannabinoid, cholecystokinin, glutamine, serotonin, melatonin, neuropeptide Y, opioid, purine, vasopressin, oxytocin, PACAP) (Eg, PACAP27, PACAP38), secretin, glucagon, calcitonin, adrenomedullin, somatostatin, GHRH, CRF, ACTH, GRP, PTH, VIP (Vasoactive Intestinal and Related Polypeptide), somatostatin, dopamine , Motilin, amylin, bradykinin, CGRP (calcitonin gene relayed peptide), leukotriene, pancreastatin, prostaglandin , Thromboxane, adenosine, adrenaline, chemokine superfamily (eg, IL-8, GROα, GROβ, GROγ, NAP-2, ENA-78, GCP-2, PF4, IP-10, Mig, PBSF / SDF-1 etc. Chemokine subfamily of MCAF / MCP-1, MCP-2, MCP-3, MCP-4, eotaxin, RANTES, MIP-1α, MIP-1β, HCC-1, MIP-3α / LARC, MIP-3β / CC chemokine subfamily such as ELC, I-309, TARC, MIPF-1, MIPF-2 / eotaxin-2, MDC, DC-CK1 / PARC, SLC; C chemokine subfamily such as lymphotoactin; CX3C chemokine subfamily such as fractionalkine Amily), endothelin, enterogastrin, histamine, neurotensin, TRH, pancreatic polypeptide, galanin, lysophosphatidic acid (LPA), sphingosine 1-phosphate, lysophosphatidylserine, sphingosylphosphorylcholine, lysophosphatidylcholine, steroids , Bile acids, isoprenoids, arachidonic acid metabolites, amines, amino acids, nucleotides, nucleosides, saturated or unsaturated fatty acids, etc., as well as, for example, humans or mammals (eg, mouse, rat, pig, cow, sheep) , Monkeys, etc.), cell culture supernatant, and the like. For example, the tissue extract, the cell culture supernatant, and the like are added to cells expressing a fusion protein of an orphan receptor and GFP, and screening is performed while measuring the cell stimulating activity and the like. Can be determined.
[0027]
Specifically, the ligand determination method A of the present invention comprises constructing an expression cell of the fusion protein, and performing a cell stimulating activity assay or a receptor binding assay using the expression cell or a cell membrane fraction thereof to obtain an orphan. It binds to the receptor and stimulates cell stimulating activity (eg, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ Release, intracellular cAMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation, activity to promote or suppress pH reduction, etc.), MAP kinase Activation, activation of transcription factors (eg, CRE, AP1, NFkB, etc.), diacylglycerol production, ion channels on the cell membrane (eg, K ⁺ , Ca ²⁺ , Na ⁺ , Cl ⁻ ), Induction of apoptosis, change of cell morphology, transfer of receptor (fusion protein) from cell membrane to cytoplasm, activation of low molecular weight G protein (eg, Ras, Rap, Rho, Rab, etc.), cell division promotion This is a method for determining a compound having an activity, a DNA synthesis promoting activity, and the like, that is, a ligand (for example, a peptide, a protein, a non-peptidic compound, a synthetic compound, a fermentation product, and the like).
The method for determining a ligand of the present invention comprises, when a test compound is brought into contact with a cell expressing a fusion protein or a cell membrane fraction thereof, for example, the amount of the test compound bound to the orphan receptor and the cell stimulating activity. It is characterized by measuring.
[0028]
More specifically, the present invention provides the following ligand determination method.
(1) When a labeled test compound is brought into contact with a cell expressing a fusion protein or a cell membrane fraction thereof, the amount of binding of the labeled test compound to the cell or a cell membrane fraction thereof is measured. A method for determining a ligand for an orphan receptor,
(2) Binding of the labeled test compound to the orphan receptor when the labeled test compound is brought into contact with the fusion protein expressed on the cell membrane by culturing a transformant containing the DNA encoding the fusion protein A method for determining a ligand for an orphan receptor, comprising measuring the amount,
(3) When the test compound is brought into contact with a cell expressing the fusion protein, the cell stimulating activity via the orphan receptor, activation of MAP kinase, transcription factors (eg, CRE, AP1, NFkB, etc.) ), Diacylglycerol production, ion channels on cell membranes (eg, K ⁺ , Ca ²⁺ , Na ⁺ , Cl ⁻ ), Induction of apoptosis, change of cell morphology, transfer of receptor (fusion protein) from cell membrane to cytoplasm, activation of low molecular weight G protein (eg, Ras, Rap, Rho, Rab, etc.), cell division promotion A method for determining a ligand for an orphan receptor, which comprises measuring its activity, DNA synthesis promoting activity, and the like, and
(4) Orphan receptor-mediated cell stimulating activity when a test compound is brought into contact with a fusion protein expressed on a cell membrane by culturing a transformant containing a DNA encoding the fusion protein, MAP kinase Activation, activation of transcription factors (eg, CRE, AP1, NFkB, etc.), diacylglycerol production, ion channels on the cell membrane (eg, K ⁺ , Ca ²⁺ , Na ⁺ , Cl ⁻ ), Induction of apoptosis, change of cell morphology, transfer of receptor (fusion protein) from cell membrane to cytoplasm, activation of low molecular weight G protein (eg, Ras, Rap, Rho, Rab, etc.), cell division promotion The present invention provides a method for determining a ligand for an orphan receptor, which comprises measuring an activity, a DNA synthesis promoting activity, and the like.
In particular, it is preferable to carry out the tests (1) and (2) and confirm that the test compound binds to the orphan receptor before conducting the tests (3) and (4).
[0029]
In some cases, the fusion protein can be isolated and purified from the above-mentioned expression cells, and used for a receptor binding assay or the like. As a fusion protein used in the method for determining a ligand, a fusion protein expressed in a large amount using the above cells is suitable.
To produce a fusion protein, the above-described expression method is used, and it is preferable to express DNA encoding the fusion protein in mammalian cells, insect cells, and the like. In order to introduce the DNA fragment encoding the fusion protein into host cells and express them efficiently, the DNA fragment must be polyhedrin promoter of nuclear polyhedrosis virus (NPV) belonging to baculovirus, It is preferable to incorporate the promoter into the downstream of an SV40-derived promoter, a retrovirus promoter, a metallothionein promoter, a human heat shock promoter, a cytomegalovirus promoter, and an SRα promoter. The amount and quality of the expressed fusion protein can be examined by a known method. For example, the literature [Nambi, P .; J. et al. Biol. Chem. , 267, 19555-19559 (1992)].
[0030]
In the ligand determination method of the present invention, it is preferable to use cells expressing the fusion protein or a cell membrane fraction thereof.
When a cell expressing a fusion protein is used in the ligand determination method of the present invention, the cell may be immobilized with glutaraldehyde, formalin, or the like. The immobilization method can be performed according to a known method.
Cells expressing the fusion protein refer to host cells that have expressed the fusion protein, and Escherichia coli, Bacillus subtilis, yeast, insect cells, animal cells, and the like are used as the host cells.
The cell membrane fraction refers to a fraction containing a large amount of cell membrane obtained by crushing cells and then by a known method. As a method for crushing the cells, a method of crushing the cells with a Potter-Elvehjem type homogenizer, crushing with a Waring blender or a polytron (manufactured by Kinematica), crushing with an ultrasonic wave, or ejecting the cells from a thin nozzle while applying pressure by a French press or the like. Crushing and the like. For fractionation of cell membranes, fractionation by centrifugal force such as fractionation centrifugation or density gradient centrifugation is mainly used. For example, the cell lysate is centrifuged at a low speed (500 rpm to 3000 rpm) for a short time (usually about 1 minute to 10 minutes), and the supernatant is further centrifuged at a high speed (15000 rpm to 30000 rpm) for usually 30 minutes to 2 hours. The precipitate is the membrane fraction. The membrane fraction is rich in the expressed fusion protein and membrane components such as cell-derived phospholipids and membrane proteins.
[0031]
The amount of the fusion protein in the cells expressing the fusion protein and its membrane fraction is 10 per cell. ³ -10 ⁸ Preferably a molecule ⁵ -10 ⁷ Preferably it is a molecule. The higher the expression level, the higher the ligand binding activity (specific activity) per membrane fraction, which makes it possible not only to construct a highly sensitive screening system, but also to measure a large number of samples in the same lot. . The expression level of this fusion protein can be roughly estimated from the GFP emission level in cells or cell membranes using a fluorescence microscope or a fluorometer.
In order to carry out the above methods (1) and (2) for determining the ligand for the orphan receptor, an appropriate cell or cell membrane fraction containing the fusion protein and a labeled test compound are required. As the fusion protein fraction, those containing a recombinant fusion receptor having the same activity as the natural orphan receptor are desirable. Here, “equivalent activity” refers to equivalent ligand binding activity, signal transduction action, and the like.
As a labeled test compound, [ ³ H], [ ¹²⁵ I], [ ¹⁴ C], [ ³⁵ S] or the like, and a compound selected from the above-mentioned ligand compound group is used.
[0032]
Specifically, to carry out the method for determining a ligand of the present invention, first, a fusion protein-expressing cell or a cell membrane fraction thereof is suspended in a buffer suitable for the determination method to prepare a fusion protein preparation. Prepare. Any buffer may be used as long as it does not inhibit the binding between the ligand and the orphan receptor, such as a phosphate buffer having a pH of 4 to 10 (preferably pH 6 to 8) or a tris-hydrochloride buffer. Further, in order to reduce non-specific binding, CHAPS, Tween-80 are used. ^TM Surfactants such as (Kao-Atlas), digitonin, deoxycholate and various proteins such as bovine serum albumin and gelatin can also be added to the buffer. Furthermore, a protease inhibitor such as PMSF, leupeptin, E-64 (manufactured by Peptide Research Laboratories), pepstatin and the like can be added for the purpose of suppressing the degradation of the receptor or ligand by the protease. Then, for example, a certain amount (5,000 cpm to 500,000 cpm) of [ ³ H], [ ¹²⁵ I], [ ¹⁴ C], [ ³⁵ S] and the like. A reaction tube containing a large excess of an unlabeled test compound is also prepared to determine the non-specific binding amount (NSB). The reaction is carried out at about 0 ° C. to 50 ° C., preferably about 4 ° C. to 37 ° C., for about 20 minutes to 24 hours, preferably for about 30 minutes to 3 hours. After the reaction, the mixture is filtered through a glass fiber filter or the like, washed with an appropriate amount of the same buffer, and the radioactivity remaining on the glass fiber filter is measured with a liquid scintillation counter or a γ-counter. A test compound in which the count (B-NSB) obtained by subtracting the non-specific binding amount (NSB) from the total binding amount (B) exceeds 0 cpm can be selected as a ligand (including an agonist) for the orphan receptor.
[0033]
In order to carry out the above method (3) or (4) for determining the ligand of the present invention, the above-mentioned cell stimulating activity via orphan receptor, for example, activation of MAP kinase, transcription factor (eg, CRE, AP1) , NFκB, etc., diacylglycerol production, ion channels on cell membranes (eg, K ⁺ , Ca ²⁺ , Na ⁺ , Cl ⁻ ), Induction of apoptosis, change of cell morphology, transfer of receptor (fusion protein) from cell membrane to cytoplasm, activation of low molecular weight G protein (eg, Ras, Rap, Rho, Rab, etc.), cell division promotion The activity, DNA synthesis promoting activity, and the like can be measured using a known method or a commercially available measurement kit. Specifically, first, cells expressing the fusion protein are cultured in a multiwell plate or the like. Prior to ligand determination, replace the cells with a fresh medium or an appropriate buffer that is not toxic to cells, add test compounds, etc., incubate for a certain period of time, and then extract cells or collect supernatant to generate The metabolites determined are quantified according to the respective methods. When the production of a substance (for example, arachidonic acid) as an indicator of cell stimulating activity is difficult due to a degrading enzyme contained in a cell, an assay may be performed by adding an inhibitor to the degrading enzyme. In addition, the activity such as cAMP production suppression can be detected as a production suppression effect on cells whose basic production amount has been increased by forskolin or the like.
[0034]
In the measurement of the “translocation of the receptor into the cytoplasm” of the cell stimulating activity, the migration of the fusion protein from the cell membrane to the cytoplasm can be observed by measuring the fluorescence of a fluorescent protein such as GFP. In order to observe the movement of the fusion protein from the cell membrane to the cytoplasm, it is suitable to use animal cells stably or transiently expressing the fusion protein. A test compound diluted to an appropriate concentration is added to the cells cultured in an appropriate incubator using a normal medium. At this time, the test compound solution diluted directly into the medium may be added, but the cells are washed with Hanks' balanced salt solution (HBSS, BSA may be added at 0 to 10%, preferably 0.1 to 1%). After that, the same solution containing the test compound may be added. After adding the ligand, the cells are allowed to stand at 4 ° C. to 37 ° C., preferably 20 ° C. to 37 ° C. for 1 minute to 6 hours, preferably 10 minutes to 2 hours, and then the migration of the fusion protein from the cell membrane to the cytoplasm is observed. The cells can be observed as they are, but may be fixed with glutaraldehyde or formalin. The fixing method can be performed according to a known method.
Observation may be performed using a normal fluorescence microscope or confocal laser microscope, but a plate reader having a function of irradiating excitation light and a function of capturing a fluorescent image may also be used. At that time, in order to detect a fusion protein of the receptor and the wild-type GFP or GFPuv represented by SEQ ID NO: 3 or SEQ ID NO: 5, excitation with ultraviolet light, preferably 395 nm, fluorescein isocyanate (FITC) or It can be observed using a commercially available filter for GFP detection. Further, in order to detect the fusion protein with GFP shown in SEQ ID NO: 1 or SEQ ID NO: 7, it is excited with excitation light of 460 to 500 nm, preferably 488 nm, and a FITC or a commercially available GFP detection filter is generally used. What is necessary is just to observe using.
Observation of microscopically observable morphological changes such as receptor aggregation (fusion protein) into the cytoplasm, as well as receptor aggregation, receptor localization, or decreased or increased receptor expression. You can also.
[0035]
When performing the ligand determination method B of the present invention, a plasmid containing a DNA encoding a reporter protein downstream of a specific enhancer / promoter, in addition to an expression vector for a fusion protein, is used to prepare a cell, preferably a cell derived from a eukaryote. It is necessary to incorporate into. This plasmid contains a promoter capable of expressing the reporter protein in a cell, preferably a cell derived from a eukaryote, and further comprises a drug resistance gene (for example, ampicillin resistance) as a selectable marker for growth in a prokaryote. Gene).
A plasmid containing a DNA encoding a reporter protein downstream of an enhancer / promoter may be a commercially available plasmid such as a plasmid that can express the reporter protein in a cell under the control of the enhancer and can be introduced into a cell. Any plasmid may be used.
As the enhancer, for example, an enhancer derived from a virus such as SV40 or papilloma virus, an LTR of a retrovirus, a cAMP response element (cAMP response element) (CRE), a TPA response element (TPA response element) (TRE), or the like is used. , Preferably a cAMP response element. Enhancers activated by the cell stimulating activity described above, which can be mediated by orphan receptors expressed by the cells, are suitable.
[0036]
As the promoter, for example, an SV40 promoter, a CMV promoter, a TATA-like promoter of the thymidine kinase gene of HSV and the like are used, and a TATA-like promoter is preferable.
As the reporter protein gene, for example, luciferase gene, β-galactosidase gene, GFP gene, alkaline phosphatase gene and the like are used. Any enzyme gene whose enzyme activity can be detected by a known method can be used as a reporter protein.
Specific examples of such a plasmid include a plasmid in which a gene encoding a TATA-like promoter and a reporter protein (eg, luciferase gene) are linked downstream of a cAMP response element, such as pCRE-Luc (Clontech).
As the cells used in the ligand determination method B, the above-described host cells are used, preferably eukaryotic cells, more preferably animal cells (eg, monkey cells COS-7, Vero, CHO cells, CHO (dhfr) ⁻ ) Cells, mouse L cells, mouse AtT-20, mouse myeloma cells, rat GH3, human FL cells, human HEK293 cells, etc.).
[0037]
The cells may express two or more (preferably two to three) fusion proteins.
When expressing two or more fusion proteins, it is preferable to use two or more orphan receptors having similar biological characteristics.
Similar characteristics include, for example, the expression level of a reporter protein when each of two or more orphan receptors to be used is independently expressed. Specifically, when two or more orphan receptors are independently expressed, (1) the basal expression level of the reporter protein and / or (2) the expression level of the reporter protein upon addition of forskolin as an index, The characteristics of each receptor protein can be distinguished.
Therefore, when a ligand is determined by expressing two or more orphan receptors, the reporter protein is classified into low, medium, and clearly high basal expression levels in advance, or forskolin is added. It is desirable to clarify a receptor protein in which the expression level of the reporter protein is hardly increased. This is because, for example, when two types of receptor proteins, a receptor protein having a high basal expression level of a reporter protein and a receptor having a low basal expression level of a reporter protein, are expressed, when the ligand is bound to the latter, the expression level of the reporter protein increases. Is difficult to detect. That is:
(1) It is preferable to avoid mixing an orphan receptor having a high basal expression level of a reporter protein and a low receptor protein,
(2) It is preferable not to mix a receptor protein in which the expression level of a reporter protein is significantly increased by addition of forskolin and an orphan receptor in which the increase is not so significant.
(3) It is preferable to express a combination of orphan receptors having similar basal expression levels of reporter proteins.
Examples of combinations of orphan receptors having similar characteristics as described above include, for example, a combination of APJ (apelin receptor; Gene, 136, 355 (1993)) and TGR-1 (Japanese Patent Application Laid-Open No. 2002-078492). Can be
[0038]
A specific example of the ligand determination method B will be described below.
The cells are seeded in a 96-well plate and cultured overnight, for example, in DMEM containing 10% fetal calf serum. Here, for example, a commercially available transfection kit is used to simultaneously introduce a fusion protein expression plasmid and a reporter plasmid into cells, and the cells are further cultured overnight to transiently express the orphan receptor in the cells. Let it. After the cells are washed and the medium is made serum-free, the test compound is added. When the enhancer is a CRE, forskolin may be added at the same time as the test compound. After a certain period of incubation, the cells are lysed and the activity of the reporter protein is measured.
In the above-described determination method, when the baseline of the reporter protein activity is high and it is difficult to detect a change in the activity by the test compound, a means for lowering the baseline may be taken. For example, when the orphan receptor is a G protein-coupled receptor protein (GPCR), a change in activity can be easily detected by adding a Gi protein that exhibits a cAMP inhibitory effect among the α subunits of the G protein. To express the Gi protein, a plasmid expressing the DNA encoding the Gi protein can be introduced into the cell along with the orphan receptor plasmid and the reporter plasmid. In this case, the mixing ratio of the three plasmids (orphan receptor plasmid: reporter plasmid: Gi plasmid) is preferably about 5 to 15: 1: 1 to 6, more preferably about 7: 1: 3.
In the ligand determination method B of the present invention, when the activity of the reporter protein increases or decreases by about 20% or more, preferably about 50% or more when a test compound is added, the test compound can be identified as a ligand. it can.
The test compound used in the ligand determination method B of the present invention is a compound selected from the aforementioned test compounds and the like.
[0039]
Furthermore, the kit for determining a ligand of the present invention contains cells capable of expressing the fusion protein of the present invention or a cell membrane fraction thereof.
Examples of the kit for determining a ligand of the present invention include the following.
1. Ligand determination reagent
(1) Measurement buffer and washing buffer
Hanks' Balanced Salt Solution (manufactured by Gibco) plus 0.05% bovine serum albumin (manufactured by Sigma).
The solution may be sterilized by filtration through a filter having a pore size of 0.45 μm and stored at 4 ° C., or may be prepared at use.
(2) Fusion protein preparation
CHO cells expressing the fusion protein were placed in a 12-well plate at 5 × 10 5 ⁵ Passage at 37 ° C, 5% CO ₂ , Cultured at 95% air for 2 days.
(3) Labeled test compound
Commercially available [ ³ H], [ ¹²⁵ I], [ ¹⁴ C], [ ³⁵ S] or a compound labeled by an appropriate method.
The aqueous solution is stored at 4 ° C. or −20 ° C., and diluted to 1 μM with a measuring buffer before use. Test compounds that are poorly soluble in water are dissolved in dimethylformamide, DMSO, methanol and the like.
(4) Unlabeled test compound
The same as the labeled compound is prepared at a concentration 100 to 1000 times higher.
[0040]
2. Measurement method
(1) CHO cells expressing the receptor protein of the present invention cultured on a 12-well tissue culture plate are washed twice with 1 ml of the measurement buffer, and then 490 μl of the measurement buffer is added to each well.
(2) 5 μl of the labeled test compound is added and reacted at room temperature for 1 hour. To determine the amount of non-specific binding, add 5 μl of unlabeled test compound.
(3) Remove the reaction solution and wash three times with 1 ml of washing buffer. The labeled test compound bound to the cells is dissolved in 0.2N NaOH-1% SDS, and mixed with 4 ml of liquid scintillator A (manufactured by Wako Pure Chemical Industries, Ltd.).
(4) Radioactivity is measured using a liquid scintillation counter (manufactured by Beckman).
[0041]
As described above, according to the ligand determination methods (A and B) of the present invention, immunofluorescence using a fluorescent protein such as GFP or immunostaining using a fluorescent protein antibody such as a GFP antibody or Western blotting can be used.
(1) It can be confirmed that the receptor protein is expressed at the protein level,
(2) it can be confirmed that the receptor protein is expressed on the cell membrane,
(3) the amount of receptor protein expression can be estimated;
(4) selecting cells that highly express the receptor protein, and
(5) The specific reaction of the receptor by the ligand can be detected as the internalization of the fusion protein of the receptor and the fluorescent protein into cells. By utilizing these characteristics, the ligand of a receptor protein (orphan receptor) whose ligand has not been determined can be efficiently determined.
The ligand determined in this way binds to the receptor protein and regulates its physiological function, and thus can be used as a prophylactic and / or therapeutic agent for diseases related to the function of the receptor protein. Furthermore, using a ligand and its receptor protein, an agonist / antagonist of the receptor can be screened.
[0042]
In the present specification and drawings, bases, amino acids, and the like are represented by abbreviations based on the abbreviations by IUPAC-IUB Commission on Biochemical Nomenclature or commonly used abbreviations in the art. When an amino acid can have an optical isomer, the L-form is indicated unless otherwise specified.
[0043]
The sequence numbers in the sequence listing in the present specification indicate the following sequences.
SEQ ID NO: 1
1 shows the amino acid sequence of GFP (hereinafter abbreviated as GFP-1) used in Example 1.
SEQ ID NO: 2
1 shows the nucleotide sequence of DNA encoding GFP used in Example 1.
SEQ ID NO: 3
2 shows the amino acid sequence of wild-type GFP.
SEQ ID NO: 4
1 shows the nucleotide sequence of DNA encoding wild-type GFP.
SEQ ID NO: 5
1 shows the amino acid sequence of GFPuv.
SEQ ID NO: 6
2 shows the nucleotide sequence of DNA encoding GFPuv.
SEQ ID NO: 7
1 shows the amino acid sequence of EGFP.
SEQ ID NO: 8
1 shows the nucleotide sequence of cDNA encoding EGFP.
SEQ ID NO: 9
1 shows the amino acid sequence of human-derived G protein-coupled receptor protein TGR5 used in Example 1.
SEQ ID NO: 10
1 shows the nucleotide sequence of cDNA encoding human-derived G protein-coupled receptor protein TGR5 used in Example 1.
SEQ ID NO: 11
2 shows the base sequence of primer 1 used in the PCR reaction in Reference Example 1.
SEQ ID NO: 12
2 shows the base sequence of primer 2 used in the PCR reaction in Reference Example 1.
SEQ ID NO: 13
1 shows the amino acid sequence of human-derived parathyroid hormone receptor (PTH-R).
SEQ ID NO: 14
1 shows the nucleotide sequence of cDNA encoding human-derived parathyroid hormone receptor (PTH-R).
SEQ ID NO: 15
1 shows the amino acid sequence of human-derived GPR40.
SEQ ID NO: 16
1 shows the nucleotide sequence of cDNA encoding human-derived GPR40.
SEQ ID NO: 17
2 shows the amino acid sequence of His-Tag.
SEQ ID NO: 18
2 shows the amino acid sequence of V5-tag.
SEQ ID NO: 19
2 shows the amino acid sequence of myc-tag.
SEQ ID NO: 20
2 shows the amino acid sequence of Xpress-tag.
SEQ ID NO: 21
2 shows the amino acid sequence of HA-tag.
SEQ ID NO: 22
2 shows the amino acid sequence of ECFP.
SEQ ID NO: 23
1 shows the nucleotide sequence of cDNA encoding ECFP.
SEQ ID NO: 24
2 shows the amino acid sequence of EYFP.
SEQ ID NO: 25
1 shows the nucleotide sequence of cDNA encoding EYFP.
SEQ ID NO: 26
2 shows the amino acid sequence of DsRED.
SEQ ID NO: 27
1 shows the nucleotide sequence of cDNA encoding DsRED.
SEQ ID NO: 28
2 shows the amino acid sequence of EBFP.
SEQ ID NO: 29
1 shows the nucleotide sequence of cDNA encoding EBFP.
SEQ ID NO: 30
2 shows the amino acid sequence of a fusion protein of orphan receptor hBL5 and GFP-1.
SEQ ID NO: 31
2 shows the amino acid sequence of a fusion protein of orphan receptor h7TBA62 and GFP-1.
SEQ ID NO: 32
3 shows the amino acid sequence of a fusion protein of orphan receptor 14273 and GFP-1.
SEQ ID NO: 33
2 shows the amino acid sequence of a fusion protein of orphan receptor EMR3 and GFP-1.
SEQ ID NO: 34
2 shows the amino acid sequence of a fusion protein of orphan receptor GPR15 and GFP-1.
SEQ ID NO: 35
2 shows the amino acid sequence of a fusion protein of orphan receptor GPR31 and GFP-1.
SEQ ID NO: 36
2 shows the amino acid sequence of a fusion protein of orphan receptor GPRC5B and GFP-1.
SEQ ID NO: 37
2 shows the amino acid sequence of a fusion protein of orphan receptor PSEC0142 and GFP-1.
SEQ ID NO: 38
2 shows the amino acid sequence of a fusion protein of orphan receptor HE6 and GFP-1.
SEQ ID NO: 39
2 shows the amino acid sequence of a fusion protein of orphan receptor GPR61 and GFP-1.
SEQ ID NO: 40
2 shows the amino acid sequence of a fusion protein of orphan receptor TGR9 and GFP-1.
SEQ ID NO: 41
2 shows the amino acid sequence of a fusion protein of orphan receptor TGR24 and GFP-1.
SEQ ID NO: 42
2 shows the amino acid sequence of a fusion protein of orphan receptor ZGPR1 and GFP-1.
SEQ ID NO: 43
2 shows the amino acid sequence of a fusion protein of orphan receptor EMR1 and GFP-1.
SEQ ID NO: 44
2 shows the amino acid sequence of a fusion protein of orphan receptor GPR25 and GFP-1.
SEQ ID NO: 45
2 shows the amino acid sequence of a fusion protein of orphan receptor GPR55 and GFP-1.
SEQ ID NO: 46
2 shows the amino acid sequence of a fusion protein of orphan receptor AXOR14 and GFP-1.
SEQ ID NO: 47
2 shows the amino acid sequence of a fusion protein of orphan receptor TM7SF1 and GFP-1.
SEQ ID NO: 48
2 shows the amino acid sequence of a fusion protein of orphan receptor PSP24B and GFP-1.
SEQ ID NO: 49
2 shows the amino acid sequence of a fusion protein of the orphan receptor SREB3 and GFP-1.
SEQ ID NO: 50
2 shows the amino acid sequence of a fusion protein of orphan receptor TGR37 and GFP-1.
SEQ ID NO: 51
3 shows the amino acid sequence of a fusion protein of orphan receptor H963 and GFP-1.
SEQ ID NO: 52
2 shows the amino acid sequence of a fusion protein of orphan receptor GPR87 and GFP-1.
SEQ ID NO: 53
2 shows the amino acid sequence of a fusion protein of orphan receptor GPR91 and GFP-1.
SEQ ID NO: 54
2 shows the amino acid sequence of a fusion protein of orphan receptor PNR and GFP-1.
SEQ ID NO: 55
2 shows the amino acid sequence of a fusion protein of orphan receptor TGR29 and GFP-1.
SEQ ID NO: 56
2 shows the amino acid sequence of a fusion protein of orphan receptor TGR36 and GFP-1.
SEQ ID NO: 57
2 shows the amino acid sequence of a fusion protein of orphan receptor H9 and GFP-1.
SEQ ID NO: 58
2 shows the amino acid sequence of a fusion protein of orphan receptor TGR18 and GFP-1.
SEQ ID NO: 59
2 shows the amino acid sequence of a fusion protein of orphan receptor TGR19 and GFP-1.
SEQ ID NO: 60
2 shows the amino acid sequence of a fusion protein of orphan receptor AM-R and GFP-1.
SEQ ID NO: 61
2 shows the amino acid sequence of a fusion protein of orphan receptor GPR19 and GFP-1.
SEQ ID NO: 62
2 shows the amino acid sequence of a fusion protein of orphan receptor GPR45 and GFP-1.
SEQ ID NO: 63
2 shows the amino acid sequence of a fusion protein of orphan receptor GPRC5D and GFP-1.
SEQ ID NO: 64
2 shows the amino acid sequence of a fusion protein of orphan receptor LGR6 and GFP-1.
SEQ ID NO: 65
2 shows the amino acid sequence of a fusion protein of orphan receptor RUP3 and GFP-1.
SEQ ID NO: 66
2 shows the amino acid sequence of a fusion protein of orphan receptor TGR14 and GFP-1.
SEQ ID NO: 67
2 shows the amino acid sequence of a fusion protein of orphan receptor TPRA40 and GFP-1.
SEQ ID NO: 68
2 shows the amino acid sequence of a fusion protein of orphan receptor GPR22 and GFP-1.
SEQ ID NO: 69
2 shows the amino acid sequence of a fusion protein of orphan receptor GPR52 and GFP-1.
SEQ ID NO: 70
2 shows the amino acid sequence of a fusion protein of orphan receptor FLH2882 and GFP-1.
SEQ ID NO: 71
2 shows the amino acid sequence of a fusion protein of the orphan receptor SNORF36 and GFP-1.
SEQ ID NO: 72
2 shows the amino acid sequence of a fusion protein of orphan receptor MRG and GFP-1.
SEQ ID NO: 73
2 shows the amino acid sequence of a fusion protein of the orphan receptor SREB2 and GFP-1.
SEQ ID NO: 74
2 shows the amino acid sequence of a fusion protein of orphan receptor GPR12 and GFP-1.
SEQ ID NO: 75
3 shows the amino acid sequence of a fusion protein of orphan receptor GPR30 and GFP-1.
SEQ ID NO: 76
2 shows the amino acid sequence of a fusion protein of orphan receptor GPR82 and GFP-1.
SEQ ID NO: 77
2 shows the amino acid sequence of a fusion protein of orphan receptor RECAP and GFP-1.
SEQ ID NO: 78
2 shows the amino acid sequence of a fusion protein of orphan receptor HB954 and GFP-1.
SEQ ID NO: 79
2 shows the amino acid sequence of a fusion protein of orphan receptor RDC1 and GFP-1.
SEQ ID NO: 80
2 shows the amino acid sequence of a fusion protein of orphan receptor TGR6 and GFP-1.
SEQ ID NO: 81
2 shows the amino acid sequence of a fusion protein of orphan receptor A-2 and GFP-1.
SEQ ID NO: 82
2 shows the amino acid sequence of a fusion protein of orphan receptor JEG18 and GFP-1.
SEQ ID NO: 83
2 shows the amino acid sequence of a fusion protein of orphan receptor GPR17 and GFP-1.
SEQ ID NO: 84
2 shows the amino acid sequence of a fusion protein of orphan receptor GPR35 and GFP-1.
SEQ ID NO: 85
2 shows the amino acid sequence of a fusion protein of orphan receptor GPRC5C and GFP-1.
SEQ ID NO: 86
2 shows the amino acid sequence of a fusion protein of orphan receptor HM74 and GFP-1.
SEQ ID NO: 87
2 shows the amino acid sequence of a fusion protein of orphan receptor RPE and GFP-1.
SEQ ID NO: 88
2 shows the amino acid sequence of a fusion protein of orphan receptor TGR13 and GFP-1.
SEQ ID NO: 89
2 shows the amino acid sequence of a fusion protein of orphan receptor TGR27 and GFP-1.
SEQ ID NO: 90
2 shows the amino acid sequence of a fusion protein of orphan receptor DEZ and GFP-1.
SEQ ID NO: 91
2 shows the amino acid sequence of a fusion protein of orphan receptor ratGPR1 and GFP-1.
SEQ ID NO: 92
2 shows the amino acid sequence of a fusion protein of orphan receptor GPR3 and GFP-1.
SEQ ID NO: 93
2 shows the amino acid sequence of a fusion protein of orphan receptor GPR6 and GFP-1.
SEQ ID NO: 94
2 shows the amino acid sequence of a fusion protein of orphan receptor RAIG1 and GFP-1.
SEQ ID NO: 95
2 shows the amino acid sequence of a fusion protein of orphan receptor TGR2-1 and GFP-1.
SEQ ID NO: 96
2 shows the amino acid sequence of a fusion protein of orphan receptor TGR2-2 and GFP-1.
SEQ ID NO: 97
2 shows the amino acid sequence of a fusion protein of orphan receptor TGR21 and GFP-1.
SEQ ID NO: 98
2 shows the amino acid sequence of a fusion protein of orphan receptor GPR56 and GFP-1.
SEQ ID NO: 99
2 shows the amino acid sequence of a fusion protein of orphan receptor KIAA0758 and GFP-1.
SEQ ID NO: 100
2 shows the amino acid sequence of a fusion protein of orphan receptor RE2 and GFP-1.
SEQ ID NO: 101
2 shows the amino acid sequence of a fusion protein of orphan receptor P40 and GFP-1.
SEQ ID NO: 102
2 shows the amino acid sequence of a fusion protein of orphan receptor GPR27 and GFP-1.
SEQ ID NO: 103
2 shows the amino acid sequence of a fusion protein of orphan receptor HG38 and GFP-1.
SEQ ID NO: 104
2 shows the amino acid sequence of a fusion protein of orphan receptor DRR1 and GFP-1.
SEQ ID NO: 105
2 shows the amino acid sequence of a fusion protein of orphan receptor TGR12 and GFP-1.
SEQ ID NO: 106
2 shows the amino acid sequence of a fusion protein of orphan receptor TGR11 and GFP-1.
SEQ ID NO: 107
2 shows the amino acid sequence of a fusion protein of orphan receptor TGR15 and GFP-1.
SEQ ID NO: 108
2 shows the amino acid sequence of a fusion protein of orphan receptor TGR8 and GFP-1.
SEQ ID NO: 109
2 shows the amino acid sequence of a fusion protein of orphan receptor GPR20 and GFP-1.
SEQ ID NO: 110
2 shows the amino acid sequence of a fusion protein of orphan receptor TGR10 and GFP-1.
SEQ ID NO: 111
2 shows the amino acid sequence of a fusion protein of orphan receptor TGR30 and GFP-1.
SEQ ID NO: 112
2 shows the amino acid sequence of a fusion protein of orphan receptor GPR18 and GFP-1.
SEQ ID NO: 113
2 shows the amino acid sequence of a fusion protein of orphan receptor TGR25 and GFP-1.
SEQ ID NO: 114
2 shows the amino acid sequence of a fusion protein of orphan receptor GPR23 and GFP-1.
SEQ ID NO: 115
2 shows the amino acid sequence of a fusion protein of orphan receptor P2Y10 and GFP-1.
SEQ ID NO: 116
2 shows the amino acid sequence of a fusion protein of orphan receptor GPR37 and GFP-1.
SEQ ID NO: 117
2 shows the amino acid sequence of a fusion protein of orphan receptor ET (B) R-LP-2 and GFP-1.
SEQ ID NO: 118
2 shows the amino acid sequence of a fusion protein of orphan receptor FPRL2 and GFP-1.
SEQ ID NO: 119
2 shows the amino acid sequence of a fusion protein of orphan receptor GPR32 and GFP-1.
SEQ ID NO: 120
2 shows the amino acid sequence of a fusion protein of orphan receptor dj287G14.2 and GFP-1.
SEQ ID NO: 121
2 shows the amino acid sequence of a fusion protein of orphan receptor BRS-3 and GFP-1.
SEQ ID NO: 122
2 shows the amino acid sequence of a fusion protein of orphan receptor GPR39 and GFP-1.
SEQ ID NO: 123
3 shows the amino acid sequence of a fusion protein of orphan receptor 63A2 and GFP-1.
SEQ ID NO: 124
2 shows the amino acid sequence of a fusion protein of orphan receptor GPR84 and GFP-1.
SEQ ID NO: 125
2 shows the amino acid sequence of a fusion protein of orphan receptor GPR21 and GFP-1.
SEQ ID NO: 126
2 shows the amino acid sequence of a fusion protein of orphan receptor GPR48 and GFP-1.
SEQ ID NO: 127
2 shows the amino acid sequence of a fusion protein of the orphan receptor SNORF1 and GFP-1.
SEQ ID NO: 128
2 shows the amino acid sequence of a fusion protein of orphan receptor BA12 and GFP-1.
SEQ ID NO: 129
2 shows the amino acid sequence of a fusion protein of orphan receptor MAS and GFP-1.
SEQ ID NO: 130
2 shows the amino acid sequence of a fusion protein of orphan receptor OT7T009 and GFP-1.
SEQ ID NO: 131
2 shows the amino acid sequence of a fusion protein of orphan receptor TGR34 and GFP-1.
SEQ ID NO: 132
1 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor hBL5 and GFP-1.
SEQ ID NO: 133
2 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor h7TBA62 and GFP-1.
SEQ ID NO: 134
The nucleotide sequence of a DNA encoding a fusion protein of orphan receptor 14273 and GFP-1 is shown.
SEQ ID NO: 135
1 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor EMR3 and GFP-1.
SEQ ID NO: 136
2 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor GPR15 and GFP-1.
SEQ ID NO: 137
1 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor GPR31 and GFP-1.
SEQ ID NO: 138
1 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor GPRC5B and GFP-1.
SEQ ID NO: 139
The nucleotide sequence of the DNA encoding the fusion protein of the orphan receptor PSEC0142 and GFP-1 is shown.
SEQ ID NO: 140
1 shows the nucleotide sequence of a DNA encoding a fusion protein of orphan receptor HE6 and GFP-1.
SEQ ID NO: 141
1 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor GPR61 and GFP-1.
SEQ ID NO: 142
1 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor TGR9 and GFP-1.
SEQ ID NO: 143
1 shows the nucleotide sequence of a DNA encoding a fusion protein of orphan receptor TGR24 and GFP-1.
SEQ ID NO: 144
1 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor ZGPR1 and GFP-1.
SEQ ID NO: 145
1 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor EMR1 and GFP-1.
SEQ ID NO: 146
2 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor GPR25 and GFP-1.
SEQ ID NO: 147
1 shows the nucleotide sequence of a DNA encoding a fusion protein of orphan receptor GPR55 and GFP-1.
SEQ ID NO: 148
1 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor AXOR14 and GFP-1.
SEQ ID NO: 149
1 shows the nucleotide sequence of a DNA encoding a fusion protein of orphan receptor TM7SF1 and GFP-1.
SEQ ID NO: 150
1 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor PSP24B and GFP-1.
SEQ ID NO: 151
1 shows the nucleotide sequence of a DNA encoding a fusion protein of orphan receptor SREB3 and GFP-1.
SEQ ID NO: 152
1 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor TGR37 and GFP-1.
SEQ ID NO: 153
2 shows the nucleotide sequence of a DNA encoding a fusion protein of orphan receptor H963 and GFP-1.
SEQ ID NO: 154
1 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor GPR87 and GFP-1.
SEQ ID NO: 155
1 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor GPR91 and GFP-1.
SEQ ID NO: 156
1 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor PNR and GFP-1.
SEQ ID NO: 157
1 shows the nucleotide sequence of a DNA encoding a fusion protein of orphan receptor TGR29 and GFP-1.
SEQ ID NO: 158
1 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor TGR36 and GFP-1.
SEQ ID NO: 159
1 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor H9 and GFP-1.
SEQ ID NO: 160
2 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor TGR18 and GFP-1.
SEQ ID NO: 161
1 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor TGR19 and GFP-1.
SEQ ID NO: 162
1 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor AM-R and GFP-1.
SEQ ID NO: 163
1 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor GPR19 and GFP-1.
SEQ ID NO: 164
1 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor GPR45 and GFP-1.
SEQ ID NO: 165
1 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor GPRC5D and GFP-1.
SEQ ID NO: 166
1 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor LGR6 and GFP-1.
SEQ ID NO: 167
1 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor RUP3 and GFP-1.
SEQ ID NO: 168
1 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor TGR14 and GFP-1.
SEQ ID NO: 169
1 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor TPRA40 and GFP-1.
SEQ ID NO: 170
1 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor GPR22 and GFP-1.
SEQ ID NO: 171
1 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor GPR52 and GFP-1.
SEQ ID NO: 172
1 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor FLH2882 and GFP-1.
SEQ ID NO: 173
1 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor SNORF36 and GFP-1.
SEQ ID NO: 174
1 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor MRG and GFP-1.
SEQ ID NO: 175
2 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor SREB2 and GFP-1.
SEQ ID NO: 176
2 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor GPR12 and GFP-1.
SEQ ID NO: 177
1 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor GPR30 and GFP-1.
SEQ ID NO: 178
1 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor GPR82 and GFP-1.
SEQ ID NO: 179
1 shows the nucleotide sequence of a DNA encoding a fusion protein of orphan receptor RECAP and GFP-1.
SEQ ID NO: 180
1 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor HB954 and GFP-1.
SEQ ID NO: 181
1 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor RDC1 and GFP-1.
SEQ ID NO: 182
1 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor TGR6 and GFP-1.
SEQ ID NO: 183
1 shows the nucleotide sequence of a DNA encoding a fusion protein of orphan receptor A-2 and GFP-1.
SEQ ID NO: 184
1 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor JEG18 and GFP-1.
SEQ ID NO: 185
1 shows the nucleotide sequence of a DNA encoding a fusion protein of orphan receptor GPR17 and GFP-1.
SEQ ID NO: 186
1 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor GPR35 and GFP-1.
SEQ ID NO: 187
2 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor GPRC5C and GFP-1.
SEQ ID NO: 188
1 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor HM74 and GFP-1.
SEQ ID NO: 189
1 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor RPE and GFP-1.
SEQ ID NO: 190
1 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor TGR13 and GFP-1.
SEQ ID NO: 191
1 shows the nucleotide sequence of a DNA encoding a fusion protein of orphan receptor TGR27 and GFP-1.
SEQ ID NO: 192
1 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor DEZ and GFP-1.
SEQ ID NO: 193
2 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor ratGPR1 and GFP-1.
SEQ ID NO: 194
1 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor GPR3 and GFP-1.
SEQ ID NO: 195
1 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor GPR6 and GFP-1.
SEQ ID NO: 196
1 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor RAIG1 and GFP-1.
SEQ ID NO: 197
1 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor TGR2-1 and GFP-1.
SEQ ID NO: 198
1 shows the nucleotide sequence of a DNA encoding a fusion protein of orphan receptor TGR2-2 and GFP-1.
SEQ ID NO: 199
1 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor TGR21 and GFP-1.
SEQ ID NO: 200
2 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor GPR56 and GFP-1.
SEQ ID NO: 201
1 shows the nucleotide sequence of a DNA encoding a fusion protein of orphan receptor KIAA0758 and GFP-1.
SEQ ID NO: 202
2 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor RE2 and GFP-1.
SEQ ID NO: 203
1 shows the nucleotide sequence of a DNA encoding a fusion protein of orphan receptor P40 and GFP-1.
SEQ ID NO: 204
2 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor GPR27 and GFP-1.
SEQ ID NO: 205
1 shows the nucleotide sequence of a DNA encoding a fusion protein of orphan receptor HG38 and GFP-1.
SEQ ID NO: 206
1 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor DRR1 and GFP-1.
SEQ ID NO: 207
2 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor TGR12 and GFP-1.
SEQ ID NO: 208
2 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor TGR11 and GFP-1.
SEQ ID NO: 209
1 shows the nucleotide sequence of a DNA encoding a fusion protein of orphan receptor TGR15 and GFP-1.
SEQ ID NO: 210
1 shows the nucleotide sequence of a DNA encoding a fusion protein of orphan receptor TGR8 and GFP-1.
SEQ ID NO: 211
1 shows the nucleotide sequence of a DNA encoding a fusion protein of orphan receptor GPR20 and GFP-1.
SEQ ID NO: 212
1 shows the nucleotide sequence of a DNA encoding a fusion protein of orphan receptor TGR10 and GFP-1.
SEQ ID NO: 213
1 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor TGR30 and GFP-1.
SEQ ID NO: 214
2 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor GPR18 and GFP-1.
SEQ ID NO: 215
2 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor TGR25 and GFP-1.
SEQ ID NO: 216
1 shows the nucleotide sequence of a DNA encoding a fusion protein of orphan receptor GPR23 and GFP-1.
SEQ ID NO: 217
1 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor P2Y10 and GFP-1.
SEQ ID NO: 218
1 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor GPR37 and GFP-1.
SEQ ID NO: 219
1 shows the nucleotide sequence of a DNA encoding a fusion protein of orphan receptor ET (B) R-LP-2 and GFP-1.
SEQ ID NO: 220
1 shows the nucleotide sequence of a DNA encoding a fusion protein of orphan receptor FPRL2 and GFP-1.
SEQ ID NO: 221
1 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor GPR32 and GFP-1.
SEQ ID NO: 222
The nucleotide sequence of the DNA encoding the fusion protein of orphan receptor dj287G14.2 and GFP-1 is shown.
SEQ ID NO: 223
1 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor BRS-3 and GFP-1.
SEQ ID NO: 224
1 shows the nucleotide sequence of a DNA encoding a fusion protein of orphan receptor GPR39 and GFP-1.
SEQ ID NO: 225
2 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor 63A2 and GFP-1.
SEQ ID NO: 226
1 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor GPR84 and GFP-1.
SEQ ID NO: 227
1 shows the nucleotide sequence of a DNA encoding a fusion protein of orphan receptor GPR21 and GFP-1.
SEQ ID NO: 228
2 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor GPR48 and GFP-1.
SEQ ID NO: 229
1 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor SNORF1 and GFP-1.
SEQ ID NO: 230
1 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor BA12 and GFP-1.
SEQ ID NO: 231
1 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor MAS and GFP-1.
SEQ ID NO: 232
1 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor OT7T009 and GFP-1.
SEQ ID NO: 233
1 shows the nucleotide sequence of DNA encoding a fusion protein of orphan receptor TGR34 and GFP-1.
[0044]
The transformant Escherichia coli JM109 / pCR4-hTGR5 obtained in Reference Example 1 has been used since April 3, 2000, 1-1, Higashi 1-1, Tsukuba City, Ibaraki Pref. 305-8566) at the National Institute of Advanced Industrial Science and Technology (AIST) under the Patent Organism Depositary Center (formerly Ministry of International Trade and Industry, National Institute of Advanced Industrial Science and Technology (NIBH)) under the deposit numbers FERM BP-7114 and 2000 (2000). It has been deposited with the Fermentation Research Institute (IFO) at 17-17-85, Jusanhoncho, Yodogawa-ku, Osaka-shi, Osaka (Postal code 532-8686) from March 23, 2003 as deposit number IFO 16410.
[0045]
【Example】
Hereinafter, the present invention will be described in more detail with reference to Reference Examples and Examples, but these examples do not limit the scope of the present invention. In addition, the genetic manipulation using Escherichia coli followed the method described in Molecular cloning (Molecular cloning).
[0046]
Reference Example 1 Cloning and nucleotide sequence of cDNA encoding human spleen G protein-coupled receptor protein
Using human spleen cDNA (Clontech) as a template, a PCR reaction was performed using two primers, primer 1 (SEQ ID NO: 11) and primer 2 (SEQ ID NO: 12). The composition of the reaction solution in the reaction was as follows: 1/10 amount of the template cDNA, 1/50 amount of Advantage-GC2 Polymerase Mix (Clontech), 0.5 μM each of primer 1 (SEQ ID NO: 11) and primer 2 (SEQ ID NO: 12). , 200 μM of dNTPs, 1/5 of the buffer attached to the enzyme product, and 1/5 of GC Melt. The final volume was 20 μl. In the PCR reaction, a cycle of 94 ° C for 30 seconds, 60 ° C for 30 seconds, and 68 ° C for 2 minutes was repeated 30 times after 94 ° C for 5 minutes, and an extension reaction was finally performed at 68 ° C for 5 minutes. The PCR reaction product was subcloned into a plasmid vector pCR4 (Invitrogen) according to the procedure of a TA cloning kit (Invitrogen). This was introduced into E. coli JM109, and a clone having the cDNA of the PCR product was selected on an LB agar medium containing ampicillin. As a result of analyzing the sequence of each clone, a cDNA sequence (SEQ ID NO: 10) encoding a novel G protein-coupled receptor protein was obtained. The novel G protein-coupled receptor protein having the amino acid sequence deduced from this cDNA sequence (SEQ ID NO: 9) was named TGR5. A transformant containing the DNA represented by SEQ ID NO: 10 was named Escherichia coli JM109 / pCR4-hTGR5.
[0047]
Reference Example 2 Detection of reporter activation by cholesterol metabolism-related substances in human HEK293 cells transiently expressing TGR5
The detection of TGR5-specific stimulatory activity by a cholesterol metabolism-related substance was performed using the expression level of a reporter gene product (luciferase) produced by induction of CRE promoter expression as an index.
Human-derived HEK293 cells were suspended in a growth medium (DMEM (Dulbecco's Modified Eagle Medium) (GibcoBRL) supplemented with 10% fetal bovine serum (GibcoBRL)) and 1 × 10 5 ⁵ Cells were plated in a Black well 96-well plate (Becton Dickinson) coated with collagen at a concentration of cells / well. 37 ° C, 5% CO ₂ After culturing overnight under the conditions, the expression vector pAKKO-111H for animal cells (Biochem. Biophys. Acta, Hinuma, S. et al., 1989) together with a reporter gene-containing plasmid pCRE-Luc (Clontech) is used in a known manner. 1219, 251-259, 1994), a TGR5 expression vector plasmid prepared by inserting the TGR5 gene into pAKKO-1.111H or the original pAKKO-111H containing no TGR5 gene. Transfections were performed as follows.
OPTI-MEM-I (GibcoBRL) and Lipofectamine ^TM Lipofectamine dilutions were prepared by mixing 2000 reagents (GibcoBRL) at 24: 1. Further, a DNA diluent is prepared by mixing OPTI-MEM-I, TGR5 expression vector plasmid or original vector plasmid (240 ng / μl) and pCRE-Luc (240 ng / μl) at 24: 0.9: 0.1. Was prepared. An equal amount of the lipofectamine diluent and the DNA diluent are mixed and left at room temperature for 20 minutes to form a complex of DNA and lipofectamine. Then, 25 μl of the solution is added to a plate on which the HEK293 cells are cultured. At 37 ° C, 5% CO ₂ The cells were cultured overnight under the conditions.
After washing the transfected HEK293 cells with an assay medium (DMEM supplemented with 0.1% bovine serum albumin), lithocholic acid (Wako Pure Chemical) and progesterone (Wako Pure Chemical) diluted with the assay medium were washed. ) Is 2 × 10 ^-5 M at 37 ° C, 5% CO ₂ The cells were cultured under the conditions for 4 hours. The culture supernatant is discarded, 50 μl of Picagene LT2.0 (Toyo Ink Mfg. Co., Ltd.), a substrate for measuring luciferase activity, is added, and the luciferase luminescence is measured using a plate reader (ARVO sx multilabel counter, Wallac). Was measured.
As a result, an increase in luciferase activity by lithocholic acid and progesterone was specifically observed in HEK293 cells into which the TGR5 gene having the nucleotide sequence represented by SEQ ID NO: 10 was introduced (FIG. 1).
[0048]
Reference Example 3 Introduction of G protein-coupled receptor protein expression plasmid and reporter plasmid into host cells
Various G protein-coupled receptor protein cDNAs prepared by known methods, ie thyroid hormone stimulating factor receptor (TRHR), neuromedin U receptor (FM-3 and TGR-1), prolactin releasing factor receptor (hGR3), apelin receptor (APJ) ) Was used to transform Escherichia coli JM109. The resulting colonies were isolated and cultured, and then the plasmids were prepared using QIAGEN Plasmid Maxi Kit (Qiagen). In addition, a reporter plasmid of pCRE-Luc (Clontech) in which a luciferase gene was linked as a reporter downstream of the cAMP response element (CRE) was prepared in the same manner.
As host cells for introducing the G protein-coupled receptor protein expression plasmid and the reporter plasmid, human HEK293 cells were coated on a 96-well black plate (Becton Dickinson) coated with type I collagen at 100,000 cells / well at a culture volume of 100 μl. Seeded and cultured overnight. CHO (dhfr ⁻ ) CHO-mock cells transformed with pAKKO-111H were seeded on a Costar 96-well black plate at 40,000 cells / well in a culture volume of 100 μl and cultured overnight. For all cells, a medium obtained by adding only 10% fetal bovine serum to DMEM (Gibco BRL) as a medium for plate culture was used.
Each of the above plasmids was diluted to a concentration of 240 ng / μl, and added to 240 μl of Opti-MEM-I (Gibco BRL) at a ratio of 9 μl of the expression plasmid for the G protein-coupled receptor protein and 1 μl of the reporter plasmid. This was mixed with an equal amount of 240 μl of Opti-MEM-I to which 10 μl of Lipofectamine 2000 (GibcoBRL) was added and mixed with the lipofectamine 2000 according to the method attached to Lipofectamine 2000. A complex was formed. For efficient screening, three types of receptor expression plasmids were added at 5 μl each at a concentration of 240 ng / μl, and the ratios of other reagents were the same as those described above. These were added at 25 μl / well to the culture solution of HEK293 or CHO-mock cells, and cultured at 37 ° C. overnight to introduce the plasmid. For the CHO-mock cells, the culture solution was replaced with an assay buffer (DMEM supplemented with 0.1% bovine serum albumin) 4 hours after the addition of the plasmid, and serum-free was performed.
[0049]
Reference example 4 Detection of ligand activity by reporter assay
For HEK293 cells, the culture solution was exchanged for the assay buffer described in Reference Example 1 one hour before the assay, and preincubation was performed. A solution prepared by dissolving a ligand or a ligand candidate compound in an assay buffer was prepared and added to the HEK293 cells or CHO-mock cells prepared in Reference Example 3. In addition, the assay under the condition that forskolin at a final concentration of 2 μM was added to the assay buffer was performed in the same manner. Incubation was performed for 4 hours after the addition of the ligand or the test compound to induce the promotion or suppression of transcription / translation of a reporter gene derived from intracellular signal transduction induced by the agonist activity of the ligand via the receptor. After the completion of the incubation, the assay buffer in each well was removed, and 50 μl of a luminescent substrate of Picker Gene LT2.0 (Toyo Ink) was added. After the cells were lysed and thoroughly mixed with the substrate, the amount of luminescence corresponding to the amount of reporter gene expression induced in each well was measured using the plate reader described in Reference Example 2.
Using expression plasmids into which various G protein-coupled receptor protein cDNAs were inserted according to the methods described in Reference Examples 3 and 4, the induction of reporter gene expression induced by ligand stimulation in HEK293 cells was measured. Regarding the CRFR coupled to Gs as an α subunit of a G protein that transmits a signal into cells, activation of the reporter gene by addition of a ligand was detected under any of the conditions in which forskolin was not added or added. In addition, for APJ conjugated to Gi, which is an inhibitory agent, suppression of reporter gene expression by addition of a ligand was detected under the conditions of forskolin addition. In addition, with respect to the receptors TRHR, FM-3, and TGR-1 coupled to Gq, promotion of the expression of the reporter gene was detected under the addition of forskolin. Similarly, for the receptor hGR3, which is coupled to both Gq and Gi, promotion of reporter gene expression was detected under the addition of forskolin (FIG. 2).
[0050]
Reference example 5 Reporter assay using inhibitory G protein α subunit Gi expression plasmid
An expression plasmid for the inhibitory G protein α subunit (Gi) was prepared in the same manner as in the G protein receptor expression plasmid shown in Reference Example 3 (Gi is not limited to animal species). 3 μl of this, 7 μl of the receptor expression plasmid and 1 μl of the reporter plasmid were added to 240 μl of Opti-MEM-I, and the DNA was introduced into HEK293 or CHO-mock cells in the same manner as in Example 2 except for the other conditions. did. When the total amount of these three plasmids is 11 μl, Gi is 1 to 6 μl, preferably 1 to 3 μl. These were assayed according to the method of Example 3 to detect ligand activity.
That is, as a result of detecting the reaction of TGR5 with lithocholic acid in the presence of Gi, in the assay of the G protein receptor TGR5 using CHO-mock cells, Gi was simultaneously expressed with TGR5, and thus when Gi ligand was not added (ligand (ligand ( -)), The luciferase activity of the ligand (lithocholic acid, 2 × 10 ^-5 M, ligand (+)) to detect an increase in activity (FIG. 3).
[0051]
Example 1 Transfer of TGR5-GFP fusion protein expressed in CHO cells into cells by addition of taurolithocholic acid
An expression plasmid for expressing a fusion protein in which Green Fluorescent Protein (GFP) isolated from Oan jellyfish was connected to the C-terminus of TGR5 was constructed. At that time, a fragment cut out from the GFP expression vector pQBI25 (Takara Shuzo) was used as GFP cDNA (SEQ ID NO: 2). The TGR5 cDNA was modified at its stop codon to a recognition sequence for the restriction enzyme NheI by PCR, and a GFP cDNA fragment was ligated thereto and inserted into the expression vector pAKKO-111H described in Example 1. The thus obtained expression vector plasmid of the fusion protein of TGR5 and GFP (hereinafter, TGR5-GFP) was transfected into CHO-mock cells by the following method. The CHO-mock cells were suspended in a growth medium [DMEM (Dulbecco's Modified Eagle Medium) (GIBCO BRL) supplemented with 10% fetal bovine serum (GIBCO BRL)], and 0.6 × 10 6 ⁵ The cells were spread at a concentration of cells / chamber on a Lab-Tek II coverglass chamber with 4 chambers (Nalgen Nunc) at 37 ° C., 5% CO 2. ₂ After overnight culture under the conditions, transfection was performed. Lipofectamine for transfection ^TM 2000 reagents (GIBCO BRL) were used. First, Lipofectamine ^TM 2000 μl of 2 μl and 50 μl of OPTI-MEM-I (GIBCO BRL) were mixed, left for 5 minutes, then mixed with 0.48 μg of DNA and 50 μl of OPTI-MEM-I, and allowed to stand at room temperature for 20 minutes. By this, a complex of DNA and Lipofectamine was formed. 100 μl of this mixed solution was added to the chamber in which the above CHO cells were cultured, and further added at 37 ° C., 5% CO 2. ₂ The cells were cultured overnight under the conditions. The medium was replaced with a confocal microscope observation medium [a suspension of 0.1% bovine albumin (Essentially Fatty Acid Free (GIBCO BRL)) in Hanks' Balanced Salt Solution (GIBCO BRL)] and a confocal microscope ( (Leica) to observe the fluorescence image of GFP. At that time, GFP was excited at 488 nm.
As a result, the TGR5-GFP fusion protein was observed on the cell membrane (FIG. 4). Taurolithocholic acid is added to the cells for 10 minutes. ^-5 It was found that the fluorescence of GFP moved to the cytoplasm, not to the cell membrane, 30 minutes after the addition to the medium so as to reach M (FIG. 5). This indicates that TGR5 is a G protein-coupled receptor expressed on the cell membrane, and that TGR5 translocated to the cytoplasm in response to taurolithocholic acid, that is, internalized.
[0052]
Example 2 Expression of parathyroid hormone receptor (PTH-R) and GFP fusion protein by transient expression in pancreatic β cell line RINm5F
In the same manner as in Example 1, an expression plasmid for expressing a fusion protein in which GFP was linked to the C-terminal of human PTH-R (SEQ ID NO: 13) was inserted into the expression vector pAKKO-111H described in Reference Example 2. An expression vector was prepared. The thus obtained expression vector plasmid of the fusion protein of PTH-R and GFP (hereinafter, PTH-GFP) was transfected into RINm5F cells by the following method. The RINm5F cells were suspended in a growth medium [RPMI1640 (GIBCO BRL) supplemented with 10% of Charcoal / Dextran-treated fetal bovine serum (Hyclone)] and 0.3 × 10 5 ⁵ At a concentration of cells / chamber, a Lab-Tek II cover glass chamber (Nalgen Nunc) with 8 chambers was spread, and 37 ° C., 5% CO 2. ₂ After overnight culture under the conditions, transfection was performed. Lipofectamine for transfection ^TM 2000 reagents (GIBCO BRL) were used. First, Lipofectamine ^TM 2000 reagent 3.3 μl and OPTI-MEM-I (GIBCO BRL) 80 μl are mixed, left for 5 minutes, and then mixed with 0.72 μg of DNA and a mixture of OPTI-MEM-I 80 μl, and room temperature for 20 minutes. To form a complex of DNA and Lipofectamine. 160 μl of this mixture was added to the chamber in which the RINm5F cells were cultured, and further added at 37 ° C., 5% CO 2. ₂ The cells were cultured overnight under the conditions. Observation with a confocal microscope was performed in the same manner as in Example 1.
As a result, expression of the PTH-GFP fusion protein on the cell membrane was observed.
[0053]
Example 3 Expression of GPCR and GFP fusion protein using insulin II promoter
In the same manner as in Example 1, a fragment obtained by connecting GFP to the C-terminus of human GPR40 (SEQ ID NO: 15) was inserted downstream of the mouse insulin II promoter cloned from the mouse genome. An expression vector for expressing a fusion protein (hereinafter, GPR40-GFP) was prepared. The thus obtained GPR40-GFP expression vector plasmid was transfected into MIN6 cells by the following method. MIN6 cells were prepared by adding a 15% final concentration of fetal bovine serum (Trace), 55 μM 2-mercaptoethanol (Invitrogen), 20 mM HEPES (Dai Nippon Pharmaceutical Co., Ltd.) to a growth medium [DMEM (containing 4.5 g / l Glucose) (Invitrogen)]. ) Were added to the suspension, and 1.2 × 10 ⁵ Spread the cells at a concentration of cells / chamber onto a Lab Tek II coverglass chamber with 4 chambers (Nalge Nunc) at 37 ° C., 5% CO 2. ₂ After transfection for two nights under the conditions, transfection was performed. Lipofectamine for transfection ^TM 2000 reagents (Invitrogen) were used. First, Lipofectamine ^TM 2000 μl of 4 μl of reagent and 100 μl of Opti-MEM medium (Invitrogen) were mixed, left for 5 minutes, mixed with 2 μg of DNA and 100 μl of Opti-MEM medium, and allowed to stand at room temperature for 20 minutes. And a lipofectamine complex. 100 μl of this mixture was added to the chamber in which the above MIN6 cells were cultured, and the mixture was added at 37 ° C., 5% CO 2. ₂ After culturing for 4 hours under the conditions, the medium was replaced with 400 μl of a new growth medium, and further cultured overnight. Observation with a confocal microscope was performed in the same manner as in Example 1.
As a result, it was observed that the GPR40-GFP fusion protein was expressed in the cell membrane of MIN6 cells.
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Example 4 Production of transformant capable of expressing each of 102 fusion proteins
According to the method of Example 1, a DNA encoding a fusion protein of 102 types of receptor proteins whose ligands have not been determined and GFP comprising the amino acid sequence represented by SEQ ID NO: 1 or its modified amino acid sequence (sequence: No. 132 to SEQ ID NO: 233) were prepared and transfected into CHO-mock cells. When these CHO cells were cultured in the same manner as in Example 1, expression of the fusion protein was observed in CHO-mock cells.
[0055]
【The invention's effect】
The method of the present invention for determining the ligand of a receptor protein for which a ligand has not been determined is simple and can be performed in a short time because various cell lines can be used.
[0056]
[Sequence list]

[Brief description of the drawings]
FIG. 1 shows the results of detection of reporter activation by a cholesterol metabolism-related substance (n = 2) in HEK293 cells into which a TGR5 expression vector has been introduced (A) and HEK293 cells into which only the original vector has been introduced (B).
FIG. 2 shows the results of reporter gene expression induction in response to ligand stimulation in HEK293 cells.
FIG. 3 shows the results of the reaction of TGR5 with lithocholic acid in the presence of Gi.
FIG. 4 shows the localization of a TGR5-GFP fusion protein expressed in CHO cells in the absence of a ligand. The white line in the figure indicates 4 μm.
FIG. 5 shows the localization of the fusion protein 30 minutes after TLCA was added to CHO cells expressing TGR5-GFP. The white line in the figure indicates 4 μm.

Claims (28)

A method for determining a ligand for a receptor protein, which comprises using a fusion protein of a fluorescent protein and a receptor protein for which a ligand has not been determined. The method for determining a ligand according to claim 1, wherein a fusion protein of a GFP and a receptor protein for which a ligand has not been determined is used. The method for determining a ligand according to claim 1, wherein the test compound is contacted with a cell expressing a fusion protein of GFP and a receptor protein for which a ligand has not been determined, or a cell membrane fraction thereof. (1) Arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release, intracellular cAMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation or pH Activity to promote or suppress reduction, (2) MAP kinase activation, (3) transcription factor activation, (4) diacylglycerol production, (5) opening and closing of ion channels on cell membrane, (6) apoptosis Induction, (7) morphological change, (8) translocation of the fusion protein from the cell membrane to the cytoplasm, (9) activation of low molecular weight G protein, (10) cell division promoting activity or (11) DNA synthesis promoting activity. The method for determining a ligand according to claim 1, wherein the measurement is performed. The method for determining a ligand according to claim 1, wherein the translocation of the fusion protein from the cell membrane to the cytoplasm is measured. The method for determining a ligand according to claim 5, wherein the translocation of the fusion protein from the cell membrane to the cytoplasm is measured by observing GFP fluorescence. A test compound is brought into contact with cells containing a plasmid that expresses a fusion protein of a receptor protein for which a ligand has not been determined and a fluorescent protein and has a DNA encoding a reporter protein downstream of a cAMP response element / promoter. 2. The method for determining a ligand according to claim 1, wherein the activity of the reporter protein is measured. (1) a plasmid containing a DNA encoding a fusion protein of a receptor protein and a fluorescent protein whose ligand has not been determined, and (2) a plasmid containing a DNA encoding a reporter protein linked downstream of a cAMP response element / promoter. 8. The method according to claim 7, wherein the cells to be cultured are cultured and contacted with a test compound to measure the activity of the reporter protein. A test compound is brought into contact with cells containing a plasmid containing a fusion protein of GFP with a receptor protein for which a ligand has not been determined and having a DNA encoding a reporter protein downstream of a cAMP response element / promoter. 3. The method for determining a ligand according to claim 2, wherein the activity of the reporter protein is measured. (1) a plasmid containing a DNA encoding a fusion protein of a receptor protein and a GFP whose ligand has not been determined, and (2) a plasmid containing a DNA encoding a reporter protein linked downstream of a cAMP response element / promoter. The method according to claim 9, wherein the cells are cultured, and the activity of the reporter protein is measured by bringing the cells into contact with a test compound. The method according to claim 1, wherein the receptor protein is a G protein-coupled receptor protein. 2. The method according to claim 1, wherein GFP is a protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7. The method according to claim 7, wherein the promoter is a TATA-like sequence. The method according to claim 7, wherein the reporter protein is luciferase. The method according to claim 7, wherein the plasmid is obtained by ligating a gene encoding a TATA-like promoter and a reporter protein downstream of the cAMP response element. 8. The method of claim 7, wherein the cells express two or more receptor proteins for which a ligand has not been determined. The method according to claim 7, wherein the cell further comprises a plasmid containing a gene encoding an inhibitory G protein α subunit Gi. The method according to claim 7, further comprising adding forskolin. 17. The method of claim 16, wherein two or more receptor proteins have similar characteristics. 20. The method according to claim 19, wherein similar characteristics are the basal expression level of the reporter protein and / or the expression level of the reporter protein upon addition of forskolin. The basal expression level of the reporter protein and / or the expression level of the reporter protein when forskolin is added when two or more receptor proteins are independently expressed are measured in advance, and the expression levels of the reporter proteins are comparable. 17. The method according to claim 16, wherein two or more receptor proteins are expressed in combination. A fusion protein of a receptor protein for which a ligand has not been determined and a fluorescent protein, or a salt thereof. 23. The fusion protein according to claim 22, wherein the fluorescent protein is GFP, or a salt thereof. A DNA containing the DNA encoding the fusion protein according to claim 22. A recombinant vector containing the DNA according to claim 22. A transformant transformed with the recombinant vector according to claim 25. Use of a fluorescent protein to determine a ligand for a receptor protein for which a ligand has not been determined. Use of GFP to determine a ligand for a receptor protein for which a ligand has not been determined.

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