| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | April 2, 2002 |
| PATENT TITLE |
Method for identifying or screening agonist and antagonist to PPAR |
| PATENT ABSTRACT | A method for identifying or screening an agonist for or antagonist to a peroxisome proliferator activated receptor (PPAR) which comprises allowing a test cell and a substance to be tested to coexist, and detecting a change in a ligand-dependent interaction between the PPAR and a coactivator in the test cells due to the substance to be tested by measuring the expression of a reporter gene as an index |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | February 28, 2000 |
| PATENT FOREIGN APPLICATION PRIORITY DATA | This data is not available for free |
| PATENT REFERENCES CITED |
Mizukami J, et al. The antidiabetic agent thiazolidinedione stimulates the interaction between PPARy and CBP. Biochem. Biophys. Res. Comm. 240:61-64, 1997.* Zhu Y, et al. Cloning and identification of mouse steroid receptor coactivator-1 (mSRC-1), as a coactivator of peroxisome proliferator-activated receptor gamma. Gene Expression 6:185-195, 1996.* Elbrecht A, et al. Molecular cloning, expression and characterization of human peroxisome proliferator activated receptors gamma1 and gamma2. Biochem. Biophys. Res. Comm. 224:431-437, 1996.* Krey, "Fatty Acids, Eicosanoids, and Hypolipidemic Agents Identified as Ligands of Peroxisome Proliferator-Activated Receptors by Coactivator-Dependent Receptor Ligand Assay", Molecular Endocrinology, 1997, 779-791, 11(6). |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
What we claimed is: 1. A method for identifying or screening an agonist for, or an antagonist to, a human peroxisome proliferator-activated receptor gamma (PPAR.gamma.), which comprises contacting a test cell with a substance to be tested, and detecting a change in a ligand-dependent interaction between the human PPAR.gamma. and a coactivator due to the substance to be tested, by measuring the expression of a reporter gene as an index, wherein the test cell contains: (i) a first extrinsic fused gene coding for a first fused protein comprising a ligand binding domain of human PPAR.gamma. and a first domain of a transcription factor, wherein the first domain of said transcription factor being a DNA binding domain or a transcriptional activation domain; (ii) a second extrinsic fused gene coding for a second fused protein comprising a human PPAR.gamma. binding domain of a coactivator which interacts with the human PPAR.gamma. and a second domain of the transcription factor, wherein the second domain of said transcription factor is a transcriptional activation domain when the first domain of the transcription factor is a DNA binding domain or is a DNA binding domain when the first domain of the transcription factor is a transcriptional activation domain; and (iii) a response element to which the DNA binding domain of said transcription factor can bind and a reporter gene linked thereto, and wherein the coactivator is CREB-binding protein (CBP). 2. The method according to claim 1, wherein the test cell is a yeast cell. 3. The method according to claim 1, wherein the ligand binding domain of human PPAR.gamma. comprises residues 174 to 475 of SEQ ID NO:6. 4. The method according to claim 1, wherein human PPAR.gamma. comprises the amino acid sequence of SEQ ID NO:6. 5. The method according to claim 1, wherein the CBP has the amino acid sequence of SEQ ID NO:8. 6. The method according to claim 1, wherein the CBP has the amino acid sequence of SEQ ID NO:10. -------------------------------------------------------------------------------- |
| PATENT DESCRIPTION |
BACKGROUND OF THE INVENTION 1. Technical Field This invention relates to a novel method for identifying or screening an agonist for and/or antagonist to peroxisome proliferator activated receptor (PPAR). 2. Background Art Peroxisome, an organelle found in the cells of animals and plants, contains a group of enzymes participating in the lipometabolism and absorption of lipids such as cholesterol. An increase in peroxisome is also induced by diet or physiological factors. It is known that a group of chemicals diversified in structure including antilipemic (fibrates), insecticides and plasticizers such as phthalic acids when they are administered dramatically increase the size and number of peroxisome in liver and kidney and at the same time elevate the ability of metabolizing fatty acids in peroxisome through intermediary of an increase in the expression of enzymes necessary for the .beta.-oxidation cycle. Hence, they are called peroxisome proliferator. Among studies on the mechanism of such a peroxisome proliferation, a nuclear receptor that is activated by the group of chemicals has been identified and named peroxisome proliferator activated receptor (PPAR). From its structure, etc., PPAR is considered to be a member of nuclear receptor (nuclear hormone receptor) super family. Like other nuclear receptors, it is activated by its binding to a ligand, and its binding to a response sequence (PPRE: peroxisome proliferator response element) existing upstream of a target gene domain activates transcription of the target gene. PPAR is known to form a heterodimer with a retinoid X receptor (RXR) and binds to PPRE in the form of the heterodimer. Also, like other nuclear receptors, PPAR is considered to have the interaction with a group of transcription coactivators (coactivators) in order to exhibit its transcription activation activity. Hitherto, three kinds of PPAR subtypes called PPAR.alpha., PPAR.delta. (or NUC-1, PEAR.beta., FAAR) and PPAR.gamma. have been identified and their genes (cDNA) have been cloned (Lemberger et al., Annu. Rev. Cell. Dev. Biol., vol. 12, pp. 335-363, 1996). Of the three kinds, PPAR.gamma. is expressed particularly in an adipose tissue and considered to be a factor that closely participates in differentiation of adipocytes (Tontonoz et al., Genes and Development, vol. 8, pp. 1224-1234, 1994; Tontonoz et al., Cell, vol. 79, pp. 1147-1156, 1994). On the other hand, various thiazolidinedione derivatives show hypoglycemic effect in a model animal of non-insulin-dependent diabetes mellitus (NIDDM) and are expected as a NIDDM remedy having an insulin resistance releasing effect. These thiazolidinedione derivatives act as ligands to PPAR.gamma. and specifically activate PPAR.gamma., which has been discovered in recent studies (Lehmann et al., Journal of Biological Chemistry, vol. 270, pp. 12953-12956, 1995). Since a strong correlation is observed between such a PPAR.gamma. activation ability of thiazolidinedione derivatives and the hypoglycemic effect in a hereditary obese mouse, PPAR.gamma. is considered to be a target molecule of the pharmaceutical effect of the thiazolidinedione derivatives (Willson et al., Journal of Medicinal Chemistry, vol. 39, pp. 665-668, 1996). This also relates to the fact that an adipose tissue where PPAR.gamma. is specifically expressed is an organ that plays an important role in maintaining energy balance. From these findings, a compound specifically acting as an agonist for PPAR.gamma. is considered to be very useful as a remedy for diabetes mellitus. However, to date, those methods known as screening methods for PPAR acting agents each involve the problems that operation is complicated and simultaneous treatment of multiple samples is difficult. For example, there has been known a method for examining PPAR activation ability of a sample using animal cells having introduced therein reporter plasmid containing a reporter gene linked to a PPAR expression vector and a PPAR response element (PPRE), with using as an index a change in the amount of expression of a reporter gene in the cells (WO 96/22884, Tontonoz et al., Genes and Development, vol. 8, pp. 1224-1234, 1994). As its improved method, there has been known a method using animal cells having introduced therein vector for expressing fused protein in which the DNA binding domain of GAL4, i.e., the transcription factor of yeast, and the ligand binding domain of PPAR linked together, along with having introduced a reporter plasmid containing a reporter gene linked to the response element of GAL4 (GAL4 binding element) (WO 96/33724, Lehmann et al., Journal of Biological Chemistry, vol. 270, pp. 12953-12956, 1995; Willson et al., Journal of Medicinal Chemistry, vol. 39, pp. 665-668, 1996). In these methods, an extrinsic gene is introduced into animal cells. Upon the introduction of gene, it is sometimes the case that the integration of a gene into a chromosome has taken place, the gene is influenced by the site where the gene is integrated. Therefore, it is necessary to use a transformed cell in which gene is not influenced by the chromosome. To acquire such a transformed animal cell and express an extrinsic gene stably are accompanied by technical difficulties. Since coactivators, RXR, etc. derived from host animal are considered to participate in the activation of transcription in these methods, there is the possibility that the action of the test substance to PPAR alone cannot be detected surely. As a method for directly detecting the binding between PPAR and a ligand without using any animal cell or reporter gene, there has been known a method for examining binding and antagonism between a fused protein comprising the ligand binding domain of PPAR and glutathione-S-transferase (GST) and a test compound labeled with a radioisotope (Willson et al., Journal of Medicinal Chemistry, vol. 39, pp. 665-668, 1996; Buckle et al., Bioorganic & Medical Chemistry Letters, vol. 6, pp. 2121-2126, 1996). Recently, it has been elucidated that like other nuclear receptor RXR, etc., PPAR interacts with SRC-1, one of coactivators, ligand-dependently. Based on this finding, Krey et al. reported a method for detecting the action of a test compound as a ligand using a fused protein comprising the ligand binding domain of PPAR and glutathione-S-transferase (GST) and SRC-1 labeled with a radioisotope (Krey et al., Molecular Endocrinology, Vol. 11, pp. 779-791, 1997). However, these methods each use a label of radioisotope and therefore it is accompanied by a danger and has a limitation in treating power since preparation of a labeled compound or coactivator on a large scale is difficult. As described above, upon screening PPAR acting agents, a screening method which is simple, high precision, and efficient has been desired. An object of this invention is to provide a novel method for identifying and screening an agonist and/or antagonist to peroxisome proliferator activated receptor (PPAR). The present inventors have uniquely found that in addition to SRC-1, one of the coactivators, that is already known to interact with PPAR, CBP (CREB-binding protein) interacts with PPAR ligand-dependently and identified the binding domain of the coactivator to PPAR. Further, based on these findings, they have completed a method for identifying or screening a novel PPAR acting agent that detects a ligand-dependent interaction between PPAR and a coactivator using a Two-hybrid system of yeast. SUMMARY OF THE INVENTION This invention relates to a method for identifying or screening an agonist for or antagonist to a peroxisome proliferator-activated receptor (PPAR), which comprises allowing a test cell and a substance to be tested to coexist, and detecting a change in a ligand-dependent interaction between the PPAR and a coactivator in the test cells due to the substance to be tested by measuring the expression of a reporter gene as an index. BRIEF DESCRIPTION OF THE DRAWINGS FIGS. 1A and 1B are schematic diagrams illustrating the constitutions of used plasmids pGBT9-PPAR.gamma.2 (FIG. 1A) and pGAD424-CBP (FIG. 1B); FIGS. 2A and 2B are diagrams illustrating ligand-dependent interaction between PPAR.gamma. and CBP (FIG. 2A), and dose-dependent effects of 15d-PGJ.sub.2 (FIG. 2B); and FIGS. 3A and 3B are diagrams illustrating action of T-174 to the interaction between PPAR.gamma. and CBP (FIG. 3A) and dose-dependent effects of T-174 (FIG. 3B). DESCRIPTION OF THE PREFERRED EMBODIMENTS In this invention, a ligand-dependent interaction between PPAR and a coactivator in the test cells is detected. PPAR changes its conformation into an activated type by binding to a ligand and the interaction with a coactivator takes place. That is, the ligand-dependent interaction is the binding of PPAR with the coactivator promoted in the presence of a ligand of PPAR. As PPAR, subtypes such as PPAR.alpha., PPAR.delta. (or NUC-1, PPAR.beta., FAAR) and PPAR.gamma. are known. In this invention, any one of these subtypes can be used. Among these, PPAR.gamma. is a target molecule of thiazolidinedione derivatives having an antidiabetic effect. A method for identifying or screening a specifically acting agent therefor is useful in research and development of a remedy for diabetes mellitus. PPAR may be derived from any species so far as it is identified as the same molecular species and exhibits its function in the organism as a nuclear receptor. For example, it includes those derived from mammalians such as human, mouse, rat, hamster, etc., and in addition those derived from clawed toad (Xenopus laevis). From the point of view of utilizing research and development of a remedy for humans, it is preferred to use human-derived one out of these. The gene sequences and amino acid sequences of PPAR.alpha. (Dreyer et al., Cell, vol. 68, pp. 879-887, 1992, Green et al., Nature, vol. 347, pp. 645-650, 1990, Goettlicher et al., Proc. Natl. Acad. Sci. USA, vol. 89, pp. 4653-4657, 1992), PPAR5 (or NUC-1, PPAR.beta., FFAR) (Dreyer et al., Cell, vol. 68, pp. 879-887, 1992, Kliewer et al., Proc. Natl. Acad. Sci. USA, vol. 91, pp. 7355-7359, 1994, Amri et al., Journal of Biological Chemistry, vol. 270, pp. 2367-2371, 1995, Xing et al., Biochem. Biophys. Res. Commun., vol. 217, pp. 1015-1025, 1995) and PPAR.gamma. (Dreyer et al., Cell, vol. 68, p. 879-887, 1992, Zhu et al., Journal of Biological Chemistry, vol. .268, pp. 26817-26820, 1993, Kliewer et al., Proc. Natl. Acad. Sci., USA, vol. 91, pp. 7355-7359, 1994, Mukherjee et al., Journal of Biological Chemistry, vol. 272, pp. 8071-8076, 1997, Elbrecht et al., Biochem. Biophys. Res. Commun., vol. 224, pp. 431-437, 1996, Chem et al., Biochem. Biophys. Res. Commun., vol. 196, pp. 671-677, 1993, Tontonoz et al., Genes & Development, vol. 8, pp. 1224-1234, 1994, Aperlo et al., Gene, vol. 162, pp. 297-302, 1995) have already been reported. PPAR.gamma. includes two kinds of isoforms, PPAR.gamma.1 and PPAR.gamma.2. PPAR.gamma.1 as compared with PPAR.gamma.2 is deleted of 30 amino acids on the N-terminal side but the other amino acid sequence is quite the same. Each is expressed in an adipose tissue. Among the reports, presuming from the homology with other nuclear receptor, etc., the ligand binding domain (LBD) of PPAR is considered to correspond to the domain including about No. 167 to 468 amino acids from the N-terminal side in the case of PPAR.alpha., to the domain including about No. 138 to 440 amino acids from the N-terminal side in the case of PPAR.delta., and the domain including about No. 174 to 475 amino acids from the N-terminal side in the case of PPAR.gamma. (corresponding to about residues 174 to 475 of SEQ ID NO:6). To detect the interaction between PPAR and coactivator, a polypeptide including at least the ligand binding domain may be used. Cut and use of a polypeptide including the ligand binding domain of PPAR can exclude nonspecific interaction and hence are preferred. The coactivator used in this invention may be any one so far as it interacts with PPAR ligand-dependently, that is, the interaction with PPAR in the presence of a ligand of PPAR is promoted. The coactivator which is considered to interact with nuclear receptor includes, for example, CBP, SRC-1, RIP140 (Cavailles et al., EMBO Journal, vol. 14, pp. 3741-3751, 1995), TIFI (Douarin et al., EMBO Journal, vol. 14, pp. 2020-2033, 1995, Vom Baur et al., EMBO Journal, vol. 15, pp. 110-124, 1996), TIF2 (Voegel et al., EMBO Journal, vol. 15, pp. 3667-3675, 1996), SUGI (Vom Baur et al., EMBO Journal, vol. 15, pp. 110-124; 1996), P300 (Chakravarti et al., Nature, vol. 383, pp. 99-103, 1996), etc. These are expected to interact also with PPAR ligand-dependently. The coactivator which is considered to interact specifically with PPAR.gamma. includes, for example, PGC-1 (PPAR gamma coactivator-1) (Puigserver et al., Cell, vol. 92, pp. 829-839, 1998), PGC-2 (PPAR gamma coactivator-2) (Cactillo et al., EMBO Journal, vol. 18, pp. 3676-3687, 1999), etc. These are expected to interact with PPAR.gamma. ligand-dependently. Among these, CBP and SRC-1, as shown in Examples in the present specification later on and in the report by Krey et al., have been confirmed to interact with PPAR and can be used advantageously in this invention. CBP (CREB-binding protein) is a protein that has been originally identified as a coactivator of transcription factor CREB (cAMP-regulated enhancer binding protein) that binds to CRE (cAMP-regulated enhancer) and both gene (SEQ ID Nb:7 for mouse and SEQ ID NO:9 for human) and amino acid (SEQ ID NO:8 for mouse and SEQ ID NO:10 for human) sequences thereof have been known (Chrivia et al. Nature, vol., 365, pp. 855-859, 1993; Kwok et al., Nature, vol. 370, pp. 223-226). Recently, it has been revealed that CBP binds not only to CREB but also to a nuclear receptor in the presence of a ligand to serve as a coactivator and that the N-terminal moiety of CBP participates in the interaction with the nuclear receptor (Kamei et al., Cell, vol. 85, pp. 403-414, 1995). That the N-terminal moiety of CBP interacts also with PPAR.gamma. ligand-dependently was found uniquely by the present inventors. SRC-1 is known to interact with nuclear receptors such as glucocorticoid receptor, estrogen receptor, thyroid hormone receptor and retinoid X receptor (RXR) ligand-dependently and serves as a coactivator. Its gene and amino acid sequence are also known (Onate et al., Science, vol. 270, pp. 1354-1357, 1995). In the Krey et al. report (Molecular Endocrinology, vol. 11, pp. 779-791, 1997), the experiment using the ligand binding domain of clawed toad (Xenopus laevis-derived PPAR and RI-labeled SRC-1 indicated that PPAR also interacts with SRC-1 ligand-dependently. Upon detecting the ligand-dependent interaction with PPAR, the whole coactivator may be used, besides, a polypeptide that contains at least PPAR binding domain (the domain that participates in binding to PPAR) may be used. Coactivators generally have large molecular weights and use of the whole sometimes result in difficulty of expression of protein and it is preferred that appropriate domain be selected and used from this point of view. The PPAR binding domain (domain participating in binding to PPAR) of a coactivator can be guessed from information on the position of its binding domain with a nuclear receptor if such an information has been reported. Also, using a system for detecting protein-protein interaction (for example, two-hybrid system of yeast), presence or absence of the interaction of a certain domain with PPAR may be examined and selection of a proper domain may be made. In the case where the coactivator is CBPthen PPAR binding domain exists near the N-terminal moiety (domain including about No. 1 to 450 amino acids). In the present invention, the ligand-dependent interaction between PPAR and coactivator is detected in test cells using the expression of a reporter gene as an index and measurement was made of a change in the interaction due to the substance to be tested. Noticing the interaction between PPAR and coactivator, the transcription activation effect of PPAR per se is not detected, so that various factors inherent to mammals participating in the expression of transcription activation ability of PPAR do not have to be present. Therefore, there is no need to use mammalian cells as test cells. Cells may be any one so far as they are eucaryotic cells. For example, there may be mentioned yeast cells, insect cells, mammalian cells, etc. Among these, yeast cells are advantageous in that their cultivation is easy and can be performed quickly and that application of genetic recombination technique such as introduction of extrinsic genes is easy. As yeast cells, there can be used cell lines of microbes belonging to the genera Saccharomyces, Schizosaccharomiyces, etc., such as Saccharomyces cerevisiae, Schizosaccharomiyces pombe, etc. As the test cells, usually those that contain extrinsic PPAR and coactivators may be used. Use of cells containing no intrinsic PPAR or coactivators interacting therewith is preferred since the influence due to intrinsic elements can be excluded. The change in the interaction between PPAR and coactivator due to the substance to be tested can be efficiently measured by a method utilizing a two-hybrid system. The two-hybrid system is a method for detecting protein-protein interaction using the expression of a reporter gene as a marker (U.S. Pat. No. 5,283,173 and Proc. Natl. Acad. Sci., USA, vol. 88, pp. 9578-9582, 1991). Many transcription factors can be divided into two domains having different functions, that is, a DNA binding domain and a transcriptional activation domain. In the two-hybrid system, for example, to examine the interaction between the two proteins X and Y, two kinds of fused protein, that is, a fused protein composed of the DNA binding domain of a transcription factor and X, and a fused protein composed of the transcriptional activation domain of a transcription factor and Y are simultaneously expressed in yeast cells. When the proteins X and Y interact with each other, the two kinds of fused proteins form by combination a transcription complex exhibiting a single function as a whole. This transcription complex combines with a response element (the site of DNA to which a transcription factor is bound specifically) in the nuclei of cells and activates transcription of a reporter gene positioned downstream. Thus, the interaction between the two proteins can be detected by the expression of the reporter gene (for example, the enzyme activity of gene products). The two-hybrid system can usually be used in the identification of unknown proteins that interact with a specific protein and generally used in qualitative evaluation of protein-protein interaction. The present inventors utilized this system, and thus, completed a method which can quantitatively measure the ligand-dependent interaction between PPAR and a coactivator, and can be applied to the identification or screening of antagonist/agonist for receptors, in which quantitative evaluation is indispensable. As one embodiment of the present invention, there may be mentioned a method for identifying an agonist for or an antagonist to PPAR, comprising: using test cells containing (i) a first fused gene coding for a first fused protein comprising at least ligand binding domain of PPAR and a first domain of a transcription factor, wherein the first domain of said transcription factor being a DNA binding domain or a transcriptional activation domain; (ii) a second fused gene coding for a second fused protein comprising at least PPAR binding domain of a coactivator which interacts with the PPAR and a second domain of the transcription factor, wherein the second domain of said transcription factor is a transcriptional activation domain when the first domain of the transcription factor is a DNA binding domain or is a DNA binding domain when the first domain of the transcription factor is a transcriptional activation domain, and (iii) a response element to which the DNA binding domain of said transcription factor can bind and a reporter gene linked thereto, making the test cells coexist with a substance to be tested, and detecting, by measuring the expression of a reporter gene as an index, a change in the ligand-dependent interaction between the peroxisome proliferator-activated receptor (PPAR) and a coactivator in the test cells occurring due to the substance to be tested. In this embodiment, the transcription factor used for detecting the interaction between the PPAR and coactivator is not limited particularly so long as it is a transcription factor (other than PPAR) of eucaryotic organism that can exhibit the function of transcriptional activation in cells. However, it is preferred to use a transcription factor derived from yeast from the viewpoint that it does not need the coactivator, etc. derived from mammalian cells to function and it independently exhibits transcriptional activation ability efficiently in yeast cells. Such a transcription factor includes yeast GAL4 protein (Keegan et al., Science, vol. 231, pp. 699-704, 1986, Ma et al., Cell, vol. 48, pp. 847-853, 1987), GCN4 protein (Hope et al., Cell, vol. 46, pp. 885-894, 1986), ADR1 protein (Thukral et al., Molecular and Cellular Biology, vol. 9, pp. 2360-2369, 1989), etc. The DNA binding domain of the transcription factor may be those having a DNA binding ability to the response element but alone having no transcriptional activation ability. Also, the transcriptional activation domain of the transcription factor may be those having a transcriptional activation ability but alone having no DNA binding ability to the response element. The DNA binding domain and transcriptional activation domain of a transcription factor, in the case of, for example, GAL4, are known to be present on the N-terminal side (a domain including about No. 1 to 147 amino acids) and C-terminal side (the domain including about No. 768 to 881 amino acids), respectively. In the case of GCN4, they are known to be present on the C-terminal side (the domain including about No. 228 to 265 amino acids) and N-terminal side (the domain including about No. 107 to 125 amino acids), respectively. In the case of ADR1, they are known to be present on the N-terminal side (the domain including about No. 76 to 172 amino acids) and the C-terminal side (the domain including about No. 250 to 1323 amino acids), respectively. As the response element, a response element corresponding to a transcription factor may be used and DNA sequences to which the DNA binding domain of the transcription factor can bind are used. The response element corresponding to a transcription factor generally exists in a domain upstream of the gene whose transcriptional activity is controlled by the transcription factor, so that such a domain may be cut out for use. If its sequence is known, corresponding oligonucleotide may be synthesized by chemical synthesis and used. For example, in case of GAL4 is used as a transcription factor, GAL4-specific DNA sequence called UASg (upstream activation site of galactose genes) may be used as the response element. UASg is contained in the domain upstream of galactose metabolism genes such as the GAL1 gene, etc., so that these domains may be used. Alternatively, a nucleotide sequence corresponding to UASg may be chemically synthesized and used. The reporter gene positioned downstream of the response element is not limited particularly so far as it is a commonly used one and it is preferred to use the gene of an enzyme which is stable and allows easy quantitative measurement of its activity, etc. Such a reporter gene includes, for example, .beta.-galactosidase gene (lacZ) derived from E. coli, chloramphenicol acetyltransferase gene (CAT) derived from bacterial transposone, luciferase gene (Luc) derived from a firefly, etc. Among these, E. coli-derived .beta.-galactosidase gene (lacZ) is preferable since it can be readily measured with visible light using a coloring substrate. The reporter gene may be a gene having an original promoter of the gene, or besides, a gene of which promoter part is replaced with one derived from of another gene may be used. The reporter gene may be enough if it is operatively linked downstream of the response element. The first fused protein contains the ligand binding domain of PPAR and the first domain of the transcription factor (DNA binding domain or transcriptional activation domain) and the second fused protein contains the PPAR binding domain of a coactivator and the second domain of transcription factor (transcriptional activation domain or DNA binding domain). The two kinds of domains constituting the fused protein may be each arranged in the upstream domain. The fused protein may have additional construction or deletion or substitution of sequence within the range that necessary functions are not damaged. The first and second domains of the transcription factor must be integrated before they can bind to the response element and play the function of activating gene transcription. For this purpose, when the first domain is a DNA binding domain, the second domain must be a transcriptional activation domain. When the first domain is a transcriptional activation domain, the second domain must be a DNA binding domain. The first and second domains do not necessarily be derived from the same transcription factor but may be derived from different transcription factors. The fused genes coding for the first and second fused proteins may be designed and constructed by using a usual genetic recombination technique. As for the DNA coding for the ligand binding domain of PPAR, PPAR binding domain of a coactivator, DNA binding domain of a transcription factor and transcriptional activation domain of a transcription factor constituting the first and second fused proteins, cDNA may be isolated from cDNA library by, for example, screening, etc., using PCR (Polymerase Chain Reaction) or a synthetic probe which uses a primer or probe designed and synthesized based on the information on the known amino acid sequence or nucleotide sequence. DNAs coding for the respective domains are linked and the resulting material is linked downstream of a suitable promoter to construct a fused gene. To each domain or DNA coding this, it may be introduced addition, deletion, substitution of sequence within the range where necessary functions are not damaged. The resulting first and second fused genes may be incorporated into a suitable vector plasmid and introduced into host cells in the form of a plasmid. The first and second fused genes may be constructed so as to be contained on the same plasmid or on separate plasmids. The response element and the reporter gene linked thereto may also be designed, constructed using usual genetic recombination technique and the construction is incorporated into the vector plasmid, and the resulting recombinant plasmid may be introduced into host cells. Alternatively, cells in which such a construction is incorporated in chromosomal DNA may be acquired and used. Test cells including all the constitution may be acquired, for example, by introducing one or more plasmids containing the first and second fused genes into host cells in which a response element along with a reporter gene linked thereto are introduced into the chromosomal DNA of the host cells. The thus obtained test cells are cultivated, for example, in the presence of a substance to be tested, and an interaction between PPAR and a coactivator is detected and measured by the expression of the reporter gene. When the substance to be tested binds to PPAR and an interaction with the coactivator occurs depending on the binding, an increase in the reporter activity is observed. Such a substance to be tested can be identified as an agonist for PPAR. For example, when the substance to be tested binds to PPAR but does not promote the interaction with the coactivator, addition of it together with true ligand or the drug identified as an agonist, a decrease in the reporter activity expressed by the true ligand or agonist is observed. Such a substance to be tested is identified as an antagonist to PPAR. Of the invention, as another embodiment of the method in which the ligand-dependent interaction with CBP is detected and the effect of a substance to be tested is measured with respect to said interaction, there is, for example, a method in which the ligand-dependent interaction between PPAR and CBP is measured directly. In this method, for example, CBP or its PPAR binding domain labeled with RI, etc. is used and the binding with a fused protein composed of a suitable tag protein, such as glutathione-S-trans-ferase (GST), protein A, .beta.-galactosidase, and maltose-binding protein (MBP), and the ligand binding domain of PPAR is directly detected in the presence of the substance to be tested. According to the method of the invention, for example, screening for an acting agent against PPAR.gamma. can be performed. As the ligand for PPAR.gamma., various types of thiazolidinedione derivatives have been identified and prostaglandin, 15d-PGJ.sub.2 (15-deoxy-.DELTA.12,14-prostaglandin J.sub.2), one of arachidonic acid metabolites, is considered to be a true ligand (Cell, vol. 83, pp. 803-812 and pp. 813-819, 1995). Therefore, upon the identification or screening of an agonist for PPAR.gamma., 15d-PGJ.sub.2 can be used as a positive control. By examining presence or absence of inhibition against ligand-dependent interaction expressed by 15d-PGJ.sub.2, the identification or screening of antagonist to PPAR.gamma. can be practiced. The agonist for PPAR.gamma. is expected as a remedy for treating diabetes having excellent hypoglycemic effect. Since PPAR.gamma. is an inducing factor for differentiation of adipocytes, the antagonist to PPAR.gamma. is expected to have effect as an anti-obese agent. Upon screening PPAR.gamma. acting agents, the effect on other subtypes, that is, PPAR.alpha. or PPAR.delta. (or NUC-1, PPAR.beta., FAAR) is inspected, whereby medicines having a high selectivity for PPAR.gamma. can be selected. EXAMPLES In the following, the invention will be explained in more detail by referring to Examples. However, the present invention is not limited thereto. In the following examples, unless otherwise specified particularly, each operation was according to the method described in "Molecular Cloning" (written by Sambrook, J., Fritsch, E. F. and Maniatis, T., published by Cold Spring Harbor laboratory Press in 1988) was followed, or when commercially available reagent or kit was used, they were used according to the commercially available specification |
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