Distinct Interaction of Cortivazol with the Ligand Binding Domain Confers Glucocorticoid Receptor Specificity
2002; Elsevier BV; Volume: 277; Issue: 7 Linguagem: Inglês
10.1074/jbc.m107946200
ISSN1083-351X
AutoresNoritada Yoshikawa, Yuichi Makino, Kensaku Okamoto, Chikao Morimoto, Isao Makino, Hirotoshi Tanaka,
Tópico(s)Adrenal Hormones and Disorders
ResumoLigand-receptor coupling is one of the important constituents of signal transduction and is essential for physiological transmission of actions of endogenous substances including steroid hormones. However, molecular mechanisms of the redundancy between glucocorticoid and mineralocorticoid actions remain unknown because of complicated cross-talk among, for example, these adrenal steroids, their cognate receptors, and target genes. Receptor-specific ligand that can distinctly modulate target gene expression should be developed to overcome this issue. In this report, we showed that a pyrazolosteroid cortivazol (CVZ) does not induce either nuclear translocation or transactivation function of the mineralocorticoid receptor (MR) but does both for the glucocorticoid receptor (GR). Moreover, deletion analysis of the C-terminal end of the GR has revealed that CVZ interacts with the distinct portion of the ligand binding domain (LBD) and differentially modulates the ligand-dependent interaction between transcription intermediary factor 2 and the LBD when compared with cortisol, dexamethasone, and aldosterone. Thus, it is indicated that CVZ may not be only a molecular probe for the analysis of the redundancy between the GR and MR in vivo but also a useful reagent to clarify structure-function relationship of the GR LBD. Ligand-receptor coupling is one of the important constituents of signal transduction and is essential for physiological transmission of actions of endogenous substances including steroid hormones. However, molecular mechanisms of the redundancy between glucocorticoid and mineralocorticoid actions remain unknown because of complicated cross-talk among, for example, these adrenal steroids, their cognate receptors, and target genes. Receptor-specific ligand that can distinctly modulate target gene expression should be developed to overcome this issue. In this report, we showed that a pyrazolosteroid cortivazol (CVZ) does not induce either nuclear translocation or transactivation function of the mineralocorticoid receptor (MR) but does both for the glucocorticoid receptor (GR). Moreover, deletion analysis of the C-terminal end of the GR has revealed that CVZ interacts with the distinct portion of the ligand binding domain (LBD) and differentially modulates the ligand-dependent interaction between transcription intermediary factor 2 and the LBD when compared with cortisol, dexamethasone, and aldosterone. Thus, it is indicated that CVZ may not be only a molecular probe for the analysis of the redundancy between the GR and MR in vivo but also a useful reagent to clarify structure-function relationship of the GR LBD. Ligand-receptor coupling is one of the important constituents of signal transduction and is essential for physiological transmission of actions of endogenous substances including hormones, cytokines, and chemokines. Of note, redundancy occurs at the level of both multiple ligands for each receptor and multiple receptors for each ligand, leading to the generation of multiple pathways to achieve similar or different cellular responses. Underlying mechanisms of redundancy, therefore, are distinct among each ligand-receptor coupling and are often tissue-specific. In any case, such redundancy is considered to play an important role in precise tuning of biological actions of ligand and receptor. From a pharmacological point of view, selective modulators with small molecular weight controlling redundancy may be of clinical versatility (1Kishimoto T. Akira S. Taga T. Science. 1992; 258: 593-597Crossref PubMed Scopus (798) Google Scholar, 2Lohnes D. Kastner P. Dierich A. Mark M. LeMeur M. Chambon P. Cell. 1993; 73: 643-658Abstract Full Text PDF PubMed Scopus (533) Google Scholar, 3Devreotes P.N. Neuron. 1994; 12: 235-241Abstract Full Text PDF PubMed Scopus (135) Google Scholar). Glucocorticoids are produced in the adrenal cortex under the strict control of the hypothalamus-pituitary-adrenal axis and exert a variety of biological actions as follows: regulation of glucose metabolism, lipolysis, immune system, cardiovascular system, electrolyte metabolism, and central nervous system (4Sapolsky R.M. Romero L.M. Munck A.U. Endocr. Rev. 2000; 21: 55-89Crossref PubMed Scopus (5611) Google Scholar). Moreover, at pharmacological doses, various glucocorticoid compounds are widely used as an anti-inflammatory and/or immunosuppressive reagent (5Cato A.C. Wade E. Bioessays. 1996; 18: 371-378Crossref PubMed Scopus (297) Google Scholar). Glucocorticoid actions are believed to be mediated by the binding to their cognate receptor, the glucocorticoid receptor (GR), 1The abbreviations used are:GRglucocorticoid receptorAF-1activation function-1ALDaldosteroneARandrogen receptor11β-HSD11β-hydroxysteroid dehydrogenaseCVZcortivazolDBDDNA binding domainDCCdextran-coated charcoalDEXdexamethasoneFcortisolFCSfetal calf serumGFPgreen fluorescent proteinGREglucocorticoid response elementhsp90heat shock protein 90LBDligand binding domainMRmineralocorticoid receptorMREmineralocorticoid response elementNIDnuclear receptor interaction domainPMAphorbol 12-myristate acetateTIF2transcription intermediary factor 2PBSphosphate-buffered salineDTTdithiothreitolCHOChinese hamster ovaryMOPS4-morpholinepropanesulfonic acidDMEMDulbecco's modified Eagle's mediumCcytoplasmicNnuclear which belongs to the steroid, thyroid, and retinoic acid receptor superfamily (6Beato M. Herrlich P. Schutz G. Cell. 1995; 83: 851-857Abstract Full Text PDF PubMed Scopus (1639) Google Scholar). The GR has a modular structure comprising several regions (6Beato M. Herrlich P. Schutz G. Cell. 1995; 83: 851-857Abstract Full Text PDF PubMed Scopus (1639) Google Scholar). The N-terminal region harbors an autonomous activation function, denoted activation function-1 (AF-1). The central DNA binding domain (DBD) is highly conserved and is composed of two zinc fingers involved in DNA binding and receptor dimerization (6Beato M. Herrlich P. Schutz G. Cell. 1995; 83: 851-857Abstract Full Text PDF PubMed Scopus (1639) Google Scholar). Nuclear targeting of receptors is directed by two nuclear localization signals, NL1, and NL2, that are mapped immediately C-terminal to the DBD and in the ligand binding domain (LBD), respectively (6Beato M. Herrlich P. Schutz G. Cell. 1995; 83: 851-857Abstract Full Text PDF PubMed Scopus (1639) Google Scholar). The LBDs of the nuclear receptors have a common fold, with 12 α-helices (numbered H1 through H12) and one β-turn arranged as an antiparallel α-helical “sandwich” in a three-layer structure and mediates numerous functions, including ligand binding, nuclear targeting, interaction with heat shock protein 90 (hsp90), dimerization, interaction with coactivators, and hormone-dependent transactivation (7Glass C.K. Rose D.W. Rosenfeld M.G. Curr. Opin. Cell Biol. 1997; 9: 222-232Crossref PubMed Scopus (600) Google Scholar, 8Moras D. Gronemeyer H. Curr. Opin. Cell Biol. 1998; 10: 384-391Crossref PubMed Scopus (710) Google Scholar, 9Glass C.K. Rosenfeld M.G. Genes Dev. 2000; 14: 121-141Crossref PubMed Google Scholar, 10Bourguet W. Germain P. Gronemeyer H. Trends Pharmacol. Sci. 2000; 21: 381-388Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar). Many coactivators for the GR and MR have been identified to date, including steroid receptor coactivator-1 (SRC-1), transcriptional intermediary factor 2 (TIF2/GRIP-1), and CBP/p300 (7Glass C.K. Rose D.W. Rosenfeld M.G. Curr. Opin. Cell Biol. 1997; 9: 222-232Crossref PubMed Scopus (600) Google Scholar, 9Glass C.K. Rosenfeld M.G. Genes Dev. 2000; 14: 121-141Crossref PubMed Google Scholar). In the absence of ligand, the GR is a part of a large protein complex, in which they interact with the hsp90, and ligand binding promotes conformational change and hsp90 release (11Simons S.S., Jr. Pratt W.B. Methods Enzymol. 1995; 251: 406-422Crossref PubMed Scopus (44) Google Scholar). The receptors then translocate into the nucleus and act as a transcription factor, binding as a homodimer to the glucocorticoid response elements (GRE), and regulate transcription with the aid of those coactivators and mediators (6Beato M. Herrlich P. Schutz G. Cell. 1995; 83: 851-857Abstract Full Text PDF PubMed Scopus (1639) Google Scholar, 9Glass C.K. Rosenfeld M.G. Genes Dev. 2000; 14: 121-141Crossref PubMed Google Scholar). Recently, part of the glucocorticoid actions are not mediated by binding to DNA but by the interaction with other protein factors. For example, the GR represses activity of the transcription factor AP-1 and NF-κB, which is now considered to be a pharmacological basis of anti-inflammatory activity of glucocorticoids (often referred as transrepression) (12Adcock I.M. Caramori G. Immunol. Cell Biol. 2001; 79: 376-384Crossref PubMed Scopus (295) Google Scholar). Moreover, the concept of ligand-based modularity of the structure and function of the GR is now experimentally challenged, and so-called dissociated glucocorticoids or selective GR modifiers are being developed to separate untoward actions from therapeutic activities of glucocorticoids (12Adcock I.M. Caramori G. Immunol. Cell Biol. 2001; 79: 376-384Crossref PubMed Scopus (295) Google Scholar, 13Vayssiere B.M. Dupont S. Choquart A. Petit F. Garcia T. Marchandeau C. Gronemeyer H. Resche-Rigon M. Mol. Endocrinol. 1997; 11: 1245-1255Crossref PubMed Scopus (299) Google Scholar, 14Vanden Berghe W. Francesconi E., De Bosscher K. Resche-Rigon M. Haegeman G. Mol. Pharmacol. 1999; 56: 797-806PubMed Google Scholar). glucocorticoid receptor activation function-1 aldosterone androgen receptor 11β-hydroxysteroid dehydrogenase cortivazol DNA binding domain dextran-coated charcoal dexamethasone cortisol fetal calf serum green fluorescent protein glucocorticoid response element heat shock protein 90 ligand binding domain mineralocorticoid receptor mineralocorticoid response element nuclear receptor interaction domain phorbol 12-myristate acetate transcription intermediary factor 2 phosphate-buffered saline dithiothreitol Chinese hamster ovary 4-morpholinepropanesulfonic acid Dulbecco's modified Eagle's medium cytoplasmic nuclear Secretion of a physiological glucocorticoid cortisol (F) is in general significantly greater than that of the other steroid hormone aldosterone (ALD) that is secreted as a mineralocorticoid from the adrenal cortex (4Sapolsky R.M. Romero L.M. Munck A.U. Endocr. Rev. 2000; 21: 55-89Crossref PubMed Scopus (5611) Google Scholar). Because glucocorticoids, under certain physiological and pharmacological conditions, can also cause mineralocorticoid-like sodium and fluid retention, the functional redundancy has been suggested between glucocorticoids and mineralocorticoids in the regulation of fluid and electrolyte homeostasis (15Fuller P.J. Lim-Tio S.S. Brennan F.E. Kidney Int. 2000; 57: 1256-1264Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). It should be noted that the mineralocorticoid receptor (MR) is highly homologous with the GR (16Arriza J.L. Weinberger C. Cerelli G. Glaser T.M. Handelin B.L. Housman D.E. Evans R.M. Science. 1987; 237: 268-275Crossref PubMed Scopus (1651) Google Scholar), and these receptors are simultaneously expressed in several tissues (15Fuller P.J. Lim-Tio S.S. Brennan F.E. Kidney Int. 2000; 57: 1256-1264Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 17De Kloet E.R. Ratka A. Reul J.M. Sutanto W. Van Eekelen J.A. Ann. N. Y. Acad. Sci. 1987; 512: 351-361Crossref PubMed Scopus (92) Google Scholar). Moreover, biochemical experiments have revealed that the GR binds not only glucocorticoids but also mineralocorticoid, and the MR binds not only mineralocorticoids but also glucocorticoids with high affinity (18Teutsch G. Costerousse G. Deraedt R. Benzoni J. Fortin M. Philibert D. Steroids. 1981; 38: 651-665Crossref PubMed Scopus (87) Google Scholar, 19Gomez-Sanchez C.E. Gomez-Sanchez E.P. Endocrinology. 1983; 113: 1004-1009Crossref PubMed Scopus (27) Google Scholar, 20Rafestin-Oblin M.E. Lombes M. Lustenberger P. Blanchardie P. Michaud A. Cornu G. Claire M. J. Steroid Biochem. 1986; 25: 527-534Crossref PubMed Scopus (31) Google Scholar, 21Reul J.M. de Kloet E.R. Endocrinology. 1985; 117: 2505-2511Crossref PubMed Scopus (2230) Google Scholar, 22Rupprecht R. Reul J.M. van Steensel B. Spengler D. Soder M. Berning B. Holsboer F. Damm K. Eur. J. Pharmacol. 1993; 247: 145-154Crossref PubMed Scopus (252) Google Scholar, 23Lim-Tio S.S. Keightley M.C. Fuller P.J. Endocrinology. 1997; 138: 2537-2543Crossref PubMed Scopus (70) Google Scholar, 24Rogerson F.M. Dimopoulos N. Sluka P. Chu S. Curtis A.J. Fuller P.J. J. Biol. Chem. 1999; 274: 36305-36311Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 25Hellal-Levy C. Couette B. Fagart J. Souque A. Gomez-Sanchez C. Rafestin-Oblin M. FEBS Lett. 1999; 464: 9-13Crossref PubMed Scopus (122) Google Scholar). Although more complicated, the GR and MR can bind common DNA sequences of the GRE on the promoter region of some but not all of the target genes (26Govindan M.V. Leclerc S. Roy R. Rathanaswami P. Xie B.X. J. Steroid Biochem. Mol. Biol. 1991; 39: 91-103Crossref PubMed Scopus (18) Google Scholar, 27Kolla V. Robertson N.M. Litwack G. Biochem. Biophys. Res. Commun. 1999; 266: 5-14Crossref PubMed Scopus (49) Google Scholar). The enzyme type 2 11β-hydroxysteroid dehydrogenase (11β-HSD2) contributes to some extent in the functional distinction of these different classes of steroid hormones, because this enzyme inactivates endogenous glucocorticoids into 11-keto congeners (28Funder J.W. Pearce P.T. Smith R. Smith A.I. Science. 1988; 242: 583-585Crossref PubMed Scopus (1494) Google Scholar). However, particularly in the brain and heart in which the GR and MR are simultaneously expressed but 11β-HSD2 is not (29Young M. Funder J.W. Trends Endocrinol. Metab. 2000; 11: 224-226Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar), the biological significance of the redundancy between glucocorticoid and mineralocorticoid remains unknown (29Young M. Funder J.W. Trends Endocrinol. Metab. 2000; 11: 224-226Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 30Funder J.W. Annu. Rev. Med. 1997; 48: 231-240Crossref PubMed Scopus (251) Google Scholar, 31Hellal-Levy C. Fagart J. Souque A. Rafestin-Oblin M.E. Kidney Int. 2000; 57: 1250-1255Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Several receptor-specific ligands that can distinctly regulate target gene expression have been developed and contribute to understanding this redundancy (19Gomez-Sanchez C.E. Gomez-Sanchez E.P. Endocrinology. 1983; 113: 1004-1009Crossref PubMed Scopus (27) Google Scholar, 32Lomax R.B. Sandle G.I. Am. J. Physiol. 1994; 267: G485-G493PubMed Google Scholar, 33Kim P.J. Cole M.A. Kalman B.A. Spencer R.L. J. Steroid Biochem. Mol. Biol. 1998; 67: 213-222Crossref PubMed Scopus (36) Google Scholar). The phenylpyrazolo glucocorticoid cortivazol (CVZ) is a synthetic glucocorticoid agonist, which has been reported to have two dissociation constants for the GR and be 40-fold more potent than the synthetic glucocorticoid dexamethasone (DEX) in inducing tyrosine aminotransferase in HTC cells (34Schlechte J.A. Schmidt T.J. J. Clin. Endocrinol. Metab. 1987; 64: 441-446Crossref PubMed Scopus (14) Google Scholar, 35Schlechte J.A. Simons S.S., Jr. Lewis D.A. Thompson E.B. Endocrinology. 1985; 117: 1355-1362Crossref PubMed Scopus (31) Google Scholar). Furthermore, CVZ has been shown more effective in raising blood pressure than other natural and several synthetic glucocorticoids in sheep. However, in contrast to F, DEX, and ALD, CVZ did not decrease plasma potassium concentration (36Spence C.D. Coghlan J.P. Denton D.A. Mills E.H. Whitworth J.A. Scoggins B.A. J. Steroid Biochem. 1986; 25: 411-415Crossref PubMed Scopus (8) Google Scholar). These results prompted us to speculate that CVZ might regulate electrolyte and fluid balance not via the MR but exclusively by the GR. In this line, we studied the mechanism of CVZ action on the GR and MR. As anticipated, CVZ did not induce either nuclear translocation or transactivation function of the MR but did both of the GR. Moreover, mutational analysis of the C-terminal end of the GR revealed that CVZ interacts with the distinct portions of the LBD and differentially modulates the ligand-dependent interaction between TIF2 and the LBD when compared with F, DEX, and ALD. We thus indicate that CVZ may be not only a molecular probe for the analysis of the redundancy between the GR and MR in vivo but also a useful reagent to clarify structure-function relationships of the GR LBD. F, DEX, ALD, and phorbol 12-myristate acetate (PMA) were purchased from Sigma. CVZ was a gift from Merck. Other chemicals were from Wako Pure Chemical (Osaka, Japan) unless otherwise specified. Monoclonal anti-hsp90 antibodies (IgG and IgM) were obtained from Affinity Bioreagents, Inc. (Golden, CO). Goat anti-mouse IgM and control mouse IgM, TEPC183, were obtained from Sigma. Monoclonal anti-GFP antibody was obtained fromCLONTECH Laboratories (Palo Alto, CA). Polyclonal anti-GRIP1/TIF2 antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). COS7, CV-1, CHO, F9, and HeLa cells were obtained from the RIKEN Cell Bank (Tsukuba Science City, Japan) and maintained in Dulbecco's modified Eagle's medium (DMEM, Iwaki Glass Inc., Chiba, Japan) supplemented with 10% fetal calf serum (FCS) and antibiotics. In all experiments, serum steroids were stripped with dextran-coated charcoal (DCC), and cells were cultured in a humidified atmosphere at 37 °C with 5% CO2. Heat shock treatment for COS7 cells was achieved by shifting flasks to another 5% CO2 incubator set at 43 °C. The expression plasmids for the wild-type human GR, pRShGRα, and wild-type human MR, pRShMR, were the kind gifts from Dr. R. M. Evans (Salk Institute, La Jolla, CA). Another expression plasmid for the wild-type human GR, pCMX-GR, was constructed by cutting out a KpnI-XhoI fragment including the entire human GR-coding sequence and the 5′- and 3′-untranslated regions from pRShGRα, and this fragment was inserted into parent pCMX (37Perlmann T. Rangarajan P.N. Umesono K. Evans R.M. Genes Dev. 1993; 7: 1411-1422Crossref PubMed Scopus (333) Google Scholar). The expression plasmids for chimeric protein of green fluorescent protein (GFP) and the wild-type human GR or MR, pCMX-GFP-GR (38Okamoto K. Tanaka H. Ogawa H. Makino Y. Eguchi H. Hayashi S. Yoshikawa N. Poellinger L. Umesono K. Makino I. J. Biol. Chem. 1999; 274: 10363-10371Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar) and pCMX-GFP-MR (39Iida T. Makino Y. Okamoto K. Yoshikawa N. Makino I. Nakamura T. Tanaka H. Kidney Int. 2000; 58: 1450-1460Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar), respectively, were described previously. The expression plasmids for chimeric protein of GAL4-DBD and the LBD of the human GR (Glu-489 to Lys-777), pCMX-GAL-L-hGR (pCMX-GAL4-GRLBD), was a kind gift from Dr. K. Umesono (University of Kyoto, Kyoto, Japan). To construct an expression plasmid for chimeric protein of VP16 transactivation domain and nuclear receptor interaction domain (NID) of the TIF2, the DNA fragment encoding 173 amino acids (Glu-594 to Leu-766) of the human TIF2 were amplified by PCR using pSG5-TIF2 (the kind gift from Dr. P. Chambon, Institut de Genetique et de Biologie Moleculaire et Cellulaire, Strasbourg, France) as a template with appropriate flanking sequences and inserted into the parent pCMX-VP16 (37Perlmann T. Rangarajan P.N. Umesono K. Evans R.M. Genes Dev. 1993; 7: 1411-1422Crossref PubMed Scopus (333) Google Scholar), resulting in pCMX-VP-TIF2NID. To construct the expression plasmids for the C-terminal truncated mutant of the GR, pCMX-GR-(1–774), pCMX-GR-(1–765), pCMX-GR-(1–750), pCMX-GFP-GR-(1–774), pCMX-GFP-GR-(1–765), and pCMX-GFP-GR-(1–750), the DNA fragments encoding corresponding amino acids (Leu-596 to Phe-774, Ser-765, or Pro-750) of the human GR were amplified by PCR with appropriate flanking sequences and inserted intoPstI-BamHI-opened parent pCMX-GR or pCMX-GFP-GR. Construction of pCMX-GRI747T, pCMX-GRL753F, pCMX-GFP-GRI747T, pCMX-GFP-GRL753F, and pCMX-GAL4-GRLBDL753F was carried out involving point mutations to generate substitution of Ile-747 by Thr or Leu-753 by Phe in the human GR with QuickChangeTM site-directed mutagenesis kit (Stratagene, La Jolla, CA) using pCMX-GR, pCMX-GFP-GR, and pCMX-GAL4-GRLBD as templates. Expression plasmid for the GFP-tagged DNA-binding deficient mutant of the GR, pCMX-GFP-D4X, was constructed by cutting out a ClaI-BamHI fragment from the plasmid phGR-D4X (the kind gift from Dr. A. C. B. Cato, Forschungszentrum Karlsruhe, Germany) (40Heck S. Kullmann M. Gast A. Ponta H. Rahmsdorf H.J. Herrlich P. Cato A.C. EMBO J. 1994; 13: 4087-4095Crossref PubMed Scopus (466) Google Scholar), and this fragment was inserted into ClaI-BamHI-opened parent pCMX-GFP-GR. The glucocorticoid/mineralocorticoid-responsive reporter plasmid pGRE/MRE-Luc, GAL4-responsive reporter plasmid tk-GALpx3-LUC (41Makino Y. Yoshikawa N. Okamoto K. Hirota K. Yodoi J. Makino I. Tanaka H. J. Biol. Chem. 1999; 274: 3182-3188Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar), and NF-κB-responsive reporter plasmid pNFκBHL (42Hiramoto M. Shimizu N. Sugimoto K. Tang J. Kawakami Y. Ito M. Aizawa S. Tanaka H. Makino I. Handa H. J. Immunol. 1998; 160: 810-819PubMed Google Scholar) were described previously. The β-galactosidase expression plasmid pCH110 (Amersham Biosciences) was used as an internal control for transfection efficiency when appropriate. COS7 cells transfected with pRShGRα or pRShMR were cultured in DMEM supplemented with 2% DCC-treated FCS in 12-well flat-bottom plastic plates (IWAKI Glass) to confluence. The cells were washed three times with phosphate-buffered saline (PBS), and medium was replaced with Opti-MEM medium (Invitrogen) and then cultured with 20 nm [3H]DEX (70–110 Ci/mmol, Amersham Biosciences) in GR-expressing cells or [3H]ALD (50–85 Ci/mmol, Amersham Biosciences) in the MR-expressing cells in the presence or absence of various concentrations of radioinert ligands for 4 h at 37 °C. The monolayer was washed three times with PBS and lysed in the whole cell extract buffer (20 mm HEPES, pH 7.9, 350 mmNaCl, 1 mm MgCl2, 0.5 mm EDTA, 0.1 mm EGTA, 1% Nonidet P40, 1 mm DTT, 0.2 mm phenylmethylsulfonyl fluoride). Aliquots were added to scintillation fluid to determine radioactivity. The difference between total and nonspecific binding gives specific GR- or MR-binding sites. Results are expressed as percent of maximum binding, which is given relative to the maximal [3H]DEX or [3H]ALD binding in the absence of competitors. Nonspecific binding was determined by the experiments run in parallel without any receptor-expressing vector. Cells were plated on 6-cm diameter culture dishes (IWAKI Glass) to 30–50% confluence, and cell culture medium was replaced with Opti-MEM medium lacking phenol red before transfection. Plasmid mixture was mixed with TransIT-LT1 transfection reagent (Panvera Corp., Madison, WI) and added to the culture. Total amount of the plasmids was kept constant by adding an irrelevant plasmid (pGEM3Z was used unless otherwise specified). After 6 h of incubation, the medium was replaced with fresh DMEM supplemented with 2% DCC-treated FCS, and the cells were further cultured in the presence or absence of various reagents for 24 h at 37 °C. After normalization of transfection efficiency by β-galactosidase expression, luciferase enzyme activity was determined using a luminometer (Berthold GmbH & Co. KG, Bad Wildbad, Germany) essentially as described before (43Makino Y. Okamoto K. Yoshikawa N. Aoshima M. Hirota K. Yodoi J. Umesono K. Makino I. Tanaka H. J. Clin. Invest. 1996; 98: 2469-2477Crossref PubMed Scopus (162) Google Scholar). For analysis of subcellular localization of the GR and MR in living cells, we transiently expressed GFP-tagged receptors in COS7 cells, and assays were performed as described previously (38Okamoto K. Tanaka H. Ogawa H. Makino Y. Eguchi H. Hayashi S. Yoshikawa N. Poellinger L. Umesono K. Makino I. J. Biol. Chem. 1999; 274: 10363-10371Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). Briefly, after 6 h of transient transfection of the expression plasmids for GFP fusion proteins, the medium was replaced with DMEM supplemented with 2% DCC-treated FCS, and the cells were cultured at 37 °C for 24 h, then at 30 °C for at least 4 h, and at 37 °C thereafter. After various treatments, cells were examined using an IX70 microscope (Olympus, Tokyo, Japan) enclosed by an incubator and equipped with a heating stage and an fluorescein isothiocyanate filter set, and photographs were taken for 8 randomly selected views. Quantitative assessment of the subcellular localization of expressed GFP fusion proteins was performed according to methods described previously (38Okamoto K. Tanaka H. Ogawa H. Makino Y. Eguchi H. Hayashi S. Yoshikawa N. Poellinger L. Umesono K. Makino I. J. Biol. Chem. 1999; 274: 10363-10371Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). In brief, blindfolded observers were asked to examine the photographs for each experimental set and classify ∼200 GFP-positive cells into four different categories: N < C for cytoplasmic dominant fluorescence; N = C, cells having equal distribution of fluorescence in the cytoplasmic (C) and nuclear (N) compartments; N > C for nuclear-dominant fluorescence; N for exclusive nuclear fluorescence. For analysis of the interaction between GFP-tagged receptors and hsp90, we transiently expressed GFP chimeric protein in COS7 cells, and the assays were performed as described previously (38Okamoto K. Tanaka H. Ogawa H. Makino Y. Eguchi H. Hayashi S. Yoshikawa N. Poellinger L. Umesono K. Makino I. J. Biol. Chem. 1999; 274: 10363-10371Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). Briefly, whole cell extracts were prepared by lysing cells, and immunoprecipitation experiments, with either the anti-hsp90 IgM antibody 3G3 or control mouse IgM antibody TEPC 183, were carried out as follows. Goat anti-mouse IgM was coupled to CNBr-activated Sepharose 4B (Amersham Biosciences) by incubating in the coupling buffer (0.1 mNaHCO3, 0.5 m NaCl, pH 8.3) overnight at 4 °C. 35 μg of either the monoclonal anti-hsp90 IgM antibody or control mouse IgM antibody were then incubated with 80 μl of a 1:1 suspension of the goat anti-mouse IgM antibody coupled to Sepharose in MENG buffer (25 mm MOPS, pH 7.5, 1 mm EDTA, 0.02% NaN3, 10% glycerol) on ice for 90 min. This Sepharose-adsorbed material was pelleted and washed successively and then resuspended in 80 μl of MENG buffer containing 20 mmsodium molybdate, 2 mm DTT, 0.25 m NaCl, and 2.5% (w/v) bovine serum albumin. In immunoprecipitation experiments, 70 μg of cellular protein was added to the suspension. The reaction mixtures were incubated on ice for 90 min, after which Sepharose beads were pelleted by centrifugation and washed three times with MENG buffer containing 20 mm sodium molybdate and 2 mm DTT. Immunoprecipitated proteins were eluted by boiling in sample buffer and analyzed by SDS-PAGE and electrically transferred to an Immobilon-NC Pure nitrocellulose membrane (Millipore, Bedford, MA). Subsequently, immunoblotting was performed with a monoclonal anti-GFP antibody diluted at 1:500, followed by horseradish peroxidase-conjugated sheep anti-mouse Ig (Amersham Biosciences) diluted at 1:1000. After stripping off the immune complexes, Western immunoblot analysis was performed on the same membrane for detection of hsp90 and GFP chimeric protein, using monoclonal mouse anti-hsp90 IgG antibody 3B6 (1:500) and a monoclonal anti-GFP antibody (1:500), followed by horseradish peroxidase-conjugated sheep anti-mouse Ig diluted at 1:1000. In parallel, 20 μg of whole cell extracts were independently used for immunodetection of GFP chimeric protein and hsp90. Antibody-protein complexes were visualized using the enhanced chemiluminescence method according to the manufacturer's protocol (Amersham Biosciences). GFP-tagged chimeric GR and TIF2-expressing COS7 cells were grown on 8-chambered sterile glass slides (Nippon Becton Dickinson, Tokyo, Japan) and further cultured for 2 h in the presence or absence of various steroid ligands. For immunostaining of TIF2, the cells were fixed in cold acetone for 2 min on ice and air-dried. After fixation, the cells were washed three times with PBS at room temperature and incubated with anti-mouse GRIP1/TIF2 polyclonal goat antibody at a dilution of 1:50 in PBS containing 0.1% Triton X-100 for 1 h at room temperature. The cells were then washed three times with PBS and incubated with rhodamine-conjugated anti-goat IgG (Santa Cruz Biotechnology) at a dilution of 1:100 in PBS containing 0.1% Triton X-100 for 1 h at room temperature. The cells were finally washed three times with PBS and mounted with GEL/MOUNT™ (Biomeda Corp.) for examination on a confocal laser scanning microscope IX70. Dual excitation was achieved from krypton-argon laser, and digital images were analyzed on FLUOVIEW FV 500 systems (Olympus). As described in the Introduction, both the GR and MR, at least in part, bind to the common palindromic DNA sequence and transactivate gene expression. To test the effect of natural and synthetic glucocorticoids and mineralocorticoid on transactivation function of the receptors, we first performed transient transfection assay using GRE/MRE-luciferase as a reporter in COS7 cells. In the absence of expression plasmids for the receptors, any of F, DEX, ALD, and CVZ did not induce significant luciferase activity (Fig. 1 A). When the human GR expression plasmid was cotransfected, not only F, DEX, and CVZ but ALD as well induced expression of the reporter plasmid (Fig.1 A). When
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