Steroid-induced Conformational Changes at Ends of the Hormone-binding Domain in the Rat Glucocorticoid Receptor Are Independent of Agonist Versus Antagonist Activity

Abstract

The underlying molecular mechanism for the expression of agonist versus antagonist activity for a given receptor-steroid complex is still not known. One attractive hypothesis, based on data from progesterone receptors, is that agonistversus antagonist binding induces unique conformations at the C terminus of receptors, which can be detected by the different fragments produced by partial proteolysis. We now report that the determinants of glucocorticoid receptor (GR)-antagonist complex activity are more complex. Steroid binding did cause a conformational change in the GR that was detected by partial trypsin digestion, as described previously (Simons, S. S., Jr., Sistare, F. D., and Chakraborti, P. K. (1989) J. Biol. Chem. 264, 14493–14497). However, there was no uniformity in the digestion patterns of unactivated or activated receptors bound by a series of six structurally different antagonists including the affinity labeling antiglucocorticoid dexamethasone 21-mesylate. A total of four resistant bands were observed on SDS-polyacrylamide gels in the range of 30–27 kDa. Using a series of point mutations and epitope-specific antibodies, it was determined that the 30-kDa species represented the entire C-terminal sequence of amino acids 518–795, whereas the other bands arose from additional N-terminal and/or C-terminal cleavages. Bioassays with GRs containing various point and deletion mutations failed to reveal any C-terminal alterations that could convert antagonists into biologically active agonists. Thus, the presence or absence of C-terminal amino acids of the GR did not uniquely determine either the appearance of smaller trypsin-resistant fragments or the nature of the biological response of receptor-bound antisteroids. When compared with the current model of the ligand-binding domain, which is based on the x-ray structures of the comparable region of thyroid and retinoic acid receptors, the present results suggest that sequences outside of the model structure are relevant for the binding and biological activity of GRs. The underlying molecular mechanism for the expression of agonist versus antagonist activity for a given receptor-steroid complex is still not known. One attractive hypothesis, based on data from progesterone receptors, is that agonistversus antagonist binding induces unique conformations at the C terminus of receptors, which can be detected by the different fragments produced by partial proteolysis. We now report that the determinants of glucocorticoid receptor (GR)-antagonist complex activity are more complex. Steroid binding did cause a conformational change in the GR that was detected by partial trypsin digestion, as described previously (Simons, S. S., Jr., Sistare, F. D., and Chakraborti, P. K. (1989) J. Biol. Chem. 264, 14493–14497). However, there was no uniformity in the digestion patterns of unactivated or activated receptors bound by a series of six structurally different antagonists including the affinity labeling antiglucocorticoid dexamethasone 21-mesylate. A total of four resistant bands were observed on SDS-polyacrylamide gels in the range of 30–27 kDa. Using a series of point mutations and epitope-specific antibodies, it was determined that the 30-kDa species represented the entire C-terminal sequence of amino acids 518–795, whereas the other bands arose from additional N-terminal and/or C-terminal cleavages. Bioassays with GRs containing various point and deletion mutations failed to reveal any C-terminal alterations that could convert antagonists into biologically active agonists. Thus, the presence or absence of C-terminal amino acids of the GR did not uniquely determine either the appearance of smaller trypsin-resistant fragments or the nature of the biological response of receptor-bound antisteroids. When compared with the current model of the ligand-binding domain, which is based on the x-ray structures of the comparable region of thyroid and retinoic acid receptors, the present results suggest that sequences outside of the model structure are relevant for the binding and biological activity of GRs. Ligand binding to the cognate intracellular receptor is the obligate first step by which steroid hormones in the circulatory system regulate gene transcription in selected cells of mammals. In general, the steroid receptors contain two transactivation domains, AF-1 and AF-2, in the amino- and carboxyl-terminal portions of the molecule, respectively (1Bocquel M.T. Kumar V. Stricker C. Chambon P. Gronemeyer H. Nucleic Acids Res. 1989; 17: 2581-2595Crossref PubMed Scopus (230) Google Scholar). The binding of agonists to the ligand-binding domain (LBD) 1The abbreviations used are: LBD, ligand-binding domain; GR, glucocorticoid receptor; rGR, rat glucocorticoid receptor; TAPS, 3-[tris(hydroxymethyl)methyl]aminopropanesulfonic acid; TBS, Tris-buffered saline; DM, dexamethasone 21-mesylate. in the carboxyl-terminal half of the receptor is thought to cause a conformational change to uncover/create the AF-2 domain that regulates the transcriptional activation of receptors bound to the appropriate hormone response element (Ref. 2Wurtz J.-M. Bourguet W. Renaud J.-P. Vivat V. Chambon P. Moras D. Gronemeyer H. Nat. Struct. Biol. 1996; 3: 87-94Crossref PubMed Scopus (685) Google Scholar; reviewed in Refs. 3Simons Jr., S.S. Vitam. Horm. 1994; 48: 49-130Crossref Scopus (53) Google Scholar and 4Simons Jr., S.S. The Molecular Biology of Steroid and Nuclear Hormone Receptors.in: Freedman L.P. Birkhaeuser Boston, Inc., Boston1997Google Scholar). Many antisteroids can also cause the binding of receptors to hormone response elements (5Miller P.A. Ostrowski M.C. Hager G.L. Simons Jr., S.S. Biochemistry. 1984; 23: 6883-6889Crossref PubMed Scopus (27) Google Scholar, 6DeLabre K. Guiochon-Mantel A. Milgrom E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4421-4425Crossref PubMed Scopus (48) Google Scholar), but the resulting complexes appear to be transcriptionally inactive (7Richard-Foy H. Sistare F.D. Riegel A.T. Simons Jr., S.S. Hager G.L. Mol. Endocrinol. 1987; 1: 659-665Crossref PubMed Scopus (36) Google Scholar, 8Webster N.J.G. Green S. Jin J.R. Chambon P. Cell. 1988; 54: 199-207Abstract Full Text PDF PubMed Scopus (442) Google Scholar, 9Meyer M.-E. Pornon A. Ji J. Bocquel M.T. Chambon P. Gronemeyer H. EMBO J. 1990; 9: 3923-3932Crossref PubMed Scopus (299) Google Scholar). This ability of antisteroids to block the action of agonist steroids makes them useful both as probes of the mechanism of steroid hormone action and as drugs. Antiestrogens are commonly used to treat estrogen-dependent breast cancers, and antiandrogens are prescribed to combat prostate cancer (10Newling D.W. Br. J. Urol. 1996; 77: 776-784Crossref PubMed Google Scholar, 11Wilson J.D. Hardman J.G.G. Limbird L.L. Gilman A. Goodman and Gilman's Pharmacological Basis of Therapeutics. 9th Ed. McGraw-Hill, New York1996: 1441-1457Google Scholar, 12Parczyk K. Schneider M.R. J. Cancer Res. Clin. Oncol. 1996; 122: 383-396Crossref PubMed Scopus (27) Google Scholar). Despite the differences in the final biological response, the initial steps for most agonist and antagonist steroids are identical. The high affinity binding site in the LBD appears to be the same for both classes of steroid, as indicated by the affinity labeling of the same amino acid of the human estrogen receptor by an estrogen agonist (ketononestrol aziridine) and antagonist (tamoxifen aziridine) (13Harlow K.W. Smith D.N. Katzenellenbogen J.A. Greene G.L. Katzenellenbogen B.S. J. Biol. Chem. 1989; 264: 17476-17485Abstract Full Text PDF PubMed Google Scholar). Both classes of steroid can cause release of hsp90 after steroid binding that is accompanied by the acquisition of high affinity DNA binding (Refs. 5Miller P.A. Ostrowski M.C. Hager G.L. Simons Jr., S.S. Biochemistry. 1984; 23: 6883-6889Crossref PubMed Scopus (27) Google Scholar and 6DeLabre K. Guiochon-Mantel A. Milgrom E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4421-4425Crossref PubMed Scopus (48) Google Scholar; reviewed in Ref. 14Pratt, W. B., and Toft, D. O. (1997) Endocr. Rev., in press.Google Scholar). Perhaps most revealing is that virtually all antagonists have been observed to exhibit partial agonist activity under some condition. The fact that even the “best” antagonists for glucocorticoid (15Simons Jr., S.S. Yen P.M. Spelsberg T.C. Kumar R. Steroid Sterol Hormone Action. M. Nijhoff, Boston, MA1987: 251-268Google Scholar, 16Hoeck W. Rusconi S. Groner B. J. Biol. Chem. 1989; 264: 14396-14402Abstract Full Text PDF PubMed Google Scholar, 17Guido E.C. Delorme E.O. Clemm D.L. Stein R.B. Rosen J. Miner J.N. Mol. Endocrinol. 1996; 10: 1178-1190PubMed Google Scholar), progesterone (18Gravanis A. Schaison G. George M. de Brux J. Satyaswaroop P.G. Baulieu E.E. Robel P. J. Clin. Endocrinol. & Metab. 1985; 60: 156-163Crossref PubMed Scopus (185) Google Scholar,19Tung L. Mohamed M.K. Hoeffler J.P. Takimoto G.S. Horwitz K.B. Mol. Endocrinol. 1993; 7: 1257-1265Google Scholar), and estrogen (8Webster N.J.G. Green S. Jin J.R. Chambon P. Cell. 1988; 54: 199-207Abstract Full Text PDF PubMed Scopus (442) Google Scholar, 20Weaver C.A. Springer P.A. Katzenellenbogen B.S. Mol. Endocrinol. 1988; 2: 936-945Crossref PubMed Scopus (38) Google Scholar, 21Sundstrom S.A. Komm B.S. Xu Q. Boundy V. Lyttle C.R. Endocrinology. 1990; 126: 1449-1456Crossref PubMed Scopus (35) Google Scholar) receptors can be made to display significant amounts of agonist activity suggests that the differences between agonist and antagonist complexes are quantitative rather than qualitative. Thus, receptors complexed with either agonists or antagonists share at least a portion of the various components required for transcriptional activation. While the distinction in biological activity between agonist and antagonist steroids may not be absolute, there usually are major differences in the amount of activity that need to be explained. Clearly, the structure of each steroid is of primary importance. Often changes in just the substituents of the basic steroid structure are sufficient to convert an agonist into an antagonist. However, the same substituent may not be equally effective within each group of agonists (22Lamontagne N. Mercier L. Pons M. Thompson E.B. Simons Jr., S.S. Endocrinology. 1984; 114: 2252-2263Crossref PubMed Scopus (34) Google Scholar). Thus, structure-activity relationships have yet to provide a satisfactory framework for predicting the properties of a given steroid. For this reason, attention has shifted from differences in the structure of the steroid to possible modifications in receptor conformation following steroid binding. The most commonly used method for detecting conformational changes has been site-selective proteolysis. This method was first used to study the tertiary structure of the DNA- and non-DNA-binding forms of glucocorticoid receptors (GRs) (23Reichman M.E. Foster C.M. Eisen L.P. Eisen H.J. Torain B.F. Simons Jr., S.S. Biochemistry. 1984; 23: 5376-5384Crossref PubMed Scopus (60) Google Scholar) and then to establish a conformational change in GRs following steroid binding (24Simons Jr., S.S. Sistare F.D. Chakraborti P.K. J. Biol. Chem. 1989; 264: 14493-14497Abstract Full Text PDF PubMed Google Scholar). These studies employed affinity labeling to identify the various receptor fragments. Since then, the utility of the method has been greatly expanded both by the availability of anti-receptor antibodies and by the use of in vitro translated, [35S]methionine-labeled receptors in the elegant studies of O'Malley and co-workers (25Allan G.F. Leng X. Tsai S.Y. Weigel N.L. Edwards D.P. Tsai M.-J. O'Malley B.W. J. Biol. Chem. 1992; 267: 19513-19520Abstract Full Text PDF PubMed Google Scholar, 26Beekman J.M. Allan G.F. Tsai S.Y. Tsai M.-J. O'Malley B.W. Mol. Endocrinol. 1993; 7: 1266-1274PubMed Google Scholar, 27Leng X. Tsai S.Y. O'Malley B.W. Tsai M.-J. J. Steroid Biochem. Mol. Biol. 1993; 46: 643-661Crossref PubMed Scopus (80) Google Scholar). Thus, protease digestion studies have confirmed that steroid binding induces conformational changes in all of the members of the steroid receptor superfamily that have been examined (reviewed in Ref. 4Simons Jr., S.S. The Molecular Biology of Steroid and Nuclear Hormone Receptors.in: Freedman L.P. Birkhaeuser Boston, Inc., Boston1997Google Scholar). Even more tantalizing were the observations that antisteroids appeared to place the receptor in a conformation that rendered the carboxyl-terminal tail of the protein more susceptible to proteolytic cleavage (25Allan G.F. Leng X. Tsai S.Y. Weigel N.L. Edwards D.P. Tsai M.-J. O'Malley B.W. J. Biol. Chem. 1992; 267: 19513-19520Abstract Full Text PDF PubMed Google Scholar). At the same time, studies with the long form of the human progesterone receptor (B form) indicated that deletion of the last 42 amino acids, which were required for the binding of progesterone but not the antiprogestin RU 486, permitted RU 486 to act as an agonist (28Vegeto E. Allan G.F. Schrader W.T. Tsai M.-J. McDonnell D.P. O'Malley B.W. Cell. 1992; 69: 703-713Abstract Full Text PDF PubMed Scopus (338) Google Scholar). Apparently similar phenomena for other receptors and additional confirmatory data have subsequently appeared (25Allan G.F. Leng X. Tsai S.Y. Weigel N.L. Edwards D.P. Tsai M.-J. O'Malley B.W. J. Biol. Chem. 1992; 267: 19513-19520Abstract Full Text PDF PubMed Google Scholar, 29Weigel N.L. Beck C.A. Estes P.A. Prendergast P. Altmann M. Christensen K. Edwards D.P. Mol. Endocrinol. 1992; 6: 1585-1597PubMed Google Scholar, 30Durand B. Saunders M. Gaudon C. Roy B. Losson R. Chambon P. EMBO J. 1994; 13: 5370-5382Crossref PubMed Scopus (316) Google Scholar, 31Keidel S. 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A. 1996; 93: 8739-8744Crossref PubMed Scopus (54) Google Scholar). Other studies, however, with partially proteolyzed complexes of steroid-bound androgen (35Zeng Z. Allan G.F. Thaller C. Cooney A.J. Tsai S.Y. O'Malley B.W. Tsai M.-J. Endocrinology. 1994; 135: 248-252Crossref PubMed Scopus (16) Google Scholar, 36Kuil C.W. Berrevoets C.A. Mulder E. J. Biol. Chem. 1995; 270: 27569-27576Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar), estrogen (37McDonnell D.P. Clemm D.L. Hermann T. Goldman M.E. Pike J.W. Mol. Endocrinol. 1995; 9: 659-669Crossref PubMed Google Scholar), mineralocorticoid (38Trapp T. Holsboer F. Biochem. Biophys. Res. Commun. 1995; 215: 286-291Crossref PubMed Scopus (54) Google Scholar), and progesterone (39Allan G.F. Lombardi E. Haynes-Johnson D. Palmer S. Kiddoe M. Kraft P. Campen C. Rybczynski P. Combs D.W. Phillips A. Mol. Endocrinol. 1996; 10: 1206-1213PubMed Google Scholar) receptors and retinoic acid receptor α (31Keidel S. LeMotte P. Apfel C. Mol. Cell. Biol. 1994; 14: 287-298Crossref PubMed Scopus (113) Google Scholar, 40Lamour F.P.Y. Lardelli P. Apfel C.M. Mol. Cell. Biol. 1996; 16: 5386-5392Crossref PubMed Google Scholar) have raised questions about the generality of this hypothesis. The purpose of this study was to address several major unanswered questions concerning the importance of the C-terminal sequences of the GR LBD in the expression of antiglucocorticoid activity. Thus, we wanted to know whether steroid-induced conformational changes in the GR were specific for agonist versus antagonist steroids and where precisely in the receptor LBD the steroid-induced conformational changes occurred. To answer these questions, we have used trypsin to probe rat GRs bound by a variety of steroids. The receptor fragments were identified with affinity labeling and/or anti-receptor antibodies. Receptor mutations were used to confirm various assignments. Unless otherwise indicated, all operations were performed at 0 °C. [1,2,4-3H]Dexamethasone was purchased from Amersham Corp. [6,7-3H]Dexamethasone 21-mesylate (DM), R5020, and Enlightning were obtained from NEN Life Science Products. TAPS, dextran, and lysyl endopeptidase C were purchased from Calbiochem. Hydrofluor was from National Diagnostics, Inc. (Atlanta, GA). Tween, acrylamide, bisacrylamide, and SDS were purchased from Bio-Rad. Deacylcortivazol, RU 486, and ZK 98,299 were gifts from Roussel-UCLAF (Romainville, France), Etienne Baulieu (INSERM U33 and Collège de France, Le Kremlin-Bicêtre, Paris), and David Henderson (Schering AG, Berlin), respectively. Dexamethasone oxetanone (41Pons M. Simons Jr., S.S. J. Org. Chem. 1981; 46: 3262-3264Crossref Scopus (33) Google Scholar) and DM (42Simons Jr., S.S. Pons M. Johnson D.F. J. Org. Chem. 1980; 45: 3084-3088Crossref Scopus (95) Google Scholar) were prepared as described. All other chemicals were obtained from Sigma, includingN α-p-tosyl-l-lysine chloromethyl ketone-treated chymotrypsin andl-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin. The polyclonal antibody aP1, which was raised against the carboxyl-terminal region of the rGR (amino acids 440–795) (16Hoeck W. Rusconi S. Groner B. J. Biol. Chem. 1989; 264: 14396-14402Abstract Full Text PDF PubMed Google Scholar), was a gift from Dr. Bernd Groner (Friedrich Miescher-Institut, Basel, Switzerland), and the antibody hGRα, which was raised against the C-terminal 19 amino acids of human (and rat or mouse) GR (43de Castro M. Elliot S. Kino T. Bamberger C. Karl M. Webster E. Chrousos G.P. Mol. Med. 1996; 2: 597-607Crossref PubMed Google Scholar), was a gift from Dr. George Chrousos (NICHD, National Institutes of Health, Bethesda, MD). The anti-GR-(788–795) antibody (44Flach H. Kaiser U. Westphal H.M. J. Steroid Biochem. Mol. Biol. 1992; 42: 467-474Crossref PubMed Scopus (11) Google Scholar) was a gift from Dr. Heinrich Westphal (University of Marburg, Marburg, Germany). TAPS buffer (25 mm TAPS, 1 mm EDTA, and 10% glycerol) was adjusted to pH 8.8 or 9.5 at 0 °C with NaOH. Tris-buffered saline (TBS) contained 20 mm Tris and 0.28 m NaCl (pH 7.5). The 2 × SDS sample buffer contained 0.6 m Tris (pH 8.85), 0.2m dithiothreitol, 2% SDS, 20% glycerol, and bromphenol blue. The Western blot transfer buffer was composed of 25 mm Tris (pH 8.3), 192 mm glycine, and 20% methanol. The K536A mutant GR was prepared using the Sculptor in vitro mutagenesis kit (Amersham Corp.) by annealing a synthetic oligonucleotide, with the underlined base changes to create the desired mutant (5′-AAAATCCTAACGCAACAATAGTTCCTG-3′), to the full-length single-stranded GR cloned in the pTZ18U plasmid (45Chakraborti P.K. Garabedian M.J. Yamamoto K.R. Simons Jr., S.S. J. Biol. Chem. 1992; 267: 11366-11373Abstract Full Text PDF PubMed Google Scholar). After the annealed oligonucleotide was extended and ligated using Klenow polymerase and T4 DNA ligase, the remaining single-stranded template was removed with T5 exonuclease. The non-mutant strand was then nicked with NciI and digested with exonuclease III. Repolymerization with DNA polymerase I and T4 DNA ligase resulted in a double-stranded homoduplex. After transformation into MV1190 cells, colonies were picked and screened for the desired mutation by DNA sequencing using Sequenase Version 2.0 (U. S. Biochemical Corp.). The CS1, CD, and CS1/CD mutant GRs were gifts from Dr. Sandro Rusconi (University of Fribourg, Fribourg, Switzerland). HTC Spinner cultures were grown in Swim's S77 medium supplemented with 5% newborn calf serum, 5% fetal calf serum, and 0.03% glutamine (46Mercier L. Thompson E.B. Simons Jr., S.S. Endocrinology. 1983; 112: 601-609Crossref PubMed Scopus (80) Google Scholar). The calcium phosphate-mediated transient transfection of wild-type (pSVL-GR) or mutant receptors into monolayer cultures of COS-7 cells was conducted as described previously (45Chakraborti P.K. Garabedian M.J. Yamamoto K.R. Simons Jr., S.S. J. Biol. Chem. 1992; 267: 11366-11373Abstract Full Text PDF PubMed Google Scholar). HTC cell cytosol containing steroid-free receptors was prepared as reported (23Reichman M.E. Foster C.M. Eisen L.P. Eisen H.J. Torain B.F. Simons Jr., S.S. Biochemistry. 1984; 23: 5376-5384Crossref PubMed Scopus (60) Google Scholar). Transfected COS-7 cell cytosol was made by resuspending the frozen cell pellet in TAPS buffer (pH 9.5), slowly thawing the pellet on ice, followed by centrifugation at × The was used as For receptors, HTC cell cytosol was first by with for at 0 °C. A of cytosol was a with of that been with TAPS buffer (pH containing The was with of the same and the receptor was in with TAPS buffer (pH containing mm The of were at °C Jr., S.S. J. Biol. Chem. 1987; Full Text PDF PubMed Google Scholar). For of HTC cell or COS-7 cell cytosol were in TAPS buffer (pH 20 mm with a of dexamethasone for at °C. steroid was removed by an of 10% binding was determined by the binding in the presence of dexamethasone from the total binding. The affinity was determined by the of bound bound steroid. digestion was performed with HTC or COS-7 cell cytosol that been with or 1 for at °C. unactivated receptor contained 20 mm complexes were activated by at 20 °C for receptors were prepared by cytosol with a of dexamethasone for at °C Jr., S.S. P.A. Biochemistry. 1984; 23: PubMed Scopus Google Scholar). The proteolytic fragments were by of steroid-bound receptors with trypsin for 1 at 0 °C. A of trypsin or was then to and the were at °C. were in 2 × SDS sample for in a and on gels in a were and in a of and for at The gels were in a of 10% and acid and then with in Enlightning for 1 followed by a 10% for at The gels were on a at °C for 2 and to at °C for at least 2 after the of the molecular with a were with 2 × SDS sample buffer and on gels as described The gels were in transfer buffer for at to transfer of receptor to in a followed by for 2 The was in and acid in to molecular with 10% in for and with containing for antibody was in for aP1, for hGRα, or for and to the for a at or the antibody by Dr. Heinrich antibody and Inc., were each for at After the with primary and the was for each with and an additional with containing after with the of was performed by using the of the (Amersham Corp.). The of the molecular were indicated by with a A total of six and two (22Lamontagne N. Mercier L. Pons M. Thompson E.B. Simons Jr., S.S. Endocrinology. 1984; 114: 2252-2263Crossref PubMed Scopus (34) Google Scholar, L. P.A. Simons Jr., S.S. J. Steroid Biochem. PubMed Scopus Google Scholar, T. J. J.-P. J. Med. Chem. 1988; PubMed Scopus Google Scholar, D. Xu M. Simons Jr., S.S. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar) of different structures were selected HTC cell receptors were by each steroid in the presence of 20 mm to unactivated complexes that not A series of fragments in the range of 30–27 were by Western after digestion with chymotrypsin and trypsin or lysyl endopeptidase C not all or more bands at were for receptors by RU 486 as to The smaller was also with ZK 98,299 but not with any other antiglucocorticoid examined 2 and data not Thus, a unique digestion with lysyl endopeptidase or trypsin appears to be a of receptors bound by steroids containing a as to of unactivated GRs bound by in receptors bound by the most Jr., S.S. Thompson E.B. Johnson D.F. Biochem. Biophys. Res. Commun. Scopus Google Scholar), the same after lysyl endopeptidase C digestion as for RU 486 not no with steroid activity the digestion patterns of unactivated receptor-steroid complexes for any of the of unactivated complexes with chymotrypsin or trypsin. of HTC cell cytosol in TAPS buffer pH at 0 with 20 mm with 1 steroid for × DM × were at 0 digested with the indicated amounts of chymotrypsin or on SDS-polyacrylamide with and by as described under and The the the molecular of the which were by of of The to the of the four major species between and kDa. 1 for of abbreviations The that steroid is bound receptor has from (reviewed in Ref. 4Simons Jr., S.S. The Molecular Biology of Steroid and Nuclear Hormone Receptors.in: Freedman L.P. Birkhaeuser Boston, Inc., Boston1997Google Scholar) and x-ray J.-P. N. M. Vivat V. Chambon P. Gronemeyer H. Moras D. 1995; PubMed Scopus Google Scholar, J.W. M.E. B.L. J.D. 1995; PubMed Scopus Google Scholar) However, of a with high of steroid F. V. D. Endocrinology. 1989; PubMed Scopus (35) Google Scholar), have attention Jr., S.S. 1996; PubMed Scopus Google Scholar) with the of P.M. Jr., J.A. 1996; PubMed Scopus Google Scholar). We were to a The