Volume 272, Number 38, Issue of September 19, 1997 pp. 23986-23994
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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

(Received for publication, May 30, 1997, and in revised form, July 7, 1997)

From the Steroid Hormones Section, NIDDK/Laboratory of Molecular and Cellular Biology, National Institutes of Health, Bethesda, Maryland 20892

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 agonist versus 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.

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