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

Kevin J Modarress,Justicia Opoku,Min Xu,Nicholas J Sarlis,S Stoney Simons

doi:10.1074/jbc.272.38.23986

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 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.

Highlights

Obligate first step by which steroid hormones in the circulatory system regulate gene transcription in selected cells of mammals
The binding of agonists to the ligand-binding domain (LBD)1 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. 2; reviewed in Refs. 3 and 4)
It has been proposed that the transcriptional inactivation by antisteroids is controlled by a steroid-induced conformational change in the C terminus of the LBD that can be detected by differential proteolysis of the individual receptor-steroid complexes [25, 34]

Summary

Introduction

Obligate first step by which steroid hormones in the circulatory system regulate gene transcription in selected cells of mammals. 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) [13]. This paper is available on line at http://www.jbc.org 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) [23] and to establish a conformational change in GRs following steroid binding [24]. It has been proposed that the transcriptional inactivation by antisteroids is controlled by a steroid-induced conformational change in the C terminus of the LBD that can be detected by differential proteolysis of the individual receptor-steroid complexes [25, 34]

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Conclusion