STEROID-INDUCED CONFORMATIONAL-CHANGES AT ENDS OF THE HORMONE-BINDINGDOMAIN IN THE RAT GLUCOCORTICOID RECEPTOR ARE INDEPENDENT OF AGONIST VERSUS ANTAGONIST ACTIVITY

The underlying molecular mechanism for the expression of agonist versu s antagonist activity for a given receptor-steroid complex is still no t known. One attractive hypothesis, based on data from progesterone re ceptors, is that agonist versus antagonist binding induces unique conf ormations at the C terminus of receptors, which can be detected by the different fragments produced by partial proteolysis. We now report th at 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 d escribed previously (Simons, S. S., Jr., Sistare, F. D., and Chakrabor ti, P. K. (1989) J. Biol. Chem. 264, 14493-14497). However, there was no uniformity in the digestion patterns of unactivated or activated re ceptors bound by a series of six structurally different antagonists in cluding the affinity labeling antiglucocorticoid dexamethasone 21-mesy late, A total of four resistant bands were observed on SDS-polyacrylam ide 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 spe cies represented the entire C-terminal sequence of amino acids 518-795 , whereas the other bands arose from additional N-terminal and/or C-te rminal cleavages, Bioassays with GRs containing various point and dele tion mutations failed to reveal any C-terminal alterations that could convert antagonists into biologically active agonists, Thus, the prese nce or absence of C-terminal amino acids of the GR did not uniquely de termine either the appearance of smaller trypsin-resistant fragments o r the nature of the biological response of receptor-bound antisteroids . When compared with the current model of the ligand-binding domain, w hich is based on the x-ray structures of the comparable region of thyr oid and retinoic acid receptors, the present results suggest that sequ ences outside of the model structure are relevant for the binding and biological activity of GRs.