LIGAND-INDUCED CONFORMATIONAL CHANGE IN THE HUMAN MINERALOCORTICOID RECEPTOR OCCURS WITHIN ITS HETEROOLIGOMERIC STRUCTURE

To determine the first steps involved in the mechanism of action of al dosterone and its antagonists, we analysed the ligand-induced structur al changes of the human mineralocorticoid receptor (hMR) translated in vitro. Limited chymotrypsin digestion of the receptor generated a 30 kDa fragment. Following binding of a ligand to hMR, the 30 kDa fragmen t became resistant to chymotrypsin proteolysis, indicating a change in the receptor conformation. Differences in sensitivity to chymotrypsin of the 30 kDa fragment were observed after binding of agonists and an tagonists to hMR, suggesting that these two classes of ligands induced different hMR conformations. Several lines of evidence allowed us to identify the 30 kDa fragment as the subregion encompassing the C-termi nal part of the hinge region and the ligand-binding domain (LBD) of hM R (hMR 711-984). (1) The 30 kDa fragment is not recognized by FD4, an antibody directed against the N-terminal region of hMR. (2) Aldosteron e remains associated with the 30 kDa fragment after chymotrypsin prote olysis of the aldosterone-hMR complex. (3) A truncated hMR, lacking th e last 40 C-terminal amino acids (hMR 1-944), yields a 26 kDa proteoly tic fragment. In addition, we showed that the unbound and the aldoster one-bound 30 kDa fragment were both associated with heat-shock protein (hsp) 90, indicating that the ligand-induced conformational change ta kes place within the hetero-oligomeric structure and that the 711-984 region is sufficient for hsp90-MR interaction. We conclude that the li gand-induced conformational change of the receptor is a crucial step i n mineralocorticoid action. It occurs within the LBD, precedes the rel ease of hsp90 from the receptor and is dependent upon the agonist/anta gonist nature of the ligand.