Aldosterone antagonists destabilize the mineralocorticosteroid receptor

To elucidate the mechanism of action of aldosterone antagonists, we studied the interaction of spironolactone with the chick mineralocorticosteroid receptor (MR). Intestinal cytosol contains specific spironolactone-binding sites (Kd approximately 3 nM; max. no. of binding sites approximately 100 fmol/mg of protein) that have been identified as MRs by competition experiments with steroid ligands and with the monoclonal anti-idiotypic antibody H10E that interacts with aldosterone-binding domain of the MR. Binding studies indicate that aldosterone and spironolactone bind to the MR through a common site that encompasses the epitope recognized by H10E. At 4 degrees C, spironolactone dissociates much more rapidly from the cytosol 8-9 S form of MR (t1/2 38 min) than does aldosterone (t1/2 3240 min). A high dissociation rate was also observed for progesterone, a natural aldosterone antagonist (t1/2 84 min). The covalent linkage of the 90 kDa heat shock protein (hsp90) to the ligand-binding subunit of MR with dimethyl pimelimidate did not notably modify the rate of dissociation of spironolactone from the receptor (t1/2 96 min), excluding the possibility that the rapid dissociation rate of the antagonist was related to hsp90 release. The effects of aldosterone and the two anti-mineralocorticosteroids on the 8-9 S heterooligomeric structure of the MR differed strikingly. Using low-salt density-gradient centrifugation analysis, aldosterone-labelled receptors were recovered as 8-9S complexes, whereas 4 S entities were detected after spironolactone and progesterone binding. This indicated that, under the experimental conditions used, aldosterone antagonists facilitate hsp90 release and thus do not stabilize the non-DNA-binding 8-9S form of MR. We propose that the combination of rapid dissociation of the ligand and a weakened hsp90-receptor interaction is involved in the anti-mineralococorticosteroid activity of aldosterone antagonists.