Biochemical and immunological evidence that an acidic domain of hsp 90 is involved in the stabilization of untransformed glucocorticoid receptor complexes.

Polyclonal antibodies (AS 232-266) have been raised against the 232-266 amino acid sequence of the mouse hsp 84. This sequence possesses 54% acidic residues. AS 232-266 react with both the denatured and the free native murine hsp 84, but not with the bound hsp 84 present in the untransformed glucocorticoid receptor complexes (GR). Both AS 232-266 and peptide 232-266 were shown to decrease [3H]dexamethasone binding by GR. Moreover synthetic peptide 232-266, when added to 7 nm untransformed GR, convert them into 5 nm hsp 84-free GR. Taken together these data suggest that the acidic 232-266 sequence of hsp 84 is involved in the stabilization of the hsp 84-GR interaction, which is known to result in 7 nm complex formation and in GR ligand binding activity improvement. Both peptide 232-266 and AS 232-266 destabilize this interaction.

Binding of RU486 and deacylcortivazol to the glucocorticoid receptor is insensitive to sulfhydryl-modifying agents.

The differential sensitivity of the rat liver glucocorticoid receptor (GR) to sulfhydryl group modifying agents when bound to various agonist and antagonist ligands was studied. [3H]Triamcinolone acetonide (TA) binding was completely abolished by previous treatment of the unbound receptor with various N-alkylmaleimides. On the contrary, [3H]RU486 binding was only slightly affected by treatment with N-ethylmaleimide (NEM) and more significantly decreased with maleimides bearing bulky substituents. Ligand exchange experiments demonstrated that, unlike the agonist TA, the antiglucocorticoid RU486 was unable to protect the GR binding site from the effect of NEM. This lack of protection would seem to be due to the presence of the bulky 11 beta-substituent in RU486 since RU26988 and RU28362, two 11 beta hydroxylated glucocorticoids bearing the same 17 alpha-propynyl side chain as RU486 but lacking the 11 beta-substituent could protect GR against NEM. The ability of a GR ligand to prevent NEM inactivation of TA binding appeared unrelated to its agonist or antagonist nature: deacylcortivazol, a potent agonist, afforded no protection whereas antagonists of the 17 beta-carboxamide series did. These data strongly suggest that compounds bearing bulky substituents on the steroid A and/or C rings, like deacylcortivazol and RU486, are positioned differently from canonical glucocorticoids in the steroid binding groove of the GR.

Study of the heteromeric structure of the untransformed glucocorticoid receptor using chemical cross-linking and monoclonal antibodies against the 90K heat-shock protein.

The untransformed rat glucocorticoid receptor is assumed to be a hetero-oligomeric complex, containing a non-steroid binding component, the 90K heat-shock protein (HSP 90). Direct measurement of its molecular weight by chemical cross-linking provides new evidence for a trimeric structure with a Mr of ca. 270,000. Resorting to an anti HSP 90 probe (AC 88), we show that the native dimeric HSP 90 possess two accessible epitopes for this monoclonal antibody, while when bound to the steroid-binding subunit, only one epitope remains accessible. These data clearly suggest that the untransformed rat glucocorticoid receptor is an asymmetrical hetero-oligomeric complex.