Differential effects of the reversible thiol-reactive agents arsenite and methyl methanethiosulfonate on steroid binding by the glucocorticoid receptor

Abstract

The hormone binding domain of the glucocorticoid receptor contains a unique vicinally spaced dithiol, and when it is bound by arsenite under conditions that are specific for reaction with vicinally spaced dithiols versus monothiols, steroid binding activity is eliminated [Simons, S. S., Jr., Chakraborti, P. K., & Cavanaugh, A. H. (1990) J. Biol. Chem. 265, 1938-1945]. The vicinally spaced dithiol lies in a region of the receptor that appears to be a contact site for hsp90, which is required for the high-affinity steroid binding conformation of the glucocorticoid receptor [Dalman, F. C., Scherrer, L. C., Taylor, L. P., Akil, H., & Pratt, W. B. (1991) J. Biol. Chem. 266, 3482-3490]. As part of a long-term project to develop a vicinal dithiol-specific agent that will permit studies of ligand-induced conformational changes in this region of the receptor, we have examined here the differential effects of two reversible thiol-reactive agents, arsenite and MMTS. At low concentration, arsenite inactivates the steroid binding activity of the unliganded receptor in a vicinal dithiol-specific manner, whereas dissociation of steroid from untransformed, transformed, or DNA-bound transformed receptors occurs only at concentrations typical of monothiol interactions. MMTS produces a unique bimodal effect on the steroid binding capacity of the unliganded receptor at pH 9 that is pH-dependent and becomes essentially unimodal at physiological pH. Whereas arsenite disrupts the dexamethasone-receptor complex more readily than the triamcinolone acetonide-receptor complex, MMTS has the opposite effect. During treatment for 1 h at 0 ° C, neither reagent causes dissociation of hsp90 from the receptor. Also pretreatment of the hsp90-free unliganded receptor with the covalent sulfhydryl-modifying agents N-ethylmalemide or iodoacetamide does not affect the ability of the receptor to be enzymatically refolded into a heterocomplex with hsp90 by reticulocyte lysate. The observations of this work are consistent with the concept that the vicinally spaced dithiol lies in a portion of the binding pocket that is critical for binding the D-ring region of the steroid, and they suggest that the labelling of the receptor with a vicinally spaced dithiol-specific derivatizing agent may allow detection of conformational changes likely to occur in a critical region of the hormone binding domain on dissociation of hsp90.