Differential effects of the reversible thiol-reactive agents arsenite and methyl methanethiosulfonate on steroid binding by the glucocorticoid receptor.
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 degree C, neither reagent causes dissociation of hsp90 from the receptor.(ABSTRACT TRUNCATED AT 250 WORDS)
Stancato
L F
LF
Department of Pharmacology, University of Michigan Medical School, Ann Arbor 48109.
Hutchison
K A
KA
Chakraborti
P K
PK
Simons
S S
SS
Pratt
W B
WB
D000818:Animals; D001151:Arsenic / Q000494:pharmacology*; D018053:Arsenites; D001665:Binding Sites; D002460:Cell Line; D003600:Cytosol / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D003907:Dexamethasone / Q000378:metabolism; D005033:Ethylmaleimide / Q000494:pharmacology; D006360:Heat-Shock Proteins / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007460:Iodoacetamide / Q000494:pharmacology; D007739:L Cells; D008741:Methyl Methanesulfonate / Q000031:analogs & derivatives* / Q000494:pharmacology; D051379:Mice; D011965:Receptors, Glucocorticoid / Q000187:drug effects / Q000378:metabolism*; D014222:Triamcinolone Acetonide / Q000378:metabolism
D018053:Arsenites; D006360:Heat-Shock Proteins; D011965:Receptors, Glucocorticoid; C014674:methyl methanethiosulfonate; D003907:Dexamethasone; D004247:DNA; D008741:Methyl Methanesulfonate; D014222:Triamcinolone Acetonide; C015001:arsenite; D001151:Arsenic; D005033:Ethylmaleimide; D007460:Iodoacetamide