METAL OXYANION STABILIZATION OF THE RAT GLUCOCORTICOID RECEPTOR IS INDEPENDENT OF THIOLS

The ability of sodium molybdate, both to stabilize the steroid binding activity of glucocorticoid receptors and to prevent the activation of receptor-steroid complexes to a DNA binding species, has long been th ought to involve thiols. Two receptor thiols in particular, Cys-656 an d Cys-661 of rat receptors, have been suspected. The requirements for the action of molybdate, as well as two other metal oxyanions (tungsta te and vanadate) known to exert the same effects as molybdate, have no w been examined with receptors in which these thiols, or a third cyste ine in the steroid binding cavity (Cys-640), have been mutated to seri ne. No mutation prevented any metal oxyanion from either stabilizing s teroid-free receptors or blocking the activation of complexes for bind ing to nonspecific or specific DNA sequences. Thus, Cys-640, Cys-656, and Cys-661 are not required for any of the effects of molybdate, tung state, or vanadate with rat glucocorticoid receptors. Studies with hyb rid receptors, and with a 16-kDa steroid binding core fragment contain ing only 3 cysteines at positions 640, 656, and 661, indicated that no cysteine of the rat receptor was needed to maintain responsiveness to molybdate. Even when all of the thiol groups in crude cytosol were bl ocked by reaction with excess methyl methanethiol-sulfonate, each meta l oxyanion was still able to stabilize the steroid binding of receptor s. These results argue that molybdate, tungstate, and vanadate each in teract with the receptor or an associated nonreceptor protein(s) in a manner that does not require thiols. An indirect mechanism of molybdat e action was evaluated in light of the recent report that the whole ce ll actions are mediated by increased levels of intracellular cGMP. Und er cell-free conditions, however, the effects of molybdate could not b e reproduced by cGMP derivatives. Evidence consistent with a direct ef fect was that molybdate, tungstate, or vanadate each modified the kine tics of proteolysis of wild type receptors at 0 degrees C by trypsin, presumably due to induced conformational changes of the receptor. This alteration of trypsin digestion constitutes yet another effect of met al oxyanions on the glucocorticoid receptor.