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 thought to involve thiols. Two receptor thiols in particular, Cys-656 and Cys-661 of rat receptors, have been suspected. The requirements for the action of molybdate, as well as two other metal oxyanions (tungstate and vanadate) known to exert the same effects as molybdate, have now been examined with receptors in which these thiols, or a third cysteine in the steroid binding cavity (Cys-640), have been mutated to serine. No mutation prevented any metal oxyanion from either stabilizing steroid-free receptors or blocking the activation of complexes for binding to nonspecific or specific DNA sequences. Thus, Cys-640, Cys-656, and Cys-661 are not required for any of the effects of molybdate, tungstate, or vanadate with rat glucocorticoid receptors. Studies with hybrid receptors, and with a 16-kDa steroid binding core fragment containing 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 blocked by reaction with excess methyl methanethiol-sulfonate, each metal oxyanion was still able to stabilize the steroid binding of receptors. These results argue that molybdate, tungstate, and vanadate each interact with the receptor or an associated nonreceptor protein(s) in a manner that does not require thiols. An indirect mechanism of molybdate action was evaluated in light of the recent report that the whole cell actions are mediated by increased levels of intracellular cGMP. Under cell-free conditions, however, the effects of molybdate could not be reproduced by cGMP derivatives. Evidence consistent with a direct effect was that molybdate, tungstate, or vanadate each modified the kinetics 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 metal oxyanions on the glucocorticoid receptor.
Modarress
K J
KJ
Steroid Hormones Section, NIDDK/LMCB, National Institutes of Health, Bethesda, Maryland 20892.
Cavanaugh
A H
AH
Chakraborti
P K
PK
Simons
S S
SS
D000345:Affinity Labels; D000818:Animals; D000838:Anions / Q000494:pharmacology; D001483:Base Sequence; D006152:Cyclic GMP / Q000031:analogs & derivatives; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D003907:Dexamethasone / Q000031:analogs & derivatives / Q000378:metabolism*; D008741:Methyl Methanesulfonate / Q000031:analogs & derivatives / Q000494:pharmacology; D008969:Molecular Sequence Data; D008982:Molybdenum / Q000494:pharmacology*; D009154:Mutation; D010446:Peptide Fragments / Q000378:metabolism; D051381:Rats; D011965:Receptors, Glucocorticoid / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013438:Sulfhydryl Compounds / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism; D017973:Tungsten Compounds / Q000494:pharmacology*; D014638:Vanadates / Q000494:pharmacology*
D000345:Affinity Labels; D000838:Anions; D010446:Peptide Fragments; D011965:Receptors, Glucocorticoid; D013438:Sulfhydryl Compounds; D017973:Tungsten Compounds; C044659:molybdate; C014674:methyl methanethiosulfonate; D014638:Vanadates; D003907:Dexamethasone; D008982:Molybdenum; D008741:Methyl Methanesulfonate; D014357:Trypsin; D006152:Cyclic GMP; D003545:Cysteine; C031710:dexamethasone 21-methanesulfonate; C045951:tungstate