Abstract
Treatment of the transformed glucocorticoid receptor with hydrogen peroxide promotes the formation of disulfide bonds and inhibits the ability of the receptor to bind to DNA (Tienrungroj, W., Meshinchi, S., Sanchez, E. R., Pratt, S. E., Grippo, J. F., Holmgren, A., and Pratt, W. B. (1987) J. Biol. Chem. 262, 6992-7000). It has not been determined whether the inhibition of DNA binding activity is due to disulfide bonds formed within the DNA binding domain or between the DNA binding domain and another region of the receptor. In this paper, we examined the ability of hydrogen peroxide to inactivate the DNA binding activity of the mouse glucocorticoid receptor. We show that inhibition of DNA binding activity caused by hydrogen peroxide can be accounted for entirely by the formation of disulfide bonds between cysteine residues lying within the 15-kDa tryptic fragment containing the DNA binding domain of the receptor. Reversal of the peroxide-induced inactivation of DNA binding activity requires both zinc and a thiol-disulfide exchange reagent, such as dithiothreitol. Peroxide also eliminates recognition of the intact receptor and the 15-kDa tryptic fragment by the BuGR monoclonal antibody, and the reactivity of the BuGR epitope is restored by reduction without a requirement for zinc. Pretreatment of the receptor with methyl methanethiosulfonate inhibits much of the peroxide-mediated inactivation of the BuGR epitope but pretreatment with N-ethylmaleimide does not. Similarly, DNA binding activity of the receptor is inhibited by methyl methanethiosulfonate but not by N-ethylmaleimide. These results are consistent with the proposal that peroxide promotes the formation of disulfide bonds between thiols that lie spatially close to one another in the 15-kDa tryptic fragment, resulting in rapid elimination of zinc. Restoration of the zinc finger structure restores DNA-binding activity but restoration of the BuGR epitope requires only reduction without restoration of the zinc fingers.
Highlights
Treatment of the transformed glucocorticoid recep- amino-terminal half of the receptor [4]
It hasnot been determined whether the inhibi-function of this receptor, it will be necessary to determine tion of DNA binding activity is due to disulfide bonds which cysteine residues areinvolved in the formation formed within the DNA binding domain or between of stable intramolecular disulfide bonds, to determine which the DNA binding domain and another region of the cysteines lie close enough to each other in tfhoelded molecule receptor
A major question that inhibits much of the peroxide-mediated inactivatioonf has not been answered is whether the disulfide bonds are the BuGR epitope but pretreatment with N-ethylmal- formed within the DNA- binding domaoirnwhether they are eimide does not
Summary
Treatment of the transformed glucocorticoid recep- amino-terminal half of the receptor [4] Both the hormone tor withhydrogen peroxide promotesthe formationof binding and DNA binding functions of the receptor can be disulfide bondsand inhibits the abilitoyf the receptor blocked in vitro by both thiol-derivatizing reagents and reto bind to DNA (Tienrungroj, W., Meshinchi, S., San- agents that promote disulfide bond formation (see Pratt ( 5 ) , chez, E. In thiswork we show that inactivation of the ate but not byN-ethylmaleimide.Theseresults are DNA binding activity of the glucocorticoid receptor caused consistent with the proposal that peroxide promotes by peroxide-induced disulfide bondformationcan be acthe formationof disulfide bonds between thiotlhs at lie counted forbydisulfidebridgingbetween cysteines lying spatially close to one another in the 15-kDa tryptic within the 15-kDa trypsin fragment containing the DNA-. Binding activity but restoration of the BuGR epitope peroxidealso inactivates the epitope for the BuGR monorequires only reduction without restorationof the zinc clonal anti-receptor antibody, but this effect is reversed by fingers
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