Localization of the vicinal dithiols involved in steroid binding to the rat glucocorticoid receptor

Our previous studies with the thiol-specific reagent methyl methanethiolsulfonate (MMTS) and the vicinal dithiol-specific reagent sodium arsenite have established that 2 spatially close thiols (i.e. vicinal dithiols) are involved in steroid binding to the intact 98 K rat glucocorticoid receptor. These 2 thiols form an intramolecular disulfide after treatment with low concentrations of MMTS. One of these thiols was proposed to by Cys-656. In an effort to identify both thiols, we have examined the effects of MMTS and arsenite on proteolytic fragments of the receptor, which contain progressively fewer cysteines. MMTS and arsenite are now found to cause the same dithiothreitol-reversible inhibition of steroid binding and affinity labeling of both the 42 K chymotrypsin fragment and the 16 K steroid-binding core fragment of the receptor as was seen for the intact receptor. Characteristic responses include a bimodal inhibition curve for steroid binding after preincubation with MMTS and an inhibition of binding by very low concentrations of arsenite. Low concentrations of MMTS could block steroid binding by forming a disulfide bond between the receptor and a tightly associated, nonreceptor protein. However, no evidence for such cross-linking was observed when intact 98 K receptors, 42 K chymotrypsin fragments, or 16 K trypsin fragments were treated with various concentrations of MMTS, separated on nonreducing sodium dodecyl sulfate-polyacrylamide gels, and visualized by Western blotting with antiheat shock protein 90 or antireceptor antibodies. One of the antireceptor antibodies (aP1) that had been raised against the rat receptor sequence 440-795 was now found to recognize at least 1 epitope in the 16 K core fragment. We conclude that the vicinal dithiols involved in steroid binding are 2 of the 3 cysteines in the sequence of Thr537-Arg673.