Identification of cysteine-644 as the covalent site of attachment of dexamethasone 21-mesylate to murine glucocorticoid receptors in WEHI-7 cells

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Abstract
Dexamethasone 21-mesylate is a highly specific synthetic glucocorticoid derivative that binds covalently to glucocorticoid receptors via sulfhydryl groups. We have identified the amino acid that reacts with the dexamethasone 21-mesylate by using enzymatic digestion and microsequencing for radiolabel. Nonactivated glucocorticoid receptors obtained from labeling intact WEHI-7 mouse thymoma cells with [3H]dexamethasone 21-mesylate were immunopurified and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified .apprx. 100-kDa steroid-binding subunit was eluted from gel slices and subjected to enzymatic digestion. Trypsin digestion followed by reversed-phase high-performance liquid chromatography (reversed-phase HPLC) produced a single [3H]dexamethasone 21-mesylate labeled peptide. Automated Edman degradation of this peptide revealed that the [3H]dexamethasone 21-mesylate was located at position 5 from the amino terminus. Dual-isotope labeling studies with [3H]dexamethasone 21-mesylate and [35S]methionine demonstrated that this peptide contained methionine. Staphylococcus aureus V8 protease digestion of [3H]dexamethasone 21-mesylate labeled steroid-binding subunits generated a different radiolabeled peptide containing label at position 7 from the amino terminus. On the basis of the published amino acid sequence of the murine glucocorticoid receptor, our data clearly identify cysteine-644 as the single residue in the steroid-binding domain that covalently binds dexamethasone 21-mesylate. We have confirmed this finding by demonstrating that a synthetic peptide representing the amino acid sequence 640-650 of the murine glucocorticoid receptor behaves in an identical manner on reversed-phase HPLC as the trypsin-generated peptide from intact cells.

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