Activation of covalent affinity labeled glucocorticoid receptor-steroid complexes

Dexamethasone 21-mesylate is an irreversible anti-glucocorticoid in rat hepatoma tissue culture (HTC) cells and can affinity label the glucocorticoid receptor in cell-free systems. We now report that dexamethasone 21-mesylate also labels glucocorticoid receptors in intact HTC cells. As a first step toward understanding the whole cell antiglucocorticoid effects of dexamethasone 21-mesylate, we have examined the ability of cell-free covalent rat liver and HTC cell receptor-steroid complexes to undergo activation to forms that bind to DNA-cellulose. After optimization of the covalent labeling conditions, greater than 90% of the available glucocorticoid receptors could be labeled covalently with [³H]dexamethasone 21-mesylate. Under conditions of ~70% labeling efficiency, those covalently labeled receptor complexes that had been subjected to activating conditions were found to bind to DNA-cellulose. Little binding of covalent complexes to DNA-cellulose occurred in the absence of activation. The ability of covalently labeled receptors to be activated to the DNA-binding state has been used to prepare labeled receptors that are almost radiochemically pure. The molecular weights of unactivated or activated rat liver on HTC cell receptors labeled by [³H]dexamethasone 21-mesylate in cell-free systems or in whole cells with or without protease inhibitors were indistinguishable by NaDodSO₄-polyacrylamide gel electrophoresis (M(r)=89,000 ± 3,000). As judged by the efficiency of activation and the NaCl elution profile of DNA cellulose-bound complexes, no significant differences could be detected between the cell-free activated forms of covalent dexamethasone 21-mesylate and noncovalent dexamethasone 21-mesylate-labeled receptors in whole HTC cells, as defined by nuclear binding, was much less efficient both than that seen under cell-free conditions and than that seen for dexamethasone-labeled receptors in whole cells.