Affinity labeling of the rat liver glucocorticoid receptor with dexamethasone 21-mesylate. Identification of covalently labeled receptor by immunochemical methods

Two convergent, independent lines of evidence are used to identify the rat liver glucocorticoid receptor. First, the electrophilic affinity label [³H]dexamethasone 21-mesylate is used to label covalently the steroid-specific binding site of the receptor. (Dexamethasone is defined as 9α-fluoro-11β,17,21-trihydroxy-16α-methyl-pregna-1,4-diene-3,20-dione). Second, antibodies to the rat liver receptor are used to isolate it from crude cytosols. A combination of these two techniques results in an immunochemical isolation of receptors covalently labeled with [³H]dexamethasone 21-mesylate. Crude rat liver cytosol containing steroid-free receptors was chromatographed on Sephadex G-100 in buffer containing 20 mM sodium molybdate in order to remove small molecular weight components that react with dexamethasone 21-mesylate. Alternatively, the crude cytosol was treated with ammonium sulfate in the presence of 20 mM sodium molybdate to precipitate the steroid-free receptor. Treatment of these steroid-free receptor solutions with [³H]dexamethasone 21-mesylate, followed by analysis on denaturing polyacrylamide gels, reveals three major peaks of radioactivity at Mr ~ 90,000, 67,000, and 52,000. The Mr~67,000 component is identified as albumin with the use of antibody to rat albumin. Competition experiments with dexamethasone demonstrate that only the Mr~90,000 moiety is saturated. The same Mr~90,000 moiety is adsorbed by immobilized antibody to the glucocorticoid receptor. Immobilized antibody can also be used to adsorb the steroid-free receptor from crude cytosols; treatment of the adsorbed receptor with [³H]dexamethasone 21-mesylate results in specific labeling of an Mr~90,000 moiety. Thus, the steroid affinity labeling experiments and the immunochemical experiments concurrently identify the Mr~90,000 moiety as the glucocorticoid receptor. These studies firmly establish the specificity of the antibody, and of the affinity-labeling steroid dexamethasone 21-mesylate for the rat liver glucocorticoid receptor.