[3H]cortivazol: a unique high affinity ligand for the glucocorticoid receptor

Cortivazol (CVZ) and deacylcortivazol (DAC) are pyrazolosteroids with potent glucocorticoid activity. In previous work we showed that DAC is 40-fold more potent than dexamethasone (DEX) in lysing leukemic lymphoblasts. To assess the interaction between these atypical steroids and the glucocorticoid receptor, we examined the binding of [3H]CVZ to cytosol from glucocorticoid-sensitive and -resistant variants of the human leukemic cell line CEM C7. In glucocorticoid-sensitive cells [3H]CVZ causes a 2-fold induction of glutamine synthetase and binds to a protein in the 4.6 S region of high salt sucrose gradients. On DEAE-cellulose chromatography, [3H]CVZ-receptor complexes show a shift from high (0.25 M KP) to low salt (0.09 M KP) eluting forms upon activation. CVZ competes for a 97,000-dalton protein labeled by [3H]dexamethasone mesylate. Scatchard analysis of the binding of [3H]CVZ in glucocorticoid-sensitive cells revealed a curvilinear plot which resolved into high (0.4 nM) and low (11 nM) affinity components. The receptor concentration of the low affinity site (0.30 pmol/mg protein) was approximately twice that of the high affinity site (0.14 pmol/mg protein). Dissociation experiments with dilution and/or excess unlabeled CVZ supported the presence of independent sites. In contrast, the binding of [3H]DEX to C7 cytosol revealed a single class of binding sites (Kd = 1.9 nM; receptor concentration, 0.46 pmol/mg protein). Examination of the binding of [3H]CVZ using 10(-5) M DEX as the competing ligand showed that DEX binds only to the low affinity site detected by [3H]CVZ. In cytosol from a glucocorticoid-resistant cell line with virtually no [3H]DEX binding, [3H]CVZ detected a single high affinity binding site that was similar in dissociation constant (0.8 nM) and receptor concentration (0.13 pmol/mg protein) to the high affinity site detected in the glucocorticoid-sensitive cell line C7.