Physical characterization of the activated and non-activated forms of the glucocorticoid-receptor complex bound to the steroid antagonist [3H]RU 486.

We have compared the physicochemical characteristics of the non-activated (molybdate stabilized) glucocorticoid-receptor complex bound either to [3H]triamcinolone acetonide or to the antagonist [3H]RU 486 with those of the activated (25 degrees C preheated) complexes. The level of activation was measured by a DNA cellulose assay. The physicochemical features of the steroid-receptor complexes were analyzed by various techniques including high-performance size exclusion chromatography, sucrose gradient sedimentation and high-performance DEAE ion exchange chromatography. All the buffers used during the analytical procedures contained sodium molybdate in order to prevent any dissociation of the steroid-receptor complex. The non-activated glucocorticoid receptor bound either to [3H]TA or to [3H]RU 486 sedimented at 9.3S on sucrose gradient, displayed at 70 A Stokes radius in high performance size exclusion chromatography on TSK G4000 SW column, and was eluted at 0.22-0.28 M KCl by anion-exchange chromatography on a DEAE 545 column. After activation the Stokes radius and the sedimentation coefficient declined to 50 A and 4.5S respectively, and the complex was eluted by 0.10-0.12 M KCl on the ion-exchange column. No qualitative difference could be detected between the characteristics of the glucocorticoid-receptor complexes bound either to [3H]TA or to [3H]RU 486. Moreover, the relative distribution of "non-activated" and "activated" forms of the glucocorticoid receptor obtained through physicochemical experiments was highly correlated with the activation level determined by DNA binding experiments. However the activation level of [3H]RU 486-glucocorticoid-receptor complex appeared markedly decreased (15%) when compared with that of the agonist [3H]TA-receptor complex (65%). Experiments done with an antagonist steroid of the 17 beta-carboxamide series of dexamethasone exhibit the same results with an activation level of 12% as compared with triamcinolone acetonide. Thus, two antihormones which have different structures show the same behavior towards the glucocorticoid-receptor complex by strikingly reducing both the activation process and the size reduction of the steroid-receptor complex which is concomitant to activation and which could constitute the first step of this process.