DIFFERENTIAL INTRACELLULAR-LOCALIZATION OF HUMAN MINERALOCORTICOSTEROID RECEPTOR ON BINDING OF AGONISTS AND ANTAGONISTS

The effect of aldosterone and antimineralocorticoids on the intracellu lar localization of human mineralocorticosteroid receptor (hMR) was st udied using a new monoclonal anti-peptide antibody FD4. This antibody was directed against the peptide hMR-(412-422). As demonstrated by ult racentrifugation analysis, immunoprecipitation assays and Western blot , FD4 recognized both the native and denatured form of the receptor ov erexpressed in the baculovirus expression system. In whole-cell assays , the amount of hMR recovered in high-salt extracts was significantly lower after exposure to the antimineralocorticoid ZK91587 than to aldo sterone, suggesting a lack of nuclear MR translocation. FD4 was also u sed for immunohistochemical studies on hMR-expressing High Five cells. In the absence of hormone, immunoreactive hMR was detected almost exc lusively in the cytoplasmic compartment of cells. After aldosterone ex posure, intense nuclear immunostaining appeared in a time-dependent ma nner, consistent with stable nuclear localization of the receptor. Imm unohistochemistry showed that antimineralo-corticosteroids (ZK91587, S C9420, 18-vinylprogesterone) predominantly maintained a cytoplasmic di stribution of hMR and inhibited its aldosterone-dependent nuclear loca lization. Thus, in our model, the nuclear/cytoplasmic partition of hMR is drastically different in the presence of antagonists from that in the presence of aldosterone. This phenomenon may contribute to their m echanism of action by preventing productive interaction of antagonist- receptor complex with specific DNA sequences in aldosterone target cel ls.