Differential intracellular localization of human mineralocorticosteroid receptor on binding of agonists and antagonists

摘要

pThe effect of aldosterone and antimineralocorticoids on the intracellular localization of human mineralocorticosteroid receptor (hMR) was studied using a new monoclonal anti-peptide antibody FD4. This antibody was directed against the peptide hMR-(412-422). As demonstrated by ultracentrifugation analysis, immunoprecipitation assays and Western blot, FD4 recognized both the native and denatured form of the receptor overexpressed 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 aldosterone, suggesting a lack of nuclear MR translocation. FD4 was also used for immunohistochemical studies on hMR-expressing High Five cells. In the absence of hormone, immunoreactive hMR was detected almost exclusively in the cytoplasmic compartment of cells. After aldosterone exposure, intense nuclear immunostaining appeared in a time-dependent manner, consistent with stable nuclear localization of the receptor. Immunohistochemistry showed that antimineralocorticosteroids (ZK91587, SC9420, 18-vinylprogesterone) predominantly maintained a cytoplasmic distribution of hMR and inhibited its aldosterone-dependent nuclear localization. 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 mechanism of action by preventing productive interaction of antagonist-receptor complex with specific DNA sequences in aldosterone target cells./p