SUBCELLULAR-LOCALIZATION OF MINERALOCORTICOID RECEPTORS IN LIVING CELLS - EFFECTS OF RECEPTOR AGONISTS AND ANTAGONISTS

Results on the subcellular localization of the mineralocorticoid recep tor (MR) have been controversial. To determine the subcellular distrib ution and trafficking of the MR in living cells after binding of agoni sts and antagonists, we expressed a MR-green fluorescent protein (GFP) chimera in mammalian cells lacking endogenous MR. The GFP-tagged MR ( GFP-MR) remained transcriptionally active, as determined in cotransfec tion experiments with the MR-responsive reporter, TAT3-LUC. The subcel lular localization of GFP-MR was monitored by fluorescence time-lapse microscopy. In the absence of hormone, MR was present both in the cyto plasm and nucleus. Aldosterone induced a rapid nuclear accumulation of the MR Aldosterone-bound GFP-MR was concentrated in prominent cluster s within the nucleus, whereas GFP-MR did not form clusters in the abse nce of hormone. Similar subnuclear distribution was observed with cort icosterone, another MR agonist. In the presence of the MR antagonists spironolactone or ZK91587 the rate of nuclear translocation was signif icantly slower and the final nuclear-to-cytoplasmic ratio in steady st ate was significantly lower than with aldosterone. In addition, MR ant agonists did not induce formation of nuclear GFP-MR clusters. MR antag onists also were able to disrupt pre-existing nuclear clusters formed in the presence of aldosterone. GFP-MR clusters were; retained in nucl ear matrix preparations after in vivo crosslinking. These data strongl y suggest that hormone-activated MRs accumulate in dynamic discrete cl usters in the cell nucleus, and this phenomenon occurs only with trans criptionally active mineralocorticoids.