Subcellular localization of mineralocorticoid receptors in living cells:: Effects of receptor agonists and antagonists
Subcellular localization of mineralocorticoid receptors in living cells:: Effects of receptor agonists and antagonists
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DOI：
10.1073/pnas.95.6.2973
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发表时间：
1998-03-17
影响因子：
11.1
通讯作者：
Náray-Fejes-Tóth, A
中科院分区：
文献类型：
--
作者：
Fejes-Tóth, G;Pearce, D;Náray-Fejes-Tóth, A
盐皮质激素受体（MR）的亚细胞定位结果一直存在争议。为了确定激动剂和拮抗剂结合后的MR在活细胞中的亚细胞分布和运输，我们在缺乏内源性MR的哺乳动物细胞中表达了MR-绿色荧光蛋白（GFP）嵌合体。GFP标记的MR（GFP-MR）保持转录活性，如在与MR响应报告基因TAT 3-LUC的共转染实验中所确定的。荧光延时显微镜观察GFP-MR的亚细胞定位。在激素缺乏的情况下，MR存在于细胞质和细胞核中。醛固酮诱导MR的快速核积累，醛固酮结合的GFP-MR集中在核内的突出簇中，而GFP-MR在不存在激素的情况下不形成簇。皮质酮（另一种MR激动剂）也观察到类似的亚核分布。在MR拮抗剂螺内酯或ZK 91587的存在下，核转位的速率显着较慢，在稳态下的最终核质比显着低于醛固酮。此外，MR拮抗剂不诱导核GFP-MR簇的形成。MR拮抗剂也能够破坏在醛固酮存在下形成的预先存在的核簇。GFP-MR簇在体内交联后保留在核基质制剂中。这些数据强烈表明，激素激活的MR在细胞核中以动态离散的簇聚集，并且这种现象仅在转录活性盐皮质激素的情况下发生。
Results on the subcellular localization of the mineralocorticoid receptor (MR) have been controversial. To determine the subcellular distribution and trafficking of the MR in living cells after binding of agonists 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 cotransfection experiments with the MR-responsive reporter, TAT3-LUC. The subcellular localization of GFP-MR was monitored by fluorescence time-lapse microscopy. In the absence of hormone, MR was present both in the cytoplasm and nucleus. Aldosterone induced a rapid nuclear accumulation of the MR Aldosterone-bound GFP-MR was concentrated in prominent clusters within the nucleus, whereas GFP-MR did not form clusters in the absence of hormone. Similar subnuclear distribution was observed with corticosterone, another MR agonist. In the presence of the MR antagonists spironolactone or ZK91587 the rate of nuclear translocation was significantly slower and the final nuclear-to-cytoplasmic ratio in steady state was significantly lower than with aldosterone. In addition, MR antagonists did not induce formation of nuclear GFP-MR clusters. MR antagonists also were able to disrupt pre-existing nuclear clusters formed in the presence of aldosterone. GFP-MR clusters were; retained in nuclear matrix preparations after in vivo crosslinking. These data strongly suggest that hormone-activated MRs accumulate in dynamic discrete clusters in the cell nucleus, and this phenomenon occurs only with transcriptionally active mineralocorticoids.