Human glucocorticoid receptor β binds RU-486 and is transcriptionally active
Human glucocorticoid receptor β binds RU-486 and is transcriptionally active
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DOI：
10.1128/mcb.01439-06
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发表时间：
2007-03-01
影响因子：
5.3
通讯作者：
Cidlowski, John A.
中科院分区：
文献类型：
--
作者：
Lewis-Tuffin, Laura J.;Jewell, Christine M.;Cidlowski, John A.
人类糖皮质激素受体(HGR)表达为两个交替剪接的C末端亚型，即α和β。与经典的HGRα不同，HGRβ是一种定位于细胞核的孤儿受体，被认为不结合配体，也不影响基因转录，而不是作为HGRα的显性负值。在这里，我们使用共聚焦显微镜来检测瞬时表达的荧光蛋白标记的hGRβ在COS-1和U-2OS细胞中的细胞定位。令人惊讶的是，应用糖皮质激素拮抗剂RU-486后，黄色荧光蛋白(YFP)-HGRβ主要定位于细胞质并转移到细胞核。RU-486的这种作用被瞬时表达的野生型HGRβ所证实。共表达的YFP-HGRβ和青色荧光蛋白-HGRα在COS-1细胞中共表达的共聚焦显微镜显示，受体独立地进入细胞核。通过配体结合实验，我们证实了HGRβ与RU-486结合，但不与HGRα配体地塞米松结合。对YFP-HGRβ对一系列57个相关化合物的细胞定位的研究表明，到目前为止，RU-486是唯一与HGRβ相互作用的配体。分子模拟和计算对接研究也支持RU-486与HGRβ的选择性相互作用。有趣的是，微阵列分析表明，在没有HGRα的情况下表达的HGRβ可以调节基因的表达，而且，尽管配体结合区缺乏螺旋12，但HGRβ与拮抗剂RU-486结合会降低这种能力。
Human glucocorticoid receptor (hGR) is expressed as two alternately spliced C-terminal isoforms, alpha and beta. In contrast to the canonical hGR alpha, hGR beta is a nucleus-localized orphan receptor thought not to bind ligand and not to affect gene transcription other than by acting as a dominant negative to hGR alpha. Here we used confocal microscopy to examine the cellular localization of transiently expressed fluorescent protein-tagged hGR beta in COS-1 and U-2 OS cells. Surprisingly, yellow fluorescent protein (YFP)-hGR beta was predominantly located in the cytoplasm and translocated to the nucleus following application of the glucocorticoid antagonist RU-486. This effect of RU-486 was confirmed with transiently expressed wild-type hGR beta. Confocal microscopy of coexpressed YFP-hGR beta and cyan fluorescent protein-hGR alpha in COS-1 cells indicated that the receptors move into the nucleus independently. Using a ligand binding assay, we confirmed that hGR beta bound RU-486 but not the hGR alpha ligand dexamethasone. Examination of the cellular localization of YFP-hGR beta in response to a series of 57 related compounds indicated that RU-486 is thus far the only identified ligand that interacts with hGR beta. The selective interaction of RU-486 with hGR beta was also supported by molecular modeling and computational docking studies. Interestingly, microarray analysis indicates that hGR beta, expressed in the absence of hGR alpha, can regulate gene expression and furthermore that occupation of hGR beta with the antagonist RU-486 diminishes that capacity despite the lack of helix 12 in the ligand binding domain.