Direct visualization of the human estrogen receptor α reveals a role for ligand in the nuclear distribution of the receptor
Direct visualization of the human estrogen receptor α reveals a role for ligand in the nuclear distribution of the receptor
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DOI：
10.1091/mbc.10.2.471
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发表时间：
1999-02-01
影响因子：
3.3
通讯作者：
Hager, GL
中科院分区：
文献类型：
--
作者：
Htun, H;Holth, LT;Hager, GL
人类雌激素受体ct(ER Ct)的氨基末端被绿色荧光蛋白(Gfp)的S65T变异体标记，通过荧光显微镜可以观察到活细胞中的亚细胞转运和定位。标记受体GFP-ER具有配体依赖性转录因子的功能，对激动剂和拮抗剂配体均有反应，并可与核基质结合。在不同的配基条件下，分析了它在四个人乳腺癌上皮细胞系，两个ER+(MCF7和T47D)和两个ER-(MDA-MB-231和MDA-MB-435A)中的细胞定位。在所有细胞系中，GFP-ER仅在缺乏配体的情况下在细胞核中观察到。当加入激动剂或拮抗剂配体后，GFP-ER在细胞核内发生了戏剧性的重新分布，从网状分布到点状分布。此外，完全拮抗剂ICI 182780改变了受体的核质区划，并在需要持续蛋白质合成的过程中导致部分积聚在细胞质中。GFP-ER的定位因细胞而异，尽管以相似的方式培养和处理。分析核荧光强度在频率分布上的变化有助于建立具有细胞系和配体特征的定位模式。在本研究过程中，在ER-而不是ER+的人乳腺癌上皮细胞系中观察到GFP-ER定位于核仁区。最后，我们的工作提供了一个“未被占据的”和配体结合的受体的可视化描述，并在配体在调节受体活性的作用的背景下进行了讨论。
The human estrogen receptor ct (ER ct) has been tagged at its amino terminus with the S65T variant of the green fluorescent protein (GFP), allowing subcellular trafficking and localization to be observed in living cells by fluorescence microscopy. The tagged receptor, GFP-ER, is functional as a ligand-dependent transcription factor, responds to both agonist and antagonist ligands, and can associate with the nuclear matrix. Its cellular localization was analyzed in four human breast cancer epithelial cell lines, two ER+ (MCF7 and T47D) and two ER- (MDA-MB-231 and MDA-MB-435A), under a variety of ligand conditions. In all cell lines, GFP-ER is observed only in the nucleus in the absence of ligand. Upon the addition of agonist or antagonist ligand, a dramatic redistribution of GFP-ER from a reticular to punctate pattern occurs within the nucleus. In addition, the full antagonist ICI 182780 alters the nucleocytoplasmic compartmentalization of the receptor and causes partial accumulation in the cytoplasm in a process requiring continued protein synthesis. GFP-ER localization varies between cells, despite being cultured and treated in a similar manner. Analysis of the nuclear fluorescence intensity for variation in its frequency distribution helped establish localization patterns characteristic of cell line and ligand. During the course of this study, localization of GFP-ER to the nucleolar region is observed for ER- but not ER+ human breast cancer epithelial cell lines. Finally, our work provides a visual description of the "unoccupied" and ligand-bound receptor and is discussed in the context of the role of ligand in modulating receptor activity.