Devin-Leclerc J, Meng X, Delahaye F, Leclerc P, Baulieu E E, Catelli M G
Institut National de la Santé et de la Recherche Médicale U33 Lab Hormones, Le Kremlin Bicêtre, France.
Mol Endocrinol. 1998 Jun;12(6):842-54. doi: 10.1210/mend.12.6.0121.
The in vivo interaction of estrogen receptor (ER) and Hsp90, demonstrated in the absence of hormone by a nuclear cotranslocation assay of the cytoplasmic Hsp90 with the karyophilic receptor, was disrupted by agonist and antagonist ligands, which, after dissociating the Hsp90, allowed the chaperone protein to be relocalized in the cytoplasm. The pure antiestrogen RU 58668 (RU), which was unable to stimulate an estrogen-dependent reporter gene and completely inhibited its estradiol-induced activity, also profoundly modified the subcellular distribution of ER in a specific time- and dose-dependent manner; ER appeared as speckled fluorescent clusters mainly located in the perinuclear region of the cytoplasm. The kinetics of appearance and reversal of the RU-dependent ER mislocalization in the presence or absence of cycloheximide demonstrated 1) that this effect was reversed by RU withdrawal or estradiol (E2) treatment, and 2) that cycloheximide with RU inhibited and reversed the ER cytoplasmic mislocalization induced by RU alone. These results point to a protein synthesis-dependent step in the mechanism of action of this antiestrogen. After RU treatment, a large portion of ER was found in the particulate fraction of the cytoplasm. However, confocal and electron microscopic analysis showed that ER clusters were not associated with specific cytoplasmic organelles or compartments. Using ER mutants, it was found that the ligand binding domain was sufficient for RU to produce receptor mislocalization, while the constitutive nuclear localization signals were dispensable. We propose that the antiestrogenic properties of RU are primarily due to the induction of an aggregation-prone receptor conformation that cannot undertake the constitutive and the ligand-induced nuclear localization function of the receptor because it is sequestered in the cytoplasm by fast turning over protein(s). We predict that antiestrogens able to block ER nuclear localization will behave as pure antihormones and will inhibit all the nuclear action of ER elicited by agonistic ligands or by ligand-independent mechanisms such as growth factor stimulation.
通过细胞质热休克蛋白90（Hsp90）与亲核受体的核共转位试验证明，在无激素情况下雌激素受体（ER）与Hsp90的体内相互作用，会被激动剂和拮抗剂配体破坏，这些配体在使Hsp90解离后，使伴侣蛋白重新定位于细胞质中。纯抗雌激素RU 58668（RU）无法刺激雌激素依赖性报告基因，并完全抑制其雌二醇诱导的活性，它还以特定的时间和剂量依赖性方式深刻改变了ER的亚细胞分布；ER呈现为斑点状荧光簇，主要位于细胞质的核周区域。在有或无环己酰亚胺的情况下，RU依赖性ER错误定位的出现和逆转动力学表明：1）这种效应可通过撤去RU或用雌二醇（E2）处理而逆转；2）环己酰亚胺与RU一起可抑制并逆转单独使用RU诱导的ER细胞质错误定位。这些结果表明，在这种抗雌激素的作用机制中存在一个蛋白质合成依赖性步骤。RU处理后，大部分ER存在于细胞质的颗粒部分。然而，共聚焦和电子显微镜分析表明，ER簇与特定的细胞质细胞器或区室无关。利用ER突变体发现，配体结合域足以使RU产生受体错误定位，而组成型核定位信号则是不必要的。我们提出，RU的抗雌激素特性主要是由于诱导了一种易于聚集的受体构象，这种构象无法承担受体的组成型和配体诱导的核定位功能，因为它被快速周转的蛋白质隔离在细胞质中。我们预测，能够阻断ER核定位的抗雌激素将表现为纯抗激素，并将抑制激动剂配体或生长因子刺激等非配体依赖性机制引发的ER的所有核作用。
