Stable and specific binding of heat shock protein 90 by geldanamycin disrupts glucocorticoid receptor function in intact cells
Stable and specific binding of heat shock protein 90 by geldanamycin disrupts glucocorticoid receptor function in intact cells
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DOI：
10.1210/me.10.6.705
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发表时间：
1996-06-01
影响因子：
--
通讯作者：
Cook, P
中科院分区：
文献类型：
--
作者：
Whitesell, L;Cook, P
糖皮质激素受体（GR）是一种从无糖蛋白条件下生长的细胞中分离出来的大的杂蛋白复合体，其中包括应激蛋白hsp 90（热休克蛋白90）。为了探讨热休克蛋白90的作用，在介导糖皮质激素的作用，我们已经研究了热休克蛋白90的选择性结合剂，格尔德霉素（GA），对GR的结构和功能的影响。使用类固醇反应的报告构建体，我们发现，GA抑制地塞米松依赖的GR在转染细胞的反式激活活性。在分子水平上，GA结合热休克蛋白90，而不是GR，在一个稳定的和特定的方式在完整的细胞。GA处理的细胞并不抑制热休克蛋白90或热休克蛋白70与GR的共沉淀，但最近描述的GR免疫沉淀物中的p23蛋白的完全丢失的结果。这种药物诱导的GR异源蛋白复合物组成的改变与地塞米松结合活性的快速（15-30分钟）非竞争性丧失相关。细胞较长时间暴露于GA（2-8小时），导致GR蛋白的细胞水平显着下降。脉冲追踪数据显示，这种下降是由于GR蛋白稳定性下降，而不是合成速率下降。GA诱导的GR蛋白水平的下降被阻断的细胞与lactacystin，20 S蛋白酶体活性的选择性抑制剂，表明可能参与的泛素-蛋白酶体途径介导GA诱导的GR蛋白丰度的下降。总体而言，这些发现提供了直接的药理学证据表明，热休克蛋白90的功能是必需的，以维持完整细胞中的GR蛋白的结合活性和稳定性，并表明热休克蛋白90的功能可能提供了一个新的目标类固醇激素信号的调制。
When isolated from cells grown under hormone-free conditions, the glucocorticoid receptor (GR) is known to exist as a large heteroprotein complex that contains, among its multiple components, the stress protein hsp90 (heat shock protein 90). To explore hsp90's role in mediating glucocorticoid hormone action, we have examined the effects of a selective hsp90-binding agent, geldanamycin (GA), on GR structure and function. Using a steroid-responsive reporter construct, we found that GA inhibited the dexamethasone-dependent transactivating activity of GR in transfected cells. At the molecular level, GA bound hsp90, but not GR, in a stable and specific manner in intact cells. GA treatment of cells did not inhibit coprecipitation of hsp90 or hsp70 with the GR but did result in a complete loss of the recently described p23 protein from GR immunoprecipitates. This drug-induced alteration in GR heteroprotein complex composition was associated with a rapid (15-30 min), noncompetitive loss of dexamethasone-binding activity. Longer exposures of cells to GA (2-8 h), resulted in a marked decline in the cellular level of GR protein. Pulse-chase data revealed that this decline resulted from a decrease in GR protein stability, not rate of synthesis. GA-induced declines in GR protein level were blocked by cotreatment of cells with lactacystin, a selective inhibitor of 20S proteasome activity, suggesting the possible involvement of the ubiquitin-proteasome pathway in mediating GA-induced decreases in GR protein abundance. Overall, these findings provide direct pharmacological evidence that hsp90 function is required to maintain both the hormone-binding activity and stability of the GR protein in intact cells and suggest that hsp90 function may provide a novel target for the modulation of steroid hormone signaling.