Galigniana M D, Scruggs J L, Herrington J, Welsh M J, Carter-Su C, Housley P R, Pratt W B
Department of Pharmacology, The University of Michigan Medical School, Ann Arbor 48109, USA.
Mol Endocrinol. 1998 Dec;12(12):1903-13. doi: 10.1210/mend.12.12.0204.
We use here a chimera of the green fluorescent protein (GFP) and the glucocorticoid receptor (GR) to test the notion that the protein chaperone heat shock protein-90 (hsp90) is required for steroid-dependent translocation of the receptor through the cytoplasm along cytoskeletal tracks. The GFP-GR fusion protein undergoes steroid-mediated translocation from the cytoplasm to the nucleus, where it is transcriptionally active. Treatment of 3T3 cells containing steroid-bound GFP-GR with geldanamycin, a benzoquinone ansamycin that binds to hsp90 and disrupts its function, inhibits dexamethasone-dependent translocation from the cytoplasm to the nucleus. The t1/2 for translocation in the absence of geldanamycin is approximately 5 min, and the t1/2 in the presence of geldanamycin is approximately 45 min. In cells treated for 1 h with the cytoskeletal disrupting agents colcemid, cytochalasin D, and beta,beta'-iminodipropionitrile to completely disrupt the microtubule, microfilament, and intermediate filament networks, respectively, the GFP-GR still translocates rapidly to the nucleus in a strictly dexamethasone-dependent manner but translocation is no longer affected by geldanamycin. After withdrawal of the cytoskeletal disrupting agents for 3 h, normal cytoskeletal architecture is restored, and geldanamycin inhibition of dexamethasone-dependent GFP-GR translocation is restored. We suggest that in cells without an intact cytoskeletal system, the GFP-GR moves through the cytoplasm by diffusion. However, under physiological conditions in which the cytoskeleton is intact, diffusion is limited, and the GFP-GR utilizes a movement machinery that is dependent upon hsp90 chaperone activity. In contrast to the GR, GFP-STAT5B, a signaling protein that is not complexed with hsp90, undergoes GH-dependent translocation to the nucleus in a manner that is not dependent upon hsp90 chaperone activity.
我们在此使用绿色荧光蛋白（GFP）与糖皮质激素受体（GR）的嵌合体，以验证蛋白质伴侣热休克蛋白90（hsp90）对于受体通过细胞质沿细胞骨架轨道进行类固醇依赖性转运是必需的这一观点。GFP-GR融合蛋白会经历类固醇介导的从细胞质到细胞核的转运，在细胞核中它具有转录活性。用格尔德霉素处理含有与类固醇结合的GFP-GR的3T3细胞，格尔德霉素是一种与hsp90结合并破坏其功能的苯醌安莎霉素，它会抑制地塞米松依赖性的从细胞质到细胞核的转运。在没有格尔德霉素的情况下转运的t1/2约为5分钟，而在有格尔德霉素的情况下t1/2约为45分钟。在用细胞骨架破坏剂秋水仙酰胺、细胞松弛素D和β,β'-亚氨基二丙腈分别处理细胞1小时以完全破坏微管、微丝和中间丝网络后，GFP-GR仍以严格的地塞米松依赖性方式迅速转运至细胞核，但转运不再受格尔德霉素影响。在撤去细胞骨架破坏剂3小时后，正常的细胞骨架结构得以恢复，格尔德霉素对地塞米松依赖性GFP-GR转运的抑制作用也得以恢复。我们认为，在没有完整细胞骨架系统的细胞中，GFP-GR通过扩散在细胞质中移动。然而，在细胞骨架完整的生理条件下，扩散受到限制，GFP-GR利用一种依赖于hsp90伴侣活性的移动机制。与GR不同，GFP-STAT5B是一种不与hsp90复合的信号蛋白，它以不依赖于hsp90伴侣活性的方式进行生长激素依赖性的向细胞核的转运。
