Yang J, DeFranco D B
Department of Biological Sciences, University of Pittsburgh, Pennsylvania 15260.
Mol Cell Biol. 1994 Aug;14(8):5088-98. doi: 10.1128/mcb.14.8.5088-5098.1994.
Nuclear import of glucocorticoid receptors (GRs) was analyzed in vitro with digitonin-permeabilized cells (S. A. Adam, R. Sterne-Marr, and L. Gerace, J. Cell Biol. 111:807-816, 1990). Indirect immunofluorescence methods were used to monitor the transport of GRs from rat hepatoma and fibroblast cell cytosol into HeLa nuclei. In vitro nuclear import of GRs was shown to be hormone dependent and to require ATP and incubation at ambient temperatures (i.e., 30 degrees C). Hormone-dependent dissociation of GR-bound proteins, such as the 90-kDa heat shock protein, hsp90, is part of an activation process that is obligatory for the expression of the receptor's DNA-binding activity. Inhibition of in vitro GR activation by Na2MoO4 blocked hormone-dependent nuclear import, demonstrating that receptor activation is required for nuclear import. The addition to GR-containing cytosol of antiserum directed against the cytosolic 70-kDa heat shock protein, hsp70, while effective in blocking the nuclear import of simian virus 40 large tumor antigen (SV40 TAg), did not affect hormone-dependent nuclear import of endogenous, full-length GRs or an exogenously added truncated GR protein (i.e., XGR556) that lacks a hormone-binding domain but possesses a constitutively active nuclear localization signal sequence (NLS). Depletion of hsp70 from HeLa cell cytosol did not affect the nuclear import of exogenously added XGR556 but led to inhibition of SV40 TAg nuclear import. Thus, two closely related NLSs, one contained within GRs and the other contained within SV40 TAg, are distinguished by their differential requirements for hsp70 in vitro.
利用洋地黄皂苷通透细胞在体外分析了糖皮质激素受体（GRs）的核输入（S. A. 亚当、R. 斯特恩 - 马尔和L. 杰拉西，《细胞生物学杂志》111:807 - 816，1990年）。采用间接免疫荧光法监测GRs从大鼠肝癌细胞和成纤维细胞胞质溶胶转运至HeLa细胞核的过程。结果表明，GRs的体外核输入依赖激素，需要ATP，并在环境温度（即30℃）下孵育。GR结合蛋白（如90 kDa热休克蛋白hsp90）的激素依赖性解离是激活过程的一部分，而该激活过程对于受体DNA结合活性的表达是必不可少的。Na2MoO4对体外GR激活的抑制作用阻断了激素依赖性核输入，表明核输入需要受体激活。向含有GR的胞质溶胶中加入针对胞质70 kDa热休克蛋白hsp70的抗血清，虽然能有效阻断猿猴病毒40大肿瘤抗原（SV40 TAg）的核输入，但不影响内源性全长GRs或外源添加的截短GR蛋白（即XGR556）的激素依赖性核输入，XGR556缺乏激素结合结构域，但具有组成型活性核定位信号序列（NLS）。从HeLa细胞胞质溶胶中耗尽hsp70并不影响外源添加的XGR556的核输入，但会导致SV40 TAg核输入受到抑制。因此，两种密切相关的NLS，一种存在于GRs中，另一种存在于SV40 TAg中，在体外对hsp70有不同的需求，以此得以区分。
