Tyagi R K, Amazit L, Lescop P, Milgrom E, Guiochon-Mantel A
Hormones et Reproduction, INSERM U135, Faculté de Médecine Paris-Sud, Le Kremlin-Bicêtre, France.
Mol Endocrinol. 1998 Nov;12(11):1684-95. doi: 10.1210/mend.12.11.0197.
Steroid hormone receptors are, in most cases, mainly nuclear proteins that undergo a continuous nucleocytoplasmic shuttling. The mechanism of the nuclear export of these proteins remains largely unknown. To approach this problem experimentally in vivo, we have prepared cell lines permanently coexpressing the wild-type nuclear progesterone receptor (PR) and a cytoplasmic receptor mutant deleted of its nuclear localization signal (NLS) [(deltaNLS)PR]. Each receptor species was deleted from the epitope recognized by a specific monoclonal antibody, thus allowing separated observation of the two receptor forms in the same cells. Administration of hormone provoked formation of heterodimers during nucleocytoplasmic shuttling and import of (deltaNLS)PR into the nucleus. Washing out of the hormone allowed us to follow the export of (deltaNLS)PR into the cytoplasm. Microinjection of BSA coupled to a NLS inhibited the export of (deltaNLS)PR. On the contrary, microinjection of BSA coupled to a nuclear export signal (NES) was without effect. Moreover, leptomycin B, which inhibits NES-mediated export, was also without effect. tsBN2 cells contain a thermosensitive RCC1 protein (Ran GTP exchange protein). At the nonpermissive temperature, the nuclear export of (deltaNLS)PR could be observed, whereas the export of NES-BSA was suppressed. Microinjection of GTPgammaS confirmed that the export of (deltaNLS)PR was not dependent on GTP hydrolysis. These experiments show that the nuclear export of PR is not NES mediated but probably involves the NLS. It does not involve Ran GTP, and it is not dependent on the hydrolysis of GTP. The nucleocytoplasmic shuttling of steroid hormone receptors thus appears to utilize mechanisms different from those previously described for some viral, regulatory, and heterogeneous ribonuclear proteins.
在大多数情况下，类固醇激素受体主要是核蛋白，它们在细胞核与细胞质之间持续穿梭。这些蛋白的核输出机制在很大程度上仍不清楚。为了在体内通过实验解决这个问题，我们制备了稳定共表达野生型核孕酮受体（PR）和缺失其核定位信号（NLS）的细胞质受体突变体[（δNLS）PR]的细胞系。每种受体都从一种特异性单克隆抗体识别的表位中缺失，从而可以在同一细胞中分别观察两种受体形式。激素处理促使在核质穿梭过程中形成异二聚体，并使（δNLS）PR导入细胞核。洗去激素后，我们能够追踪（δNLS）PR向细胞质的输出。显微注射与NLS偶联的牛血清白蛋白（BSA）可抑制（δNLS）PR的输出。相反，显微注射与核输出信号（NES）偶联的BSA则没有效果。此外，抑制NES介导的输出的雷帕霉素B也没有效果。tsBN2细胞含有一种温度敏感的RCC1蛋白（Ran GTP交换蛋白）。在非允许温度下，可以观察到（δNLS）PR的核输出，而NES-BSA的输出则受到抑制。显微注射GTPγS证实（δNLS）PR的输出不依赖于GTP水解。这些实验表明，PR的核输出不是由NES介导的，而是可能涉及NLS。它不涉及Ran GTP，也不依赖于GTP水解。因此，类固醇激素受体的核质穿梭似乎利用了与先前描述的某些病毒、调节和异质性核糖核蛋白不同的机制。
