Glucocorticoid receptor binding to rat liver nuclei occurs without nuclear transport.
Glucocorticoid receptor binding to rat liver nuclei occurs without nuclear transport.
复制标题
糖皮质激素受体与大鼠肝细胞核的结合发生在没有核转运的情况下。
DOI：
10.1016/0960-0760(93)90220-q
复制
发表时间：
1993
期刊：
影响因子：
--
通讯作者：
SimonsJr,SS
中科院分区：
文献类型：
--
作者：
Miyashita,Y;Miller,M;Yen,PM;Harmon,JM;Hanover,JA;SimonsJr,SS
已经在支持或不支持通过核孔进行大分子核转位的条件下检查了来自 HTC 细胞的无细胞活化糖皮质激素受体-类固醇复合物与 HTC 和大鼠肝细胞核的各种制剂的结合。据我们所知，这是首次针对具有功能活性的分离核进行此类研究。传统制备的 HTC 核被发现是多孔的，这是因为它们在 4°C 下无法排除荧光大分子藻红蛋白 (PE)。因此，活化复合物与这些核的核结合不能涉及核转位。使用从大鼠肝脏制备的密封核的既定条件进行的进一步研究表明，含有共价连接的真实核转位序列的 PE 的核转位可以在 22°C 下获得，但不能在 4°C 下获得。然而，在相同条件下，活化的糖皮质激素复合物在两种温度下均表现出相同水平的核结合。因此，我们得出结论，当前完整大鼠肝细胞核的易位条件不足以重现在完整细胞中观察到的糖皮质激素复合物的核运输。在完整的大鼠肝细胞核中观察到的核结合不受金精三羧酸的影响，金精三羧酸选择性地抑制蛋白质-核酸相互作用。抗体 AP-64 对大鼠糖皮质激素受体核易位序列的氨基酸 506-514 具有特异性，它显然是通过阻断受体进入核膜来抑制活化复合物的核结合。总的来说，这些数据表明，与能够进行核易位的核结合的活化复合物仅涉及与核膜成分（例如核孔）的关联。因此，该系统和这些试剂可能有助于未来研究活化复合物与核孔的结合。
The binding of cell-free activated glucocorticoid receptor-steroid complexes from HTC cells to various preparations of HTC and rat liver nuclei has been examined under conditions that did or did not support the nuclear translocation of macromolecules via nuclear pores. To the best of our knowledge, this is the first such study with functionally active isolated nuclei. Conventionally prepared HTC nuclei were found to be porous, as determined from their inability to exclude the fluorescent macromolecule phycoerythrin (PE) at 4°C. Thus the nuclear binding of activated complexes to these nuclei can not involve nuclear translocation. Further studies, using established conditions with sealed nuclei prepared from rat liver, revealed that nuclear translocation of PE containing a covalently linked, authentic nuclear translocation sequence could be obtained at 22°C, but not at 4°C. However, under the same conditions, activated glucocorticoid complexes displayed equal levels of nuclear binding at both temperatures. We therefore conclude that the current translocation conditions with intact rat liver nuclei are not sufficient to reproduce the nuclear transport of glucocorticoid complexes observed in intact cells. The nuclear binding that was seen with intact rat liver nuclei was not affected by aurintricarboxylic acid, which selectively inhibits protein-nucleic acid interactions. The antibody AP-64, shown to be specific for amino acids 506–514 of the nuclear translocation sequence of the rat glucocorticoid receptor, inhibited the nuclear binding of activated complexes, apparently by blocking receptor access to the nuclear membrane. Collectively, these data argue that activated complex binding to nuclei capable of nuclear translocation involves only an association with nuclear membrane components such as nuclear pores. Thus this system, and these reagents, may be useful in future studies of activated complex binding to nuclear pores.