STEROID RECEPTOR TRANSLOCATION AND HEAT SHOCK

这项建议的长期目标是研究 糖皮质激素引起其受体从 不活跃状态转变为能主动调节特定基因表达的状态。 这项拟议的研究由两种方法组成。在第一个阶段，一个 将开发检测糖皮质激素受体的实验系统 (GR)从细胞质转运到细胞核。在第二种情况下， 细胞热休克反应与糖皮质激素受体的关系 功能将被研究。在我和威廉博士一起做博士后期间 Pratt，我们证明了GR存在于90 kDa热休克的复合体中 蛋白质(J.Biol.Chem.13810、1985年和J.Biol。化学。260：12398,1985年)， 激素依赖的hsp9O从GR解离在转化之前 GR到DNA状态(J.Biol.Chem.262：6986,1987)，并删除 GR内较短的保守区将导致hsp9O结合的丧失 (J.Biol.Chem.265：12778,1990年)。我们的报告称，完整细胞内的hsp9O 可能与微管结合(Mol.Endocrinol.2：756,1988和Euro.J.Cell 比奥尔。58：66,1989)，提示细胞成分在GR运动中的作用。 在本方案中，描述了一种系统，完整细胞中的GR 对荷尔蒙的反应缓慢地进入细胞核以识别 受体相关蛋白参与：1)转运到细胞核，2) 通过核孔的运输，以及3)GR到ITS的定位 特定基因转录的最终位置。我们已经证明了 在CHO细胞中转基因的小鼠GR定位于 与hsp70有关，而L细胞的GR是胞质的，而不是 与hsp7O相关(J.Biol.Chem.265：20123,1990年)。建议的GR 运输系统可用于确定CHO细胞中的GR 代表易位途径中的中间状态。我们有 结果表明，IM-9细胞的GR复合体含有一种独特的56 kDa蛋白 氨基酸序列(生物化学29：5145,1990)。自从来到MCO以来，我 结果表明，该蛋白也是一种新的热休克蛋白hsp56(J. 比奥尔。化学。265：22067,1990年)。这让我猜测， 应激反应和糖皮质激素的作用在功能上是相关的。 在提交给《自然》杂志的一封信中，我展示了L的热休克治疗 细胞将诱导去连接的GR的核定位。这项建议 试图进一步阐述这一现象的细胞和分子意义 观察。

The long-term objective of this proposal is to study the mechanism by which glucocorticoid hormone causes transformation of its receptor from an inactive state to one which can actively regulate specific gene expression. The proposed research is composed of two approaches. In the first, an experimental system will be developed to examine glucocorticoid receptor (GR) transport from the cytoplasm to the nucleus. In the second, the relationship of the cellular heat shock response to glucocorticoid receptor function will be studied. During my post-doctoral tenure with Dr. William Pratt, we showed that GR exists in a complex with the 90-kDa heat shock protein (J.Biol.Chem. 260:13810, 1985 and J.Biol. Chem. 260:12398, 1985), that hormone-dependent dissociation of hsp9O from GR precedes conversion of GR to the DNA-state (J.Biol.Chem. 262:6986, 1987), and that deletion of a short, conserved region within the GR will cause loss of hsp9O binding (J.Biol.Chem. 265:12778, 1990). Our reports that hsp9O within intact cells may be bound to microtubules (Mol.Endocrinol. 2:756, 1988 and Euro.J.Cell Biol. 58:66, 1989), suggests a role for cellular elements in GR movement. In this proposal, a system is described by which GR in intact cells is slowly driven into the nucleus in response to hormone to identify receptor-associated proteins involved in: 1) transport to the nucleus, 2) transport across nuclear pores, and 3) localization of the GR to its ultimate site of specific gene transcription. We have shown that transfected mouse GR in CHO cells is localized to the nucleus in association with hsp7O, while GR of L cells is cytoplasmic and not associated with hsp7O (J.Biol.Chem. 265:20123, 1990). The proposed GR transport system may serve to determine if the GR in the CHO cells represents an intermediate state in the translocation pathway. We have shown that the GR complex of IM-9 cells contains a 56kDa protein of unique amino acid sequence (Biochemistry 29:5145, 1990). Since arriving at MCO, I have shown that this protein is also a novel heat shock protein, hsp56 (J. Biol. Chem. 265:22067, 1990). This has led me to speculate that the stress response and glucocorticoid hormone action are functionally related. In a letter submitted to Nature, I show that heat shock treatment of L cells will induce nuclear localization of the unliganded GR. This proposal seeks to expand on the cellular and molecular significance of this observation.