STEROID RECEPTOR TRANSLOCATION AND HEAT SHOCK

这项建议的长远目标，是研究 糖皮质激素引起其受体从一种 非活性状态转变为可以主动调节特定基因表达的状态。 拟议的研究是由两种方法。 在第一，一个 建立了检测糖皮质激素受体的实验系统 (GR)从细胞质到细胞核的运输。 在第二 细胞热休克反应与糖皮质激素受体的关系 功能将被研究。 在我和威廉博士的博士后任期内， Pratt，我们发现GR与90-kDa的热休克蛋白存在于一个复合物中， 蛋白质（J.Biol.Chem. 260：13810，1985和J. Biol. Chem. 260：12398，1985）， Hsp 9 O从GR上的依赖性解离先于Hsp 9 O的转化， GR到DNA-状态（J.Biol.Chem. 262：6986，1987），并且缺失一个 GR内的一个短的保守区域将导致hsp 9 O结合的丧失 （J.Biol.Chem.265：12778，1990）。 我们的报告表明，完整细胞内的hsp 9 O 可以与微管结合（Mol.Endocrinol. 2：756，1988和Euro.J.Cell Biol. 58：66，1989），表明细胞元件在GR运动中的作用。 在该提议中，描述了一种系统，通过该系统，完整细胞中的GR被 在激素的作用下慢慢进入细胞核 受体相关蛋白参与：1）转运至细胞核，2） 运输通过核孔，和3）本地化的GR到其 特定基因转录的最终位点。 我们已经证明 CHO细胞中转染的小鼠GR定位于细胞核， GR与hsp 7 O结合，而L细胞的GR是胞浆型的， 与hsp 7 O相关（J.Biol.Chem. 265：20123，1990）。 建议的GR 转运系统可用于确定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复合物含有一个56 kDa的独特的蛋白质， 氨基酸序列（Biochemistry 29：5145，1990）。 自从来到MCO，我 已经表明这种蛋白质也是一种新的热休克蛋白，hsp 56（J. Biol. 265：22067，1990）。 这让我推测， 应激反应和糖皮质激素作用在功能上相关。 在给《自然》杂志的一封信中，我表明，热休克处理L 细胞将诱导未配体GR的核定位。 试图扩大对细胞和分子的意义， 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.