GLUCOCORTICOID RECEPTOR--STRUCTURE AND FUNCTION

类固醇和甲状腺激素受体基因家族的成员是配体 激活的转录调节因子。 因此，它们在以下方面发挥着关键作用： 控制分化和发育，在两者的生长中， 正常和转化的细胞以及生物体的反应能力 外部刺激。 糖皮质激素受体（GR）是第一个 类固醇受体被克隆，它已经被深入研究， 一些实验室。 这导致了对整体的理解， 结构域的结构和功能的GR。然而，激素的细节 结合、激活、DNA结合和转录激活是远 从被解决。 该建议的重点是激素结合结构域的 GR，旨在阐明以下问题。 1)激素结合的结构决定因素是什么 网站？ 2)激动剂结合如何激活受体，以及为什么 拮抗剂与受体结合但不能激活它？ 这个问题将从两个方面来处理。 首先，混合受体将 其中GR激素结合结构域的小区域具有 被孕酮或孕酮的相应序列取代， 雌激素受体 将分析这些受体的能力， 结合糖皮质激素、各种糖皮质激素拮抗剂（包括 孕酮）和雌激素。 当绑定发生时， 将受体激活为特异性DNA结合形式激素将 研究了 因此，使用这种被称为同源扫描的技术， 诱变，我们应该能够研究的结构特征， 激素结合域是识别特异性 类固醇和受体激活。 作为一个长期的项目，激素 GR的结合结构域和选择的杂交体将在E.杆菌 以纯化足够的蛋白质用于结构研究。 第二 一种方法是在组织培养中选择含有突变体的细胞， 受体a）变得不依赖激素，或B）需要减少 激素水平，或c）可以被化合物有效地激活， 通常是部分激动剂和/或拮抗剂。 这些受体将 鉴定、cDNA克隆和序列分析。

Members of the steroid and thyroid hormone receptor gene family are ligand activated transcriptional modulators. As such they play a crucial role in the control of differentiation and development, in the growth of both normal and transformed cells and in the ability of an organism to respond to external stimuli. The glucocorticoid receptor (GR) was the first steroid receptor to be cloned and it has since been studied intensively by a number of laboratories. This has led to an understanding of the overall domain structure and function of the GR. However the details of hormone binding, activation, DNA binding and transcriptional activation are far from being solved. This proposal focuses on the hormone binding domain of the GR and is designed to shed light on the following questions. 1) What are the structural determinants of the hormone binding site? 2) How does agonist binding activate the receptor, and why do antagonists bind to the receptor but fail to activate it? The problem will be approached in two ways. First, hybrid receptors will be constructed in which small regions of the GR hormone binding domain have been replaced by the corresponding sequence from either the progesterone or estrogen receptors. These receptors will be analyzed for their ability to bind to glucocorticoids, to various glucocorticoid antagonists (including progesterone) and to estrogen. When binding occurs, the ability of the hormone to activate receptor to a specific DNA binding form will be studied. Thus using this technique, which has been termed homolog-scanning mutagenesis, we should be able to study the structural characteristics of the hormone binding domain which are required for recognition of specific steroids and for receptor activation. As a long term project, the hormone binding domain of the GR and selected hybrids will be expressed in E. coli in order to purify enough protein for structural studies. The second approach is to select for cells in tissue culture that contain mutant receptors that have a) become hormone independent, or b) require reduced levels of hormone, or c) can be activated efficiently by compounds that are usually partial agonists and/or antagonists. These receptors will be characterized, the cDNAs cloned and the sequences analyzed.