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 激活的转录调节器。因此，它们在 控制分化和发展，在两者的成长中 正常和转化的细胞以及有机体的反应能力 对外界刺激的反应。糖皮质激素受体(GR)是第一个 类固醇受体将被克隆，自那以来，它已被广泛研究 一些实验室。这导致了对总体上的 GR的领域结构和功能。然而，激素的细节 结合、激活、DNA结合和转录激活 不会被解决。这项建议集中在荷尔蒙结合区域 GR和旨在阐明以下问题。 1)激素结合部位的结构决定因素是什么？ 2)激动剂结合是如何激活受体的，为什么？ 拮抗剂能与受体结合却不能激活它吗？ 这个问题将通过两种方式解决。首先，混合受体将 其中GR激素结合域的小区域具有 被来自黄体酮或黄体酮的相应序列所取代 雌激素受体。这些受体将被分析为它们的能力 与糖皮质激素、各种糖皮质激素拮抗剂(包括 黄体酮)和雌激素。当绑定发生时， 激素激活受体以特定的DNA结合形式将 学习。因此使用这种被称为同源扫描的技术 突变后，我们应该能够研究其结构特征 识别特定基因所需的激素结合域 类固醇和受体激活。作为一个长期项目，荷尔蒙 GR的结合域和所选的杂交体将在大肠杆菌中表达 以提纯足够的蛋白质用于结构研究。第二 方法是在组织培养中选择含有突变的细胞 受体a)变得不依赖激素，或b)需要减少 激素水平，或c)可以被以下化合物有效地激活 通常是部分激动剂和/或拮抗剂。这些受体将是 对其进行了鉴定、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 First, hybrid receptors will be constructed in which small regions of the GR hormone binding domain have been replaced by the corresponding sequence from either the progesterone or estrogen receptors.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.