GLUCOCORTICOID ACTION IN SYNCHRONIZED CELLS

该项目的目标是阐明细胞机制， 控制糖皮质激素受体基因的表达和功能， 蛋白 具体来说，我们将把研究重点放在分子水平上， 糖皮质激素的同源下调机制 受体（GR）的糖皮质激素或拮抗剂，以及相关的过程， 受体再循环 虽然类固醇和其他激素的下调 受体对配体的反应是一种常见的生物学过程， 被认为是细胞适应机制的组成部分， 这一假设的直接证据很少。 在本申请中，我们寻求 基金来检验糖皮质激素的下调 糖皮质激素受体是一个关键组成部分的过程中， 减弱体内类固醇反应。 为了验证这一假设，我们建议 遗传学研究描述糖皮质激素和拮抗剂（RU486） 控制糖皮质激素受体基因的表达， 受体蛋白的代谢。 了解这些机制将 允许我们通过基因工程“抗下调” 糖皮质激素受体基因和蛋白质，并评估它们在 vivo. 为实现这些目标，提出了以下具体目标： 1)为了定义和功能评估新的“基因内下调”， 在hGR cDNA内的“调节元件”。 2)为了确定贡献 糖皮质激素受体基因启动子和3'端非翻译区的转录水平， 同源下调。 3)评价糖皮质激素的疗效 对GR mRNA稳定性和翻译成功能性受体的影响。 4)到 设计下调缺陷型hGR基因并确定hGR的作用 在激素反应减弱中mRNA下调。 5)到 结合受体突变和免疫组化分析， 配体对核摄取、滞留和再循环影响 糖皮质激素受体 6)为了确定分子信号， 糖皮质激素受体的核出口。 7)来描述 糖皮质激素受体被降解， 蛋白质，研究它们的功能。 总之，这些研究应该 显著提高了我们对GR基因表达和蛋白质 代谢，并确定下调的作用， 信号转导

The goal of this project is to elucidate the cellular mechanisms that control the expression and function of the glucocorticoid receptor gene and protein. Specifically, we will focus our studies on the molecular mechanisms involved in homologous down regulation of glucocorticoid receptors (GR) by glucocorticoid or antagonists, and the related process of receptor recycling. Although down regulation of steroid and other receptors in response to ligands is a common biological process which is thought to be a component of the cellular adaptive mechanisms, there is little direct proof of this supposition. In this application, we seek funds to test the hypothesis that the down regulation of glucocorticoid receptors by glucocorticoids is a critical component in the process of attenuating steroid responses in vivo. To test this hypothesis, we propose genetic studies to delineate how glucocorticoids and antagonists (RU486) control the expression of the glucocorticoid receptor gene and the metabolism of the receptor protein. Knowledge of these mechanisms will permit us to genetically engineer "down regulation resistant" glucocorticoid receptor genes and proteins and evaluate their function in vivo. The following specific aims are proposed to accomplish these goals: 1) To define and functionally evaluate the novel "intragenic down regulatory elements" within the hGR cDNA. 2) To determine the contribution of the glucocorticoid receptor gene promoter and 3' untranslated region in homologous down regulation. 3) To evaluate the effect of glucocorticoids on GR mRNA stability and translation into a functional receptor. 4) To engineer a down regulation deficient hGR gene and determine the role of hGR mRNA down regulation in the attenuation of hormone response. 5) To analyze, by combining receptor mutagenesis and immunohistochemistry, the influence of ligand on a nuclear uptake, retention and recycling of glucocorticoid receptors. 6) To define the molecular signals necessary for nuclear exit of glucocorticoid receptors. 7) To delineate how glucocorticoid receptors are degraded, engineer degradation resistant GR proteins and study their function. Together, these studies should significantly enhance our knowledge of GR gene expression and protein metabolism and determine the role of down regulation in hormone-dependent signal transduction.