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.
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