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 First, hybrid receptors will be constructed in which small regions of the GR hormone binding domain have been replaced by the corresponding sequence from either the progesterone or estrogen receptors.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.
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