We wish to investigate the functional consequences of mutations in the AF1/taul I/enh2 transcription transactivation domain of the human glucocorticoid receptor (GR). We hypothesize that this domain is naturally unfolded. Under DK58829 we are studying aspects of AF1 folding in vitro and are developing mutants that define intra- and extra-AF1 amino acids or regions important for AF1 to gain structure. Exposure to the osmolyte trimethylamine oxide or binding of the GR's DNA-binding domain impart structure to AF1, and when thus structured, it binds several nuclear proteins, including the TATA box binding protein and CBP. The review of the proposal funded as DK58829 criticized the lack of in vivo cellular studies to complement the in vitro work. This request for supplemental funding is in response to that criticism. Under this supplemental funding, we will pursue the Specific Aim of testing in cells the functional consequences of GR mutations that may alter the folding of AF1. We will transfect two types of cells - lymphoid and epithelioid - in which we will compare the transcription-regulating efficacy of mutants affecting AF1 function in both holo GR constructs and those lacking a ligand binding domain. Simple and more complex promoters controlling a reporter gene will be compared in responsiveness. In the lymphoid cells regulation of both induced and repressed endogenous genes will be evaluated, and the bioendpoint of lymphoid apoptosis will be followed. Each mutant will also be tested for folding in vitro, thus the experiments will directly test the correlation between AF1 folding and its functions. With this supplement, our results will provide a comprehensive evaluation of AF1 function, with relevance to both basic biochemical mechanisms and the practical use of hormones and other ligands in medical applications.
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