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.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Endocrinology Study Section (END)
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Margolis, Ronald N
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University of Texas Medical Br Galveston
Schools of Medicine
United States
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Kumar, R; Thompson, E B (2012) Folding of the glucocorticoid receptor N-terminal transactivation function: dynamics and regulation. Mol Cell Endocrinol 348:450-6
Kumar, Raj; McEwan, Iain J (2012) Allosteric modulators of steroid hormone receptors: structural dynamics and gene regulation. Endocr Rev 33:271-99
Khan, Shagufta H; Ling, Jun; Kumar, Raj (2011) TBP binding-induced folding of the glucocorticoid receptor AF1 domain facilitates its interaction with steroid receptor coactivator-1. PLoS One 6:e21939
Garza, Anna S; Khan, Shagufta H; Moure, Carmen M et al. (2011) Binding-folding induced regulation of AF1 transactivation domain of the glucocorticoid receptor by a cofactor that binds to its DNA binding domain. PLoS One 6:e25875
Khan, Shagufta H; Arnott, John A; Kumar, Raj (2011) Naturally occurring osmolyte, trehalose induces functional conformation in an intrinsically disordered activation domain of glucocorticoid receptor. PLoS One 6:e19689
Garza, Anna M S; Khan, Shagufta H; Kumar, Raj (2010) Site-specific phosphorylation induces functionally active conformation in the intrinsically disordered N-terminal activation function (AF1) domain of the glucocorticoid receptor. Mol Cell Biol 30:220-30
Kumar, Raj; Litwack, Gerald (2009) Structural and functional relationships of the steroid hormone receptors' N-terminal transactivation domain. Steroids 74:877-83
Kumar, Raj (2008) Osmolyte-induced folding of an intrinsically disordered activation function subdomain of glucocorticoid receptor. J Recept Signal Transduct Res 28:465-74
Kumar, R; Serrette, J M; Khan, S H et al. (2007) Effects of different osmolytes on the induced folding of the N-terminal activation domain (AF1) of the glucocorticoid receptor. Arch Biochem Biophys 465:452-60
Copik, Alicja J; Webb, M Scott; Miller, Aaron L et al. (2006) Activation function 1 of glucocorticoid receptor binds TATA-binding protein in vitro and in vivo. Mol Endocrinol 20:1218-30

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