Lack of knowledge of the structure of its major transactivation domain (AF1) has hampered full understanding of the mechanism by which the glucocorticoid receptor functions. It is presumed that AF1 interacts directly, or through intermediary proteins, with the "transcription machinery" complex of proteins. This requires surface-to-surface interactions between proteins and the structure to produce them. When expressed separately as a recombinant protein, however, AR displays little or no structure and binds weakly at best with a few such proteins. We have made two observations that offer exciting promise for understanding how AF1 obtains the structure necessary for its presumed function and for discovering its natural binding partners. First, we found that in the presence of an osmolyte, trimethylamine oxide (TMAO), the AF1 of the human GR acquires native-like structure and shows greatly enhanced binding to certain other proteins. We will use TMAO and other osmolytes to study the structure of AF1 and to find its binding partners. Second, we have shown that when a two-domain fragment of the GR containing AF1 and the DNA-binding domain is bound to its cognate DNA binding site, AR seems to acquire structure and be able to bind certain other proteins. In this project, we will determine the protein and DNA parameters that control this event. We will compare the AF1 structure and the protein binding partners uncovered by the two techniques. One of the nuclear proteins shown to bind AF1 under the conditions we employ appears to be a member of the nuclear receptor co- activator family of proteins. Methods used will include circular dichroism, intrinsic fluorescence emission spectroscopy, peptide mapping, thermodynamically rigorous studies of protein:DNA binding NMR and if crystals are obtained, X-ray diffraction.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
3R01DK058829-04S1
Application #
6836899
Study Section
Endocrinology Study Section (END)
Program Officer
Margolis, Ronald N
Project Start
2001-02-15
Project End
2005-09-29
Budget Start
2004-02-01
Budget End
2005-09-29
Support Year
4
Fiscal Year
2004
Total Cost
$98,150
Indirect Cost
Name
University of Texas Medical Br Galveston
Department
Biochemistry
Type
Schools of Medicine
DUNS #
800771149
City
Galveston
State
TX
Country
United States
Zip Code
77555
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 :
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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