The long-term goal of this ongoing grant is to understand the relationship between structure and function of the glucocorticoid receptor (GR). The precise mechanism by which the GR controls regulation of specific genes is not known. This is in part due to lack of knowledge of the functional structure of GR's N-terminal activation function domain AF. Until recently, it was difficult to determine AF1 structure because in solution it exists as a random ensemble of conformers. Our findings under this ongoing grant have for the first time given an opportunity to determine functional folded AF1 structure. Using a variety of biophysical/proteomics techniques, we have shown that: i) naturally occurring osmolytes can induce structure in recombinant AF1; and ii) AF1 acquires folded structure through inter-domain signaling when the DMA-binding domain binds to its cognate glucocorticoid response element. Under in vitro conditions, this folded form of AF1 selectively binds specific coregulatory proteins, known to be important for GR's action. We therefore believe that this induced conformation in AF1 is functional. However, as for all the steroid receptors, the 3-D structure of the GR AF1 is lacking. Also unknown is whether in the holo-GR the AF1 domain is still unstructured. Based on our published and new preliminary results, we hypothesize that under physiological conditions, AF1 acquires partial or fully folded conformation due to its direct interaction with other cofactor protein(s), and/or intra-molecular signals passed by ligand/GRE binding. During the next funding period of this grant, we will: 1) test the effects of interactions of specific coregulatory protein(s) on the structure of AF1; 2) determine the 3-D structure of the GR AF1 domain under conditions that fold it into a functionally active form; 3) examine the structure of AF1 in the holo-GR; 4) test the effect of AF1 folding on its interaction with other coregulatory proteins under in vivo conditions. Successfully completing our Specific Aims will not only provide pivotal knowledge on the structure and functions of AF1, but will also give important new general insights into how the GR--and related transcription factors--transmit the transcriptional signal from ligand to specific target gene(s). ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
2R01DK058829-05A1
Application #
7033647
Study Section
Molecular and Cellular Endocrinology Study Section (MCE)
Program Officer
Margolis, Ronald N
Project Start
2001-02-15
Project End
2008-07-31
Budget Start
2005-09-30
Budget End
2006-07-31
Support Year
5
Fiscal Year
2005
Total Cost
$271,800
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 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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