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). ? ?
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