Structure:function studies of the glucocorticoid receptor

描述(由申请人提供)：这项正在进行的资助的长期目标是了解糖皮质激素受体(GR)结构和功能之间的关系。GR控制特定基因调控的确切机制尚不清楚。这部分是由于缺乏对GR N端激活功能结构域AF的功能结构的了解。直到最近，还很难确定AF1的结构，因为在溶液中，它以随机的构象系综的形式存在。我们在这项正在进行的资助下的发现第一次给了我们一个机会来确定AF1的功能折叠结构。利用各种生物物理/蛋白质组学技术，我们已经证明：i)天然存在的渗透压可以诱导重组AF1的结构；ii)当DMA结合结构域与其同源糖皮质激素反应元件结合时，AF1通过结构域间信号获得折叠结构。在体外条件下，这种折叠形式的AF1选择性地结合特定的共调节蛋白，这对GR的作用是重要的。因此，我们认为AF1的这种诱导构象是有功能的。然而，对于所有的类固醇受体来说，GR AF1的三维结构是缺乏的。同样未知的是，在HOLO-GR中，AF1结构域是否仍然是非结构化的。根据我们已发表的和新的初步结果，我们假设在生理条件下，AF1由于与其他辅因子蛋白(S)的直接相互作用和/或通过配体/Gre结合传递的分子内信号而获得部分或完全折叠的构象。在这笔赠款的下一个资助期，我们将：1)测试特定的共调控蛋白(S)对AF1结构的相互作用；2)在将其折叠成功能活性形式的条件下确定GR AF1结构域的三维结构；3)研究AF1在全息GR中的结构；4)在体内条件下测试AF1折叠对其与其他共调控蛋白相互作用的影响。成功完成我们的特定目标不仅将提供关于AF1结构和功能的关键知识，还将为GR-及其相关转录因子如何将转录信号从配体传递到特定的靶基因(S)提供重要的新的一般性见解。

DESCRIPTION (provided by applicant): 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).