Structure and Functions of Steroid Hormone Receptors

描述(申请人提供)：本研究的长期目标是揭示糖皮质激素受体(GR)和盐皮质激素受体(MR)介导的激素作用的结构机制。GR和MR是类固醇激素受体家族的成员，调节从免疫/炎症反应到代谢动态平衡和血压控制等广泛的人体生理过程。重要的是，GR和MR都是公认的药物靶点，它们的一些配体目前被用于治疗包括炎症和高血压在内的各种疾病。GR和MR通过激素结合到其C末端配体结合域(LBD)和将目的基因的启动子结合到其中间的DMA结合结构域(DBD)来调节它们的作用。这两种受体都含有一个有效的N-末端激活结构域(AF-1)。直到最近，只有单独的DBD或单独使用单一激动剂(地塞米松)的GR LBD的结构被解决。显然，考虑到GR和MR在正常生理和疾病中的重要性，这种有限的结构信息是一个严重的缺陷。在这项研究中，我们建议通过解决各种GR和MR络合物的晶体结构来填补这一知识空白。我们的具体目标是1)MR LBD的结晶和结构测定，2)GR LBD与科维拉唑的络合物，科维拉唑是一种有效的抗炎配体，与GR结合的亲和力是地塞米松的40倍，3)MR DBD-LBD片段与MR共识DNA结合，以及4)包含GR AF-1结构域及其DBD的GR AF-1-DBD/DNA复合体。针对我们特定目标的假设是，在这些晶体结构中观察到的分子相互作用(蛋白质-蛋白质、蛋白质-DNA和蛋白质-激素)将对于从机制上理解GR和MR的激素作用至关重要。在结构确定后，我们将通过仔细观察和分析结构来确定关键结构元素，并且我们将在定点突变和基于细胞的转录分析方面与Stoney Simons(NIDDK)、Brad Thompson和Raj Kumar(UTMB，Galveston)密切合作，以评估已确定的关键功能的功能意义。意义：本应用中产生的结构信息将显著增强我们对GR和MR激素作用的分子机制的理解，并应成为针对这两种受体治疗哮喘、高血压和心脏病的药物开发的合理模板。

DESCRIPTION (provided by applicant): The long-term objective of this study is to reveal the structural mechanisms of hormone actions mediated by the glucocorticoid receptor (GR) and the mineralococorticoid receptor (MR). GR and MR are members of the steroid hormone receptor family that regulate broad spectrum of human physiology ranging from immune/inflammatory responses to metabolic homeostasis and control of blood pressure. Importantly, both GR and MR are well-established drug targets, with a number of their ligands currently being used to treat diverse diseases including inflammation and hypertension. GR and MR mediate their actions through hormone binding to their C-terminal ligand-binding domain (LBD) and promoter binding of target genes to their middle DMA-binding domain (DBD). Both receptors also contain a potent N-terminal activation domain (AF-1). Until recently, only structures of the isolated DBD or the isolated GR LBD with a single agonist (dexamethasone) had been solved. Clearly, this limited amount of structural information is a serious deficiency considering the importance of GR and MR in normal physiology and in disease. In this study, we propose to fill in that knowledge gap by solving the crystal structures of various GR and MR complexes. Our specific aims are focused on crystallization and structural determination of 1) the MR LBD, 2) the GR LBD in complex with cortivazol, a potent anti-inflammatory ligand that binds to GR with 40-fold higher affinity than dexamethasone, 3) a MR DBD-LBD fragment bound to the MR consensus DNA site, and 4) the GR AF-1-DBD/DNA complex that contains the GR AF-1 domain and its DBD. The hypothesis for our specific aims is that the molecular interactions (protein-protein, protein-DNA, and protein-hormone) observed in these crystal structures will be crucial for mechanistic understanding of the hormone actions of GR and MR. After structural determination, we will identify key structural elements by scrutinizing and analyzing the structures, and we will collaborate closely with Stoney Simons (NIDDK), Brad Thompson, and Raj Kumar (UTMB, Galveston), in site-directed mutagenesis and cell-based transcriptional assays to assess the functional significance of the key features identified. Significance: The structural information generated in this application will significantly enhance our understanding of the molecular mechanisms of hormone actions by GR and MR, and should serve as rational templates for drug discovery that targets these 2 receptors for the treatment of asthma, hypertension, and heart disease.