Structural studies of human extracellular calcium-sensing receptor

 描述(申请人提供)：人细胞外钙敏感受体(CaSR)是一种G蛋白偶联受体，通过调节甲状旁腺激素的分泌来维持钙稳态。CaSR也在与钙平衡无关的生物过程中发挥重要作用，如胎儿发育。CaSR基因的异常与一系列的钙稳态紊乱有关，包括潜在威胁生命的高钙血症。CaSR的表达变化也与其他疾病有关，包括癌症和阿尔茨海默病。Cinacalcet是CaSR的变构调节剂，临床上用于治疗骨和矿物质代谢障碍。因此，阐明CaSR的结构有助于设计有价值的治疗药物。CaSR作为二硫键连接的同源二聚体发挥作用。它以一种配体特异性的方式激活一系列不同的信号通路。除了其主要的激动剂细胞外钙离子外，CaSR还对多种激动剂做出反应，其活性受正负变构调节剂的调节。该建议的第一部分旨在了解CASR在保持结合亲和力和特异性的同时识别结构上不同的配体的机制。我们建议解决CaSR胞外区域与不同激动剂和变构调节剂结合的晶体结构。第二个目的是基于CaSR胞外结构域在静息和活动状态下的结构，识别与受体激活相关的构象变化。我们计划测量基于这些结构设计的突变的功能效应，以探测配体结合位点和同源二聚体界面。本研究的第三部分是通过解决全长受体及其与下游信号分子的复合体的结构来阐明CaSR的信号转导机制。结合CaSR的结构和功能分析，将有助于我们对细胞外钙感应的分子基础和GPCR二聚体的一般激活机制的理解。

 DESCRIPTION (provided by applicant): Human extracellular calcium-sensing receptor (CaSR) is a G-protein coupled receptor that maintains Ca2+ homeostasis through the regulation of parathyroid hormone secretion. CaSR also plays important roles in biological processes unrelated to Ca2+ balance such as fetal development. Abnormalities in the CaSR gene are associated with a vareity of Ca2+ homeostatic disorders including potentially life threatening hypercalcaemic conditions. Altered expression of CaSR has also been implicated in other diseases including cancer and Alzheimer's disease. Cinacalcet, an allosteric modulator of CaSR, is used clinically to treat disorders of bone and mineral metabolism. Therefore, elucidating the structure of CaSR may assist the design of valuable therapeutic agents. CaSR functions as a disulfide-tethered homodimer. It activates a diverse array of signaling pathways in a ligand-specific manner. In addition to its principal agonist, extracellular Ca2+, CaSR responds to a variety of agonists, and its activity is regulated by postive and negative allosteric modulators. The first part of this proposal is aimed at understanding the mechanisms by which CaSR recognizes structurally distinct ligands while maintaining binding affinity and specificity. We propose to solve the crystal structures of CaSR ectodomain bound to different agonists and allosteric modulators. The second aim is to identify the conformational changes associated with receptor activation based on the structures of CaSR ectodomain in the resting and active states. We plan to measure the functional effects of mutations that are designed based on these structures to probe the ligand binding sites and homodimer interface. The third part of the proposed research is to elucidate the signal transduction mechanism of CaSR by solving the structures of full-length receptor and its complexes with downstream signaling molecules. A combination of structural and functional analysis of CaSR will advance our understanding of the molecular basis of extracellular Ca2+ sensing and the general activation mechanisms of GPCR dimers.