Structural Biology of receptor-mediated extracellular calcium signaling

描述(由申请人提供)：本研究项目的长期目标是了解钙离子介导的信号转导的机制，以及与钙离子稳态改变相关的疾病的分子基础。细胞外钙([Ca~(2+)]_o)被认为是触发多种细胞过程的第一信使。钙敏感受体(CARS)是一类对细胞内钙离子变化作出反应并激活多条信号通路的受体。CARS作为人体内[Ca~(2+)]_o的“恒温器”，在[Ca~(2+)]_o的稳态调节中起着核心作用，是重要的治疗靶点。进一步了解钙离子在调节CARS中的作用的一个主要障碍是缺乏关于它们的钙结合位点的位置和这类膜蛋白的结构信息的足够信息。在合作的多位点系统中遇到的复杂性阻碍了获得自然产生的蛋白质的特定位点的钙结合亲和力。CAR和相关蛋白质中钙结合位点的描述进一步受到结晶条件的限制、低钙结合亲和力导致的快速下降速度以及相互平衡的多重构象的存在的阻碍。本建议的近期目标是1)探索钙离子感受器中的钙结合位点，2)通过将特定部位和结构域的钙结合信息与W.T的生物活性相关联来验证我们对特定钙结合位点的预测。哺乳动物细胞中的受体以及在这些钙结合部位发生突变的受体。我们提出的工作结果将对理解钙调节受体进行生物学活动的机制产生重大影响。这些研究将为识别CAR和相关蛋白质中的钙结合位点提供新的方法，从而克服在可视化弱结合亲和力的钙结合位点方面遇到的主要障碍。成功识别钙结合部位并阐明钙是如何调控CAR的，不仅有助于理解钙作为细胞外信使的功能，还将为与该受体相关的临床疾病的分子基础提供深入的见解。我们成功地将金属结合部位设计和设计成任意蛋白质，也可能为诊断测试和化疗开发有价值的试剂带来新的方法。

DESCRIPTION (provided by applicant): The long-term goal of this research project is to understand the mechanisms underlying Ca2+-mediated signaling and the molecular basis for diseases associated with alterations in Ca2+ homeostasis. Extracellular Ca2+ ([Ca2+]o) has been proposed to function as a first messenger to trigger diverse cellular processes. Ca2+-sensing receptors (CaRs) represent a class of receptors that respond to changes in [Ca2+]o and activate multiple signaling pathways. By serving as the body's "thermostats" for [Ca2+]o, CaRs play a central role in the regulation of [Ca2+]o homeostasis and represent important therapeutic targets. A major barrier to advancing our understanding of the role of Ca2+ in regulating CaRs is the lack of adequate information about the location of their Ca2+-binding sites and the structural information of this class of membrane proteins. Obtaining site-specific Ca2+-binding affinities of naturally-occurring proteins is hampered by the complexities encountered in cooperative, multi-site systems. The delineation of the Ca2+-binding sites in the CaR and related proteins is further hindered by limitations of crystallization conditions, rapid off-rates owing to low Ca2+-binding affinities and the existence of multiple conformations that are in equilibrium with one another.The immediate goals of this proposal are to 1) probe Ca2+-binding sites in the Ca2+-sensing receptors and 2) verify our prediction of specific Ca2+-binding sites by correlating the site-specific and domain-specific Ca2+-binding information with the biological activity of the w.t. receptor in mammalian cells as well as receptors with mutations in these Ca2+-binding sites. Results from our proposed work will have a major impact on the understanding of the mechanisms underlying the biological activities carried out by Ca2+-modulated receptors. These proposed investigations will provide novel methods for identifying Ca2+- binding sites in the CaR and related proteins, thus overcoming the major obstacles encountered in visualizing Ca2+-binding sites with weak binding affinities. Success in identifying Ca2+-binding sites and clarifying how Ca2+ regulates the CaR will not only promote an understanding of how Ca2+ functions as an extracellular messenger, but will also provide insights into the molecular basis of the clinical disorders associated with this receptor. Our success in designing and engineering metal-binding sites into arbitrary proteins could also lead to new ways of developing valuable reagents for diagnostic tests and chemotherapy.