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Ca2+介导的信号传导机制以及与Ca2+稳态改变相关疾病的分子基础。细胞外Ca2+ ([Ca2+]o)被认为是触发多种细胞过程的第一信使。Ca2+感应受体（CaRs）是一类响应[Ca2+]o变化并激活多种信号通路的受体。通过作为体内[Ca2+]o的“恒温器”，car在[Ca2+]o稳态调节中发挥核心作用，并代表重要的治疗靶点。促进我们对Ca2+在调节CaRs中的作用的理解的一个主要障碍是缺乏关于它们的Ca2+结合位点的位置和这类膜蛋白的结构信息的足够信息。获得天然存在的蛋白质的位点特异性Ca2+结合亲和力受到合作，多位点系统中遇到的复杂性的阻碍。CaR和相关蛋白中Ca2+结合位点的描绘进一步受到结晶条件的限制，由于Ca2+结合亲和力低而导致的快速脱靶率以及相互平衡的多种构象的存在的阻碍。本提案的直接目标是1)探测Ca2+感应受体中的Ca2+结合位点，2)通过将位点特异性和区域特异性Ca2+结合信息与哺乳动物细胞中w.t.受体的生物活性以及这些Ca2+结合位点突变的受体相关联，验证我们对特异性Ca2+结合位点的预测。我们提出的工作结果将对理解Ca2+调节受体进行生物活动的机制产生重大影响。这些拟议的研究将为鉴定CaR中Ca2+结合位点和相关蛋白提供新的方法，从而克服可视化弱结合亲和力Ca2+结合位点所遇到的主要障碍。成功识别Ca2+结合位点并阐明Ca2+如何调节CaR不仅将促进对Ca2+如何作为细胞外信使的理解，而且还将提供与该受体相关的临床疾病的分子基础的见解。我们在设计和工程金属结合位点到任意蛋白质上的成功，也可能导致开发诊断测试和化疗有价值的试剂的新方法。

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.