Biochemistry and Regulation of Cadherin Activity

描述(由申请人提供)：钙粘附素介导的黏附调节是许多形态发生过程的基础。了解胚胎中钙粘附素的调控对包括出生缺陷在内的许多发育疾病具有重要意义，对于再生医学的进步也将是重要的。钙粘附素的调节也存在于成人组织中，并参与许多病理生理过程，包括上皮运输、炎症和血管重塑的改变。特别是，在上皮向间充质转化(EMT)过程中，E-钙粘附素功能下调或黏附连接重塑有助于肿瘤转移，减缓这一过程的能力可能为癌症治疗提供新的途径。近年来，人们对细胞过程有了很多了解(例如。膜运输、细胞骨架动力学、蛋白质周转)和影响钙粘附素介导的黏附和黏附连接重塑的信号通路。在另一个不同的研究领域，人们已经了解了大量关于钙粘蛋白高亲和键的分子结构。该项目的目标是弥合这两个区域之间的差距，并阐明这些不同的细胞过程和生长因子信号通路对粘附性的调节如何控制钙粘附素粘附键的结构和活性状态。新的工具将是该项目的关键是钙粘蛋白激活的单抗(MAbbs)和构象选择性单抗针对不同的钙粘蛋白结构和功能状态。其具体目的是：A.分析细胞表面E-钙粘蛋白的活性状态与E-钙粘蛋白内化的关系，粘着上皮-间充质-转化(EMT)过程中的连接重构或解体，以及人上皮性肿瘤细胞的生长和扩散；B.确定旁轴原钙粘附素(PAPC)如何调节C-钙粘附素介导的细胞黏附和细胞分选；C.探索钙粘附素的构象和与功能状态相关的三维结构，包括构象选择性和激活单抗所定义的二聚化和活性状态；D.分析控制细胞表面钙粘素活性状态的细胞和分子机制，以响应激活的单抗和生长因子信号；使用高分辨率荧光成像，分析钙粘附素-连环蛋白复合体的结构-功能分析，并鉴定与活性状态特定相关的钙粘附素相互作用蛋白。一个需要检验的重要假说是，细胞表面钙粘附素活性状态的内向外调节参与了许多与黏附连接重塑和钙粘附素下调相关的细胞事件。公共卫生相关性：钙粘附素是一种细胞黏附蛋白，其功能是在组织和器官中将细胞聚集在一起，钙粘附素功能障碍可导致胚胎发育缺陷以及成年肿瘤的生长和转移。我们正试图了解钙粘附素蛋白分子在细胞表面开启和关闭的方式，以控制它们在组织中的黏附功能。此外，我们正在开发检测和控制细胞上钙粘附素功能状态的方法，这些方法有可能被用作许多疾病的诊断或治疗方法，包括癌症。

DESCRIPTION (provided by applicant): The regulation of cadherin-mediated adhesion underlies many morphogenetic processes. Understanding cadherin regulation in embryos has important implications for many developmental diseases, including birth defects, and it will be important for advances in regenerative medicine. The regulation of cadherins also occurs in adult tissues and is involved in many pathophysiological processes including alterations in epithelial transport, inflammation, and vascular remodeling. In particular, downregulation of E-cadherin function or remodeling of adherens junction during the epithelial to mesenchymal transition (EMT) contributes to tumor metastasis, and the ability to slow this process could provide approaches for cancer therapy. Much has been learned in recent years about the cellular processes (eg. membrane trafficking, cytoskeletal dynamics, protein turnover) and signaling pathways that impact on cadherin-mediated adhesion and adherens junction remodeling. In a different area of investigation, a great deal has been learned about the molecular structure of the cadherin homophilic bond. The goals of this proposed project are to bridge the gap between these two areas and elucidate how regulation of adhesion by these various cellular processes and growth factor signaling pathways controls the structure and activity state of the cadherin adhesive bond. Novel tools that will be key to this project are cadherin activating monoclonal antibodies (mAbs) and conformation selective mAbs for different cadherin structural and functional states. The specific aims are: A. Analyze the relationship between the activity state of the E-cadherin at the cell surface and E-cadherin internalization, adherens junction remodeling or disassembly during the epithelial-mesenchymal-transition (EMT), and the growth and spread of human epithelial tumor cells; B. Determine how Paraxial Protocadherin (PAPC) regulates C-cadherin- mediated cell adhesion and cell sorting in the early Xenopus embryo; C. Explore cadherin conformations and three-dimensional structures associated with functional states, including dimerization and activity state, as defined by conformation selective and activating mAbs; D. Analyze cellular and molecular mechanisms controlling cadherin activity states at the cell surface in response to activating monoclonal antibodies and growth factor signaling; using high resolution fluorescence imaging, a structure-function analysis of the cadherin-catenin complex, and the identification of cadherin interacting proteins specifically associated with activity state. An important hypothesis to be tested is that inside-out regulation of the activity state of cadherins at the cell surface participates in many of the cellular events associated with adherens junction remodeling and cadherin downregulation. PUBLIC HEALTH RELEVANCE: Cadherins are cell adhesion proteins, which function to hold cells together in tissues and organs, and cadherin dysfunction can lead to development defects in embryos as well as tumor growth and metastasis in adults. We are trying to understand the way that cadherin protein molecules are switched on and off at the surface of the cell to control their adhesive functions in tissues. Moreover, we are developing methods to detect and control the functional state of cadherins on the cell, which have the potential to be used as diagnostic or therapeutic approaches for the treatment of many diseases, including cancer.