Biochemistry and Regulation of Cadherin Activity

描述（由申请人提供）：钙粘蛋白介导的粘附调节是许多形态发生过程的基础。了解胚胎中的钙粘蛋白调控对包括出生缺陷在内的许多发育性疾病具有重要意义，并将对再生医学的进步具有重要意义。钙粘蛋白的调节也发生在成人组织中，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，上皮细胞向间质转化（EMT）过程中E-cadherin功能的下调或粘附连接的重塑有助于肿瘤转移，而减缓这一过程的能力可能为癌症治疗提供途径。近年来，人们对细胞过程(如：膜运输，细胞骨架动力学，蛋白质周转)和信号通路影响钙粘蛋白介导的粘附和粘附连接重塑。在另一个研究领域，人们对钙粘蛋白亲同性键的分子结构已经有了大量的了解。本项目的目标是弥合这两个领域之间的差距，并阐明通过这些不同的细胞过程和生长因子信号通路对粘附的调节如何控制钙粘蛋白粘附的结构和活性状态。新工具将是这个项目的关键是钙粘蛋白激活单克隆抗体（mab）和构象选择性mab针对不同的钙粘蛋白结构和功能状态。具体目的为：A.分析细胞表面E-cadherin的活性状态与上皮-间质转化（epithelial- mesenchyal -transition， EMT）过程中E-cadherin内化、粘附连接重构或解体、人上皮肿瘤细胞生长和扩散的关系；B.确定傍轴原钙粘蛋白（PAPC）如何调节早期爪蟾胚胎中c -钙粘蛋白介导的细胞粘附和细胞分选；C.探索与功能状态相关的钙粘蛋白构象和三维结构，包括二聚化和活性状态，由构象选择性和激活单克隆抗体定义；D.分析单克隆抗体和生长因子信号激活下细胞表面钙粘蛋白活性状态的细胞和分子机制；利用高分辨率荧光成像，对钙粘蛋白-连环蛋白复合物进行结构-功能分析，并鉴定与活性状态特异性相关的钙粘蛋白相互作用蛋白。需要验证的一个重要假设是，细胞表面钙粘蛋白活性状态的内向外调节参与了许多与粘附连接重塑和钙粘蛋白下调相关的细胞事件。公共卫生相关性：钙粘蛋白是细胞粘附蛋白，其功能是将组织和器官中的细胞结合在一起，钙粘蛋白功能障碍可导致胚胎发育缺陷以及成人肿瘤生长和转移。我们正试图了解钙粘蛋白分子在细胞表面的开启和关闭方式，以控制它们在组织中的粘附功能。此外，我们正在开发检测和控制细胞上钙粘蛋白功能状态的方法，这些方法有可能用于包括癌症在内的许多疾病的诊断或治疗方法。

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.