BIOCHEMISTRY AND REGULATION OF CADHERIN ACTIVITY

钙粘附素细胞黏附分子在血管生成中起着非常重要的作用。 固体组织的发展和维护。钙粘附素的调节 细胞表面的黏附活性是一种重要的机制 控制组织形态发生。钙粘蛋白胞浆尾巴， 相关的连环蛋白多肽和肌动蛋白细胞骨架是重要的 对于钙粘附素介导的黏附，但这些元件如何控制 胞外亲和结合域的黏附活性不是 明白了。 拟议项目的总体目标是了解这些机制 决定钙粘附素细胞黏附蛋白如何组装形成 细胞表面的功能性黏附复合体及其黏附方式 细胞表面的功能受细胞质因子的调节。这个 具体目标旨在解决这些问题，并在越来越多的 复杂性；首先定义了基本的生物化学和同嗜性结合 钙粘蛋白胞外区的性质，然后确定 寡聚和细胞骨架锚定对钙粘附素的作用 黏附活性，最后分析潜在的机制 钙粘附素结合和细胞黏附的调节。 地球表面高分辨率三维结构的最新发现 N-末端钙粘蛋白胞外区(EC1)，展示了 存在具有两个粘附性界面的平行“链”二聚体， 为许多拟议的研究提供框架。C-钙粘素，即 非洲爪哇早期胚胎中的主要黏附蛋白将是 本研究的主要课题。我们过度表达和提纯了 毫克量的功能活性的可溶胞外域 C-钙粘素。这种蛋白质及其突变形式将被用于许多 所提议的实验。 具体目的是：1)分析其亲性结合特性 C-钙粘素；2)确定平行的“链二聚体”的作用 C-蛋白结合特性和细胞黏附功能中的相互作用 3)确定C-钙粘蛋白的5个胞外钙粘蛋白结构域中的哪一个。 钙粘附素有助于其亲性结合相互作用；4)研究 细胞骨架锚定、寡聚和连环蛋白结合的作用 在控制细胞表面C-钙粘附素的黏附功能方面； 5)研究胶粘剂调节的机制 C-钙粘附素在非洲爪哇卵裂球和培养细胞中的作用 确定E-钙粘附素的具体要求是否完整 非洲爪哇外胚层的大小取决于它的胞外区或 它的细胞质尾巴。 从这些研究中获得的信息应该会增进我们的理解 钙粘附素在组织形态发生中的作用。调查结果 将对理解粘附性行为的变化产生影响 与形态发生缺陷相关的肿瘤进展过程中的细胞数量 人类先天疾病。

The cadherin cell adhesion molecules play very important roles in the development and maintenance of solid tissues. The regulation of cadherin adhesion activity at the cell surface is an important mechanism for controlling tissue morphogenesis. The cadherin cytoplasmic tail, the associated catenin polypeptides, and the actin cytoskeleton are important for cadherin-mediated adhesion, but how these elements control the adhesive activity of the extracellular homophilic binding domain is not understood. The overall goals of the proposed project are to understand the mechanisms that determine how cadherin cell adhesion proteins assemble to form a functional adhesive complex at the cell surface and how their adhesive functions at the cell surface are regulated by cytoplasmic factors. The specific aims are designed to address these issues at increasing levels of complexity; first to define the basic biochemical and homophilic-binding properties of cadherin extracellular domains, then to determine the contributions of oligomerization and cytoskeletal anchoring to cadherin adhesion activity, and finally to analyze the mechanisms underlying the regulation of cadherin binding and cell adhesion. Recent findings of the high resolution three dimensional structure of the N-terminal cadherin extracellular domain (EC1), demonstrating the existence of a parallel "strand" dimer with two adhesive interfaces, provide a framework for many of the proposed studies. C-cadherin, the major adhesion protein in the very early Xenopus embryo, will be the primary subject of this study. We have overexpressed and purified milligram amounts of a functionally active, soluble extracellular domain of C-cadherin. This protein and mutated forms of it will be used in many of the proposed experiments. The specific aims are to: 1) Analyze the homophilic binding properties of C-cadherin; 2) Determine the role of the parallel "strand dimer" interaction in the binding properties and cell adhesion functions of C- cadherin; 3) Determine which of the 5 extracellular cadherin domains of C- cadherin contribute to its homophilic binding interactions; 4) Investigate the roles of cytoskeletal anchoring, oligomerization, and catenin-binding in the control of the adhesive function of C-cadherin at the cell surface; 5) Investigate the mechanisms underlying the regulation of the adhesive function of C-cadherin in Xenopus blastomeres and cultured cells; 6) Determine whether the specific requirement for E-cadherin in the integrity of the ectoderm in Xenopus is determined by its extracellular domain or its cytoplasmic tail. The information gained from these studies should improve our understanding of the functions of the cadherins in tissue morphogenesis. The findings will have implications for understanding changes in the adhesive behavior of cells during tumor progression in morphogenetic defects associated with human congenital diseases.