PTPLB CONTROL OF CADHERIN FUNCTION & RETINA DEVELOPMENT

我们的长期目标是描述这些机制和 黏附分子调节变化的发育分支 对发育中的视网膜起作用。这项建议的重点是监管 非受体对N-钙粘附素在视网膜发育中作用的影响 蛋白酪氨酸磷酸酶PTPlB。钙粘附素的功能依赖于其 与含有细胞骨架的肌动蛋白的连接，一种由 α-和β-连环蛋白。酪氨酸残基在β-位上的磷酸化 连环蛋白与钙粘附素-细胞骨架连接的丧失有关， 以及随之而来的粘接能力的丧失。PTPlB绑定到 N-钙粘蛋白的胞质结构域和维持肌动蛋白的连接 去磷酸化β-连环蛋白。我们的具体目标是 N-钙粘附素与钙粘附素相互作用的分子基础 PTPlB，以及这种相互作用在视网膜中所起的作用 形态发生。我们的具体目标是定义分子 N-钙粘蛋白与PTPlB相互作用的基础及其作用 这种相互作用在视网膜形态发生中起作用。我们会1. 识别和功能分析与之相互作用的N-钙粘附素 通过体外结合和原位结合分析PTP1B。要本地化 我们将构建N-钙粘附素的嵌套缺失。我们还将 开发模拟N-钙粘附素结合域的细胞通透性多肽 从而干扰PTPlB与钙粘附素的原位结合。2. 通过以下方法确定靶向N-钙粘素所必需的PTP1B结构域 PTPlB与钙粘附素结合的体外和原位观察 酪氨酸残基的定点突变及其构建 嵌套删除。3.分析比较钙粘蛋白-PTPlB的作用 与其他N-钙粘附素效应器相互作用的作用 轴突生长和细胞黏附。我们将确定 模拟细胞质不同区域的细胞通透性多肽 N-钙粘附素在几种底物上对轴突生长的影响 它们对β-连环蛋白磷酸化的影响及其对细胞完整性的影响 钙粘素与细胞骨架的连接。4.分析暂时性的影响 钙粘附素/PTPlB相互作用在完整细胞形态发生中的特异性丢失 视网膜。干扰N-受体相互作用的细胞通透性多肽 与PTPlB结合的钙粘附素将与那些破坏结合的钙粘蛋白进行比较 β-连环蛋白对完整神经形态发生的影响 在视网膜发育过程中的特定时间添加之后的视网膜。

Our long-term objectives are to characterize the mechanisms and developmental ramifications of regulated changes in adhesion molecule function the developing retina. This proposal focuses on the regulation of N-cadherin function in the developing retina by the non-receptor protein tyrosine phosphatase PTPlB. Cadherin function depends on its linkage to the actin containing cytoskeleton, a linkage mediated by alpha- and beta-catenin. Phosphorylation of tyrosine residues on beta- catenin is associated with loss of the cadherin-cytoskeletal connection, and consequent loss of adhesive competence. PTPlB is bound to the cytoplasmic domain of N-cadherin and maintain the actin connection by dephosphorylating beta-catenin. Our specific aims are directed at defining the molecular basis for the interaction between N-cadherin and PTPlB, and the role that this interaction plays in retinal morphogenesis. Our specific aims are directed at defining the molecular basis for the interaction between N-cadherin and PTPlB, and the role that this interaction plays in retinal morphogenesis. We will 1. identify and functionally analyze the N-cadherin which interacts with PTP1B through analysis of binding in vitro and in situ. To localize the site, we will construct nested deletions of N-cadherin. We will also develop cell permeable peptides that mimic the N-cadherin binding domain thus interfere with the binding of PTPlB to cadherin in situ. 2. Identify the PTP1B domain essential for targeting to N-cadherin by analyzing binding of PTPlB to cadherin in vitro and in situ following site specific mutagenesis of tyrosine residues and construction of nested deletions. 3. Analyze and compare the role of cadherin-PTPlB interactions with the role of other N-cadherin effector interactions on neurite outgrowth and cell adhesion. We will determine the effect of cell permeable peptides which mimic different regions of the cytoplasmic of N-cadherin on neurite outgrowth on several substrates, as well as their effect on phosphorylation of beta-catenin and the integrity of the cadherin cytoskeletal connection. 4. Analyze the effect of temporally specific loss of cadherin/PTPlB interactions on morphogenesis of intact retina. Cell permeable peptides which perturb the interaction of N- cadherin with PTPlB will be compared to those which disrupt the binding of beta-catenin for their effect on morphogenesis of intact neural retina following addition at specific times during retina development.