PTPLB CONTROL OF CADHERIN FUNCTION & RETINA DEVELOPMENT

我们的长期目标是确定这些机制的特点， 粘附分子调节变化的发育分支 发育中的视网膜。该提案的重点是监管 在视网膜发育过程中， 蛋白酪氨酸磷酸酶PTP 1B。钙粘蛋白的功能取决于其 连接到含有肌动蛋白的细胞骨架，由 α-和β-连环蛋白β-氨基丁酸上酪氨酸残基的磷酸化 连环蛋白与钙粘蛋白-细胞骨架连接的丧失有关， 以及随之而来的粘合能力的损失。PTP 1B结合至 细胞质结构域的N-钙粘蛋白，并保持肌动蛋白连接， 使β-连环蛋白去磷酸化。我们的具体目标是 定义N-钙粘蛋白和 PTPlB，以及这种相互作用在视网膜病变中的作用。 形态发生我们的具体目标是定义分子 N-钙粘蛋白和PTP 1B之间相互作用的基础， 这种相互作用在视网膜形态发生中的作用。我们将1。 鉴定和功能分析与之相互作用的N-钙粘蛋白， 通过体外和原位结合分析PTP 1B。本地化 位点，我们将构建N-cadherin的巢式缺失。我们还将 开发模拟N-钙粘蛋白结合结构域的细胞渗透性肽 从而干扰PTP 1B与钙粘蛋白的原位结合。2. 通过以下方法鉴定靶向N-钙粘蛋白所必需的PTP 1B结构域： 分析PTP 1B与钙粘蛋白的体外和原位结合， 酪氨酸残基的位点特异性诱变和 嵌套删除3.分析比较钙粘蛋白-PTP 1B的作用 与其他N-钙粘蛋白效应物相互作用的作用， 突起生长和细胞粘附。我们将确定 模拟细胞质不同区域的细胞渗透性肽 N-钙粘蛋白对神经突生长的影响，以及 它们对β-连环蛋白磷酸化和细胞膜完整性的影响 钙粘蛋白细胞骨架连接。4.分析时间上的影响 钙粘蛋白/PTP 1B相互作用对完整的 视网膜。细胞渗透性肽干扰N- 将钙粘蛋白与PTP 1B的结合与破坏结合的那些进行比较 β-连环蛋白对完整神经元形态发生的影响 在视网膜发育期间的特定时间添加后的视网膜。

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.