Dysfunction of the endothelial cell barrier has been implicated in the etiology of a variety of disease states including atherosclerosis, microvascular hyperpermeability with diabetes and pulmonary edema following acute lung injury. VE-cadherin is a cell-cell adhesion protein that participates in the regulation of a number of endothelial cell functions. The cytoplasmic domain of VE-cadherin binds to the armadillo family of proteins called catenins to form the adherens junction (AJ). Changes in the phosphorylation of AJ proteins have been implicated in regulating barrier function. For example, phosphorylation of AJ proteins occurs during the formation of edema following stroke and myocardial infarction, a process that is dependent on activation of Src kinase. This proposalfocuses on p120, a catenin that binds to the juxtamembrane of VE- cadherin where it serves a scaffolding role for a number of proteins. Interestingly, p120 is a target for Src kinase and changes in the phosphorylation state of p120 are associated with decreased endothelial barrier function following treatment with inflammatory mediators. In addition, our recent publications have shown that the interaction of p120 with VE-cadherin is required for maintaining VE-cadherin levels in endothelial cells. Within this context, we hypothesize that the juxtamembrane region of VE-cadherin and its interaction with p120 plays a critical role in regulating cell-cell adhesion and barrier function in endothelial cell monolayers. This hypothesis will be investigated by performing three specific aims: 1) identify the regions of VE-cadherin that are responsible for maintaining VE-cadherin levels and barrier function in endothelial cell monolayers;2) determine if p120 serves as a signaling molecule when released from the membrane into the cytoplasm;and 3) identify the domains of p120 required for the regulation of endothelial cell-cell adhesion and barrier function. We will use expression of chimeric molecules of VE and N-cadherin as well as deletion mutants of p120 to perform structure/function analysis of p120 and VE-cadherin and their effects on endothelial cell-cell adhesion and barrier function. Expression of these molecules will be performed in the absence of endogenous cadherin or p120 that will be depleted using siRNA. These studies will provide new insights into the cellular and molecular mechanisms controlling endothelial barrier function and potentially identify new sites for intervention.