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.
