The central dogma of cell-cell adhesion is that E-cadherin binds to beta-catenin which binds to alpha-catenin which binds to the cytoskeleton. Since the original elucidation of this process, it has been seen that interruption of this connection at any of these intermediates resulted in loss of adhesion. The consensus was that homotypic cadherin interactions would trigger recruitment of these proteins into a stabile adhesive complex. There is now evidence suggest that alternative pathways may exist. In this proposal we will show preliminary evidence that the cadherin complex may be constructed and possibly regulated by interactions and modifications of cytoplasmic proteins. We use a recombinant cadherin probe for homotypic interaction to contrast static , strong or stable adhesion with dynamic , weak or transcient adhesion. These experiments suggest that different protein components are present in each complex. In particular, we find that vinculin appears to participate in the dynamic complexes, in a direct interaction with cadherin complexes containing E-cadherin and beta- catenin but not alpha-catenin. We hypothesize that vinculin and alpha- catenin, by their differential interactions with cytoskeletal elements and beta-catenin, play a critical role in establishment and regulation of cell-cell adhesion. We propose three main approaches for testing this hypothesis. First we will determine the mechanism of interaction of vinculin with the cadherin complex, testing its interaction with beta-catenin or determining how it is able to interact with the complex. Secondly, we will define the strength and temporal sequence of adhesion mediated by vinculin compared to that mediated by alpha-catenin. Finally, we will further define the connections of the cadherin complex to the cytoskeleton, comparing that mediated by vinculin with that mediated by alpha-catenin. This proposal describes a fusion of the extracellular domain of E-cadherin with the Fc region of IgG and shows it is capable of participating in the Ca++ sensitive homotypic interaction like a native cadherin molecular. We use this tool throughout this proposal in in vitro adhesion assays and also as a unique probe for the homotypic interaction, as a mechanism to assess the co-precipitable components in the adhesion complex.
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