This grant builds on our novel discovery that E-cadherin at epithelial cell-cell junctions transduces mechanical signals, by activating a kinase cascade via the epidermal growth factor receptor (EGFR). E-cadherin is an essential adhesion protein at epithelial cell-cell junctions, and E-cadherin complexes also transduce force, to regulate cell shape and epithelial barrier integrity. These new findings suggest that E-cadherin force- transduction also activates signals that regulate cell proliferation, morphogenesis, and disease. The broad goal of this program is to identify initial steps in the mechanical activation of EGFR by E-cadherin, and to establish the broader physiological implications of this mechanism. Our preliminary data also demonstrate that this force-activated signaling pathway regulates the cytoskeletal reinforcement of stressed cell-cell junctions by α−catenin in E-cadherin complexes. This unexpected finding supports the hypothesis that EGFR and E- cadherin are essential components in the core force-transduction machinery at epithelial cell junctions. In this program, Specific Aim 1 tests the hypothesis that mechanically stimulated E-cadherin activates EGFR phosphorylation, by triggering the disruption of putative E-cadherin/EGFR complexes. Specific Aim 2 will use innovative fluorescence-based methodology, developed by collaborator Hristova (Johns Hopkins) to investigate direct interactions between E-cadherin and EGFR at the plasma membrane. Proposed studies are based on substantial preliminary data, which reveal direct protein-protein association. Biophysical studies will establish the molecular requirements for this association, using a subset of E-cadherin and EGFR mutants. Specific Aim 3 will test the physiological implications of these findings in a three-dimensional, organotypic model of human mammary epithelial tissue, in collaboration with Weaver (UCSF). Studies will determine whether E-cadherin/EGFR complexes are indeed central force-sensing units that coordinate with integrins to tune morphogenesis and malignancy, in response to tissue mechanics. 3D cultures of breast epithelial cells engineered to express E-cadherin mutants (Aim 2) will determine the impact of E-cadherin/EGFR complex disruption on proliferation, morphogenesis, and invasion, as a function of matrix rigidity. Integrins are well known to coordinate with EGFR to regulate breast tissue development and tumor progression. These studies would potentially establish E-cadherin as an essential component in this force-sensitive network.