Alpha5beta1 is a member of the integrin family of cell adhesion receptors which serve both the mechanical function of adhering cells and a signaling function in the control of many cell processes. It is likely that the mechanical and signaling aspects of alpha5beta1 function are linked since some alpha5beta1 signaling functions cannot be replaced by ligand binding alone. To examine the mechanical aspects of alpha5beta1 function and regulation of alpha5beta1 mediated cell adhesion, a spinning disc device has been engineered to provide a measurement of the force required to detach cells from a substrate. By applying concepts from chemical binding analysis it has been possible to convert the adhesion data to a model of monovalent receptor-ligand binding and demonstrate that there is a linear relationship between the number of integrin-extracellular matrix bonds and the force required to detach the cell. The proposal considers models to explain activation of alpha5beta1 binding to fibronectin: agonist activation, induced-fit activation, clustering activation, and tension activation. Each of these models makes different predictions for ligand, cytoskeletal, and energy requirements. These requirements would be tested using biochemical predictions. Inhibitors, and genetic analysis of fibronectin mutants, beta1 cytoplasmic domain splice variants, and beta1 cytoplasmic domain mutants. The analysis views the adhesion connection as one from fibronectin to alpha5beta1 to actin cytoskeleton and these connections, either ligand binding or cytoskeletal binding, can control the adhesion strength. Procedures have been developed to assay these connections separately. Their approach would be extended to the examination of long term (or steady-state) adhesion, including analysis of the increase in the number of alpha5beta1 receptors bound, and the role of clustering n the adhesion strengthening-cell spreading process.
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