The objective of this proposal is to examine the role of src in the control of integrin mediated cell adhesion to fibronectin. Fibronectin is the dominant ligand for adhesion of fibroblasts and other mesenchymal cell types, and adhesion to fibronectin plays a central role in the control of the proliferation and differentiation of these cells. The experiments focus on the isolation of the different molecular mechanisms which contribute to cell adhesion strength both at early and later times in the adhesion process. The experiments are based on a novel spanning this device to measure adhesion strength and an analytic system based on a K562 cell model and a collection of monoclonal antibodies which provide the basis for the measurement of the specific receptor-ligand binding strength for different fibronectin receptors and for different activation states of those receptors. In the first two specific aims v- src (or activated c-src) transformed cells and cells from src-/- and FAK-/- cell lines will be examined in combination with specific src and FAK mutants to examine the possible role(s) for these genes in the control of integrin activation states. The third and fourth specific aims focus on the connections between integrin and cytoskeleton which may be regulated by the action of src. The level of phosphorylation of b1 integrin is raised in v-src transformed cells; the possibility that this is also true for b3 will be tested. The role of phosphorylation of b1 and b3 integrin will be examined using specific mutants to determine how and if this phosphorylation regulates the functional interaction of integrin and cytoskeleton. The spinning disc analysis will be adapted to analysis of longer term adhesion and the development of a general method to examine the interplay between the strength of integrin-ligand and integrin-cytoskeletal linkages is proposed. This will be applied to analysis of the role of src in regulation of cytoskeletal structure and the linkage of cytoskeleton to integrin.
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