Continued support is requested for an analysis of the integrin-dependent assembly of a fibronectin (Fn)matrix. The applicant characterized the role of talin in integrin activation and in forming the integrin-mediated linkages between Fn and the cytoskeleton that are essential for assembly. He hypothesizes that integrin p cytoplasmic domain interactions with CD98hc play important roles in integrin-dependent cellular functions. He will therefore use CD98hc null cells to characterize the role of this protein in matrix assembly, cell migration and biochemical signaling. He will map the sites of interactions between CD98hc and integrins and use that information to test the role of the interactions in integrin function. Lastly, conditional CD98hc-deficient mice will be generated to assess the functional role of CD98hc in endothelium. The applicant hypothesizes that interaction of integrins with Src family kinases (SFK) and talin are mediated by divergent sites in integrin P tails and contribute to dissimilar integrin-dependent biological functions. He will therefore to identify key residues that control the specificity of P cytoplasmic domain binding to SFK.He will use mutants of the integrins that specifically perturb binding to SFK, talin, or CD98hc to examine the roles of each interaction in integrin functions. Conversely, integrin binding-defective mutants of SFK, talin, and CD98hc will be used to reconstitute deficient cells to address the same issues. Finally, the sub endothelial matrix and endothelial cytoskeleton are shaped and oriented by flowing blood. The applicant hypothesizes that spatially restricted Protein Kinase A (PKA)-mediated phosphorylation of the a4 integrin imposes polarity on Rac activation in endothelial cells leading to this alignment. He will use pharmacological and reverse genetic approaches combined with live cell microscopy with a PKA activation biosensor to evaluate the role of PKA phosphorylation of a4 on the shear-mediated alignment of endothelial cells, their cytoskeleton, and their matrix. He will also examine the role of PKA phosphorylation of a4 on thrombogenicity of matrices formed by cultured endothelial cells in vitro. These fundamental studies will provide novel insight into the regulation of Fn matrix assembly and will test and advance paradigms that apply to many integrin-dependent biological processes.
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