Examining the Effects of VAP on Vinculin

Sites where cells adhere to the extracellular matrix are rich in integrins, the major cell surface adhesion receptors. Integrins are coupled to the actin cytoskeleton by a series of actin-binding proteins including vinculin. Maintenance of a connection to the underlying actin cytoskeleton is critical for integrin function; interruptions in this linkage contribute to the formation and progression of numerous diseased states including cancer, leukocyte adhesion deficiency, heart disease, and some muscular dystrophies. In spite of the critical role of integrins, effective targets for manipulating integrin function are still lacking. We have recently identified a short vinculin activating peptide (VAP) that increases integrin-mediated adhesion to fibronectin or collagen. The increased adhesion is both vinculin- and integrin-dependent. An analysis of the vinculin activating peptide reveals that it is comprised of three vinculin binding sites. Using a series of truncation mutants, we have mapped the residues sufficient for binding vinculin. We find that any one of the three binding sites in VAP is sufficient for vinculin binding and co-localization with vinculin in focal adhesions. We are investigating the mechanism by which the vinculin activating peptide increases adhesion, with an emphasis on which residues in vinculin are required for VAP to exert its effects, and how these effects regulate integrin activity. These studies provide novel insight into how vinculin activation regulates integrin function and will open up novel therapeutic possibilities.