Abstract 397: The Interaction of Integrin beta1 to Galpha13 Mediates RhoA Inhibition and Cell Migration

Background: Integrin-dependent cell migration is critically important in many physiological and pathological processes such as angiogenesis, inflammation, wound healing, and atherosclerosis. However, the underlying signaling mechanisms remain unclear. Hypothesis: We have recently shown that Ga13 directly interacts with the cytoplasmic domain of integrin b3 subunits in platelets and is a proximal mechanism of integrin outside-in signaling. We further showed that the binding site for Ga13 requires a conserved ExE motif also present in b1 integrins important in adhesion and migration of nearly all vascular cells. We hypothesize that the direct binding of Ga13 to the ExE motif in integrin b1 plays an important role in mediates b1-dependent cell migration. Results: To specifically study the role of b1 integrin in cell migration, we compared cell migration of GD25 cells that are deficient in integrin b1 subunit with cells that re-constitute b1 expression using a scratch wound healing assay and a transwell migration assay. Cell migration was abolished in b1-deficient cells, indicating a critical role for b1 in these assays. To study whether Ga13-integrin interaction is important in cell migration, we constructed b1 mutants that were deficient in binding to Ga13 by mutating the critically important glutamic acid residues to alanine. Co-immunoprecipitation experiments indicate the ExE to alanine mutants abolished Ga13-integrin interaction. The b1-dependent cell migration is dramatically inhibited in the ExE to alanine mutant b1-expressing cells. Importantly, a small peptide based on the ExE motif similarly inhibited cell migration. Furthermore, Ga13 binding-deficient b1 mutant cells showed diminished b1 integrin-dependent Src activation and accelerated RhoA activation during cell spreading on fibronectin, suggesting that the integrin-dependent transient inhibition of RhoA was abolished. Conclusions: Ga13 mediates integrin-dependent cell migration by direct binding to the ExE motif of integrin b1 and mediating c-Src activation and RhoA inhibition.