The Role of Galpha13 in Integrin Signaling and Function

Integrins are known to transmit signaling in two directions: intracellular signaling stimulates conformational changes in the extracellular domain, leading to increased ligand affinity (inside-out signaling). Conversely, ligand binding in the extracellular domain of integrins transmit signals into the cell, leading to a cascade of signaling events that stimulate cell spreading, retraction, migration, and proliferation (outside-in signaling). The mechanism of integrin outside-in signaling has been unclear. We showed here that Galpha13 directly bound to the integrin beta3 cytoplasmic domain and that Galpha13-integrin interaction was promoted by ligand binding to the integrin alphaIIbbeta3 and by GTP loading of Galpha13. We showed that integrin ligation mediates thrombus formation and outside-in signaling, which requires Galpha13 and greatly expands thrombi. Our studies also demonstrated that Galpha13 and talin bind to mutually exclusive but distinct sites within the integrin beta3 cytoplasmic domain in opposing waves. The first talin-binding wave mediates inside-out signaling and also ligand-induced integrin activation, but is not required for outside-in signaling. Integrin ligation induces transient talin dissociation and Galpha13 binding to an EXE motif, which selectively mediates outside-in signaling and platelet spreading. The second talin-binding wave is associated with clot retraction. An EXE-motif-based inhibitor of Galpha13–integrin interaction selectively abolishes outside-in signaling without affecting integrin ligation, and suppresses occlusive arterial thrombosis without affecting bleeding time. Thus, we have discovered a new mechanism for the directional switch of integrin signaling and, on the basis of this mechanism, designed a new antithrombotic drug that does not cause bleeding. Secondly, we showed that the Galpha13 binding is important in integrin-dependent cell migration. We showed that interaction between Galpha13 and the beta1 subunit plays a critical role in beta1-dependent cell migration. We further showed that the Galpha13-beta1 interaction mediates beta1 integrin–dependent Src activation and transient RhoA inhibition during initial cell adhesion, which is in contrast to the role of Galpha13 in mediating GPCR-dependent RhoA activation. These data indicate that Galpha13 plays dynamic roles in both stimulating RhoA via a GPCR pathway and inhibiting RhoA via an integrin signaling pathway. This dynamic regulation of RhoA activity is critical for cell migration on beta1 integrin ligands.