Rap1 defines site and activation signals for collagen phagocytosis

Collagen phagocytosis is a crucial, alpha2 beta1 integrin-dependent process that mediates extracellular matrix remodeling by fibroblasts. We have shown previously that gelsolin is required for Rac1 activation in the initial binding step of collagen phagocytosis. Objective: To define the roles of the Ras related GTPase, Rap1, and the GTP exchange factor Vav2, in regulation of Rac1 during collagen phagocytosis. Methods: Cultured mouse fibroblasts were incubated with collagen beads to ligate alpha2 beta1 integrins. Cells were transfected with plasmids expressing small GTPase regulatory proteins and studied by confocal microscopy, immunoblotting and Rac1 activation assays. Results: Collagen bead binding required Rac activation but not cdc42 or RhoA. Rac activity was also increased following collagen bead binding. Accordingly, we focused on the upstream regulation of Rac in the collagen binding step of phagocytosis. Collagen bead binding promoted phosphorylation of Vav2 at Y172. Cells transfected with constitutively active Vav2 exhibited Rac activation and enhanced collagen bead binding while dominant-negative Vav2 blocked Rac activity and bead binding. Recruitment of active rac to bound collagen beads required active Vav2. Immunoprecipitation studies showed interactions between constitutively endogenous Vav2 and active Rap1. Transfection with dominant negative Rap1, with or without co-transfection with constitutively active Vav2, inhibited collagen bead binding. In contrast, cells co-transfected with constitutively active Rap1 and constitutively active Vav2, exhibited enhanced collagen bead binding. In cells transfected with constitutively active, but not dominant negative Rap1, incubation with collagen beads promoted translocation of constitutively active Vav2 to collagen bead binding sites. Conclusion: Rap1 interacts with constitutively active Vav2 to localize Vav2 activity to integrins and to there enhance collagen binding by activating Rac.