Interaction of kindlin-2 with integrin β3 promotes outside-in signaling responses by the αVβ3 vitronectin receptor

The bidirectional signaling and hemostatic functions of platelet alpha IIb beta 3 are regulated by kindlin-3 through interactions with the beta 3 cytoplasmic tail. Little is known about kindlin regulation of the related vitronectin receptor, alpha V beta 3. These relationships were investigated in endothelial cells, which express alpha V beta 3 and kindlin-2 endogenously. beta 3 Delta RGT knock-in mice lack the 3 C-terminal beta 3 tail residues, whereas in beta 3/beta 1(EGK) mice, RGT is replaced by the corresponding residues of beta 1. The wild-type beta 3 tail pulled down kindlin-2 and c-Src in vitro, whereas beta 3 Delta RGT bound neither protein and beta 3/beta 1(EGK) bound kindlin-2, but not c-Src. beta 3 Delta RGT endothelial cells, but not beta 3/beta 1(EGK) endothelial cells, exhibited migration and spreading defects on vitronectin and reduced sprouting in 3-dimensional fibrin. Short hairpin RNA silencing of kindlin-2, but not c-Src, blocked sprouting by beta 3 wild-type endothelial cells. Moreover, defective sprouting by beta 3 Delta RGT endothelial cells could be rescued by conditional, forced interaction of alpha V beta 3 Delta RGT with kindlin-2. Stimulation of beta 3 Delta RGT endothelial cells led to normal extracellular ligand binding to alpha V beta 3, pin-pointing their defect to one of outside-in alpha V beta 3 signaling. beta 3 Delta RGT mice, but not beta 3/beta 1(EGK) mice, exhibited defects in both developmental and tumor angiogenesis, responses that require endothelial cell function. Thus, the beta 3/kindlin-2 interaction promotes outside-in alpha V beta 3 signaling selectively, with biological consequences in vivo.