Proximal, selective, and dynamic interactions between integrin αIIbβ3 and protein tyrosine kinases in living cells

Abstract

Stable platelet aggregation, adhesion, and spreading during hemostasis are promoted by outside-in αIIbβ3 signals that feature rapid activation of c-Src and Syk, delayed activation of FAK, and cytoskeletal reorganization. To evaluate these αIIbβ3-tyrosine kinase interactions at nanometer proximity in living cells, we monitored bioluminescence resonance energy transfer between GFP and Renilla luciferase chimeras and bimolecular fluorescence complementation between YFP half-molecule chimeras. These techniques revealed that αIIbβ3 interacts with c-Src at the periphery of nonadherent CHO cells. After plating cells on fibrinogen, complexes of αIIbβ3-c-Src, αIIbβ3-Syk, and c-Src-Syk are observed in membrane ruffles and focal complexes, and the interactions involving Syk require Src activity. In contrast, FAK interacts with αIIbβ3 and c-Src, but not with Syk, in focal complexes and adhesions. All of these interactions require the integrin β3 cytoplasmic tail. Thus, αIIbβ3 interacts proximally, if not directly, with tyrosine kinases in a coordinated, selective, and dynamic manner during sequential phases of αIIbβ3 signaling to the actin cytoskeleton.