Adhesion-Induced Unclasping of Cytoplasmic Tails of Integrin αIIbβ3,

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Integrin alpha(IIb)beta(3) plays a pivotal role in hemostasis and thrombosis by mediating adhesive interactions of platelets. Binding of alpha(IIb)beta(3) to its physiological ligands, immobilized fibrinogen and fibrin, induces outside-in signaling in platelets, leading to their adhesion and spreading even without prior stimulation by agonists. Implicit in these phenomena is a requirement for the linkage between integrins' cytoplasmic tails and intracellular proteins. However, the nature of the initiating signal has not been established. In this study, we examined whether binding of alpha(IIb)beta(3) to immobilized fibrin(ogen), per se, triggers interaction of the integrin with cytoplasmic proteins. Using the integrin-binding skelemin fragment as a marker of exposure of residues involved in the clasp between alpha(IIb) and beta(3) cytoplasmic tails, we showed that its binding site in the membrane-proximal beta(3) 715-730 segment is cryptic and becomes exposed as a result of binding of isolated alpha(IIb)beta(3) to immobilized ligands. Furthermore, the skelemin-like protein present in platelets and CHO cells does not associate with alpha(IIb)beta(3) in resting platelets or suspended alpha(IIb)beta(3)-expressing CHO cells but is recruited to integrin during cell adhesion. In addition, not only beta(3) but also the membrane-proximal 989-1000 segment of the alpha(IIb) cytoplasmic tail binds the skelemin fragment. Finally, the same residues, alpha(IIb) Val(990), alpha(IIb) Arg(995), and beta(3) His(722), involved in the formation of the clasp between the tails are also required for skelemin binding. These studies suggest that ligation of alpha(IIb)beta(3) by immobilized ligands during platelet adhesion induces a transmembrane conformation change in the integrin, resulting in unclasping of the complex between the membrane-proximal parts of cytoplasmic tails, thereby unmasking residues involved in binding the skelemin-like protein. Thus, the junction between alpha(IIb) and beta(3) cytoplasmic tails may contain the critical structural information for the initiation of outside-in signaling.