Critical roles for the COOH-terminal NITY and RGT sequences of the integrin β(3) cytoplasmic domain in inside-out and outside-in signaling

Bidirectional signaling of integrin α(IIb)β(3) requires the β(3) cytoplasmic domain. To determine the sequence in the β(3) cytoplasmic domain that is critical to integrin signaling, cell lines were established that coexpress the platelet receptor for von Willebrand factor (vWF), glycoprotein Ib-IX, integrin α(IIb), and mutants of β(3) with truncations at sites COOH terminal to T(741), Y(747), F(754), and Y(759). Truncation at Y(759) did not affect integrin activation, as indicated by vWF-induced fibrinogen binding, but affected cell spreading and stable adhesion. Thus, the COOH-terminal RGT sequence of β(3) is important for outside-in signaling but not inside-out signaling. In contrast, truncation at F(754), Y(747), or T(741) completely abolished integrin activation. A point mutation replacing Y(759) with alanine also abolished integrin activation. Thus, the T(755)NITY(759) sequence of β(3), containing an NXXY motif, is critical to inside-out signaling, whereas the intact COOH terminus is important for outside-in signaling. In addition, we found that the calcium-dependent protease calpain preferentially cleaves at Y(759) in a population of β(3) during platelet aggregation and adhesion, suggesting that calpain may selectively regulate integrin outside-in signaling.