Critical roles for the COOH-terminal NITY and RGT sequences of the integrin β3 cytoplasmic domain in inside-out and outside-in signaling

Bidirectional signaling of integrin alpha(IIb)beta(3) requires the beta(3) cytoplasmic domain. To determine the sequence in the beta(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 alpha(IIb), and mutants of beta(3) with truncations at sites COOH terminal to T-741, Y-754, 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 33 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 (TNITY759)-N-755 sequence of beta(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 beta(3) during platelet aggregation and adhesion, suggesting that calpain may selectively regulate integrin outside-in signaling.