Ga13 and Talin Coordinate integrin Inside-out and Outside-In Signaling by “time-sharing” of b3 cytoplasmic Domain

Abstract Abstract 162 The bidirectional signaling of integrins mediates cell adhesion, spreading, retraction and migration. The binding of talin and kindlins to the cytoplasmic domain of integrin b3 subunit transmits inside-out signals to induce integrin activation. Ligand-induced outside-in signaling requires the binding of a G protein subunit, Ga13, and a tyrosine kinase, c-Src, to the b3 cytoplasmic domain. It is unclear how the short cytoplasmic domain of b3 accommodates these molecules and allows coordinated bidirectional signaling. Here we show that Ga13 and talin are mutually exclusive in binding to b3 both in vivo and in vitro. Increasing expression level of talin head or full-length talin in CHO123 cell decreases Ga13-b3 association. Ga13 also competes with talin head for GST-b3 binding in purified binding system. More importantly, talin is associated with b3 only in inside-out signaling during platelet aggregation. Following integrin ligation, however, Ga13 binds to b3, replacing talin. The Ga13 binding site located between K729-T741 within the talin binding region. However, Ga13 binding and signaling require a distinct ExE733 motif (EEE in b3) conserved in most integrin b subunits that is not required for talin binding but flanked by talin binding sequences on both sides. Interference of Ga13 binding to integrin b3 cytoplasmic domain by myristorylated b3 peptide (Myr-EEERA735) or by point-mutating the EEE motif to AAA selectively inhibits outside-in signaling, thus inhibited cell spreading on fibrinogen, accelerated RhoA activation and inhibited c-Src activity. But they have no effect on talin-dependent inside-out signaling judged by fibrinogen binding assay. In conclusion, our data suggest that the timed share of binding sites in b3 between Ga13 and talin coordinates bidirectional integrin signaling. Disclosures: No relevant conflicts of interest to declare.