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

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.