Identification of Shc as the primary protein binding to the tyrosine-phosphorylated β3 subunit of αIIbβ3 during outside-in integrin platelet signaling

Outside-in signaling mediated by the integrin alpha (IIb)beta (3) (GPIIbIIIa) is critical to platelet function and has been shown to involve the phosphorylation of tyrosine residues on the cytoplasmic tail of beta (3). To identify proteins that bind directly to phosphorylated beta (3), we utilized an affinity column consisting of a peptide modeled on the tyrosine-phosphorylated cytoplasmic domain of beta (3). Tandem mass spectrometric sequencing and immunoblotting demonstrated that She was the primary protein binding to phosphorylated beta (3). To determine the involvement of Shc in outside-in alpha (IIb)beta (3) signaling, the phosphorylation of Shc during platelet aggregation was examined; transient She phosphorylation was observed when thrombin-stimulated platelets were allowed to aggregate or when aggregation was induced by an LIBS (ligand-induced binding site) antibody, D3. Moreover, She was co-immunoprecipitated with tyrosine-phosphorylated beta (3) in detergent lysates of aggregated platelets. Using purified, recombinant protein, it was found that the binding of Shc to monophosphorylated (C-terminal tyrosine) and diphosphorylated beta (3) peptides was direct, demonstrating She recognition motifs on phospho-beta (3). Aggregation-induced She phosphorylation was also observed to be robust in platelets from wild-type mice, but not in those from mice expressing (Y747F,Y759F) beta (3), which are defective in outside-in alpha (IIb)beta (3) signaling. Thus, Shc is the primary downstream signaling partner of beta (3) in its tyrosine phosphorylation outside-in signaling pathway.