Identification of Shc as the primary protein binding to the tyrosine-phosphorylated beta(3) subunit of alpha(IIb)beta(3) during outside-in integrin platelet signaling

Outside-in signaling mediated by the integrin alpha (IIb)beta (3) (GPIIbIII a) is critical to platelet function and has been shown to involve the phosp horylation of tyrosine residues on the cytoplasmic tail of beta (3). To ide ntify proteins that bind directly to phosphorylated beta (3), we utilized a n affinity column consisting of a peptide modeled on the tyrosine-phosphory lated 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 t hrombin-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 de tergent lysates of aggregated platelets. Using purified, recombinant protei n, it was found that the binding of Shc to monophosphorylated (C-terminal t yrosine) and diphosphorylated beta (3) peptides was direct, demonstrating S he recognition motifs on phospho-beta (3). Aggregation-induced She phosphor ylation was also observed to be robust in platelets from wild-type mice, bu t not in those from mice expressing (Y747F,Y759F) beta (3), which are defec tive in outside-in alpha (IIb)beta (3) signaling. Thus, Shc is the primary downstream signaling partner of beta (3) in its tyrosine phosphorylation ou tside-in signaling pathway.