Abstract
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 Shc 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 Shc 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, Shc 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 Shc recognition motifs on phospho-beta(3). Aggregation-induced Shc 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.
Highlights
Widely distributed ␣v3 integrin, suggest that ␣IIb3 signaling serves as a prototype for the elucidation of signaling pathways within the integrin family of adhesion receptors
Because conserved integrin cytoplasmic tyrosine (ICY) domain sequences are found in the  subunits of several integrins, including 1, 3, 6, and 7, and because tyrosine phosphorylation appears to be involved in the migration of cells on 1 integrins, it is anticipated that information concerning the mechanisms of signaling through ␣IIb3 might be applicable to other integrins
These proteins include: 3 endonexin [11], which interacts with the NITY motif at residues 756 –759 on the distal end of the 3 cytoplasmic tail [12], in a phosphorylation-independent manner; calcium- and integrin-binding protein [13], which binds in a calcium-dependent manner to the cytoplasmic tail of the ␣IIb subunit [14]; talin [15], a cytoskeletal protein that binds directly with the cytoplasmic tail of both ␣IIb3 subunits, integrin-associated protein [16], a protein that laterally associates with ␣IIb3 and activates this integrin in response to thrombospondin [17]; and myosin, Shc, and Grb2, which have previously been shown to bind tyrosine-phosphorylated 3 peptides [9, 10]
Summary
We discovered that Shc was the primary protein associating with the phosphorylated 3 peptide and showed that Shc becomes transiently phosphorylated during platelet aggregation and can be co-immunoprecipitated with 3 from lysates in which outside-in ␣IIb3 signaling occurs. Analysis of platelets from the diYF mouse expressing (Y747F,Y759F) 3 showed that, in platelets where 3 tyrosine phosphorylation cannot occur and where outside-in ␣IIb3 signaling is defective, the level of aggregation-induced Shc phosphorylation was abrogated. These data implicate Shc as playing a major role in outside-in ␣IIb3 signaling in platelets by serving as a direct signaling partner for tyrosine-phosphorylated 3
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