OUTSIDE-IN INTEGRIN SIGNAL-TRANSDUCTION - ALPHA(IIB)BETA(3)-(GP IIB-IIIA) TYROSINE PHOSPHORYLATION-INDUCED BY PLATELET-AGGREGATION

alpha(IIb)beta(3)-(GPIIb-IIIa) is the most abundant integrin expressed on platelets and plays a critical role in platelet aggregation and no rmal hemostasis. In response to platelet stimulation by agonists such as thrombin, alpha(IIb)beta(3) becomes a receptor for the adhesive pro teins fibrinogen, von Willebrand factor, vitronectin, and fibronectin. Binding of extracellular matrix ligands allows the integrin to transm it a signal to the inside of the cell, but the exact mechanisms whereb y integrins transduce these signals remain unclear. In this paper we d emonstrate that the beta(3) subunit of alpha(IIb)beta(3) was phosphory lated on tyrosine residues in response to thrombin induced platelet ag gregation. However, tyrosine phosphorylation was not observed when pla telets were stimulated by thrombin in the presence of an inhibitor of aggregation. Phosphotyrosine was only detected when platelets were sol ubilized under protein-denaturing conditions, A peptide corresponding to residues 740-762 of the beta(3) cytoplasmic domain was capable of b inding the signaling proteins SHC and GRB2, GRB2 binding occurred only when both tyrosine residues (Tyr-747 and Tyr-759) were phosphorylated . SHC binding also occurred to a peptide monophosphorylated at Tyr-759 , The data suggest that tyrosine phosphorylation of an integrin beta s ubunit may be important in initiating outside in signaling cascades by inducing association of signaling components directly with the integr in.