Glycoproteins GPVI and GPIb-IX-V stimulate robust tyrosine phosphorylation of Syk and PLCγ2 (phospholipase Cγ2) in washed platelets, but only the former stimulates pronounced activation of phospholipase. Using phospho-specific antibodies, we demonstrate that GPVI, but not GPIb-IX-V, stimulates significant tyrosine phosphorylation of Syk at the autophosphorylation site pY525/526, a marker of Syk activity. In addition, GPVI stimulates tyrosine phosphorylation of PLCγ2 at Tyr753 and Tyr759, whereas GPIb-IX-V only induces significant phosphorylation at Tyr753. Both receptors stimulate tyrosine phosphorylation of Btk at the regulatory Tyr223 and Tyr551. Syk and Btk phosphorylate peptides from PLCγ2 containing Tyr753 and Tyr759 respectively, suggesting that they may stimulate phosphorylation at these sites in phospholipase. Studies using PLCγ2-deficient platelets demonstrated that phospholipase is not required for the activation of integrin αIIbβ3 by GPIb-IX-V. Our results demonstrate fundamental differences between GPVI and GPIb-IX-V in the regulation of tyrosine phosphorylation of Syk and PLCγ2 consistent with the functional impairment of phospholipase in signalling by GPIb-IX-V.
- blood platelet
- glycoprotein VI (GPVI)
- glycoprotein Ib-IX-V (GPIb-IX-V)
- phospholipase Cγ2 (PLCγ2)
- von Willebrand factor (vWF)
↵2 Present address: Pathway Discovery, Genomic and Proteomic Sciences, GlaxoSmithKline, Medicines Research Centre, Gunnels Wood Road, Stevenage SG1 2NY, U.K.
↵3 Present address: Ahringer Laboratory, Wellcome/CR U.K. Institute, Tennis Court Road, Cambridge CB2 1QR, U.K.
Abbreviations used: GP, glycoprotein; ITAM, immunoreceptor tyrosine-based activation motif; mAb, monoclonal antibody; pAb, polyclonal antibody; PLCγ2, phospholipase Cγ2; SLP-76, SH2-containing leucocyte phosphoprotein of 76 kDa; vWF, von Willebrand factor.
- The Biochemical Society, London ©2004