Fc gamma RII tyrosine phosphorylation differs between Fc gamma RII cross-linking and platelet-activating anti-platelet monoclonal antibodies

Using glutathione S-transferase Syk fusion proteins, we evaluated the mode of platelet Fc gamma RII tyrosine phosphorylation induced by Fc gamma RII c ross-linking or anti-CD9 monoclonal antibodies (mAb). The N-terminal SH2 do main of Syk (Syk-NSH2), the C-terminal SH2 domain of Syk (Syk-C-SH2), and t he domain having both the N- and C-terminal SH2 of Syk (Syk-NC-SH2) all bou nd to tyrosine-phosphorylated Fc gamma RII with FcyRII cross-linking. In th e case of anti-CD9 mAb-induced platelet activation, only Syk-C-SH2 and Syk- NC-SH2. bound to tyrosine-phosphorylated FcyRII. Since the SH2 domain is sp ecific for a particular structure containing phosphotyrosine, these finding s suggest that only one tyrosine residue in the immunoreceptor tyrosine-bas ed activation motif (ITAM) is phosphorylated with anti-CD9 mAb, and that bo th are phosphorylated with FcyRII cross-linking. Synthetic peptides corresp onding to the ITAM of human platelet FcyRII with the N-terminal tyrosine re sidue phosphorylated (N-P) or the C-terminal tyrosine residue phosphorylate d (C-P), were used. N-P more potently dissociated Syk-C-SH2 from tyrosine-p hosphorylated FcyRII than C-P, suggesting that the N-terminal tyrosine resi due is phosphorylated upon anti-CD9 mAb-induced activation. Furthermore, th ese findings imply that Syk-N SH2 binds to the phosphorylated C-terminal ty rosine residue of ITAM, and Syk-C-SH2 to the N-terminal tyrosine. Taken tog ether, our findings suggest that Fc gamma RII-dependent platelet activation without FcyRII dimerization, such as with anti-CDB mAb, is distinct from t hat induced by FcyRII cross-linking. (C) 1999 Elsevier Science B.V. All rig hts reserved.