Evidence that phospholipase C-gamma 2 interacts with SLP-76, Syk, Lyn, LATand the Fc receptor gamma-chain after stimulation of the collagen receptorglycoprotein VI in human platelets

Platelet activation by collagen is mediated by the sequential tyrosine phos phorylation of the Fc receptor gamma-chain (FcR gamma-chain), which is part of the collagen receptor glycoprotein VI, the tyrosine kinase Syk and phos pholipase C-gamma 2 (PLC-gamma 2). In this study tyrosine-phosphorylated pr oteins that associate with PLC-gamma 2 after stimulation by a collagen-rela ted peptide (CRP) were characterized using glutathione S-transferase fusion proteins of PLC-gamma 2 Src homology (SK) domains and by immunoprecipitati on of endogenous PLC-gamma 2. The majority of the tyrosine-phosphorylated p roteins that associate with PLC-gamma 2 bind to its C-terminal SH2 domain. These were found to include PLC-gamma 2, Syk, SH2-domain-containing leucocy te protein of 76 kDa (SLP-76), Lyn, linker for activation of T cells (LAT) and the FcR gamma-chain. Direct association was detected between PLC-gamma 2 and SLP-76, and between PLC-gamma 2 and LAT upon CRP stimulation of plate lets by far-Western blotting. FcR gamma-chain and Lyn were found to co-immu noprecipitate with PLC-gamma 2 as well as with unidentified 110-kDa and 75- kDa phosphoproteins. The absence of an in vivo association between Syk and PLC-gamma 2 in platelets is in contrast with that for PLC-gamma 1 and Syk i n B cells. The in vivo function of PLC-gamma 2 SH2 domains was examined thr ough measurement of Ca2+ increases in mouse megakaryocytes that had been mi croinjected with recombinant proteins. This revealed that the C-terminal SH 2 domain is involved in the regulation of PLC-gamma 2. These data indicate that the C-terminal SH2 domain of PLC-gamma 2 is important for PLC-gamma 2 regulation through possible interactions with SLP-76, Syk, Lyn, LAT and the FcR gamma-chain.