Evidence that phospholipase C-gamma2 interacts with SLP-76, Syk, Lyn, LAT and the Fc receptor gamma-chain after stimulation of the collagen receptor glycoprotein VI in human platelets

• Published in: European journal of biochemistry Vol. 263; no. 3; pp. 612 - 623
• Main Authors: Gross, B S, Melford, S K, Watson, S P
• Format: Journal Article
• Language: English
• Published: England 01.08.1999
• Subjects: Adaptor Proteins, Signal Transducing; Animals; Blood Platelets - metabolism; Calcium - metabolism; Carrier Proteins - metabolism; Enzyme Precursors - metabolism; Glutathione Transferase - metabolism; Humans; Intracellular Signaling Peptides and Proteins; Isoenzymes - blood; Megakaryocytes - metabolism; Membrane Proteins - metabolism; Mice; Phospholipase C gamma; Phosphoproteins - isolation & purification; Phosphoproteins - metabolism; Phosphorylation; Polymerase Chain Reaction; Protein-Tyrosine Kinases - metabolism; Receptors, IgG - metabolism; Recombinant Fusion Proteins - metabolism; src Homology Domains; src-Family Kinases - metabolism; Syk Kinase; Thrombin - metabolism; Type C Phospholipases - blood
• ISSN: 0014-2956

Platelet activation by collagen is mediated by the sequential tyrosine phosphorylation of the Fc receptor gamma-chain (FcR gamma-chain), which is part of the collagen receptor glycoprotein VI, the tyrosine kinase Syk and phospholipase C-gamma2 (PLC-gamma2). In this study tyrosine-phosphorylated proteins that associate with PLC-gamma2 after stimulation by a collagen-related peptide (CRP) were characterized using glutathione S-transferase fusion proteins of PLC-gamma2 Src homology (SH) domains and by immunoprecipitation of endogenous PLC-gamma2. The majority of the tyrosine-phosphorylated proteins that associate with PLC-gamma2 bind to its C-terminal SH2 domain. These were found to include PLC-gamma2, Syk, SH2-domain-containing leucocyte 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-gamma2 and SLP-76, and between PLC-gamma2 and LAT upon CRP stimulation of platelets by far-Western blotting. FcR gamma-chain and Lyn were found to co-immunoprecipitate with PLC-gamma2 as well as with unidentified 110-kDa and 75-kDa phosphoproteins. The absence of an in vivo association between Syk and PLC-gamma2 in platelets is in contrast with that for PLC-gamma1 and Syk in B cells. The in vivo function of PLC-gamma2 SH2 domains was examined through measurement of Ca2+ increases in mouse megakaryocytes that had been microinjected with recombinant proteins. This revealed that the C-terminal SH2 domain is involved in the regulation of PLC-gamma2. These data indicate that the C-terminal SH2 domain of PLC-gamma2 is important for PLC-gamma2 regulation through possible interactions with SLP-76, Syk, Lyn, LAT and the FcR gamma-chain.