Open AccessEvidence that phospholipase C‐γ2 interacts with SLP‐76, Syk, Lyn, LAT and the Fc receptor γ‐chain after stimulation of the collagen receptor glycoprotein VI in human platelets
Author(s) -
Gross Barbara S.,
Melford Steven K.,
Watson Stephen P.
Publication year - 1999
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1327.1999.00560.x
Subject(s) - syk , lyn , tyrosine phosphorylation , sh2 domain , proto oncogene tyrosine protein kinase src , phosphorylation , platelet activation , tyrosine kinase , tyrosine , phospholipase c , microbiology and biotechnology , biochemistry , biology , chemistry , receptor , platelet , immunology
Platelet activation by collagen is mediated by the sequential tyrosine phosphorylation of the Fc receptor γ‐chain (FcR γ‐chain), which is part of the collagen receptor glycoprotein VI, the tyrosine kinase Syk and phospholipase C‐γ2 (PLC‐γ2). In this study tyrosine‐phosphorylated proteins that associate with PLC‐γ2 after stimulation by a collagen‐related peptide (CRP) were characterized using glutathione S‐transferase fusion proteins of PLC‐γ2 Src homology (SH) domains and by immunoprecipitation of endogenous PLC‐γ2. The majority of the tyrosine‐phosphorylated proteins that associate with PLC‐γ2 bind to its C‐terminal SH2 domain. These were found to include PLC‐γ2, Syk, SH2‐domain‐containing leucocyte protein of 76 kDa (SLP‐76), Lyn, linker for activation of T cells (LAT) and the FcR γ‐chain. Direct association was detected between PLC‐γ2 and SLP‐76, and between PLC‐γ2 and LAT upon CRP stimulation of platelets by far‐Western blotting. FcR γ‐chain and Lyn were found to co‐immunoprecipitate with PLC‐γ2 as well as with unidentified 110‐kDa and 75‐kDa phosphoproteins. The absence of an in vivo association between Syk and PLC‐γ2 in platelets is in contrast with that for PLC‐γ1 and Syk in B cells. The in vivo function of PLC‐γ2 SH2 domains was examined through measurement of Ca 2+ 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‐γ2. These data indicate that the C‐terminal SH2 domain of PLC‐γ2 is important for PLC‐γ2 regulation through possible interactions with SLP‐76, Syk, Lyn, LAT and the FcR γ‐chain.