A global proteomics approach identifies novel phosphorylated signaling proteins in GPVI-activated platelets: involvement of G6f, a novel platelet Grb2-binding membrane adapter.
Collagen-related peptide (CRP) stimulates powerful activation of platelets through the glycoprotein VI (GPVI)-FcR gamma-chain complex. We have combined proteomics and traditional biochemistry approaches to study the proteome of CRP-activated platelets, focusing in detail on tyrosine phosphorylation. In two separate approaches, phosphotyrosine immunoprecipitations followed by 1-D-PAGE, and 2-DE, were used for protein separation. Proteins were identified by MS. By following these approaches, 96 proteins were found to undergo PTM in response to CRP in human platelets, including 11 novel platelet proteins such as Dok-1, SPIN90, osteoclast stimulating factor 1, and beta-Pix. Interestingly, the type I transmembrane protein G6f was found to be specifically phosphorylated on Tyr-281 in response to platelet activation by CRP, providing a docking site for the adapter Grb2. G6f tyrosine phoshporylation was also found to take place in response to collagen, although not in response to the G protein-coupled receptor agonists, thrombin and ADP. Further, we also demonstrate for the first time that Grb2 and its homolog Gads are tyrosine-phosphorylated in CRP-stimulated platelets. This study provides new insights into the mechanism of platelet activation through the GPVI collagen receptor, helping to build the basis for the development of new drug targets for thrombotic disease.
García
Angel
A
Department of Biochemistry, Oxford Glycobiology Institute, University of Oxford, Oxford, UK. agarcia@usc.es
Senis
Yotis A
YA
Antrobus
Robin
R
Hughes
Craig E
CE
Dwek
Raymond A
RA
Watson
Steve P
SP
Zitzmann
Nicole
N
D000595:Amino Acid Sequence; D001792:Blood Platelets / Q000737:chemistry / Q000378:metabolism*; D002097:C-Reactive Protein / Q000502:physiology; D002478:Cells, Cultured; D051380:GRB2 Adaptor Protein / Q000378:metabolism* / Q000502:physiology; D006801:Humans; D007136:Immunoglobulins / Q000378:metabolism* / Q000502:physiology; D008969:Molecular Sequence Data; D010766:Phosphorylation; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism* / Q000502:physiology; D011485:Protein Binding / Q000502:physiology; D020543:Proteome / Q000032:analysis / Q000378:metabolism* / Q000502:physiology; D040901:Proteomics; D014443:Tyrosine / Q000378:metabolism
D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D007136:Immunoglobulins; C477999:LY6G6F protein, human; D010980:Platelet Membrane Glycoproteins; D020543:Proteome; C098341:platelet membrane glycoprotein VI; D014443:Tyrosine; D002097:C-Reactive Protein