TNF receptor-associated factor 4 (TRAF4) is a novel binding partner of glycoprotein Ib and glycoprotein VI in human platelets.
Reactive oxygen species generation is one consequence of ligand engagement of platelet glycoprotein (GP) receptors GPIb-IX-V and GPVI, which bind VWF/collagen and initiate thrombosis at arterial shear; however, the precise molecular mechanism coupling redox pathway activation to engagement of these receptors is unknown.The objective of this study was to identify ... novel binding partners for GPIb-IX-V and GPVI that could provide a potential link between redox pathways and early platelet signaling events.Using protein array analysis and affinity-binding assays, we demonstrated that the orphan TNF receptor-associated factor (TRAF) family member, TRAF4, selectively binds cytoplasmic sequences of GPIbβ and GPVI. TRAF4, p47(phox) [of the NADPH oxidase (Nox2) enzyme complex] and other redox relevant signaling proteins such as Hic-5, co-immunoprecipitate with GPIb/GPVI from human platelet lysates whilst MBP-TRAF4 or MBP-p47(phox) fusion proteins specifically pull-down GPIb/GPVI. GPIb- or GPVI-selective agonists induce phosphorylation of the TRAF4-associated proteins, Hic-5 and Pyk2, with phosphorylation attenuated by Nox2 inhibition.These results describe the first direct association of TRAF4 with a receptor, and identify a novel binding partner for GPIb-IX-V and GPVI, providing a potential link between these platelet receptors and downstream TRAF4/Nox2-dependent redox pathways.
Mesh Terms:
Blood Platelets, Chromatography, Affinity, Focal Adhesion Kinase 2, Humans, Immunoprecipitation, Intracellular Signaling Peptides and Proteins, LIM Domain Proteins, NADPH Oxidase, Oxidation-Reduction, Phosphorylation, Platelet Glycoprotein GPIb-IX Complex, Platelet Membrane Glycoproteins, Protein Array Analysis, Protein Binding, Protein Interaction Domains and Motifs, Protein Interaction Mapping, Recombinant Fusion Proteins, Signal Transduction, TNF Receptor-Associated Factor 4
Blood Platelets, Chromatography, Affinity, Focal Adhesion Kinase 2, Humans, Immunoprecipitation, Intracellular Signaling Peptides and Proteins, LIM Domain Proteins, NADPH Oxidase, Oxidation-Reduction, Phosphorylation, Platelet Glycoprotein GPIb-IX Complex, Platelet Membrane Glycoproteins, Protein Array Analysis, Protein Binding, Protein Interaction Domains and Motifs, Protein Interaction Mapping, Recombinant Fusion Proteins, Signal Transduction, TNF Receptor-Associated Factor 4
J. Thromb. Haemost.
Date: Jan. 01, 2011
PubMed ID: 20946164
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