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The C-type lectin receptor CLEC-2 activates platelets through Src and Syk tyrosine kinases, leading to tyrosine phosphorylation of downstream adapter proteins and effector enzymes, including phospholipase-C gamma2. Signaling is initiated through phosphorylation of a single conserved tyrosine located in a YxxL sequence in the CLEC-2 cytosolic tail. The signaling pathway used by CLEC-2 shares many similarities with that used by receptors that have 1 or more copies of an immunoreceptor tyrosine-based activation motif, defined by the sequence Yxx(L/I)x(6-12)Yxx(L/I), in their cytosolic tails or associated receptor chains. Phosphorylation of the conserved immunoreceptor tyrosine-based activation motif tyrosines promotes Syk binding and activation through binding of the Syk tandem SH2 domains. In this report, we present evidence using peptide pull-down studies, surface plasmon resonance, quantitative Western blotting, tryptophan fluorescence measurements, and competition experiments that Syk activation by CLEC-2 is mediated by the cross-linking through the tandem SH2 domains with a stoichiometry of 2:1. In support of this model, cross-linking and electron microscopy demonstrate that CLEC-2 is present as a dimer in resting platelets and converted to larger complexes on activation. This is a unique mode of activation of Syk by a single YxxL-containing receptor.

Original publication

DOI

10.1182/blood-2009-08-237834

Type

Journal article

Journal

Blood

Publication Date

08/04/2010

Volume

115

Pages

2947 - 2955

Keywords

Amino Acid Motifs, Blood Platelets, Enzyme Activation, Humans, Intracellular Signaling Peptides and Proteins, Lectins, C-Type, Membrane Glycoproteins, Phospholipase C gamma, Platelet Activation, Protein Multimerization, Protein-Tyrosine Kinases, Signal Transduction, Syk Kinase, src Homology Domains