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 ␥2. 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.
IntroductionThe C-type lectin receptor CLEC-2 is expressed on platelets and on a subpopulation of other hematopoietic cells, including mouse neutrophils and dendritic cells. 1-3 CLEC-2 is a receptor for the snake venom toxin rhodocytin 4 and the transmembrane protein podoplanin, 5,6 which is expressed on the leading edge of tumor cells and on kidney podocytes, lung type 1 alveolar cells, and lymphatic endothelium. In addition, recent evidence suggests that activated platelets express or release a ligand for CLEC-2 that supports platelet aggregation at arteriolar rates of flow. 7 Mice pretreated with a specific antibody to CLEC-2 exhibit a selective loss of the C-type lectin receptor and impaired platelet activation on collagen at high shear in vitro or in vivo. 7 Cross-linking of CLEC-2 by rhodocytin, podoplanin, or specific antibodies elicits powerful platelet aggregation and secretion. 4,6 CLEC-2 signals through Src-and Syk-dependent tyrosine kinases, leading to phosphorylation of a series of adapter and effector proteins that culminate in activation of phospholipase-C ␥2 (PLC␥2) and platelet activation. 4 This mechanism of platelet activation resembles that used by the immunoglobulin collagen receptor, glycoprotein VI (GPVI), which is constitutively associated with the FcR␥ chain at the platelet surface. Cross-linking of GPVI by collagen or specific agonists, such as the snake venom toxin convulxin or antibodies, leads to Src kinase-dependent phosphorylation of 2 conserved tyrosines in the FcR␥-chain immunoreceptor tyrosine-based activation motif (ITAM). 8,9 ITAMs are present in a variety of hematopoietic receptors, including T-and B-cell antigen receptors and th...