CLEC-2 activates Syk through dimerization

Abstract

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...

Fucoidan Is a Novel Platelet Agonist for the C-type Lectin-like Receptor 2 (CLEC-2)

“…CLEC-2, a C-type lectin receptor, has been shown to signal independently of the FcR␥ chain in platelets (16,34). The presence of Syk phosphorylation (38) and detectable levels of Lat phosphorylation in platelets activated with fucoidan ( Fig. 3) suggested that CLEC-2 (39) may be a possible receptor candidate for fucoidan on platelets.…”

“…2-4). Downstream of the GPVI or CLEC-2 receptor, Syk kinase has been shown to play an important role in activating other effectors, which ultimately leads to the activation of ␣IIb␤3 (38,44). Inhibition of Syk following GPVI stimulation shuts down all functional responses.…”

A novel interaction between FlnA and Syk regulates platelet ITAM-mediated receptor signaling and function

“…Active PLC-2 hydrolyzes membrane bound polyphosphoinositides, notably phosphatidylinositol 4,5-bisphosphate, to form soluble inositol 1,4,5-trisphosphate, which releases cytosolic calcium from the internal stores, and membrane-bound 1,2-diacylglycerol, which activates protein kinase C. Signals are also sent to phosphoinositide 3-kinase, generating D3-containing polyphosphoinositides (Pasquet et al, 1999). CLEC-2 only contains a single YXXL motif but appears to be active as a dimer (Watson et al, 2009;Hughes et al, 2010) and signals through many of the same components as GPVI because platelets lacking LAT, SLP-76, or PLC-2 are not receptive to ligation of this receptor with rhodocytin (SuzukiInoue et al, 2006;Fuller et al, 2007).…”

Src family kinases: at the forefront of platelet activation

“…65 Unlike the FcR g-chain and FcgRIIA that contain tandem tyrosine residues in their ITAMs, CLEC-2 contains a single tyrosine residue in its hemi-ITAM that binds a single Syk SH2 domain with low affinity when phosphorylated. 66 Tandem phosphorylated CLEC-2 receptors are thus required to provide a high-affinity docking site for a single Syk molecule. 66 Rhodocytinmediated tyrosine phosphorylation of CLEC-2 and downstream signaling proteins is inhibited in human platelets treated with PP2, which suggests an important role of SFKs in initiating CLEC-2 signaling.…”

“…66 Tandem phosphorylated CLEC-2 receptors are thus required to provide a high-affinity docking site for a single Syk molecule. 66 Rhodocytinmediated tyrosine phosphorylation of CLEC-2 and downstream signaling proteins is inhibited in human platelets treated with PP2, which suggests an important role of SFKs in initiating CLEC-2 signaling. 67 However, phosphorylation of the CLEC-2 is normal in rhodocytin-stimulated Src-, Lyn-and Fyn-deficient mouse platelets.…”