Phosphorylation of CLEC-2 is dependent on lipid rafts, actin polymerization, secondary mediators, and Rac

Abstract

The C-type lectin-like receptor 2 (CLEC-2) activates platelets through Src and Syk tyrosine kinases via a single cytoplasmic YxxL motif known as a hem immunoreceptor tyrosine-based activation motif (hemI-TAM). Here, we demonstrate using sucrose gradient ultracentrifugation and methyl-␤-cyclodextrin treatment that CLEC-2 translocates to lipid rafts upon ligand engagement and that translocation is essential for hemITAM phosphorylation and signal initiation. HemITAM phosphorylation, but not translocation, is also critically dependent on actin polymerization, Rac1 activation, and release of ADP and thromboxane A 2 (TxA 2 ). The role of ADP and TxA 2 in mediating phosphorylation is dependent on ligand engagement and rac activation but is independent of platelet aggregation. In contrast, tyrosine phosphorylation of the GPVIFcR␥-chain ITAM, which has 2 YxxL motifs, is independent of actin polymerization and secondary mediators. These results reveal a unique series of proximal events in CLEC-2 phosphorylation involving actin polymerization, secondary mediators, and Rac activation. (Blood. 2010; 115(14):2938-2946) IntroductionDectin-1, C-type lectin-like receptor 2 (CLEC-2), and CLEC-9A are C-type lectin receptors which have been shown to signal through an immunoreceptor tyrosine-based activation motif (ITAM)-like pathway via a single YxxL in their short cytoplasmic tails known as a hemITAM. [1][2][3][4] Tyrosine phosphorylation of the conserved tyrosine and both SH2 domains of the tyrosine kinase Syk have been shown to be essential for activation of Syk by CLEC-2 and dectin-1, favoring a model in which the tyrosine kinase is activated by the cross-linking of 2 receptors. 2 This is consistent with the observations that CLEC-9A is a disulphide-linked dimer and that CLEC-2 is expressed as a noncovalent dimer. 1,5 CLEC-2 is highly expressed in platelets and at lower levels in other hematopoietic cells, including neutrophils and dendritic cells. 6,7 It is a type II transmembrane protein and a nonclassical C-type lectin that lacks the conserved amino acids that mediate binding to glycans. 7 CLEC-2 was identified by affinity chromatography as a ligand for the snake venom protein rhodocytin, purified from the Malayan pit viper, Calloselasma rhodostoma. 3 Independent crystal structures of rhodocytin show that it assembles as a tetramer, which leads to the suggestion that it mediates activation of CLEC-2 through clustering. 8,9 Consistent with this clustering model of activation, whole antibodies and F(ab) 2 fragments have been reported to activate CLEC-2, whereas antibody Fab fragments were inhibitory. 3,10,11 Physiologic roles of CLEC-2 are now beginning to emerge. Recently, CLEC-2 has been implicated in tumor metastasis via its interaction with the type I sialoglycoprotein podoplanin, which is also expressed on the surface of kidney podocytes, lung type I alveolar cells, and lymphatic endothelium. 12-14 CLEC-2 has also been shown to play a role in neutrophil phagocytosis. 15 Evidence obtained using antibody depletion of CLEC-2...

RhoG Protein Regulates Glycoprotein VI-Fc Receptor γ-Chain Complex-mediated Platelet Activation and Thrombus Formation

“…Previous studies have shown that Rac1 is activated in platelets by a number of agonists (45)(46)(47) and plays an important role in aggregation, secretion, and clot retraction. Interestingly, Rac1 is known to be essential for the regulation of PLC␥2 in platelets downstream of GPVI or CLEC-2 (48,49). In the presence of pharmacological inhibitors of Rac, as well as in Rac1-deficient murine platelets, agonistinduced dense granule secretion is inhibited (50).…”

New Fundamentals in Hemostasis

“…Clustering of CLEC-2 evokes a similar set of signaling events as seen with GPVI, including tyrosine phosphorylation by SFK, Syk, and Tec-family kinases and signal complex assembly around scaffold proteins (313). Platelet stimulation via CLEC-2 relies on autocrine effects via release of secondary mediators like ADP and thromboxane A 2 (233). There is still discussion on the possible existence of a physiological ligand for CLEC-2 receptors on platelets, which could potentially be CLEC-2 itself (281).…”

HemITAM: A single tyrosine motif that packs a punch

“…However, a later study showed that neither CLEC-2 phosphorylation nor subsequent Syk activation depends on lipid raft integrity, leading to the proposal that lipid raft disruption inhibits the actions of secondary mediators necessary for CLEC-2-triggered platelet aggregation rather than directly interfering with CLEC-2 phosphorylation (85). In addition, CLEC-2 phosphorylation and activation of Syk were also inhibited when actin polymerization was blocked by cytochalasin D, whereas Syk activation stimulated by the ITAM-coupled GPVI receptor was unaffected (84). It remains to be shown whether membrane localization and actin polymerization also affect the function of other hemITAM receptors.…”

“…CLEC-2 tyrosine phosphorylation and subsequent platelet aggregation were originally reported to require translocation of CLEC-2 into lipid rafts upon ligand engagement (84). However, a later study showed that neither CLEC-2 phosphorylation nor subsequent Syk activation depends on lipid raft integrity, leading to the proposal that lipid raft disruption inhibits the actions of secondary mediators necessary for CLEC-2-triggered platelet aggregation rather than directly interfering with CLEC-2 phosphorylation (85).…”