The snake venom rhodocytin has been reported to bind to integrin alpha2beta1 and glycoprotein (GP) Ibalpha on platelets, but it is also able to induce activation independent of the 2 receptors and of GPVI. Using rhodocytin affinity chromatography, we have identified a novel C-type lectin receptor, CLEC-2, in platelets that confers signaling responses to rhodocytin when expressed in a cell line. CLEC-2 has a single tyrosine residue in a YXXL motif in its cytosolic tail, which undergoes tyrosine phosphorylation upon platelet activation by rhodocytin or an antibody to CLEC-2, but not to collagen, thrombin receptor agonist peptide (TRAP), or convulxin. Tyrosine phosphorylation of CLEC-2 and other signaling proteins by rhodocytin is inhibited by the Src family kinase inhibitor PP2. Further, activation of murine platelets by rhodocytin is abolished in the absence of Syk and PLCgamma2, and partially reduced in the absence of LAT, SLP-76, and Vav1/Vav3. These findings define a novel signaling pathway in platelets whereby activation of CLEC-2 by rhodocytin leads to tyrosine phosphorylation of its cytosolic tail, binding of Syk and initiation of downstream tyrosine phosphorylation events, and activation of PLCgamma2. CLEC-2 is the first C-type lectin receptor to be found on platelets which signals through this novel pathway.

The two lectin receptors, CLEC-2 and Dectin-1, have been shown to signal through a Syk-dependent pathway, despite the presence of only a single YXXL in their cytosolic tails. In this study, we show that stimulation of CLEC-2 in platelets and in two mutant cell lines is dependent on the YXXL motif and on proteins that participate in signaling by immunoreceptor tyrosine-based activation motif receptors, including Src, Syk, and Tec family kinases, and on phospholipase C␥. Strikingly, mutation of either Src homology (SH) 2 domain of Syk blocks signaling by CLEC-2 despite the fact that it has only a single YXXL motif. Furthermore, signaling by CLEC-2 is only partially dependent on the BLNK/SLP-76 family of adapter proteins in contrast to that of immunoreceptor tyrosine-based activation motif receptors. The C-type lectin receptor, Dectin-1, which contains a YXXL motif preceded by the same four amino acids as for CLEC-2 (DEDG), signals like CLEC-2 and also requires the two SH2 domains of Syk and is only partially dependent on the BLNK/SLP-76 family of adapters. In marked contrast, the C-type lectin receptor, DC-SIGN, which has a distinct series of amino acids preceding a single YXXL, signals independent of this motif. A mutational analysis of the DEDG sequence of CLEC-2 revealed that the glycine residue directly upstream of the YXXL tyrosine is important for CLEC-2 signaling. These results demonstrate that CLEC-2 and Dectin-1 signal through a single YXXL motif that requires the tandem SH2 domains of Syk but is only partially dependent on the SLP-76/BLNK family of adapters.The C-type lectin superfamily of transmembrane proteins consists of at least 70 members in the human genome (1). The superfamily can be divided into "classical" C-type lectins, which contain a carbohydrate recognition domain and bind sugars in a calcium-dependent manner, and the "nonclassical" C-type lectin-like proteins, which contain a C-type lectin-like domain, homologous to a carbohydrate recognition domain, but lacks the consensus sequence for binding sugars and calcium (2). Protein ligands for a number of classical and nonclassical C-type lectin receptors have been described.C-type lectin-like receptor 2 (CLEC-2) 6 is a type II transmembrane protein and a nonclassical C-type lectin (3). The C-type lectin-like domain in CLEC-2 is supported by a 41-amino acid neck region, a single transmembrane domain, and 31-amino acid cytoplasmic domain (3). CLEC-2 mRNA has been identified in liver and in blood cells, mostly of myeloid origin, including monocytes, granulocytes, and dendritic cells (3). Recently, we have identified expression of CLEC-2 in platelets and have shown that it functions as a receptor for the snake venom toxin rhodocytin (also known as aggretin), which elicits powerful platelet activation (4). Rhodocytin, however, also binds to several other platelet receptors (5, 6), making it unclear whether CLEC-2 is sufficient to mediate activation alone and thereby hampering analysis of the mechanism of activation.The cytosolic domain of CLEC-2 con...

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