CLEC-2 is a 32 kDa C-type lectin-like immune receptor (Colonna et al. 2000; O’Callaghan 2009). CLEC-2 (gene name CLEC1B) is part of the NK (natural killer) gene cluster found on human chromosome 12 and mouse chromosome 6. Within this cluster, CLEC-2 is part of the Dectin-1 subfamily which consists of all type 2 transmembrane proteins with extracellular C-type lectin-like domains (CTLDs) and immune or homeostatic roles. Other members of this cluster include: CLEC-1, Dectin-1, CLEC8A (Lox-1), CLEC9A, CLEC12A, and CLEC12B.
CLEC-2 is expressed on the surface of platelets and ligand binding results in platelet activation and aggregation (Suzuki-Inoue et al. 2006). CLEC-2 is also expressed on megakaryocytes and liver sinusoidal endothelial cells (Chaipan et al. 2006), and transcripts have been reported in peripheral blood cells, including NK cells, monocytes, granulocytes, and dendritic cells (Colonna et al. 2000; Suzuki-Inoue et al. 2006; Fuller et al. 2007; Christou et al. 2008). CLEC-2 has also been shown to be a phagocytic receptor on mouse neutrophils. There are two currently identified ligands for CLEC-2: the snake venom protein rhodocytin and the endogenous protein podoplanin (Suzuki-Inoue et al. 2006, 2007; Christou et al. 2008). There is an increasing appreciation of the role of CLEC-2 in the lymphatic system and in development.
Sugar-binding C-type lectins typically coordinate calcium ions as part of their interaction with a sugar. However, CLEC-2 does not contain the residues required for calcium coordination, and no Ca2+ ions were seen in the CLEC-2 crystal structure, (Watson et al. 2007). The CLEC-2 ligand rhodocytin is unglycosylated, indicating that this interaction is not mediated by sugars. CLEC-2 has been shown to be found at the cell surface as a nondisulphide-linked homodimer (Watson et al. 2009). CLEC-2 has been crystallized in complex with rhodocytin and separately in complex with a short glycosylated peptide derived from podoplanin (Nagae et al. 2014). Both ligands share one region of interaction with CLEC-2, but each also has a further site of interaction that is not shared by the other ligand.
CLEC-2 has a single conserved YxxL motif in its cytoplasmic domain (Colonna et al. 2000). This YxxL motif is preceded by three acidic residues and has been termed a hemiTAM. As CLEC-2 is dimeric at the cell surface, two such motifs will be in close proximity (Watson et al. 2009) (Fig. 1).
Binding of ligand or antibody to CLEC-2 triggers phosphorylation of the tyrosine residue in the YxxL motif (Suzuki-Inoue et al. 2006), and mutation of this tyrosine prevents signaling (Fuller et al. 2007). Within CLEC-2, a triacidic amino acid sequence is required for phosphorylation of the YxxL motif (Hughes et al. 2013). Upon ligand engagement, CLEC-2 translocates to lipid rafts, and this translocation appears essential for phosphorylation and, therefore, signaling (Pollitt et al. 2010). Tyrosine phosphorylation is mediated by Src kinases, and Src kinase inhibition prevent signaling (Fuller et al. 2007). CLEC-2 phosphorylation results in binding to, and phosphorylation of, Syk (spleen tyrosine kinase) (Suzuki-Inoue et al. 2006; Fuller et al. 2007). This initiates a signaling cascade culminating in phosphorylation of PLCγ2 (phospholipase-Cγ2) (Suzuki-Inoue et al. 2006; Fuller et al. 2007). Both of the SH2 (Src-homology domain 2) domains of Syk are required for productive binding to phosphorylated CLEC-2 (Fuller et al. 2007). Since CLEC-2 only has a single YxxL motif, these two SH2 domains must therefore bind to two chains of CLEC-2 (Hughes et al. 2010b), although whether this is achieved through binding to two YxxLs within a single CLEC-2 dimer or to one from each of two dimers is not currently understood. Downstream phosphorylation has additionally been shown of Vav3, LAT, Btk, and SLP-76 (Src-homology 2 domain-containing leukocyte protein of 76 kDa), although activation through CLEC-2 is only partially dependent upon the SLP-76/Blnk family of adapter molecules (Suzuki-Inoue et al. 2006; Fuller et al. 2007).
Two ligands, rhodocytin and podoplanin, have been identified for CLEC-2 (Suzuki-Inoue et al. 2006; Suzuki-Inoue et al. 2007; Christou et al. 2008). Rhodocytin (also known as aggretin) is a venom protein from the Malayan pit viper Calloselasma rhodostoma that was known to activate platelets; CLEC-2 was identified as its ligand by affinity purification and mass spectrometry (Suzuki-Inoue et al. 2006). The crystal structure of rhodocytin shows it to be a tetramer, consisting of two heterodimers each composed of an α and a β chain (Watson et al. 2007; Hooley et al. 2008). These α and β chains each contain C-type lectin-like domains. Rhodocytin binds directly to CLEC-2 and is not glycosylated (Watson et al. 2007).
Podoplanin (also known as aggrus) is an endogenous ligand for CLEC-2 and binds directly to CLEC-2 (Suzuki-Inoue et al. 2007; Christou et al. 2008). Podoplanin was identified as a ligand for CLEC-2 when similarities were noted between the signaling profiles of platelets stimulated with rhodocytin and podoplanin (Watanabe et al. 1990; Suzuki-Inoue et al. 2006). Podoplanin is a heavily O-glycosylated type 1 membrane protein with a short cytoplasmic region. However, it seems unlikely that CLEC-2 could recognize a carbohydrate ligand in addition to the nonglycosylated protein rhodocytin. A recent study indicates that the state of glycosylation of podoplanin does not have a significant effect on the interaction with CLEC-2 (Cueni et al. 2010).
Podoplanin is expressed on podocytes in the kidney (from where it takes its name) and on lymphatic endothelial cells, but not upon vascular endothelial cells. Podoplanin expression has also been reported in skeletal tissue, muscle, type I alveolar cells in the lung, heart, myofibroblasts in breast and salivary glands, osteoblasts, certain mesothelial cells, and follicular dendritic cells. Podoplanin is expressed by several tumor types, and expression at the leading edge of some tumors has been observed. CLEC-2 has been identified as an attachment factor for HIV-1, facilitating capture of the virus by platelets (Chaipan et al. 2006). CLEC-2 appears to bind to podoplanin incorporated into the virion from the virus-producing cells (Chaipan et al. 2010). Expression of podoplanin has been shown on inflammatory macrophages which can activate murine platelets through CLEC-2 (Kerrigan et al. 2012). Podoplanin expression was identified in smooth muscle cells and macrophages in human atherosclerotic lesions, and expression was increased in advanced lesions (Hatakeyama et al. 2012). The interaction of CLEC-2 on dendritic cells with podoplanin on stromal cells plays a role in dendritic cell motility (Acton et al. 2012).
A number of groups have generated murine CLEC-2 knockout models which are embryonic/neonatal lethal (Bertozzi et al. 2010; Hughes et al. 2010a; Suzuki-Inoue et al. 2010; Tang et al. 2010). Knockout results in severe defects in lymphatic development with blood-filled lymphatics, indicating a failure of blood/lymphatic separation. Similarly, mice deficient in podoplanin die at birth from respiratory failure and also have a failure of blood/lymphatic separation (Schacht et al. 2003). Indeed, the phenotypes of CLEC-2, podoplanin, Syk, SLP76, PLCγ2, and platelet-defective mice all share similar characteristics of a failure in blood/lymphatic separation (Abtahian et al. 2003; Schacht et al. 2003; Ichise et al. 2009; Bertozzi et al. 2010; Hughes et al. 2010a; Suzuki-Inoue et al. 2010; Tang et al. 2010). This suggests that the developmental interaction between CLEC-2 on platelets and podoplanin on the future lymphatic endothelial cells may induce separation of the vascular and lymphatic systems by triggering platelet aggregation, so sealing the future lymphatic vessels. The development and maintenance of normal lymph nodes required CLEC-2 expression (Benezech et al. 2014). Lymph node expansion is diminished in the absence of CLEC-2 (Acton et al. 2014). Antibody-mediated depletion of CLEC-2 from circulating platelets results in normal adhesion, but reduced aggregation, of platelets under flow conditions and is associated with increased bleeding times and inhibition of occlusive arterial thrombus formation (May et al. 2009). One of the knockout studies in which irradiated wild-type mice were reconstituted with CLEC-2-deficient fetal liver cells and so have CLEC-2-deficient platelets also showed a decrease in aggregation under flow and observed defects in thrombosis formation following injury, supporting a role for CLEC-2 in stabilizing thrombus formation (Suzuki-Inoue et al. 2010). However, a second, independent, knockout study observed no decrease in platelet aggregation under shear conditions (Hughes et al. 2010a). Neither of these mouse knockout studies reported statistically significant decreases in tail bleeding times (Hughes et al. 2010a; Suzuki-Inoue et al. 2010), in contrast to the study in which CLEC-2 expression was reduced by antibody administration (May et al. 2009), and the role of CLEC-2 in hemostasis and thrombosis remains unclear. Conditional knockout of CLEC-2 in the megakaryocyte lineage causes abnormalities of both brain vascular and lymphatic development (Finney et al. 2012).
CLEC-2 is an important platelet receptor, which may also have an immune role. Ligand engagement of CLEC-2 results in tyrosine phosphorylation in its cytoplasmic YXXL motif and triggers platelet activation and aggregation. Knockout models reveal a key role for CLEC-2 in blood/lymphatic separation during development. For the function of CLEC-2 beyond platelets to be understood, its cellular expression pattern and the regulation of this expression need further study, especially in immune cells.
The involvement of CLEC-2 in platelet aggregation makes it a key medical target in a variety of contexts, including myocardial infarction and ischemic stroke (O’Callaghan 2009). The structure of podoplanin should accelerate the development of reagents to inhibit the CLEC-2 axis.
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