The Platelet Receptor CLEC-2 Is Active as a Dimer

Abstract

The platelet receptor CLEC-2 binds to the snake venom toxin rhodocytin and the tumor cell surface protein podoplanin. Binding of either of these ligands promotes phosphorylation of a single tyrosine residue in the YXXL motif in the intracellular domain of CLEC-2. Phosphorylation of this tyrosine initiates binding of spleen tyrosine kinase (Syk) and triggers further downstream signaling events and ultimately potent platelet activation and aggregation. However, it is unclear how a single YXXL motif can interact efficiently with Syk, which usually recognizes two tandem YXXL repeats presented as an immunoreceptor tyrosine-based activation motif (ITAM). Using bioluminescence resonance energy transfer, coimmunopreciptitation, recombinant protein expression and analytical gel filtration chromatography, surface plasmon resonance, Western blotting, multiangle light scattering (MALS), and analytical ultracentrifugation, we show that CLEC-2 exists as a non-disulfide-linked homodimer which could allow each Syk molecule to interact with two YXXL motifs, one from each CLEC-2 monomer.

Structural Flexibility of the Macrophage Dengue Virus Receptor CLEC5A

“…The BRETeff maxima for the CLEC5A BRET pairs reside between the documented BRETeff maxima for known monomers and for disulfide-linked homodimers, and the curve shape is typical of nonconstitutive homodimerization (20). These curves resemble those we previously reported for the nonconstitutive homodimer and related C-type lectin-like protein CLEC-2 and indicate that human CLEC5A homodimerizes at the cell surface and that this dimerization occurs independently of DAP12 (20,32). A model of dimeric CLEC5A based on the CLEC5A crystal structure and standard dimerization arrangement for related molecules (Fig.…”

“…CLEC5A Forms Homodimers At Cell Surface-Many C-type lectin-like molecules bind their ligands as dimers (26,29,30,32,33). Additionally, all DAP12-associated C-type lectin-like molecules are dimeric (34).…”

“…CLEC5A crystallized as a monomer, and the site on its surface equivalent to the homodimerization site of many C-type lectin-like proteins does not possess a surface distribution of hydrophobic patches sufficient to dictate constitutive dimerization as observed in some family members, such as the immune receptor NKG2D and members of the Ly49 receptor family (26,29). However, we have recently shown that the related receptor CLEC-2, which similarly lacks a prominent "dimerization patch," does indeed form dimers in the plasma membrane, and this dimerization is central to its function (32). This and the homodimeric nature of DAP12 prompted us to investigate whether CLEC5A can similarly homodimerize in the membrane.…”

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

“…These data suggest that fucoidan does not mediate its effects primarily through the GPVI or Fc␥RIIA receptor. 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.…”

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).…”