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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Cell Line
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Cell Membrane / metabolism
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Chromatography, Gel
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Cystine / analysis
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Cystine / chemistry
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Fluorescence Resonance Energy Transfer
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Humans
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Immunoprecipitation
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Jurkat Cells
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Lectins, C-Type / chemistry*
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Lectins, C-Type / genetics
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Lectins, C-Type / metabolism*
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Light
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Mass Spectrometry
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Membrane Glycoproteins / chemistry*
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Membrane Glycoproteins / genetics
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Membrane Glycoproteins / metabolism*
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Molecular Dynamics Simulation
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Peptide Fragments / chemistry
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Peptide Fragments / genetics
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Peptide Fragments / metabolism
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Protein Binding / physiology
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Protein Multimerization / physiology*
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RNA Interference
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Recombinant Fusion Proteins / chemistry
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / metabolism
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Scattering, Radiation
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Surface Plasmon Resonance
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Transfection
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Ultracentrifugation
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Viper Venoms / chemistry
Substances
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CLEC2B protein, human
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Lectins, C-Type
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Membrane Glycoproteins
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PDPN protein, human
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Peptide Fragments
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Recombinant Fusion Proteins
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Viper Venoms
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rhodocytin protein, Calloselasma rhodostoma
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Cystine