The platelet receptor CLEC-2 is active as a dimer.
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.
Watson
Aleksandra A
AA
Henry Wellcome Building for Molecular Physiology, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK.
Christou
Charita M
CM
James
John R
JR
Fenton-May
Angharad E
AE
Moncayo
Gerald E
GE
Mistry
Anita R
AR
Davis
Simon J
SJ
Gilbert
Robert J C
RJ
Chakera
Aron
A
O'Callaghan
Chris A
CA
D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002850:Chromatography, Gel; D003553:Cystine / Q000032:analysis / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D047468:Immunoprecipitation; D019169:Jurkat Cells; D037181:Lectins, C-Type / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008027:Light; D013058:Mass Spectrometry; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D056004:Molecular Dynamics Simulation; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D055503:Protein Multimerization / Q000502:physiology*; D034622:RNA Interference; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012542:Scattering, Radiation; D020349:Surface Plasmon Resonance; D014162:Transfection; D014461:Ultracentrifugation; D014757:Viper Venoms / Q000737:chemistry; D000098853:Podoplanin
D003553:Cystine; D037181:Lectins, C-Type; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D014757:Viper Venoms; C104778:CLEC2B protein, human; C117468:PDPN protein, human; D000098853:Podoplanin; C112292:rhodocytin protein, Calloselasma rhodostoma