Receptor homodimerization significantly prolongs the lifetime of ligand-induced cross-linking of CLEC-2 but not GPVI.

The platelet immunoreceptor tyrosine-based activation motif receptors, glycoprotein VI (GPVI) and C-type lectin-like receptor 2 (CLEC-2), are activated by a diversity of ligands that bind to distinct epitopes, indicating that competitive antagonists will not block activation by all stimuli. In this study, we used 2-color single-particle tracking to investigate the dynamic nanoscale organization of the 2 receptors in the cell membrane to identify new strategies for inhibition. The studies were performed in CHO-K1 cells that lack the tyrosine kinase Syk. The results show that, when expressed at low level, GPVI and CLEC-2 diffuse over the cell surface as monomers with the presence of dimers due to random collisions. The addition of divalent and trivalent nanobody ligands induces homodimerization and cessation of movement of both receptors proportionate to ligand valency. The dimers of CLEC-2 are longer-lived than those of GPVI, despite a lower affinity of the monomeric nanobody that forms the ligand backbone. The dimerization of recombinant monomeric CLEC-2 but not GPVI was detected by surface plasmon resonance with an affinity constant of 18.5μM. The results suggest that the prolonged lifetime of the CLEC-2 interactions is due to synergy between ligand-induced cross-linking and receptor homodimerization. Blocking dimerization may be an effective way to inhibit the activation of CLEC-2 by its diverse range of ligands.