Glycoprotein VI oligomerization in cell lines and platelets

Berlanga O, Bori-Sanz T, James JR, Frampton J, Davis SJ, Tomlinson MG, Watson SP. (2007), J Thromb Haemost. 5, 1026-33

Abstract

BACKGROUND: Glycoprotein VI (GPVI) is a physiologic receptor for collagen expressed at the surface of platelets and megakaryocytes. Constitutive dimerization of GPVI has been proposed as being necessary for the interaction with collagen, although direct evidence of dimerization has not been reported in cell lines or platelets.

OBJECTIVES: To investigate oligomerization of GPVI in transfected cell lines and in platelets under non-stimulated conditions.

METHODS AND RESULTS: By using a combination of molecular and biochemical techniques, we demonstrate that GPVI association occurs at the surface of transfected 293T cells under basal conditions, through an interaction at the extracellular domain of the receptor. Bioluminescence resonance energy transfer was used to confirm oligomerization of GPVI under these conditions. A chemical crosslinker was used to detect constitutive oligomeric forms of GPVI at the surface of platelets, which contain the Fc receptor (FcR) gamma-chain.

CONCLUSIONS: The present results directly demonstrate GPVI-FcR gamma-chain oligomerization at the surface of the platelet, and thereby add to the growing evidence that oligomerization of GPVI may be a prerequisite for binding of the receptor to collagen, and therefore for proper functioning of platelets upon vascular damage.

Key figure: Bioluminescence resonance energy transfer (BRET) analysis of 293T cells transfected with glycoprotein VI (GPVI)

(A) Cells were cotransfected with GPVI_GFP, GPVI_Luc and the Fc receptor (FcR) γ-chain (GPVI_BP), to demonstrate the dependence of BRET_eff values on the acceptor/donor ratio. These values were compared with the known monomer, CD2, and the disulfide-linked homodimer, CTLA-4. Cotransfection of GPVI_GFP with CD2_Luc showed an independence from acceptor/donor ratio, as expected for random interactions. All data are shown fitted to the most appropriate oligomerization model. BRET_eff values from GPVI_BP – expressing cells in the presence or absence of FcRγ-chain were measured to assess the effect of the latter on GPVI dimerization. (C) Incubation with collagen (20 μg mL⁻¹) or convulxin (10 μg mL⁻¹) did not increase the level of dimerization compared to a phosphate-buffered saline (PBS) control after 15 min. Results from three experiments are combined.