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Glycoprotein VI (GPVI) is a physiological receptor for collagen expressed at the surface of platelets and megakaryocytes. Constitutive dimerisation of GPVI has been proposed as necessary for the interaction with collagen, although direct evidence of dimerisation has not been reported in cell lines or platelets.
To investigate oligomerisation of GPVI in transfected cell lines and in platelets under nonstimulated conditions.
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 extra-cellular domain of the receptor. Bioluminescence resonance energy transfer was used to confirm oligomerisation of GPVI under these conditions. A chemical cross-linker was used to detect constitutive oligomeric forms of GPVI at the surface of platelets, which contain the FcR γ-chain.
The present results directly demonstrate GPVI-FcR γ-chain oligomerisation at the surface of the platelet, and thereby add to the growing evidence that oligomerisation of GPVI may be a pre-requisite for binding of the receptor to collagen, and therefore for proper functioning of platelets upon vascular damage.
The extracellular matrix protein collagen is the major and most thrombogenic component of the vessel wall. Circulating platelets adhere to exposed collagen and undergo activation, leading to thrombus formation. The interaction with collagen is mediated through two distinct receptor classes on the platelet surface, the integrin α2β1 and the GPVI-FcR γ-chain receptor complex . Blocking of either receptor in vitro using specific antibodies inhibits and delays collagen-induced platelet aggregation [2-4], respectively. Similarly, platelets deficient for α2β1 or GPVI-FcR γ-chain show loss of reactivity towards collagen in vitro [5-8]. Further, mice deficient for GPVI-FcR γ-chain are protected against lethal thromboembolism [8,9], illustrating the crucial role that the receptor plays in vivo under pathological conditions.
Although the intracellular signalling events mediated by α2β1 in platelets have remained elusive [10,11], the mechanism of action of GPVI has been well documented and remains an area of intense research. GPVI mediates platelet activation in response to collagen through a pathway that shares many features with those used by immune receptors such as FcεRI, and T and B cell antigen receptors . Since GPVI has no intrinsic signalling capacity, it is widely recognised that it must be co-expressed in association with the FcR γ-chain, which acts as the signalling partner. Further, this association is a pre-requisite for surface expression of GPVI on mouse platelets .
From a structure/function point of view, there are several lines of circumstantial evidence to suggest that GPVI functions as a dimer on the platelet surface. Moroi and co-workers have demonstrated, using recombinant protein, that collagen binds to the dimeric but not monomeric form of GPVI, and that only the former was able to attenuate collagen-induced platelet aggregation . In contrast, both monomeric and dimeric forms of GPVI bind to immobilised convulxin and inhibited platelet aggregation induced by the snake toxin with similar concentration dependencies . The possibility that GPVI functions as a dimer is strongly reinforced by studies analysing the ability of a series of synthetic peptides with differentially spaced GPVI-recognition motifs to activate the collagen receptor in platelets . Finally, structural studies of the two immunoglobulin (Ig) domains of human GPVI reveal formation of a back-to-back dimer in the crystal structure, which is mediated through the more membrane-proximal of the two Ig domains .
Since the FcR γ-chain is present as a disulphide-linked homodimer, it has been proposed that each chain associates independently with GPVI . In light of a recent report indicating that the two chains of the FcR γ-chain are necessary for binding a single GPVI molecule , this model needs to be reviewed. Further, direct evidence that GPVI is expressed at the cell surface as a dimer or possibly as a larger complex is not available. We set out to investigate this in platelets and in transfected cell lines using distinct biochemical and molecular approaches. Our results confirm that GPVI is capable of undergoing oligomerisation in transfected cells and forming oligomers in platelets, and indicate that a modified version of the current model for GPVI dimerisation may be necessary.
Convulxin was purchased from Latoxan (Valence, France). Anti-convulxin was a kind gift from Dr. Mireille Leduc (Institute Pasteur, Paris, France). Anti-CD2 antibody was kindly supplied by Dr. Vaclav Horejsi (Institute of Molecular Genetics, Academy of Sciences, Prague, Czech Republic). Anti FcR γ-chain was from Upstate Biotechnology (Buckingham, UK). Anti-Flag (M2) was from Sigma (Dorset, UK). Anti-Myc (9B11) was from Cell Signalling Technology (Hertfordshire, UK). All other reagents were from previously described sources  unless otherwise stated.
Human blood was taken from drug-free volunteers on the day of experiment using acidic citrate dextrose (120 mM sodium citrate, 110 mM glucose, 80 mM citric acid). Platelet-rich plasma was obtained by centrifugation at 200g for 20 min, and platelets were isolated by centrifugation at 1000g for 10 min in the presence of prostacyclin (0.1 μg/ml). Platelets were resuspended in modified Tyrode’s-Hepes buffer (134 mM NaCl, 2.9 mM KCl, 0.34 mM Na2HPO4.12H2O, 12 mM NaHCO3, 20 mM Hepes, 1 mM MgCl2, 5 mM glucose, pH 7.3 at 37°C) in the presence of prostacyclin (0.1 μg/ml), re-centrifuged at 1000 g for 10 min, and resuspended in the above buffer to a density of 5×108 cells/ml.
293T cells were grown in DMEM medium supplemented with 100 U/ml of penicillin, 100 μg/ml streptomycin and 10% heat-inactivated FBS under 5% CO2/95% air in a humidified incubator. Cells were kept at exponential phase of growth.
Cells were transfected using the calcium phosphate method as previously described , and incubated in complete medium for 48 hours prior to experimentation. For stable transfections, plasmid DNA was cut with an appropriate restriction enzyme, and after transfection following the above method, divided in 24 wells, and the required antibiotic for selection added. Approximately 10 days later individual clones of cells were selected and placed in 96-well plates for expansion.
GPVI-Flag and (Δ288)GPVI-Flag were subcloned into pRc plasmid, whereas FcR γ-chain was subcloned into pMG plasmid, as previously described (19). GPVI-Myc tagged was obtained by standard PCR using a vector primer (T7) and GPVI-Myc primer (5’-CCCTAAGCGGCCGCTCACAGATCCTCTTCTGAGATGAGTTTTTGTTCTGAACATAACCCGCGGC-3’). The final amplified product was digested with HindIII and NotI and inserted into similarly cut mammalian expression vector pcDNA 3.1. CD2 extra cellular domain was fused to transmembrane region and cytoplasmic tail of GPVI using standard overlapping PCR techniques. Extra cellular domain of CD2 was amplified using oligo 1 (5’-CCCTAAAAGCTTACCATGAGCTTTCCATGTAAATTT-3’) and oligo 2 (5’-GGTGTAGTAGTCCAGACCTTTCTCTGGACA-3’), and a fragment containing the transmembrane and intracellular domains of GPVI was amplified using oligo 3 (5’-GGTCTGGACTACTACACCAAGGGCAACCTG-3’) and a vector primer (sp6). The two fragments were subsequently mixed together, and oligos 1 and sp6 added to perform a second overlap PCR. The final amplified product, encoding for a chimaeric protein containing the extra cellular part of CD2 and the transmembrane and intracellular domains of GPVI, was digested with HindIII and XbaI and inserted into similarly cut mammalian expression vector pcDNA 3.1. GPVI-GFP and GPVI-luciferase constructs used for BRET analysis were generated by excision of GPVI from pRc-GPVI-Flag, followed by cloning into pGFP2-N3 and pRluc-N3 (PerkinElmer, UK), respectively. CD2 and CTLA-4 BRET constructs were as described . The integrity and authenticity of constructs was confirmed by nucleotide sequencing.
Stimulations were terminated by the addition of an equal volume of ice-cold lysis buffer (2% NP-40, 300 mM NaCl, 20 mM Tris, 10 mM EDTA, 2 mM Na3VO4, 1 mM PMSF, 10 μg/ml leupeptin, 10 μg/ml aprotinin, and 1 μg/ml pepstatin A, pH 7.4). Insoluble cell debris was removed by centrifugation for 15 min at 12000 g at 4°C. Samples were immunoprecipitated using an appropriate antibody overnight, then 30 μl of 50% (v/v) protein-A- or protein-G-sepharose added for 2 hours. Beads were washed with lyses buffer and resuspended in 20 μl SDS sample buffer.
Samples were separated by SDS-PAGE and transferred to PVDF membranes, then blocked with TBS-T (0.5M Tris, 1.5M NaCl, 0.1% (v/v) Tween-20, pH 7.4) containing 10% (w/v) BSA for at least 1 hour. Blots were incubated with appropriate antibodies and developed using an enhanced chemiluminescence (ECL) detection system. For GPVI ligand blotting with convulxin, membranes were incubated with 10 μg/ml of convulxin for 1 hour at room temperature and incubated with anti-convulxin antibody. Pre-stained molecular weight protein markers were from Bio-Rad (Hemel Hempstead, UK) and New England Biolabs (Hertfordshire, UK).
Cells were resuspended in PBS buffer containing 1 mg/ml of BSA. Cells were incubated with 10 μg/ml of primary antibody for 15 minutes, washed and incubated with fluorescein isothiocyanate (FITC)-conjugated secondary antibody for a further 15 min. Stained cells were analysed immediately using FACScalibur (Becton Dickinson). Data were recorded and analysed using CellQuest software.
Cross-linking reaction was performed on washed platelets resuspended in PBS at room temperature. Sulfo-EGS is a water-soluble analogue of a homobifunctional N-hydroxysuccimide ester (NHS-ester), EGS. Primary amines are principle targets for NHS-esters. Platelets were incubated with freshly prepared cross-linker solution at a concentration of 1 mM, 1.5 mM and 2 mM of sulfo-EGS in PBS for 30 or 60 min at room temperature. The reaction was quenched by adding Tris-HCl (pH 7.5) at a final concentration of 50 mM for 20 min. Samples were lysed and separated by SDS-PAGE under non-reducing conditions.
The BRET analysis was carried out essentially as described [James et al, 2006]. Briefly, FuGene (Roche) was used to transfect 293T cells, with varying ratios of the GFP and luciferase constructs. Cells were harvested 24 hours post-transfection and, for each transfection, 10 μM DeepBlueC (final concentration) was added to 100 μl cells in a 96-well plate and light emission in the 410±40 nm (LU-A) and 515±15 nm (LU-B) wavelength ranges was collected immediately. GFP and luciferase expression was measured in a separate well and converted to a ratio of concentrations. BRETeff values were calculated, after background subtraction, as LU-B/LU-A, corrected for luciferase expression alone (typically 7% of LU-A). To assess the ability of ligands to alter GPVI oligomerisation, 10 μl of convulxin (10 μg/ml), collagen (20 μg/ml) or PBS (control) were incubated with GPVI-transfected cells for 15 minutes before assaying for BRET as above.
We sought to investigate the possible dimerisation or formation of higher oligomers of GPVI in a transfected cell line via immunoprecipitation. Plasmid constructs were generated encoding for GPVI coupled to either Myc or Flag tags at the cytosolic C-terminus (Fig. 1A) and transiently transfected into 293T cells, a human kidney cell line with a high-efficiency of transfection. The expression of both forms of GPVI was detected by western blotting for Myc and Flag (not shown). The two forms of GPVI were expressed with similar levels at the surface of the cells in the absence of FcR γ-chain, as demonstrated by flow cytometry using an anti-GPVI antibody (Fig. 1B).
In order to demonstrate association, 293T cells were transiently co-transfected with both GPVI constructs, either with or without the FcR γ-chain. After two days in culture, the cells were lysed and protein extracts were immunoprecipitated using an anti-Flag antibody. Subsequent western blot analysis using an anti-Myc antibody demonstrated association between the two tagged forms of GPVI (Fig. 1C), independent of FcR γ-chain. Co-transfection of the latter did not increase the yield of association (Fig. 1D). Significantly, the experiments shown in Figures Figures1C1C and and1D1D were performed on the same day and the ECL exposures are time-matched. Ligand blots using convulxin revealed similar levels of expression of GPVI in the co-transfected cells. In order to rule out the possibility that association was an artefact of this experimental approach, lysates from cells with either GPVI-Flag or GPVI-Myc were combined and subjected to immunoprecipitation and western blotting as above. Under these conditions, no association was detected (data not shown). These observations confirm that GPVI is able to undergo oligomerisation in transfected cells, and that this is not dependent on the FcR γ-chain.
Further studies were performed to establish whether the intracellular or extracellular regions of GPVI are required for the homophilic interaction. For these studies, a chimaeric and a mutant protein were generated (Fig. 2A). The chimaeric protein consisted of the extracellular domain of the Ig surface protein CD2 coupled to the transmembrane and intracellular domains of GPVI, and tagged with Flag (CD2-GPVI-Flag) (Fig. 2A). For the mutant protein, GPVI was truncated at the interface of the transmembrane and cytoplasmic domains, and tagged with Flag at the C-terminus ((Δ288)GPVI-Flag) (Fig. 2A). Similar levels of expression of the two constructs was demonstrated by transient transfection into 293T cells followed by western or ligand blotting (not shown), and by flow cytometry using specific antibodies for CD2 or GPVI (Fig. 2B).
The two modified forms of GPVI were co-transfected with Myc-tagged wild type GPVI (GPVI-Myc) in 293T cells. Protein association was detected by immunoprecipitation with an antibody to Flag and western blotting for Myc (Fig. 2C). (Δ288)GPVI-Flag was found to co-precipitate with wild type GPVI-Myc, demonstrating that the GPVI cytosolic tail is not required for the GPVI-GPVI interaction. In contrast, there was no association between CD2-GPVI and wild type GPVI-Myc, confirming that the extracellular portion of GPVI is essential for the homophilic interaction.
Bioluminescence resonance energy transfer (BRET) relies on the transfer of energy from a donor molecule (luciferase) to acceptor (GFP) that is only effective at distances of < -10nm. Proteins of interest can be genetically fused to these fluorophores, permitting the analysis of protein interactions in live cells, with the level of energy transfer (BRETeff) and its dependence on the acceptor/donor ratio allowing the assignment of stoichiometry . Human 293T cells were transiently co-transfected with GPVI-GFP (GPVIGFP) and GPVI-luciferase (GPVILuc) expressing plasmids as a ‘BRET pair’ (GPVIBP), along with the FcR γ-chain. Subsequent analysis demonstrated that GPVI was capable of oligomerisation, with the dependence of BRETeff values on the acceptor/donor ratio fitting best to a dimer model (Fig. 3A, ,3B).3B). The level of BRET observed was intermediate between that of the known monomer, CD2 and the disulphide-linked homodimer, CTLA-4, implying that GPVI dimers are likely to be in equilibrium with the monomeric moiety at the cell surface (Fig. 3A, ,3B),3B), as observed previously for CD80 . Expression of GPVI and CD2 as a BRET pair gave BRETeff values that were lower than those of CD2 and exhibited an independence from the acceptor/donor ratio, which is a characteristic of random interactions at the cell surface . GPVI dimerisation was observed regardless of FcR γ-chain co-expression, although the level of dimerisation was slightly enhanced in its presence (Fig 3A). Incubation of GPVI-transfected cells with either collagen or convulxin did not increase the level of BRETeff observed for GPVI in the absence of ligand (Fig. 3C), with no effect observed over 30 minutes (not shown). This suggests that oligomerisation of GPVI by these two ligands does not bring the luciferase and GFP tags to within the 10 nm required for BRET.
To determine the molecular organisation of GPVI on the platelet surface, platelets were treated with a chemical cross-linker that stabilises pre-existing structures, including the presence of homodimers and associations with other membrane proteins. The membrane impermeable cross-linker sulfo-EGS was chosen for these studies, as it has been previously used in similar studies [21,22]. Three concentrations (1, 1.5 and 2 mM) of sulfo-EGS were assessed for incubation times of 30 and 60 min as recommended by the manufacturer. Similar data were obtained using all combinations, as illustrated following a 30 min incubation with 1.5 mM sulfo-EGS (Fig. 4), with no major differences between the 30 and 60 min time points. The subsequent separation of whole cell lysates by SDS-PAGE under non-reducing conditions, combined with ligand blotting using the snake venom toxin, convulxin, identified three major molecular weight bands of approximately 55, 70 and 220 kDa in sulfo-EGS-treated cells, in comparison to a single major band of around 55 kDa in control samples. In addition, several uncharacterised, minor bands could also be seen which could be due to additional GPVI complexes, non-specific binding of convulxin, or presence of contaminants in the snake toxin preparation. The major band of 55 kDa corresponds to the GPVI monomer, as confirmed by western blotting with an antibody to GPVI (not shown). The possibility that the 70 kDa band corresponds to a complex of GPVI and a dimer of the FcR γ-chain, which would be preserved under the non-reducing conditions of the experiment, was supported by the appearance of a specific co-migrating band in sulfo-EGS-treated cells upon western blotting for FcR γ-chain (Fig. 4B). Similarly, the band of 220 kDa was detected by western blotting for FcR γ-chain, suggesting that it may represent a trimeric form of the GPVI-FcR γ-chain dimer association (i.e. [GPVI-FcR γ-chain]3). Alternatively, it could represent the formation of a complex between GPVI-FcR γ-chain and one or more other proteins on the platelet surface, or an alternative combination of GPVI and the FcR γ-chain. The efficiency of the cross-linker was investigated by heterodimerisation of the platelet integrin subunits αIIb and β3 (Fig. 4C). We show that under the same conditions, treatment with sulfo-EGS induces formation of a high molecular complex of greater than 200 kDa, which appears to contain two prominent and other more minor bands. The size of this complex corresponds to that of the αIIbβ3 heterodimer, with the multiple bands possibly reflecting differential glycosylation. Significantly, however, as with GPVI and other platelet receptors , the monomeric form of αIIb was the predominant band in the presence of the cross-linking reagent, which might reflect the inefficiency of the cross-linking process.
The organisation of the GPVI-FcR γ-chain receptor complex at the surface of platelets is of considerable interest. GPVI has been proposed as a possible target in the fight against thrombus-related diseases. GPVI plays a recognised role in platelet activation upon vascular injury under normal physiological conditions, and its absence has been linked to mild bleeding disorders in humans and to protection against thrombus formation [24,25]. Further, it has been proposed that GPVI antibodies and soluble GPVI dimers can protect against thrombus formation in animal models, although the latter has raised contradictory results between research groups [26,27].
It is widely accepted that GPVI must dimerise under basal conditions in order to bind collagen in view of the low affinity of the monomeric form for the matrix protein. Further, the concept of dimerisation of GPVI is supported by a recent report showing that the crystal structure of GPVI contains back-to-back dimers of the receptor through an interaction in the extracellular domain . The accepted molecular model for GPVI dimerisation proposes that an FcR γ-chain homodimer binds a molecule of GPVI on either side , with the site for interaction taking place in the transmembrane region, between the aspartic acid of the FcR γ-chain and the arginine residue of GPVI . However, this model has recently been challenged by a study showing that the two aspartic residues in the FcR γ-chain homodimer are necessary for association to the single arginine residue in the transmembrane region of GPVI . This model is energetically stable since prior protonisation of at least one of the carboxyl groups in the aspartic acid may occur, so that there is not a charge imbalance in the assembled structure [28,29]. Furthermore, this seems to be a distinctive but common assembly mechanism for a number of activating immune receptors, which are structurally similar to GPVI .
Our results suggest that GPVI dimerises at the surface of GPVI-transfected 293T cells through an interaction that takes place in the extracellular domain. The FcR γ-chain is not necessary for the dimerisation, as it is not expressed in these cells. This would agree with the proposed model for GPVI dimerisation from the recent crystallisation study, which is independent of FcR γ-chain. Interestingly, in platelets, GPVI requires association to FcR γ-chain for expression on the platelet surface. However, this is not the case in the majority of transfected cell lines that have been studied, as demonstrated previously not only for GPVI, but also for other immune receptors with similar structural organisations which require the FcR γ-chain for expression in the host cell [19,30,31]. Although the FcR γ-chain is not essential for surface expression and dimerisation of GPVI in 293T cell line, it may help to stabilise expression, which may explain the difference in dimerisation with cells that express the FcR γ-chain, as demonstrated by BRET.
Interestingly, experiments using platelets pre-treated with a chemical cross-linker that preserves surface complexes, followed by western blotting for either GPVI or FcR γ-chain, demonstrate the presence of two oligomeric structures that contain both proteins. The two bands of approximately 70 and 220 kDa containing GPVI and FcR γ-chain could correspond to a single GPVI molecule with two FcR γ-chains and a trimer of this complex, respectively. Alternatively, the latter could represent a protein multicomplex containing GPVI, FcR γ-chain and possibly one or more other protein(s). The possibility that a GPVI trimer may be present in this oligomeric structure is important in the context that dimers show high affinity to fibrous collagen and inhibit platelet adhesion and aggregation to the injured vessel wall in vivo [14,17,26].
An important question is the extent to which activation of GPVI by multivalent ligands is mediated by oligomerisation. The BRET results obtained indicate that neither convulxin nor collagen bring GPVI molecules to within less than 10 nm of each other, and therefore within the resolution of BRET. The recent crystal structures of convulxin and GPVI provide an explanation for this [16,32]. The crystal structures indicate that only a monomer of convulxin can fit into the binding site of GPVI. Further the tetrameric shape of convulxin would not allow a GPVI dimer to form across the binding sites because they are in the wrong orientation. This suggests that convulxin binds to GPVI as a monomer. On the other hand, the presence of multiple binding sites for GPVI in convulxin would enable the snake toxin to crosslink GPVI molecules and thereby induce a signal into the cell. However, the results from the BRET study demonstrates that the distance between the binding sites for convulxin on the GPVI molecules must be greater than 10 nm. This underlines the fundamental difference between basal receptor dimerisation/oligomerisation, which appears to be required for collagen binding, and agonist-induced receptor crosslinking/clustering that is sufficient to induce signalling but not a close enough interaction for detection by BRET.
In light of our own results as shown here, and those recently published elsewhere [16,18], we propose a modified version of the current model for GPVI dimerisation at the platelet surface, whereby two GPVI molecules dimerise through their respective membrane-proximal Ig domains in the extracellular domain, and each GPVI molecule binds a FcR γ-chain homodimer in the transmembrane region. The two FcR γ-chain homodimers must be kept apart during basal conditions in order to avoid activation, but the latter can be achieved upon collagen binding to initiate a signalling response.
Overall, we demonstrate that surface oligomerisation of GPVI in living cells occurs, and provide evidence that this may also take place in platelets, where a protein multi-complex is formed that contains GPVI, the FcR γ-chain and possibly one or more other component(s). The correct expression and assembly of this multi-molecular structure may be a pre-requisite for formation of a receptor complex that retains the capacity to bind collagen with high affinity and trigger a signal cascade inside the cell. Establishing the organisation of this complex has important consequences in our understanding of platelet regulation by collagen and in possible therapeutic strategies to fight cardiovascular disease.
OB was supported by the BHF. TBS was supported by the University of Birmingham (UK). JRJ and SJD are funded by the Wellcome Trust. MGT is a MRC research fellow. SPW holds a BHF chair.