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Glycoprotein VI Oligomerization in Cell Lines and Platelets

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Glycoprotein VI Oligomerization in Cell Lines and Platelets Journal of Thrombosis and Haemostasis, 5: 1026–1033 ORIGINAL ARTICLE Glycoprotein VI oligomerization in cell lines and platelets O. BERLANGA,1 *T.BORI-SANZ,1 *J.R.JAMES, J. FRAMPTON,* S. J. DAVIS, M. G. TOMLINSON* and S . P . W A T S O N * *Institute of Biomedical Research, Medical School, University of Birmingham, Edgbaston, Birmingham; and Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Headington, University of Oxford, Oxford, UK To cite this article: Berlanga O, Bori-Sanz T, James JR, Frampton J, Davis SJ, Tomlinson MG, Watson SP. Glycoprotein VI oligomerization in cell lines and platelets. J Thromb Haemost 2007; 5: 1026–33. mediated through two distinct receptor classes on the platelet Summary. Background: Glycoprotein VI (GPVI) is a physio- surface, the integrin aIIb1 and the glycoprotein VI (GPVI)–FC logic receptor for collagen expressed at the surface of platelets receptor (FcR) c-chain receptor complex [1]. Blocking of either and megakaryocytes. Constitutive dimerization of GPVI has receptor in vitro using specific antibodies inhibits or delays, been proposed as being necessary for the interaction with respectively, collagen-induced platelet aggregation [2–4]. Simi- collagen, although direct evidence of dimerization has not been larly, platelets deficient in aIIb1 or GPVI–FcR c-chain show reported in cell lines or platelets. Objectives: To investigate loss of reactivity towards collagen in vitro [5–8]. Furthermore, oligomerization of GPVI in transfected cell lines and in platelets mice deficient in GPVI–FcR c-chain are protected against under non-stimulated conditions. Methods and results: By lethal thromboembolism [8,9], illustrating the crucial role that using a combination of molecular and biochemical techniques, the receptor plays in vivo under pathologic conditions. we demonstrate that GPVI association occurs at the surface of Although the intracellular signaling events mediated by aIIb1 transfected 293T cells under basal conditions, through an in platelets have remained elusive [10,11], the mechanism of interaction at the extracellular domain of the receptor. action of GPVI has been well documented and remains an area Bioluminescence resonance energy transfer was used to confirm of intense research. GPVI mediates platelet activation in oligomerization of GPVI under these conditions. A chemical response to collagen through a pathway that shares many crosslinker was used to detect constitutive oligomeric forms of features with those used by immune receptors such as FceRI, GPVI at the surface of platelets, which contain the Fc receptor and T-cell and B-cell antigen receptors [12]. As GPVI has no (FcR) c-chain. Conclusions: The present results directly dem- intrinsic signaling capacity, it is widely recognized that it must be onstrate GPVI–FcR c-chain oligomerization at the surface of coexpressed in association with the FcR c-chain, which acts as the platelet, and thereby add to the growing evidence that the signaling partner. Furthermore, this association is a prere- oligomerization of GPVI may be a prerequisite for binding of quisite for surface expression of GPVI on mouse platelets [13]. the receptor to collagen, and therefore for proper functioning of Froma structure–function pointofview,thereare several lines platelets upon vascular damage. of circumstantial evidence to suggest that GPVI functions as a dimer on the platelet surface. Moroi and coworkers have Keywords: BRET, dimerization, glycoprotein VI, GPVI, olig- demonstrated, using recombinant protein, that collagen binds omerization, platelets. to the dimeric but not the monomeric form of GPVI, and that only the former is able to attenuate collagen-induced platelet Introduction aggregation [14]. In contrast, both the monomeric and dimeric formsofGPVIbindtoimmobilizedconvulxinandinhibitplatelet The extracellular matrix protein collagen is the major and most aggregation induced by the snake toxin with similar concentra- thrombogenic component of the vessel wall. Circulating tion dependencies [14]. The possibility that GPVI functions as a platelets adhere to exposed collagen and undergo activation, dimer is strongly reinforced by studies analyzing the ability of a leading to thrombus formation. The interaction with collagen is series of synthetic peptides with differentially spaced GPVI- recognition motifs to activate the collagen receptor in platelets Correspondence: Steve P. Watson, Institute of Biomedical Research, [15]. Finally, structural studies of the two immunoglobulin (Ig) Medical School, University of Birmingham, Edgbaston, Birmingham domains of human GPVI have revealed the formation of a back- B15 2TT, UK. to-back dimer in the crystal structure, which is mediated through Tel.: +44 0 121 414 6514; fax: +44 0 121 415 8817; e-mail: s.p. the more membrane-proximal of the two Ig domains [16]. [email protected] As the FcR c-chain is present as a disulfide-linked homo- 1These authors contributed equally to this work. dimer, it has been proposed that each chain associates independently with GPVI [17]. In light of a recent report Received 13 May 2006, accepted 9 February 2007 indicating that the two chains of the FcR c-chain are necessary Ó 2007 International Society on Thrombosis and Haemostasis Platelet glycoprotein VI oligomerization 1027 for binding a single GPVI molecule [18], this model needs to be Approximately 10 days later, individual clones of cells were reviewed. Furthermore, direct evidence that GPVI is expressed selected and placed in 96-well plates for expansion. 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 Plasmid constructs transfected cell lines using distinct biochemical and molecular approaches. Our results confirm that GPVI is capable of GPVI–Flag and (D288)GPVI–Flag were subcloned into the pRc undergoing oligomerization in transfected cells and forming plasmid, whereas the FcR c-chain was subcloned into the pMG oligomers in platelets, and indicate that a modified version of plasmid, as previously described [19]. GPVI–Myc was obtained the current model for GPVI dimerization may be necessary. by standard PCR using a vector primer (T7) and GPVI–Myc primer (5¢-CCCTAAGCGGCCGCTCACAGATCCTCTT- CTGAGATGAGTTTTTGTTCTGAACATAACCCGCGG- Materials and methods C-3¢). The final amplified product was digested with HindIII and NotI, and inserted into the similarly cut mammalian expression Reagents and antibodies vector pcDNA 3.1. The CD2 extracellular domain was fused to Convulxin was purchased from Latoxan (Valence, France). the transmembrane region and cytoplasmic tail of GPVI using Anticonvulxin was a kind gift from M. Leduc (Institute standard overlapping PCR techniques. The extracellular Pasteur, Paris, France). Anti-CD2 antibody was kindly domain of CD2 was amplified using oligo 1 (5¢-CCCTAA- supplied by V. Horejsi (Institute of Molecular Genetics, AAGCTTACCATGAGCTTTCCATGTAAATTT-3¢)and Academy of Sciences, Prague, Czech Republic). Anti-FcR c- oligo 2 (5¢-GGTGTAGTAGTCCAGACCTTTCTCTGGA- chain was obtained from Upstate Biotechnology (Buckingham, CA-3¢), and a fragment containing the transmembrane and UK). Anti-Flag (M2) was obtained from Sigma (Dorset, UK). intracellular domains of GPVI was amplified using oligo 3 (5¢- Anti-Myc (9B11) was obtained from Cell Signalling Technol- GGTCTGGACTACTACACCAAGGGCAACCTG-3¢)and ogy (Hertfordshire, UK). All other reagents were obtained a vector primer (sp6). The two fragments were subsequently from previously described sources [19] unless otherwise stated. mixed together, and oligo 1 and sp6 were added to perform a second overlap PCR. The final amplified product, encoding a chimeric protein containing the extracellular part of CD2 and Platelet preparation the transmembrane and intracellular domains of GPVI, was Human blood was taken from drug-free volunteers on the day digested with HindIII and XbaI and inserted into the similarly of experiment using acidic citrate dextrose (120 mM sodium cut mammalian expression vector pcDNA 3.1. GPVI–green citrate, 110 mM glucose, 80 mM citric acid). Platelet-rich fluorescent protein (GFP) and GPVI–luciferase constructs used plasma was obtained by centrifugation at 200 · g for 20 min, for bioluminescence resonance energy transfer (BRET) analysis and platelets were isolated by centrifugation at 1000 · g for were generated by excision of GPVI from pRc–GPVI–Flag, ) 10 min in the presence of prostacyclin (0.1 lgmL 1). Platelets followed by cloning into pGFP2-N3 and pRluc-N3 (PerkinEl- were resuspended in modified TyrodeÕs/HEPES buffer mer, UK), respectively. CD2 and cytotoxic T-lymphocyte- (134 mM NaCl, 2.9 mM KCl, 0.34 mM Na2HPO4.12H2O, associated antigen-4 (CTLA-4) BRET constructs were prepared 12 mM NaHCO3,20mM HEPES, 1 mM MgCl2,5 mM glucose, as previously described [20]. The integrity and authenticity of ) pH 7.3, at 37 °C) in the presence of prostacyclin (0.1 lgmL 1), constructs was confirmed by nucleotide sequencing. recentrifuged at 1000 · g for 10 min, and resuspended in the ) above buffer to a density of 5 · 108 cells mL 1. Immunoprecipitation Stimulations were terminated by the addition of an equal Cell culture volume of ice-cold lysis buffer (2% Nonidet P-40, 300 mM 293T cells were grown in DMEM supplemented with NaCl, 20 mM Tris, 10 mM EDTA, 2 mM Na3VO4,1mM ) ) 100 U mL 1 penicillin, 100 lgmL 1 streptomycin and 10% phenylmethanesulfonyl fluoride, 10 lgmL)1 leupeptin, )1 )1 heat-inactivated fetal bovine serum under 5% CO2/95% air in 10 lgmL aprotinin, and 1 lgmL pepstatin A, pH 7.4).
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