Chimeric Fc receptors identify ligand binding regions in human glycoprotein VI.
Glycoprotein (GP) VI, a key receptor for collagen-induced platelet activation, recently emerged as a major target for developing new antithrombotics. However, little is known about its functional domains, which is a disadvantage for the rational development of antagonists. Our aim was to identify the structures determining GPVI specificity. GPVI presents homologies with members of the Ig superfamily (in particular with FcalphaRI) whose extracellular parts present two domains, D1 and D2 linked by a hinge interdomain. To identify the respective role of these domains in GPVI, we have substituted D1 and D2 by their FcalphaRI homologue in a soluble GPVI fusion protein (GPVI-Fc) and have modified the linker motif by mutagenesis. Proteins were tested for their binding to ligands and antibodies specific for GPVI and FcalphaRI. We demonstrate for the first time that D2 plays a specific and significant role in GPVI binding to collagen and that the hinge interdomain is critical for the binding to convulxin. Furthermore, binding to CRP requires elements of D1 and of the linker motif. Our results indicate that GPVI is unique amongst the receptors of its family as it uses different structural domains to interact with several agonists and provide evidence that different sites on GPVI constitute targets to develop antagonists of GPVI.
Dumont
Bénédicte
B
Institut National de la Santé et de la Recherche Médicale, INSERM, U698, Université Paris 7, Hôpital Bichat, France.
Minullina
Izida
I
Loyau
Stéphane
S
Monteiro
Renato C
RC
Lacapere
Jean-Jacques
JJ
Arocas
Véronique
V
Jandrot-Perrus
Martine
M
D000911:Antibodies, Monoclonal / Q000276:immunology; D015703:Antigens, CD / Q000378:metabolism*; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism; D003435:Crotalid Venoms / Q000378:metabolism; D006801:Humans; D007070:Immunoglobulin A / Q000378:metabolism; D037181:Lectins, C-Type / Q000378:metabolism; D008024:Ligands; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D010455:Peptides / Q000378:metabolism; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011961:Receptors, Fc / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D002352:Carrier Proteins; D003435:Crotalid Venoms; C036832:Fc(alpha) receptor; D007070:Immunoglobulin A; D037181:Lectins, C-Type; D008024:Ligands; D010455:Peptides; D010980:Platelet Membrane Glycoproteins; D011961:Receptors, Fc; D011993:Recombinant Fusion Proteins; C435908:collagen-related peptide; C098341:platelet membrane glycoprotein VI; C028890:convulxin; D003094:Collagen