Identification of residues within human glycoprotein VI involved in the binding to collagen: evidence for the existence of distinct binding sites.
Glycoprotein VI (GPVI) has a crucial role in platelet responses to collagen. Still, little is known about its interaction with its ligands. In binding assays using soluble or cell-expressed human GPVI, we observed that (i) collagen, and the GPVI-specific ligands collagen-related peptides (CRP) and convulxin, competed with one another for the binding to GPVI and (ii) monoclonal antibodies directed against the extracellular part of the human receptor displayed selective inhibitory properties on GPVI interaction with its ligands. Monoclonal antibody 9E18 strongly reduced the binding of GPVI to collagen/CRP, 3F8 inhibited its interaction with convulxin, whereas 9O12 prevented all three interactions. These observations suggest that ligand-binding sites are distinct, exhibiting specific features but at the same time also sharing some common residues participating in the recognition of these ligands. The epitope of 9O12 was mapped by phage display, along with molecular modeling of human GPVI, which allowed the identification of residues within GPVI potentially involved in ligand recognition. Site-directed mutagenesis revealed that valine 34 and leucine 36 are critical for GPVI interaction with collagen and CRP. The loop might thus be part of a collagen/CRP-binding site.
Lecut
Christelle
C
INSERM, E348, Faculté de Médecine Xavier Bichat, Paris Cedex 18, France.
Arocas
Véronique
V
Ulrichts
Hans
H
Elbaz
Anthony
A
Villeval
Jean-Luc
JL
Lacapère
Jean-Jacques
JJ
Deckmyn
Hans
H
Jandrot-Perrus
Martine
M
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000378:metabolism; D003094:Collagen / Q000378:metabolism*; D003435:Crotalid Venoms / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D018604:Epitope Mapping; D006801:Humans; D066298:In Vitro Techniques; D037181:Lectins, C-Type / Q000378:metabolism; D007930:Leucine / Q000737:chemistry; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000378:metabolism; D010980:Platelet Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014633:Valine / Q000737:chemistry
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D003435:Crotalid Venoms; D018076:DNA, Complementary; D037181:Lectins, C-Type; D008024:Ligands; D010455:Peptides; D010980:Platelet Membrane Glycoproteins; D011994:Recombinant Proteins; C435908:collagen-related peptide; C098341:platelet membrane glycoprotein VI; C028890:convulxin; D003094:Collagen; D007930:Leucine; D014633:Valine