A novel gain-of-function mutation of the integrin alpha2 VWFA domain.
Integrin alpha2beta1 is the major receptor for collagens in human tissues, being involved in cell adhesion and the control of collagen and collagenase gene expression. The collagen binding site of alpha2beta1 has been localized to the alpha2 von Willebrand Factor type A (VWFA) domain (A-domain or I-domain) and the residues responsible for the interaction with collagen have been mapped. We report a study of alpha2 VWFA domain in which residue E318, which lies outside the collagen binding site, is mutated to tryptophan, showing that this is a gain-of-function mutation. Recombinant alpha2-E318W VWFA domain showed elevated and specific binding to collagen I compared with the wild-type. Side chain hydrophobicity was important for the gain-of-function as elevated binding was seen with E318I and E318Y, but not with E318R. The E318W mutation had additional effects on VWFA domain properties as alpha2-E318W VWFA domain differed from the wild-type in its cation preferences for ligand binding and in binding to monoclonal antibody JA203, which bound at a site distal to E318. The gain-of-function effect was not restricted to binding to collagen I as alpha2-E318W also showed elevated binding to collagen IV, collagen I C-propeptide, laminin and E-cadherin. Binding to these ligands was inhibited by collagen peptide containing the GFOGER motif, indicating that these bound to the VWFA domain by a similar mechanism to collagen I. These data indicate that residue E318 plays a novel and important role in modulating alpha2 VWFA domain--ligand binding and may be involved in the conformational changes associated with its regulation.
Aquilina
Alexis
A
School of Biological Sciences, University of Manchester, Manchester, UK.
Korda
Michelle
M
Bergelson
Jeffrey M
JM
Humphries
Martin J
MJ
Farndale
Richard W
RW
Tuckwell
Danny
D
D000595:Amino Acid Sequence; D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism*; D015820:Cadherins / Q000378:metabolism; D003094:Collagen / Q000378:metabolism; D018698:Glutamic Acid / Q000235:genetics; D006801:Humans; D039421:Integrin alpha2; D016023:Integrins / Q000235:genetics / Q000378:metabolism*; D007797:Laminin / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D014364:Tryptophan / Q000235:genetics; D014841:von Willebrand Factor / Q000235:genetics
D015703:Antigens, CD; D015820:Cadherins; D039421:Integrin alpha2; D016023:Integrins; D007797:Laminin; D014841:von Willebrand Factor; D018698:Glutamic Acid; D014364:Tryptophan; D003094:Collagen