Mapping the collagen-binding site in the I domain of the glycoprotein Ia/IIa (integrin alpha(2)beta(1)).
The I domain present within the alpha2 chain of the integrin alpha(2)beta(1) (GPIa/IIa) contains the principal collagen-binding site. Based on the crystal structure of the alpha2-I domain, a hypothetical model was proposed in which collagen binds to a groove on the upper surface of the I domain (Emsley, J., King, S. L., Bergelson, J. M., and Liddington, R. C. (1997) J. Biol. Chem. 272, 28512-28517). We have introduced point mutations into 13 residues on the upper surface of the domain. Recombinant mutant proteins were assayed for binding to monoclonal antibodies 6F1 and 12F1, to collagen under static conditions, and for the ability to retain adhesive activity under flow conditions. The mutations to residues surrounding the metal ion-dependent adhesion site that caused the greatest loss of collagen binding under both static and flow conditions are N154S in the betaA-alpha1 turn, N190D in the betaB-betaC turn, D219R in the alpha3-alpha4 turn, and E256V and H258V in the betaD-alpha5 turn. Mutation in one of the residues that coordinate the metal binding, S155A, completely lost the adhesive activity under flow but bound normally under static conditions, whereas the mutation Y285F had the converse effect. We conclude that the upper surface of the domain, including the metal ion-dependent adhesion site motif, defines the collagen recognition site.
Smith
C
C
Hematology Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.
Estavillo
D
D
Emsley
J
J
Bankston
L A
LA
Liddington
R C
RC
Cruz
M A
MA
D000911:Antibodies, Monoclonal / Q000378:metabolism; D001665:Binding Sites; D015815:Cell Adhesion Molecules / Q000737:chemistry / Q000235:genetics; D003094:Collagen / Q000378:metabolism*; D006801:Humans; D016023:Integrins / Q000737:chemistry* / Q000235:genetics; D008863:Microspheres; D008958:Models, Molecular; D009154:Mutation; D011137:Polystyrenes; D011485:Protein Binding; D038981:Receptors, Collagen; D011994:Recombinant Proteins
D000911:Antibodies, Monoclonal; D015815:Cell Adhesion Molecules; D016023:Integrins; D011137:Polystyrenes; D038981:Receptors, Collagen; D011994:Recombinant Proteins; D003094:Collagen