Multiple binding sites in collagen type I for the integrins α1β1 and α2β1

Integrins alpha (1)beta (1) and alpha (2)beta (1) are two major collagen receptors on the surface of eukaryotic cells. Binding to collagen is primarily due to an A-domain near the N terminus of the alpha chains. Previously, we reported that recombinant A-domain of alpha (1)beta (1) (alpha (1)A) had at least two affinity classes of binding sites in type I collagen (Rich, R. L., ct al. (1999) J. Biol. Chem. 274, 24906-24913), Here, we compared the binding of the recombinant A-domain of alpha (2)beta (1) (alpha (2)A) to type I collagen with that of alpha (1)A using surface plasmon resonance and showed that alpha (2)A exhibited only one detectable class of binding sites in type I collagen, with a K-D of similar to 10 muM at similar to3 binding sites per collagen molecule. We further demonstrated that alpha (1)A and alpha (2)A competed with each other for binding to type I collagen in enzyme-linked immunosorbent assay (ELISA), suggesting that the binding sites in collagen for the two A-domains overlap or are adjacent to each other. By using rotary shadowing, the complexes of alpha (1)A- and alpha (2)A-procollagen were visualized. Morphometric analyses indicated three major binding regions (near the N terminus, in the central part, and near the C terminus) along the type I procollagen molecule for both A-domains. The positions of the respective binding regions for alpha (1)A and alpha (2)A were overlapping with or adjacent to each other, consistent with the ELISA results. Analysis of the sequences of type I collagen revealed that GER or GER-like motifs are present at each of the binding regions, and notably, the central region contains the GFOGER sequence, which was previously identified as a high affinity site for both alpha (1)A and alpha (2)A (Knight, C. G., ct al. (2000) J. Biol. Chem. 275, 35-40). Peptides containing GLOGERGRO (peptide I, near the N terminus), GFOGERGVQ (peptide II, central), and GASGERGPO (peptide III, near the C terminus) were synthesized. Peptides I and II effectively inhibited the binding of cli,A and alpha (2)A to type I collagen, while peptide III did so moderately, The N-terminal site in type I collagen has the sequence GLOGER in all three chains. Thus, it seems that peptide I represents a newly discovered native high affinity site for alpha (1)A and alpha (2)A.