Multiple Binding Sites in Collagen Type I for the Integrins α1β1 and α2β1 *

Integrins α1β1 and α2β1 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 α chains. Previously, we reported that recombinant A-domain of α1β1(α1A) had at least two affinity classes of binding sites in type I collagen (Rich, R. L., et al. (1999)J. Biol. Chem. 274, 24906–24913). Here, we compared the binding of the recombinant A-domain of α2β1 (α2A) to type I collagen with that of α1A using surface plasmon resonance and showed that α2A exhibited only one detectable class of binding sites in type I collagen, with a K D of ∼10 μm at ∼3 binding sites per collagen molecule. We further demonstrated that α1A and α2A 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 α1A- and α2A-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 α1A and α2A 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 α1A and α2A (Knight, C. G., et 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 α1A and α2A 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 α1A and α2A.