title: Multiple binding sites in collagen type I for the integrins alpha1beta1 and alpha2beta1.
abstract: 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., et 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 approximately 10 microm at approximately 3 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., 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 alpha(1)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.
authors.affiliation: Center for Extracellular Matrix Biology, Institute of Biosciences and Technology, Texas A & M University, Houston, Texas 77030, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Xu
authors.forename: S
authors.initials: S
authors.lastname: Gurusiddappa
authors.forename: R L
authors.initials: RL
authors.lastname: Rich
authors.forename: R T
authors.initials: RT
authors.lastname: Owens
authors.forename: D R
authors.initials: DR
authors.lastname: Keene
authors.forename: R
authors.initials: R
authors.lastname: Mayne
authors.forename: A
authors.initials: A
authors.lastname: Höök
authors.forename: M
authors.initials: M
authors.lastname: Höök
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002417:Cattle; D002645:Chickens; D003001:Cloning, Molecular; D003094:Collagen / Q000737:chemistry* / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D015723:Gene Library; D006801:Humans; D039222:Integrin alpha1beta1; D016023:Integrins / Q000378:metabolism*; D007700:Kinetics; D008854:Microscopy, Electron; D008954:Models, Biological; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D017434:Protein Structure, Tertiary; D038981:Receptors, Collagen; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D020349:Surface Plasmon Resonance; D013997:Time Factors
chemical_list: D039222:Integrin alpha1beta1; D016023:Integrins; D010455:Peptides; D038981:Receptors, Collagen; D011994:Recombinant Proteins; D003094:Collagen