title: The integrin alpha1 A-domain is a ligand binding site for collagens and laminin.
abstract: The integrin alpha1beta1 is a cell surface receptor for collagens and laminin. The alpha1 subunit contains an A-domain, and the A-domains of other integrins are known to mediate ligand binding. To determine the role of the alpha1 A-domain in ligand binding and the extent to which it reproduced the ligand binding activity and specificity of the parent molecule, we produced recombinant alpha1 A-domain and tested its ability to bind collagens and laminin. In solid phase assays, the A-domain from alpha1 was found to bind to collagen I, collagen IV, and laminin in a largely cation-dependent manner. The alpha2 A-domain, from the alpha2beta1 integrin, also bound to these ligands, but the binding hierarchy differed from that seen for alpha1. This is the first demonstration of laminin binding by A-domains. Specificity of A-domain-ligand binding was further investigated using the triple-helical proteolytic fragment of collagen IV, CB3, and its subfragments, F1 and F4. alpha1 A-domain bound to all three fragments, while the alpha2 A-domain bound CB3 less well and exhibited little binding to F1 and no binding to F4. These differences mirror previous reports of distinct integrin binding sites in collagen IV and for the first time identify a limited proteolytic fragment of a ligand that contains integrin A-domain binding activity. To gain insight into the contribution that the A-domain makes to ligand binding within the whole integrin heterodimer, we measured binding constants for A-domain-collagen interactions using surface plasmon resonance biosensor technology. The values obtained were similar to those reported for intact integrin binding, suggesting that the A-domain is the major collagen binding site in the alpha1beta1 and alpha2beta1 integrins.
authors.affiliation: Wellcome Trust Centre for Cell-Matrix Research, School of Biological Sciences, University of Manchester, Manchester, M13 9PT, United Kingdom.
authors.forename: D A
authors.initials: DA
authors.lastname: Calderwood
authors.forename: D S
authors.initials: DS
authors.lastname: Tuckwell
authors.forename: J
authors.initials: J
authors.lastname: Eble
authors.forename: K
authors.initials: K
authors.lastname: Kühn
authors.forename: M J
authors.initials: MJ
authors.lastname: Humphries
mesh_terms: D000818:Animals; D015703:Antigens, CD / Q000378:metabolism*; D001665:Binding Sites; D002122:Calcium Chloride / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism; D003094:Collagen / Q000378:metabolism*; D005340:Fibrinogen / Q000378:metabolism; D006801:Humans; D039423:Integrin alpha1; D016023:Integrins / Q000378:metabolism*; D007700:Kinetics; D007797:Laminin / Q000378:metabolism*; D015636:Magnesium Chloride / Q000378:metabolism; D017895:Manganese Compounds / Q000378:metabolism; D051379:Mice; D016133:Polymerase Chain Reaction
chemical_list: D015703:Antigens, CD; D015815:Cell Adhesion Molecules; D002712:Chlorides; D039423:Integrin alpha1; D016023:Integrins; D007797:Laminin; D017895:Manganese Compounds; D015636:Magnesium Chloride; D005340:Fibrinogen; D003094:Collagen; D002122:Calcium Chloride; C025340:manganese chloride