title: Role of ADMIDAS cation-binding site in ligand recognition by integrin alpha 5 beta 1.
abstract: Integrin-ligand interactions are regulated in a complex manner by divalent cations, and multiple cation-binding sites are found in both alpha and beta integrin subunits. A key cation-binding site that lies in the beta subunit A-domain is known as the metal-ion dependent adhesion site (MIDAS). Recent x-ray crystal structures of integrin alpha V beta 3 have identified a novel cation binding site in this domain, known as the ADMIDAS (adjacent to MIDAS). The role of this novel site in ligand recognition has yet to be elucidated. Using the interaction between alpha 5 beta 1 and fibronectin as a model system, we show that mutation of residues that form the ADMIDAS site inhibits ligand binding but this effect can be partially rescued by the use of activating monoclonal antibodies. The ADMIDAS mutants had decreased expression of activation epitopes recognized by 12G10, 15/7, and HUTS-4, suggesting that the ADMIDAS is important for stabilizing the active conformation of the integrin. Consistent with this suggestion, the ADMIDAS mutations markedly increased the dissociation rate of the integrin-fibronectin complex. Mutation of the ADMIDAS residues also reduced the allosteric inhibition of Mn2+-supported ligand binding by Ca2+, suggesting that the ADMIDAS is a Ca2+-binding site involved in the inhibition of Mn2+-supported ligand binding. Mutations of the ADMIDAS site also perturbed transduction of a conformational change from the MIDAS through the C-terminal helix region of the beta A domain to the underlying hybrid domain, implying an important role for this site in receptor signaling.
authors.affiliation: Wellcome Trust Centre for Cell-Matrix Research, School of Biological Sciences, University of Manchester, Manchester M13 9PT, United Kingdom. paul.mould@man.ac.uk
authors.forename: A Paul
authors.initials: AP
authors.lastname: Mould
authors.forename: Stephanie J
authors.initials: SJ
authors.lastname: Barton
authors.forename: Janet A
authors.initials: JA
authors.lastname: Askari
authors.forename: Susan E
authors.initials: SE
authors.lastname: Craig
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Humphries
mesh_terms: D000494:Allosteric Regulation; D019943:Amino Acid Substitution; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D003001:Cloning, Molecular; D005353:Fibronectins / Q000378:metabolism; D006801:Humans; D039081:Integrin alpha5beta1 / Q000235:genetics / Q000378:metabolism*; D008024:Ligands; D008345:Manganese / Q000378:metabolism; D008670:Metals / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D014162:Transfection
chemical_list: D005353:Fibronectins; D039081:Integrin alpha5beta1; D008024:Ligands; D008670:Metals; D008345:Manganese; D002118:Calcium