title: Distinct ligand binding sites in the I domain of integrin alpha M beta 2 that differentially affect a divalent cation-dependent conformation.
abstract: The I domains of the leukocyte beta 2 integrins have been shown to be essential for ligand recognition. Amino acid substitutions of Asp140 and Ser142, which reside in a conserved cluster of oxygenated residues, abrogate divalent cation ligand binding function of alpha M beta 2. Presently, we evaluated the role of two I domain regions in alpha M beta 2 ligand recognition: 1) the conserved cluster of oxygenated residues (Asp134, Asp140, Ser142, and Ser144) and 2) a 7-amino acid region (Phe246-Tyr252), conserved in alpha M and alpha X but absent in alpha L of the beta 2 integrins. Recombinant alpha M beta 2 was expressed on COS-7 cells, and function was assessed by iC3b recognition. Alanine substitution at position Asp140, Asp140/Ser142, Ser142, or Ser144 produced a complete loss in the capacity of alpha M beta 2 to recognize iC3b and attenuated the binding of a divalent cation-dependent epitope recognized by monoclonal antibody 24. Moreover, alanine substitution at Asp248 or Tyr252 or deletion of Phe246-Tyr252 abolished iC3b ligand recognition as well as the binding of a blocking antibody. In contrast, these mutations did not affect the binding of the cation-dependent epitope. These data implicate a second region within the I domain important for alpha M beta 2 ligand binding function and suggest that this region does not affect a divalent cation-dependent conformation of alpha M beta 2.
authors.affiliation: Upjohn Laboratories, Kalamazoo, Michigan 49001, USA.
authors.forename: S L
authors.initials: SL
authors.lastname: McGuire
authors.forename: M L
authors.initials: ML
authors.lastname: Bajt
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002413:Cations, Divalent / Q000494:pharmacology; D002448:Cell Adhesion; D002478:Cells, Cultured; D003179:Complement C3b / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes; D005434:Flow Cytometry; D008024:Ligands; D016177:Macrophage-1 Antigen / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011233:Precipitin Tests; D011485:Protein Binding; D011487:Protein Conformation / Q000187:drug effects; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D012397:Rosette Formation; D017386:Sequence Homology, Amino Acid; D014162:Transfection
chemical_list: D002413:Cations, Divalent; D000939:Epitopes; D008024:Ligands; D016177:Macrophage-1 Antigen; D011994:Recombinant Proteins; D003179:Complement C3b