title: Amino acid mutagenesis within ligand-binding loops in alpha v confers loss-of-function or gain-of-function phenotype on integrin alpha v beta 3.
abstract: The crystal structure of alpha(v)beta(3) in complex with a cyclic RGD-containing ligand has recently been demonstrated. However, the functional significance of each residue within ligand binding loops has not been fully elucidated. Here, by employing alanine-scanning mutagenesis, we have examined the functional role of ligand contact residues in alpha(v). Tyr178 --> Ala substitution (Tyr178Ala) and Asp218Ala abolished a monovalent ligand, WOW-1 Fab binding as well as soluble fibrinogen binding, which is in perfect agreement with the crystallography. However, Asp150Ala showed no or only a modest inhibition of ligand binding. In contrast, Tyr substitution at Ala215 (Ala215Tyr) increased WOW-1 Fab binding, suggesting that the substitution increased the integrin affinity. The adhesion assay to immobilized fibrinogen showed essentially the same data as obtained using soluble ligands. Our present data indicate that Tyr178 and Asp218, but not Asp150 in alpha(v) is critically involved in ligand-binding and that Ala215 could regulate the affinity of alpha(v)beta(3).
authors.affiliation: Department of Internal Medicine and Molecular Science, Graduate School of Medicine, Osaka University, Japan.
authors.forename: Shigenori
authors.initials: S
authors.lastname: Honda
authors.forename: Hirokazu
authors.initials: H
authors.lastname: Kashiwagi
authors.forename: Teruo
authors.initials: T
authors.lastname: Kiyoi
authors.forename: Hisashi
authors.initials: H
authors.lastname: Kato
authors.forename: Satoru
authors.initials: S
authors.lastname: Kosugi
authors.forename: Masamichi
authors.initials: M
authors.lastname: Shiraga
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Kurata
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Tomiyama
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D005340:Fibrinogen / Q000378:metabolism; D007140:Immunoglobulin Fab Fragments / Q000378:metabolism; D039564:Integrin alphaV / Q000737:chemistry / Q000235:genetics*; D039302:Integrin alphaVbeta3 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D016296:Mutagenesis; D010641:Phenotype; D011485:Protein Binding
chemical_list: D007140:Immunoglobulin Fab Fragments; D039564:Integrin alphaV; D039302:Integrin alphaVbeta3; D005340:Fibrinogen