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Shigenori Honda, Hirokazu Kashiwagi, Teruo Kiyoi, Hisashi Kato, Satoru Kosugi, Masamichi Shiraga, Yoshiyuki Kurata, Yoshiaki Tomiyama
Amino acid mutagenesis within ligand-binding loops in v confers loss-of-function or gain-of-function phenotype on integrin v3
The crystal structure of v3 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 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 v is critically involved in ligand-binding and that Ala215 could regulate the affinity of v3.
Thrombosis and Haemostasis, Schattauer
Print ISSN: 0340-6245
Volume: 92, 11/2004
Pages: 1092 - 1098