Amino acid mutagenesis within ligand-binding loops in αv confers loss-of-function or gain-of-function phenotype on integrin αvβ3

Shigenori Honda; Hirokazu Kashiwagi; Teruo Kiyoi; Hisashi Kato; Satoru Kosugi; Masamichi Shiraga; Yoshiyuki Kurata; Yoshiaki Tomiyama

doi:10.1160/TH04-04-0257

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2004; 92(05): 1092-1098
DOI: 10.1160/TH04-04-0257

Endothelium and Vascular Development

Schattauer GmbH

Amino acid mutagenesis within ligand-binding loops in α_v confers loss-of-function or gain-of-function phenotype on integrin α_vβ₃

Authors

Shigenori Honda

¹Department of Internal Medicine and Molecular Science, Graduate School of Medicine, Osaka University, Japan
Hirokazu Kashiwagi

¹Department of Internal Medicine and Molecular Science, Graduate School of Medicine, Osaka University, Japan
Teruo Kiyoi

¹Department of Internal Medicine and Molecular Science, Graduate School of Medicine, Osaka University, Japan
Hisashi Kato

¹Department of Internal Medicine and Molecular Science, Graduate School of Medicine, Osaka University, Japan
Satoru Kosugi

¹Department of Internal Medicine and Molecular Science, Graduate School of Medicine, Osaka University, Japan
Masamichi Shiraga

¹Department of Internal Medicine and Molecular Science, Graduate School of Medicine, Osaka University, Japan
Yoshiyuki Kurata

²Department of Blood Transfusion, Osaka University Hospital, Japan
Yoshiaki Tomiyama

¹Department of Internal Medicine and Molecular Science, Graduate School of Medicine, Osaka University, Japan

Abstract

Summary

The crystal structure of α_vβ₃ in complex with a cyclic RGDcontaining ligand has recently been demonstrated. However, the functional significance of each residue within ligand binding loops has not been fully elucidated. Here, by employing alaninescanning mutagenesis, we have examined the functional role of ligand contact residues in α_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 α_v is critically involved in ligand-binding and that Ala215 could regulate the affinity of α_vβ₃.

Keywords

β-propeller - cation-π interaction - gain-of-function - ligand-binding site - loss-of-function

Full Text

References

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