Molecular Basis of the Ligand Binding Specificity of αvβ8 Integrin

Akio Ozawa,Yuya Sato,Tsukasa Imabayashi,Toshihiko Uemura,Junichi Takagi,Kiyotoshi Sekiguchi

doi:10.1074/jbc.m116.719138

Abstract

αvβ8 is an integrin that recognizes an Arg-Gly-Asp (RGD) motif and interacts with fibronectin, vitronectin, and latent TGF-β1. We comprehensively determined the binding activity of the αvβ8 integrin toward 25 secreted proteins having an RGD motif. The αvβ8 integrin strongly bound to latent TGF-β1 but showed marginal activity for other RGD-containing proteins, including fibronectin and vitronectin. Site-directed mutagenesis of latent TGF-β1 demonstrated that the high affinity binding of αvβ8 integrin to latent TGF-β1 was defined by Leu-218 immediately following the RGD motif within the latency-associated peptide of TGF-β1. Consistent with the critical role of Leu-218 in latent TGF-β1 recognition by αvβ8 integrin, a 9-mer synthetic peptide containing an RGDL sequence strongly inhibited interactions of latent TGF-β1 with αvβ8 integrin, whereas a 9-mer peptide with an RGDA sequence was ∼60-fold less inhibitory. Because αvβ3 integrin did not exhibit strong binding to latent TGF-β1 or distinguish between RGDL- and RGDA-containing peptides, we explored the mechanism by which the integrin β8 subunit defines the high affinity binding of latent TGF-β1 by αvβ8 integrin. Production of a series of swap mutants of integrin β8 and β3 subunits indicated that the high affinity binding of αvβ8 integrin with latent TGF-β1 was ensured by interactions between the Leu-218 residue and the β8 I-like domain, with the former serving as an auxiliary recognition residue defining the restricted ligand specificity of αvβ8 integrin toward latent TGF-β1. In support of this conclusion, high affinity binding toward the αvβ8 integrin was conferred on fibronectin by substitution of its RGDS motif with an RGDL sequence.

Highlights

␣5␤1 integrin binds to fibronectin through the bipartite recognition of an RGD motif in the 10th type III repeat, together with the PHSRN sequence and several basic residues within the 9th type III repeat, the latter serving as a “synergy site” [22, 23]. ␣8␤1 integrin selectively binds to nephronectin via a bipartite interaction with the RGD motif and LFEIFEIER sequence, the latter located at the C-terminal ϳ10 amino acids from the RGD motif [24]
Accumulating evidence indicates that the binding affinities of RGD-containing ligands toward integrins are potentiated by sequences residing outside the RGD motif
Nephronectin contains an auxiliary sequence LFEIFEIER required for the high affinity binding of nephronectin to the ␣8␤1 integrin, which functions in concert with an RGD motif [24]

Summary

Introduction

18 ␣ and 8 ␤ subunits have been identified, and combinations of these subunits give rise to at least 24 distinct integrin heterodimers, among which 18 isoforms function as ECM receptors Based on their ligand binding specificities, ECM-binding integrins are classified into three major groups as follows: laminin-, collagen-, and Arg-Gly-Asp (RGD)-binding integrins [1, 2], of which the RGD-binding integrins have been most extensively investigated. The RGD-binding integrins include ␣5␤1, ␣8␤1, ␣IIb␤3, and ␣v-containing integrins, which interact with a variety of ECM ligands containing RGD motifs with distinct binding specificities. The binding capability of the ␣v␤8 integrin to other RGD-containing proteins has not been comprehensively analyzed at the molecular level, suggesting unknown proteins containing the RGD motif might serve as ligands that interact with the ␣v␤8 integrin. The mechanism by which the ␣v␤8 integrin recognizes the Leu-218 residue was investigated by constructing a series of swap mutants between integrin ␤8 and ␤3 subunits

Methods

Results

Conclusion