Molecular Basis of the Ligand Binding Specificity of αvβ8 Integrin

alpha v beta 8 is an integrin that recognizes an Arg-Gly-Asp (RGD) motif and interacts with fibronectin, vitronectin, and latent TGF-beta 1. We comprehensively determined the binding activity of the alpha v beta 8 integrin toward 25 secreted proteins having an RGD motif. The alpha v beta 8 integrin strongly bound to latent TGF-beta 1 but showed marginal activity for other RGD-containing proteins, including fibronectin and vitronectin. Site-directed mutagenesis of latent TGF-beta 1 demonstrated that the high affinity binding of alpha v beta 8 integrin to latent TGF-beta 1 was defined by Leu-218 immediately following the RGD motif within the latency-associated peptide of TGF-beta 1. Consistent with the critical role of Leu-218 in latent TGF-beta 1 recognition by alpha v beta 8 integrin, a 9-mer synthetic peptide containing an RGDL sequence strongly inhibited interactions of latent TGF-beta 1 with alpha v beta 8 integrin, whereas a 9-mer peptide with an RGDA sequence was similar to 60-fold less inhibitory. Because alpha v beta 3 integrin did not exhibit strong binding to latent TGF-beta 1 or distinguish between RGDL- and RGDA-containing peptides, we explored the mechanism by which the integrin beta 8 subunit defines the high affinity binding of latent TGF-beta 1 by alpha v beta 8 integrin. Production of a series of swap mutants of integrin beta 8 and beta 3 subunits indicated that the high affinity binding of alpha v beta 8 integrin with latent TGF-beta 1 was ensured by interactions between the Leu-218 residue and the beta 8 I-like domain, with the former serving as an auxiliary recognition residue defining the restricted ligand specificity of alpha v beta 8 integrin toward latent TGF-beta 1. In support of this conclusion, high affinity binding toward the alpha v beta 8 integrin was conferred on fibronectin by substitution of its RGDS motif with an RGDL sequence.