title: Molecular basis of the recognition of nephronectin by integrin alpha8beta1.
abstract: Integrin alpha8beta1 interacts with a variety of Arg-Gly-Asp (RGD)-containing ligands in the extracellular matrix. Here, we examined the binding activities of alpha8beta1 integrin toward a panel of RGD-containing ligands. Integrin alpha8beta1 bound specifically to nephronectin with an apparent dissociation constant of 0.28 +/- 0.01 nm, but showed only marginal affinities for fibronectin and other RGD-containing ligands. The high-affinity binding to alpha8beta1 integrin was fully reproduced with a recombinant nephronectin fragment derived from the RGD-containing central "linker" segment. A series of deletion mutants of the recombinant fragment identified the LFEIFEIER sequence on the C-terminal side of the RGD motif as an auxiliary site required for high-affinity binding to alpha8beta1 integrin. Alanine scanning mutagenesis within the LFEIFEIER sequence defined the EIE sequence as a critical motif ensuring the high-affinity integrin-ligand interaction. Although a synthetic LFEIFEIER peptide failed to inhibit the binding of alpha8beta1 integrin to nephronectin, a longer peptide containing both the RGD motif and the LFEIFEIER sequence was strongly inhibitory, and was approximately 2,000-fold more potent than a peptide containing only the RGD motif. Furthermore, trans-complementation assays using recombinant fragments containing either the RGD motif or LFEIFEIER sequence revealed a clear synergism in the binding to alpha8beta1 integrin. Taken together, these results indicate that the specific high-affinity binding of nephronectin to alpha8beta1 integrin is achieved by bipartite interaction of the integrin with the RGD motif and LFEIFEIER sequence, with the latter serving as a synergy site that greatly potentiates the RGD-driven integrin-ligand interaction but has only marginal activity to secure the interaction by itself.
authors.affiliation: Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Yuya
authors.initials: Y
authors.lastname: Sato
authors.forename: Toshihiko
authors.initials: T
authors.lastname: Uemura
authors.forename: Keisuke
authors.initials: K
authors.lastname: Morimitsu
authors.forename: Ryoko
authors.initials: R
authors.lastname: Sato-Nishiuchi
authors.forename: Ri-Ichiroh
authors.initials: R
authors.lastname: Manabe
authors.forename: Junichi
authors.initials: J
authors.lastname: Takagi
authors.forename: Masashi
authors.initials: M
authors.lastname: Yamada
authors.forename: Kiyotoshi
authors.initials: K
authors.lastname: Sekiguchi