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.
Highlights
14524 JOURNAL OF BIOLOGICAL CHEMISTRY in embryonic development and the maintenance of tissue architectures by providing essential links between cells and the extracellular matrix (ECM) [1]
To date, ␣81 integrin has been shown to bind to fibronectin, vitronectin, osteopontin, latency-associated peptide of transforming growth factor-1, tenascin-W, and nephronectin [7,8,9,10,11,12,13], among which nephronectin is believed to be an ␣81 integrin ligand involved in kidney development [10]
linker segment (LS)/391– 407 and LS/395– 407, both lacking the RGD motif but retaining the (D)LFEIFEIER sequence, were barely active in binding to ␣81 integrin, whereas LS/381– 407, an N-terminal deletion mutant possessing both the RGD motif and the DLFEIFEIER sequence, was fully active in binding to ␣81 integrin (Fig. 4C). These results indicate that both the RGD motif and the DLFEIFEIER sequence at 10 residues to the C-terminal side of the RGD motif are required for the high-affinity binding of nephronectin to ␣81 integrin
Summary
Cells and Reagents—HT1080 cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum. For expression of a panel of nephronectin deletion mutants in mammalian cells, a cDNA fragment encoding a mouse nephronectin 5Ј-non-translated region and the signal peptide (nucleotides Ϫ61 to 69) was amplified by PCR with the primer set 5Ј-GAATTCGAGATCCCGGGACGC-3Ј (forward) and 5Ј-GGGCCCGTCGAAGTCGGCAGC-3Ј (reverse) using the full-length nephronectin cDNA as a template This fragment was digested with EcoRI and ApaI, and fused in-frame with cDNAs encoding the RGD-linker segment or the MAM domain. The plates were washed with TBS containing 1 mM MnCl2, 0.1% BSA, and 0.02% Tween 20 with or without 10 mM EDTA, followed by quantification of bound integrins by an enzyme-linked immunosorbent assay using a biotinylated rabbit anti-Velcro (ACID/BASE coiledcoil) antibody and horseradish peroxidase-conjugated streptavidin. After lysis in 1% SDS, the attached cells were quantified by their absorption at 595 nm
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