Molecular Basis of the Recognition of Nephronectin by Integrin α8β1

Abstract

Integrin ␣8␤1 interacts with a variety of Arg-Gly-Asp (RGD)-containing ligands in the extracellular matrix. Here, we examined the binding activities of ␣8␤1 integrin toward a panel of RGD-containing ligands. Integrin ␣8␤1 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 ␣8␤1 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 ␣8␤1 integrin. Alanine scanning mutagenesis within the LFEIFEIER sequence defined the EIE sequence as a critical motif ensuring the high-affinity integrinligand interaction. Although a synthetic LFEIFEIER peptide failed to inhibit the binding of ␣8␤1 integrin to nephronectin, a longer peptide containing both the RGD motif and the LFEIF-EIER sequence was strongly inhibitory, and was ϳ2,000-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 ␣8␤1 integrin. Taken together, these results indicate that the specific high-affinity binding of nephronectin to ␣8␤1 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.Integrins are a family of adhesion receptors that interact with a variety of extracellular ligands, typically cell-adhesive proteins in the extracellular matrix (ECM).2 They play mandatory roles in embryonic development and the maintenance of tissue architectures by providing essential links between cells and the ECM (1). Integrins are composed of two non-covalently associated subunits, termed ␣ and ␤. In mammals, 18 ␣ and 8 ␤ subunits have been identified, and combinations of these subunits give rise to at least 24 distinct integrin heterodimers.Based on their ligand-binding specificities, ECM-binding integrins are classified into three groups, namely laminin-, collagen-and RGD-binding integrins (2, 3), 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, and have been shown to interact with a variety of ECM ligands, such as fibronectin and vitronectin, with distinct binding specificities. The ␣8 integrin subunit was originally identified in chick nerves (4). Integrin ␣8␤1 is expressed in the metanephric mesenchyme and plays a crucial role in epithelial-mesenchymal interactions during the early stages of kidney morphogenesis.

Molecular Basis of the Ligand Binding Specificity of αvβ8 Integrin

“…The occurrence of such an auxiliary binding sequence was originally proposed in the central cellbinding domain of fibronectin, where a set of residues within the 9th type III repeat (designated synergy site) potentiates the ␣5␤1 integrin-mediated cell adhesive activity of the RGD motif within the 10th type III repeat, although electron microscopic analyses failed to confirm a direct interaction of the 9th type III module harboring the synergy site with ␣5␤1 integrin (49). Nephronectin also 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). DiCara et al (25) reported that the interaction of latent TGF-␤1 with ␣v␤6 integrin is determined by an LXXI sequence immediately C-terminal to the RGD motif, in which two hydrophobic residues Leu-218 and Ile-221 are required for the high affinity binding of latent TGF-␤1 to the ␣v␤6 integrin.…”

“…For example, ␣5␤1 integrin specifically 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). The high affinity binding of ␣v␤6 integrin to its ligands, foot-and-mouth disease virus and latent TGF-␤1, requires the RGD motif and an LXX(L/I) sequence, of which the latter forms an ␣-helix to align the two conserved hydrophobic residues along the length of the helix (25,26).…”

Polydom/SVEP1 Is a Ligand for Integrin α9β1

“…Cell Adhesion Assays-Cell adhesion assays were performed as described (30). For the assays involving blocking antibodies, RD cells were preincubated with the specified mAbs (10 g/ml) for 15 min at room temperature and plated on 96-well plates coated with recombinant polydom proteins or fibronectin.…”

Kif26b , a kinesin family gene, regulates adhesion of the embryonic kidney mesenchyme

“…The primary antibodies used were: anti-Sall1 (31) (Perseus Proteomics); anti-cleaved caspase-3 (Cell Signaling); anti-pan-cytokeratin (Sigma); anti-Ecadherin (BD Transduction Laboratories); anti-phosphorylated Erk (Cell Signaling); anti-α8 integrin (1); anti-N-cadherin (Santa Cruz Biotechnology); anti-MYH10 (Cell Signaling or Developmental Studies Hybridoma Bank); and antiacetylated α-tubulin (Sigma). A polyclonal antibody against mouse nephronectin was produced by immunizing rabbits with FLAG-tagged recombinant mouse nephronectin (33). The antibody was purified by affinity chromatography using columns of 6× His-tagged mouse nephronectin immobilized on CNBr-activated Sepharose 4B (GE Healthcare).…”