| E-cadherin, which is expressed on epithelial cells, is classically described as a homophilic adhesion molecule critical in embryonic development and later in maintaining tissue integrity via adherens junctions. Recently, E-cadherin has been described as a ligand for the integrin αEβ 7 that is selectively expressed on a subpopulation of T lymphocytes located within the epithelium termed intraepithelial lymphocytes. Prior determination of the crystal structures of the murine E- and N-cadherin revealed that the cadherin domain consists of seven β-strands arranged in two β-sheets. The β-strands are connected by loops that make up the majority of the solvent-exposed residues. The realization that the cadherin domain is similar to the immunoglobulin (Ig) fold suggested that integrin recognition of E-cadherin may be similar to recognition of Ig super-family ligands (ICAMs, MAdCAM-1 and VCAM-1) or fibronectin that shares an Ig-like topology. In order to understand the molecular basis of αEβ7 recognition of E-cadherin, we developed a cell-to-substrate adhesion assay to measure adhesion of α Eβ7-expressing cells to an immobilized, recombinant E-cadherin fusion protein consisting of the five extracellular domains of human E-cadherin coupled to the Fc region of human IgG1. Using a model of domain 1 of human E-cadherin, we selected solvent-exposed residues for site-directed mutagenesis. Mutations E31A, E31D and KEG30-32RDT completely abrogated the adhesion of αEβ7-expressing cells and localized integrin recognition of E-cadherin to Glu31 at the tip of the BC loop on top of domain 1. These mutants retained the ability to function in homophilic adhesion providing the first direct evidence that the integrin α Eβ7 binding site is distinct from the homophilic binding site. Further mutagenesis identified that the sidechains of Asn27, Lys30 and Glu89, which are predicted to project from the face formed by the BC and FG loops, could also contribute to a site of interaction with the integrin α Eβ7. Our studies extend the paradigm that an Ig-like fold can serve as a scaffold for integrin recognition and that integrin ligands have a critical acidic residue that may be coordinated to a metal ion bound to the integrin MIDAS motif. However, the site of integrin recognition of E-cadherin at the BC loop is distinct from the recognition of ICAMs, VCAM-1 and MAdCAM-1 that takes place on the C-strand or CD-loop and recognition of fibronectin that takes place on the FG loop. |
