Mapping of mutual binding sites in the hemidesmosomal proteins BPAG1e and α6ß4 integrin

Abstract

BPAG1e is a member of the plakin family of cytolinkers present in the hemidesmosomes (HDs), junctional multiprotein complexes that mediate the stable adhesion and provide resistance to mechanical stress by linking the extracellular matrix with the intermediate filament system of the cytoskeleton. BPAG1e binds to the transmembrane receptor integrin α6ß4. The BPAG1e-α6ß4 interaction occurs through a region near the amino terminus of BPAG1e and the third and fourth FnIII domains (FnIII-3,4) of the ß4 cytoplasmic moiety. In this work, we have mapped in detail the binding sites in BPAG1e and ß4. The interaction was analysed in vitro by measuring changes in the fluorescence anisotropy of fluorescein and Oregon Green labelled-samples. By using deletion BPAG1e mutants, a region of ~30 residues was identified to be sufficient to support bind to ß4 integrin. On other hand, by using a broad collection of structure-based ß4 integrin point mutants, the BPAG1e-binding site has been mapped to a groove near the interface between the FnIII-3 and FnIII-4 domains of ß4. Collectively, our results have implications for the regulation of the HDs and for signaling processes mediated by integrin α6ß4.