β-catenin’s interaction with the membrane binding domain (MBD) of Protein 4.1R
Protein 4.1R is a crucial cytoskeletal protein that links the spectrin-actin cytoskeleton to transmembrane proteins in erythrocytes. In endothelial cells, 4.1R co-localizes and associates in a complex with the adherens junction proteins β-catenin and VE-cadherin. Adherens junctions (AJ) are intercellular junctions essential for providing strong mechanical attachments between adjacent cells in a wide variety of tissues. Previous work from our lab has shown that the MBD of 4.1R is responsible for its direct interaction with the armadillo domain of β-catenin. Indeed, 4.1R knockdown induces a decrease of β-catenin protein level in the cell, resulting in functional disorder of AJ. When β-catenin was sequenced, it was found to have multiple copies of the armadillo repeat domain that is specialized for protein-protein binding. We used a Yeast Two-Hybrid assay to map precisely which of these armadillo repeat(s) is responsible for its interaction with the MBD of 4.1R. Plasmids carrying different repeats were expressed as fusions to the GAL4 DNA-binding domain and co-transformed with fusions of the MBD of 4.1R to the GAL4 transcriptional activation domain. We observed interactions most notably in the 1-2 armadillo domain of β-catenin, supported by its consistent secretion of α-galactosidase in response to GAL4 activation. We believe the association between 4.1R and β-catenin contributes to the stabilization of the AJ complex in endothelial cells and functions in the organization of the junctional complex. The elucidation of the functional role of 4.1R in endothelial cells will further our understanding towards the etiology of functional AJ assembly disorder.