In Vitro and In Vivo Analysis of Protein-Protein Interactions Involved in the Formation of Epithelial Adherens Junctions

Abstract

Adherens junctions are a main cell-cell adhesion structure found in epithelial cells. The stability of adherens junctions is attributed to various protein-signaling cascades and importantly the interaction between the transmembrane protein E-cadherin and cytoplasmic p120 catenin. This interaction is critical for cell adhesion and prevention of uncontrolled growth in normal cells. The interaction interface between these two binding partners was previously determined to comprise p120's Armadillo repeat domain (p120Arm) and Ecadherin's cytoplasmic juxtamembrane domain (Ecadc). Based on this information, peptide aptamers were derived from p120Arm and their interaction with Ecadc was tested in vitro. We reasoned that those could be expressed in vivo to stabilize adherens junctions at the cell-cell junction. In this study, we established protein-protein interaction assays to demonstrate p120Arm's ability to bind Ecadc and then used these assays to determine if p120Arm-derived peptides may competitively bind Ecadc. We demonstrated the interaction between p120Arm and Ecadc using assays that were not previously used such as: co-precipitation, analytical gel filtration and the bacterial-2-hybrid assay. However, the p120Arm-derived peptides did not bind to Ecadc or compete its interaction with p120Arm. This may be due to the nature of the assays that may not reflect competitive binding or the aptamers may not adopt the native conformation preventing binding to Ecadc.