Assembly of the cadherin-catenin complex in vitro with recombinant proteins

Abstract

The cytoplasmic domain of classical cadherins is tightly associated with three proteins termed α-, β- and γ-catenin. These accessory proteins are of central importance for the adhesive properties of this class of cell adhesion molecules. In order to examine the molecular architecture of the cadherin-catenin complex in more detail we have expressed the catenins and the cytoplasmic domain of E-cadherin as fusion proteins in Escherichia coli, and analyzed the interaction of purified recombinant cadherin and catenins in combinatorial protein-protein interaction experiments. The cytoplasmic domain of E-cadherin cannot directly associate with α-catenin but interacts with high affinity with β-catenin, whereas the binding of γ-catenin (plakoglobin) to E-cadherin is less efficient. α- and β-catenin assemble into a 1:1 heterodimeric complex. The analysis of various truncated β-catenins revealed that an α-catenin binding site in β-catenin is localized between amino acid positions 120 and 151. The central role of β-catenin for the assembly of the heterotrimeric E-cadherin/α-catenin/β-catenin complex in mixing experiments with all components was demonstrated. The reconstitution in vitro of the cadherin-catenin complex should allow the study of the interaction with signalling molecules and with the actinbased cytoskeleton.