A Microtiter Assay for Quantifying Protein-Protein Interactions Associated with Cell-Cell Adhesion

Abstract

Cell-cell adhesions are a hallmark of epithelial tissues, and the disruption of these contacts plays a critical role in both the early and late stages of oncogenesis. The interaction between the transmembrane protein E-cadherin and the intracellular protein beta-catenin plays a crucial role in the formation and maintenance of epithelial cell-cell contacts and is known to be downregulated in many cancers. The authors have developed a protein complex enzyme-linked immunosorbent assay (ELISA) that can quantify the amount of beta-catenin bound to E-cadherin in unpurified whole-cell lysates with a Z' factor of 0.74. The quantitative nature of the E-cadherin:beta-catenin ELISA represents a dramatic improvement over the low-throughput assays currently used to characterize endogenous E-cadherin:beta-catenin complexes. In addition, the protein complex ELISA format is compatible with standard sandwich ELISAs for parallel measurements of total levels of endogenous E-cadherin and beta-catenin. In 2 case studies closely related to cancer cell biology, the authors use the protein complex ELISA and traditional sandwich ELISAs to provide a detailed, quantitative picture of the molecular changes occurring within adherens junctions in vivo. Because the E-cadherin: beta-catenin protein complex plays a crucial role in oncogenesis, this protein complex ELISA may prove to be a valuable quantitative prognostic marker of tumor progression.