The EMARS Method: A Tool for Molecular Interactome

Abstract

Important biological events associated with plasma membranes, such as signal transduction, cell adhesion, and protein trafficking, are mediated through lipid rafts. These biological events involve interactions between functional molecules. It is, therefore, important to know crucial molecular interactions required for biological outputs. We have developed a novel method termed enzyme-mediated activation of radical sources (EMARS) to identify coclustering molecules on the cell surface under living conditions, which is featured by a radical formation of the labeling reagent by horseradish peroxidase (HRP). Spatiotemporally regulated interaction between β1 integrin and ErbB4 involved in fibronectin-dependent cell migration and therapeutic antibody-stimulated interaction between FGFR3 and CD20 have been found using this method. Furthermore, this method has identified molecules coclustering with a glycosylphosphatidylinositol (GPI)-anchored HRP fusion protein in individual raft domains. Thus, the EMARS method is useful to address the cell surface “molecular interactome” and provides a clue to study functional molecular interactions in biological events.