Locating a protein-protein interaction in living cells via split Renilla luciferase complementation

For spatial and quantitative kinetic analysis of protein-protein interactions (PPIs) in living mammalian cells, a method was developed in which PPI-induced complementation of split Renilla luciferase triggers spontaneous emission of luminescence using a cell membrane permeable substrate, coelenterazine. This split Renilla luciferase complementation readout was shown to work for locating a PPI between the tyrosine-phosphorylated peptide (Y941) of IRS-1 and the SH2 domain of PI3K among insulin signaling pathways in living Chinese hamster ovary cells overexpressing human insulin receptors (CHO-HIR). It was thereby found that the insulin-stimulated interaction occurred near the plasma membrane in the cytosol.