Rapid and High-Sensitivity Cell-Based Assays of Protein−Protein Interactions Using Split Click Beetle Luciferase Complementation: An Approach to the Study of G-Protein-Coupled Receptors Naomi Misawa A. K. M. Kafi Mitsuru Hattori Kenji Miura Kenji Masuda Takeaki Ozawa 10.1021/ac100104q.s002 https://acs.figshare.com/articles/media/Rapid_and_High_Sensitivity_Cell_Based_Assays_of_Protein_Protein_Interactions_Using_Split_Click_Beetle_Luciferase_Complementation_An_Approach_to_the_Study_of_G_Protein_Coupled_Receptors/2784355 To identify biologically relevant compounds in basic biology and drug discovery processes, rapid quantitative methods for elucidating protein−protein interactions have become necessary. We describe a novel optical technique for monitoring protein−protein interactions in living cells based on complementation of split luciferase fragments from click beetle (Brazilian <i>Pyrearinus termitilluminans</i>). A new pair of amino-terminal and carboxy-terminal fragments of the luciferase was identified using semirational library screening, demonstrating achieved markedly higher sensitivity and signal-to-background ratio. The identified fragments were applied to the study of five G-protein coupled receptors (GPCR) that interact with β-arrestin on the plasma membrane. By generating cell lines stably expressing the GPCRs and β-arrestin connected with the luciferase fragments, we demonstrated rapid and sensitive screening of potential chemicals that act on GPCRs using a 96-well microtiter plate format. The screening time was reduced to 5−10 min after ligand stimulation. The maximum response became more than 15-fold higher than the background signal. This luciferase complementation method also enabled accurate spatial and temporal analyses of interactions in single living cells using bioluminescence microscopy. These GPCR assays will facilitate developments of high-throughput screening systems in a multiwell plate format. Furthermore, using specific proteins of interest, the novel fragments of luciferase will provide different assay methods for the study of many intracellular signals in living cells and animals. 2010-03-15 00:00:00 split luciferase fragments microtiter plate format cell lines stably background signal novel fragments Click Beetle Luciferase Complementation multiwell plate format ligand stimulation intracellular signals GPCR assays interaction screening time luciferase complementation method drug discovery processes semirational library screening plasma membrane assay methods arrestin click beetle Brazilian Pyrearinus termitilluminans luciferase fragments protein bioluminescence microscopy