Anal. Chem., 75 (16), 4176 -4181, 2003. 10.1021/ac0300800 S0003-2700(03)00080-5
Web Release Date: July 15, 2003

Copyright © 2003 American Chemical Society

Locating a Protein-Protein Interaction in Living Cells via Split Renilla Luciferase Complementation

Asami Kaihara, Yasutoshi Kawai, Moritoshi Sato, Takeaki Ozawa, and Yoshio Umezawa*

Department of Chemistry, School of Science, The University of Tokyo and Japan Science and Technology Corporation (JST), Hongo, Bunkyo-ku, Tokyo, Japan 113-0033

Received for review March 3, 2003. Accepted May 7, 2003.

Abstract:

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.

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