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.
Kaihara
Asami
A
Department of Chemistry, School of Science, The University of TokyoHongo, Bunkyo-ku, Tokyo, Japan 113-0033.
Kawai
Yasutoshi
Y
Sato
Moritoshi
M
Ozawa
Takeaki
T
Umezawa
Yoshio
Y
D000595:Amino Acid Sequence; D000818:Animals; D037421:Anthozoa / Q000201:enzymology; D016466:CHO Cells; D002462:Cell Membrane / Q000737:chemistry; D006224:Cricetinae; D005816:Genetic Complementation Test; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D008156:Luciferases / Q000032:analysis / Q000737:chemistry* / Q000235:genetics; D008163:Luminescent Measurements; D008969:Molecular Sequence Data; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D019154:Protein Splicing; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013997:Time Factors
D007328:Insulin; D011993:Recombinant Fusion Proteins; D008156:Luciferases