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.
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 Pyrearinus termitilluminans). 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 beta-arrestin on the plasma membrane. By generating cell lines stably expressing the GPCRs and beta-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.
Misawa
Naomi
N
Department of Chemistry, School of Science, The University of Tokyo, 7-3-1 Bunkyo-ku, Hongo, Tokyo 113-0033, Japan.
Kafi
A K M
AK
Hattori
Mitsuru
M
Miura
Kenji
K
Masuda
Kenji
K
Ozawa
Takeaki
T
D000818:Animals; D019390:Arrestins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D001517:Coleoptera / Q000201:enzymology*; D015870:Gene Expression; D006801:Humans; D008156:Luciferases / Q000737:chemistry / Q000378:metabolism*; D025941:Protein Interaction Mapping / Q000379:methods*; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism*; D012680:Sensitivity and Specificity; D000071557:beta-Arrestins
D019390:Arrestins; D043562:Receptors, G-Protein-Coupled; D000071557:beta-Arrestins; D008156:Luciferases