title: Visualization and quantitative analysis of G protein-coupled receptor-β-arrestin interaction in single cells and specific organs of living mice using split luciferase complementation.
abstract: Methods used to assess the efficacy of potentially therapeutic reagents for G protein-coupled receptors (GPCRs) have been developed. Previously, we demonstrated sensitive detection of the interaction of GPCRs and β-arrestin2 (ARRB2) using 96-well microtiter plates and a bioluminescence microscope based on split click beetle luciferase complementation. Herein, using firefly luciferase emitting longer wavelength light, we demonstrate quantitative analysis of the interaction of β2-adrenergic receptor (ADRB2), a kind of GPCR, and ARRB2 in a 96-well plate assay with single-cell imaging. Additionally, we showed bioluminescence in vivo imaging of the ADRB2-ARRB2 interaction in two systems: cell implantation and hydrodynamic tail vein (HTV) methods. Specifically, in the HTV method, the luminescence signal from the liver upon stimulation of an agonist for ADRB2 was obtained in the intact systems of mice. The results demonstrate that this method enables noninvasive screening of the efficacy of chemicals at the specific organ in in vivo testing. This in vivo system can contribute to effective evaluation in pharmacokinetics and pharmacodynamics and expedite the development of new drugs for GPCRs.
authors.affiliation: Department of Chemistry, School of Science, The University of Tokyo, 7-3-1 Hongo, Tokyo 113-0033, Japan.
authors.forename: Hideo
authors.initials: H
authors.lastname: Takakura
authors.forename: Mitsuru
authors.initials: M
authors.lastname: Hattori
authors.forename: Masaki
authors.initials: M
authors.lastname: Takeuchi
authors.forename: Takeaki
authors.initials: T
authors.lastname: Ozawa
mesh_terms: D000818:Animals; D019390:Arrestins / Q000032:analysis / Q000378:metabolism*; D002460:Cell Line; D048888:Fireflies / Q000201:enzymology; D006801:Humans; D049409:Luciferases, Firefly / Q000032:analysis; D049408:Luminescent Agents / Q000032:analysis; D008163:Luminescent Measurements / Q000379:methods*; D051379:Mice; D025941:Protein Interaction Mapping / Q000379:methods*; D018343:Receptors, Adrenergic, beta-2 / Q000032:analysis / Q000378:metabolism*; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins
chemical_list: C000605254:ARRB2 protein, human; C000605258:Arrb2 protein, mouse; D019390:Arrestins; D049408:Luminescent Agents; D018343:Receptors, Adrenergic, beta-2; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins; D049409:Luciferases, Firefly