title: Monitoring G protein-coupled receptor and β-arrestin trafficking in live cells using enhanced bystander BRET.
abstract: Endocytosis and intracellular trafficking of receptors are pivotal to maintain physiological functions and drug action; however, robust quantitative approaches are lacking to study such processes in live cells. Here we present new bioluminescence resonance energy transfer (BRET) sensors to quantitatively monitor G protein-coupled receptors (GPCRs) and β-arrestin trafficking. These sensors are based on bystander BRET and use the naturally interacting chromophores luciferase (RLuc) and green fluorescent protein (rGFP) from Renilla. The versatility and robustness of this approach are exemplified by anchoring rGFP at the plasma membrane or in endosomes to generate high dynamic spectrometric BRET signals on ligand-promoted recruitment or sequestration of RLuc-tagged proteins to, or from, specific cell compartments, as well as sensitive subcellular BRET imaging for protein translocation visualization. These sensors are scalable to high-throughput formats and allow quantitative pharmacological studies of GPCR trafficking in real time, in live cells, revealing ligand-dependent biased trafficking of receptor/β-arrestin complexes.
authors.affiliation: Department of Medicine, Research Institute of the McGill University Health Center (RI-MUHC), McGill University, Montréal, Québec, Canada H4A 3J1.
authors.forename: Yoon
authors.identifier: 0000-0002-3085-2066
authors.initials: Y
authors.lastname: Namkung
authors.affiliation: Department of Biochemistry and Institute for Research in Immunology and Cancer (IRIC), Université de Montréal, Montréal, Québec, Canada H3C 1J4.
authors.forename: Christian
authors.initials: C
authors.lastname: Le Gouill
authors.affiliation: Department of Biochemistry and Institute for Research in Immunology and Cancer (IRIC), Université de Montréal, Montréal, Québec, Canada H3C 1J4.
authors.forename: Viktoria
authors.initials: V
authors.lastname: Lukashova
authors.affiliation: Department of Biochemistry and Institute for Research in Immunology and Cancer (IRIC), Université de Montréal, Montréal, Québec, Canada H3C 1J4.
authors.forename: Hiroyuki
authors.identifier: 0000-0003-4965-0883
authors.initials: H
authors.lastname: Kobayashi
authors.affiliation: Department of Biochemistry and Institute for Research in Immunology and Cancer (IRIC), Université de Montréal, Montréal, Québec, Canada H3C 1J4.
authors.forename: Mireille
authors.initials: M
authors.lastname: Hogue
authors.affiliation: Department of Medicine, Research Institute of the McGill University Health Center (RI-MUHC), McGill University, Montréal, Québec, Canada H4A 3J1.
authors.forename: Etienne
authors.initials: E
authors.lastname: Khoury
authors.affiliation: Department of Medicine, Research Institute of the McGill University Health Center (RI-MUHC), McGill University, Montréal, Québec, Canada H4A 3J1.
authors.forename: Mideum
authors.initials: M
authors.lastname: Song
authors.affiliation: Department of Biochemistry and Institute for Research in Immunology and Cancer (IRIC), Université de Montréal, Montréal, Québec, Canada H3C 1J4.
authors.forename: Michel
authors.identifier: 0000-0003-1128-0100
authors.initials: M
authors.lastname: Bouvier
authors.affiliation: Department of Medicine, Research Institute of the McGill University Health Center (RI-MUHC), McGill University, Montréal, Québec, Canada H4A 3J1.|Department of Pharmacology and Therapeutics, McGill University, Montréal, Québec, Canada H3G 1Y6.|Department of Anatomy and Cell Biology, McGill University, Montréal, Québec, Canada H3A 0C7.
authors.forename: Stéphane A
authors.initials: SA
authors.lastname: Laporte
mesh_terms: D060807:Bioluminescence Resonance Energy Transfer Techniques / Q000379:methods*; D002462:Cell Membrane / Q000378:metabolism*; D011992:Endosomes / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D057809:HEK293 Cells; D006801:Humans; D008156:Luciferases; D021381:Protein Transport; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D000071557:beta-Arrestins / Q000378:metabolism*
chemical_list: D043562:Receptors, G-Protein-Coupled; D000071557:beta-Arrestins; D049452:Green Fluorescent Proteins; D008156:Luciferases