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Subcellular Imaging of Dynamic Protein Interactions by Bioluminescence Resonance Energy Transfer

Vincent Coulon ^* , Martin Audet , Vincent Homburger ^*, Joël Bockaert ^*, Laurent Fagni ^*, Michel Bouvier  and Julie Perroy ^*

^* Institut de Génomique fonctionnelle, CNRS UMR5203, INSERM U661, University of Montpellier, Montpellier, France; Laboratoire de Dermatologie Moléculaire-EA3754, UFR Médecine Site NORD UPM/IURC, Montpellier, France; and Department of Biochemistry and Groupe de Recherche Universitaire sur le Médicament, Institute of Research in Immunology and Cancer, Université de Montréal, Montréal, QC, Canada

Correspondence: Address reprint requests to Julie Perroy, IGF, 141 rue de la Cardonille, 34094 Montpellier Cedex 05, France. Tel.: 33-467142960; Fax: 33-467542432; E-mail: julie.perroy{at}igf.cnrs.fr.

Despite the fact that numerous studies suggest the existence of receptor multiprotein complexes, visualization and monitoring of the dynamics of such protein assemblies remain a challenge. In this study, we established appropriate conditions to consider spatiotemporally resolved images of such protein assemblies using bioluminescence resonance energy transfer (BRET) in mammalian living cells. Using covalently linked Renilla luciferase and yellow fluorescent proteins, we depicted the time course of dynamic changes in the interaction between the V2-vasopressin receptor and β-arrestin induced by a receptor agonist. The protein-protein interactions were resolved at the level of subcellular compartments (nucleus, plasma membrane, or endocytic vesicules) and in real time within tens-of-seconds to tens-of-minutes time frame. These studies provide a proof of principle as well as experimental parameters and controls required for high-resolution dynamic studies using BRET imaging in single cells.

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