Real-time monitoring of receptor and G-protein interactions in living cells Ce ´line Gale ´s 1 , R Victor Rebois 2 , Mireille Hogue 1 , Phan Trieu 3 , Andreas Breit 1 , Terence E He ´bert 1,3 & Michel Bouvier 1 G protein–coupled receptors (GPCRs) represent the largest family of proteins involved in signal transduction. Here we present a bioluminescence resonance energy transfer (BRET) assay that directly monitors in real time the early interactions between human GPCRs and their cognate G-protein subunits in living human cells. In addition to detecting basal precoupling of the receptors to Ga-, Gb- and Gc-subunits, BRET measured very rapid ligand-induced increases in the interaction between receptor and Gabc-complexes (t 1/2 B 300 ms) followed by a slower (several minutes) decrease, reflecting receptor desensitization. The agonist-promoted increase in GPCR-Gbc interaction was highly dependent on the identity of the Ga-subunit present in the complex. Therefore, this G protein– activity biosensor provides a novel tool to directly probe the dynamics and selectivity of receptor-mediated, G-protein activation-deactivation cycles that could be advantageously used to identify ligands for orphan GPCRs. GPCRs represent the largest family of cell surface receptors involved in signal transduction across biological membranes. These recep- tors control many diverse physiological processes and, conse- quently, are important targets for the development of drugs with wide clinical applications. In recent years, increased knowledge about GPCRs has facilitated the development and screening of many new therapeutically active molecules. However, our under- standing of the molecular events determining receptor signaling efficacy remains rudimentary. Although recent advances in cellular biology have led to the identification of a broad range of proteins directly interacting with GPCRs 1 , coupling with abg-trimeric G proteins remains the common benchmark of all GPCR family members. Thus, these proteins constitute one of the most important and earliest plasma membrane transducers, relaying information from activated cell surface GPCRs to intracellular signalling molecules. It is generally accepted that agonist binding promotes or stabilizes specific con- formational states of the receptors that favor the engagement of the Gabg-complex. The ensuing exchange of guanosine diphosphate (GDP) for guanosine triphosphate (GTP) on the Ga-subunit is then believed to precipitate complex disassembly, leading to free GTP-bound Ga- and Gbg-subunits that can in turn interact and modulate downstream effectors. The termination of the signal results from the reassociation of the Gabg-inactive heterotrimer after GTP hydrolysis by the Ga-subunit 2–4 . Despite much evidence supporting this activation-inactivation cycle model, no method permits direct assessment of the real-time interactions between receptors and G proteins in living cells. Such assays would be particularly relevant since the dynamics of protein interactions can be influenced by multiple spatiotemporal factors not easily recapitulated in vitro. The emergence of resonance energy transfer–based approaches for the study of protein-protein interactions 5,6 offers new oppor- tunities to probe the dynamics of signal transduction in living cells 7–9 . Herein, we present the development of a BRET-based approach that directly monitors real-time interactions between GPCRs and their cognate G proteins in living mammalian cells. This provides a reliable biosensor to directly monitor the kinetics and selectivity of G-protein engagement upon receptor activation. In addition to offering a new generally applicable method to probe dynamic protein interactions, the assay allows monitoring of the activation of distinct G proteins using a single assay mode. RESULTS BRET between receptors and Gabc fusion proteins The prototypical family 1 GPCR, b 2 -adrenoceptor (b 2 AR), was used as our main model to probe the interaction with its preferred Ga-subunit, Ga s , and the ubiquitous Gb 1 - and Gg 2 - subunits after receptor activation. For this purpose, we developed a proximity- based BRET 2 assay (Fig. 1) that relies on the nonradiative transfer of energy between the energy donor Renilla reniformis luciferase (Rluc) and the energy acceptor GFP10 10 , a blue-shifted variant of the Aequorea victoria green fluorescent protein. The receptor–G protein interaction assay was conceived by fusing Rluc to the C terminus of the b 2 AR. For Ga s , GFP10 was inserted within the linker 1 region between the helical and GTPase domains as previously described 11 , whereas the Gb 1 - and Gg 2 -subunits were fused to GFP10 at their N termini. All fusion proteins were found to be functional. PUBLISHED ONLINE 17 FEBRUARY 2005; DOI:10.1038/NMETH743 1 Department of Biochemistry, Universite ´ de Montre ´al, P.O. Box 6128 Down-town station, Montre ´al, H3C 3J7, Canada. 2 Laboratory of Cellular Biology, 5 Research Court, Room 2A11, National Institutes of Health, Rockville, Maryland 20850-3211, USA. 3 Institut de Cardiologie de Montre ´ al et De ´partement d’Anesthe ´siologie, Universite ´ de Montre ´al, 5000 Be ´langer St. East, Montre ´al, H1T 1C8, Canada. Correspondence should be addressed to M.B. (michel.bouvier@umontreal.ca). NATURE METHODS | VOL.2 NO.3 | MARCH 2005 | 177 ARTICLES © 2005 Nature Publishing Group http://www.nature.com/naturemethods