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Monitoring Ligand-Dependent Assembly of Receptor Ternary Monitoring ligand-dependent assembly of receptor PNAS PLUS ternary complexes in live cells by BRETFect David Cotnoir-Whitea,1, Mohamed El Ezzya, Pierre-Luc Boulaya, Marieke Rozendaala, Michel Bouviera,b, Etienne Gagnona,c,1, and Sylvie Madera,b,d,1 aInstitute for Research in Immunology and Cancer, Université de Montréal, Montréal, QC H3T 1J4, Canada; bDépartement de Biochimie et Médecine Moléculaire, Université de Montréal, Montréal, QC H3T 1J4, Canada; cDépartement de Microbiologie, Infectiologie et Immunologie, Université de Montréal, Montréal, QC H3T 1J4, Canada; and dCentre de Recherche du Centre Hospitalier Universitaire de Montréal, Université de Montréal, Montréal, QC H2X 0A9, Canada Edited by Pierre Chambon, Institut de Génétique et de Biologie Moléculaire et Cellulaire, Strasbourg, France, and approved January 19, 2018 (received for review September 24, 2017) There is currently an unmet need for versatile techniques to monitor false negative results (3). Carriba et al. (4) developed an approach the assembly and dynamics of ternary complexes in live cells. termed “sequential resonance energy transfer,” basedonsequential Here we describe bioluminescence resonance energy transfer with transfer of energy from a donor to an intermediate by BRET and fluorescence enhancement by combined transfer (BRETFect), a high- subsequently to an acceptor by FRET, to measure heteromeric throughput technique that enables robust spectrometric detection interactions between more than two neurotransmitter receptors. of ternary protein complexes based on increased energy transfer However, potential shortcomings include signal contamination, from a luciferase to a fluorescent acceptor in the presence of a necessitating resolution by spectral unmixing, and inefficient cou- fluorescent intermediate. Its unique donor–intermediate–acceptor pling between chromophores, reducing the output signal, limiting relay system is designed so that the acceptor can receive energy the use of serial resonance energy transfer (SRET) as a versatile, either directly from the donor or indirectly via the intermediate in high-throughput–amenable spectrometric detection method for a combined transfer, taking advantage of the entire luciferase emis- heteromeric complexes. sion spectrum. BRETFect was used to study the ligand-dependent To overcome the limitations of current ternary complex de- cofactor interaction properties of the estrogen receptors ERα and tection methods, we developed a RET assay that entails the use ERβ, which form homo- or heterodimers whose distinctive regula- of an intermediate fluorescence protein tailored to ensure a PHARMACOLOGY tory properties are difficult to dissect using traditional methods. strong increase in output signal when the ternary complex is BRETFect uncovered the relative capacities of hetero- vs. homo- formed while minimizing artifactual signals. We termed this dimers to recruit receptor-specific cofactors and regulatory proteins, method “BRET with fluorescence enhancement by combined and to interact with common cofactors in the presence of receptor- transfer” (BRETFect) and demonstrate using several examples specific ligands. BRETFect was also used to follow the assembly of that it reliably enables spectrometric detection of ternary com- ternary complexes between the V2R vasopressin receptor and two plex formation in live cells without the need for spectral different intracellular effectors, illustrating its use for dissection of unmixing. We validated this approach for the study of homo- – ternary protein protein interactions engaged by G protein-coupled dimers formed by the estrogen receptors ERα or ERβ, which receptors. Our results indicate that BRETFect represents a powerful function as dimeric ligand-dependent transcription factors that and versatile technique to monitor the dynamics of ternary interac- recruit a variety of cofactors via two transcriptional activation tions within multimeric complexes in live cells. Significance bioluminescence resonance energy transfer | ternary complexes | nuclear receptors | G protein-coupled receptors | heterodimers Many biological processes, including signal transduction pathways and gene expression regulation, require the assembly of multi- he need to interrogate multimeric protein complex formation subunit protein complexes in a temporally coordinated fashion. Tin live cells is crucial for our understanding of a multitude of However, approaches to studying the dynamics and properties of cellular processes, including signal transduction and transcrip- multimeric complex assembly currently remain limited. We have tional regulation. Ternary complexes can be detected by fluo- developed a method to monitor assembly of trimeric complexes in rescence resonance energy transfer (FRET), based on sequential live cells, using a specific combination of bioluminescence- and transfer between three fluorophores in a three-color or triple- fluorescence-based energy transfer. We illustrate the potential of FRET assay (1, 2). However, this approach suffers from constraints this technology to reveal ligand-activated assembly of ternary due to photobleaching and contaminating cross-excitation re- protein complexes involving nuclear receptors or G protein- quiring spectral unmixing, typically yielding low signal output and a coupled receptors (GPCRs) and their effectors or regulators. We restricted dynamic range. The need for an advanced microscopy show that this methodology uniquely enables the functional setup has also limited implementation in high-throughput screens. characterization of nuclear receptor heterodimers and the dem- Bioluminescence resonance energytransfer(BRET)assaysdonot onstration of corecruitment of effector proteins to a GPCR. require use of fluorescence excitation, reducing the need for compensation in the acceptor channel due to cross-excitation. The Author contributions: D.C.-W., E.G., and S.M. designed research; D.C.-W., M.E.E., and M.R. enhanced cell survival also extends the potential duration of dy- performed research; M.B. contributed new reagents/analytic tools; D.C.-W., P.-L.B., M.B., namic follow-up. BRET interrogates interactions between two E.G., and S.M. analyzed data; and D.C.-W., E.G., and S.M. wrote the paper. protein partners, but combination with either a protein comple- The authors declare no conflict of interest. mentation assay (PCA) or FRET has been used to a limited extent This article is a PNAS Direct Submission. to detect ternary complexes (3, 4). Reconstitution of a green Published under the PNAS license. fluorescent protein or a luciferase from separate fragments in a 1To whom correspondence may be addressed. Email: [email protected], PCA (5, 6) enables combination with BRET for spectrometric [email protected], or [email protected]. detection of complexes involving three proteins but can affect This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. the kinetics of assembly and stability of complexes as well as 1073/pnas.1716224115/-/DCSupplemental. introduce conformational constraints, leading to a high rate of Published online February 27, 2018. www.pnas.org/cgi/doi/10.1073/pnas.1716224115 PNAS | vol. 115 | no. 11 | E2653–E2662 Downloaded by guest on September 30, 2021 domains, AF1 and AF2 (7–10). ERs mediate estrogenic effects Venus fluorescence emission. However, less than half of the in a wide range of physiological processes and pathologies, in- emission spectrum of RLucII/coel-h overlaps with the Venus cluding breast cancer (7, 11). ERα and ERβ have distinct func- excitation spectrum (Fig. 1B, blue and yellow dotted lines, re- tional properties, including differential roles in estrogen-induced spectively), indicating that many of the energetic states arising breast cancer cell growth (11–14) and in response to synthetic from coel-h oxidation are not compatible with RET. antiestrogens used in the clinic to block estrogen signaling (15, A similar observation is made when considering the spectral 16). ERα and ERβ have overlapping tissue-specific expression overlap between RLucII and another BRET partner, monomeric patterns and can form heterodimers when coexpressed, but the TFP1 (mTFP1) (Fig. 1C, blue and cyan dotted lines, re- patterns of specific cofactor recruitment by heterodimers in the spectively). Interestingly, mTFP1 (20) and Venus do not com- presence of various ER ligands remain underexplored due to a pete for resonant energy transfer, due to the minimal overlap lack of suitable assays (12). Using BRETFect, we revealed that between their excitation spectra (Fig. 1D, cyan and yellow dotted the ERα partner in heterodimers is sufficient to trigger recruitment lines, respectively). This suggests that mTFP1 can act as an en- of an ERα AF1-specific cofactor domain in the presence of ergy relay between RLucII/coel-h and Venus for the portion of agonists, or of small ubiquitin-like modifier (SUMO) modification the emission spectrum that is less favorable for direct transfer to marks in the presence of pure antiestrogens, and demonstrated that Venus (i.e., the 400- to 460-nm window) (Fig. 1D). Indeed, en- receptor-selective ligands induced recruitment of common cofactors ergy transfer from RLucII/coel-h to mTFP1 would lead to an to heterodimers with intermediate potencies. Finally, we documented emission peaking around 492 nm, which represents more favor- the versatility of BRETFect by detecting ternary complexes formed able energetic states for resonant energy transfer to Venus
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