Review
Special Issue: Illuminating GPCRs in Living Cells
Intramolecular and Intermolecular FRET Sensors for GPCRs – Monitoring Conformational Changes and Beyond

https://doi.org/10.1016/j.tips.2017.10.011Get rights and content

Trends

GPCRs play an outstanding role in therapeutic drug development. Hence, a thorough understanding of receptor dynamics upon ligand binding and signaling events is of great importance for biomedical research.

Receptor fluorescence and bioluminescence resonance energy transfer (FRET and BRET) sensors provide high spatial and temporal resolution, and can be applied in near-native systems.

RET sensor technologies are not only interesting for academic research, but also for industrial approaches with high-throughput screening applications for substance library screening.

Recently, an influence of different G-protein expression levels on receptor conformational changes was reported and will be discussed here.

Within the past decade, a large increase in structural knowledge from crystallographic studies has significantly fostered our understanding of the structural biology of G protein-coupled receptors (GPCRs). However, information on dynamic events upon receptor activation or deactivation is not yet readily accessed by these structural approaches. GPCR-based fluorescence resonance energy transfer or bioluminescence resonance energy transfer sensors or sensors for interacting proteins (e.g., G proteins or arrestins) can in part cover this gap. The principal design of such sensors was reported 15 years ago. Since then, sensors for almost 20 different GPCRs have been designed. If used with necessary controls and cautious interpretation, such sensors can contribute significantly to our understanding of the basic mechanisms of GPCR function and beyond. In this review, we will discuss the recent developments in this area of GPCR dynamics.

Section snippets

G Protein-Coupled Receptors

G protein-coupled receptors (GPCRs) recognize extracellular stimuli and translate this encoded information across the plasma membrane to give rise to multiplexed cellular responses. The almost unlimited diversity of interacting partners, ranging from single photons to large protein domains, is reflected by more than 800 expressed genes for GPCRs all over the human body [1]. Understanding these transmembrane (TM) connectors in greater detail has been a major aim of biomedical research for

Labeling Techniques and RET Sensor Design

The first receptor sensors were designed by introducing cyan or yellow derivatives of GFP into the receptor structure, mostly into a truncated third intracellular loop and shortened C terminus [17]. These first sensors can be regarded as milestones in sensor design, but they proved suboptimal because both of these receptor domains have crucial impact on functionality, leading to hampered downstream signaling capability 17, 19. The downstream signaling of these sensors was significantly improved

Dynamic Receptor Movements

For a long period, the GPCR activation process was understood as a two-state model. The origin of this classical point of view was the well-studied activation mechanism of rhodopsin that is triggered by a cis/trans isomerization of retinal. Nowadays, it appears that rhodopsin rather remains the exception in GPCR dynamics than the rule. Recent studies indicate that GPCRs behave as a highly dynamic system, adopting various conformations of different free energy without favoring a distinct

Can Intermolecular FRET Sensor Signals Be Influenced by G-Protein Coupling?

The initially designed FRET sensors, for the α2A-AR, parathyroid (PTH-R), and adenosine A2A receptor, had been intensively studied for factors other than the conformational changes within the 7TM domains that might cause a change in FRET signal and contribute to the signal. A possible contribution of G proteins was investigated by preparing membranes, which were subjected to 6M urea treatment and treatment with Pertussis toxin, to block endogenous Gi proteins 17, 19. Since none of these

Intramolecular RET Sensors to Study Immediate Events in Receptor Signaling

In this section, we will briefly discuss very recent achievements in the design of sensors to study the immediate activation of G proteins or β-arrestin as molecules with direct contact to the receptor. Sivaraj Sivaramakrishnan and his group 50, 51 have published information on promising novel sensors of GPCRs that report upon signaling specific conformational changes induced by a ligand. Such sensors are based on a dual labeling of the respective receptor C terminus with cerulean and citrine

RET Sensor Applications and Receptor Oligomers

Receptor dimerization has recently been reviewed in the light of novel resonance energy-based approaches and other technical features and is certainly a topic of its own right [57]. In the previous sections, we have briefly touched a few reports that discussed the use of FRET-based conformational sensors to study receptor oligomers. Not many reports currently exist on this research topic. Here, we would like to mention a few studies that have particularly addressed issues of receptor

High-Throughput Applications

For screening substance libraries, a single-cell method is not really feasible because of the relatively intense effort and time-consuming experimental setup. High-throughput screening (HTS) assays are performed in at least 96-well plates, but most frequently in a 384- or 1536-well format. At this point we want to mention that the benefits of high-throughput assays are often counterbalanced by losing important spatial and temporal information and we refer to Box 2 for a comparison of HTS versus

Concluding Remarks

Although the principle of these conformational sensors for GPCRs was described almost 15 years ago, we are still witnessing the development of novel areas for their applications. The recent developments of 96-well assay formats for ligand screening and the proof of principle of fragment-based screening at single cells are encouraging news. Allosteric modulators can now be studied in living cells without a radioactive tracer and with different orthosteric probes to investigate probe dependency

Disclaimer Statement

The authors declare that there is no personal conflict of interest. The University of Würzburg does hold a patent on this technology: WO2004057333 A1.

Acknowledgments

This work was supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – TRR 166: project C2 (C.H.), the Marie Curie Initial Training Networks (ITN) ‘WntsApp’ grant agreement number 608180 (C.H.), as well as the international doctoral college ‘Receptor Dynamics: Emerging Paradigms for Novel Drugs’ fund within the framework of the Elite Network of Bavaria (C.H.).

References (73)

  • R. Schrage et al.

    Functional selectivity and dualsteric/bitopic GPCR targeting

    Curr. Opin. Pharmacol.

    (2017)
  • M. Tateyama et al.

    Binding of Gq protein stabilizes the activated state of the muscarinic receptor type 1

    Neuropharmacology

    (2013)
  • R.U. Malik

    Detection of G protein-selective G protein-coupled receptor (GPCR) conformations in live cells

    J. Biol. Chem.

    (2013)
  • P. Hein

    GS activation is time-limiting in initiating receptor-mediated signaling

    J. Biol. Chem.

    (2006)
  • B. Szalai

    Allosteric interactions within the AT(1) angiotensin receptor homodimer: role of the conserved DRY motif

    Biochem. Pharmacol.

    (2012)
  • H. Bazin

    Time resolved amplification of cryptate emission: a versatile technology to trace biomolecular interactions

    J. Biotechnol.

    (2002)
  • N. Boute

    The use of resonance energy transfer in high-throughput screening: BRET versus FRET

    Trends Pharmacol. Sci.

    (2002)
  • R. Fredriksson

    The G-protein-coupled receptors in the human genome form five main families. Phylogenetic analysis, paralogon groups, and fingerprints

    Mol. Pharmacol.

    (2003)
  • S.G. Rasmussen

    Crystal structure of the beta2 adrenergic receptor-Gs protein complex

    Nature

    (2011)
  • Y. Kang

    Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser

    Nature

    (2015)
  • Y. Zhang

    Cryo-EM structure of the activated GLP-1 receptor in complex with a G protein

    Nature

    (2017)
  • M.J. Lohse

    Fluorescence/bioluminescence resonance energy transfer techniques to study G-protein-coupled receptor activation and signaling

    Pharmacol. Rev.

    (2012)
  • H. Tian

    Labeling and single-molecule methods to monitor G protein-coupled receptor dynamics

    Chem. Rev.

    (2016)
  • L.A. Stoddart

    Application of BRET to monitor ligand binding to GPCRs

    Nat. Methods

    (2015)
  • L.A. Stoddart

    Fluorescence- and bioluminescence-based approaches to study GPCR ligand binding

    Br. J. Pharmacol.

    (2016)
  • E. Christiansen

    Development and characterization of a potent free fatty acid receptor 1 (FFA1) fluorescent tracer

    J. Med. Chem.

    (2016)
  • A.H. Hansen

    Development and characterization of a fluorescent tracer for the free fatty acid receptor 2 (FFA2/GPR43)

    J. Med. Chem.

    (2017)
  • A.D. Stumpf et al.

    Optical probes based on G protein-coupled receptors – added work or added value?

    Br. J. Pharmacol.

    (2016)
  • G. Vauquelin et al.

    G protein-coupled receptors: a count of 1001 conformations

    Fundam. Clin. Pharmacol.

    (2005)
  • M.J. Lohse et al.

    Spatial and temporal aspects of signaling by G-protein-coupled receptors

    Mol. Pharmacol.

    (2015)
  • T. Förster

    Zwischenmolekulare energiewanderung und ffluoreszenz

    Ann. Phys.

    (1948)
  • J.P. Vilardaga

    Measurement of the millisecond activation switch of G protein-coupled receptors in living cells

    Nat. Biotechnol.

    (2003)
  • C. Hoffmann

    Conformational changes in G-protein-coupled receptors-the quest for functionally selective conformations is open

    Br. J. Pharmacol.

    (2008)
  • C. Hoffmann

    A FlAsH-based FRET approach to determine G protein-coupled receptor activation in living cells

    Nat. Methods

    (2005)
  • S. Nuber

    beta-Arrestin biosensors reveal a rapid, receptor-dependent activation/deactivation cycle

    Nature

    (2016)
  • K. Bourque

    Distinct conformational dynamics of three G protein-coupled receptors measured using FlAsH-BRET biosensors

    Front. Endocrinol. (Lausanne)

    (2017)
  • Cited by (51)

    • The romantic age of pharmacological science

      2022, Pharmacology Biochemistry and Behavior
    • Computational and experimental approaches to probe GPCR activation and signaling

      2022, Progress in Molecular Biology and Translational Science
    View all citing articles on Scopus
    View full text