Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

HTS-compatible FRET-based conformational sensors clarify membrane receptor activation

Nature Chemical Biology volume 13, pages 372–380 (2017)Cite this article

Subjects

Abstract

Cell surface receptors represent a vast majority of drug targets. Efforts have been conducted to develop biosensors reporting their conformational changes in live cells for pharmacological and functional studies. Although Förster resonance energy transfer (FRET) appears to be an ideal approach, its use is limited by the low signal-to-noise ratio. Here we report a toolbox composed of a combination of labeling technologies, specific fluorophores compatible with time-resolved FRET and a novel method to quantify signals. This approach enables the development of receptor biosensors with a large signal-to-noise ratio. We illustrate the usefulness of this toolbox through the development of biosensors for various G-protein-coupled receptors and receptor tyrosine kinases. These receptors include mGlu, GABAB, LH, PTH, EGF and insulin receptors among others. These biosensors can be used for high-throughput studies and also revealed new information on the activation process of these receptors in their cellular environment.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: A toolbox for the development of cell surface receptor biosensors.
Figure 2: Cell surface receptor biosensors for class C GPCRs.
Figure 3: Sensing heterodimeric mGlu receptor activation.
Figure 4: Cell surface biosensors for class A and class B GPCRs.
Figure 5: Sensing receptor tyrosine kinases activation.
Figure 6: Biosensors are compatible with high-throughput screening.

Similar content being viewed by others

References

  1. Rask-Andersen, M., Masuram, S. & Schiöth, H.B. The druggable genome: evaluation of drug targets in clinical trials suggests major shifts in molecular class and indication. Annu. Rev. Pharmacol. Toxicol. 54, 9–26 (2014).

    Article  CAS  PubMed  Google Scholar 

  2. Thomsen, W., Frazer, J. & Unett, D. Functional assays for screening GPCR targets. Curr. Opin. Biotechnol. 16, 655–665 (2005).

    CAS  PubMed  Google Scholar 

  3. Lemmon, M.A. & Schlessinger, J. Cell signaling by receptor tyrosine kinases. Cell 141, 1117–1134 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Boute, N., Jockers, R. & Issad, T. The use of resonance energy transfer in high-throughput screening: BRET versus FRET. Trends Pharmacol. Sci. 23, 351–354 (2002).

    Article  CAS  PubMed  Google Scholar 

  5. Lohse, M.J., Maiellaro, I. & Calebiro, D. Kinetics and mechanism of G-protein-coupled receptor activation. Curr. Opin. Cell Biol. 27, 87–93 (2014).

    Article  CAS  PubMed  Google Scholar 

  6. Ramanoudjame, G., Du, M., Mankiewicz, K.A. & Jayaraman, V. Allosteric mechanism in AMPA receptors: a FRET-based investigation of conformational changes. Proc. Natl. Acad. Sci. USA 103, 10473–10478 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Tateyama, M., Abe, H., Nakata, H., Saito, O. & Kubo, Y. Ligand-induced rearrangement of the dimeric metabotropic glutamate receptor 1alpha. Nat. Struct. Mol. Biol. 11, 637–642 (2004).

    Article  CAS  PubMed  Google Scholar 

  8. Nikolaev, V.O., Hoffmann, C., Bünemann, M., Lohse, M.J. & Vilardaga, J.P. Molecular basis of partial agonism at the neurotransmitter alpha-2A-adrenergic receptor and Gi-protein heterotrimer. J. Biol. Chem. 281, 24506–24511 (2006).

    Article  CAS  PubMed  Google Scholar 

  9. Vilardaga, J.P., Steinmeyer, R., Harms, G.S. & Lohse, M.J. Molecular basis of inverse agonism in a G protein-coupled receptor. Nat. Chem. Biol. 1, 25–28 (2005).

    Article  CAS  PubMed  Google Scholar 

  10. Tateyama, M. & Kubo, Y. Dual signaling is differentially activated by different active states of the metabotropic glutamate receptor 1alpha. Proc. Natl. Acad. Sci. USA 103, 1124–1128 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Vogel, S.S., Thaler, C. & Koushik, S.V. Fanciful FRET. Sci. STKE 2006, re2 (2006).

    PubMed  Google Scholar 

  12. Angers, S. et al. Detection of beta 2-adrenergic receptor dimerization in living cells using bioluminescence resonance energy transfer (BRET). Proc. Natl. Acad. Sci. USA 97, 3684–3689 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. De, A., Ray, P., Loening, A.M. & Gambhir, S.S. BRET3: a red-shifted bioluminescence resonance energy transfer (BRET)-based integrated platform for imaging protein-protein interactions from single live cells and living animals. FASEB J. 23, 2702–2709 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Xu, Y., Piston, D.W. & Johnson, C.H. A bioluminescence resonance energy transfer (BRET) system: application to interacting circadian clock proteins. Proc. Natl. Acad. Sci. USA 96, 151–156 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Bazin, H., Trinquet, E. & Mathis, G. Time resolved amplification of cryptate emission: a versatile technology to trace biomolecular interactions. J. Biotechnol. 82, 233–250 (2002).

    CAS  PubMed  Google Scholar 

  16. Selvin, P.R. Principles and biophysical applications of lanthanide-based probes. Annu. Rev. Biophys. Biomol. Struct. 31, 275–302 (2002).

    Article  CAS  PubMed  Google Scholar 

  17. Lakowicz, J.R. Principles of Fluorescence Spectroscopy. 3rd ed. (Springer, 2006).

  18. Encell, L.P. et al. Development of a dehalogenase-based protein fusion tag capable of rapid, selective and covalent attachment to customizable ligands. Curr. Chem. Genomics 6, 55–71 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  19. Gautier, A. et al. An engineered protein tag for multiprotein labeling in living cells. Chem. Biol. 15, 128–136 (2008).

    Article  CAS  PubMed  Google Scholar 

  20. George, N., Pick, H., Vogel, H., Johnsson, N. & Johnsson, K. Specific labeling of cell surface proteins with chemically diverse compounds. J. Am. Chem. Soc. 126, 8896–8897 (2004).

    Article  CAS  PubMed  Google Scholar 

  21. Keppler, A. et al. A general method for the covalent labeling of fusion proteins with small molecules in vivo. Nat. Biotechnol. 21, 86–89 (2003).

    Article  CAS  PubMed  Google Scholar 

  22. Yin, J. et al. Genetically encoded short peptide tag for versatile protein labeling by Sfp phosphopantetheinyl transferase. Proc. Natl. Acad. Sci. USA 102, 15815–15820 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Fernández-Suárez, M. et al. Redirecting lipoic acid ligase for cell surface protein labeling with small-molecule probes. Nat. Biotechnol. 25, 1483–1487 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Brun, M.A. et al. Semisynthesis of fluorescent metabolite sensors on cell surfaces. J. Am. Chem. Soc. 133, 16235–16242 (2011).

    Article  CAS  PubMed  Google Scholar 

  25. Doumazane, E. et al. A new approach to analyze cell surface protein complexes reveals specific heterodimeric metabotropic glutamate receptors. FASEB J. 25, 66–77 (2011).

    Article  CAS  PubMed  Google Scholar 

  26. Hinner, M.J. & Johnsson, K. How to obtain labeled proteins and what to do with them. Curr. Opin. Biotechnol. 21, 766–776 (2010).

    Article  CAS  PubMed  Google Scholar 

  27. Masharina, A., Reymond, L., Maurel, D., Umezawa, K. & Johnsson, K. A fluorescent sensor for GABA and synthetic GABA(B) receptor ligands. J. Am. Chem. Soc. 134, 19026–19034 (2012).

    Article  CAS  PubMed  Google Scholar 

  28. Maurel, D. et al. Cell-surface protein–protein interaction analysis with time-resolved FRET and snap-tag technologies: application to GPCR oligomerization. Nat. Methods 5, 561–567 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Monnier, C. et al. Trans-activation between 7TM domains: implication in heterodimeric GABAB receptor activation. EMBO J. 30, 32–42 (2011).

    Article  CAS  PubMed  Google Scholar 

  30. Doumazane, E. et al. Illuminating the activation mechanisms and allosteric properties of metabotropic glutamate receptors. Proc. Natl. Acad. Sci. USA 110, E1416–E1425 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Olofsson, L. et al. Fine tuning of sub-millisecond conformational dynamics controls metabotropic glutamate receptors agonist efficacy. Nat. Commun. 5, 5206 (2014).

    Article  CAS  PubMed  Google Scholar 

  32. Geng, Y., Bush, M., Mosyak, L., Wang, F. & Fan, Q.R. Structural mechanism of ligand activation in human GABA(B) receptor. Nature 504, 254–259 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Yin, S. et al. Selective actions of novel allosteric modulators reveal functional heteromers of metabotropic glutamate receptors in the CNS. J. Neurosci. 34, 79–94 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Brock, C. et al. Activation of a dimeric metabotropic glutamate receptor by intersubunit rearrangement. J. Biol. Chem. 282, 33000–33008 (2007).

    Article  CAS  PubMed  Google Scholar 

  35. Lagerström, M.C. & Schiöth, H.B. Structural diversity of G-protein-coupled receptors and significance for drug discovery. Nat. Rev. Drug Discov. 7, 339–357 (2008).

    Article  CAS  PubMed  Google Scholar 

  36. Rasmussen, S.G. et al. Crystal structure of the β2 adrenergic receptor-Gs protein complex. Nature 477, 549–555 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Guo, W., Shi, L., Filizola, M., Weinstein, H. & Javitch, J.A. Crosstalk in G protein-coupled receptors: changes at the transmembrane homodimer interface determine activation. Proc. Natl. Acad. Sci. USA 102, 17495–17500 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Xue, L. et al. Major ligand-induced rearrangement of the heptahelical domain interface in a GPCR dimer. Nat. Chem. Biol. 11, 134–140 (2015).

    Article  CAS  PubMed  Google Scholar 

  39. Fan, Q.R. & Hendrickson, W.A. Structure of human follicle-stimulating hormone in complex with its receptor. Nature 433, 269–277 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Jiang, X. et al. Structure of follicle-stimulating hormone in complex with the entire ectodomain of its receptor. Proc. Natl. Acad. Sci. USA 109, 12491–12496 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  41. Li, S. et al. Structural basis for inhibition of the epidermal growth factor receptor by cetuximab. Cancer Cell 7, 301–311 (2005).

    Article  CAS  PubMed  Google Scholar 

  42. Mattoon, D., Klein, P., Lemmon, M.A., Lax, I. & Schlessinger, J. The tethered configuration of the EGF receptor extracellular domain exerts only a limited control of receptor function. Proc. Natl. Acad. Sci. USA 101, 923–928 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Ozcan, F., Klein, P., Lemmon, M.A., Lax, I. & Schlessinger, J. On the nature of low- and high-affinity EGF receptors on living cells. Proc. Natl. Acad. Sci. USA 103, 5735–5740 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Croll, T.I. et al. Higher-resolution structure of the human insulin receptor ectodomain: multi-modal inclusion of the insert domain. Structure 24, 469–476 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Longo, F.M. & Massa, S.M. Small-molecule modulation of neurotrophin receptors: a strategy for the treatment of neurological disease. Nat. Rev. Drug Discov. 12, 507–525 (2013).

    Article  CAS  PubMed  Google Scholar 

  46. Couturier, C. & Jockers, R. Activation of the leptin receptor by a ligand-induced conformational change of constitutive receptor dimers. J. Biol. Chem. 278, 26604–26611 (2003).

    Article  CAS  PubMed  Google Scholar 

  47. Landes, C.F., Rambhadran, A., Taylor, J.N., Salatan, F. & Jayaraman, V. Structural landscape of isolated agonist-binding domains from single AMPA receptors. Nat. Chem. Biol. 7, 168–173 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. El Moustaine, D. et al. Distinct roles of metabotropic glutamate receptor dimerization in agonist activation and G-protein coupling. Proc. Natl. Acad. Sci. USA 109, 16342–16347 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  49. Bono, F. et al. Inhibition of tumor angiogenesis and growth by a small-molecule multi-FGF receptor blocker with allosteric properties. Cancer Cell 23, 477–488 (2013).

    Article  CAS  PubMed  Google Scholar 

  50. De Smet, F., Christopoulos, A. & Carmeliet, P. Allosteric targeting of receptor tyrosine kinases. Nat. Biotechnol. 32, 1113–1120 (2014).

    Article  CAS  PubMed  Google Scholar 

  51. Chung, I. et al. Spatial control of EGF receptor activation by reversible dimerization on living cells. Nature 464, 783–787 (2010).

    Article  CAS  PubMed  Google Scholar 

  52. Ward, C.W., Lawrence, M.C., Streltsov, V.A., Adams, T.E. & McKern, N.M. The insulin and EGF receptor structures: new insights into ligand-induced receptor activation. Trends Biochem. Sci. 32, 129–137 (2007).

    Article  CAS  PubMed  Google Scholar 

  53. Zwier, J.M., Bazin, H., Lamarque, L. & Mathis, G. Luminescent lanthanide cryptates: from the bench to the bedside. Inorg. Chem. 53, 1854–1866 (2014).

    Article  CAS  PubMed  Google Scholar 

  54. Rives, M.L. et al. Crosstalk between GABAB and mGlu1a receptors reveals new insight into GPCR signal integration. EMBO J. 28, 2195–2208 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Chen, T.W. et al. Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature 499, 295–300 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

The authors thank A. Jean and M. Allali from Cisbio Bioassays for help in the uHTS assays and the synthesis of compound 6, respectively, and G. Stewart, X. Rovira Algans and T. Durroux for helpful discussions. We thank H. Liu for the CaSR experiments. The authors also thank D. Maurel, L. Prézeau and the ARPEGE Pharmacology Screening-Interactome platform facility at the Institute for Functional Genomics (Montpellier, France). This work was supported by grants from Centre National de la Recherche Scientifique, the Institut National de la Santé et de la Recherche Médicale, Cisbio Bioassays, the Agence Nationale pour la Recherche (ANR-09-BIOT-018) (JPP and PR) the Fondation pour la Recherche Médicale (Equipe FRM DEQ20130326522) (JPP) and the Fondation Bettencourt Schueller (JPP). P.S. was supported by a CIFRE fellowship. E.D. was supported by a fellowship from the Fondation pour la Recherche Médicale and from the Association Schizo Oui.

Author information

Author notes

  1. David Moreno-Delgado and Nathalie Lecat-Guillet: These authors contributed equally to this work.

Authors and Affiliations

  1. Institut de Génomique Fonctionnelle, (IGF), CNRS, INSERM, Université de Montpellier, Montpellier, France

    Pauline Scholler, David Moreno-Delgado, Nathalie Lecat-Guillet, Etienne Doumazane, Carine Monnier, Ludovic Fabre, Philippe Rondard & Jean-Philippe Pin

  2. Cisbio Bioassays, Codolet, France

    Pauline Scholler, Fabienne Charrier-Savournin, Ludovic Fabre, Cédric Chouvet, Stéphanie Soldevila, Laurent Lamarque, Geoffrey Donsimoni, Thomas Roux, Jurriaan M Zwier & Eric Trinquet

Authors
  1. Pauline Scholler
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. David Moreno-Delgado
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Nathalie Lecat-Guillet
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Etienne Doumazane
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Carine Monnier
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Fabienne Charrier-Savournin
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Ludovic Fabre
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Cédric Chouvet
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Stéphanie Soldevila
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Laurent Lamarque
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Geoffrey Donsimoni
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Thomas Roux
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Jurriaan M Zwier
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Eric Trinquet
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Philippe Rondard
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Jean-Philippe Pin
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

P.S., D.M.-D., N.L.-G., E.D., C.M., F.C.-S., L.F., C.C., G.D., T.R., J.M.Z., E.T., P.R. and J.-P.P. designed experiments; P.S., D.M.-D., E.D. and L.F. performed the mGluR and class A and B GPCR experiments; P.S., N.L.-G. and C.M. performed the GABAB experiments; P.S. and C.C. performed the RTK experiments; L.F. and G.D. performed the HTS experiments; S.S. and L.L. synthesized the compounds needed for this study; P.S., J.M.Z., P.R. and J.-P.P. wrote the manuscript.

Corresponding authors

Correspondence to Philippe Rondard or Jean-Philippe Pin.

Ethics declarations

Competing interests

Cisbio Bioassays is the manufacturer and the provider of most of the SNAP-tag and HaloTag reagents used in this study, and ACP-tag reagents may be commercialized in the future. A patent has been filled for the VFT sensor technology WO2010125314(A1).

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Tables 1–2 and Supplementary Figures 1–6. (PDF 1010 kb)

Supplementary Note

Synthetic Procedures. (PDF 498 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Scholler, P., Moreno-Delgado, D., Lecat-Guillet, N. et al. HTS-compatible FRET-based conformational sensors clarify membrane receptor activation. Nat Chem Biol 13, 372–380 (2017). https://doi.org/10.1038/nchembio.2286

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.2286

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research