Time-Resolved FRET-Based Assays to Characterize G Protein-Coupled Receptor Hetero-oligomer Pharmacology

  • Joyce Heuninck
  • Candide Hounsou
  • Elodie Dupuis
  • Eric Trinquet
  • Bernard Mouillac
  • Jean-Philippe Pin
  • Dominique BonnetEmail author
  • Thierry DurrouxEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1947)


Although G protein-coupled receptor (GPCR) oligomerization is a matter of debate, it has been shown that the nature of the GPCR partners within the oligomers can influence the pharmacological properties of the receptors. Therefore, finding specific ligands for homo- or hetero-oligomers opens new perspectives for drug discovery. However, no efficient experimental strategy to screen for such ligands existed yet. Indeed, conventional binding strategies do not discriminate ligand binding on GPCR monomers, homo- or hetero-oligomers. To address this issue, we recently developed a new assay based on a time-resolved FRET method that is easy to implement and that can focus on ligand binding specifically on the hetero-oligomer.

Key words

Tag-lite® screening G protein-coupled receptor Fluorescent ligand HTRF® Time-resolved FRET Lanthanide Terbium Self-labeling enzyme Binding experiment Homo- and hetero-oligomerization 



The development of the technique has been supported by the Fonds Unique Interministériel and OSEO in a collaborative program named “CELL2Lead”. Thanks are due to the plateforme Arpège of the Institut de Génomique Fonctionnelle. This work has been funded by the European Consortium Oncornet (HORIZON 2020 MSCA–ITN–2014–ETN–Project 641833 ONCORNET) (to J.H., and J.-P.P. and T.D.).


  1. 1.
    Overington JP, Al-Lazikani B, Hopkins AL (2006) How many drug targets are there? Nat Rev Drug Discov 5:993–996CrossRefGoogle Scholar
  2. 2.
    Hounsou C, Margathe J-F, Oueslati N, Belhocine A, Dupuis E, Thomas C, Mann A, Ilien B, Rognan D, Trinquet E, Hibert M, Pin J-P, Bonnet D, Durroux T (2015) Time-resolved FRET binding assay to investigate hetero-oligomer binding properties: proof of concept with dopamine D1/D3 heterodimer. ACS Chem Biol 10:466–474CrossRefGoogle Scholar
  3. 3.
    Zwier JM, Roux T, Cottet M, Durroux T, Douzon S, Bdioui S, Gregor N, Bourrier E, Oueslati N, Nicolas L, Tinel N, Boisseau C, Yverneau P, Charrier-Savournin F, Fink M, Trinquet E (2010) A fluorescent ligand-binding alternative using Tag-lite® technology. J Biomol Screen 15:1248–1259CrossRefGoogle Scholar
  4. 4.
    Loison S, Cottet M, Orcel H, Adihou H, Rahmeh R, Lamarque L, Trinquet E, Kellenberger E, Hibert M, Durroux T, Mouillac B, Bonnet D (2012) Selective fluorescent nonpeptidic antagonists for vasopressin V2 GPCR: application to ligand screening and oligomerization assays. J Med Chem 55:8588–8602CrossRefGoogle Scholar
  5. 5.
    Cottet M, Faklaris O, Falco A, Trinquet E, Pin J-P, Mouillac B, Durroux T (2013) Fluorescent ligands to investigate GPCR binding properties and oligomerization. Biochem Soc Trans 41:148–153CrossRefGoogle Scholar
  6. 6.
    Emami-Nemini A, Roux T, Leblay M, Bourrier E, Lamarque L, Trinquet E, Lohse MJ (2013) Time-resolved fluorescence ligand binding for G protein-coupled receptors. Nat Protoc 8:1307–1320CrossRefGoogle Scholar
  7. 7.
    Oueslati N, Hounsou C, Belhocine A, Rodriguez T, Dupuis E, Zwier JM, Trinquet E, Pin J-P, Durroux T (2015) Time-resolved FRET strategy to screen GPCR ligand library. Methods Mol Biol 1272:23–36CrossRefGoogle Scholar
  8. 8.
    Valencia C, Dujet C, Margathe J-F, Iturrioz X, Roux T, Trinquet E, Villa P, Hibert M, Dupuis E, Llorens-Cortes C, Bonnet D (2017) A time-resolved FRET cell-based binding assay for the apelin receptor. ChemMedChem 12:925–931CrossRefGoogle Scholar
  9. 9.
    Hounsou C, Baehr C, Gasparik V, Alili D, Belhocine A, Rodriguez T, Dupuis E, Roux T, Mann A, Heissler D, Pin J-P, Durroux T, Bonnet D, Hibert M (2018) From the promiscuous asenapine to potent fluorescent ligands acting at a series of aminergic G-protein-coupled receptors. J Med Chem 61:174–188CrossRefGoogle Scholar
  10. 10.
    Vogel SS, Thaler C, Koushik SV (2006) Fanciful FRET. Sci STKE 2006:re2PubMedGoogle Scholar
  11. 11.
    Selvin PR (2002) Principles and biophysical applications of lanthanide-based probes. Annu Rev Biophys Biomol Struct 31:275–302CrossRefGoogle Scholar
  12. 12.
    Mathis G, Bazin H (2011) Stable luminescent chelates and macrocyclic compounds. In: Hänninen P, Härmä HD (eds) In lanthanide luminescence. Springer, Berlin, pp 47–88Google Scholar
  13. 13.
    Albizu L, Teppaz G, Seyer R, Bazin H, Ansanay H, Manning M, Mouillac B, Durroux T (2007) Toward efficient drug screening by homogeneous assays based on the development of new fluorescent vasopressin and oxytocin receptor ligands. J Med Chem 50:4976–4985CrossRefGoogle Scholar
  14. 14.
    Keppler A, Gendreizig S, Gronemeyer T, Pick H, Vogel H, Johnsson K (2003) A general method for the covalent labeling of fusion proteins with small molecules in vivo. Nat Biotechnol 21:86–89CrossRefGoogle Scholar
  15. 15.
    Juillerat A, Gronemeyer T, Keppler A, Gendreizig S, Pick H, Vogel H, Johnsson K (2003) Directed evolution of O6-alkylguanine-DNA alkyltransferase for efficient labeling of fusion proteins with small molecules in vivo. Chem Biol 10:313–317CrossRefGoogle Scholar
  16. 16.
    Juillerat A, Heinis C, Sielaff I, Barnikow J, Jaccard H, Kunz B, Terskikh A, Johnsson K (2005) Engineering substrate specificity of O6-alkylguanine-DNA alkyltransferase for specific protein labeling in living cells. Chembiochem 6:1263–1269CrossRefGoogle Scholar
  17. 17.
    Gronemeyer T, Chidley C, Juillerat A, Heinis C, Johnsson K (2006) Directed evolution of O6-alkylguanine-DNA alkyltransferase for applications in protein labeling. Protein Eng Des Sel 19:309–316CrossRefGoogle Scholar
  18. 18.
    Gautier A, Juillerat A, Heinis C, Corrêa IR, Kindermann M, Beaufils F, Johnsson K (2008) An engineered protein tag for multiprotein labeling in living cells. Chem Biol 15:128–136CrossRefGoogle Scholar
  19. 19.
    Zhang Y, So M-K, Loening AM, Yao H, Gambhir SS, Rao J (2006) HaloTag protein-mediated site-specific conjugation of bioluminescent proteins to quantum dots. Angew Chem Int Ed Engl 45:4936–4940CrossRefGoogle Scholar
  20. 20.
    Maurel D, Comps-Agrar L, Brock C, Rives M-L, Bourrier E, Ayoub MA, Bazin H, Tinel N, Durroux T, Prézeau L, Trinquet E, Pin J-P (2008) Cell-surface protein-protein interaction analysis with time-resolved FRET and snap-tag technologies: application to GPCR oligomerization. Nat Methods 5:561–567CrossRefGoogle Scholar
  21. 21.
    Durroux T, Peter M, Turcatti G, Chollet A, Balestre MN, Barberis C, Seyer R (1999) Fluorescent pseudo-peptide linear vasopressin antagonists: design, synthesis, and applications. J Med Chem 42:1312–1319CrossRefGoogle Scholar
  22. 22.
    Terrillon S, Cheng LL, Stoev S, Mouillac B, Barberis C, Manning M, Durroux T (2002) Synthesis and characterization of fluorescent antagonists and agonists for human oxytocin and vasopressin V(1)(a) receptors. J Med Chem 45:2579–2588CrossRefGoogle Scholar
  23. 23.
    Albizu L, Cottet M, Kralikova M, Stoev S, Seyer R, Brabet I, Roux T, Bazin H, Bourrier E, Lamarque L, Breton C, Rives M-L, Newman A, Javitch J, Trinquet E, Manning M, Pin J-P, Mouillac B, Durroux T (2010) Time-resolved FRET between GPCR ligands reveals oligomers in native tissues. Nat Chem Biol 6:587–594CrossRefGoogle Scholar
  24. 24.
    Margathe J-F, Iturrioz X, Regenass P, Karpenko IA, Humbert N, de Rocquigny H, Hibert M, Llorens-Cortes C, Bonnet D (2016) Convenient access to fluorescent probes by chemoselective acylation of hydrazinopeptides: application to the synthesis of the first far-red ligand for apelin receptor imaging. Chem Eur J 22:1399–1405CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Joyce Heuninck
    • 1
    • 2
    • 3
  • Candide Hounsou
    • 1
    • 2
    • 3
  • Elodie Dupuis
    • 4
  • Eric Trinquet
    • 4
  • Bernard Mouillac
    • 1
    • 2
    • 3
  • Jean-Philippe Pin
    • 1
    • 2
    • 3
  • Dominique Bonnet
    • 5
    Email author
  • Thierry Durroux
    • 1
    • 2
    • 3
    Email author
  1. 1.CNRS, UMR 5203, Institut de Génomique FonctionnelleMontpellierFrance
  2. 2.INSERM, U. 1191MontpellierFrance
  3. 3.Université de MontpellierMontpellierFrance
  4. 4.CisbioCodoletFrance
  5. 5.Laboratoire d’Innovation Thérapeutique, Faculté de PharmacieUMR7200 CNRS/Université de Strasbourg, LabEx MEDALISIllkirchFrance

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