Time Resolved FRET Strategy with Fluorescent Ligands to Analyze Receptor Interactions in Native Tissues: Application to GPCR Oligomerization

  • Martin Cottet
  • Laura Albizu
  • Laetitia Comps-Agrar
  • Eric Trinquet
  • Jean-Philippe Pin
  • Bernard Mouillac
  • Thierry DurrouxEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 746)


G protein-coupled receptors (GPCRs) play a key role in the regulation of physiological functions. Deregulation of their activities often results in pathological disorders and therefore these receptors constitute major targets for drug development. The emergence of new concepts such as GPCR oligomerization has modified our understanding of these proteins, and identifying the role of receptor complexes is probably a major challenge for the next decade. Various experimental strategies have been developed to study GPCR oligomers and energy transfer experiments between partners within a complex constitute one of the most convenient approaches. These experimental strategies usually require receptor fusion to tags or fluorescent or luminescent proteins and therefore cannot be easily applied to native tissues. We developed a new experimental approach based on the labeling of receptors with high affinity fluorescent ligands compatible with time-resolved energy transfer measurements. Because of the very high signal-to-noise ratio of the time-resolved fluorescent energy transfer (TR-FRET) signals, this approach constitutes a breakthrough since it allows the direct identification of wild-type GPCR oligomers in native tissues.

Key words

Fluorescent ligands G protein-coupled receptor GPCR Time-resolved FRET Europium Terbium 



Thanks are due to Dr. L. Prezeau for his critical reading of the manuscript. This work was supported by research grants from CNRS, INSERM, ACI Molécules Cibles et Thérapeutiques (no. 240 and 355), ANR (06-Blanc-0087-03). Thanks to Plate-forme de Pharmacologie-Criblage Interactome of Montpellier and the Region Languedoc-Roussillon for making this work possible.


  1. 1.
    Milligan, G. (2004) G protein-coupled ­receptor dimerization: function and ligand pharmacology. Mol. Pharmacol. 66, 1–7.PubMedCrossRefGoogle Scholar
  2. 2.
    Terrillon, S. and Bouvier, M. (2004) Roles of G-protein-coupled receptor dimerization. EMBO Rep 5, 30–34.PubMedCrossRefGoogle Scholar
  3. 3.
    Gomes, I., Gupta, A., Filipovska, J., Szeto, H.H., Pintar, J.E., and Devi, L.A. (2004) A role for heterodimerization of mu and delta opiate receptors in enhancing morphine analgesia. Proc. Natl. Acad. Sci. U. S. A. 101, 5135–5139.PubMedCrossRefGoogle Scholar
  4. 4.
    Fotiadis, D., Jastrzebska, B., Philippsen, A., Muller, D.J., Palczewski, K., and Engel, A. (2006) Structure of the rhodopsin dimer: a working model for G-protein-coupled receptors. Curr. Opin. Struct. Biol. 16, 252–259.PubMedCrossRefGoogle Scholar
  5. 5.
    Roess, D.A., Horvat, R.D., Munnelly, H., and Barisas, B.G. (2000) Luteinizing hormone receptors are self-associated in the plasma membrane. Endocrinology 141, 4518–4523.PubMedCrossRefGoogle Scholar
  6. 6.
    Urizar, E., Montanelli, L., Loy, T., Bonomi, M., Swillens, S., Gales, C., Bouvier, M., Smits, G., Vassart, G., and Costagliola, S. (2005) Glycoprotein hormone receptors: link between receptor homodimerization and negative cooperativity. EMBO J. 24, 1954–1964.PubMedCrossRefGoogle Scholar
  7. 7.
    Waldhoer, M., Fong, J., Jones, R.M., Lunzer, M.M., Sharma, S.K., Kostenis, E., Portoghese, P.S., and Whistler, J.L. (2005) A heterodimer-selective agonist shows in vivo relevance of G protein-coupled receptor dimers. Proc. Natl. Acad. Sci. U. S. A. 102, 9050–9055.PubMedCrossRefGoogle Scholar
  8. 8.
    Wreggett, K.A. and Wells, J.W. (1995) Cooperativity manifest in the binding properties of purified cardiac muscarinic receptors. J. Biol. Chem. 270, 22488–22499.PubMedCrossRefGoogle Scholar
  9. 9.
    Angers, S., Salahpour, A., and Bouvier, M. (2002) Dimerization: an emerging concept for G protein-coupled receptor ontogeny and function. Annu. Rev. Pharmacol. Toxicol. 42, 409–435.Google Scholar
  10. 10.
    Bazin, H., Trinquet, E., and Mathis, G. (2002) Time resolved amplification of cryptate emission: a versatile technology to trace biomolecular interactions. J. Biotechnol. 82, 233–250.Google Scholar
  11. 11.
    Albizu, L., Balestre, M.N., Breton, C., Pin, J-P., Manning, M., Mouillac, B., Barberis, C., and Durroux, T. (2006) Probing the existence of G protein-coupled receptor dimers by positive and negative ligand-dependent cooperative binding. Mol. Pharmacol. 70, 1783–1791.Google Scholar
  12. 12.
    Maurel, D., Kniazeff, J., Mathis, G., Trinquet, E., Pin, J.P., and Ansanay, H. (2004) Cell ­surface detection of membrane protein interaction with homogeneous time-resolved fluorescence resonance energy transfer technology. Anal. Biochem. 329, 253–262.Google Scholar
  13. 13.
    Maurel, D., Comps-Agrar, L., Brock, C., Rives, M-L., Bourrier, E., Ayoub, M.A., Bazin, H., Tinel, N., Durroux, T., Prézeau, L., Trinquet, E., and 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, 561–567.Google Scholar
  14. 14.
    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. and Durroux, T. (2010) Time-resolved FRET between GPCR ligands reveals oligomers in native tissues. Nat. Chem. Biol. 6, 587–594.Google Scholar
  15. 15.
    Middleton, R.J. and Kellam, B. (2005) Fluorophore-tagged GPCR ligands. Curr. Opin. Chem. Biol. 9, 517–525.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Martin Cottet
  • Laura Albizu
  • Laetitia Comps-Agrar
  • Eric Trinquet
  • Jean-Philippe Pin
  • Bernard Mouillac
  • Thierry Durroux
    • 1
    Email author
  1. 1.Institut de Genomique Fonctionnelle, Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche MédicaleUniversity of Montpellier 1 and 2MontpellierFrance

Personalised recommendations