Cell-Surface Protein–Protein Interaction Analysis with Time-Resolved FRET and Snap-Tag Technologies: Application to G Protein-Coupled Receptor Oligomerization

  • Laëtitia Comps-Agrar
  • Damien Maurel
  • Philippe Rondard
  • Jean-Philippe Pin
  • Eric Trinquet
  • Laurent Prézeau
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 756)

Abstract

G protein-coupled receptors (GPCRs) are key players in cell–cell communication, the dysregulation of which has often deleterious effects leading to pathologies such as psychiatric and neurological diseases. Consequently, GPCRs represent excellent drug targets, and as such are the object of intense research in drug discovery for therapeutic application. Recently, the GPCR field has been revolutionized by the demonstration that GPCRs are part of large protein complexes that control their pharmacology, activity, and signaling. Moreover, in these complexes, one GPCR can either associate with itself, forming homodimers or homooligomers, or with other receptor types, forming heterodimeric or heterooligomeric receptor entities that display new receptor features. These features include alterations in ligand cooperativity and selectivity, the activation of novel signaling pathways, and novel processes of desensitization. Thus, it has become necessary to identify GPCR-associated protein complexes of interest at the cell surface, and to determine the state of oligomerization of these receptors and their interactions with their partner proteins. This is essential to understand the function of GPCRs in their native environment, as well as ways to either modulate or control receptor activity with appropriate pharmacological tools, and to develop new therapeutic strategies. This requires the development of technologies to precisely address protein–protein interactions between oligomers at the cell surface. In collaboration with Cisbio Bioassay, we have developed such a technology, which combines TR-FRET detection with a new labeling method called SnapTag. This technology has allowed us to address the oligomeric state of many GPCRs.

Key words

Fluorescence resonance energy transfer G protein-coupled receptor Dimerization SnapTag GABAB receptor 

References

  1. 1.
    Bouvier, M. (2001) Oligomerization of G-protein-coupled transmitter receptors. Nature Rev 2, 27486.CrossRefGoogle Scholar
  2. 2.
    Milligan, G. (2006) G-protein-coupled receptor heterodimers: pharmacology, function and relevance to drug discovery. Drug Discov Today 11, 541–9.PubMedCrossRefGoogle Scholar
  3. 3.
    Pin, J. P., Neubig, R., Bouvier, M., Devi, L., Filizola, M., Javitch, J. A., Lohse, M. J., Milligan, G., Palczewski, K., Parmentier, M., and Spedding, M. (2007) International Union of Basic and Clinical Pharmacology. LXVII. Recommendations for the recognition and nomenclature of G protein-coupled receptor heteromultimers. Pharmacol Rev 59, 5–13.PubMedCrossRefGoogle Scholar
  4. 4.
    Romano, C., Yang, W. L., and O’Malley, K. L. (1996) Metabotropic glutamate receptor 5 is a disulfide-linked dimer. J Biol Chem 271, 286126.PubMedCrossRefGoogle Scholar
  5. 5.
    White, J. H., Wise, A., Main, M. J., Green, A., Fraser, N. J., Disney, G. H., Barnes, A. A., Emson, P., Foord, S. M., and Marshall, F. H. (1998) Heterodimerization is required for the formation of a functional GABA(B) receptor. Nature 396, 67982.PubMedCrossRefGoogle Scholar
  6. 6.
    Angers, S., Salahpour, A., Joly, E., Hilairet, S., Chelsky, D., Dennis, M., and Bouvier, M. (2000) Detection of β2-adrenergic receptor dimerization in living cells using bioluminescence resonance energy transfer (BRET). Proc Natl Acad Sci U S A 97, 3684–9.PubMedGoogle Scholar
  7. 7.
    Overton, M. C., and Blumer, K. J. (2000) G-protein-coupled receptors function as oligomers in vivo. Curr Biol 10, 3414.PubMedCrossRefGoogle Scholar
  8. 8.
    Rocheville, M., Lange, D. C., Kumar, U., Patel, S. C., Patel, R. C., and Patel, Y. C. (2000) Receptors for dopamine and somatostatin: formation of hetero-oligomers with enhanced functional activity. Science 288, 154–7.PubMedCrossRefGoogle Scholar
  9. 9.
    Bazin, H., Trinquet, E., and Mathis, G. (2002) Time resolved amplification of cryptate emission: a versatile technology to trace biomolecular interactions. Rev Mol Biotech 82, 23350.CrossRefGoogle Scholar
  10. 10.
    Mathis, G. (1995) Probing molecular interactions with homogeneous techniques based on rare earth cryptates and Xuorescence energy transfer. Clin Chem 41, 1391–7.PubMedGoogle Scholar
  11. 11.
    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, 253–62.PubMedCrossRefGoogle Scholar
  12. 12.
    Keppler, A., Gendreizig, S., Gronemeyer, T., Pick, H., Vogel, H., and Johnsson, K. A (2003) General method for the covalent labeling of fusion proteins with small molecules in vivo. Nat Biotechnol 21, 86–9.PubMedCrossRefGoogle 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, 561–7.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Laëtitia Comps-Agrar
    • 1
  • Damien Maurel
    • 1
  • Philippe Rondard
    • 1
  • Jean-Philippe Pin
    • 1
  • Eric Trinquet
    • 2
  • Laurent Prézeau
    • 1
  1. 1.Institut de Génomique FonctionnelleUniversity of Montpellier 1 and 2MontpellierFrance
  2. 2.Cisbio BioassaysBagnols-sur-Cèze CedexFrance

Personalised recommendations