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