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Cell-surface protein-protein interaction analysis with time-resolved FRET and snap-tag technologies: application to GPCR oligomerization

Nature Methods volume 5, pages 561–567 (2008)Cite this article

Abstract

Cell-surface proteins are important in cell-cell communication. They assemble into heterocomplexes that include different receptors and effectors. Elucidation and manipulation of such protein complexes offers new therapeutic possibilities. We describe a methodology combining time-resolved fluorescence resonance energy transfer (FRET) with snap-tag technology to quantitatively analyze protein-protein interactions at the surface of living cells, in a high throughput–compatible format. Using this approach, we examined whether G protein–coupled receptors (GPCRs) are monomers or assemble into dimers or larger oligomers—a matter of intense debate. We obtained evidence for the oligomeric state of both class A and class C GPCRs. We also observed different quaternary structure of GPCRs for the neurotransmitters glutamate and γ-aminobutyric acid (GABA): whereas metabotropic glutamate receptors assembled into strict dimers, the GABAB receptors spontaneously formed dimers of heterodimers, offering a way to modulate G-protein coupling efficacy. This approach will be useful in systematic analysis of cell-surface protein interaction in living cells.

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Figure 1: The use of snap tags to label cell-surface proteins with TR-FRET–compatible fluorophores.
Figure 2: Detection of GABAB heteromers at the surface of COS-7 cells using snap tags and TR-FRET.
Figure 3: Detection of cell-surface protein oligomers using snap-tag fusions and TR-FRET.
Figure 4: Detection of GABAB dimers of dimers at the cell surface.
Figure 5: Functional correlate of the association between GABAB heterodimers.

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Acknowledgements

We thank C. Vol for expert assistance with the GPCR functional assays; F. Maurin (Cisbio) for his participation in the synthesis of BG derivatives; and K. Johnsson (Ecole Polytechnique Fédérale de Lausanne) for his support to this project, his critical reading of the manuscript and for providing us with snap-tag tools. We also thank P. Rondard and R. Jockers for their comments on the manuscript. Confocal imaging was performed at the Centre de Ressources en Imagerie Cellulaire, Montpellier, with the help of N. Lautredou. This work was made possible thanks to the screening facilities of the Institut Fédératif de Recherche 3. This work was supported by CNRS, INSERM, Cisbio and by grants from the French Ministry of Research, Action Concertée Incitative “Biologie Cellulaire Moléculaire et Structurale” (ACI-BCMS 328), the Agence Nationale de la Recherche (ANR-05-PRIB-02502, ANR-BLAN06-3_135092 and ANR-05-NEUR-035) and by an unrestricted grant from Senomyx. D.M. was supported by the Association pour le Dévelopement de l'Enseignement et de la Recherche Languedoc Roussillon and C.B. by the Fondation Recherche Médicale.

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  1. Damien Maurel

    Present address: Present address: Ecole Polytechnique Fédérale de Lausanne, Institute of Chemical Sciences and Engineering, CH-1015 Lausanne, Switzerland.,

  2. Damien Maurel and Laëtitia Comps-Agrar: These authors contributed equally to this work.

Authors and Affiliations

  1. Centre National de la Recherche Scientifique (CNRS), UMR 5203, Institut de Génomique Fonctionnelle, 141 Rue de la Cardonille, Montpellier, F-34000, France

    Damien Maurel, Laëtitia Comps-Agrar, Carsten Brock, Marie-Laure Rives, Mohammed Akli Ayoub, Thierry Durroux, Laurent Prézeau & Jean-Philippe Pin

  2. Institut National de la Santé et de la Recherche Médicale (INSERM), U661, 141 Rue de la Cardonille, Montpellier F-34000, France and Université Montpellier 1 and Université Montpellier 2, 141 Rue de la Cardonille, Montpellier, F-34000, France

    Damien Maurel, Laëtitia Comps-Agrar, Carsten Brock, Marie-Laure Rives, Mohammed Akli Ayoub, Thierry Durroux, Laurent Prézeau & Jean-Philippe Pin

  3. Cisbio, Parc technologique Marcel Boiteux, Bagnols/Cèze, F-30204, cedex, France

    Damien Maurel, Emmanuel Bourrier, Hervé Bazin, Norbert Tinel & Eric Trinquet

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Contributions

D.M. and L.C.A. executed most of the experiments and participated in the writing of the manuscript; C.B. developed the system to control subunit composition and performed the confocal experiments; M.L.R. performed the experiments with mGlu1 receptor and the initial experiments with class A receptors; E.B. and H.B. synthesized the BG derivatives; M.A. participated in the BRET experiments; N.T. and E.T. supervised the work at Cisbio; T.D. and L.P. supervised some aspects of the work at the IGF; and J.P.P. supervised the project and wrote the manuscript.

Corresponding author

Correspondence to Jean-Philippe Pin.

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Competing interests

E.B., H.B., N.T. and E.T. are employees of Cisbio, which develops products combining its htrf technology with Covalys SNAP-tag protein labeling system, and therefore may gain financially through publication of this paper. Part of the work of CNRS UMR 5203 has been financially supported by Cisbio and by an unrestricted grant from Senomyx.

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Maurel, D., Comps-Agrar, L., Brock, C. 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). https://doi.org/10.1038/nmeth.1213

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