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Real-time monitoring of receptor and G-protein interactions in living cells

Nature Methods volume 2, pages 177–184 (2005)Cite this article

Abstract

G protein–coupled receptors (GPCRs) represent the largest family of proteins involved in signal transduction. Here we present a bioluminescence resonance energy transfer (BRET) assay that directly monitors in real time the early interactions between human GPCRs and their cognate G-protein subunits in living human cells. In addition to detecting basal precoupling of the receptors to Gα-, Gβ- and Gγ-subunits, BRET measured very rapid ligand-induced increases in the interaction between receptor and Gαβγ-complexes (t1/2 ∼ 300 ms) followed by a slower (several minutes) decrease, reflecting receptor desensitization. The agonist-promoted increase in GPCR-Gβγ interaction was highly dependent on the identity of the Gα-subunit present in the complex. Therefore, this G protein–activity biosensor provides a novel tool to directly probe the dynamics and selectivity of receptor-mediated, G-protein activation-deactivation cycles that could be advantageously used to identify ligands for orphan GPCRs.

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Figure 1: Illustration of the BRET2 assay between receptor and G-protein subunits.
Figure 2: BRET measurements of GPCRs and Gαsβ1γ2 interactions in living cells.
Figure 3: BRET signals between β2AR and Gβγ reflect receptor-mediated G-protein activation.
Figure 4: Kinetics analysis of receptor-Gβ1γ2 interactions.

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Acknowledgements

We thank M. Lagacé for critical reading of the manuscript. The pcDNA-CD8-βARK-C, α2AAR-Rluc and Gαs-EGFP were kindly provided by S. Gütkind, H. Paris and M. Rasenick, respectively. The GFP-Gβ1 and GFP-Gγ2 constructs were generously provided by BioSignal-Packard Biosciences and SNSR-4-Rluc was a gift from AstraZeneca Research Center Montréal. This work was supported by grants from the Canadian Institute of Health Research and the Heart and Stroke Foundation of Quebec to M.B. and T.E.H. C.G. held a fellowship from Institut National de la Santé et de la Recherche Médicale (INSERM), T.E.H. is a MacDonald Scholar of the Heart and Stroke Foundation of Canada, and M.B. holds a Canada Research Chair in Molecular Pharmacology and Signal Transduction.

Author information

Affiliations

  1. Department of Biochemistry, Université de Montréal, P.O. Box 6128 Down-town station, Montréal, H3C 3J7, Canada

    Céline Galés, Mireille Hogue, Andreas Breit, Terence E Hébert & Michel Bouvier

  2. Laboratory of Cellular Biology, 5 Research Court, Room 2A11, National Institutes of Health, Rockville, 20850-3211, Maryland, USA

    R Victor Rebois

  3. Institut de Cardiologie de Montréal et Département d'Anesthésiologie, Université de Montréal, 5000 Bélanger St. East, Montréal, H1T 1C8, Canada

    Phan Trieu & Terence E Hébert

Authors
  1. Céline Galés
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  2. R Victor Rebois
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  3. Mireille Hogue
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  4. Phan Trieu
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  5. Andreas Breit
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  6. Terence E Hébert
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  7. Michel Bouvier
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Corresponding author

Correspondence to Michel Bouvier.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Plasma membrane targeting of CD8-Rluc fusion protein. (PDF 155 kb)

Supplementary Fig. 2

Agonist-promoted BRET signal between β2AR-Rluc and GFPGγ2 is insensitive to pH. (PDF 139 kb)

Supplementary Fig. 3

Vasopressin-selective agonist AVP is unable to modulate BRET signal between β2ARRluc and GFP-Gγ2 or GFP-Gβ1. (PDF 133 kb)

Supplementary Fig. 4

Interactions between Gβ1 and Gγ2. (PDF 147 kb)

Supplementary Fig. 5

BRET signal between β2ARRluc and GFP-Gβ1 or GFP-Gγ2 is GTP sensitive. (PDF 142 kb)

Supplementary Fig. 6

Basal BRET signal between β2AR-Rluc and GFP-Gγ2 is sensitive to a β2AR-inverse agonist. (PDF 123 kb)

Supplementary Fig. 7

Kinetics analysis of β2AR and Gγ2 interactions. (PDF 131 kb)

Supplementary Methods (PDF 109 kb)

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Galés, C., Rebois, R., Hogue, M. et al. Real-time monitoring of receptor and G-protein interactions in living cells. Nat Methods 2, 177–184 (2005). https://doi.org/10.1038/nmeth743

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