Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Probing the activation-promoted structural rearrangements in preassembled receptor–G protein complexes

Nature Structural & Molecular Biology volume 13, pages 778–786 (2006)Cite this article

Abstract

Activation of heterotrimeric G proteins by their cognate seven transmembrane domain receptors is believed to involve conformational changes propagated from the receptor to the G proteins. However, the nature of these changes remains unknown. We monitored the conformational rearrangements at the interfaces between receptors and G proteins and between G protein subunits by measuring bioluminescence resonance energy transfer between probes inserted at multiple sites in receptor–G protein complexes. Using the data obtained for the α2AAR–Gαi1β1γ2 complex and the available crystal structures of Gαi1β1γ2, we propose a model wherein agonist binding induces conformational reorganization of a preexisting receptor–G protein complex, leading the Gα-Gβγ interface to open but not dissociate. This conformational change may represent the movement required to allow nucleotide exit from the Gα subunit, thus reflecting the initial activation event.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Configurations of BRET partners.
Figure 2: BRET measurements of GPCR and Gαi1β1γ2 interactions in living cells.
Figure 3: Assessment of the dynamic nature of receptor-Gαi1β1γ2 interactions.
Figure 4: BRET measurements of Gαi1β1γ2 subunit interactions in living cells.
Figure 5: Different receptors promote similar Gαi1β1γ2 structural rearrangements.
Figure 6: Structural model of G protein activation.

Similar content being viewed by others

Accession codes

Accessions

Protein Data Bank

References

  1. Gilman, A.G. G proteins: transducers of receptor-generated signals. Annu. Rev. Biochem. 56, 615–649 (1987).

    CAS  Google Scholar 

  2. Bourne, H.R. How receptors talk to trimeric G proteins. Curr. Opin. Cell Biol. 9, 134–142 (1997).

    Article  CAS  Google Scholar 

  3. Cabrera-Vera, T.M. et al. Insights into G protein structure, function, and regulation. Endocr. Rev. 24, 765–781 (2003).

    Article  CAS  Google Scholar 

  4. Rebois, R.V., Warner, D.R. & Basi, N.S. Does subunit dissociation necessarily accompany the activation of all heterotrimeric G proteins? Cell. Signal. 9, 141–151 (1997).

    Article  CAS  Google Scholar 

  5. Klein, S., Reuveni, H. & Levitzki, A. Signal transduction by a nondissociable heterotrimeric yeast G protein. Proc. Natl. Acad. Sci. USA 97, 3219–3223 (2000).

    Article  CAS  Google Scholar 

  6. Bunemann, M., Frank, M. & Lohse, M.J. Gi protein activation in intact cells involves subunit rearrangement rather than dissociation. Proc. Natl. Acad. Sci. USA 100, 16077–16082 (2003).

    Article  Google Scholar 

  7. Frank, M., Thumer, L., Lohse, M.J. & Bunemann, M. G protein activation without subunit dissociation depends on a Gαi-specific region. J. Biol. Chem. 280, 24584–24590 (2005).

    Article  CAS  Google Scholar 

  8. Gales, C. et al. Real-time monitoring of receptor and G-protein interactions in living cells. Nat. Methods 2, 177–184 (2005).

    Article  CAS  Google Scholar 

  9. Sprang, S.R. G protein mechanisms: insights from structural analysis. Annu. Rev. Biochem. 66, 639–678 (1997).

    Article  CAS  Google Scholar 

  10. Noel, J.P., Hamm, H.E. & Sigler, P.B. The 2.2 Å crystal structure of transducin-α complexed with GTPγS. Nature 366, 654–663 (1993).

    Article  CAS  Google Scholar 

  11. Sondek, J., Lambright, D.G., Noel, J.P., Hamm, H.E. & Sigler, P.B. GTPase mechanism of Gproteins from the 1.7-Å crystal structure of transducin α-GDP-AIF-4. Nature 372, 276–279 (1994).

    Article  CAS  Google Scholar 

  12. Ceruso, M.A., Periole, X. & Weinstein, H. Molecular dynamics simulations of transducin: interdomain and front to back communication in activation and nucleotide exchange. J. Mol. Biol. 338, 469–481 (2004).

    Article  CAS  Google Scholar 

  13. Cherfils, J. & Chabre, M. Activation of G-protein Gα subunits by receptors through Gα–Gβ and Gα–Gγ interactions. Trends Biochem. Sci. 28, 13–17 (2003).

    Article  CAS  Google Scholar 

  14. Iiri, T., Farfel, Z. & Bourne, H.R. G-protein diseases furnish a model for the turn-on switch. Nature 394, 35–38 (1998).

    Article  CAS  Google Scholar 

  15. Rondard, P. et al. Mutant G protein α subunit activated by Gβγ: a model for receptor activation? Proc. Natl. Acad. Sci. USA 98, 6150–6155 (2001).

    Article  CAS  Google Scholar 

  16. Miyawaki, A. Visualization of the spatial and temporal dynamics of intracellular signaling. Dev. Cell 4, 295–305 (2003).

    Article  CAS  Google Scholar 

  17. Pfleger, K.D. & Eidne, K.A. Monitoring the formation of dynamic G-protein-coupled receptor-protein complexes in living cells. Biochem. J. 385, 625–637 (2005).

    Article  CAS  Google Scholar 

  18. Charest, P.G., Terrillon, S. & Bouvier, M. Monitoring agonist-promoted conformational changes of β-arrestin in living cells by intramolecular BRET. EMBO Rep. 6, 334–340 (2005).

    Article  CAS  Google Scholar 

  19. Schaufele, F. et al. The structural basis of androgen receptor activation: intramolecular and intermolecular amino-carboxy interactions. Proc. Natl. Acad. Sci. USA 102, 9802–9807 (2005).

    Article  CAS  Google Scholar 

  20. Tateyama, M., Abe, H., Nakata, H., Saito, O. & Kubo, Y. Ligand-induced rearrangement of the dimeric metabotropic glutamate receptor 1α. Nat. Struct. Mol. Biol. 11, 637–642 (2004).

    Article  CAS  Google Scholar 

  21. Tsuboi, T., Lippiat, J.D., Ashcroft, F.M. & Rutter, G.A. ATP-dependent interaction of the cytosolic domains of the inwardly rectifying K+ channel Kir6.2 revealed by fluorescence resonance energy transfer. Proc. Natl. Acad. Sci. USA 101, 76–81 (2004).

    Article  CAS  Google Scholar 

  22. Vilardaga, J.P., Bunemann, M., Krasel, C., Castro, M. & Lohse, M.J. Measurement of the millisecond activation switch of G protein-coupled receptors in living cells. Nat. Biotechnol. 21, 807–812 (2003).

    Article  CAS  Google Scholar 

  23. Gibson, S.K. & Gilman, A.G. Giα and Gβ subunits both define selectivity of G protein activation by α2-adrenergic receptors. Proc. Natl. Acad. Sci. USA 103, 212–217 (2006).

    Article  CAS  Google Scholar 

  24. Kozasa, T. & Gilman, A.G. Purification of recombinant G proteins from Sf9 cells by hexahistidine tagging of associated subunits. Characterization of α12 and inhibition of adenylyl cyclase by αz . J. Biol. Chem. 270, 1734–1741 (1995).

    Article  CAS  Google Scholar 

  25. Posner, B.A., Mukhopadhyay, S., Tesmer, J.J., Gilman, A.G. & Ross, E.M. Modulation of the affinity and selectivity of RGS protein interaction with Gα subunits by a conserved asparagine/serine residue. Biochemistry 38, 7773–7779 (1999).

    Article  CAS  Google Scholar 

  26. Nakafuku, M., Itoh, H., Nakamura, S. & Kaziro, Y. Occurrence in Saccharomyces cerevisiae of a gene homologous to the cDNA coding for the alpha subunit of mammalian G proteins. Proc. Natl. Acad. Sci. USA 84, 2140–2144 (1987).

    Article  CAS  Google Scholar 

  27. Breit, A., Lagace, M. & Bouvier, M. Hetero-oligomerization between β2- and β3-adrenergic receptors generates a β-adrenergic signaling unit with distinct functional properties. J. Biol. Chem. 279, 28756–28765 (2004).

    Article  CAS  Google Scholar 

  28. Daaka, Y., Luttrell, L.M. & Lefkowitz, R.J. Switching of the coupling of the β2-adrenergic receptor to different G proteins by protein kinase A. Nature 390, 88–91 (1997).

    Article  CAS  Google Scholar 

  29. Xiao, R.P. β-adrenergic signaling in the heart: dual coupling of the β2-adrenergic receptor to Gs and Gi proteins. Sci. STKE 2001, RE15 (2001).

    CAS  PubMed  Google Scholar 

  30. Arcaro, A. et al. Essential role of CD8 palmitoylation in CD8 coreceptor function. J. Immunol. 165, 2068–2076 (2000).

    Article  CAS  Google Scholar 

  31. Huang, C. et al. Organization of G proteins and adenylyl cyclase at the plasma membrane. Mol. Biol. Cell 8, 2365–2378 (1997).

    Article  CAS  Google Scholar 

  32. Head, B.P. et al. G-protein-coupled receptor signaling components localize in both sarcolemmal and intracellular caveolin-3-associated microdomains in adult cardiac myocytes. J. Biol. Chem. 280, 31036–31044 (2005).

    Article  CAS  Google Scholar 

  33. Mercier, J.F., Salahpour, A., Angers, S., Breit, A. & Bouvier, M. Quantitative assessment of β1- and β2-adrenergic receptor homo- and heterodimerization by bioluminescence resonance energy transfer. J. Biol. Chem. 277, 44925–44931 (2002).

    Article  CAS  Google Scholar 

  34. Ramsay, D. et al. High-affinity interactions between human α1A-adrenoceptor C-terminal splice variants produce homo- and heterodimers but do not generate the α1L-adrenoceptor. Mol. Pharmacol. 66, 228–239 (2004).

    Article  CAS  Google Scholar 

  35. Oakley, R.H., Laporte, S.A., Holt, J.A., Barak, L.S. & Caron, M.G. Molecular determinants underlying the formation of stable intracellular G protein-coupled receptor-β-arrestin complexes after receptor endocytosis. J. Biol. Chem. 276, 19452–19460 (2001).

    Article  CAS  Google Scholar 

  36. Perroy, J., Pontier, S., Charest, P.G., Aubry, M. & Bouvier, M. Real-time monitoring of ubiquitination in living cells by BRET. Nat. Methods 1, 203–208 (2004).

    Article  CAS  Google Scholar 

  37. Yu, J.Z. & Rasenick, M.M. Real-time visualization of a fluorescent Gαs: dissociation of the activated G protein from plasma membrane. Mol. Pharmacol. 61, 352–359 (2002).

    Article  CAS  Google Scholar 

  38. Neubig, R.R. Membrane organization in G-protein mechanisms. FASEB J. 8, 939–946 (1994).

    Article  CAS  Google Scholar 

  39. Rebois, R.V. & Hebert, T.E. Protein complexes involved in heptahelical receptor-mediated signal transduction. Receptors Channels 9, 169–194 (2003).

    Article  CAS  Google Scholar 

  40. Hein, P., Frank, M., Hoffmann, C., Lohse, M.J. & Bunemann, M. Dynamics of receptor/G protein coupling in living cells. EMBO J. 24, 4106–4114 (2005).

    Article  CAS  Google Scholar 

  41. Nobles, M., Benians, A. & Tinker, A. Heterotrimeric G proteins precouple with G protein-coupled receptors in living cells. Proc. Natl. Acad. Sci. USA 102, 18706–18711 (2005).

    Article  CAS  Google Scholar 

  42. Janetopoulos, C., Jin, T. & Devreotes, P. Receptor-mediated activation of heterotrimeric G-proteins in living cells. Science 291, 2408–2411 (2001).

    Article  CAS  Google Scholar 

  43. Yi, T.M., Kitano, H. & Simon, M.I. A quantitative characterization of the yeast heterotrimeric G protein cycle. Proc. Natl. Acad. Sci. USA 100, 10764–10769 (2003).

    Article  CAS  Google Scholar 

  44. Milligan, G. Applications of bioluminescence- and fluorescence resonance energy transfer to drug discovery at G protein-coupled receptors. Eur. J. Pharm. Sci. 21, 397–405 (2004).

    Article  CAS  Google Scholar 

  45. Gales, C. et al. Mutation of Asn-391 within the conserved NPXXY motif of the cholecystokinin B receptor abolishes Gq protein activation without affecting its association with the receptor. J. Biol. Chem. 275, 17321–17327 (2000).

    Article  CAS  Google Scholar 

  46. Krieger, E., Koraimann, G. & Vriend, G. Increasing the precision of comparative models with YASARA NOVA–a self-parameterizing force field. Proteins 47, 393–402 (2002).

    Article  CAS  Google Scholar 

  47. Weng, G., Jordan, J. & Chen, Y. Structural basis for the function of the heterotrimeric G-proteins. Semin. Neurosci. 9, 175–188 (1998).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank M. Lagacé and E. Urizar for critical reading of the manuscript, R. Sunahara, M. Coinçon, J.P. Pin and M. Ayoub for helpful discussions and B. Lorazo from Direction Générale des Technologies de l'Information et de la Communication (University of Montreal) for informatics support. This work was supported by grants from the Canadian Institute of Health Research and the Heart and Stroke Foundation of Quebec to M.B. C.G. was the recipient of a fellowship from INSERM. M.B. holds a Canada Research Chair in Molecular Pharmacology and Signal Transduction.

Author information

Authors and Affiliations

  1. Department of Biochemistry and Groupe de Recherche Universitaire sur le Médicament, Institute for Research in Immunology and Cancer, Université de Montréal, P.O. Box 6128, Downtown station, Montreal, H3C 3J7, Quebec, Canada

    Céline Galés, Stéphanie Pontier, Yann Percherancier, Martin Audet & Michel Bouvier

  2. Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire, Université Libre de Bruxelles, Campus Erasme, C.P. 602, route de Lennik 808, Brussels, 1070, Belgium

    Joost J J Van Durm

  3. INSERM U388, Institut Louis Bugnard, CHU Rangueil, BP 84225, Toulouse, 31432 Cedex 4, France

    Stéphane Schaak & Hervé Paris

Authors
  1. Céline Galés
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Joost J J Van Durm
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Stéphane Schaak
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Stéphanie Pontier
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yann Percherancier
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Martin Audet
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Hervé Paris
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Michel Bouvier
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

C.G. coconceived the project, established the overall experimental strategy, did most of the experiments, analyzed and interpreted data and cowrote the manuscript. J.J.J.V.D. contributed to the molecular dynamics study, created three-dimensional representations of receptors and G protein and helped conceive the structural model. S.S. helped construct and functionally characterize the α2AR BRET fusion proteins. S.P. collected and interpreted confocal microscopy data on β2AR and raft-marker localization in HEK293T cells (Supplementary Fig. 7) and designed the figure and figure legend. Y.P. collected and interpreted biochemical data on β2AR and raft-marker distribution in HEK293T cells (Supplementary Fig. 7). M.A. contributed to the molecular dynamics study. H.P. helped construct and functionally characterize the α2AR BRET fusion proteins (Supplementary Fig. 3) and contributed to the writing of the manuscript. M.B. coconceived the project, helped establish the overall experimental strategy, analyzed and interpreted data and cowrote the manuscript.

Corresponding author

Correspondence to Michel Bouvier.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

View of the structures of Gαi1, Gαi1-91Rluc and Gαi1-122Rluc. (PDF 346 kb)

Supplementary Fig. 2

Plasma membrane targeting of Gαi1-91Rluc and Gαi1-122Rluc fusion proteins. (PDF 166 kb)

Supplementary Fig. 3

Functionality of Gαi1-91Rluc and Gαi1-122Rluc fusion proteins. (PDF 145 kb)

Supplementary Fig. 4

Configurations of the different BRET assays used to probe receptor-mediated G protein activation. (PDF 218 kb)

Supplementary Fig. 5

Pertussis toxin sensitivity of receptor-mediated G protein activation. (PDF 132 kb)

Supplementary Fig. 6

Trypsin cleavage pattern of Gαi1 and Gαi1-122Rluc. (PDF 164 kb)

Supplementary Fig. 7

Analysis of β2AR submembraneous localization by detergent extraction and confocal microscopy. (PDF 231 kb)

Supplementary Fig. 8

BRET measurements of α2BAR and Gαi1 interaction in living cells. (PDF 122 kb)

Supplementary Fig. 9

Kinetic analysis of the agonist-promoted BRET increase between β2AR-GFP10 and QL-Gαi1-122Rluc. (PDF 123 kb)

Supplementary Methods

Constructs and methods of the Supplementary Figures. (PDF 27 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Galés, C., Van Durm, J., Schaak, S. et al. Probing the activation-promoted structural rearrangements in preassembled receptor–G protein complexes. Nat Struct Mol Biol 13, 778–786 (2006). https://doi.org/10.1038/nsmb1134

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb1134

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing