Letter

Single-molecule imaging reveals receptor–G protein interactions at cell surface hot spots

Received:
Accepted:
Published online:

Abstract

G-protein-coupled receptors mediate the biological effects of many hormones and neurotransmitters and are important pharmacological targets1. They transmit their signals to the cell interior by interacting with G proteins. However, it is unclear how receptors and G proteins meet, interact and couple. Here we analyse the concerted motion of G-protein-coupled receptors and G proteins on the plasma membrane and provide a quantitative model that reveals the key factors that underlie the high spatiotemporal complexity of their interactions. Using two-colour, single-molecule imaging we visualize interactions between individual receptors and G proteins at the surface of living cells. Under basal conditions, receptors and G proteins form activity-dependent complexes that last for around one second. Agonists specifically regulate the kinetics of receptor–G protein interactions, mainly by increasing their association rate. We find hot spots on the plasma membrane, at least partially defined by the cytoskeleton and clathrin-coated pits, in which receptors and G proteins are confined and preferentially couple. Imaging with the nanobody Nb37 suggests that signalling by G-protein-coupled receptors occurs preferentially at these hot spots. These findings shed new light on the dynamic interactions that control G-protein-coupled receptor signalling.

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Acknowledgements

We thank S. P. Watson, M. Sauer, C. Manzo and E. Cocucci for discussions. This work was supported by grants from the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich/Transregio 166–Project C1 to D.C., CA 1014/1-1 to D.C. and FZT82 Rudolf Virchow Center to M.J.L.), the IZKF Würzburg (grant B-281 to D.C.), the European Research Council (Advanced Grant 232944–TOPAS to M.J.L.) and the Polish National Science Center (Maestro Grant No. 2012/06/A/ST1/00258 to K.B. and A.W.). T.S. was in part supported by an Alexander-von-Humboldt/Bayer Foundation fellowship.

Author information

Affiliations

  1. Institute of Pharmacology and Toxicology, University of Würzburg, Versbacher Straße 9, 97078 Würzburg, Germany

    • Titiwat Sungkaworn
    • , Marie-Lise Jobin
    • , Martin J. Lohse
    •  & Davide Calebiro
  2. Bio-Imaging Center/Rudolf Virchow Center, University of Würzburg, Versbacher Straße 9, 97078 Würzburg, Germany

    • Titiwat Sungkaworn
    • , Marie-Lise Jobin
    • , Martin J. Lohse
    •  & Davide Calebiro
  3. Faculty of Pure and Applied Mathematics, Hugo Steinhaus Center, Wroclaw University of Science and Technology, Wyspianskiego 27, 50-370 Wroclaw, Poland

    • Krzysztof Burnecki
    •  & Aleksander Weron
  4. Max Delbrück Center for Molecular Medicine, Robert-Rössle-Straße 10, 13125 Berlin, Germany

    • Martin J. Lohse
  5. Institute of Metabolism and Systems Research, University of Birmingham, Birmingham B15 2TT, UK

    • Davide Calebiro
  6. Centre of Membrane Proteins and Receptors (COMPARE), Universities of Birmingham and Nottingham, Birmingham B15 2TT, UK

    • Davide Calebiro

Authors

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Contributions

T.S. and M.-L.J. performed the experiments. T.S., M.-L.J., K.B. and D.C. analysed the data. K.B., A.W. and D.C. developed the mathematical analyses. D.C. wrote the software. D.C., T.S. and M.J.L. wrote the manuscript. All authors discussed the results. D.C. and M.J.L. developed the project. D.C. conceived and supervised the study.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Davide Calebiro.

Reviewer Information Nature thanks J. Levitz, S. Marullo and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    This file contains Supplementary Data, Supplementary Discussion and Supplementary Methods.

  2. 2.

    Reporting Summary

Videos

  1. 1.

    Single-molecule imaging of individual receptors and G proteins at the surface of living cells

    Shown are individual α2A-ARs (green) and Gαi subunits (magenta), imaged by fast two-colour TIRF microscopy. Frames were acquired every 28 ms.

  2. 2.

    Single-particle tracking of receptors and G proteins

    Shown are individual α2A-AR (green) and Gαi (magenta) trajectories. Frames were acquired every 28 ms.

  3. 3.

    Receptors stopping at areas of high localization density.

    Shown are individual receptor trajectories (α2A-ARs; different colours) overlaid on the corresponding localization density map. The playback is accelerated to capture receptor accumulation at high density areas.

  4. 4.

    Relationship between receptor trajectories and underlying microtubules

    Shown are individual α2A-AR trajectories (green) over a TIRF image of microtubules (magenta). Frames were acquired every 28 ms.

  5. 5.

    Relationship between receptor trajectories and underlying actin fibres

    Shown are individual α2A-AR trajectories (green) over a TIRF image of actin fibres (magenta). Frames were acquired every 28 ms.

  6. 6.

    Relationship between receptor trajectories and underlying clathrin-coated pits (CCPs)

    Shown are individual α2A-AR trajectories (green) over a TIRF image of CCPs. Frames were acquired every 28 ms.

  7. 7.

    Combined single-particle tracking of receptors and superresolution imaging of actin fibres

    Shown are individual α2A-AR trajectories (green) over a PALM image of actin fibres (orange). Frames were acquired every 28 ms.

  8. 8.

    Combined single-particle tracking of G proteins and superresolution imaging of actin fibres

    Shown are individual Gαi trajectories (green) over a PALM image of actin fibres (orange). Frames were acquired every 28 ms.

  9. 9.

    Receptor and G protein stopping during an apparent interaction

    Shown are single-molecule images and corresponding trajectories of an α2A-AR (green) and a Gαi subunit (magenta) diffusing and then stopping during an apparent interaction. The trajectories are coloured in blue during the interaction. Afterwards, the receptor resumes its movement, whereas the G protein remains immobile until it disappears due to fluorophore photobleaching. Frames were acquired every 28 ms.