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Activation of the A2A adenosine G-protein-coupled receptor by conformational selection

Nature 533, 265–268 (12 May 2016) | Download Citation

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Abstract

Conformational selection and induced fit are two prevailing mechanisms1,2 to explain the molecular basis for ligand-based activation of receptors. G-protein-coupled receptors are the largest class of cell surface receptors and are important drug targets. A molecular understanding of their activation mechanism is critical for drug discovery and design. However, direct evidence that addresses how agonist binding leads to the formation of an active receptor state is scarce3. Here we use 19F nuclear magnetic resonance to quantify the conformational landscape occupied by the adenosine A2A receptor (A2AR), a prototypical class A G-protein-coupled receptor. We find an ensemble of four states in equilibrium: (1) two inactive states in millisecond exchange, consistent with a formed (state S1) and a broken (state S2) salt bridge (known as ‘ionic lock’) between transmembrane helices 3 and 6; and (2) two active states, S3 and S3′, as identified by binding of a G-protein-derived peptide. In contrast to a recent study of the β2-adrenergic receptor4, the present approach allowed identification of a second active state for A2AR. Addition of inverse agonist (ZM241385) increases the population of the inactive states, while full agonists (UK432097 or NECA) stabilize the active state, S3′, in a manner consistent with conformational selection. In contrast, partial agonist (LUF5834) and an allosteric modulator (HMA) exclusively increase the population of the S3 state. Thus, partial agonism is achieved here by conformational selection of a distinct active state which we predict will have compromised coupling to the G protein. Direct observation of the conformational equilibria of ligand-dependent G-protein-coupled receptor and deduction of the underlying mechanisms of receptor activation will have wide-reaching implications for our understanding of the function of G-protein-coupled receptor in health and disease.

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Acknowledgements

This work was supported by the Natural Sciences and Engineering Research Council of Canada, research discovery award grant number 261980 (to R.S.P.) and the Canada Excellence Research Chair Program (to O.P.E., who is the Anne and Max Tanenbaum Chair in Neuroscience at the University of Toronto). We thank T. Kobayashi and R. Grisshammer for providing plasmids with A2AR sequence. We thank J. Wells, S. Larda, and F. Huang from the University of Toronto, as well as S. Furness, B. K. Kobilka, and R. Sunahara for their suggestions and comments.

Author information

Affiliations

  1. Department of Chemistry, University of Toronto, UTM, 3359 Mississauga Road North, Mississauga, Ontario L5L 1C6, Canada

    • Libin Ye
    •  & R. Scott Prosser

  2. Department of Biochemistry, University of Toronto, 1 King’s College Circle, Toronto, Ontario M5S 1A8, Canada

    • Libin Ye
    • , Ned Van Eps
    • , Marco Zimmer
    • , Oliver P. Ernst
    •  & R. Scott Prosser

  3. Department of Technical Biochemistry, University of Stuttgart, 31 Allmandring, Stuttgart, Baden-Württemberg, D-70569, Germany

    • Marco Zimmer

  4. Department of Molecular Genetics, University of Toronto, 1 King’s College Circle, Toronto, Ontario M5S 1A8, Canada

    • Oliver P. Ernst

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Contributions

L.Y., O.P.E., and R.S.P. designed the research. L.Y. performed the molecular biology work, generated high-yield transformants, and optimized receptor expression and purification. L.Y. also performed NMR and EPR labelling, NMR experiments, and analysed spectroscopy data. N.V.E. performed and analysed data from EPR experiments. M.Z. assisted with cell culture and receptor purification. R.S.P., L.Y., and O.P.E. prepared the manuscript. O.P.E. and R.S.P. supervised the project.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Oliver P. Ernst or R. Scott Prosser.

Extended data

Extended data figures

  1. 1.

    Comparison of inactive and active GPCR crystal structures.

  2. 2.

    Secondary structure and topology of C-terminally truncated A2AR-V229C.

  3. 3.

    Labelling efficiency of A2AR-V229C.

  4. 4.

    Car–Purcell–Meiboom–Gill (CPMG) relaxation dispersion experiment to evaluate dynamics of S1–2.

  5. 5.

    Comparison of two- and three-state models of 19F-labelled A2AR-V229C.

  6. 6.

    19F NMR spectra of 19F-labelled A2AR-V229C in the presence of 50- or 100-fold excess of different ligands.

  7. 7.

    The role of HMA in the receptor activation process.

  8. 8.

    Saturation transfer experiments of 19F-labelled A2AR-V229C.

Extended data tables

  1. 1.

    Primers/gene fragments used to construct plasmids for this study

Supplementary information

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  1. 1.

    Supplementary Information

    This file contains Supplementary Text and Data.

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DOI

https://doi.org/10.1038/nature17668

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