Abstract

G-protein-coupled receptor (GPCR)-mediated signal transduction is central to human physiology and disease intervention, yet the molecular mechanisms responsible for ligand-dependent signalling responses remain poorly understood. In class A GPCRs, receptor activation and G-protein coupling entail outward movements of transmembrane helix 6 (TM6). Here, using single-molecule fluorescence resonance energy transfer imaging, we examine TM6 movements in the β2 adrenergic receptor (β2AR) upon exposure to orthosteric ligands with different efficacies, in the absence and presence of the Gs heterotrimer. We show that partial and full agonists differentially affect TM6 motions to regulate the rate at which GDP-bound β2AR–Gs complexes are formed and the efficiency of nucleotide exchange leading to Gs activation. These data also reveal transient nucleotide-bound β2AR–Gs species that are distinct from known structures, and provide single-molecule perspectives on the allosteric link between ligand- and nucleotide-binding pockets that shed new light on the G-protein activation mechanism.

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Acknowledgements

We thank M. Howarth for the gift of trans-divalent streptavidin, and C. Stern in the laboratory of J. Chodera for constructing the CHARMM-consistent parameters for the dyes used in the molecular dynamics simulations. Computational resources are gratefully acknowledged: an XSEDE allocation at the Texas Advanced Computing Center at the University of Texas at Austin (Stampede supercomputer, project TG MCB120008), support from resources at the Oak Ridge Leadership Computing Facility (ALCC allocation BIP109) at the Oak Ridge National Laboratory that is supported by the Office of Science of the US Department of Energy under contract no. DE-AC05-00OR22725; and the resources of the David A. Cofrin Center for Biomedical Information in the HRH Prince Alwaleed Bin Talal Bin Abdulaziz Alsaud Institute for Computational Biomedicine at Weill Cornell Medicine. This work was supported in part by National Institutes of Health (NIH) grants GM098859 (S.C.B.), R21DA0354585 (J.A.J., S.C.B. and G.G.G.), K05DA022413 and R01 MH54137 (J.A.J.), R01GM083118 and R01NS028471 (B.K.K.), and U54GM087519 (H.W. and J.M.P.-A.), the German Academic Exchange Service (DAAD) (D.H.), the American Heart Association Postdoctoral fellowship (15POST22700020) (M.M.), and the Novo Nordisk Foundation Center for Basic Metabolic Research (M.H.).

Author information

Author notes

    • Jose Manuel Perez-Aguilar

    Present address: IBM Thomas J. Watson Research Center, Yorktown Heights, New York, USA.

    • G. Glenn Gregorio
    • , Matthieu Masureel
    •  & Daniel Hilger

    These authors contributed equally to this work.

Affiliations

  1. Department of Physiology and Biophysics, Weill Cornell Medicine, New York, New York, USA

    • G. Glenn Gregorio
    • , Daniel S. Terry
    • , Manuel Juette
    • , Hong Zhao
    • , Zhou Zhou
    • , Jose Manuel Perez-Aguilar
    • , Harel Weinstein
    •  & Scott C. Blanchard
  2. Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California, USA

    • Matthieu Masureel
    • , Daniel Hilger
    •  & Brian K. Kobilka
  3. Department of Psychiatry, Columbia University College of Physicians and Surgeons, New York, New York, USA

    • Maria Hauge
    • , Signe Mathiasen
    •  & Jonathan A. Javitch
  4. Division of Molecular Therapeutics, New York State Psychiatric Institute, New York, New York, USA

    • Maria Hauge
    • , Signe Mathiasen
    •  & Jonathan A. Javitch
  5. Laboratory for Molecular Pharmacology, Department of Neuroscience and Pharmacology, University of Copenhagen, Blegdamsvej 3, Copenhagen 2200, Denmark

    • Maria Hauge
  6. NNF Center for Basic Metabolic Research, University of Copenhagen, Blegdamsvej 3, Copenhagen 2200, Denmark

    • Maria Hauge
  7. Department of Pharmacology, Columbia University College of Physicians and Surgeons, New York, New York, USA

    • Jonathan A. Javitch
  8. The HRH Prince Alwaleed Bin Talal Bin Abdulaziz Alsaud Institute for Computational Biomedicine, Weill Cornell Medical College of Cornell University, New York, New York, USA

    • Harel Weinstein

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Contributions

G.G.G., M.M., D.H., B.K.K. and S.C.B. designed single-molecule experiments. G.G.G. labelled receptor and performed all single-molecule experiments. G.G.G. analysed single-molecule data, with support from D.S.T. M.J. and D.S.T. developed the imaging and analysis platform. M.M. expressed, purified and characterized receptor constructs. D.H. expressed, purified and biotinylated Gs, and performed GTP turnover assays. H.Z. and Z.Z. synthesized the fluorophores. J.M.P.-A. performed molecular dynamics simulations under the supervision of H.W. M.H. and S.M. performed cell-based G-protein-coupling assays under the supervision of J.A.J. G.G.G., M.M., D.H., J.A.J., H.W., B.K.K. and S.C.B. interpreted all the data and wrote the manuscript. B.K.K. and S.C.B. provided overall project supervision.

Competing interests

S.C.B. has an equity interest in Lumidyne Technologies.

Corresponding authors

Correspondence to Brian K. Kobilka or Scott C. Blanchard.

Reviewer Information Nature thanks M. Lohse 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.

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https://doi.org/10.1038/nature22354

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