Letter | Published:

Conformational changes in the G protein Gs induced by the β2 adrenergic receptor

Nature volume 477, pages 611615 (29 September 2011) | Download Citation

Abstract

G protein-coupled receptors represent the largest family of membrane receptors1 that instigate signalling through nucleotide exchange on heterotrimeric G proteins. Nucleotide exchange, or more precisely, GDP dissociation from the G protein α-subunit, is the key step towards G protein activation and initiation of downstream signalling cascades. Despite a wealth of biochemical and biophysical studies on inactive and active conformations of several heterotrimeric G proteins, the molecular underpinnings of G protein activation remain elusive. To characterize this mechanism, we applied peptide amide hydrogen–deuterium exchange mass spectrometry to probe changes in the structure of the heterotrimeric bovine G protein, Gs (the stimulatory G protein for adenylyl cyclase) on formation of a complex with agonist-bound human β2 adrenergic receptor (β2AR). Here we report structural links between the receptor-binding surface and the nucleotide-binding pocket of Gs that undergo higher levels of hydrogen–deuterium exchange than would be predicted from the crystal structure of the β2AR–Gs complex. Together with X-ray crystallographic and electron microscopic data of the β2AR–Gs complex (from refs 2, 3), we provide a rationale for a mechanism of nucleotide exchange, whereby the receptor perturbs the structure of the amino-terminal region of the α-subunit of Gs and consequently alters the ‘P-loop’ that binds the β-phosphate in GDP. As with the Ras family of small-molecular-weight G proteins, P-loop stabilization and β-phosphate coordination are key determinants of GDP (and GTP) binding affinity.

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Acknowledgements

We thank J.J.G. Tesmer and G. Skiniotis for discussions. This work was supported by an American Lung Association senior research training fellowship (RT-166882-N, to K.Y.C.), the Lundbeck Foundation (a junior group leader fellowship, to S.G.F.R.), a National Institute of General Medical Sciences (NIGMS) molecular biophysics training grant (GM008270, to B.T.D), a National Institute of Neural Disorders and Stroke grant (NS28471, to B.K.K.), a NHLBI grant (HL071078, to B.K.K.), the Mather Charitable Foundation (B.K.K.), NIGMS grants GM083118 (to B.K.K. and R.K.S.) and GM068603 (to R.K.S.), a Michigan Diabetes Research and Training Center grant, the National Institute of Diabetes and Digestive and Kidney Diseases (P60DK-20572, to R.K.S.), the University of Michigan Biological Sciences Scholars Program (R.K.S.), and by the NIH (grants AI076961, AI081982, AI2008031, CA118595, GM20501, GM066170, GM093325 and RR029388 (to V.L.W.)).

Author information

Author notes

    • Ka Young Chung
    • , Søren G. F. Rasmussen
    •  & Tong Liu

    These authors contributed equally to this work.

    • Pil Seok Chae

    Present address: Department of Bionano Engineering, Hanyang University, Ansan 426-791, Korea.

Affiliations

  1. Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305, USA

    • Ka Young Chung
    • , Søren G. F. Rasmussen
    •  & Brian K. Kobilka
  2. Department of Neuroscience and Pharmacology, The Panum Institute, University of Copenhagen, 2200 Copenhagen N, Denmark

    • Søren G. F. Rasmussen
  3. Department of Medicine, Biomedical Sciences Graduate Program and UCSD DXMS Proteomics Resource, University of California San Diego, La Jolla, California 92023, USA

    • Tong Liu
    • , Sheng Li
    •  & Virgil L. Woods Jr
  4. Department of Pharmacology, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA

    • Brian T. DeVree
    • , Diane Calinski
    •  & Roger K. Sunahara
  5. Department of Chemistry, University of Wisconsin, Madison, Wisconsin 53706, USA

    • Pil Seok Chae

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Contributions

K.Y.C. prepared nucleotide-treated samples for DXMS, performed hydrogen–deuterium exchange of prepared samples, analysed mass spectrometry data, and performed cross-linking studies. S.G.F.R. performed the final stages of β2AR purification, assisted with β2AR and Gs protein virus production and expression in insect cell cultures, and worked out conditions to form, stabilize and purify the β2AR–Gs complex using agonist BI-167107 and detergent MNG-3. T.L. was responsible for the overall data analysis. S.L. ran prepared samples on the mass spectrometer. B.T.D. managed Gs heterotrimer subunit virus production and titration, expressed and purified Gs protein, and, with K.Y.C. and T.L., analysed mass spectrometry data. P.S.C. provided MNG-3 detergent for stabilization of the β2AR–Gs complex. D.C. assisted with Gs heterotrimer expression and purification. B.K.K. was responsible for overall project strategy and management, and assisted with manuscript preparation. V.L.W. oversaw design, execution and data analysis of DXMS experiments, and assisted with manuscript preparation. R.K.S. supervised Gs protein production, provided ideas and insights into Gs structure and function, and wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Brian K. Kobilka or Virgil L. Woods Jr or Roger K. Sunahara.

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DOI

https://doi.org/10.1038/nature10488

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