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Structural basis of G protein–coupled receptor–G protein interactions

Nature Chemical Biology volume 6, pages 541–548 (2010)Cite this article

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Abstract

The interaction of G protein–coupled receptors (GPCRs) with heterotrimeric G proteins represents one of the most fundamental biological processes. However, the molecular architecture of the GPCR–G protein complex remains poorly defined. In the present study, we applied a comprehensive GPCR–G protein α subunit (Gα) chemical cross-linking strategy to map a receptor-Gα interface, both before and after agonist-induced receptor activation. Using the M3 muscarinic acetylcholine receptor (M3R)-Gαq system as a model system, we examined the ability of ~250 combinations of cysteine-substituted M3R and Gαq proteins to undergo cross-link formation. We identified many specific M3R-Gαq contact sites, in both the inactive and active receptor conformations, allowing us to draw conclusions regarding the basic architecture of the M3R-Gαq interface and the nature of the conformational changes following receptor activation. As heterotrimeric G proteins as well as most GPCRs share a high degree of structural homology, our findings should be of broad general relevance.

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Figure 1: Introduction of cysteine substitutions into the M3R and Gαq.
Figure 2: Cross-linking of Gαq subunits containing C-terminal cysteine substitutions with cysteine-substituted mutant M3Rs.
Figure 3: Co-immunoprecipitation experiments confirming the identity of a representative Gαq–M3R complex.
Figure 4: Cross-link formation between a cysteine residue introduced into the αN helix of Gαq (R31C) and a cysteine residue substituted into the i2 loop of the M3R (L173C).
Figure 5: Agonist-induced cross-link formation between a cysteine residue introduced into the α4-β6 loop of Gαq (D321C) and cysteine residues substituted into the N-terminal segment of H8 of the M3R.
Figure 6: Molecular model of the M3R–Gq complex.

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Acknowledgements

This research was supported by the Intramural Research Program of the US National Institute of Diabetes and Digestive and Kidney Diseases (US National Institutes of Health) and used the high-performance computational capabilities of the Biowulf Linux cluster at the US National Institutes of Health, Bethesda, Maryland, USA (http://biowulf.nih.gov).

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Authors and Affiliations

  1. Molecular Signaling Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA

    Jianxin Hu, Yan Wang, Xiaohong Zhang, Jian Hua Li & Jürgen Wess

  2. Mass Spectrometry Group, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA

    John R Lloyd

  3. Laboratory of Biological Modeling, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA

    Joel Karpiak & Stefano Costanzi

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Contributions

J.H. designed and conducted most of the biochemical experiments. Y.W., X.Z. and J.L. were involved in the pharmacological characterization of the mutant proteins. J.R.L. carried out the mass spectrometry studies. S.C. designed the molecular modeling studies. S.C. and J.K. carried out the molecular modeling studies. J.W. designed the experiments, and J.H., S.C. and J.W. wrote the paper.

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Correspondence to Jianxin Hu or Jürgen Wess.

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Hu, J., Wang, Y., Zhang, X. et al. Structural basis of G protein–coupled receptor–G protein interactions. Nat Chem Biol 6, 541–548 (2010). https://doi.org/10.1038/nchembio.385

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