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Mechanism of the receptor-catalyzed activation of heterotrimeric G proteins

Nature Structural & Molecular Biology volume 13, pages 772–777 (2006)Cite this article

Abstract

Heptahelical receptors activate intracellular signaling pathways by catalyzing GTP for GDP exchange on the heterotrimeric G protein α subunit (Gα). Despite the crucial role of this process in cell signaling, little is known about the mechanism of G protein activation. Here we explore the structural basis for receptor-mediated GDP release using electron paramagnetic resonance spectroscopy. Binding to the activated receptor (R*) causes an apparent rigid-body movement of the α5 helix of Gα that would perturb GDP binding at the β6-α5 loop. This movement was not observed when a flexible loop was inserted between the α5 helix and the R*-binding C terminus, which uncouples R* binding from nucleotide exchange, suggesting that this movement is necessary for GDP release. These data provide the first direct observation of R*-mediated conformational changes in G proteins and define the structural basis for GDP release from Gα.

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Figure 1: Characterization of spin-labeled mutants.
Figure 2: EPR spectra demonstrate receptor activation–dependent conformational changes in the α5 helix.
Figure 3: Movement of the α5 helix is necessary for GDP release.
Figure 4: DEER distance measurements support the proposed rotation-translation of the α5 helix.

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Acknowledgements

This work was supported by grants from the US National Institutes of Health (W.L.H. and H.E.H.), a Public Health Service Award for the Medical Scientist Training Program (W.M.O.), the PhRMA Foundation (W.M.O.) and the Jules Stein Professorship (W.L.H.).

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Author notes

  1. William M Oldham and Ned Van Eps: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, 37232-6600, Tennessee, USA

    William M Oldham, Anita M Preininger & Heidi E Hamm

  2. Jules Stein Eye Institute and the Departments of Ophthalmology and Chemistry & Biochemistry, University of California, Los Angeles, 90095-7008, California, USA

    Ned Van Eps & Wayne L Hubbell

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Correspondence to Wayne L Hubbell or Heidi E Hamm.

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Supplementary information

Supplementary Fig. 1

Fluorescence assay of nucleotide exchange. (PDF 129 kb)

Supplementary Fig. 2

Rhodopsin binding assay. (PDF 125 kb)

Supplementary Fig. 3

Full EPR spectra for single mutants. (PDF 627 kb)

Supplementary Fig. 4

Biochemical characterization of the 5G insertion mutant. (PDF 24 kb)

Supplementary Fig. 5

Structural changes upon GTPγS addition to the R*-G protein complex. (PDF 142 kb)

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Oldham, W., Van Eps, N., Preininger, A. et al. Mechanism of the receptor-catalyzed activation of heterotrimeric G proteins. Nat Struct Mol Biol 13, 772–777 (2006). https://doi.org/10.1038/nsmb1129

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