Abstract

G-protein-coupled receptors (GPCRs) remain the primary conduit by which cells detect environmental stimuli and communicate with each other1. Upon activation by extracellular agonists, these seven-transmembrane-domain-containing receptors interact with heterotrimeric G proteins to regulate downstream second messenger and/or protein kinase cascades1. Crystallographic evidence from a prototypic GPCR, the β2-adrenergic receptor (β2AR), in complex with its cognate G protein, Gs, has provided a model for how agonist binding promotes conformational changes that propagate through the GPCR and into the nucleotide-binding pocket of the G protein α-subunit to catalyse GDP release, the key step required for GTP binding and activation of G proteins2. The structure also offers hints about how G-protein binding may, in turn, allosterically influence ligand binding. Here we provide functional evidence that G-protein coupling to the β2AR stabilizes a ‘closed’ receptor conformation characterized by restricted access to and egress from the hormone-binding site. Surprisingly, the effects of G protein on the hormone-binding site can be observed in the absence of a bound agonist, where G-protein coupling driven by basal receptor activity impedes the association of agonists, partial agonists, antagonists and inverse agonists. The ability of bound ligands to dissociate from the receptor is also hindered, providing a structural explanation for the G-protein-mediated enhancement of agonist affinity, which has been observed for many GPCR–G-protein pairs. Our data also indicate that, in contrast to agonist binding alone, coupling of a G protein in the absence of an agonist stabilizes large structural changes in a GPCR. The effects of nucleotide-free G protein on ligand-binding kinetics are shared by other members of the superfamily of GPCRs, suggesting that a common mechanism may underlie G-protein-mediated enhancement of agonist affinity.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    , & Seven-transmembrane receptors. Nature Rev. Mol. Cell Biol. 3, 639–650 (2002)

  2. 2.

    et al. Crystal structure of the β2 adrenergic receptor–Gs protein complex. Nature 477, 549–555 (2011)

  3. 3.

    et al. The sequence of the human genome. Science 291, 1304–1351 (2001)

  4. 4.

    G protein mechanisms: insights from structural analysis. Annu. Rev. Biochem. 66, 639–678 (1997)

  5. 5.

    et al. Conformational changes in the G protein Gs induced by the β2 adrenergic receptor. Nature 477, 611–615 (2011)

  6. 6.

    et al. Structural flexibility of the Gαs α-helical domain in the β2-adrenoceptor Gs complex. Proc. Natl Acad. Sci. USA 108, 16086–16091 (2011)

  7. 7.

    , & An agonist-specific effect of guanine nucleotides on binding to the beta adrenergic receptor. Mol. Pharmacol. 12, 335–339 (1976)

  8. 8.

    , , , & Relationship between the β-adrenergic receptor and adenylate cyclase. J. Biol. Chem. 252, 5761–5775 (1977)

  9. 9.

    , & A ternary complex model explains the agonist-specific binding properties of the adenylate cyclase-coupled β-adrenergic receptor. J. Biol. Chem. 255, 7108–7117 (1980)

  10. 10.

    et al. The effect of ligand efficacy on the formation and stability of a GPCR–G protein complex. Proc. Natl Acad. Sci. USA 106, 9501–9506 (2009)

  11. 11.

    et al. Structure of a nanobody-stabilized active state of the β2 adrenoceptor. Nature 469, 175–180 (2011)

  12. 12.

    et al. Conformational biosensors reveal GPCR signalling from endosomes. Nature 495, 534–538 (2013)

  13. 13.

    & Catecholamine binding to the β-adrenergic receptor. Proc. Natl Acad. Sci. USA 74, 515–519 (1977)

  14. 14.

    et al. Adrenaline-activated structure of β2-adrenoceptor stabilized by an engineered nanobody. Nature 502, 575–579 (2013)

  15. 15.

    et al. Ligand-specific regulation of the extracellular surface of a G-protein-coupled receptor. Nature 463, 108–112 (2010)

  16. 16.

    , , & The role of the seventh transmembrane region in high affinity binding of a β2-selective agonist TA-2005. Mol. Pharmacol. 53, 128–134 (1998)

  17. 17.

    et al. Pathway and mechanism of drug binding to G-protein-coupled receptors. Proc. Natl Acad. Sci. USA 108, 13118–13123 (2011)

  18. 18.

    et al. Structure and function of an irreversible agonist–β2 adrenoceptor complex. Nature 469, 236–240 (2011)

  19. 19.

    et al. The structural basis for agonist and partial agonist action on a β1-adrenergic receptor. Nature 469, 241–244 (2011)

  20. 20.

    , , & A mutation-induced activated state of the β2-adrenergic receptor. Extending the ternary complex model. J. Biol. Chem. 268, 4625–4636 (1993)

  21. 21.

    , , & The cubic ternary complex receptor-occupancy model. I. Model description. J. Theor. Biol. 178, 151–167 (1996)

  22. 22.

    , & Reciprocal modulation of agonist and antagonist binding to muscarinic cholinergic receptor by guanine nucleotide. Proc. Natl Acad. Sci. USA 79, 1732–1736 (1982)

  23. 23.

    , , & A robust GTP-induced shift in α2-adrenoceptor agonist affinity in tissue sections from rat brain. J. Neurosci. Methods 105, 159–166 (2001)

  24. 24.

    , , & Indirect effect of guanine nucleotides on antagonist binding to A1 adenosine receptors: occupation of cryptic binding sites by endogenous vesicular adenosine. Mol. Pharmacol. 42, 765–772 (1992)

  25. 25.

    , & Multiple agonist-affinity states of opioid receptors: regulation of binding by guanyl nucleotides in guinea pig cortical, NG108-15, and 7315c cell membranes. Mol. Pharmacol. 33, 423–431 (1988)

  26. 26.

    et al. Structure of the human M2 muscarinic acetylcholine receptor bound to an antagonist. Nature 482, 547–551 (2012)

  27. 27.

    et al. Activation and allosteric modulation of a muscarinic acetylcholine receptor. Nature 504, 101–106 (2013)

  28. 28.

    et al. Crystal structure of the μ-opioid receptor bound to a morphinan antagonist. Nature 485, 321–326 (2012)

  29. 29.

    et al. Structural insights into μ-opioid receptor activation. Nature 524, 315–321 (2015)

  30. 30.

    et al. Allosteric sodium in class A GPCR signaling. Trends Biochem. Sci. 39, 233–244 (2014)

  31. 31.

    & Purification of recombinant G proteins from Sf9 cells by hexahistidine tagging of associated subunits. Characterization of α12 and inhibition of adenylyl cyclase by αz. J. Biol. Chem. 270, 1734–1741 (1995)

  32. 32.

    et al. A monomeric G protein-coupled receptor isolated in a high-density lipoprotein particle efficiently activates its G protein. Proc. Natl Acad. Sci. USA 104, 7682–7687 (2007)

  33. 33.

    et al. Structure of the agonist-bound neurotensin receptor. Nature 490, 508–513 (2012)

  34. 34.

    , , & Structural prerequisites for G-protein activation by the neurotensin receptor. Nature Commun. 6, 7895–7895 (2015)

Download references

Acknowledgements

We thank T. S. Kobilka for preparation of affinity chromatography reagents and F. S. Thian for help with cell culture. We thank J. Traynor and J. Tesmer for their support and use of their laboratory space for J.P.M. This work was supported by the Lundbeck Foundation (Junior Group Leader Fellowship to S.G.F.R.); Fund for Scientific Research of Flanders (FWO-Vlaanderen) and the Institute for the encouragement of Scientific Research and Innovation of Brussels (ISRIB) (E.P. and J.S.); National Institute of Neural Disorders and Stroke grant RO1-NS28471 (B.K.K.); the Mather Charitable Foundation (B.K.K.); National Institute of General Medical Sciences grants RO1-GM083118 and U19-GM106990 (B.K.K. and R.K.S.) and RO1-GM068603 (R.K.S.); National Institutes of Drug Abuse R21-031418 (B.K.K. and R.K.S.); Michigan Diabetes Research and Training Center Grant, National Institute of Diabetes and Digestive and Kidney Diseases, P60DK-20572 (R.K.S.); University of Michigan Biological Sciences Scholars Program (R.K.S.) and the Rackham School of Graduate Studies (B.T.D.); Molecular Biophysics Training Grant T32GM008270 (B.T.D.); Cell and Molecular Biology Training Grant T32GM007315 (G.A.V.-R.) and Pharmacological Sciences Training Program T32GM007767 (J.P.M.); and AHA Midwest Affiliate Predoctoral Fellowship 13PRE17110027 (J.P.M.).

Author information

Author notes

    • Brian T. DeVree
    •  & Jacob P. Mahoney

    These authors contributed equally to this work.

Affiliations

  1. Department of Pharmacology, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA

    • Brian T. DeVree
    • , Jacob P. Mahoney
    • , Gisselle A. Vélez-Ruiz
    • , Adam J. Kuszak
    • , Elin Edwald
    •  & Roger K. Sunahara
  2. Department of Cellular and Molecular Physiology, Stanford University, Palo Alto, California 94305, USA

    • Soren G. F. Rasmussen
    • , Juan-Jose Fung
    • , Aashish Manglik
    • , Matthieu Masureel
    • , Yang Du
    • , Rachel A. Matt
    •  & Brian K. Kobilka
  3. Structural Biology Research Center, VIB, Vrije Universiteit Brussel (VUB), Pleinlaan 2, 1050 Brussels, Belgium

    • Els Pardon
  4. Structural Biology Brussels, Vrije Universiteit Brussel (VUB), Pleinlaan 2, 1050 Brussels, Belgium

    • Jan Steyaert
  5. Department of Pharmacology, University of California San Diego School of Medicine, 9500 Gilman Drive, La Jolla, California 92093, USA

    • Roger K. Sunahara

Authors

  1. Search for Brian T. DeVree in:

  2. Search for Jacob P. Mahoney in:

  3. Search for Gisselle A. Vélez-Ruiz in:

  4. Search for Soren G. F. Rasmussen in:

  5. Search for Adam J. Kuszak in:

  6. Search for Elin Edwald in:

  7. Search for Juan-Jose Fung in:

  8. Search for Aashish Manglik in:

  9. Search for Matthieu Masureel in:

  10. Search for Yang Du in:

  11. Search for Rachel A. Matt in:

  12. Search for Els Pardon in:

  13. Search for Jan Steyaert in:

  14. Search for Brian K. Kobilka in:

  15. Search for Roger K. Sunahara in:

Contributions

B.T.D., J.P.M., G.A.V.-R., B.K.K. and R.K.S. designed the experiments. B.T.D., J.P.M., G.A.V.-R. and A.J.K. performed research; S.G.F.R., E.E., J.-J.F., A.M., M.M., Y.D., R.A.M., E.P. and J.S. contributed valuable reagents/analytic tools; B.T.D., J.P.M., G.A.V.-R., B.K.K. and R.K.S. analysed data; and B.T.D., J.P.M., B.K.K. and R.K.S. wrote the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Roger K. Sunahara.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    This file contains Supplementary Methods, a Supplementary Discussion and additional references.

Videos

  1. 1.

    Activation of the β2AR

    Morph of the β2AR in its inactive conformation bound to inverse agonist carazolol (PDB: 2RH1) and the β2AR in its active conformation bound to agonist BI-167607 and nanobody Nb80 (PDB: 3P0G). For reference, epinephrine is modeled in the orthosteric binding site. Morphs were generated using Chimera (Pettersen, E,F., et al. UCSF Chimera--a visualization system for exploratory research and analysis. J Comput Chem. 2004 25, 1605-12) and rendered with Pymol (The PyMOL Molecular Graphics System, Schrödinger, LLC). Highlighted are Phe193ECL2 and Tyr3087.35.

  2. 2.

    Comparison of β1AR and β2AR

    The closed, active conformation is stabilized by the G protein. Morph of the β2AR in its inactive conformation bound to inverse agonist carazolol (cyan, PDB: 2RH1) and the β2AR in its active conformation bound to agonist BI-167607 and nanobody Nb80 (PDB: 3P0G). Superimposed on top is a similar morph transitioning between the carazolol-boundββ1-adrenergic receptor (β1AR, lime green, PDB: 2YCW) and isoproterenol-bound (but not G protein- or Nb-bound, PDB: 2Y03). While the β2AR adopts a closed conformation stabilized by agonist and G protein, the β1AR bound only to isoproterenol does not. For reference, epinephrine is modeled in the orthosteric binding site. Note that nanobody Nb80 has been omitted from the animation for simplicity. Highlighted are Phe193ECL2 and Tyr3087.35 on β2AR and the conserved residues Phe201ECL2 and Phe3527.35 on β1AR. Morphs were generated using Chimera (Pettersen, E,F., et al. UCSF Chimera--a visualization system for exploratory research and analysis. J Comput Chem. 2004 25, 1605-12) and rendered with Pymol (The PyMOL Molecular Graphics System, Schrödinger, LLC).

  3. 3.

    Activation of the μ-opioid receptor, MOPr

    The ribbon structure of the μ-opioid receptor (MOPr) in its inactive conformation bound to β-funaltrexamine (cyan, PDB: 4DKL) and the MOPr in its active conformation bound to agonist BU72 and Nb39 (orange, PDB: 5C1M). For reference only BU72 is displayed as spheres. Note that Nb39 is not illustrated for simplicity. Morph between the inactive and active conformations was generated using Chimera (Pettersen, E,F., et al. UCSF Chimera--a visualization system for exploratory research and analysis. J Comput Chem. 2004 25, 1605-12) and rendered with Pymol (The PyMOL Molecular Graphics System, Schrödinger, LLC).

  4. 4.

    Activation of the M2 muscarinic receptor, M2R

    Top view of the ribbon structure of the M2 muscarinic receptor (M2R) in its inactive conformation bound to antagonist 3-quinuclidinyl benzilate, (QNB, PDB: 3UON) and the M2R in its active conformation bound to agonist iperoxo and nanobody Nb9-8 (PDB: 4MQS). Illustrated are sidechain residues Y1043.33, Y4036.38 and Y4267.39 to highlight the ‘lid-like’ structure over the orthosteric site. Acetylcholine is modeled into the iperoxo binding site and illustrated in stick figure for reference purposes. Note that Nb9-8 is not depicted for simplicity. Morphs were generated between the inactive and active conformations using Chimera (Pettersen, E,F., et al. UCSF Chimera--a visualization system for exploratory research and analysis. J Comput Chem. 2004 25, 1605-12) and rendered with Pymol (The PyMOL Molecular Graphics System, Schrödinger, LLC).

  5. 5.

    Activation of the M2R side view

    Side view of the ribbon structure of the M2R (above) to highlight the ‘lid-like’ structure over the orthosteric site. Illustrated are side chain residues Y4036.38 and Y4267.39 moving toward Y1043.33 during the formation of the active conformation. Note that TM5 was removed from the rendering so that the tyrosine residues may be easily viewed. Morphs were generated between the inactive and active conformations using Chimera (Pettersen, E,F., et al. UCSF Chimera--a visualization system for exploratory research and analysis. J Comput Chem. 2004 25, 1605-12) and rendered with Pymol (The PyMOL Molecular Graphics System, Schrödinger, LLC).

  6. 6.

    Stabilization of the active state of rhodopsin by G protein

    Top view of the ribbon structure of bovine rhodopsin in its inactive conformation (PDB:1F88) (Palczewski, K. et al. Crystal structure of rhodopsin: A G protein-coupled receptor Science 289: 739-745 (2000)) and the photoactivated meta-stable form of rhodopsin bound to the C-terminal fragment of the G protein alpha subunit, transducin (transducin not shown) (PDB:3PAR) (Choe H. W. et al. Crystal structure of metarhodopsin II. Nature 471, 651–655 (2011)). Note the pre-existing ‘lid-like’ structure over the orthosteric site formed by the ECL2 and N-terminus. Photoisomerization of 11-cis retinal is illustrated in magenta. Morphs were generated between the inactive and active conformations using Chimera (Pettersen, E,F., et al. UCSF Chimera--a visualization system for exploratory research and analysis. J Comput Chem. 2004 25, 1605-12) and rendered with Pymol (The PyMOL Molecular Graphics System, Schrödinger, LLC).

  7. 7.

    Stabilization of the active state of rhodopsin by arrestin

    Top view of the ribbon structure of bovine rhodopsin in its inactive conformation (PDB:1F88) (Palczewski, K. et al. Crystal structure of rhodopsin: A G protein-coupled receptor Science 289: 739-745 (2000)) and an active mutant of opsin bound to activated arrestin (arrestin not shown) (PDB:4ZWJ) (Kang, Y. et al. Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser. Nature 523, 561-567 (2015)). Note the similarities in conformational changes as with metarhodopsin bound to the C-terminal helix of transducin. Photoisomerization of 11-cis retinal, based on the metarhodopsin structures (Extended data: Movie SM6) has been modeled into the opsin-arrestin structure and is illustrated in magenta. Morphs were generated between inactive and arrestin-bound conformations of rhodopsin using Chimera (Pettersen, E,F., et al. UCSF Chimera--a visualization system for exploratory research and analysis. J Comput Chem. 2004 25, 1605-12.) and rendered with Pymol (The PyMOL Molecular Graphics System, Schrödinger, LLC).

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nature18324

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.