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Molecular control of δ-opioid receptor signalling

Nature volume 506, pages 191196 (13 February 2014) | Download Citation


Opioids represent widely prescribed and abused medications, although their signal transduction mechanisms are not well understood. Here we present the 1.8 Å high-resolution crystal structure of the human δ-opioid receptor (δ-OR), revealing the presence and fundamental role of a sodium ion in mediating allosteric control of receptor functional selectivity and constitutive activity. The distinctive δ-OR sodium ion site architecture is centrally located in a polar interaction network in the seven-transmembrane bundle core, with the sodium ion stabilizing a reduced agonist affinity state, and thereby modulating signal transduction. Site-directed mutagenesis and functional studies reveal that changing the allosteric sodium site residue Asn 131 to an alanine or a valine augments constitutive β-arrestin-mediated signalling. Asp95Ala, Asn310Ala and Asn314Ala mutations transform classical δ-opioid antagonists such as naltrindole into potent β-arrestin-biased agonists. The data establish the molecular basis for allosteric sodium ion control in opioid signalling, revealing that sodium-coordinating residues act as ‘efficacy switches’ at a prototypic G-protein-coupled receptor.

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Protein Data Bank

Data deposits

The coordinates and the structure factors have been deposited in the Protein Data Bank under accession code 4N6H.


  1. 1.

    Opioids and their receptors: are we there yet? Neuropharmacology 76, 198–203 (2014)

  2. 2.

    , & Structure-function of the G protein-coupled receptor superfamily. Annu. Rev. Pharmacol. Toxicol. 53, 531–556 (2013)

  3. 3.

    , & Emerging paradigms in GPCR allostery: implications for drug discovery. Nature Rev. Drug Discov. 12, 630–644 (2013)

  4. 4.

    , & The structure and function of G-protein-coupled receptors. Nature 459, 356–363 (2009)

  5. 5.

    , & Opiate agonists and antagonists discriminated by receptor binding in brain. Science 182, 1359–1361 (1973)

  6. 6.

    , , & Opiate receptor-mediated inhibition of adenylate cyclase in rat striatal plasma membranes. J. Neurochem. 38, 1164–1167 (1982)

  7. 7.

    , , & Application of the message-address concept in the design of highly potent and selective non-peptide δ opioid receptor antagonists. J. Med. Chem. 31, 281–282 (1988)

  8. 8.

    et al. Structure of the δ-opioid receptor bound to naltrindole. Nature 485, 400–404 (2012)

  9. 9.

    & Integrated methods for the construction of three-dimensional models and computational probing of structure-function relations in G protein-coupled receptors. Methods Neurosci. 25, 366–428 (1995)

  10. 10.

    et al. Structure of the adenosine A(2A) receptor in complex with ZM241385 and the xanthines XAC and caffeine. Structure 19, 1283–1293 (2011)

  11. 11.

    , , , & Crystal structure of the ligand-free G-protein-coupled receptor opsin. Nature 454, 183–187 (2008)

  12. 12.

    et al. Structure of the nociceptin/orphanin FQ receptor in complex with a peptide mimetic. Nature 485, 395–399 (2012)

  13. 13.

    , & Selectivity of μ-opioid receptor determined by interfacial residues near third extracellular loop. Eur. J. Pharmacol. 403, 37–44 (2000)

  14. 14.

    & Grand opening of structure-guided design for novel opioids. Trends Pharmacol. Sci. 34, 6–12 (2013)

  15. 15.

    et al. Discovery of positive allosteric modulators and silent allosteric modulators of the μ-opioid receptor. Proc. Natl Acad. Sci. USA 110, 10830–10835 (2013)

  16. 16.

    et al. Structural basis for allosteric regulation of GPCRs by sodium ions. Science 337, 232–236 (2012)

  17. 17.

    & Restructuring G-protein-coupled receptor activation. Cell 151, 14–23 (2012)

  18. 18.

    et al. Structure of an agonist-bound human A2A adenosine receptor. Science 332, 322–327 (2011)

  19. 19.

    , , & Effects of sodium on agonist efficacy for G-protein activation in μ-opioid receptor-transfected CHO cells and rat thalamus. Br. J. Pharmacol. 130, 987–996 (2000)

  20. 20.

    & Regulation of μ-opioid receptor in neural cells by extracellular sodium. J. Neurochem. 68, 1053–1061 (1997)

  21. 21.

    et al. An aspartate conserved among G-protein receptors confers allosteric regulation of alpha 2-adrenergic receptors by sodium. J. Biol. Chem. 265, 21590–21595 (1990)

  22. 22.

    , , & Spontaneous association between opioid receptors and GTP-binding regulatory proteins in native membranes: specific regulation by antagonists and sodium ions. Mol. Pharmacol. 37, 383–394 (1990)

  23. 23.

    & Allosteric modulation of A(2A) adenosine receptors by amiloride analogues and sodium ions. Biochem. Pharmacol. 60, 669–676 (2000)

  24. 24.

    Regulation of dopamine D2 receptors by sodium and pH. Mol. Pharmacol. 39, 570–578 (1991)

  25. 25.

    & G protein-coupled receptor allosterism and complexing. Pharmacol. Rev. 54, 323–374 (2002)

  26. 26.

    , , , & BW373U86: a nonpeptidic delta-opioid agonist with novel receptor-G protein-mediated actions in rat brain membranes and neuroblastoma cells. Mol. Pharmacol. 44, 827–834 (1993)

  27. 27.

    et al. Identification of an efficacy switch region in the ghrelin receptor responsible for interchange between agonism and inverse agonism. J. Biol. Chem. 282, 15799–15811 (2007)

  28. 28.

    et al. Biased and constitutive signaling in the CC-chemokine receptor CCR5 by manipulating the interface between transmembrane helices 6 and 7. J. Biol. Chem. 288, 12511–12521 (2013)

  29. 29.

    & Delta opioid modulation of the binding of guanosine-5′-O-(3-[35S]thio)triphosphate to NG108–15 cell membranes: characterization of agonist and inverse agonist effects. J. Pharmacol. Exp. Ther. 283, 1276–1284 (1997)

  30. 30.

    & Chronic agonist treatment converts antagonists into inverse agonists at δ-opioid receptors. J. Pharmacol. Exp. Ther. 302, 1070–1079 (2002)

  31. 31.

    et al. Redesign of a four-helix bundle protein by phage display coupled with proteolysis and structural characterization by NMR and X-ray crystallography. J. Mol. Biol. 323, 253–262 (2002)

  32. 32.

    & Gene splicing and mutagenesis by PCR-driven overlap extension. Nature Protocols 2, 924–932 (2007)

  33. 33.

    et al. Structures of the CXCR4 chemokine GPCR with small-molecule and cyclic peptide antagonists. Science 330, 1066–1071 (2010)

  34. 34.

    et al. Conserved binding mode of human β2 adrenergic receptor inverse agonists and antagonist revealed by X-ray crystallography. J. Am. Chem. Soc. 132, 11443–11445 (2010)

  35. 35.

    & Crystallizing membrane proteins using lipidic mesophases. Nature Protocols 4, 706–731 (2009)

  36. 36.

    , , , & A robotic system for crystallizing membrane and soluble proteins in lipidic mesophases. Acta Crystallogr. D 60, 1795–1807 (2004)

  37. 37.

    & Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997)

  38. 38.

    et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007)

  39. 39.

    , , & Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010)

  40. 40.

    , & Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D 53, 240–255 (1997)

  41. 41.

    et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010)

  42. 42.

    et al. The genetic design of signaling cascades to record receptor activation. Proc. Natl Acad. Sci. USA 105, 64–69 (2008)

  43. 43.

    et al. Discovery of β-arrestin-biased dopamine D2 ligands for probing signal transduction pathways essential for antipsychotic efficacy. Proc. Natl Acad. Sci. USA 108, 18488–18493 (2011)

  44. 44.

    et al. Ligand discovery from a dopamine D3 receptor homology model and crystal structure. Nature Chem. Biol. 7, 769–778 (2011)

  45. 45.

    et al. Structural features for functional selectivity at serotonin receptors. Science 340, 615–619 (2013)

  46. 46.

    et al. Automated design of ligands to polypharmacological profiles. Nature 492, 215–220 (2012)

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This work was supported by the National Institutes of Health Common Fund grant P50 GM073197 for technology development (V.C. and R.C.S.), PSI:Biology grant U54 GM094618 for biological studies and structure production (target GPCR-39) (V.K., V.C. and R.C.S.), R01 DA017204 and the NIMH Psychoactive Drug Screening Program (P.G., X.-P.H., B.L.R.) and the Michael Hooker Chair for Protein Therapeutics and Translational Proteomics to B.L.R. We thank J. Velasquez for help with molecular biology, T. Trinh and M. Chu for help with baculovirus expression, G.W. Han for help with structure analysis and quality control review, E. Abola for help with sodium site analysis, A. Walker for assistance with manuscript preparation and J. Smith, R. Fischetti and N. Sanishvili for assistance in development and use of the minibeam and beamtime at beamline 23-ID at the Advanced Photon Source, which is supported by National Cancer Institute grant Y1-CO-1020 and National Institute of General Medical Sciences grant Y1-GM-1104.

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Author notes

    • Gustavo Fenalti
    •  & Patrick M. Giguere

    These authors contributed equally to this work.


  1. Department of Integrative Structural and Computational Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA

    • Gustavo Fenalti
    • , Vsevolod Katritch
    • , Aaron A. Thompson
    • , Vadim Cherezov
    •  & Raymond C. Stevens
  2. National Institute of Mental Health Psychoactive Drug Screening Program and Department of Pharmacology and Division of Chemical Biology and Medicinal Chemistry, University of North Carolina Chapel Hill Medical School, Chapel Hill, North Carolina 27599, USA

    • Patrick M. Giguere
    • , Xi-Ping Huang
    •  & Bryan L. Roth


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G.F. designed, optimized and purified δ-OR receptor constructs for structural studies, crystallized the receptor in LCP, collected and processed diffraction data, determined the structure, analysed the data and wrote the paper. P.M.G. performed mutagenesis and signalling studies, analysed the data and wrote the paper. X.-P.H. performed ligand binding and signalling studies, analysed the data and wrote the paper. V.K. analysed the data and wrote the paper. A.A.T. designed and cloned initial δ-OR constructs. V.C. analysed the data and wrote the paper. B.L.R. supervised the pharmacology and mutagenesis studies, analysed the data and wrote the paper. R.C.S. was responsible for the overall project strategy and management, analysed the data and wrote the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Bryan L. Roth or Raymond C. Stevens.

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