Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

A biased ligand for OXE-R uncouples Gα and Gβγ signaling within a heterotrimer

Abstract

Differential targeting of heterotrimeric G protein versus β-arrestin signaling are emerging concepts in G protein–coupled receptor (GPCR) research and drug discovery, and biased engagement by GPCR ligands of either β-arrestin or G protein pathways has been disclosed. Herein we report on a new mechanism of ligand bias to titrate the signaling specificity of a cell-surface GPCR. Using a combination of biomolecular and virtual screening, we identified the small-molecule modulator Gue1654, which inhibits Gβγ but not Gα signaling triggered upon activation of Gαi-βγ by the chemoattractant receptor OXE-R in both recombinant and human primary cells. Gue1654 does not interfere nonspecifically with signaling directly at or downstream of Gβγ. This hitherto unappreciated mechanism of ligand bias at a GPCR highlights both a new paradigm for functional selectivity and a potentially new strategy to develop pathway-specific therapeutics.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Gue1654 is a functional antagonist of OXE-R in human recombinant and primary cells.
Figure 2: Gue1654 inhibits Gα16- but not Gαi-dependent signaling of OXE-R in recombinant and primary cells.
Figure 3: Gue1654 is an efficacious antagonist of 5-oxo-ETE–dependent Ca2+ flux in human neutrophils and eosinophils.
Figure 4: Gue1654 is a specific inhibitor of OXE-R–Gβγ signaling but does not interfere nonspecifically with signaling at or downstream of Gβγ.
Figure 5: Gue1654 spatially separates OXE-R from the Gβγ complex.

Similar content being viewed by others

Accession codes

Accessions

Protein Data Bank

References

  1. Rosenbaum, D.M., Rasmussen, S.G.F. & Kobilka, B.K. The structure and function of G-protein-coupled receptors. Nature 459, 356–363 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Kenakin, T. & Miller, L.J. Seven transmembrane receptors as shapeshifting proteins: the impact of allosteric modulation and functional selectivity on new drug discovery. Pharmacol. Rev. 62, 265–304 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Overington, J.P., Al-Lazikani, B. & Hopkins, A.L. How many drug targets are there? Nat. Rev. Drug Discov. 5, 993–996 (2006).

    Article  CAS  PubMed  Google Scholar 

  7. Bourne, H.R. How receptors talk to trimeric G proteins. Curr. Opin. Cell Biol. 9, 134–142 (1997).

    Article  CAS  PubMed  Google Scholar 

  8. Rajagopal, S., Rajagopal, K. & Lefkowitz, R.J. Teaching old receptors new tricks: biasing seven-transmembrane receptors. Nat. Rev. Drug Discov. 9, 373–386 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Whalen, E.J., Rajagopal, S. & Lefkowitz, R.J. Therapeutic potential of β-arrestin- and G protein-biased agonists. Trends Mol. Med. 17, 126–139 (2011).

    Article  CAS  PubMed  Google Scholar 

  10. Azzi, M. et al. β-arrestin–mediated activation of MAPK by inverse agonists reveals distinct active conformations for G protein–coupled receptors. Proc. Natl. Acad. Sci. USA 100, 11406–11411 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Bosier, B. & Hermans, E. Versatility of GPCR recognition by drugs: from biological implications to therapeutic relevance. Trends Pharmacol. Sci. 28, 438–446 (2007).

    Article  CAS  PubMed  Google Scholar 

  12. Kenakin, T.P. Cellular assays as portals to seven-transmembrane receptor–based drug discovery. Nat. Rev. Drug Discov. 8, 617–626 (2009).

    Article  CAS  PubMed  Google Scholar 

  13. Smith, N.J., Bennett, K.A. & Milligan, G. When simple agonism is not enough: emerging modalities of GPCR ligands. Mol. Cell Endocrinol. 331, 241–247 (2011).

    Article  CAS  PubMed  Google Scholar 

  14. Urban, J.D. et al. Functional selectivity and classical concepts of quantitative pharmacology. J. Pharmacol. Exp. Ther. 320, 1–13 (2007).

    Article  CAS  PubMed  Google Scholar 

  15. Kendall, R.T. et al. The β-arrestin pathway–selective type 1A angiotensin receptor (AT1A) agonist [Sar1,Ile4,Ile8]angiotensin II regulates a robust G protein–independent signaling network. J. Biol. Chem. 286, 19880–19891 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Antony, J. et al. Dualsteric GPCR targeting: a novel route to binding and signaling pathway selectivity. FASEB J. 23, 442–450 (2009).

    Article  CAS  PubMed  Google Scholar 

  17. Mathiesen, J.M. et al. Identification of indole derivatives exclusively interfering with a G protein–independent signaling pathway of the prostaglandin D2 receptor CRTH2. Mol. Pharmacol. 68, 393–402 (2005).

    CAS  PubMed  Google Scholar 

  18. Galandrin, S., Oligny-Longpré, G. & Bouvier, M. The evasive nature of drug efficacy: implications for drug discovery. Trends Pharmacol. Sci. 28, 423–430 (2007).

    Article  CAS  PubMed  Google Scholar 

  19. Dowal, L. et al. Identification of an antithrombotic allosteric modulator that acts through helix 8 of PAR1. Proc. Natl. Acad. Sci. USA 108, 2951–2956 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Baker, J.G. & Hill, S.J. Multiple GPCR conformations and signalling pathways: implications for antagonist affinity estimates. Trends Pharmacol. Sci. 28, 374–381 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Maillet, E.L. et al. A novel, conformation-specific allosteric inhibitor of the tachykinin NK2 receptor (NK2R) with functionally selective properties. FASEB J. 21, 2124–2134 (2007).

    Article  CAS  PubMed  Google Scholar 

  22. Hosoi, T. et al. Identification of a novel human eicosanoid receptor coupled to G(i/o). J. Biol. Chem. 277, 31459–31465 (2002).

    Article  CAS  PubMed  Google Scholar 

  23. Jones, C.E. et al. Expression and characterization of a 5-oxo-6E,8Z,11Z,14Z-eicosatetraenoic acid receptor highly expressed on human eosinophils and neutrophils. Mol. Pharmacol. 63, 471–477 (2003).

    Article  CAS  PubMed  Google Scholar 

  24. Grant, G.E., Rokach, J. & Powell, W.S. 5-Oxo-ETE and the OXE receptor. Prostaglandins Other Lipid Mediat. 89, 98–104 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Irwin, J.J. & Shoichet, B.K. ZINC—a free database of commercially available compounds for virtual screening. J. Chem. Inf. Model. 45, 177–182 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Trott, O. & Olson, A.J. AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J. Comput. Chem. 31, 455–461 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. O'Flaherty, J.T., Taylor, J.S. & Kuroki, M. The coupling of 5-oxo-eicosanoid receptors to heterotrimeric G proteins. J. Immunol. 164, 3345–3352 (2000).

    Article  CAS  PubMed  Google Scholar 

  28. Wu, D., Huang, C.K. & Jiang, H.P. Roles of phospholipid signaling in chemoattractant-induced responses. J. Cell Sci. 113, 2935–2940 (2000).

    CAS  PubMed  Google Scholar 

  29. Jiang, H. et al. Pertussis toxin–sensitive activation of phospholipase C by the C5a and fMet-Leu-Phe receptors. J. Biol. Chem. 271, 13430–13434 (1996).

    Article  CAS  PubMed  Google Scholar 

  30. Smrcka, A.V. G protein βγ subunits: central mediators of G protein–coupled receptor signaling. Cell. Mol. Life Sci. 65, 2191–2214 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Jiang, H. et al. Roles of phospholipase C β2 in chemoattractant-elicited responses. Proc. Natl. Acad. Sci. USA 94, 7971–7975 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Li, Z. et al. Roles of PLC-β2 and -β3 and PI3Kγ in chemoattractant-mediated signal transduction. Science 287, 1046–1049 (2000).

    Article  CAS  PubMed  Google Scholar 

  33. Bonacci, T.M. et al. Differential targeting of Gβγ-subunit signaling with small molecules. Science 312, 443–446 (2006).

    Article  CAS  PubMed  Google Scholar 

  34. Logothetis, D.E., Kurachi, Y., Galper, J., Neer, E.J. & Clapham, D.E. The βγ subunits of GTP-binding proteins activate the muscarinic K+ channel in heart. Nature 325, 321–326 (1987).

    Article  CAS  PubMed  Google Scholar 

  35. Sadja, R., Alagem, N. & Reuveny, E. Gating of GIRK channels: details of an intricate, membrane-delimited signaling complex. Neuron 39, 9–12 (2003).

    Article  CAS  PubMed  Google Scholar 

  36. Niswender, C.M. et al. A novel assay of Gi/o-linked G protein–coupled receptor coupling to potassium channels provides new insights into the pharmacology of the group III metabotropic glutamate receptors. Mol. Pharmacol. 73, 1213–1224 (2008).

    Article  CAS  PubMed  Google Scholar 

  37. Galés, C. et al. Probing the activation-promoted structural rearrangements in preassembled receptor-G protein complexes. Nat. Struct. Mol. Biol. 13, 778–786 (2006).

    Article  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Galés, C. et al. Real-time monitoring of receptor and G-protein interactions in living cells. Nat. Methods 2, 177–184 (2005).

    Article  PubMed  Google Scholar 

  40. Bünemann, M., Frank, M. & Lohse, M.J. Gi protein activation in intact cells involves subunit rearrangement rather than dissociation. Proc. Natl. Acad. Sci. USA 100, 16077–16082 (2003).

    Article  PubMed  PubMed Central  Google Scholar 

  41. Cherfils, J. & Chabre, M. Activation of G-protein Gα subunits by receptors through Gα-Gβ and Gα-Gγ interactions. Trends Biochem. Sci. 28, 13–17 (2003).

    Article  CAS  PubMed  Google Scholar 

  42. Schröder, R. et al. Deconvolution of complex G protein–coupled receptor signaling in live cells using dynamic mass redistribution measurements. Nat. Biotechnol. 28, 943–949 (2010).

    Article  PubMed  Google Scholar 

  43. Christiansen, E. et al. Discovery of potent and selective agonists for the free fatty acid receptor 1 (FFA(1)/GPR40), a potential target for the treatment of type II diabetes. J. Med. Chem. 51, 7061–7064 (2008).

    Article  CAS  PubMed  Google Scholar 

  44. Schröder, R. et al. The C-terminal tail of CRTH2 is a key molecular determinant that constrains Gαi and downstream signaling cascade activation. J. Biol. Chem. 284, 1324–1336 (2009).

    Article  PubMed  Google Scholar 

  45. Schmidt, J. et al. Selective orthosteric free fatty acid receptor 2 (FFA2) agonists: identification of the structural and chemical requirements for selective activation of FFA2 versus FFA3. J. Biol. Chem. 286, 10628–10640 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Schröder, R. et al. Applying label-free dynamic mass redistribution technology to frame signaling of G protein-coupled receptors noninvasively in living cells. Nat. Protoc. 6, 1748–1760 (2011).

    Article  PubMed  Google Scholar 

  47. Hartnell, A. et al. Identification of selective basophil chemoattractants in human nasal polyps as insulin-like growth factor-1 and insulin-like growth factor-2. J. Immunol. 173, 6448–6457 (2004).

    Article  CAS  PubMed  Google Scholar 

  48. Schuligoi, R. et al. PGD2 metabolism in plasma: kinetics and relationship with bioactivity on DP1 and CRTH2 receptors. Biochem. Pharmacol. 74, 107–117 (2007).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank U. Rick and M. Vasmer-Ehses for excellent technical assistance and Corning Inc. for their support on the Epic system. C.D.W. acknowledges E. Days and Y. Du for valuable technical assistance with cell culture and transient transfections. All thallium flux assays were performed in the Vanderbilt Institute of Chemical Biology High-Throughput Screening Facility. This work was funded by a grant from the Austrian Science Funds FWF (P-22521 to A.H.), a grant from the Spanish Ministerio de Ciencia e Innovación (MICINN, SAF2010-22198-C01-02 to L. Pardo), a L'Oreal Fellowship to P.L. and a fellowship of the German Research Foundation (Graduate College 804) to P.A.O.

Author information

Authors and Affiliations

Authors

Contributions

S.B. designed and performed experiments and provided important ideas. L. Peters, P.A.O., A.B., V.K., C.D.W., R.S., P.L., J.G. and S.H. designed and performed experiments. A.G. and L. Pardo created the receptor model, performed the virtual screening and contributed to discussion. R.T. and T.U. established synthesis of 5-oxo-ETE, edited the manuscript and contributed to discussion. C.D.W., L. Pardo, K.M., M.G. and A.H. designed research, contributed to discussion and edited the manuscript. E.K. designed research and wrote the manuscript.

Corresponding authors

Correspondence to Akos Heinemann or Evi Kostenis.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Methods and Supplementary Results (PDF 1464 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Blättermann, S., Peters, L., Ottersbach, P. et al. A biased ligand for OXE-R uncouples Gα and Gβγ signaling within a heterotrimer. Nat Chem Biol 8, 631–638 (2012). https://doi.org/10.1038/nchembio.962

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.962

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing