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Multiple ligand-specific conformations of the β2-adrenergic receptor

Nature Chemical Biology volume 7, pages 692700 (2011) | Download Citation

Abstract

Seven-transmembrane receptors (7TMRs), also called G protein–coupled receptors (GPCRs), represent the largest class of drug targets, and they can signal through several distinct mechanisms including those mediated by G proteins and the multifunctional adaptor proteins β-arrestins. Moreover, several receptor ligands with differential efficacies toward these distinct signaling pathways have been identified. However, the structural basis and mechanism underlying this 'biased agonism' remains largely unknown. Here, we develop a quantitative mass spectrometry strategy that measures specific reactivities of individual side chains to investigate dynamic conformational changes in the β2-adrenergic receptor occupied by nine functionally distinct ligands. Unexpectedly, only a minority of residues showed reactivity patterns consistent with classical agonism, whereas the majority showed distinct patterns of reactivity even between functionally similar ligands. These findings demonstrate, contrary to two-state models for receptor activity, that there is significant variability in receptor conformations induced by different ligands, which has significant implications for the design of new therapeutic agents.

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References

  1. 1.

    Seven transmembrane receptors: something old, something new. Acta Physiol. (Oxf.) 190, 9–19 (2007).

  2. 2.

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

  3. 3.

    & Structural diversity of G protein-coupled receptors and significance for drug discovery. Nat. Rev. Drug Discov. 7, 339–357 (2008).

  4. 4.

    , , , & beta-Arrestin: a protein that regulates beta-adrenergic receptor function. Science 248, 1547–1550 (1990).

  5. 5.

    & The role of beta-arrestins in the termination and transduction of G-protein-coupled receptor signals. J. Cell Sci. 115, 455–465 (2002).

  6. 6.

    , , , & Desensitization, internalization, and signaling functions of beta-arrestins demonstrated by RNA interference. Proc. Natl. Acad. Sci. USA 100, 1740–1744 (2003).

  7. 7.

    , , , & Negative antagonists promote an inactive conformation of the beta 2-adrenergic receptor. Mol. Pharmacol. 45, 390–394 (1994).

  8. 8.

    Collateral efficacy in drug discovery: taking advantage of the good (allosteric) nature of 7TM receptors. Trends Pharmacol. Sci. 28, 407–415 (2007).

  9. 9.

    & Transduction of receptor signals by beta-arrestins. Science 308, 512–517 (2005).

  10. 10.

    & Beta-arrestin-biased ligands at seven-transmembrane receptors. Trends Pharmacol. Sci. 28, 416–422 (2007).

  11. 11.

    , & Teaching old receptors new tricks: biasing seven-transmembrane receptors. Nat. Rev. Drug Discov. 9, 373–386 (2010).

  12. 12.

    Functional selectivity and biased receptor signaling. J. Pharmacol. Exp. Ther. 336, 296–302 (2011).

  13. 13.

    et al. Independent beta-arrestin 2 and G protein-mediated pathways for angiotensin II activation of extracellular signal-regulated kinases 1 and 2. Proc. Natl. Acad. Sci. USA 100, 10782–10787 (2003).

  14. 14.

    et al. Distinct beta-arrestin- and G protein-dependent pathways for parathyroid hormone receptor-stimulated ERK1/2 activation. J. Biol. Chem. 281, 10856–10864 (2006).

  15. 15.

    et al. A unique mechanism of beta-blocker action: carvedilol stimulates beta-arrestin signaling. Proc. Natl. Acad. Sci. USA 104, 16657–16662 (2007).

  16. 16.

    et al. Distinct conformational changes in beta-arrestin report biased agonism at seven-transmembrane receptors. Proc. Natl. Acad. Sci. USA 105, 9988–9993 (2008).

  17. 17.

    et al. Global phosphorylation analysis of beta-arrestin-mediated signaling downstream of a seven transmembrane receptor (7TMR). Proc. Natl. Acad. Sci. USA 107, 15299–15304 (2010).

  18. 18.

    et al. A beta-arrestin-biased agonist of the parathyroid hormone receptor (PTH1R) promotes bone formation independent of G protein activation. Sci. Transl. Med. 1, 1ra1 (2009).

  19. 19.

    , & Therapeutic potential of beta-arrestin- and G protein-biased agonists. Trends. Mol. Med. 17, 126–139 (2010).

  20. 20.

    et al. Selectively engaging beta-arrestins at the angiotensin II type 1 receptor reduces blood pressure and increases cardiac performance. J. Pharmacol. Exp. Ther. 335, 572–579 (2010).

  21. 21.

    , , & Differential signaling of the endogenous agonists at the beta2-adrenergic receptor. J. Biol. Chem. 285, 36188–36198 (2010).

  22. 22.

    et al. Coupling ligand structure to specific conformational switches in the beta2-adrenoceptor. Nat. Chem. Biol. 2, 417–422 (2006).

  23. 23.

    et al. Functionally different agonists induce distinct conformations in the G protein coupling domain of the beta 2 adrenergic receptor. J. Biol. Chem. 276, 24433–24436 (2001).

  24. 24.

    et al. Crystal structure of rhodopsin: A G protein-coupled receptor. Science 289, 739–745 (2000).

  25. 25.

    et al. Crystal structure of the human beta2 adrenergic G-protein-coupled receptor. Nature 450, 383–387 (2007).

  26. 26.

    et al. GPCR engineering yields high-resolution structural insights into beta2-adrenergic receptor function. Science 318, 1266–1273 (2007).

  27. 27.

    et al. High-resolution crystal structure of an engineered human beta2-adrenergic G protein-coupled receptor. Science 318, 1258–1265 (2007).

  28. 28.

    et al. Crystal structure of opsin in its G-protein-interacting conformation. Nature 455, 497–502 (2008).

  29. 29.

    et al. Structure of a nanobody-stabilized active state of the beta(2) adrenoceptor. Nature 469, 175–180 (2011).

  30. 30.

    et al. The structural basis for agonist and partial agonist action on a beta(1)-adrenergic receptor. Nature 469, 241–244 (2011).

  31. 31.

    et al. Cloning of the gene and cDNA for mammalian beta-adrenergic receptor and homology with rhodopsin. Nature 321, 75–79 (1986).

  32. 32.

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

  33. 33.

    & Multiple reactive sulfhydryl groups modulate the function of adenylate cyclase coupled beta-adrenergic receptors. Mol. Pharmacol. 16, 709–718 (1979).

  34. 34.

    et al. Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat. Biotechnol. 17, 994–999 (1999).

  35. 35.

    & Mass spectrometry-based proteomics turns quantitative. Nat. Chem. Biol. 1, 252–262 (2005).

  36. 36.

    & The Behavior of Biological Macromolecules##915–916 (Freeman, 1980).

  37. 37.

    & Potassium channel gating observed with site-directed mass tagging. Nat. Struct. Biol. 10, 280–284 (2003).

  38. 38.

    , , , & The conserved seven-transmembrane sequence NP(X)2,3Y of the G-protein-coupled receptor superfamily regulates multiple properties of the beta 2-adrenergic receptor. Biochemistry 34, 15407–15414 (1995).

  39. 39.

    et al. Functional role of the NPxxY motif in internalization of the type 2 vasopressin receptor in LLC-PK1 cells. Am. J. Physiol. Cell Physiol. 285, C750–C762 (2003).

  40. 40.

    et al. Role of the conserved NPxxY(x)5,6F motif in the rhodopsin ground state and during activation. Proc. Natl. Acad. Sci. USA 100, 2290–2295 (2003).

  41. 41.

    et al. Functional role of internal water molecules in rhodopsin revealed by X-ray crystallography. Proc. Natl. Acad. Sci. USA 99, 5982–5987 (2002).

  42. 42.

    et al. A constitutively active mutant beta 2-adrenergic receptor is constitutively desensitized and phosphorylated. Proc. Natl. Acad. Sci. USA 91, 2699–2702 (1994).

  43. 43.

    , , & A mutation-induced activated state of the beta 2-adrenergic receptor. Extending the ternary complex model. J. Biol. Chem. 268, 4625–4636 (1993).

  44. 44.

    et al. Identification of two distinct inactive conformations of the beta2-adrenergic receptor reconciles structural and biochemical observations. Proc. Natl. Acad. Sci. USA 106, 4689–4694 (2009).

  45. 45.

    , , , & A beta-arrestin binding determinant common to the second intracellular loops of rhodopsin family G protein-coupled receptors. J. Biol. Chem. 281, 2932–2938 (2006).

  46. 46.

    , , & The interaction with the cytoplasmic loops of rhodopsin plays a crucial role in arrestin activation and binding. J. Neurochem. 84, 1040–1050 (2003).

  47. 47.

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

  48. 48.

    et al. beta2-adrenergic receptor signaling and desensitization elucidated by quantitative modeling of real time cAMP dynamics. J. Biol. Chem. 283, 2949–2961 (2008).

  49. 49.

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

  50. 50.

    & Operational models of pharmacological agonism. Proc. R. Soc. Lond. B Biol. Sci. 220, 141–162 (1983).

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Acknowledgements

R.J.L. is an investigator with the Howard Hughes Medical Institute. This work was supported in part by grants from the US National Institutes of Health (HL16037 and HL70631) to R.J.L. T.G.O. is supported by a grant from the US National Institute of General Medical Sciences (5RO1GM081666). We gratefully acknowledge T. Haystead (Duke University) and D. Loiselle for valuable assistance with the mass spectrometry experiments, and Theravance, Inc. (South San Francisco, California, USA) for the supply of THRX-144877; we are also grateful to R.T. Strachan, J. Kovacs, R. Dror (D.E. Shaw Research, New York, New York, USA), C.H. Borchers (University of Victoria, Victoria, British Columbia, Canada), I.A. Kaltashov (University of Massachusetts), B. Donald (Duke University) and members of his laboratory for stimulating ideas and helpful discussions; we also thank X. Jiang and C.M. Lam for excellent technical assistance and D. Addison and Q. Lennon for secretarial assistance.

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Author notes

    • Alem W Kahsai
    •  & Kunhong Xiao

    These authors contributed equally to this work.

Affiliations

  1. Department of Medicine, Duke University Medical Center, Durham, North Carolina, USA.

    • Alem W Kahsai
    • , Kunhong Xiao
    • , Sudarshan Rajagopal
    • , Seungkirl Ahn
    • , Arun K Shukla
    • , Jinpeng Sun
    •  & Robert J Lefkowitz
  2. Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina, USA.

    • Alem W Kahsai
    • , Arun K Shukla
    •  & Robert J Lefkowitz
  3. Department of Biochemistry, Duke University Medical Center, Durham, North Carolina, USA.

    • Terrence G Oas
    •  & Robert J Lefkowitz

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Contributions

A.W.K., K.X., T.G.O. and R.J.L. designed the experiments; A.W.K., K.X., S.A. and A.K.S. conducted experiments; A.W.K., K.X., S.R., S.A., A.K.S., J.S., T.G.O. and R.J.L. analyzed data; A.W.K., K.X. and R.J.L. wrote the paper; all authors read, edited and discussed the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Kunhong Xiao or Robert J Lefkowitz.

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https://doi.org/10.1038/nchembio.634

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