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:

Conformationally selective RNA aptamers allosterically modulate the β2-adrenoceptor

Abstract

G-protein-coupled receptor (GPCR) ligands function by stabilizing multiple, functionally distinct receptor conformations. This property underlies the ability of 'biased agonists' to activate specific subsets of a given receptor's signaling profile. However, stabilizing distinct active GPCR conformations to enable structural characterization of mechanisms underlying GPCR activation remains difficult. These challenges have accentuated the need for receptor tools that allosterically stabilize and regulate receptor function through unique, previously unappreciated mechanisms. Here, using a highly diverse RNA library combined with advanced selection strategies involving state-of-the-art next-generation sequencing and bioinformatics analyses, we identify RNA aptamers that bind a prototypical GPCR, the β2-adrenoceptor (β2AR). Using biochemical, pharmacological, and biophysical approaches, we demonstrate that these aptamers bind with nanomolar affinity at defined surfaces of the receptor, allosterically stabilizing active, inactive, and ligand-specific receptor conformations. The discovery of RNA aptamers as allosteric GPCR modulators significantly expands the diversity of ligands available to study the structural and functional regulation of GPCRs.

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: Generation of conformation-specific RNA aptamers against the β2AR.
Figure 2: Aptamers distinguish between inactive and active conformations of the β2AR.
Figure 3: Selectivity of aptamers for specific β2AR conformations correlates with receptor ligand efficacy or ligand specificity.
Figure 4: Influence of aptamers on conformational changes conferred by ligands.
Figure 5: Functional effect of aptamers on β2AR-mediated activation of Gαs and adenylyl cyclase.
Figure 6: EM analysis and molecular architecture of β2AR–aptamer complexes.

Similar content being viewed by others

Accession codes

Accessions

Protein Data Bank

References

  1. Pierce, K.L., Premont, R.T. & Lefkowitz, R.J. Seven-transmembrane receptors. Nat. Rev. Mol. Cell Biol. 3, 639–650 (2002).

    CAS  PubMed  Google Scholar 

  2. Lefkowitz, R.J. A brief history of G-protein coupled receptors (Nobel Lecture). Angew. Chem. Int. Edn. Engl. 52, 6366–6378 (2013).

    CAS  Google Scholar 

  3. Lagerström, M.C. & Schiöth, H.B. Structural diversity of G protein-coupled receptors and significance for drug discovery. Nat. Rev. Drug Discov. 7, 339–357 (2008).

    PubMed  Google Scholar 

  4. Kobilka, B.K. Amino and carboxyl terminal modifications to facilitate the production and purification of a G protein-coupled receptor. Anal. Biochem. 231, 269–271 (1995).

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Violin, J.D., Crombie, A.L., Soergel, D.G. & Lark, M.W. Biased ligands at G-protein-coupled receptors: promise and progress. Trends Pharmacol. Sci. 35, 308–316 (2014).

    CAS  PubMed  Google Scholar 

  7. Soergel, D.G., Subach, R.A., Cowan, C.L., Violin, J.D. & Lark, M.W. First clinical experience with TRV027: pharmacokinetics and pharmacodynamics in healthy volunteers. J. Clin. Pharmacol. 53, 892–899 (2013).

    CAS  PubMed  Google Scholar 

  8. Soergel, D.G. et al. Biased agonism of the μ-opioid receptor by TRV130 increases analgesia and reduces on-target adverse effects versus morphine: a randomized, double-blind, placebo-controlled, crossover study in healthy volunteers. Pain 155, 1829–1835 (2014).

    CAS  PubMed  Google Scholar 

  9. Kahsai, A.W. et al. Multiple ligand-specific conformations of the β2-adrenergic receptor. Nat. Chem. Biol. 7, 692–700 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Liu, J.J., Horst, R., Katritch, V., Stevens, R.C. & Wüthrich, K. Biased signaling pathways in β2-adrenergic receptor characterized by 19F-NMR. Science 335, 1106–1110 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Manglik, A. et al. Structural insights into the dynamic process of β2-adrenergic receptor signaling. Cell 161, 1101–1111 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Granier, S. & Kobilka, B. A new era of GPCR structural and chemical biology. Nat. Chem. Biol. 8, 670–673 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Ghosh, E., Kumari, P., Jaiman, D. & Shukla, A.K. Methodological advances: the unsung heroes of the GPCR structural revolution. Nat. Rev. Mol. Cell Biol. 16, 69–81 (2015).

    CAS  PubMed  Google Scholar 

  16. Kobilka, B. The structural basis of G-protein-coupled receptor signaling (Nobel Lecture). Angew. Chem. Int. Edn. Engl. 52, 6380–6388 (2013).

    CAS  Google Scholar 

  17. Weichert, D. et al. Covalent agonists for studying G protein-coupled receptor activation. Proc. Natl. Acad. Sci. USA 111, 10744–10748 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Rosenbaum, D.M. et al. Structure and function of an irreversible agonist-β(2) adrenoceptor complex. Nature 469, 236–240 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Que-Gewirth, N.S. & Sullenger, B.A. Gene therapy progress and prospects: RNA aptamers. Gene Ther. 14, 283–291 (2007).

    CAS  PubMed  Google Scholar 

  20. Ng, E.W. et al. Pegaptanib, a targeted anti-VEGF aptamer for ocular vascular disease. Nat. Rev. Drug Discov. 5, 123–132 (2006).

    CAS  PubMed  Google Scholar 

  21. Rusconi, C.P. et al. RNA aptamers as reversible antagonists of coagulation factor IXa. Nature 419, 90–94 (2002).

    CAS  PubMed  Google Scholar 

  22. Lincoff, A.M. et al. REGULATE-PCI Investigators. Effect of the REG1 anticoagulation system versus bivalirudin on outcomes after percutaneous coronary intervention (REGULATE-PCI): a randomised clinical trial. Lancet 387, 349–356 (2016).

    CAS  PubMed  Google Scholar 

  23. Ratner, M. Next-generation AMD drugs to wed blockbusters. Nat. Biotechnol. 32, 701–702 (2014).

    CAS  PubMed  Google Scholar 

  24. Zhou, J. et al. Cell-specific RNA aptamer against human CCR5 specifically targets HIV-1 susceptible cells and inhibits HIV-1 infectivity. Chem. Biol. 22, 379–390 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Daniels, D.A., Sohal, A.K., Rees, S. & Grisshammer, R. Generation of RNA aptamers to the G-protein-coupled receptor for neurotensin, NTS-1. Anal. Biochem. 305, 214–226 (2002).

    CAS  PubMed  Google Scholar 

  26. Lee, G. et al. RNA based antagonist of NMDA receptors. ACS Chem. Neurosci. 5, 559–567 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Pratico, E.D., Sullenger, B.A. & Nair, S.K. Identification and characterization of an agonistic aptamer against the T cell costimulatory receptor, OX40. Nucleic Acid Ther. 23, 35–43 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Vinkenborg, J.L., Karnowski, N. & Famulok, M. Aptamers for allosteric regulation. Nat. Chem. Biol. 7, 519–527 (2011).

    CAS  PubMed  Google Scholar 

  29. Tesmer, J.J. Crystallographic pursuit of a protein-RNA aptamer complex. Methods Mol. Biol. 1380, 151–160 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Oberthür, D. et al. Crystal structure of a mirror-image L-RNA aptamer (Spiegelmer) in complex with the natural L-protein target CCL2. Nat. Commun. 6, 6923 (2015).

    PubMed  Google Scholar 

  31. Tuerk, C. & Gold, L. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249, 505–510 (1990).

    CAS  PubMed  Google Scholar 

  32. Ellington, A.D. & Szostak, J.W. In vitro selection of RNA molecules that bind specific ligands. Nature 346, 818–822 (1990).

    CAS  PubMed  Google Scholar 

  33. Ozer, A., Pagano, J.M. & Lis, J.T. New technologies provide quantum changes in the scale, speed, and success of SELEX methods and aptamer characterization. Mol. Ther. Nucleic Acids 3, e183 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Shendure, J. & Ji, H. Next-generation DNA sequencing. Nat. Biotechnol. 26, 1135–1145 (2008).

    CAS  PubMed  Google Scholar 

  35. Chae, P.S. et al. Maltose-neopentyl glycol (MNG) amphiphiles for solubilization, stabilization and crystallization of membrane proteins. Nat. Methods 7, 1003–1008 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Wang, J. et al. Synthesis of β2-AR agonist BI-167107. Youji Huaxue 33, 634–639 (2013).

    CAS  Google Scholar 

  37. Whorton, M.R. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Weiss, D.R. et al. Conformation guides molecular efficacy in docking screens of activated β-2 adrenergic G protein coupled receptor. ACS Chem. Biol. 8, 1018–1026 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  40. De Lean, A., Stadel, J.M. & Lefkowitz, R.J. A ternary complex model explains the agonist-specific binding properties of the adenylate cyclase-coupled beta-adrenergic receptor. J. Biol. Chem. 255, 7108–7117 (1980).

    CAS  PubMed  Google Scholar 

  41. Hoffman, B.B. & Lefkowitz, R.J. Adrenergic receptors in the heart. Annu. Rev. Physiol. 44, 475–484 (1982).

    CAS  PubMed  Google Scholar 

  42. Staus, D.P. et al. Regulation of β2-adrenergic receptor function by conformationally selective single-domain intrabodies. Mol. Pharmacol. 85, 472–481 (2014).

    PubMed  PubMed Central  Google Scholar 

  43. Jiang, J. et al. The architecture of Tetrahymena telomerase holoenzyme. Nature 496, 187–192 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Peisley, A. & Skiniotis, G. 2D projection analysis of GPCR complexes by negative stain electron microscopy. Methods Mol. Biol. 1335, 29–38 (2015).

    PubMed  Google Scholar 

  45. Kahsai, A.W., Rajagopal, S., Sun, J. & Xiao, K. Monitoring protein conformational changes and dynamics using stable-isotope labeling and mass spectrometry. Nat. Protoc. 9, 1301–1319 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Steyaert, J. & Kobilka, B.K. Nanobody stabilization of G protein-coupled receptor conformational states. Curr. Opin. Struct. Biol. 21, 567–572 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Hino, T. et al. G-protein-coupled receptor inactivation by an allosteric inverse-agonist antibody. Nature 482, 237–240 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Adams, J.J. & Sidhu, S.S. Synthetic antibody technologies. Curr. Opin. Struct. Biol. 24, 1–9 (2014).

    CAS  PubMed  Google Scholar 

  49. Ivetac, A. & McCammon, J.A. Mapping the druggable allosteric space of G-protein coupled receptors: a fragment-based molecular dynamics approach. Chem. Biol. Drug Des. 76, 201–217 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Wootten, D., Christopoulos, A. & Sexton, P.M. Emerging paradigms in GPCR allostery: implications for drug discovery. Nat. Rev. Drug Discov. 12, 630–644 (2013).

    CAS  PubMed  Google Scholar 

  51. Denisov, I.G., Grinkova, Y.V., Lazarides, A.A. & Sligar, S.G. Directed self-assembly of monodisperse phospholipid bilayer nanodiscs with controlled size. J. Am. Chem. Soc. 126, 3477–3487 (2004).

    CAS  PubMed  Google Scholar 

  52. Bompiani, K.M., Monroe, D.M., Church, F.C. & Sullenger, B.A. A high affinity, antidote-controllable prothrombin and thrombin-binding RNA aptamer inhibits thrombin generation and thrombin activity. J. Thromb. Haemost. 10, 870–880 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Shenoy, S.K. et al. beta-arrestin-dependent, G protein-independent ERK1/2 activation by the beta2 adrenergic receptor. J. Biol. Chem. 281, 1261–1273 (2006).

    CAS  PubMed  Google Scholar 

  54. Williams, R.J. & Kelly, E. Measurement of adenylyl cyclase activity in cell membranes. Methods Mol. Biol. 41, 63–77 (1995).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

R.J.L. is an investigator with the Howard Hughes Medical Institute (HHMI). This work was supported in part by grants from the US National Institutes of Health to R.J.L. (HL16037) and to B.A.S. (R01HL65222). We gratefully acknowledge B. Kobilka (Stanford University, Stanford, CA) and G. Skiniotis (University of Michigan, Ann Arbor, Michigan) for stimulating ideas and helpful discussions; we are also grateful to S. Rajagopal and J.C. Snyder of Duke University for discussions and critical reading of the manuscript; we thank O. Fedrigo and N. Hoang at the Genome Sequencing and Analysis Core Resource (Duke University) for library preparation support and quality control analysis and for performing next-generation DNA sequencing; we also acknowledge the use of transmission electron microscopy at the Shared Materials Instrumentation Facility (Duke University). T.J.C. is supported by an NHLBI grant of the National Institutes of Health (F30HL129803). We also thank X. Chen (Changzhou University, Jiangsu, China) for the supply of BI167107; A.K. Shukla (IIT, Kanpur, India), E. Pratico (Duke University), J. Kim (Duke University), and K. Xiao (University of Pittsburg) for valuable assistance with reagents; X. Jiang and W. Capel for excellent technical assistance; and D. Addison and Q. Lennon for secretarial assistance.

Author information

Authors and Affiliations

Authors

Contributions

A.W.K., J.W.W., S.A., B.A.S., and R.J.L. conceived the study. A.W.K., J.W.W., K.M.B., and H.D. performed selection and enrichment analysis. A.W.K., J.W.W., L.M.W., B.A.S., and R.J.L. designed NGS strategies. A.W.K., J.L., and H.D. performed Illumina-NGS library construction, preparation, and quality control analysis. A.W.K., J.L., H.D., X.Q., and L.M.W. participated in writing custom scripts and NGS data analysis. A.W.K. and J.L. performed aptamer synthesis, biotinylation, and fluorescence studies. S.M.D., K.M.A., and S.M.A. conducted BLI experiments. A.W.K., J.W.W., J.L., D.P.S., B.P., A.R.B.T., H.D., and R.T.S. participated in binding studies, receptor functionality tests, and reconstitution in HDL particles. S.A. conducted functional experiments. T.J.C. and A.R.B.T., with assistance from A.W.K. and J.L., performed EM imaging and particle analysis. A.W.K., B.A.S., and R.J.L. wrote the manuscript. All authors contributed to the preparation and editing of the manuscript.

Corresponding author

Correspondence to Robert J Lefkowitz.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Figures 1–9 and Supplementary Table 1. (PDF 1333 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kahsai, A., Wisler, J., Lee, J. et al. Conformationally selective RNA aptamers allosterically modulate the β2-adrenoceptor. Nat Chem Biol 12, 709–716 (2016). https://doi.org/10.1038/nchembio.2126

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.2126

This article is cited by

Search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research