Skip to main content

Thank you for visiting 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.

β-Arrestin biosensors reveal a rapid, receptor-dependent activation/deactivation cycle


(β-)Arrestins are important regulators of G-protein-coupled receptors (GPCRs)1,2,3. They bind to active, phosphorylated GPCRs and thereby shut off ‘classical’ signalling to G proteins3,4, trigger internalization of GPCRs via interaction with the clathrin machinery5,6,7 and mediate signalling via ‘non-classical’ pathways1,2. In addition to two visual arrestins that bind to rod and cone photoreceptors (termed arrestin1 and arrestin4), there are only two (non-visual) β-arrestin proteins (β-arrestin1 and β-arrestin2, also termed arrestin2 and arrestin3), which regulate hundreds of different (non-visual) GPCRs. Binding of these proteins to GPCRs usually requires the active form of the receptors plus their phosphorylation by G-protein-coupled receptor kinases (GRKs)1,3,4. The binding of receptors or their carboxy terminus as well as certain truncations induce active conformations of (β-)arrestins that have recently been solved by X-ray crystallography8,9,10. Here we investigate both the interaction of β-arrestin with GPCRs, and the β-arrestin conformational changes in real time and in living human cells, using a series of fluorescence resonance energy transfer (FRET)-based β-arrestin2 biosensors. We observe receptor-specific patterns of conformational changes in β-arrestin2 that occur rapidly after the receptor–β-arrestin2 interaction. After agonist removal, these changes persist for longer than the direct receptor interaction. Our data indicate a rapid, receptor-type-specific, two-step binding and activation process between GPCRs and β-arrestins. They further indicate that β-arrestins remain active after dissociation from receptors, allowing them to remain at the cell surface and presumably signal independently. Thus, GPCRs trigger a rapid, receptor-specific activation/deactivation cycle of β-arrestins, which permits their active signalling.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: FRET sensors for the β-arrestin2–receptor interaction and receptor-dependent conformational changes in β-arrestin2.
Figure 2: Kinetics of the interaction of β-arrestin2 with β2AR and its conformational movements.
Figure 3: Kinetics of β-arrestin2 translocation between cytosol and cell membrane after β2AR stimulation.


  1. 1

    Lohse, M. J. & Hoffmann, C. Arrestin interactions with G protein-coupled receptors. Handb. Exp. Pharmacol. 219, 15–56 (2014)

    CAS  Article  Google Scholar 

  2. 2

    Shukla, A. K., Xiao, K. & Lefkowitz, R. J. Emerging paradigms of β-arrestin-dependent seven transmembrane receptor signaling. Trends Biochem. Sci. 36, 457–469 (2011)

    CAS  Article  Google Scholar 

  3. 3

    Lohse, M. J., Benovic, J. L., Codina, J., Caron, M. G. & Lefkowitz, R. J. β-Arrestin: a protein that regulates β-adrenergic receptor function. Science 248, 1547–1550 (1990)

    ADS  CAS  Article  Google Scholar 

  4. 4

    Wilden, U., Hall, S. W. & Kühn, H. Phosphodiesterase activation by photoexcited rhodopsin is quenched when rhodopsin is phosphorylated and binds the intrinsic 48-kDa protein of rod outer segments. Proc. Natl Acad. Sci. USA 83, 1174–1178 (1986)

    ADS  CAS  Article  Google Scholar 

  5. 5

    Goodman, O. B. et al. β-Arrestin acts as a clathrin adaptor in endocytosis of the β2-adrenergic receptor. Nature 383, 447–450 (1996)

    ADS  CAS  Article  Google Scholar 

  6. 6

    Ferguson, S. S. et al. Role of β-arrestin in mediating agonist-promoted G protein-coupled receptor internalization. Science 271, 363–366 (1996)

    ADS  CAS  Article  Google Scholar 

  7. 7

    Kang, D. S., Tian, X. & Benovic, J. L. β-Arrestins and G protein-coupled receptor trafficking. Methods Enzymol. 521, 91–108 (2013)

    CAS  Article  Google Scholar 

  8. 8

    Shukla, A. K. et al. Structure of active β-arrestin-1 bound to a G-protein-coupled receptor phosphopeptide. Nature 497, 137–141 (2013)

    ADS  CAS  Article  Google Scholar 

  9. 9

    Kim, Y. J. et al. Crystal structure of pre-activated arrestin p44. Nature 497, 142–146 (2013)

    ADS  CAS  Article  Google Scholar 

  10. 10

    Kang, Y. et al. Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser. Nature 523, 561–567 (2015)

    ADS  CAS  Article  Google Scholar 

  11. 11

    Schleicher, A., Kühn, H. & Hofmann, K. P. Kinetics, binding constant, and activation energy of the 48-kDa protein-rhodopsin complex by extra-metarhodopsin II. Biochemistry 28, 1770–1775 (1989)

    CAS  Article  Google Scholar 

  12. 12

    Gurevich, V. V. & Gurevich, E. V. The structural basis of arrestin-mediated regulation of G-protein-coupled receptors. Pharmacol. Ther. 110, 465–502 (2006)

    CAS  Article  Google Scholar 

  13. 13

    Hanson, S. M. et al. Differential interaction of spin-labeled arrestin with inactive and active phosphorhodopsin. Proc. Natl Acad. Sci. USA 103, 4900–4905 (2006)

    ADS  CAS  Article  Google Scholar 

  14. 14

    Vishnivetskiy, S. A. et al. The role of arrestin α-helix I in receptor binding. J. Mol. Biol. 395, 42–54 (2010)

    CAS  Article  Google Scholar 

  15. 15

    Xiao, K., Shenoy, S. K., Nobles, K. & Lefkowitz, R. J. Activation-dependent conformational changes in β-arrestin 2. J. Biol. Chem. 279, 55744–55753 (2004)

    CAS  Article  Google Scholar 

  16. 16

    Charest, P. G., Terrillon, S. & Bouvier, M. Monitoring agonist-promoted conformational changes of β-arrestin in living cells by intramolecular BRET. EMBO Rep. 6, 334–340 (2005)

    CAS  Article  Google Scholar 

  17. 17

    Lohse, M. J., Nuber, S. & Hoffmann, C. Fluorescence/bioluminescence resonance energy transfer techniques to study G-protein-coupled receptor activation and signaling. Pharmacol. Rev. 64, 299–336 (2012)

    CAS  Article  Google Scholar 

  18. 18

    Hanson, S. M. & Gurevich, V. V. The differential engagement of arrestin surface charges by the various functional forms of the receptor. J. Biol. Chem. 281, 3458–3462 (2006)

    CAS  Article  Google Scholar 

  19. 19

    Hoffmann, C. et al. Fluorescent labeling of tetracysteine-tagged proteins in intact cells. Nature Protocols 5, 1666–1677 (2010)

    CAS  Article  Google Scholar 

  20. 20

    Klenk, C. et al. Formation of a ternary complex among NHERF1, β-arrestin, and parathyroid hormone receptor. J. Biol. Chem. 285, 30355–30362 (2010)

    Article  Google Scholar 

  21. 21

    Krasel, C., Bünemann, M., Lorenz, K. & Lohse, M. J. β-Arrestin binding to the β2-adrenergic receptor requires both receptor phosphorylation and receptor activation. J. Biol. Chem. 280, 9528–9535 (2005)

    CAS  Article  Google Scholar 

  22. 22

    Sykes, D. A. et al. Observed drug-receptor association rates are governed by membrane affinity: the importance of establishing “micro-pharmacokinetic/pharmacodynamic relationships” at the β2-adrenoceptor. Mol. Pharmacol. 85, 608–617 (2014)

    Article  Google Scholar 

  23. 23

    Krasel, C. et al. Dual role of the β2-adrenergic receptor C terminus for the binding of β-arrestin and receptor internalization. J. Biol. Chem. 283, 31840–31848 (2008)

    CAS  Article  Google Scholar 

  24. 24

    Söhlemann, P., Hekman, M., Puzicha, M., Buchen, C. & Lohse, M. J. Binding of purified recombinant β-arrestin to guanine-nucleotide-binding-protein-coupled receptors. Eur. J. Biochem. 232, 464–472 (1995)

    Article  Google Scholar 

  25. 25

    Violin, J. D., Ren, X.-R. & Lefkowitz, R. J. G-protein-coupled receptor kinase specificity for β-arrestin recruitment to the β2-adrenergic receptor revealed by fluorescence resonance energy transfer. J. Biol. Chem. 281, 20577–20588 (2006)

    CAS  Article  Google Scholar 

  26. 26

    Hein, P. & Bünemann, M. Coupling mode of receptors and G proteins. Naunyn Schmiedebergs Arch. Pharmacol. 379, 435–443 (2009)

    CAS  Article  Google Scholar 

  27. 27

    Lohse, M. J. et al. Optical techniques to analyze real-time activation and signaling of G-protein-coupled receptors. Trends Pharmacol. Sci. 29, 159–165 (2008)

    MathSciNet  CAS  Article  Google Scholar 

  28. 28

    Lee, M.-H. et al. The conformational signature of β-arrestin2 predicts its trafficking and signalling functions. Nature (this issue)

  29. 29

    Lohse, M. J. & Calebiro, D. Cell biology: Receptor signals come in waves. Nature 495, 457–458 (2013)

    ADS  CAS  Article  Google Scholar 

  30. 30

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

    ADS  CAS  Article  Google Scholar 

  31. 31

    Gurevich, V. V. & Gurevich, E. V. The new face of active receptor bound arrestin attracts new partners. Structure 11, 1037–1042 (2003)

    CAS  Article  Google Scholar 

  32. 32

    Vishnivetskiy, S. A. et al. Few residues within an extensive binding interface drive receptor interaction and determine the specificity of arrestin proteins. J. Biol. Chem. 286, 24288–24299 (2011)

    CAS  Article  Google Scholar 

  33. 33

    Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K. & Pease, L. R. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77, 51–59 (1989)

    CAS  Article  Google Scholar 

  34. 34

    Hoffmann, C. et al. A FlAsH-based FRET approach to determine G protein-coupled receptor activation in living cells. Nature Methods 2, 171–176 (2005)

    ADS  CAS  Article  Google Scholar 

  35. 35

    Zürn, A. et al. Site-specific, orthogonal labeling of proteins in intact cells with two small biarsenical fluorophores. Bioconjug. Chem. 21, 853–859 (2010)

    Article  Google Scholar 

  36. 36

    Vilardaga, J.-P., Bünemann, M., Krasel, C., Castro, M. & Lohse, M. J. Measurement of the millisecond activation switch of G protein-coupled receptors in living cells. Nature Biotechnol. 21, 807–812 (2003)

    CAS  Article  Google Scholar 

  37. 37

    Hein, P., Frank, M., Hoffmann, C., Lohse, M. J. & Bünemann, M. Dynamics of receptor/G protein coupling in living cells. EMBO J. 24, 4106–4114 (2005)

    CAS  Article  Google Scholar 

  38. 38

    Shenoy, S. K. et al. β-Arrestin-dependent, G protein-independent ERK1/2 activation by the β2-adrenergic receptor. J. Biol. Chem. 281, 1261–1273 (2006)

    CAS  Article  Google Scholar 

Download references


We thank N. Ziegler, N. Yurdagül-Hemmrich and M. Fischer for technical assistance and C. Krasel for discussions. This work was supported by the Deutsche Forschungsgemeinschaft grants SFB-487 TPA1 and SFB-TR166 (M.J.L. and C.H.), the Bundesministerium für Bildung und Forschung grant OptiMAR (M.J.L.), the ERC grants Topas and Fresca and the NIH grant 1 R01 DA038882 (M.J.L.), the Biotechnology and Biological Sciences Research Council (grant BB/K019864/1 to G.M.)

Author information




Contributed new reagents or analytical tools: S.N., U.Z., A.N., G.M. and A.B.T. Conducted experiments: S.N. (FRET, microscopy), U.Z. (cloning) and K.L. (kinase assays). Performed data analysis: S.N., A.N. and K.L. Wrote and contributed to writing of the manuscript: S.N., M.J.L. and C.H. Participated in research design: S.N., M.J.L. and C.H. Initiation of the project: C.H.

Corresponding authors

Correspondence to Martin J. Lohse or Carsten Hoffmann.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Specific labelling of FRET-based β-arrestin2 biosensors in intact cells with FlAsH.

HEK293 cells were transfected with one of the CFP-tagged β-arrestin2 biosensors, labelled with FlAsH and analysed by laser scanning microscopy. Confocal images show overlapping intracellular staining in both the CFP (blue) and the FlAsH (yellow) channels.

Extended Data Figure 2 Translocation of the β-arrestin2 biosensors.

HEK293 cells were transiently transfected with PTH1R–CFP and either wild-type β-arrestin2–YFP or one of the eight β-arrestin2–FlAsH–YFP sensors. a, Increase in membrane fluorescence 10 min after stimulation with 1 μM PTH 1–34 (N-terminal fragment) expressed as percentage increase of the initial fluorescence at t = 0 min. Data represent mean ± s.e.m. values of the indicated number of independent experiments. #P < 0.01 versus no effect (Kruskal–Wallis followed by Mann–Whitney U post-hoc analysis). b, Confocal images of the CFP-tagged PTH1R (left) and wild-type β-arrestin2–YFP (top), or β-arrestin2–FlAsH2–YFP (bottom) before (middle) and 10 min after PTH stimulation (right).

Extended Data Figure 3 β-Arrestin-dependent ERK1/2 phosphorylation.

HEK293 cells were transiently transfected with the indicated constructs or control vector (pcDNA3; Con) and treated without or with isoproterenol for 10 min (10 μM) as indicated. Cell lysates were analysed for pERK1/2 and ERK1/2 by immunoblot analysis. Data represent mean ± s.e.m., n = 6 independent experiments. *P < 0.05 versus unstimulated samples; #P < 0.05 versus isoproterenol-stimulated control (Kruskal–Wallis followed by Mann–Whitney U post-hoc analysis).

Extended Data Figure 4 Conformational changes in the β-arrestin2–FlAsH2 biosensor after FFA4R stimulation.

Representative traces of docosahexaenoic acid (DHA)-induced changes in the normalized FRET ratio (FFlAsH/FCFP) and the corresponding CFP (FCFP, cyan) or FlAsH (FFlAsH, yellow) emission recorded from one single HEK293 cell expressing the FFA4R and the FlAsH-labelled β-arrestin2–FlAsH2–CFP sensor. Application of 100 μM DHA is indicated. Representative trace of 10 experiments.

Extended Data Figure 5 β-Arrestin-mediated downstream signalling to kinases for M2-muscarinic, β2-adrenergic and FFA4 receptors.

HEK293 cells were transfected with β2AR, M2-muscarinic or FFA4 receptors and stimulated with respective agonists at saturating concentrations (isoproterenol, 100 μM; carbachol (CCH), 100 μM; docosahexaenoic acid, 10 μM) for 10 min. β-Arrestin downstream signalling was evaluated by phospho-specific antibodies for pSrc, pERK1/2 and pJNK. Gβ was used as loading control. Data represent mean ± s.e.m. of n = 12 independent experiments. *P < 0.05 versus unstimulated control; #P < 0.05 versus indicated column (Kruskal–Wallis followed by Mann–Whitney U post-hoc analysis).

Extended Data Figure 6 Concentration dependency of the kinetics of the conformational changes in β-arrestin upon β2AR stimulation.

HEK293 cells were co-transfected with the β2AR and β-arrestin2–FlAsH2–CFP and stimulated with different concentrations of isoproterenol. Kinetics of the agonist evoked intramolecular FRET changes were analysed by curve fitting according to Fig. 2. The bar graph shows the rate constants τ (s) for conformational changes detected with the β-arrestin2–FlAsH2 sensor upon stimulation with 1, 10, 30 or 100 μM isoproterenol, respectively. Data represent mean ± s.e.m. of n ≥ 3 independent experiments. The values are not significantly different (P < 0.05).

Extended Data Table 1 FRET β-arrestin2 sensor constructs used in this study

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Nuber, S., Zabel, U., Lorenz, K. et al. β-Arrestin biosensors reveal a rapid, receptor-dependent activation/deactivation cycle. Nature 531, 661–664 (2016).

Download citation

Further reading


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.


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