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

Abstract

(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.

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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.

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Acknowledgements

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.)

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Contributions

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.

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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

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Nuber, S., Zabel, U., Lorenz, K. et al. β-Arrestin biosensors reveal a rapid, receptor-dependent activation/deactivation cycle. Nature 531, 661–664 (2016). https://doi.org/10.1038/nature17198

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