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β-Arrestin drives MAP kinase signalling from clathrin-coated structures after GPCR dissociation

Nature Cell Biology volume 18, pages 303–310 (2016)Cite this article

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Abstract

β-Arrestins critically regulate G-protein-coupled receptor (GPCR) signalling, not only ‘arresting’ the G protein signal but also modulating endocytosis and initiating a discrete G-protein-independent signal through MAP kinase1,2,3. Despite enormous recent progress towards understanding biophysical aspects of arrestin function4,5, arrestin cell biology remains relatively poorly understood. Two key tenets underlie the prevailing current view: β-arrestin accumulates in clathrin-coated structures (CCSs) exclusively in physical complex with its activating GPCR, and MAP kinase activation requires endocytosis of formed GPCR–β-arrestin complexes6,7,8,9. We show here, using β1-adrenergic receptors, that β-arrestin-2 (arrestin 3) accumulates robustly in CCSs after dissociating from its activating GPCR and transduces the MAP kinase signal from CCSs. Moreover, inhibiting subsequent endocytosis of CCSs enhances the clathrin- and β-arrestin-dependent MAP kinase signal. These results demonstrate β-arrestin ‘activation at a distance’, after dissociating from its activating GPCR, and signalling from CCSs. We propose a β-arrestin signalling cycle that is catalytically activated by the GPCR and energetically coupled to the endocytic machinery.

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Figure 1: β1ARs cluster and internalize poorly, but initiate β-arrestin-2-dependent activation of ERK1/2.
Figure 2: β1ARs drive β-arrestin-2 trafficking to CCSs separately from receptors.
Figure 3: β1ARs drive β-arrestin-2 trafficking to CCSs even when laterally immobilized.
Figure 4: Surface lifetime of CCSs regulates magnitude and duration of the β-arrestin-2-dependent ERK1/2 signal.
Figure 5: Relationship between β-arrestin signalling and CME dynamics.

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Acknowledgements

We thank M. Mettlen, G. Danuser and S. Schmid for valuable discussion and help with the cmeAnalysis package used for measuring CCP lifetime, and J. Benovic, V. Gurevich and B. Shoichet for valuable discussion. We also thank R. Irannejad, G. Peng, N. Michael, K. Ashrafi, O. Weiner, H. Bourne, and other members of the von Zastrow laboratory for useful advice and critical discussion. All live cell imaging experiments were performed in the Nikon Imaging Center at UCSF; we thank K. Thorn and D. Larsen for essential advice and assistance. This work was supported by grants from the US National Institutes of Health (DA 010711 and 012864 to M.v.Z.). K.E. is a recipient of a National Science Foundation Graduate Research Fellowship.

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Authors and Affiliations

  1. Program in Biochemistry and Molecular Biology, University of California, San Francisco, California 94158, USA

    K. Eichel

  2. Department of Psychiatry, University of California, San Francisco School of Medicine, San Francisco, California 94158, USA

    D. Jullié & M. von Zastrow

  3. Department of Cellular and Molecular Pharmacology, University of California, San Francisco School of Medicine, San Francisco, California 94158, USA

    M. von Zastrow

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  1. K. Eichel
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  3. M. von Zastrow
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Contributions

K.E. conceived and designed the experiments, performed all experiments, analysed the data, and wrote the paper. D.J. developed software for analysis of TIR-FM image series. M.v.Z. conceived and designed the experiments, analysed the data, and wrote the paper.

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Correspondence to M. von Zastrow.

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Integrated supplementary information

Supplementary Figure 1 β1AR and β2AR comparably activate ERK1/2 in expression matched cells.

(a) Flow cytometric analysis of FLAG-tagged receptor surface expression in separate stably expressing cell clones. (b) Representative western blots showing phosphorylated ERK1/2 and total ERK1/2 signal in extracts prepared from β1AR or β2AR stable clones treated in parallel with 10 μM isoproterenol. (c) Quantification of ERK1/2 activation from western blots in (b). Phosphorylated ERK1/2 band intensity is normalized to ERK1/2 total band intensity and shown as fold activation observed in each independent experiment. (d) Fixed cell flow cytometric analysis of phosphorylated ERK1/2 normalized to total ERK1/2 and shown as fold over basal for cells treated with siRNA for clathrin heavy chain 17, and showing rescue of the knockdown effect using an siRNA-resistant non-targetable mutant (NTM) expression construct (∗p = 0.0209 by a two-tailed t-test). (e) Similar analysis as in (d) showing the effect of knocking down β-arrestin-2 and rescue by a β-arrestin-2 non-targetable mutant (NTM) expression construct (∗p = 0.0126 by a two-tailed t-test). (f) % knockdown of β-arrestin-2 or clathrin heavy chain 17 in cells treated with siRNA as measured by RT-qPCR. (g) FLAG-β1ARs surface expression after 72 hours of siRNA knockdown as measured by flow cytometric analysis (n = 3 independent experiments; 30,000 cells per experiment). Error bars correspond to SEM. For a and g, n = 3 independent experiments with 15,000 cells per experiment, for panels b,c and f, n = 3 independent experiments, and n values for d and e are specified in the figure. Raw data of independent repeats are provided in Supplementary Table 1, and uncropped original scans of western blots are shown in Supplementary Fig. 6.

Supplementary Figure 2 β-arrestin-2 is not recruited to internal clathrin structures but to pre-existing and forming CCSs.

(a) Representative time lapse series showing β-arrestin-2 clustering into pre-existing CCSs. Images were collected at 0.5 Hz and, for brevity of presentation, one in three frames (6 sec interval) are shown. Scale bar = 500 nm. (b) Average fluorescence intensity of the image series shown in (a) (c) Representative time lapse series showing β-arrestin-2 accumulation at forming CCSs. Images were collected at 0.5 Hz and, for brevity of presentation, one in five frames (10 sec interval) are shown. Scale bar = 500 nm. (d) Average fluorescence intensity of the image series shown in (c). (e) β-arrestin-2 is recruited to diffraction limited clathrin spots but not larger internal clathrin structures (see arrow) that briefly visit the TIR-FM evanescent field. (f) Representative time lapse series showing β-arrestin-2 and clathrin disappear from the TIR-FM evanescent field. Images were collected at 0.5 Hz and, for brevity of presentation, one in five frames (10 sec interval) are shown. Scale bar = 500 nm. (g) Average fluorescence intensity of the image series shown in (f). Panels a–g are representative data from 2 independent experiments.

Supplementary Figure 3 β-arrestin-2 is recruited to a heterogeneous population of CCSs as revealed by super resolution microscopy.

(a,b) Representative images from photoactivated localization microscopy (PALM) of β-arrestin-2-photoactivatable-mCherry1 expressed in HEK 293 (A) or COS (B) cells. Unlabeled structures are less than 100 nm as measured by full width at half max (FWHM) analysis of the super resolved images. Arrows indicate β-arrestin-2 structures that are larger than 100 nm. Unmarked structures are less than 100 nm. Scale bar = 100 nm. (c) Representative example taken from (b) of a β-arrestin-2 structure that is smaller than 100 nm as determined by FWHM calculations. Scale bar = 100 nm (d) Representative example taken from (b) of a β-arrestin-2 structure that is larger than 100 nm as determined by FWHM calculations. Scale bar = 100 nm (e) Population size distributions of β-arrestin-2 and clathrin light chain in HEK 293 cells as determined from PALM super resolution images. (n = 300 structures pooled across 3 independent experiments) (f) Representative images from structured illumination microscopy (SIM) of β-arrestin-2-GFP (green) and clathrin light chain-DsRed (red) expressing HEK 293 cells. Large image scale bar = 500 nm; inset scale bar = 200 nm. (g) Line scan analysis of fluorescence intensity profiles along line shown in (f). (h) Representative images from structured illumination microscopy (SIM) of β-arrestin-2-GFP (green) and clathrin light chain-DsRed (red) expressing COS cells. Large image scale bar = 500 nm; inset scale bar = 200 nm. (i) Line scan analysis of fluorescence intensity profiles along line shown in (h). Panels a–g are representative data from 3 independent experiments. Raw data for (e) is provided in Supplementary Table 1.

Supplementary Figure 4 Polyclonal antibodies generate clusters of immobilized SEP-β1ARs.

(a) SEP-β1ARs without antibody immobilization were photobleached after 10 seconds and rapidly recover after photobleaching (top row). SEP-β1ARs treated with 1:100 polyclonal antibody are grouped into immobilized clusters which recover much more slowly after photobleaching (bottom row). (b) Analysis of SEP-β1ARs recovery after photobleaching (n = 12 cells for non-immobilized receptors or n = 10 for immobilized receptors pooled across 3 independent experiments). Raw data of independent repeats are provided in the statistics source data Supplementary Table 1.

Supplementary Figure 5 Dyngo-4a does not increase basal ERK1/2 activity but prolongs ERK1/2 signaling kinetics and stalls β-arrestin-2 associated CCSs.

(a) Quantification of ERK1/2 activation at 5 minutes after agonist from western blots as shown in Figure 1e. Phosphorylated ERK1/2 band intensity is normalized to ERK1/2 total band intensity and shown as fold activation over untreated samples (n = 3 independent experiments). (b) Quantification of ERK1/2 activation time course from western blots as shown in Fig. 4a, c. Phosphorylated ERK1/2 band intensity is normalized to ERK1/2 total band intensity and shown as percent of 5 minute ERK1/2 activation response observed in each independent experiment (n = 4 independent experiments). Error bars correspond to SEM. (c) Representative western blot showing phosphorylated ERK1/2 and total ERK1/2 signal in extracts prepared after pre-incubating cells in the absence or presence of 30 μM Dyngo-4a, as indicated. (d) Quantification of ERK1/2 activation at 5 minutes after agonist from representative western blot shown in in Fig. 4a, c. Phosphorylated ERK1/2 band intensity is normalized to ERK1/2 total band intensity (n = 4 independent experiments). (e) Quantification of ERK1/2 activation time course from representative western blot shown in Fig. 4a, c. Phosphorylated ERK1/2 band intensity is normalized to ERK1/2 total band intensity and shown as percent of 5 minute ERK1/2 activation response observed in each independent experiment (n = 4 independent experiments). (f) Kymograph analysis of TIR-FM images of cells without (top) and with (bottom) 30 μM Dyngo-4a treatment. At 20 seconds into the image series, cells were treated with 10 μM dobutamine to selectively activate FLAG-β1ARs, thus promoting β-arrestin-2 recruitment. Error bars correspond to SEM. Panels (c) and (f) are representative data from 3 independent experiments. Uncropped original scans of western blots are shown in Supplementary Fig. 6.

Supplementary Figure 6 Unprocessed scans of western blots accompanied by size markers.

Black boxes delineate the region presented in the indicated figures. Images were obtained using a Licor Odyssey scanner, acquired using Image Studio 2.0, and analyzed using ImageStudioLite.

Supplementary information

Supplementary Information

Supplementary Information (PDF 985 kb)

Supplementary Table 1

Supplementary Information (XLSX 561 kb)

β1ARs drive β-arrestin-2 trafficking to CCSs separately from receptors (HEK cells).

This video shows TIR-FM image series of HEK 293 cells expressing FLAG-β1AR (blue), β-arrestin-2-GFP (green), and clathrin light chain-DsRed (red). 10 μM dobutamine is added at 0s. This video corresponds to Fig. 2a. (AVI 14254 kb)

β1ARs drive β-arrestin-2 trafficking to CCSs separately from receptors (COS cells).

This video shows TIR-FM image series of COS cells expressing FLAG-β1AR (blue), β-arrestin-2-GFP (green), and clathrin light chain-DsRed (red). 10 μM dobutamine is added at 0s. This video corresponds to Fig. 2d. (AVI 14206 kb)

β-arrestin-2 is recruited to diffraction limited clathrin spots but not larger, quickly moving internal structures.

This video shows TIR-FM image series of COS cells expressing FLAG-β1AR (blue), β-arrestin-2-GFP (green), and clathrin light chain-DsRed (red). 10 μM dobutamine is added at 0s. Arrow indicates an internal structure (visible in the clathrin channel) that is larger than the diffraction limit and briefly visits the TIR-FM field before abruptly leaving. This video corresponds to Supplementary Fig. 2e. (AVI 3123 kb)

Immobilized β1ARs promote robust recruitment of β-arrestin-2 to CCSs without themselves moving to CCSs.

This video shows TIR-FM image series of a cell expressing SEP-β1AR (blue), β-arrestin-2-mApple (green), and clathrin light chain-TagBFP (red). SEP-β1ARs have been immobilized with polyclonal antibody pretreatment. 10 μM dobutamine is added at 0s. This video corresponds to Fig. 3h. (AVI 33129 kb)

Enlarged example of immobilized β1ARs promoting robust recruitment of β-arrestin-2 to CCSs without themselves moving to CCSs.

This video shows TIR-FM image series of a cell expressing immobilized SEP-β1AR (blue), β-arrestin-2-mApple (green), and clathrin light chain-TagBFP (red). SEP-β1ARs have been immobilized with polyclonal antibody pretreatment. 10 μM dobutamine is added at 0s. This video corresponds to Fig. 3h and Supplementary Video 4 . (MOV 171 kb)

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Eichel, K., Jullié, D. & von Zastrow, M. β-Arrestin drives MAP kinase signalling from clathrin-coated structures after GPCR dissociation. Nat Cell Biol 18, 303–310 (2016). https://doi.org/10.1038/ncb3307

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