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Non-canonical β-adrenergic activation of ERK at endosomes

Abstract

G-protein-coupled receptors (GPCRs), the largest family of signalling receptors, as well as important drug targets, are known to activate extracellular-signal-regulated kinase (ERK)—a master regulator of cell proliferation and survival1. However, the precise mechanisms that underlie GPCR-mediated ERK activation are not clearly understood2,3,4. Here we investigated how spatially organized β2-adrenergic receptor (β2AR) signalling controls ERK. Using subcellularly targeted ERK activity biosensors5, we show that β2AR signalling induces ERK activity at endosomes, but not at the plasma membrane. This pool of ERK activity depends on active, endosome-localized Gαs and requires ligand-stimulated β2AR endocytosis. We further identify an endosomally localized non-canonical signalling axis comprising Gαs, RAF and mitogen-activated protein kinase kinase, resulting in endosomal ERK activity that propagates into the nucleus. Selective inhibition of endosomal β2AR and Gαs signalling blunted nuclear ERK activity, MYC gene expression and cell proliferation. These results reveal a non-canonical mechanism for the spatial regulation of ERK through GPCR signalling and identify a functionally important endosomal signalling axis.

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Fig. 1: GPCR-stimulated ERK activity is spatially compartmentalized.
Fig. 2: GPCR-mediated endosomal ERK activity is regulated by GPCR endocytosis and endosomal Gαs activity.
Fig. 3: Gαs interacts with RAF1 and MEK1 to regulate endosomal GPCR-mediated ERK activity.
Fig. 4: Endosomal Gαs regulates nuclear ERK activity and function.

Data availability

 The data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank A. Inoue for providing the GNAS-knockout HEK293A and ARRB1/2 -double-knockout cell lines; D. Mayer for comments on the manuscript; the members of the Nikon Imaging Center at UC San Diego for help with imaging experiments; the members of the Human Embryonic Stem Cell Core Facility for assistance of the proliferation assay; and L. D. Lavis and the staff at the HHMI Janelia Research Campus and for providing the JF646–SNAP ligand. This work was supported by the NIH (R35 CA197622 and R01 DK073368 to J.Z.) and the University of California Tobacco-Related Disease Research Program (T32FT4691 to Y.K.).

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Authors and Affiliations

Authors

Contributions

R.K.S. and J.Z. conceived the project. Y.K., R.K.S. and J.Z. designed the experiments. Y.K., M.C., G.W., H.N.L. and Y.Z. performed the experiments. Y.K. and S.M. analysed data. R.K.S. and J.Z. coordinated the study and provided guidance. Y.K., S.M. and J.Z. wrote the paper. All of the authors discussed the results and approved the final version of the manuscript.

Corresponding author

Correspondence to Jin Zhang.

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Extended data figures and tables

Extended Data Fig. 1 Characterization of subcellularly targeted EKAR4.

(a—d) Representative yellow/cyan (Y/C) emission ratio time courses from HEK293T cells expressing (a) cytoEKAR4 (EGF: n = 27; Mock: n = 12), (b) pmEKAR4 (EGF: n = 11; Mock: n = 9), (c) nucEKAR4 (EGF: n = 27; Mock: n = 11), or (d) endoEKAR4 (n = 27 each) and stimulated with or without 100 ng/mL EGF. n indicates the number of cells from a representative experiment over 2 independent experiments. Domain schematics (top) and epifluorescence images showing biosensor localization (inset) are also shown. (e) Maximum-intensity-projected super-resolution fluorescence images of HEK293T cells expressing endoEKAR (left) and stained for EEA1 (middle, endosomes; goat anti-rabbit Alexa Fluor 647 × rabbit anti-EEA1) and merged (right) image (representative of 3 independent experiments). (f) Representative ratio images (pseudocolored) before and after EGF treatment. Pseudocolor images where warmer colours represent higher emission ratios show that emission ratio changes are confined to endosomes (representative of 4 independent experiments). (g) Representative yellow/cyan (Y/C) emission ratio time courses comparing the responses from a single endosome (boxed region in f) or whole HEK293T cell expressing endoEKAR4 and stimulated with 100 ng/mL EGF, indicating that endoEKAR4 responses specifically come from endosomes. Data shown represent 3 independent experiments (e-g). Solid lines in time courses indicate the mean, and shaded areas show SEM. Scale bars, 10 μm.

Source data

Extended Data Fig. 2 Compartmentalized GPCR signalling.

(a) Live-cell epifluorescence images of HEK293T cells expressing SNAP-β2AR and labelled with JF646 before (top) and 30 min after (bottom) 10 μM adrenaline (Adr) stimulation. Data shown represents 3 independent experiments. (b) Left: Representative yellow/cyan (Y/C) emission ratio time course from HEK293T cells co-expressing pmEKAR4 plus SNAP-β2AR stimulated with EGF or adrenaline. Right: Maximum pmEKAR4 responses. (c) Representative time courses showing average adrenaline-promoted emission ratio changes from plasma membrane targeted PKA activity biosensor AKAR4, cAMP biosensor Epac2-camps, and EKAR4. (d and e) Left: Representative time courses showing the 10 μM adrenaline-stimulated yellow/cyan (Y/C) emission ratio changes in HEK293T cells co-expressing SNAP-β2AR, PKI or vector and (d) pmEKAR4 or (e) endoEKAR4. Right: Maximum EKAR4 responses. (f) Left: Representative time courses showing the normalized cyan/yellow (C/Y) emission ratio changes in cells co-expressing another cAMP biosensor ICUE4 plus vector, SNAP-β2AR or SNAP-β2AR341T upon stimulation with 10 μM adrenaline followed by 50 μM Fsk and 100 μM IBMX (Fsk/IBMX). Middle: Total adrenaline-stimulated ICUE4 responses. ’n’ in b-f indicates the number of cells from a representative experiment over 2 (d, f), 3 (b, c) and 4 (e) independent experiments. Statistics in b and d—f were derived from the number of cells (shown in brackets) analysed over 2 (d, f), 3 (b) and 4 (e) independent experiments. (g, h) Western blot analysis of phospho-ERK (pERK) and total ERK (tERK) levels in HEK293T cells expressing vector, SNAP-β2AR or SNAP-β2AR-341T following stimulation with adrenaline for 0, 5, 15, 30 (g), and 0, 2, 5 (h) min. Representative blot (left) and quantification of normalized pERK/ tERK levels at each time point (right, n = 4 biologically independent experiments). Full blots are shown in Supplementary Fig. 1. (i) Maximum-intensity-projected super-resolution fluorescence images of HEK293T cells expressing SNAP-β2AR with (lower) or without (upper) adrenaline stimulation and stained for phospho-ERK (pERK; goat anti-mouse Atto488 × mouse anti-pERK), EEA1 (endosomes; goat anti-rabbit Alexa Fluor 647 × rabbit anti-EEA1), and DAPI (nuclei). Data shown represent 3 independent experiments. (j) Histogram of the Pearson correlation coefficient for EEA1 and pERK colocalization in HEK293T cells expressing SNAP-β2AR with or without adrenaline stimulation (n = 7 and 15 cells from 3 independent experiments for − and + adrenaline, respectively). The precise p values were shown in Source data. ns, not significant, *P < 0.05, ****P < 0.0001; two-tailed Mann-Whitney U-test (b), unpaired two-tailed Student t-test without (d, e, and j) or with (f right) Welch’s correction, ordinary one-way ANOVA followed by Holm-Šídák’s multiple-comparison test (f middle and g), ordinary one-way ANOVA followed by Dunnett’s multiple-comparisons test (h). Solid lines in time courses indicate the mean, and shaded areas show SEM. Lines in scatter plots represent mean ± SEM. Scale bars, 10 μm. Negative control curves in (d and e) are reproduced from Fig. 1a,b.

Source data

Extended Data Fig. 3 GPCR-mediated endosomal ERK activity is regulated by GPCR endocytosis and endosomal Gαs activity.

(a, d and f) Left: Representative time courses showing the 10 μM adrenaline-stimulated yellow/cyan (Y/C) emission ratio changes in cells co-expressing endoEKAR4 and SNAP-β2AR (a) with or without preincubation of 5 μM Dyngo4a in HEK293T cells or (d) β-arr1/2 dKO HEK293S cells additionally transfected with vector or β-arr2 or (f) HEK293T cells additionally transfected with vector or PTX. Right (a, f) or middle (d): Maximum EKAR4 responses. Right (d): Intensity of JF646-labelled SNAP-β2AR. ’n’ in a, d and f indicates the number of cells from a representative experiment over 3 (a, d), and 2 (f) independent experiments. (b) Maximum response of pmEKAR4 upon EGF stimulation with or without Dyngo4a pretreatment. (c) Intensity of JF646-labelled SNAP-β2AR; corresponds to Fig. 2b. (e) Maximum decrease of ICUE4 cyan/yellow emission ratio (ΔR/R) upon stimulation with 500 nM LPA in HEK293T cells preincubated with 50 μM Fsk and expressing vector or PTX. Statistics in a-e were derived from the number of cells (shown in brackets) analysed over 3 (a—d), and 2 (e—f) independent experiments. The precise p values were shown in Source data. ns, not significant, ****P < 0.0001; unpaired two-tailed Student’s t test without (a, d right) or with (d middle) Welch’s correction, Kruskal–Wallis test followed by Dunn’s multiple-comparison test (c), Two-tailed Mann-Whitney U-test (b, e, f). Solid lines in time courses indicate the mean, and shaded areas show SEM. Lines in scatter plots represent mean ± SEM. (g—i) Epifluorescence (left) and maximum-intensity-projected super-resolution fluorescence images (right) of HEK293T cells transfected with (g) control siRNA, (h) siRNAs targeting β-arr1/2, and (i) siRNAs targeting β-arr1/2 plus β-arr2-mCherry cDNA. Individual epifluorescence channel images show (upper) JF646-SNAP-β2AR or (lower) Gαs-mEos3.2 before (left) and after (right) adrenaline stimulation. Line intensity profile plots corresponding to the dashed lines on the images are shown to the right of the images (light blue: 0 min; green: 30 min after adrenaline). Individual maximum-intensity-projected super-resolution fluorescence channel images of (left) Gαs, (middle) EEA1 (endosomes; goat anti-rabbit Alexa Fluor 568 × rabbit anti-EEA1) and (right) merged channels. Numbers represent average Manders’ tM1 overlap coefficient from 10 (g), 4 (h) and 8 (i) different cells after 30 min adrenaline stimulation. Data shown represent 2 independent experiments (g–i). Scale bars, 10 μm (epifluorescence images) and 5 μm (super-resolution images). Negative control curve and data points in (a and f) are reproduced from Fig. 1c.

Source data

Extended Data Fig. 4 Localization of endosome-targeted nanobodies and GsCT and the effect on ERK.

(a-c) Epifluorescence microscopy analysis of (a) endo-Nb37, (b) endo-GsCT, and (c) endo-Nb80 colocalization with the endosomal marker EEA1 in HEK293T cells. Individual channel images of (upper left) mCherry fluorescence from endo-Nb37, endo-GsCT, and endo-Nb80 and (upper right) Alexa Fluor 647 fluorescence (goat anti-rabbit Alexa Fluor 647 × rabbit anti-EEA1), as well as (lower left) merged channels are shown. Line intensity profile plots corresponding to the dashed yellow lines on the merge images are shown to the lower right. Data shown represent 3 independent experiments. (d) Maximum-intensity-projected super-resolution fluorescence images of HEK293T cells co-expressing SNAP-β2AR plus endo-Nb37 with (lower) or without (upper) adrenaline stimulation and stained for phospho-ERK (pERK; goat anti-mouse Atto488 × mouse anti-pERK), EEA1 (endosomes; goat anti-rabbit Alexa Fluor 647 × rabbit anti-EEA1), and DAPI (nuclei). Data shown represent 3 independent experiments. (e, f) Histograms showing (e) the total intensity from the 488 nm (pERK) channel and (f) the Pearson correlation coefficient for EEA1 and pERK colocalization in HEK293T cells co-expressing SNAP-β2AR plus endo-Nb37 with (+) or without (−) adrenaline stimulation (n = 6 and 9 cells for − and + adrenaline, respectively from 3 independent experiments). Scale bars, 10 μm. P = 7.60 × 10−5 (e) and 9.04 × 10−4 (f), ***P < 0.001, ****P < 0.0001; unpaired two-tailed Student’s t-test. Lines in scatter plots represent mean ± SEM.

Source data

Extended Data Fig. 5 Endosomal Gαs interacts with RAF1 and MEK1 and regulates nuclear ERK activity and function.

(a) Quantification of PLA particle density for SNAP-β2AR-expressing HEK293T cells incubated with rabbit anti-Gαs plus normal mouse IgG, mouse anti-RAF1 or mouse anti-MEK1 (n = 6, 7, and 5 different randomly selected fields of view for IgG, RAF1 and MEK1, respectively from 3 independent experiments) following stimulation with 10 μm adrenaline. Bars correspond to the mean ± SEM. (b, c) Left: Representative 10 μM adrenaline-stimulated yellow/cyan (Y/C) emission ratio changes from HEK293T cells co-expressing nucEKAR4, SNAP-β2AR and (b) Nb37 in the presence or absence of 100 μM barbadin or 5 μM Dyngo4a preincubation or (c) additionally transfected with control siRNA or siRNA targeting β-arr1 and 2 plus vector or β-arr2. (b) Right, (c) middle: Maximum nucEKAR4 responses under the indicated conditions. (c) Right: SNAP-β2AR fluorescence intensity under the indicated conditions. (d and e) Left: Representative time courses showing the 10 μM adrenaline-stimulated yellow/cyan (Y/C) emission ratio changes of (d) cytoEKAR4 or (e) pmEKAR4 in HEK293T cells co-expressing SNAP-β2AR with or without endoGsCT or preincubation with 100 μM barbadin. n indicates the number of cells from a representative experiment over 2 independent experiments. Right: Maximum EKAR4 responses. ’n’ in b-e indicates the number of cells from a representative experiment over 2 (b, d and e), and 3 (c) independent experiments. Statistics in a—e were derived from the number of cells (shown in brackets) analysed over 2 (b, d and e), and 3 (c) independent experiments. The precise p values were shown in Source data.The precise p values were shown in Source data. ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; Kruskal–Wallis test followed by Dunn’s multiple-comparison test (b, c middle), ordinary one-way ANOVA followed by Holm-Šidák’s multiple-comparison test (c right), Welch ANOVA followed by Dunnett’s T3 multiple-comparison test (d, e). Solid lines in time courses indicate the mean, and shaded areas show SEM. Lines in scatter plots represent mean ± SEM. Negative control curve and data points in (d and e) are reproduced from Fig. 1a, c, respectively.

Source data

Extended Data Fig. 6 Endogenous β-adrenergic-stimulated ERK activation.

Left: Representative 10 μM adrenaline-stimulated yellow/cyan (Y/C) emission ratio changes from (a—c) HEK293T cells or (d-f) H9c2 cells expressing (a, d) pmEKAR4, (b, e) endoEKAR4, or (c, f) nucEKAR4 with or without 5 μM Dyngo4a or (b, e) with 10 μM ICI-118,551 preincubation. n indicates the number of cells from a representative experiment over 2 independent experiments. Right: Maximum EKAR4 responses under the indicated conditions. Solid lines in time courses indicate the mean, and shaded areas show SEM. Lines in scatter plots represent mean ± SEM. n = 25, 18 (a, - and + dyngo4a, respectively), 23, 17, 15 (b, ctrl, dyngo4a, and ICI, respectively), and 33, 23 (c, - and + dyngo4a, respectively), 8, 8 (d, - and + dyngo4a, respectively), 18, 11, 15 (e, ctrl, dyngo4a, and ICI, respectively), and 12, 12 (f, - and + dyngo4a, respectively) from over 2 independent experiments. (g) Western blot analysis of phospho-ERK (pERK) and total ERK (tERK) levels in HEK293T cells stimulated with adrenaline for 0, 2, 5, 15, and 30 min with or without Dyngo4a preincubation. Representative blot (left, data shown represent 4 independent experiments) and quantification of normalized pERK/tERK levels at each time point (right, n = 4 independent biological experiments). Full blots are shown in Supplementary Fig. 1. P = 0.1899 (a); 4.22 × 10−8 Dyngo vs no treatment (NT) and 6.42 × 10−7 ICI vs NT (b); 3.86 × 10−5 (c); 0.4872 (d); 7.43 × 10−6 Dyngo vs NT and 0.0011 ICI vs NT (e); 0.0013 (f); 0.2599 (0 min), 0.0021 (2 min), 0.0351 (5 min), 0.9324 (15 min), and 0.4467 (30 min) (g); ns, not significant, **P < 0.01, ***P < 0.001, ****P < 0.0001; Two-tailed Mann-Whitney U-test (a) or unpaired two-tailed Student’s t-test without (c, d) or with (f,g) Welch’s correction, or ordinary one-way ANOVA followed by Holm-Šídák’s multiple-comparison test (b, e).

Source data

Extended Data Fig. 7 Compartmentalized ERK activation via multiple types of GPCRs.

Left: Representative yellow/cyan (Y/C) emission ratio changes of HEK293T cells co-expressing (a–c) EP4, (d-f) D1DR, or (g—i) V2R plus (a, d, g) diffusible, (b, e, h) endo- or (c, f, i) nucEKAR4 upon stimulation with 10 μM (a—c) PGE2, (d—f) 100 μM DA or (g—i) 100 nM DESMO with or without preincubation with 5 μM Dyngo4a or 100 μM barbadin. n indicates the number of cells from a representative experiment over 2 independent experiments. Right: Maximum EKAR4 responses under the indicated conditions. n = 16, 19, 12 (a, P, E, N, respectively), 19, 19, 11 (b, ctrl, Barb, dyngo4a, respectively), 12, 19, 17 (c, ctrl, Barb, dyngo4a, respectively), 23, 13, 44 (d, P, E, N, respectively), 13, 17, 15 (e, ctrl, Barb, dyngo4a, respectively), 44, 42, 14 (f, ctrl, Barb, dyngo4a, respectively), 23, 34, 46 (g, P, E, N, respectively), 34, 27, 20 (h, ctrl, Barb, dyngo4a, respectively), 46, 15, 25 (i, ctrl, Barb, dyngo4a, respectively) from over 2 independent experiments. P = 1.51 × 10−5 endo vs pm and 6.06 × 10−4 nuc vs pm (a); 4.97 × 10−8 Barb vs NT and 6.93 × 10−4 Dyngo vs NT (b); 2.15 × 10−5 Barb vs NT and 6.70 × 10−5 Dyngo vs NT (c); 5.15 × 10−5 endo vs pm and 7.58 × 10−9 nuc vs pm (d); 0.0030 Barb vs NT and 2.85 × 10−5 Dyngo vs NT (e); 6.94 × 10−4 Barb vs NT and 3.14 × 10−8 Dyngo vs NT (f); 1.33 × 10−5 endo vs pm and 9.36 × 10−15 nuc vs pm (g); 1.34 × 10−4 Barb vs NT and 4.56 × 10−10 Dyngo vs NT (h); 5.41 × 10−7 Barb vs NT and 2.95 × 10−10 Dyngo vs NT (i), **P < 0.01, ***P < 0.001, ****P < 0.0001; Kruskal–Wallis test followed by Dunn’s multiple-comparison test. Solid lines in time courses indicate the mean, and shaded areas show SEM. Lines in scatter plots represent mean ± SEM.

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Extended Data Fig. 8 Growth factor-induced ERK target gene expression.

(a) qPCR analysis of c-Myc, c-fos and cyclin D1 mRNA in EGF-treated HEK293T cells with or without U0126 preincubation. (n = 3 independent biological experiments). Error bars represent mean ± SEM. *P < 0.05, **P < 0.01, ****P < 0.0001 vs. Mock or EGF + U0126; ordinary one-way ANOVA followed by Tukey’s multiple-comparison test. (b, c) Left: Representative yellow/cyan (Y/C) emission ratio traces from GNAS KO HEK293 cells co-expressing (b) endo or (c) nucEKAR4 and additionally expressing vector, Gαs-L or Gαs-S with or without 10 μM adrenaline stimulation. n indicates the number of cells from a representative experiment over 3 independent experiments. Right: Maximum EKAR4 responses. n = 27, 53, 29 (b, vec, long, short, respectively), 24, 47, 32 (c, vec, long, short, respectively) from 3 independent experiments. P = 0.0477 EGF vs Mock and 0.0209 EGF+U0126 vs EGF (cMyc), 0.0045 EGF vs Mock and 0.0033 EGF+U0126 vs EGF (cFos), 0.0016 EGF vs Mock and 0.0019 EGF+U0126 vs EGF (cyclinD1) (a); 9.16 × 10−6s-L vs vec, 0.2994 Gαs-S vs vec, 7.01 × 10−11s-L vs Gαs-S (b); 2.00 × 10−4s-L vs vec, >0.9999 Gαs-S vs vec, 6.30 × 10−4s-L vs Gαs-S (c); ns, not significant, ***P < 0.001, ****P < 0.0001; Kruskal–Wallis test followed by Dunn’s multiple-comparison test (b, c). Solid lines in b and c indicate the mean, and shaded areas show SEM. Horizonal lines in b and c depict the mean ± SEM. Negative control and Gαs-L curves and data points in (b) are reproduced from Fig. 2c.

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Supplementary information

Supplementary Information

Supplementary Fig. 1: Images of uncropped Western blots; Supplementary Fig. 2: Gating strategy for flow cytometry-based cell proliferation assay; Supplementary Table 1: Detailed transfection information for all of the Figures; and Supplementary Table 2: Detailed replication information for all of the Extended Data Figures.

Reporting Summary

Supplementary Video 1

Time-lapse epifluorescence video of SNAP–β2AR. HEK293T cells expressing SNAP–β2AR and labelled with JF646 stimulated with 10 μM adrenaline. Endocytosis of JF646-labelled SNAP–β2AR is shown. This is a representative video from at least 3 independent experiments. Scale bar, 10 μm.

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Kwon, Y., Mehta, S., Clark, M. et al. Non-canonical β-adrenergic activation of ERK at endosomes. Nature 611, 173–179 (2022). https://doi.org/10.1038/s41586-022-05343-3

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