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:

Non-canonical β-adrenergic activation of ERK at endosomes

Nature volume 611, pages 173–179 (2022)Cite this article

Subjects

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

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.

Similar content being viewed by others

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.

References

  1. Smith, J. S., Lefkowitz, R. J. & Rajagopal, S. Biased signalling: from simple switches to allosteric microprocessors. Nat. Rev. Drug Discov. 17, 243–260 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. O’Hayre, M. et al. Genetic evidence that β-arrestins are dispensable for the initiation of β2-adrenergic receptor signaling to ERK. Sci. Signal. 10, eaal3395 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  3. Grundmann, M. et al. Lack of beta-arrestin signaling in the absence of active G proteins. Nat. Commun. 9, 341 (2018).

    Article  PubMed  PubMed Central  ADS  Google Scholar 

  4. Luttrell, L. M. et al. Manifold roles of β-arrestins in GPCR signaling elucidated with siRNA and CRISPR/Cas9. Sci. Signal. 11, eaat7650 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  5. Keyes, J. et al. Signaling diversity enabled by Rap1-regulated plasma membrane ERK with distinct temporal dynamics. eLife 9, e57410 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Smith, J. S. et al. Noncanonical scaffolding of Gαi and β-arrestin by G protein-coupled receptors. Science 371, eaay1833 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Lavoie, H., Gagnon, J. & Therrien, M. ERK signalling: a master regulator of cell behaviour, life and fate. Nat. Rev. Mol. Cell Biol. 21, 607–632 (2020).

    Article  CAS  PubMed  Google Scholar 

  8. Eichel, K. & von Zastrow, M. Subcellular organization of GPCR signaling. Trends Pharmacol. Sci. 39, 200–208 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  ADS  Google Scholar 

  10. Tsvetanova, N. G. & von Zastrow, M. Spatial encoding of cyclic AMP signaling specificity by GPCR endocytosis. Nat. Chem. Biol. 10, 1061–1065 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Kotowski, S. J., Hopf, F. W., Seif, T., Bonci, A. & von Zastrow, M. Endocytosis promotes rapid dopaminergic signaling. Neuron 71, 278–290 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. de Rooij, J. et al. Epac is a Rap1 guanine-nucleotide-exchange factor directly activated by cyclic AMP. Nature 396, 474–477 (1998).

    Article  PubMed  ADS  Google Scholar 

  13. Kolch, W. et al. Protein kinase Cα activates RAF-1 by direct phosphorylation. Nature 364, 249–252 (1993).

    Article  CAS  PubMed  ADS  Google Scholar 

  14. Daaka, Y. et al. Essential role for G protein-coupled receptor endocytosis in the activation of mitogen-activated protein kinase. J. Biol. Chem. 273, 685–688 (1998).

    Article  CAS  PubMed  Google Scholar 

  15. Pierce, K. L., Maudsley, S., Daaka, Y., Luttrell, L. M. & Lefkowitz, R. J. Role of endocytosis in the activation of the extracellular signal-regulated kinase cascade by sequestering and nonsequestering G protein-coupled receptors. Proc. Natl Acad. Sci. USA 97, 1489–1494 (2000).

    Article  CAS  PubMed  PubMed Central  ADS  Google Scholar 

  16. Gurevich, V. V. & Gurevich, E. V. Arrestin-mediated signaling: is there a controversy? World J. Biol. Chem. 9, 25–35 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  17. Luttrell, L. M. et al. Activation and targeting of extracellular signal-regulated kinases by β-arrestin scaffolds. Proc. Natl Acad. Sci. USA 98, 2449–2454 (2001).

    Article  CAS  PubMed  PubMed Central  ADS  Google Scholar 

  18. Tohgo, A. et al. The stability of the G protein-coupled receptor-beta-arrestin interaction determines the mechanism and functional consequence of ERK activation. J. Biol. Chem. 278, 6258–6267 (2003).

    Article  CAS  PubMed  Google Scholar 

  19. Schmitt, D. L. et al. Spatial regulation of AMPK signaling revealed by a sensitive kinase activity reporter. Nat. Commun. 13, 3856 (2021).

  20. Chen, M., Sun, T., Zhong, Y., Zhou, X. & Zhang, J. A highly sensitive fluorescent Akt biosensor reveals lysosome-selective regulation of lipid second messengers and kinase activity. ACS Cent. Sci. 7, 2009–2020 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Zhou, X. et al. Dynamic visualization of mTORC1 activity in living cells. Cell Rep. 10, 1767–1777 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Allen, M. D. & Zhang, J. Subcellular dynamics of protein kinase A activity visualized by FRET-based reporters. Biochem. Biophys. Res. Commun. 348, 716–721 (2006).

    Article  CAS  PubMed  Google Scholar 

  23. Miyamoto, T. et al. Compartmentalized AMPK signaling illuminated by genetically encoded molecular sensors and actuators. Cell Rep. 11, 657–670 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Vaidyanathan, H. et al. ERK MAP kinase is targeted to RSK2 by the phosphoprotein PEA-15. Proc. Natl Acad. Sci. USA 104, 19837–19842 (2007).

    Article  CAS  PubMed  PubMed Central  ADS  Google Scholar 

  25. Boned Del Rio, I. et al. SHOC2 complex-driven RAF dimerization selectively contributes to ERK pathway dynamics. Proc. Natl. Acad. Sci. USA 116, 13330–13339 (2019).

    Article  PubMed  PubMed Central  ADS  Google Scholar 

  26. Ariotti, N. et al. Modular detection of GFP-labeled proteins for rapid screening by electron microscopy in cells and organisms. Dev. Cell 35, 513–525 (2015).

    Article  CAS  PubMed  Google Scholar 

  27. Gerits, N., Kostenko, S., Shiryaev, A., Johannessen, M. & Moens, U. Relations between the mitogen-activated protein kinase and the cAMP-dependent protein kinase pathways: comradeship and hostility. Cell Signal 20, 1592–1607 (2008).

    Article  CAS  PubMed  Google Scholar 

  28. Eichel, K. et al. Catalytic activation of β-arrestin by GPCRs. Nature 557, 381–386 (2018).

    Article  CAS  PubMed  PubMed Central  ADS  Google Scholar 

  29. Peng, G. E., Pessino, V., Huang, B. & von Zastrow, M. Spatial decoding of endosomal cAMP signals by a metastable cytoplasmic PKA network. Nat. Chem. Biol. 17, 558–566 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Luttrell, L. M. et al. β-Arrestin-dependent formation of β2 adrenergic receptor-Src protein kinase complexes. Science 283, 655–661 (1999).

    Article  CAS  PubMed  ADS  Google Scholar 

  31. Pierce, K. L., Luttrell, L. M. & Lefkowitz, R. J. New mechanisms in heptahelical receptor signaling to mitogen activated protein kinase cascades. Oncogene 20, 1532–1539 (2001).

    Article  CAS  PubMed  Google Scholar 

  32. Luttrell, L. M. ‘Location, location, location’: activation and targeting of MAP kinases by G protein-coupled receptors. J. Mol. Endocrinol. 30, 117–126 (2003).

    Article  CAS  PubMed  Google Scholar 

  33. Beautrait, A. et al. A new inhibitor of the β-arrestin/AP2 endocytic complex reveals interplay between GPCR internalization and signalling. Nat. Commun. 8, 15054 (2017).

    Article  CAS  PubMed  PubMed Central  ADS  Google Scholar 

  34. Martin, B. R. & Lambert, N. A. Activated G protein galphas samples multiple endomembrane compartments. J. Biol. Chem. 291, 20295–20302 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Lazar, A. M. et al. G protein-regulated endocytic trafficking of adenylyl cyclase type 9. eLife 9, e58039 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Zhou, X. et al. Location-specific inhibition of Akt reveals regulation of mTORC1 activity in the nucleus. Nat. Commun. 11, 6088 (2020).

    Article  CAS  PubMed  PubMed Central  ADS  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  ADS  Google Scholar 

  38. Feldman, D. S. et al. Selective inhibition of heterotrimeric Gs signaling. Targeting the receptor-G protein interface using a peptide minigene encoding the Gαs carboxyl terminus. J. Biol. Chem. 277, 28631–28640 (2002).

    Article  CAS  PubMed  Google Scholar 

  39. Irannejad, R. et al. Functional selectivity of GPCR-directed drug action through location bias. Nat. Chem. Biol. 13, 799–806 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Plotnikov, A., Zehorai, E., Procaccia, S. & Seger, R. The MAPK cascades: signaling components, nuclear roles and mechanisms of nuclear translocation. Biochim. Biophys. Acta 1813, 1619–1633 (2011).

    Article  CAS  PubMed  Google Scholar 

  41. Zhang, W. & Liu, H. T. MAPK signal pathways in the regulation of cell proliferation in mammalian cells. Cell Res. 12, 9–18 (2002).

    Article  CAS  PubMed  Google Scholar 

  42. Wheeler, E. C. et al. Integrative RNA-omics discovers GNAS alternative splicing as a phenotypic driver of splicing factor-mutant neoplasms. Cancer Discov. https://doi.org/10.1158/2159-8290.CD-21-0508 (2021).

  43. Weis, W. I. & Kobilka, B. K. The molecular basis of G protein-coupled receptor activation. Annu. Rev. Biochem. 87, 897–919 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Jones, A. J. Y., Gabriel, F., Tandale, A. & Nietlispach, D. Structure and dynamics of GPCRs in lipid membranes: physical principles and experimental approaches. Molecules 25, 4729 (2020).

    Article  CAS  PubMed Central  Google Scholar 

  45. Arumugam, S. & Kaur, A. The lipids of the early endosomes: making multimodality work. ChemBioChem 18, 1053–1060 (2017).

    Article  CAS  PubMed  Google Scholar 

  46. Xiao, K. et al. Functional specialization of β-arrestin interactions revealed by proteomic analysis. Proc. Natl Acad. Sci. USA 104, 12011–12016 (2007).

    Article  CAS  PubMed  PubMed Central  ADS  Google Scholar 

  47. Wu, V. et al. Illuminating the Onco-GPCRome: novel G protein-coupled receptor-driven oncocrine networks and targets for cancer immunotherapy. J. Biol. Chem. 294, 11062–11086 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Patra, K. C. et al. Mutant GNAS drives pancreatic tumourigenesis by inducing PKA-mediated SIK suppression and reprogramming lipid metabolism. Nat. Cell Biol. 20, 811–822 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Arang, N. & Gutkind, J. S. G Protein-coupled receptors and heterotrimeric G proteins as cancer drivers. FEBS Lett. 594, 4201–4232 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Insel, P. A. et al. GPCRomics: GPCR expression in cancer cells and tumors identifies new, potential biomarkers and therapeutic targets. Front. Pharmacol. 9, 431 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

Download references

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.).

Author information

Authors and Affiliations

  1. Department of Pharmacology, University of California, San Diego, La Jolla, CA, USA

    Yonghoon Kwon, Sohum Mehta, Mary Clark, Geneva Walters, Yanghao Zhong, Ha Neul Lee, Roger K. Sunahara & Jin Zhang

  2. Department of Bioengineering, University of California, San Diego, La Jolla, CA, USA

    Jin Zhang

  3. Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, USA

    Jin Zhang

Authors
  1. Yonghoon Kwon
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sohum Mehta
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mary Clark
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Geneva Walters
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yanghao Zhong
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ha Neul Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Roger K. Sunahara
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Jin Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

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.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature thanks the anonymous reviewers for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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.

Source data

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 × 10−6 Gαs-L vs vec, 0.2994 Gαs-S vs vec, 7.01 × 10−11 Gαs-L vs Gαs-S (b); 2.00 × 10−4 Gαs-L vs vec, >0.9999 Gαs-S vs vec, 6.30 × 10−4 Gαs-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.

Source data

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.

Source data

Rights and permissions

Springer Nature or its licensor holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41586-022-05343-3

This article is cited by

Comments

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.

Search

Advanced search

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