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Endosome positioning coordinates spatially selective GPCR signaling

Nature Chemical Biology volume 20, pages 151–161 (2024)Cite this article

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Abstract

G-protein-coupled receptors (GPCRs) can initiate unique functional responses depending on the subcellular site of activation. Efforts to uncover the mechanistic basis of compartmentalized GPCR signaling have concentrated on the biochemical aspect of this regulation. Here we assess the biophysical positioning of receptor-containing endosomes as an alternative salient mechanism. We devise a strategy to rapidly and selectively redistribute receptor-containing endosomes ‘on command’ in intact cells without perturbing their biochemical composition. Next, we present two complementary optical readouts that enable robust measurements of bulk- and gene-specific GPCR/cyclic AMP (cAMP)-dependent transcriptional signaling with single-cell resolution. With these, we establish that disruption of native endosome positioning inhibits the initiation of the endosome-dependent transcriptional responses. Finally, we demonstrate a prominent mechanistic role of PDE-mediated cAMP hydrolysis and local protein kinase A activity in this process. Our study, therefore, illuminates a new mechanism regulating GPCR function by identifying endosome positioning as the principal mediator of spatially selective receptor signaling.

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Fig. 1: CID approach for redirecting endosomes.
Fig. 2: Intact β2-AR trafficking into and out of repositioned endosomes.
Fig. 3: β2-AR on repositioned endosomes adopts an active conformation and stimulates Gas/cAMP signaling.
Fig. 4: Single-cell optical readouts of GPCR-dependent transcriptional signaling.
Fig. 5: Endosome positioning is critical for site-selective signaling.
Fig. 6: Endosomes precisely position receptors to enable signal compartmentalization and spatially biased GPCR signaling.

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Data availability

All relevant data supporting the findings are available within the paper and the Supplementary Data. Source data are provided with the manuscript. Additional information and reagents are available from the corresponding author upon reasonable request. Source data are provided with this paper.

References

  1. Sorkin, A. & von Zastrow, M. Endocytosis and signalling: intertwining molecular networks. Nat. Rev. Mol. Cell Biol. 10, 609–622 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Thomsen, A. R. B., Jensen, D. D., Hicks, G. A. & Bunnett, N. W. Therapeutic targeting of endosomal G-protein-coupled receptors. Trends Pharmacol. Sci. 39, 879–891 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Tsvetanova, N. G., Irannejad, R. & von Zastrow, M. G protein-coupled receptor (GPCR) signaling via heterotrimeric G proteins from endosomes. J. Biol. Chem. 290, 6689–6696 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Vilardaga, J. P., Jean-Alphonse, F. G. & Gardella, T. J. Endosomal generation of cAMP in GPCR signaling. Nat. Chem. Biol. 10, 700–706 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Godbole, A., Lyga, S., Lohse, M. J. & Calebiro, D. Internalized TSH receptors en route to the TGN induce local Gs-protein signaling and gene transcription. Nat. Commun. 8, 443 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  6. 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 

  7. Tsvetanova, N. G. et al. Endosomal cAMP production broadly impacts the cellular phosphoproteome. J. Biol. Chem. 297, 100907 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Calebiro, D. et al. Persistent cAMP-signals triggered by internalized G-protein-coupled receptors. PLoS Biol. 7, e1000172 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  9. Ferrandon, S. et al. Sustained cyclic AMP production by parathyroid hormone receptor endocytosis. Nat. Chem. Biol. 5, 734–742 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. 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 

  11. Nash, C. A., Wei, W., Irannejad, R. & Smrcka, A. V. Golgi localized β1-adrenergic receptors stimulate golgi PI4P hydrolysis by PLCε to regulate cardiac hypertrophy. eLife 8, e48167 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Purgert, C. A. et al. Intracellular mGluR5 can mediate synaptic plasticity in the hippocampus. J. Neurosci. 34, 4589–4598 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  13. Vincent, K. et al. Intracellular mGluR5 plays a critical role in neuropathic pain. Nat. Commun. 7, 10604 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. White, A. D. et al. Spatial bias in cAMP generation determines biological responses to PTH type 1 receptor activation. Sci. Signal 14, eabc5944 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Okazaki, M. et al. Prolonged signaling at the parathyroid hormone receptor by peptide ligands targeted to a specific receptor conformation. Proc. Natl Acad. Sci. USA 105, 16525–16530 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Feinstein, T. N. et al. Noncanonical control of vasopressin receptor type 2 signaling by retromer and arrestin. J. Biol. Chem. 288, 27849–27860 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Kuna, R. S. et al. Glucagon-like peptide-1 receptor-mediated endosomal cAMP generation promotes glucose-stimulated insulin secretion in pancreatic β-cells. Am. J. Physiol. Endocrinol. Metab. 305, E161–E170 (2013).

    Article  CAS  PubMed  Google Scholar 

  18. Tian, X. et al. The α-arrestin ARRDC3 regulates the endosomal residence time and intracellular signaling of the β2-adrenergic receptor. J. Biol. Chem. 291, 14510–14525 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Thomsen, A. R. et al. GPCR-G protein-β-arrestin super-complex mediates sustained G protein signaling. Cell 166, 907–919 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Varandas, K. C., Irannejad, R. & von Zastrow, M. Retromer endosome exit domains serve multiple trafficking destinations and regulate local G protein activation by GPCRs. Curr. Biol. 26, 3129–3142 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Bowman, S. L., Shiwarski, D. J. & Puthenveedu, M. A. Distinct G protein-coupled receptor recycling pathways allow spatial control of downstream G protein signaling. J. Cell Biol. 214, 797–806 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Madamanchi, A. β-Adrenergic receptor signaling in cardiac function and heart failure. McGill J. Med. 10, 99 (2007).

    PubMed  PubMed Central  Google Scholar 

  23. Mutlu, G. M. & Factor, P. Alveolar epithelial β2-adrenergic receptors. Am. J. Respir. Cell Mol. Biol. 38, 127–134 (2008).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  25. Mayr, B. & Montminy, M. Transcriptional regulation by the phosphorylation-dependent factor CREB. Nat. Rev. Mol. Cell Biol. 2, 599–609 (2001).

    Article  CAS  PubMed  Google Scholar 

  26. 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 

  27. Schuster, M., Lipowsky, R., Assmann, M.-A., Lenz, P. & Steinberg, G. Transient binding of dynein controls bidirectional long-range motility of early endosomes. Proc. Natl Acad. Sci. USA 108, 3618–3623 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Reck-Peterson, S. L., Redwine, W. B., Vale, R. D. & Carter, A. P. The cytoplasmic dynein transport machinery and its many cargoes. Nat. Rev. Mol. Cell Biol. 19, 382–398 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Korolchuk, V. I. et al. Lysosomal positioning coordinates cellular nutrient responses. Nat. Cell Biol. 13, 453–460 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Sainath, R. & Gallo, G. The dynein inhibitor Ciliobrevin D inhibits the bidirectional transport of organelles along sensory axons and impairs NGF-mediated regulation of growth cones and axon branches. Dev. Neurobiol. 75, 757–777 (2015).

    Article  CAS  PubMed  Google Scholar 

  31. Violin, J. D. et al. β2-Adrenergic receptor signaling and desensitization elucidated by quantitative modeling of real time cAMP dynamics. J. Biol. Chem. 283, 2949–2961 (2008).

    Article  CAS  PubMed  Google Scholar 

  32. Temkin, P. et al. SNX27 mediates retromer tubule entry and endosome-to-plasma membrane trafficking of signalling receptors. Nat. Cell Biol. 13, 715–721 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  33. Odaka, H., Arai, S., Inoue, T. & Kitaguchi, T. Genetically-encoded yellow fluorescent cAMP indicator with an expanded dynamic range for dual-color imaging. PLoS ONE 9, e100252 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  34. Herskovits, J. S., Burgess, C. C., Obar, R. A. & Vallee, R. B. Effects of mutant rat dynamin on endocytosis. J. Cell Biol. 122, 565–578 (1993).

    Article  CAS  PubMed  Google Scholar 

  35. Bayle, J. H. et al. Rapamycin analogs with differential binding specificity permit orthogonal control of protein activity. Chem. Biol. 13, 99–107 (2006).

    Article  CAS  PubMed  Google Scholar 

  36. Nagendran, M., Riordan, D. P., Harbury, P. B. & Desai, T. J. Automated cell-type classification in intact tissues by single-cell molecular profiling. eLife 7, e30510 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  37. Semesta, K. M., Tian, R., Kampmann, M., von Zastrow, M. & Tsvetanova, N. G. A high-throughput CRISPR interference screen for dissecting functional regulators of GPCR/cAMP signaling. PLoS Genet. 16, e1009103 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Gillooly, D. J. et al. Localization of phosphatidylinositol 3-phosphate in yeast and mammalian cells. EMBO J. 19, 4577–4588 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Zhang, J. Z. et al. Phase separation of a PKA regulatory subunit controls cAMP compartmentation and oncogenic signaling. Cell 182, 1531–1544 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Harootunian, A. T. et al. Movement of the free catalytic subunit of cAMP-dependent protein kinase into and out of the nucleus can be explained by diffusion. Mol. Biol. Cell 4, 993–1002 (1993).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Smith, F. D. et al. Local protein kinase A action proceeds through intact holoenzymes. Science 356, 1288–1293 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Zhang, J.-F. et al. An ultrasensitive biosensor for high-resolution kinase activity imaging in awake mice. Nat. Chem. Biol. 17, 39–46 (2021).

    Article  CAS  PubMed  Google Scholar 

  43. Bock, A. et al. Optical mapping of cAMP signaling at the nanometer scale. Cell 182, 1519–1530 e1517 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Mo, G. C. et al. Genetically encoded biosensors for visualizing live-cell biochemical activity at super-resolution. Nat. Methods 14, 427–434 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Bers, D. M., Xiang, Y. K. & Zaccolo, M. Whole-cell cAMP and PKA activity are epiphenomena, nanodomain signaling matters. Physiology (Bethesda) 34, 240–249 (2019).

    CAS  PubMed  Google Scholar 

  46. Nigg, E., Hilz, H., Eppenberger, H. & Dutly, F. Rapid and reversible translocation of the catalytic subunit of cAMP‐dependent protein kinase type II from the Golgi complex to the nucleus. EMBO J. 4, 2801–2806 (1985).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Anton, S. E. et al. Receptor-associated independent cAMP nanodomains mediate spatiotemporal specificity of GPCR signaling. Cell 185, 1130–1142 (2022).

    Article  CAS  PubMed  Google Scholar 

  48. Koschinski, A. & Zaccolo, M. Activation of PKA in cell requires higher concentration of cAMP than in vitro: implications for compartmentalization of cAMP signalling. Sci. Rep. 7, 14090 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  49. English, A. R. & Voeltz, G. K. Endoplasmic reticulum structure and interconnections with other organelles. Cold Spring Harb. Perspect. Biol. 5, a013227 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  50. Jia, R. & Bonifacino, J. S. Lysosome positioning influences mTORC2 and AKT signaling. Mol. Cell 75, 26–38 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Riccio, A., Pierchala, B. A., Ciarallo, C. L. & Ginty, D. D. An NGF-TrkA-mediated retrograde signal to transcription factor CREB in sympathetic neurons. Science 277, 1097–1100 (1997).

    Article  CAS  PubMed  Google Scholar 

  52. Scerra, G. et al. Lysosomal positioning diseases: beyond substrate storage. Open Biol. 12, 220155 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Murphy, J. E., Padilla, B. E., Hasdemir, B., Cottrell, G. S. & Bunnett, N. W. Endosomes: a legitimate platform for the signaling train. Proc. Natl Acad. Sci. USA 106, 17615–17622 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Kwon, Y. et al. Non-canonical β-adrenergic activation of ERK at endosomes. Nature 611, 173–179 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Inda, C. et al. Different cAMP sources are critically involved in G protein-coupled receptor CRHR1 signaling. J. Cell Biol. 214, 181–195 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. 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 

  57. Legland, D., Arganda-Carreras, I. & Andrey, P. MorphoLibJ: integrated library and plugins for mathematical morphology with ImageJ. Bioinformatics 32, 3532–3534 (2016).

    Article  CAS  PubMed  Google Scholar 

  58. Rizk, A. et al. Segmentation and quantification of subcellular structures in fluorescence microscopy images using Squassh. Nat. Protoc. 9, 586–596 (2014).

    Article  CAS  PubMed  Google Scholar 

  59. Kamentsky, L. et al. Improved structure, function and compatibility for CellProfiler: modular high-throughput image analysis software. Bioinformatics 27, 1179–1180 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank members of the Tsvetanova Lab and R. Irannejad (UCSF) for valuable discussions of the project and feedback on the manuscript. Immunofluorescence microscopy imaging was performed using resources from the Duke Light Microscopy Core Facility, with specific training under L. Cameron, Y. Gao and B. Carlson. PKARegIIB-mCherry and EGFP-SNX27 were gifts from R. Irannejad (UCSF). SSF-β2-AR, RAB11a-GFP, DynK44E-mCherry, pcDNA3.0, eGFP-C1 and Endo-bPAC were gifts from M. von Zastrow (UCSF). MK1200 and dCas9-BFP-KRAB were gifts from M. Kampmann (UCSF). pBa.Kif1a 1-396.GFP was a gift from G. Banker and M. Bentley (Addgene plasmid, 45058; http://n2t.net/addgene:45058; RRID:Addgene_45058). pBa-KIF5C 559-tdTomato-FKBP was a gift from G. Banker and M. Bentley (Addgene plasmid, 64211; http://n2t.net/addgene:64211; RRID:Addgene_64211). pEGFP-FRB was a gift from K. Hahn (Addgene plasmid, 25919; http://n2t.net/addgene:25919; RRID:Addgene_25919). GFP-EEA1 wt was a gift from S. Corvera (Addgene plasmid, 42307; http://n2t.net/addgene:42307; RRID:Addgene_42307). pmCherry-N1-GalT was a gift from L. Lu (Addgene plasmid, 87327; http://n2t.net/addgene:87327; RRID:Addgene_87327). Flamindo2 and nlsFlamindo2 were gifts from T. Kitaguchi (Addgene plasmid, 73938; http://n2t.net/addgene:73938; RRID:Addgene_73938 and Addgene plasmid, 73939; http://n2t.net/addgene:73939; RRID:Addgene_73939). Figures 1a and 6f were created using BioRender. Research reported in this publication was supported by the National Institutes of Health (R01NS127847 and R35GM142640 to N.G.T., R01DK073368 to J.Z. and F31NS120567 to B.K.A.W.) and the American Heart Association (Predoctoral Fellowship 834472 to J.F.Z.).

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Authors and Affiliations

  1. Department of Pharmacology and Cancer Biology, Duke University, Durham, NC, USA

    Blair K. A. Willette & Nikoleta G. Tsvetanova

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

    Jin-Fan Zhang & Jin Zhang

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

    Jin-Fan Zhang & Jin Zhang

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

    Jin Zhang

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Contributions

N.G.T. supervised the project. N.G.T. and B.K.A.W. conceived the project and designed experiments. B.K.A.W. and J.F.Z. performed and analyzed all experiments. J.Z. supervised and coordinated experiments involving the generation and characterization of nuclear ExRai-AKAR2 sensor. N.G.T., B.K.A.W. and J.F.Z. interpreted results. N.G.T. and B.K.A.W. wrote the manuscript. All authors edited the manuscript.

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Correspondence to Nikoleta G. Tsvetanova.

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Nature Chemical Biology thanks Davide Calebiro and the other, anonymous, reviewers for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–14 and uncropped western blot for Supplementary Fig. 8c.

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Supplementary Data

Supporting data for Supplementary Figs. 1–14.

Supplementary Video 1

Live-cell imaging of HEK293 cells expressing CID components. The video shows endosome distribution after 5 min of AP21967 (rapalog) treatment.

Supplementary Video 2

Live-cell imaging of HEK293 cells expressing Flamindo2-FRB-EEA1 and Kif1a-tdTomato-FKBP. Cells were pretreated with AP21967 (rapalog) for 30 min.

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Numerical source data for Fig. 6.

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Willette, B.K.A., Zhang, JF., Zhang, J. et al. Endosome positioning coordinates spatially selective GPCR signaling. Nat Chem Biol 20, 151–161 (2024). https://doi.org/10.1038/s41589-023-01390-7

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