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.

A general approach for engineering RTKs optically controlled with far-red light

Nature Methods volume 19, pages 871–880 (2022)Cite this article

Subjects

Abstract

Regulation of receptor tyrosine kinase (RTK) activity is necessary for studying cell signaling pathways in health and disease. We developed a generalized approach for engineering RTKs optically controlled with far-red light. We targeted the bacterial phytochrome DrBphP to the cell surface and allowed its light-induced conformational changes to be transmitted across the plasma membrane via transmembrane helices to intracellular RTK domains. Systematic optimization of these constructs has resulted in optically regulated epidermal growth factor receptor, HER2, TrkA, TrkB, FGFR1, IR1, cKIT and cMet, named eDrRTKs. eDrRTKs induced downstream signaling in mammalian cells in tens of seconds. The ability to activate eDrRTKs with far-red light enabled spectral multiplexing with fluorescent probes operating in a shorter spectral range, allowing for all-optical assays. We validated eDrTrkB performance in mice and found that minimally invasive stimulation in the neocortex with penetrating via skull far-red light-induced neural activity, early immediate gene expression and affected sleep patterns.

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

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Learn more

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

Fig. 1: Workflow of the eDrRTK design.
Fig. 2: Induction of Elk-1-dependent luciferase reporter expression by eDrRTKs.
Fig. 3: Phosphorylation of eDrRTKs and ERK1/2 on short-term action of FR light.
Fig. 4: Activation of eDrTrkB in neuronal cells.
Fig. 5: Amounts of NREM sleep increased in mice expressing eDrTrkB in the cerebral cortex following exposure to FR light.
Fig. 6: NeuN, mCherry and cFos expression in the somatosensory cortex of mice injected with AAV9 encoding mCherry-eDrTrkB-CW3SL.

Data availability

The main data supporting the findings of this study are available within the article and its supplementary information. The main resultant plasmids encoding eDrRTKs are deposited in the Addgene depository (nos. 183994–184001). The code used in this study for LED switching is available at https://docs.arduino.cc/built-in-examples/basics/Blink. Source data are provided with this paper.

References

  1. Lemmon, M. A. & Schlessinger, J. Cell signaling by receptor tyrosine kinases. Cell 141, 1117–1134 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Leopold, A. V. & Verkhusha, V. V. Light control of RTK activity: from technology development to translational research. Chem. Sci. 11, 10019–10034 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Grusch, M. et al. Spatio-temporally precise activation of engineered receptor tyrosine kinases by light. EMBO J. 33, 1713–1726 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Huang, P. et al. Optical activation of TrkB signaling. J. Mol. Biol. 432, 3761–3770 (2020).

    Article  CAS  PubMed  Google Scholar 

  5. Piatkevich, K. D., Subach, F. V. & Verkhusha, V. V. Engineering of bacterial phytochromes for near-infrared imaging, sensing, and light-control in mammals. Chem. Soc. Rev. 42, 3441–3452 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Shcherbakova, D. M. Near-infrared and far-red genetically encoded indicators of neuronal activity. J. Neurosci. Meth. 362, 109314 (2021).

    Article  Google Scholar 

  7. Manoilov, K. Y., Verkhusha, V. V. & Shcherbakova, D. M. A guide to the optogenetic regulation of endogenous molecules. Nat. Methods 18, 1027–1037 (2021).

    Article  CAS  PubMed  Google Scholar 

  8. Kaberniuk, A. A., Baloban, M., Monakhov, M. V., Shcherbakova, D. M. & Verkhusha, V. V. Single-component near-infrared optogenetic systems for gene transcription regulation. Nat. Commun. 12, 3859 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Reichhart, E., Ingles-Prieto, A., Tichy, A. M., McKenzie, C. & Janovjak, H. A phytochrome sensory domain permits receptor activation by red light. Angew. Chem. Int. Ed. Engl. 55, 6339–6342 (2016).

    Article  CAS  PubMed  Google Scholar 

  10. Arkhipov, A. et al. Architecture and membrane interactions of the EGF receptor. Cell 152, 557–569 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Maruyama, I. N. Mechanisms of activation of receptor tyrosine kinases: monomers or dimers. Cells 3, 304–330 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  12. Shen, J. et al. Extracellular juxtamembrane motif critical for TrkB preformed dimer and activation. Cells 8, 932 (2019).

  13. Sparrow, L. G. et al. The disulfide bonds in the C-terminal domains of the human insulin receptor ectodomain. J. Biol. Chem. 272, 29460–29467 (1997).

    Article  CAS  PubMed  Google Scholar 

  14. Bell, C. A. et al. Rotational coupling of the transmembrane and kinase domains of the Neu receptor tyrosine kinase. Mol. Biol. Cell 11, 3589–3599 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Leopold, A. V., Pletnev, S. & Verkhusha, V. V. Bacterial phytochrome as a scaffold for engineering of receptor tyrosine kinases controlled with near-infrared light. J. Mol. Biol. 432, 3749–3760 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Leopold, A. V., Chernov, K. G., Shemetov, A. A. & Verkhusha, V. V. Neurotrophin receptor tyrosine kinases regulated with near-infrared light. Nat. Commun. 10, 1129 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  17. Takala, H., Lehtivuori, H., Hammaren, H., Hytonen, V. P. & Ihalainen, J. A. Connection between absorption properties and conformational changes in Deinococcus radiodurans phytochrome. Biochemistry 53, 7076–7085 (2014).

    Article  CAS  PubMed  Google Scholar 

  18. Burgie, E. S., Zhang, J. & Vierstra, R. D. Crystal structure of Deinococcus phytochrome in the photoactivated state reveals a cascade of structural rearrangements during photoconversion. Structure 24, 448–457 (2016).

    Article  CAS  PubMed  Google Scholar 

  19. Bjorling, A. et al. Structural photoactivation of a full-length bacterial phytochrome. Sci. Adv. 2, e1600920 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  20. Gotoh, N. Regulation of growth factor signaling by FRS2 family docking/scaffold adaptor proteins. Cancer Sci. 99, 1319–1325 (2008).

    Article  CAS  PubMed  Google Scholar 

  21. Gasser, C. et al. Engineering of a red-light-activated human cAMP/cGMP-specific phosphodiesterase. Proc. Natl Acad. Sci. USA 111, 8803–8808 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Gupta, V. K., You, Y., Gupta, V. B., Klistorner, A. & Graham, S. L. TrkB receptor signalling: implications in neurodegenerative, psychiatric and proliferative disorders. Int. J. Mol. Sci. 14, 10122–10142 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  23. Honarvar, H. et al. Evaluation of HER2-specific peptide ligand for its employment as radiolabeled imaging probe. Sci. Rep. 8, 2998 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  24. Singh, B., Carpenter, G. & Coffey, R. J. EGF receptor ligands: recent advances. F1000Res. 5, 2270 (2016).

  25. Gutierrez, C. & Schiff, R. HER2: biology, detection, and clinical implications. Arch. Pathol. Lab. Med. 135, 55–62 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  26. D’Uva, G. et al. ERBB2 triggers mammalian heart regeneration by promoting cardiomyocyte dedifferentiation and proliferation. Nat. Cell Biol. 17, 627–638 (2015).

    Article  PubMed  CAS  Google Scholar 

  27. Barash, S., Wang, W. & Shi, Y. Human secretory signal peptide description by hidden Markov model and generation of a strong artificial signal peptide for secreted protein expression. Biochem. Biophys. Res. Commun. 294, 835–842 (2002).

    Article  CAS  PubMed  Google Scholar 

  28. Barlowe, C. Signals for COPII-dependent export from the ER: what’s the ticket out? Trends Cell Biol. 13, 295–300 (2003).

    Article  CAS  PubMed  Google Scholar 

  29. Ma, D. et al. Golgi export of the Kir2.1 channel is driven by a trafficking signal located within its tertiary structure. Cell 145, 1102–1115 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Lu, P. et al. Accurate computational design of multipass transmembrane proteins. Science 359, 1042–1046 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Joo, D. et al. Biphasic activation of extracellular signal-regulated kinase (ERK) 1/2 in epidermal growth factor (EGF)-stimulated SW480 colorectal cancer cells. BMB Rep. 49, 220–225 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Huang, Z. et al. Two FGF receptor kinase molecules act in concert to recruit and transphosphorylate phospholipase cgamma. Mol. Cell 61, 98–110 (2016).

    Article  CAS  PubMed  Google Scholar 

  33. Chen, T. W. et al. Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature 499, 295–300 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Minichiello, L. TrkB signalling pathways in LTP and learning. Nat. Rev. Neurosci. 10, 850–860 (2009).

    Article  CAS  PubMed  Google Scholar 

  35. Bjorkholm, C. & Monteggia, L. M. BDNF—a key transducer of antidepressant effects. Neuropharmacology 102, 72–79 (2016).

    Article  PubMed  CAS  Google Scholar 

  36. Faraguna, U., Vyazovskiy, V. V., Nelson, A. B., Tononi, G. & Cirelli, C. A causal role for brain-derived neurotrophic factor in the homeostatic regulation of sleep. J. Neurosci. 28, 4088–4095 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Huber, R., Tononi, G. & Cirelli, C. Exploratory behavior, cortical BDNF expression, and sleep homeostasis. Sleep 30, 129–139 (2007).

    Article  PubMed  Google Scholar 

  38. Hairston, I. S. et al. Sleep deprivation effects on growth factor expression in neonatal rats: a potential role for BDNF in the mediation of delta power. J. Neurophysiol. 91, 1586–1595 (2004).

    Article  CAS  PubMed  Google Scholar 

  39. Taishi, P. et al. Conditions that affect sleep alter the expression of molecules associated with synaptic plasticity. Am. J. Physiol. Regul. Integr. Comp. Physiol. 281, R839–R845 (2001).

    Article  CAS  PubMed  Google Scholar 

  40. Kushikata, T., Fang, J. & Krueger, J. M. Brain-derived neurotrophic factor enhances spontaneous sleep in rats and rabbits. Am. J. Physiol. 276, R1334–R1338 (1999).

    CAS  PubMed  Google Scholar 

  41. Bachmann, V. et al. The BDNF Val66Met polymorphism modulates sleep intensity: EEG frequency- and state-specificity. Sleep 35, 335–344 (2012).

    PubMed  PubMed Central  Google Scholar 

  42. Uda, Y. et al. Efficient synthesis of phycocyanobilin in mammalian cells for optogenetic control of cell signaling. Proc. Natl Acad. Sci. USA 114, 11962–11967 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Dehkhoda, F., Lee, C. M. M., Medina, J. & Brooks, A. J. The growth hormone receptor: mechanism of receptor activation, cell signaling, and physiological aspects. Front Endocrinol. 9, 35 (2018).

    Article  Google Scholar 

  44. Watowich, S. S. The erythropoietin receptor: molecular structure and hematopoietic signaling pathways. J. Investig. Med 59, 1067–1072 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Maghazachi, A. A. Insights into seven and single transmembrane-spanning domain receptors and their signaling pathways in human natural killer cells. Pharm. Rev. 57, 339–357 (2005).

    Article  CAS  PubMed  Google Scholar 

  46. Botos, I., Segal, D. M. & Davies, D. R. The structural biology of Toll-like receptors. Structure 19, 447–459 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Fleishman, S. J., Harrington, S., Friesner, R. A., Honig, B. & Ben-Tal, N. An automatic method for predicting transmembrane protein structures using cryo-EM and evolutionary data. Biophys. J. 87, 3448–3459 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Unterreitmeier, S. et al. Phenylalanine promotes interaction of transmembrane domains via GxxxG motifs. J. Mol. Biol. 374, 705–718 (2007).

    Article  CAS  PubMed  Google Scholar 

  49. Bathina, S. & Das, U. N. Brain-derived neurotrophic factor and its clinical implications. Arch. Med Sci. 11, 1164–1178 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Kraemer, B. R., Yoon, S. O. & Carter, B. D. The biological functions and signaling mechanisms of the p75 neurotrophin receptor. Handb. Exp. Pharmacol. 220, 121–164 (2014).

    Article  CAS  PubMed  Google Scholar 

  51. Numakawa, T. & Odaka, H. Brain-derived neurotrophic factor signaling in the pathophysiology of Alzheimer’s disease: beneficial effects of flavonoids for neuroprotection. Int. J. Mol. Sci. 22, 5719 (2021).

  52. Sungur, A. O. et al. Aberrant cognitive phenotypes and altered hippocampal BDNF expression related to epigenetic modifications in mice lacking the post-synaptic scaffolding protein SHANK1: implications for autism spectrum disorder. Hippocampus 27, 906–919 (2017).

    Article  CAS  PubMed  Google Scholar 

  53. Castren, E. & Monteggia, L. M. Brain-derived neurotrophic factor signaling in depression and antidepressant action. Biol. Psychiatry 90, 128–136 (2021).

    Article  CAS  PubMed  Google Scholar 

  54. Zielinski, M. R., Gerashchenko, L., Karpova, S. A. & Gerashchenko, D. A novel telemetric system to measure polysomnographic biopotentials in freely moving animals. J. Neurosci. Methods 216, 79–86 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  55. Morairty, S. R. et al. A role for cortical nNOS/NK1 neurons in coupling homeostatic sleep drive to EEG slow wave activity. Proc. Natl Acad. Sci. USA 110, 20272–20277 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Prerau, M. J., Brown, R. E., Bianchi, M. T., Ellenbogen, J. M. & Purdon, P. L. Sleep neurophysiological dynamics through the lens of multitaper spectral analysis. Physiol. 32, 60–92 (2017).

    Article  Google Scholar 

Download references

Acknowledgements

We thank J. Ihalainen (University of Jyväskylä, Finland) for the DrBphP gene and D. Lindholm (University of Helsinki, Finland) for the cell lines. This work was supported by the grants GM122567, AG061774 and NS106406 from the US National Institutes of Health and 322226 from the Academy of Finland.

Author information

Authors and Affiliations

  1. Medicum, Faculty of Medicine, University of Helsinki, Helsinki, Finland

    Anna V. Leopold & Vladislav V. Verkhusha

  2. Department of Psychiatry, Harvard Medical School, West Roxbury, MA, USA

    Stephen Thankachan, Chun Yang & Dmitry Gerashchenko

  3. Department of Genetics and Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, NY, USA

    Vladislav V. Verkhusha

Authors
  1. Anna V. Leopold
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Stephen Thankachan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Chun Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Dmitry Gerashchenko
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Vladislav V. Verkhusha
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

A.V.L. engineered and characterized the optogenetic constructs. S.T. and C.Y. performed animal and histochemical studies. V.V.V. planned and directed the project and together with A.V.L. and D.G. designed the experiments, analyzed the data and wrote the manuscript. All authors revised the manuscript.

Corresponding author

Correspondence to Vladislav V. Verkhusha.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Methods thanks Bianxiao Cui, Benjamin Rost and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Rita Strack was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Additional information

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

Extended data

Extended Data Fig. 1 Structural predeterminants of opto-RTK engineering.

(a) Various hypotheses of RTK activation. Top: RTK dimerization: inactive RTKs exist as monomers and ligand binding causes their dimerization and consequent activation. Bottom: Rotational coupling: inactive RTKs exist as preformed dimers and ligand binding causes conformational changes resulting in RTK activation. (b) Structural changes occurring in EGFR receptor. Left: EGFR receptor exists as a preformed inactive dimer. Right: EGF ligand binding causes conformational changes and EGFR activation. Ab initio models of EGFR in active and inactive states are modified with permission from15. (c) Light-induced conformational changes in DrBphP–PCM obligate dimer cause distance increase between C-termini of DrBphP-PCM protomers17. (d) Schematic representation of proposed opto-RTK design (top) and mechanism of its activation (bottom). DrBphP-PCM targeted to the extracellular surface by Igκ signaling peptide is connected to cytoplasmic RTK domain (cytoRTK) via transmembrane (tmRTK) domain. In darkness or near-infrared light, opto-RTK remains inactive. Far-red light causes DrBphP-PCM conformational changes, which are transmitted to cytoplasmic RTK domains, causing their re-orientation and trans-phosphorylation.

Extended Data Fig. 2 Engineering and characterization of opto-EGFR and opto-HER2 prototypes.

(a) Left: Ligand binding causes RTK autophosphorylation, interaction with Grb2 and SOS, and results in activation of ERK1/2 pathway consisting of RAS, RAF, MEK, and ERK1/2 kinases. Consequently, ERK1/2 activation leads to immediate early gene (IEG) expression driven by transcription factor Elk-1. Right: Likewise, activation of chimeric opto-RTK with far-red light leads to activation of ERK1/2 pathway and induction of Elk-1 dependent IEG expression. (b) Scheme of luciferase reporter assay. Top: ERK1/2 is inactive, and Elk-1 fused to Gal4DBD is monomeric and inactive. Bottom: ERK1/2 is active, and activated ERK1/2 phosphorylates Elk-1. Phosphorylated Elk-1-Gal4DBD fusion dimerizes, binds to 5x UAS sequence, and drives Firefly luciferase (FLuc) reporter expression. (c, d) Light-induced activation of Elk-1-dependent Firefly luciferase reporter expression by opto-EGFR (c) and opto-HER2 (d) prototypes in PC6-3 cells. Renilla luciferase (RLuc) was co-transfected as a normalization control. In the darkness, Firefly luciferase expression is suppressed. 660 nm light activates eDrRTKs and upregulates Firefly luciferase expression. Expression of Firefly and Renilla luciferases was analyzed after 24 h of illumination. Relative luciferase expression activity (RLA) is shown as a ratio of the Firefly luciferase expression relative bioluminescence units to the Renilla luciferase expression relative bioluminescence units (FLuc/RLuc). 25 µM BV was added to the culture medium in all experiments. Data are presented as mean values, error bars represent standard deviation (s.d.), (n≥3, transfection experiments).

Source data

Extended Data Fig. 3 Regulation of PLCγ signaling by eDrRTKs.

(a) Top: eDrRTK activates PLCγ. PLCγ catalyzes PIP2 hydrolysis and formation of IP3 and DAG. IP3 interacts with IP3R channels in ER after which they become permeable to Ca2+. In turn, Ca2+ interacts with ORAI channels in the plasma membrane and induces Ca2+ entry from the extracellular space. Bottom: Scheme of the plasmid encoding eDrRTK and GCaMP6m Ca2+ indicator via IRES2. (b) Western blots of phospho-PLCγ, total PLCγ and GAPDH in HEK293 cell lysates and quantification of lane intensities of phospho-PLCγ normalized to GAPDH lane intensities (LIs). (c) Representative HEK293 cells co-transfected with eDrRTKs and GCaMP6m imaged before, during, and after 25 s illumination with 660 nm FR light, and their corresponding calcium flux traces are shown. Red arrows indicate the time when 660 nm light-activation started. After 25 s of 660 nm illumination, inactivating 780 nm light was switched on. Black arrows 1, 2 and 3 indicate time-points when confocal images before illumination, at 660 nm illumination, and after illumination were taken, respectively. 25 µM BV was added to the culture medium in all experiments. Scale bars, 10 µm. Data are presented as mean values, error bars represent standard deviation (s.d.), (n≥3, transfection experiments).

Source data

Supplementary information

Supplementary Information

Supplementary Figs. 1–21, Table 1 and References.

Reporting Summary

Supplementary Data

A workbook with tabs, containing statistical source data for the Supplementary Information.

Source data

Source Data Fig. 2

Statistical source data.

Source Data Fig. 3

Unprocessed western blots and statistical source data.

Source Data Fig. 4

Unprocessed western blots and statistical source data.

Source Data Fig. 5

Statistical source data.

Source Data Extended Data Fig. 2

Statistical source data.

Source Data Extended Data Fig. 3

Unprocessed western blots and statistical source data.

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Leopold, A.V., Thankachan, S., Yang, C. et al. A general approach for engineering RTKs optically controlled with far-red light. Nat Methods 19, 871–880 (2022). https://doi.org/10.1038/s41592-022-01517-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41592-022-01517-z

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