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ZNRF3 promotes Wnt receptor turnover in an R-spondin-sensitive manner

Nature volume 485, pages 195–200 (2012)Cite this article

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Abstract

R-spondin proteins strongly potentiate Wnt signalling and function as stem-cell growth factors. Despite the biological and therapeutic significance, the molecular mechanism of R-spondin action remains unclear. Here we show that the cell-surface transmembrane E3 ubiquitin ligase zinc and ring finger 3 (ZNRF3) and its homologue ring finger 43 (RNF43) are negative feedback regulators of Wnt signalling. ZNRF3 is associated with the Wnt receptor complex, and inhibits Wnt signalling by promoting the turnover of frizzled and LRP6. Inhibition of ZNRF3 enhances Wnt/β-catenin signalling and disrupts Wnt/planar cell polarity signalling in vivo. Notably, R-spondin mimics ZNRF3 inhibition by increasing the membrane level of Wnt receptors. Mechanistically, R-spondin interacts with the extracellular domain of ZNRF3 and induces the association between ZNRF3 and LGR4, which results in membrane clearance of ZNRF3. These data suggest that R-spondin enhances Wnt signalling by inhibiting ZNRF3. Our study provides new mechanistic insights into the regulation of Wnt receptor turnover, and reveals ZNRF3 as a tractable target for therapeutic exploration.

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Figure 1: ZNRF3 negatively modulates Wnt signalling.
Figure 2: ZNRF3 regulates the level of Wnt receptors on the cell surface.
Figure 3: ZNRF3 regulates the stability of LRP6 and frizzled through ubiquitylation.
Figure 4: RSPO1 increases the cell-surface level of frizzled proteins and functionally interacts with the extracellular domain of ZNRF3.
Figure 5: RSPO1 increases the interaction between ZNRF3 and LGR4 and induces membrane clearance of ZNRF3.
Figure 6: ZNRF3 regulates both Wnt/β-catenin and Wnt/PCP signalling in vivo.

References

  1. Clevers, H. Wnt/β-catenin signaling in development and disease. Cell 127, 469–480 (2006)

    Article  CAS  Google Scholar 

  2. MacDonald, B. T., Tamai, K. & He, X. Wnt/β-catenin signaling: components, mechanisms, and diseases. Dev. Cell 17, 9–26 (2009)

    Article  CAS  Google Scholar 

  3. Simons, M. & Mlodzik, M. Planar cell polarity signaling: from fly development to human disease. Annu. Rev. Genet. 42, 517–540 (2008)

    Article  CAS  Google Scholar 

  4. Kazanskaya, O. et al. R-Spondin2 is a secreted activator of Wnt/β-catenin signaling and is required for Xenopus myogenesis. Dev. Cell 7, 525–534 (2004)

    Article  CAS  Google Scholar 

  5. Kim, K. A. et al. Mitogenic influence of human R-spondin1 on the intestinal epithelium. Science 309, 1256–1259 (2005)

    Article  CAS  ADS  Google Scholar 

  6. Kim, K. A. et al. R-Spondin family members regulate the Wnt pathway by a common mechanism. Mol. Biol. Cell 19, 2588–2596 (2008)

    Article  CAS  Google Scholar 

  7. Ohkawara, B., Glinka, A. & Niehrs, C. Rspo3 binds syndecan 4 and induces Wnt/PCP signaling via clathrin-mediated endocytosis to promote morphogenesis. Dev. Cell 20, 303–314 (2011)

    Article  CAS  Google Scholar 

  8. Aoki, M. et al. R-spondin3 is required for mouse placental development. Dev. Biol. 301, 218–226 (2007)

    Article  CAS  Google Scholar 

  9. Blaydon, D. C. et al. The gene encoding R-spondin 4 (RSPO4), a secreted protein implicated in Wnt signaling, is mutated in inherited anonychia. Nature Genet. 38, 1245–1247 (2006)

    Article  CAS  Google Scholar 

  10. Kazanskaya, O. et al. The Wnt signaling regulator R-spondin 3 promotes angioblast and vascular development. Development 135, 3655–3664 (2008)

    Article  CAS  Google Scholar 

  11. Parma, P. et al. R-spondin1 is essential in sex determination, skin differentiation and malignancy. Nature Genet. 38, 1304–1309 (2006)

    Article  CAS  Google Scholar 

  12. Sato, T. et al. Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts. Nature 469, 415–418 (2011)

    Article  CAS  ADS  Google Scholar 

  13. Ootani, A. et al. Sustained in vitro intestinal epithelial culture within a Wnt-dependent stem cell niche. Nature Med. 15, 701–706 (2009)

    Article  CAS  Google Scholar 

  14. Zhao, J. et al. R-Spondin1 protects mice from chemotherapy or radiation-induced oral mucositis through the canonical Wnt/β-catenin pathway. Proc. Natl Acad. Sci. USA 106, 2331–2336 (2009)

    Article  CAS  ADS  Google Scholar 

  15. Nam, J. S., Turcotte, T. J., Smith, P. F., Choi, S. & Yoon, J. K. Mouse cristin/R-spondin family proteins are novel ligands for the Frizzled 8 and LRP6 receptors and activate β-catenin-dependent gene expression. J. Biol. Chem. 281, 13247–13257 (2006)

    Article  CAS  Google Scholar 

  16. Wei, Q. et al. R-spondin1 is a high affinity ligand for LRP6 and induces LRP6 phosphorylation and β-catenin signaling. J. Biol. Chem. 282, 15903–15911 (2007)

    Article  CAS  Google Scholar 

  17. Binnerts, M. E. et al. R-Spondin1 regulates Wnt signaling by inhibiting internalization of LRP6. Proc. Natl Acad. Sci. USA 104, 14700–14705 (2007)

    Article  CAS  ADS  Google Scholar 

  18. Carmon, K. S., Gong, X., Lin, Q., Thomas, A. & Liu, Q. R-spondins function as ligands of the orphan receptors LGR4 and LGR5 to regulate Wnt/β-catenin signaling. Proc. Natl Acad. Sci. USA 108, 11452–11457 (2011)

    Article  CAS  ADS  Google Scholar 

  19. de Lau, W. et al. Lgr5 homologues associate with Wnt receptors and mediate R-spondin signalling. Nature 476, 293–297 (2011)

    Article  CAS  ADS  Google Scholar 

  20. Glinka, A. et al. LGR4 and LGR5 are R-spondin receptors mediating Wnt/b-catenin and Wnt/PCP signalling. EMBO Rep. 12, 1055–1061 (2011)

    Article  CAS  Google Scholar 

  21. Chen, B. et al. Small molecule-mediated disruption of Wnt-dependent signaling in tissue regeneration and cancer. Nature Chem. Biol. 5, 100–107 (2009)

    Article  CAS  ADS  Google Scholar 

  22. Gonzalez-Sancho, J. M., Brennan, K. R., Castelo-Soccio, L. A. & Brown, A. M. Wnt proteins induce dishevelled phosphorylation via an LRP5/6- independent mechanism, irrespective of their ability to stabilize beta-catenin. Mol. Cell. Biol. 24, 4757–4768 (2004)

    Article  CAS  Google Scholar 

  23. Gurney, A. L. Frizzled-binding agents and uses thereof. US patent 201037041. (2011)

  24. Mukai, A. et al. Balanced ubiquitylation and deubiquitylation of Frizzled regulate cellular responsiveness to Wg/Wnt. EMBO J. 29, 2114–2125 (2010)

    Article  CAS  Google Scholar 

  25. Kim, C. H. et al. Repressor activity of Headless/Tcf3 is essential for vertebrate head formation. Nature 407, 913–916 (2000)

    Article  CAS  ADS  Google Scholar 

  26. Nasevicius, A. et al. Evidence for a frizzled-mediated wnt pathway required for zebrafish dorsal mesoderm formation. Development 125, 4283–4292 (1998)

    CAS  PubMed  Google Scholar 

  27. Smith, A. N., Miller, L. A., Song, N., Taketo, M. M. & Lang, R. A. The duality of β-catenin function: a requirement in lens morphogenesis and signaling suppression of lens fate in periocular ectoderm. Dev. Biol. 285, 477–489 (2005)

    Article  CAS  Google Scholar 

  28. Kreslova, J. et al. Abnormal lens morphogenesis and ectopic lens formation in the absence of β-catenin function. Genesis 45, 157–168 (2007)

    Article  CAS  Google Scholar 

  29. Machon, O. et al. Lens morphogenesis is dependent on Pax6-mediated inhibition of the canonical Wnt/β-catenin signaling in the lens surface ectoderm. Genesis 48, 86–95 (2010)

    CAS  PubMed  Google Scholar 

  30. Wang, J. et al. Dishevelled genes mediate a conserved mammalian PCP pathway to regulate convergent extension during neurulation. Development 133, 1767–1778 (2006)

    Article  CAS  Google Scholar 

  31. Wu, J. et al. Whole-exome sequencing of neoplastic cysts of the pancreas reveals recurrent mutations in components of ubiquitin-dependent pathways. Proc. Natl Acad. Sci. USA 108, 21188–21193 (2011)

    Article  CAS  ADS  Google Scholar 

  32. Huang, S. M. et al. Tankyrase inhibition stabilizes axin and antagonizes Wnt signalling. Nature 461, 614–620 (2009)

    Article  CAS  ADS  Google Scholar 

  33. Zhang, Y. et al. RNF146 is a poly(ADP-ribose)-directed E3 ligase that regulates axin degradation and Wnt signalling. Nature Cell Biol. 13, 623–629 (2011)

    Article  CAS  Google Scholar 

  34. Nusslein-Volhard, C. & Dahm, R. Zebrafish. A Practical Approach. (Oxford Univ. Press, 2002)

    Google Scholar 

  35. Westerfield, M. The Zebrafish Book: a Guide for the Laboratory Use of Zebrafish (Brachydanio rerio). (Univ. Oregon Press, 1995)

    Google Scholar 

  36. Goentoro, L. & Kirschner, M. W. Evidence that fold-change, and not absolute level, of β-catenin dictates Wnt signaling. Mol. Cell 36, 872–884 (2009)

    Article  CAS  Google Scholar 

  37. Gerdes, J. M. et al. Disruption of the basal body compromises proteasomal function and perturbs intracellular Wnt response. Nature Genet. 39, 1350–1360 (2007)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank G. Yu, T. Lewis, Q. Song, J. Garver, J. Wang, B. Lu, B. Guo, Q. Fang, X. Shi, J. Sprunger and R.Freeman for technical assistance, R.-F. Kwong and T. Fleming for generating ZNRF3 antibodies, K. Lee and J. Halupowski for mouse maintenance, and J. Tchorz, A. Jaffe, N. Kubica, M. Hild, J. Solomon, Y. Yang, J. Tchorz, E. Wiellette, G. Michaud, D. Cutis and K. Seuwen for comments and advice. We also thank S. Goto for providing FZD4 and FZD4 K0 plasmids.

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Author notes

  1. Yue Zhang

    Present address: Present address: AstraZeneca, 35 Gatehouse Drive, Waltham, Massachusetts 02451, USA.,

  2. Huai-Xiang Hao and Yang Xie: These authors contributed equally to this work.

Authors and Affiliations

  1. Novartis Institutes for Biomedical Research, 250 Massachusetts Avenue, Cambridge, 02139, Massachusetts, USA

    Huai-Xiang Hao, Yang Xie, Yue Zhang, Olga Charlat, Emma Oster, Monika Avello, Hong Lei, Craig Mickanin, Dong Liu, Xiaohong Mao, Qicheng Ma, Raffaella Zamponi, Peter M. Finan, Jeffery A. Porter, Fabrizio C. Serluca & Feng Cong

  2. Novartis Institutes for Biomedical Research, Novartis Pharma AG, Postfach CH-4002 Basel, Switzerland,

    Heinz Ruffner & Tewis Bouwmeester

  3. Department of Systems Biology, Harvard Medical School, Boston, 02115, Massachusetts, USA

    Marc W. Kirschner

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Contributions

H.-X.H. initiated the project, characterized the function of ZNRF3 in cultured cells and mice, and identified ZNRF3 antagonistic antibodies. H.-X.H. and Y.X. discovered the R-spondin and ZNRF3 link. Y.X. led mechanistic studies on R-spondin, LGR4 and ZNRF3. H.-X.H., Y.X., Y.Z., H.L., C.M., D.L., H.R., X.M., Q.M., T.B., P.M.F., M.W.K., J.A.P., F.C.S. and F.C. conceived and designed the study. H.-X.H., Y.X., Y.Z., O.C., E.O., M.A., H.L., C.M., D.L., H.R., X.M., Q.M., R.Z., F.C.S. and F.C. designed and implemented experiments. H.-X.H., Y.X., Y.Z. and F.C. wrote the manuscript.

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Correspondence to Feng Cong.

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Hao, HX., Xie, Y., Zhang, Y. et al. ZNRF3 promotes Wnt receptor turnover in an R-spondin-sensitive manner. Nature 485, 195–200 (2012). https://doi.org/10.1038/nature11019

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