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


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.


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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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Authors and Affiliations



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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The authors declare no competing financial interests.

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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).

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