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.
MeSH terms
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Animals
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Cell Polarity / physiology
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Colorectal Neoplasms / genetics
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DNA-Binding Proteins / deficiency
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DNA-Binding Proteins / genetics
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DNA-Binding Proteins / metabolism
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Feedback, Physiological
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Female
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Frizzled Receptors / metabolism
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HEK293 Cells
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Humans
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Low Density Lipoprotein Receptor-Related Protein-6 / metabolism
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Male
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Mice
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Mice, Knockout
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Oncogene Proteins / deficiency
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Oncogene Proteins / genetics
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Oncogene Proteins / metabolism
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Protein Stability
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Protein Structure, Tertiary
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Receptors, G-Protein-Coupled / deficiency
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Receptors, G-Protein-Coupled / genetics
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Receptors, G-Protein-Coupled / metabolism
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Receptors, Wnt / metabolism*
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Thrombospondins / metabolism*
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Ubiquitin-Protein Ligases / chemistry
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Ubiquitin-Protein Ligases / deficiency*
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Ubiquitin-Protein Ligases / genetics
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Ubiquitin-Protein Ligases / metabolism*
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Ubiquitination
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Wnt Signaling Pathway
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Xenopus
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Zebrafish
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beta Catenin / metabolism
Substances
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DNA-Binding Proteins
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Frizzled Receptors
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LGR4 protein, human
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LRP6 protein, human
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Low Density Lipoprotein Receptor-Related Protein-6
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Oncogene Proteins
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RSPO1 protein, human
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Receptors, G-Protein-Coupled
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Receptors, Wnt
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Thrombospondins
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beta Catenin
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RNF43 protein, human
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Ubiquitin-Protein Ligases
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ZNRF3 protein, human
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Znrf3 protein, mouse