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. ZNRF3 and RNF43 are identified as negative feedback regulators of Wnt signalling; the stem-cell growth factor R-spondin is shown to potentiate Wnt signalling by inhibiting ZNRF3. ZNRF3 protein inhibits Wnt signalling The R-spondin proteins are secreted molecules that function as stem-cell growth factors and potentiate Wnt signalling by binding LGR4 family receptors, but their precise mechanism of action remains unclear. Here, the transmembrane E3 ubiquitin ligase ZNRF3 is identified as an inhibitor of Wnt signalling that acts by promoting the turnover of Wnt receptors. R-spondin potentiates Wnt signalling by inhibiting ZNRF3 in a mechanism dependent on LGR4, resulting in the accumulation of Wnt receptors. Given the importance of Wnt signalling in cancer, ZNRF3 may be a target for therapeutic intervention.