USP42 protects ZNRF3/RNF43 from R-spondin-dependent clearance and inhibits Wnt signalling

The tumour suppressors RNF43 and ZNRF3 play a central role in development and tissue homeostasis by promoting the turnover of the Wnt receptors LRP6 and Frizzled (FZD). The stem cell growth factor R-spondin induces auto-ubiquitination and membrane clearance of ZNRF3/RNF43 to promote Wnt signalling. However, the deubiquitinase stabilising ZNRF3/RNF43 at the plasma membrane remains unknown. Here, we show that the USP42 antagonises R-spondin by protecting ZNRF3/RNF43 from ubiquitin-dependent clearance. USP42 binds to the Dishevelled interacting region (DIR) of ZNRF3 and stalls the R-spondin-LGR4-ZNRF3 ternary complex by deubiquitinating ZNRF3. Accordingly, USP42 increases the turnover of LRP6 and Frizzled (FZD) receptors and inhibits Wnt signalling. Furthermore, we show that USP42 functions as a roadblock for paracrine Wnt signalling in colon cancer cells and mouse small intestinal organoids. We provide new mechanistic insights into the regulation R-spondin and conclude that USP42 is crucial for ZNRF3/RNF43 stabilisation at the cell surface. SYNOPSIS Clearance of the Wnt receptors by RNF43 and ZNRF3 has emerged as the main mechanism modulating Wnt/β-catenin signalling in adult stem cells. To ensure stem cell renewal, RNF43 and ZNRF3 are ubiquitinated and removed from the plasma membrane via R-spondin and LGR4 - a process that is counteracted by USP42. The deubiquitinase USP42 forms a tug-of-war with R-spondin and LGR4 to control the plasma membrane residence of ZNRF3 and RNF43 USP42 is a negative regulator of Wnt/β-catenin signalling that promotes clearance of the Wnt receptors USP42 functions as a roadblock for Wnt-driven growth and EMT in colon cancer cells Loss of USP42 renders intestinal organoids hypersensitive to paracrine Wnt signalling Graphical Abstract The deubiquitinase USP42 inhibits Wnt signaling by stabilizing RNF43 and ZNRF3 at the plasma membrane.