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

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.

Interaction with both ZNRF3 and LGR4 is required for the signalling activity of R‐spondin

“…These data suggest that RSPO1 binds to ZNRF3 and LGR4 through distinct domains; the FU1 domain is involved in ZNRF3 binding, whereas the FU2 domain is involved in LGR4 binding. This result is consistent with our previous findings that RSPO1 can bind to both ZNRF3 and LGR4 and induce their dimerization [18]. RNF43 is a functional homologue of ZNRF3 and mediates ubiquitination and degradation of Frizzled [18,19].…”

“…To define the signalling mechanism of R-spondin, we mutated all conserved residues within two furinlike domains of RSPO1 and tested their binding to proposed RSPO1 receptors. Consistent with results of other labs [13,14], we could not detect an interaction between R-spondin and Frizzled, LRP6, or Kremen [18], and, therefore, we focused on LGR4 and ZNRF3. All conserved residues within the furin-like domains of RSPO1-GFP were individually mutated to alanine (supplementary Fig S1 online), and RSPO1-GFP mutants were tested for their interaction with LGR4 and ZNRF3 in a cell-based binding assay (supplementary Table S1 online).…”

“…Various membrane proteins have been reported to bind to R-spondin, including Wnt receptors Frizzled [10] and LRP6 [10,11], Kremen [12], Syndecan 4 [13], LGR4/5 [14][15][16][17] and membrane E3 ubiquitin ligases ZNRF3/RNF43 [18]. Relative contribution of these proteins to R-spondin signalling is not clear.…”

Targeting Wnts at the Source—New Mechanisms, New Biomarkers, New Drugs

“…1; Table 1). RNF43 and ZNRF3 negatively regulate Wnt signaling by promoting ubiquitination, internalization, and degradation of the Wnt receptors Frizzled and LRP5/6 (42,43). Recent studies (ref.…”

“…RSPOs normally inhibit the activity of RNF43 and ZNRF3 by sequestering them into a heterotrimeric complex with LGR4/5/6 (Leucine-rich repeat containing G-protein-coupled receptors 4, 5, or 6). This complex results in the inhibition and membrane clearance of these E3 ubiquitin ligases, leading to increased Frizzled and LRP5/6 proteins on the cell surface (43,54). The LGR RSPO complex may also enhances b-catenin-dependent signaling by promoting MEK1/2-mediated phosphorylation of LRP5/6 and b-catenin-independent signaling through regulation of actin dynamics (55).…”