eLife Assessment

This study presents important findings demonstrating that FZD5 and FZD8, two of the ten Frizzled proteins, undergo Wnt-mediated endocytosis. The E3 ubiquitin ligases RNF43/ZNRF3 regulate their degradation in a Wnt-dependent manner, a mechanism that was not previously recognized. The evidence supporting the claims of the authors is solid. This research will be of interest to biologists specializing in Wnt signaling, cancer, and regenerative medicine.

Reviewer #1 (Public review):

Summary:

The mechanism by which WNT signals are received and transduced into the cell has been the topic of extensive research. Cell surface levels of the WNT receptors of the FZD family are subject to tight control and it's well established that the transmembrane ubiquitin ligases ZNRF3 and RNF43 target FZDs for degradation and that proteins of the R-spondin family block this effect. This manuscript explores the role that WNT proteins play in receptor internalization, recycling and degradation, and the authors provide evidence that WNTs promote interactions of FZD with the ubiquitin ligases. Using cells mutant in all 3 DVL genes, the authors demonstrate that this effect of WNT on FZD is DVL-independent.

Strengths:

Overall, the data are of good quality and support the authors' hypothesis. Strengths of this study are the use of CRISPR-mutated cell lines to establish genetic requirements for the various components. The finding that FZD internalization and degradation is WNT dependent and does not involve DVL is novel.

Weaknesses:

Weaknesses of the work include a heavy reliance on overexpression and monitoring the effects in a single cell line, HEK293. In addition, the claim of specificity - only FZD5 and FZD8 participate in this process - is not strongly supported.

Reviewer #2 (Public review):

In this manuscript Luo et al uncover that the ZNRF3/RNF43 E3 ubiquitin ligases participate in the selective endocytosis and degradation of FZD5/8 receptors in response to Wnt stimulation. Interestingly, DVL proteins have previously been shown to be important for RNF43/ZNRF3-dependent ubiquitination of Frizzled receptors but in this study the authors show that DVL proteins are only important for ligand and RNF43/ZNRF3-independent FZD endocytosis. Although it is well established that ZNRF3 and RNF43 promote the endocytosis and degradation of FZD receptors as part of a negative regulatory loop to dampened B-catenin signaling, the dependency of Wnt stimulation for this process and the specificity of this degradation for different FZD receptors remained poorly characterized.

In my opinion there are two significant findings of this study: 1) Wnt proteins are required for ZNRF3/RNF43 mediated endocytosis and degradation of FZD receptors and this constitutes an important negative regulatory loop. 2) The ZNRF3/RNF43 substrate selectivity for FZD5/8 over the other 8 Frizzleds. Of course, many questions remain, and new ones emerge as is often the case, but these findings challenge our dogmatic view on how the ZNRF3/RNF43 regulate Wnt signaling and emphasize their role in Wnt-dependent Frizzled endocytosis/degradation and beta-catenin signaling. Below I have suggestions to strengthen the manuscript.

(1) Given their results the authors conclude that upregulation of Frizzled on the plasma membrane is not sufficient to explain the stabilization of beta-catenin seen in the ZNRF3/RNF43 mutant cells. This interpretation is sound, and they suggest in the discussion that ZNRF3/RNF43-mediated ubiquitination could serve as a sorting signal to sort endocytosed FZD to lysosomes for degradation and that absence or inhibition of this process would promote FZD recycling. This should be relatively easy to test using surface biotinylation experiments and would considerably strengthen the manuscript.
(2) The authors show that the FZD5 CRD domain is required for endocytosis since a mutant FZD5 protein in which the CRD is removed does not undergo endocytosis. This is perhaps not surprising since this is the site of Wnt binding, but the authors show that a chimeric FZD5CRD-FZD4 receptor can confer Wnt-dependent endocytosis to an otherwise endocytosis incompetent FZD4 protein. Since the linker region between the CRD and the first TM differs between FZD5 and FZD4 it would be interesting to understand whether the CRD specifically or the overall arrangement (such as the spacing) is the most important determinant.
(3) I find it surprising that only FZD5 and FZD8 appear to undergo endocytosis or be stabilized at the cell surface upon ZNRF3/RNF43 knockout. Is this consistent with previous literature? Is that a cell-specific feature? These findings should be tested in a different cell line, with possibly different relative levels of ZNRF3 and RNF43 expression.
(4) If FZD7 is not a substrate of ZNRF3/RNF43 and therefore is not ubiquitinated and degraded, how do the authors reconcile that its overexpression does not lead to elevated cytosolic beta-catenin levels in Figure 5B?
(5) For Figure 5B, it would be interesting if the authors could evaluate whether overexpression of FZD5 in the ZNRF3/RNF43 double knockout lines would synergize and lead to further increase in cytosolic beta-catenin levels. As control if the substrate selectivity is clear FZD7 overexpression in that line should not do anything.
(6) In Figure 6G, the authors need to show cytosolic levels of beta-catenin in the absence of Wnt in all cases.
(7) Since the authors show that DVL is not involved in the Wnt and ZRNF3-dependent endocytosis they should repeat the proximity biotinylation experiment in figure 7 in the DVL triple KO cells. This is an important experiment since previous studies showed that DVL was required for the ZRNF3/RNF43-mediated ubiqtuonation of FZD.

Author response:

Reviewer #2 (Public review):

(1) Given their results the authors conclude that upregulation of Frizzled on the plasma membrane is not sufficient to explain the stabilization of beta-catenin seen in the ZNRF3/RNF43 mutant cells. This interpretation is sound, and they suggest in the discussion that ZNRF3/RNF43-mediated ubiquitination could serve as a sorting signal to sort endocytosed FZD to lysosomes for degradation and that absence or inhibition of this process would promote FZD recycling. This should be relatively easy to test using surface biotinylation experiments and would considerably strengthen the manuscript.

Thank you for your valuable suggestions and comments. We will perform cell surface biotinylation experiments.

(2) The authors show that the FZD5 CRD domain is required for endocytosis since a mutant FZD5 protein in which the CRD is removed does not undergo endocytosis. This is perhaps not surprising since this is the site of Wnt binding, but the authors show that a chimeric FZD5CRD-FZD4 receptor can confer Wnt-dependent endocytosis to an otherwise endocytosis incompetent FZD4 protein. Since the linker region between the CRD and the first TM differs between FZD5 and FZD4 it would be interesting to understand whether the CRD specifically or the overall arrangement (such as the spacing) is the most important determinant.

Our results in Fig. 1F-G clearly show that the CRD of FZD5 specifically is both necessary and sufficient for Wnt3a/5a-induced FZD5 endocytosis, as replacing the CRD alone in FZD5 with the CRD from either FZD4 or FZD7 completely abolished Wnt-induced endocytosis, whereas replacing the CRD alone in FZD4 or FZD7 with the FZD5 CRD alone could confer Wnt-induced endocytosis.

(3) I find it surprising that only FZD5 and FZD8 appear to undergo endocytosis or be stabilized at the cell surface upon ZNRF3/RNF43 knockout. Is this consistent with previous literature? Is that a cell-specific feature? These findings should be tested in a different cell line, with possibly different relative levels of ZNRF3 and RNF43 expression.

Thank you for your comments and suggestions. Our finding that ZNRF3/RNF43 specifically regulates FZD5/8 degradation is consistent with recent published studies in which FZD5 is required for the survival of RNF43-mutant PDAC or colorectal cancer cells (Nature Medicine, 2017, PMID: 27869803) and FZD5 is required for the maintenance of intestinal stem cells (Developmental Cell, 2024, PMID: 39579768 and 39579769), and in both cases, FZDs other than FZD5/8 are also expressed but not sufficient to compensate for the function of FZD5. The mechanism by which Wnt3a/5a specifically induces FZD5/8 endocytosis and degradation is currently unknown and needs to be explored in the future. We speculate that Wnt binding to FZD5/8 may recruit another protein on the cell surface to specifically facilitate FZD5/8 endocytosis. On the other hand, we cannot exclude the possibility that Wnts other than Wnt3a/5a may induce the endocytosis and degradation of FZDs other than FZD5/8 since there are 19 Wnts and 10 FZDs in humans. We will perform flow cytometry experiments using FZD5/8-specific antibodies to examine whether Wnt3a/5a induces FZD5/8 endocytosis in more cell lines.

(4) If FZD7 is not a substrate of ZNRF3/RNF43 and therefore is not ubiquitinated and degraded, how do the authors reconcile that its overexpression does not lead to elevated cytosolic beta-catenin levels in Figure 5B?

We are currently not sure of the mechanism underlying this result. Considering that most FZDs are expressed in 293A cells, we do not know how much of the mature form of overexpressed FZD7 was presented to the plasma membrane.

(5) For Figure 5B, it would be interesting if the authors could evaluate whether overexpression of FZD5 in the ZNRF3/RNF43 double knockout lines would synergize and lead to further increase in cytosolic beta-catenin levels. As control if the substrate selectivity is clear FZD7 overexpression in that line should not do anything.

We will perform these experiments as you suggested.

(6) In Figure 6G, the authors need to show cytosolic levels of beta-catenin in the absence of Wnt in all cases.

We did not add Wnt CM in this experiment. RSPO1 activity, which relies on endogenous Wnt, has been well documented in previous studies.

(7) Since the authors show that DVL is not involved in the Wnt and ZRNF3-dependent endocytosis they should repeat the proximity biotinylation experiment in figure 7 in the DVL triple KO cells. This is an important experiment since previous studies showed that DVL was required for the ZRNF3/RNF43-mediated ubiqtuonation of FZD.

Thank you for your valuable suggestions. We will perform the proximity biotinylation experiment in DVL TKO cells.