{"gene":"RNF43","run_date":"2026-06-10T06:43:37","timeline":{"discoveries":[{"year":2012,"finding":"RNF43 is a transmembrane E3 ubiquitin ligase that selectively ubiquitinates frizzled (FZD) receptors on the cell surface, targeting them for lysosomal degradation and thereby reducing Wnt signaling. Expression of RNF43 in HEK293T cells blocks Wnt responses and targets surface-expressed frizzled receptors to lysosomes; reconstitution of RNF43 in RNF43-mutant HCT116 cells removes their response to exogenous Wnt.","method":"Overexpression and reconstitution in cell lines, ubiquitination assays, lysosomal trafficking assays, mouse intestinal epithelium conditional knockout","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal functional assays in multiple cell lines and in vivo mouse KO, widely replicated foundational study","pmids":["22895187"],"is_preprint":false},{"year":2013,"finding":"RNF43 inhibits Wnt/β-catenin signaling by reducing the membrane level of Frizzled in pancreatic cancer cells, serving as a negative feedback mechanism. Inactivating mutations in RNF43 increase cell-surface Frizzled and render pancreatic cancer cells dependent on autocrine/paracrine Wnt signaling, demonstrated by sensitivity to the Porcupine inhibitor LGK974 selectively in RNF43-mutant lines.","method":"Loss-of-function cell culture assays, cell-surface Frizzled measurement, wild-type RNF43 reconstitution, xenograft models, pharmacological Wnt inhibition","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (reconstitution, surface receptor measurement, xenograft) in a single focused study","pmids":["23847203"],"is_preprint":false},{"year":2013,"finding":"Crystal structure of the RNF43 ectodomain in complex with R-spondin 2 (Rspo2 Fu1-Fu2 fragment) reveals that a prominent loop in the Fu1 domain of Rspo2 clamps into a groove on the RNF43 ectodomain surface, forming a ternary complex with LGR4/5. LGR5 does not directly contact RNF43 but increases RSPO1 affinity for RNF43, supporting LGR5 as an engagement receptor and RNF43 as an effector receptor.","method":"X-ray crystallography, biophysical binding assays, mutational analysis","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structures of RNF43 ectodomain alone and in complex with R-spondin, validated by biophysical and cellular assays","pmids":["24225776"],"is_preprint":false},{"year":2013,"finding":"Crystal structure of RSPO1 bound simultaneously to both LGR5 and RNF43 ectodomains confirms physical linkage; RSPO1 is sandwiched by LGR5 and RNF43, with its CRD rod module contacting LGR5 and a hairpin inserted into the RNF43 ectodomain groove. Disease mutations map to the RSPO1-RNF43 interface.","method":"X-ray crystallography of ternary RSPO1-LGR5-RNF43 complex","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure of ternary complex with disease mutation mapping","pmids":["23756651"],"is_preprint":false},{"year":2014,"finding":"RNF43 (and ZNRF3) are encoded by Wnt target genes and constitute a negative Wnt feedback loop; the LGR5/R-spondin complex neutralizes RNF43/ZNRF3 activity, thereby amplifying Wnt signaling in stem cells. This module controls removal of Wnt receptors from the stem cell surface.","method":"Review integrating genetic epistasis and binding studies (mechanistic synthesis of prior experimental work)","journal":"Genes & development","confidence":"Medium","confidence_rationale":"Tier 3 / Strong — review synthesizing replicated experimental findings; cited as mechanistic consensus","pmids":["24532711"],"is_preprint":false},{"year":2014,"finding":"RNF43 expression is directly regulated by the canonical Wnt/β-catenin pathway via two Wnt-responsive elements (WREs) in intron 2 that associate with the TCF4/β-catenin complex, establishing RNF43 as a direct transcriptional target of TCF4/β-catenin and part of a negative feedback loop.","method":"Reporter gene assay with WRE mutagenesis, siRNA knockdown of β-catenin, chromatin immunoprecipitation (ChIP)","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP and reporter assays with mutagenesis in a single lab","pmids":["24466159"],"is_preprint":false},{"year":2015,"finding":"Dishevelled (DVL) acts as a dual-function adaptor that recruits RNF43/ZNRF3 to Wnt receptors. DVL knockout increases cell-surface FZD and LRP6; DVL is required for RNF43/ZNRF3-mediated ubiquitination and degradation of FZD. Physical interaction between DVL and ZNRF3/RNF43 is essential for their Wnt-inhibitory activity, and the DEP domain of DVL is required for FZD binding and downregulation.","method":"DVL knockout cells, co-immunoprecipitation, ubiquitination assays, domain fusion rescue experiments","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal co-IP, KO phenotype, domain mapping, and rescue experiments in one focused study","pmids":["25891077"],"is_preprint":false},{"year":2015,"finding":"RNF43 suppresses both Wnt/β-catenin (canonical) and noncanonical Wnt signaling by distinct mechanisms. Canonical suppression requires interaction between the extracellular PA domain of RNF43 and the CRD of Frizzled, plus the intracellular RING domain. Noncanonical suppression requires the C-terminal cytoplasmic region of RNF43 interacting with the PDZ domain of Dishevelled. Missense mutations in the extracellular domain change RNF43 localization from endosome to ER, abolishing canonical but not noncanonical Wnt suppression.","method":"Domain deletion/mutation analysis, co-immunoprecipitation, immunofluorescence localization, Wnt reporter assays","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple domain mutants, localization and functional readouts in one study","pmids":["25825523"],"is_preprint":false},{"year":2015,"finding":"RNF43 physically interacts with TCF4 in the nucleus and tethers TCF4 to the nuclear membrane, silencing TCF4 transcriptional activity even in the presence of constitutively active β-catenin mutants. This nuclear inhibitory mechanism is disrupted by tumor-associated RNF43 mutations, and RING domain mutation of RNF43 transactivates Wnt target genes in cells and Xenopus embryos.","method":"Co-immunoprecipitation, immunofluorescence (nuclear localization), TCF4 transcriptional reporter assays, Xenopus embryo functional assays, RNF43 RING domain mutagenesis","journal":"Science signaling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP and functional reporter with Xenopus validation; nuclear localization later questioned by antibody artifact paper (PMID 37023034), lowering confidence","pmids":["26350900"],"is_preprint":false},{"year":2015,"finding":"Paracrine Wnt3 secretion from Paneth cells is an essential driver of Rnf43/Znrf3-double-knockout intestinal tumor growth; removal of Paneth cells by Math1 mutation or deletion of Wnt3 inhibits tumor formation. Treatment with the Porcupine inhibitor C59 strongly inhibits RZ−/− neoplasia growth while sparing adjacent normal crypts.","method":"Genetic epistasis (Math1 KO, Wnt3 KO in Rnf43/Znrf3 DKO mice), pharmacological Porcupine inhibitor treatment in vivo","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo genetic epistasis with two independent gene deletions and pharmacological validation","pmids":["26023187"],"is_preprint":false},{"year":2008,"finding":"RNF43 protein resides in the endoplasmic reticulum and at least partially in the nuclear envelope/inner nuclear membrane. Recombinant RNF43 has autoubiquitylation activity in a cell-free system. RNF43 interacts with HAP95 (a chromatin-associated nuclear envelope protein) by yeast two-hybrid and co-immunoprecipitation, but HAP95 is not a ubiquitination substrate of RNF43.","method":"Immunofluorescence, biochemical fractionation, sucrose density gradient, cell-free autoubiquitylation assay, yeast two-hybrid, co-immunoprecipitation","journal":"Experimental cell research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro autoubiquitylation plus binding partner identification; single lab, and localization findings partially contradicted by later antibody artifact study","pmids":["18313049"],"is_preprint":false},{"year":2008,"finding":"RNF43 physically interacts with the PSF/p54nrb heterodimer (nuclear RNA-binding proteins); co-expression of PSF relocates RNF43 from the nuclear periphery to the nucleoplasm.","method":"Pull-down assay with mass spectrometry identification, co-immunoprecipitation, immunofluorescence","journal":"Proteomics","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single pulldown/MS identification with co-IP confirmation; functional consequence not established; localization claims subject to antibody artifact caveat","pmids":["18655028"],"is_preprint":false},{"year":2010,"finding":"RNF43 interacts with NEDL1 (a p53-activating E3 ligase) and with p53 itself; RNF43 suppresses p53 transcriptional activity in H1299 cells and attenuates UV-induced apoptosis.","method":"Yeast two-hybrid screening, co-immunoprecipitation, p53 transcriptional reporter assay, apoptosis assay after UV irradiation","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — yeast two-hybrid confirmed by co-IP, functional readout (reporter + apoptosis); single lab","pmids":["21108931"],"is_preprint":false},{"year":2015,"finding":"RNF43 promotes growth of colorectal cancer cells in an autocrine manner; the RNF43 protein is secreted and conditioned medium from RNF43-transfected cells enhances NIH3T3 cell growth. Exogenous RNF43 expression confers growth-promoting effects; suppression by siRNA retards colon cancer cell growth.","method":"Overexpression in COS7 and NIH3T3 cells, conditioned medium assay, siRNA knockdown, growth assays","journal":"International journal of oncology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, single overexpression/knockdown approach; secreted growth-promoting function not confirmed by later mechanistic work, which instead established RNF43 as a tumor suppressor","pmids":["15492824"],"is_preprint":false},{"year":2015,"finding":"The nucleoprotein (NP) of influenza A virus (IAV) interacts with RNF43 (identified by yeast two-hybrid). IAV infection attenuates RNF43 transcript and protein levels; RNF43-depleted cells show escalated viral replication. RNF43 polyubiquitinates p53, leading to p53 destabilization. NP of IAV targets RNF43 to prevent p53 ubiquitination, causing p53 stabilization and enhanced apoptosis.","method":"Yeast two-hybrid, RNF43 depletion (viral replication assay), ubiquitination assay of p53, viral infection models","journal":"Cell death & disease","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, yeast two-hybrid interaction, ubiquitination assay; p53 as RNF43 substrate needs independent confirmation","pmids":["25996295"],"is_preprint":false},{"year":2016,"finding":"The RSPO-LGR4/5-ZNRF3/RNF43 module controls Wnt/β-catenin-mediated metabolic liver zonation and hepatic growth. Liver-specific ZNRF3/RNF43 loss-of-function expands the hepatic Wnt/β-catenin signaling gradient; RSPO1 increases liver size and improves regeneration in an LGR4/5-dependent manner.","method":"Liver-specific conditional knockout mice, recombinant RSPO1 treatment, LGR4/5 LOF, liver size and regeneration readouts","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple genetic models (LOF and GOF) with clear in vivo phenotypic readouts, validated by pharmacological RSPO1","pmids":["27088858"],"is_preprint":false},{"year":2016,"finding":"Genome-wide CRISPR-Cas9 screens in RNF43-mutant pancreatic cancer cells identify a specific requirement for the Wnt receptor FZD5 for proliferation. Antibodies against FZD5 (and FZD8) inhibit growth of RNF43-mutant PDAC in vitro and as xenografts. This specificity could not be explained by FZD5 expression patterns alone.","method":"Genome-wide CRISPR-Cas9 screen, recombinant anti-FZD antibodies, in vitro growth assays, xenograft models, patient-derived organoids","journal":"Nature medicine","confidence":"High","confidence_rationale":"Tier 2 / Strong — genome-wide genetic screen with orthogonal antibody validation in vitro and in vivo","pmids":["27869803"],"is_preprint":false},{"year":2019,"finding":"RNF43 ubiquitinates E-cadherin phosphorylated at tyrosine 797 by c-Src (at lysine 816), leading to E-cadherin degradation, nuclear β-catenin translocation and EMT in lung adenocarcinoma cells. This ubiquitination requires Frizzled 8 (FZD8) interaction with RNF43 but not the PA domain of RNF43.","method":"Protein antibody microarray, E3 ligase profiling, immunoprecipitation, immunoblotting, immunofluorescence, shRNA knockdown, xenograft models, site-directed mutagenesis of E-cadherin phosphorylation and ubiquitination sites","journal":"BMC cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — site-specific mutagenesis plus in vivo xenograft; single lab; contradicted by another study (PMID 32103169) that could not confirm E-cadherin as RNF43 substrate","pmids":["31286874"],"is_preprint":false},{"year":2019,"finding":"RNF43 C-terminal truncation mutants (including G659fs), when expressed at endogenous levels via CRISPR knock-in, retain the ability to suppress β-catenin signaling and Wnt-receptor turnover equivalently to wild-type RNF43, and retain DVL-binding. Only N-terminal RNF43 mutations convey Wnt-dependency onto cancer cells. (NOTE: this finding partially contradicts other studies that report C-terminal truncations as loss-of-function.)","method":"CRISPR-Cas9 endogenous knock-in/knockout in HEK293T and KM12 cells, β-catenin reporter assay, Wnt-receptor surface flow cytometry, co-immunoprecipitation","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — endogenous CRISPR models with multiple readouts; single lab; contradicted by other CRISPR-based studies","pmids":["32103169"],"is_preprint":false},{"year":2020,"finding":"RNF43 activity requires phosphorylation at a conserved triplet of serines (phospho-switch). This phospho-regulation is required for zebrafish development and mouse intestinal organoid growth. Cancer-associated mutations that abrogate RNF43 phosphorylation cooperate with active Ras to promote tumorigenesis by abolishing Wnt-inhibitory function of RNF43 while maintaining p53-inhibitory function. Phosphomimetic substitutions of the serine trio restore tumor-suppressive activity of extracellular oncogenic mutants.","method":"Site-directed mutagenesis of serine phosphosites, phosphomimetic substitutions, zebrafish developmental assays, mouse intestinal organoids, co-expression with active Ras, Wnt reporter assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — mutagenesis, multiple organism models (zebrafish, mouse organoids), and rescue experiments in one study","pmids":["32934222"],"is_preprint":false},{"year":2020,"finding":"A class of RNF43 truncating cancer mutations (C-terminal truncations) retains Wnt receptor downregulation activity but interferes with a ubiquitin-independent suppressor role of the RNF43 cytosolic tail involving Casein kinase 1 (CK1) binding and phosphorylation. Truncated RNF43 variants trap CK1 at the plasma membrane, preventing β-catenin turnover and driving ligand-independent Wnt/β-catenin transcription. Gene editing in human colon stem cells shows these truncations cooperate with p53 loss for niche-independent self-renewal.","method":"CRISPR gene editing of patient-derived mutations, CK1 co-immunoprecipitation, phosphorylation assays, β-catenin reporter assays, PORCN inhibitor sensitivity assays, human colon organoids","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — endogenous CRISPR mutations, biochemical CK1 interaction, functional organoid assays in one focused study","pmids":["32965059"],"is_preprint":false},{"year":2020,"finding":"Systematic functional assay of 119 missense and 45 truncating patient-derived RNF43 mutations reveals: RING domain missense mutations and a subset of extracellular domain mutations hyperactivate Wnt/β-catenin by forming inactive dimers with endogenous RNF43 or ZNRF3. C-terminal truncation mutants (including G659fs) are loss-of-function at endogenous levels, increase cell-surface Frizzled and Wnt/β-catenin signaling, and render xenografts responsive to PORCN inhibition.","method":"Cell-based reporter assays, genome editing, flow cytometry for surface Frizzled, immunofluorescence, patient-derived xenografts and cell lines","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 2 / Strong — systematic mutagenesis with multiple orthogonal methods and in vivo validation","pmids":["33067269"],"is_preprint":false},{"year":2021,"finding":"USP42 deubiquitinase antagonizes R-spondin by protecting ZNRF3/RNF43 from ubiquitin-dependent membrane clearance. USP42 binds to the Dishevelled interacting region (DIR) of ZNRF3 and stalls the R-spondin-LGR4-ZNRF3 ternary complex by deubiquitinating ZNRF3, increasing LRP6 and FZD turnover and inhibiting Wnt signaling.","method":"Co-immunoprecipitation, ubiquitination assays, deubiquitination assays, Wnt reporter assays, colon cancer cells and mouse small intestinal organoids","journal":"EMBO reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — biochemical interaction and functional rescue; single lab","pmids":["33786993"],"is_preprint":false},{"year":2021,"finding":"ZNRF3 deletion alone promotes hepatocyte proliferation, which becomes limited by compensatory RNF43 upregulation. Concomitant deletion of both ZNRF3 and RNF43 in hepatocytes results in metabolic reprogramming of periportal hepatocytes, clonal expansion, and liver tumor development, demonstrating cooperative roles of the two ligases in restricting WNT/β-catenin activity to balance metabolic function and proliferation.","method":"Hepatocyte-specific conditional double knockout mice (ZNRF3 and RNF43), single-cell RNA sequencing, organoid culture, chromatin accessibility analysis","journal":"Cell stem cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional KO in vivo with scRNA-seq and organoid validation","pmids":["34129813"],"is_preprint":false},{"year":2021,"finding":"RNF43 is a negative regulator of noncanonical WNT5A signaling. RNF43 interacts with noncanonical Wnt receptor complex components ROR1, ROR2, VANGL1, and VANGL2 (identified by BioID and immunoprecipitation). RNF43 triggers VANGL2 ubiquitination and proteasomal degradation and induces clathrin-dependent internalization of ROR1 receptor and inhibits ROR2 activation, thereby blocking pro-metastatic WNT5A signaling in melanoma.","method":"BioID proximity labeling, co-immunoprecipitation, ubiquitination assays, receptor internalization assays, in vitro and in vivo invasion assays, BRAF/MEK inhibitor resistance models","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 / Strong — BioID plus co-IP, ubiquitination, internalization assays, and in vivo validation in one study","pmids":["34702444"],"is_preprint":false},{"year":2021,"finding":"Rnf43 (but not Znrf3) is potently activated by Wnt signaling in oligodendrocyte progenitor cells (OPCs) and promotes OPC differentiation specifically in injury contexts by negatively regulating Wnt signal strength at the level of Fzd1 receptor presentation on the OPC cell surface. Inhibition of Fzd1 using UM206 promotes remyelination following demyelinating injury.","method":"Conditional Rnf43 knockout in OPCs, neonatal hypoxic injury and adult demyelination models, Fzd1 inhibition, in vivo and ex vivo remyelination assays","journal":"Neuron","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional KO with injury models and pharmacological Fzd1 inhibition as mechanistic confirmation","pmids":["34390652"],"is_preprint":false},{"year":2022,"finding":"RNF43_p.G659fs promotes cancer cell growth independent of Wnt signaling. RNF43-G659fs mutant binds the PI3K regulatory subunit p85, leading to increased PI3K signaling through p85 ubiquitination and degradation. PI3K/mTOR inhibitors selectively kill RNF43-G659fs cells in isogenic lines and xenografts, and reduce interferon response gene expression which is reversed by PI3K/mTOR inhibition.","method":"Drug repurposing library screen, isogenic cell lines, co-immunoprecipitation (G659fs:p85 interaction), ubiquitination assay, xenograft models, patient-derived organoids, RNA-seq","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — biochemical interaction plus multiple functional models; single lab; novel finding requiring independent replication","pmids":["35676246"],"is_preprint":false},{"year":2022,"finding":"RNF43/ZNRF3 hepatocyte-specific double knockout results in steatohepatitis, accumulation of unsaturated lipids, defective hepatocyte regeneration upon injury, and liver cancer development. Differentiation defects and lipid alterations are, in part, cell-autonomous, demonstrated by hepatocyte-, hepatoblast-, and ductal cell-derived organoids.","method":"Hepatocyte-specific conditional Rnf43/Znrf3 double KO mice, organoid cultures from multiple liver cell types, lipidomic analysis","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional KO with multiple orthogonal organoid models and metabolic readouts","pmids":["35039505"],"is_preprint":false},{"year":2023,"finding":"RNF43 is a negative feedback regulator of PAR2 (protease-activated receptor 2, a GPCR): RNF43 co-associates with PAR2 and promotes its membrane elimination and polyubiquitination, reducing PAR2-induced β-catenin signaling. This degradation is rescued by R-spondin2 in the presence of LGR5.","method":"Co-immunoprecipitation, cell-surface biotinylation assay, polyubiquitination assay, β-catenin reporter assays, mouse AOM/DSS colon cancer model with Par2 KO","journal":"FASEB journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP, ubiquitination assay, and in vivo mouse model; single lab","pmids":["36468684"],"is_preprint":false},{"year":2023,"finding":"RNF43 directly interacts with γH2AX (phosphorylated H2AX) and its loss impairs DNA damage response (DDR) activation in gastric cells, conferring resistance to γ-radiation and chemotherapy by dampening DDR and preventing apoptosis.","method":"Co-immunoprecipitation (RNF43:γH2AX), RNF43 depletion, γ-radiation and chemotherapy sensitivity assays, Helicobacter pylori infection in Rnf43ΔEx8 mice","journal":"Cellular and molecular gastroenterology and hepatology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct interaction by co-IP with multiple functional readouts in vitro and in vivo; single lab","pmids":["33188943"],"is_preprint":false},{"year":2024,"finding":"RNF43 preferentially targets FZD1, FZD5, and FZD7 for endocytosis, while the related ZNRF3 preferentially targets FZD6. The RNF43 transmembrane domain (TMD) is a key molecular determinant for FZD5 endocytosis specificity; swapping the TMD between RNF43 and ZNRF3 redirects their FZD preferences.","method":"Endocytosis assays comparing RNF43 vs. ZNRF3 for each FZD, TMD domain-swap mutagenesis, flow cytometry for surface FZD levels","journal":"Life science alliance","confidence":"High","confidence_rationale":"Tier 2 / Strong — domain-swap mutagenesis with functional endocytosis readouts for multiple FZD members","pmids":["38969364"],"is_preprint":false},{"year":2024,"finding":"RNF43 ubiquitinates B-RAF at K499 to promote proteasome-dependent B-RAF degradation, reducing MEK activity and proliferative ability in cancer cells. Phosphorylation of B-RAF at T491 suppresses this ubiquitination by decreasing the RNF43:B-RAF interaction. MEK and Wnt inhibitors synergistically suppress growth of RNF43-mutated pancreatic cancer cells in vitro and in vivo.","method":"Co-immunoprecipitation (RNF43:B-RAF), ubiquitination assay, site-directed mutagenesis (K499, T491), MEK activity assays, xenograft models, combination drug studies","journal":"Advanced science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — site-specific mutagenesis and ubiquitination assay with in vivo validation; single lab; novel finding","pmids":["38225722"],"is_preprint":false},{"year":2024,"finding":"ZNRF3 and RNF43 interact with EGFR via their extracellular domains, leading to EGFR ubiquitination and subsequent degradation via the RING E3 ligase domain. Overexpression of ZNRF3 reduces EGFR levels and suppresses cancer cell growth; knockout of ZNRF3/RNF43 upregulates EGFR signaling and promotes tumorigenesis.","method":"Biochemical co-immunoprecipitation (extracellular domain interaction), ubiquitination assay, ZNRF3 overexpression, ZNRF3/RNF43 double knockout, in vitro and in vivo tumor growth assays, proteogenomic correlation","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP, ubiquitination assay, KO phenotype, and in vivo models; preprint, not yet peer-reviewed","pmids":["38260423"],"is_preprint":true},{"year":2023,"finding":"RNF43 interacts with PD-L1 in gastric cancer cells and augments both K48- and K63-linked ubiquitination of PD-L1, reducing PD-L1 surface expression and enhancing T cell antitumor activity.","method":"Co-immunoprecipitation (RNF43:PD-L1), ubiquitination assay (K48/K63 linkage-specific), flow cytometry for PD-L1 surface levels, T cell co-culture killing assay","journal":"Scandinavian journal of immunology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single co-IP and ubiquitination assay; single lab; limited mechanistic follow-up","pmids":["39007965"],"is_preprint":false},{"year":2024,"finding":"m6A methylation (mediated by METTL3/IGF2BP2 axis) regulates RNF43 mRNA stability. RNF43 protein directly interacts with and ubiquitinates NDUFS1 (NADH dehydrogenase Fe-S protein 1), promoting its proteasomal degradation and suppressing oxidative phosphorylation in endometrial stromal cells.","method":"m6A modification assay, METTL3/IGF2BP2 knockdown, co-immunoprecipitation (RNF43:NDUFS1), ubiquitination assay, NDUFS1 siRNA knockdown, cell viability/migration assays, rat endometriosis model","journal":"Journal of cellular physiology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — co-IP and ubiquitination assay for NDUFS1; single lab; novel substrate, limited orthogonal validation","pmids":["38988031"],"is_preprint":false},{"year":2020,"finding":"The extracellular Protease Associated (PA) domain of RNF43 is not necessary for suppression of Wnt/β-catenin signaling. RNF43ΔPA mutant still inhibits LRP6 phosphorylation, DVL2/DVL3 phosphorylation, and β-catenin-dependent gene expression. RNF43ΔPA is not sensitive to R-spondin1 treatment, confirming that R-spondin inhibits RNF43 function through the PA domain.","method":"TetON controlled overexpression, CRISPR/Cas9 RNF43/ZNRF3 double KO rescue, Western blot, TOPflash dual luciferase assay","journal":"Cell communication and signaling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CRISPR rescue in defined KO background, multiple signaling readouts; single lab","pmids":["32527265"],"is_preprint":false},{"year":2023,"finding":"Commercial RNF43 antibodies targeting exons 8-9 epitopes yield non-specific signals in immunoblotting, immunofluorescence, and immunohistochemistry; nuclear staining patterns attributed to RNF43 are antibody artifacts. Endogenous RNF43 localization to the nucleus (as reported in earlier studies) is not supported by validated antibody data.","method":"CRISPR deletion of RNF43 exons 8-9, immunoblotting, immunofluorescence, immunohistochemistry with knockout-validated negative controls for four commercial antibodies","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CRISPR-validated antibody controls; directly refutes prior nuclear localization claims; single lab","pmids":["37023034"],"is_preprint":false},{"year":2025,"finding":"Wnt ligands specifically induce FZD5/8 endocytosis and degradation in a ZNRF3/RNF43-dependent manner; ZNRF3/RNF43 selectively target FZD5/8 for Wnt-stimulated degradation. DVL proteins promote ligand-independent FZD endocytosis but are dispensable for Wnt-induced FZD5/8 degradation. Wnt promotes the interaction between FZD5 and RNF43.","method":"FZD endocytosis assays with Wnt stimulation, ZNRF3/RNF43 knockdown/knockout, DVL knockout, co-immunoprecipitation (FZD5:RNF43), receptor internalization fluorescence assays","journal":"eLife","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO validation, co-IP, and functional endocytosis assays; single lab","pmids":["41070826"],"is_preprint":false},{"year":2023,"finding":"LGR4 (but not LGR5) forms a 2:2 complex with RNF43/ZNRF3 that accommodates bivalent RSPO binding, providing high-affinity RSPO recruitment. Co-expression of ZNRF3 with LGR4 dramatically increases monovalent RSPO2 binding affinity, whereas co-expression of ZNRF3 with LGR5 has no effect, explaining why only LGR4 mediates the RSPO-RNF43/ZNRF3 inhibitory complex.","method":"Whole-cell binding affinity measurements with monovalent and bivalent RSPO2 furin domains, co-expression of LGR4/5 with ZNRF3 in cells, structural modeling","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — systematic binding measurements in whole cells with controls; single lab","pmids":["37402772"],"is_preprint":false}],"current_model":"RNF43 is a transmembrane RING-type E3 ubiquitin ligase that functions primarily as a negative regulator of Wnt signaling by ubiquitinating Frizzled receptors (preferentially FZD1/5/7) and LRP6 for lysosomal/proteasomal degradation, a process requiring Dishevelled as an adaptor and phosphorylation of a conserved serine triplet; its activity is inhibited when R-spondin ligands recruit LGR4 to form a ternary complex with the RNF43 ectodomain, and is stabilized at the membrane by the deubiquitinase USP42; beyond canonical Wnt, RNF43 also suppresses noncanonical WNT5A signaling by ubiquitinating VANGL2 and internalizing ROR1/ROR2, targets additional substrates including B-RAF and PAR2 for ubiquitin-mediated degradation, and contains a cytosolic tail that sequesters Casein kinase 1 (CK1) to promote β-catenin turnover through a ubiquitin-independent mechanism; loss-of-function mutations in RNF43 increase surface Frizzled abundance, rendering tumors dependent on paracrine Wnt signaling and sensitive to upstream Wnt inhibitors."},"narrative":{"mechanistic_narrative":"RNF43 is a transmembrane RING-type E3 ubiquitin ligase that operates as a negative-feedback brake on Wnt signaling, limiting the abundance of Wnt receptors at the cell surface [PMID:22895187, PMID:23847203]. It ubiquitinates Frizzled receptors and targets them for lysosomal degradation, with member-selective preference for FZD1/5/7 dictated by its transmembrane domain [PMID:22895187, PMID:38969364]; receptor engagement is mediated by Dishevelled, which acts as an adaptor recruiting RNF43 to Frizzled/LRP6 and is required for receptor downregulation [PMID:25891077, PMID:25825523]. Catalytic activity is gated by phosphorylation of a conserved serine triplet, and cancer mutations that abolish this phospho-switch eliminate Wnt-inhibitory function while sparing other activities [PMID:32934222]. RNF43 is itself a direct transcriptional target of the TCF4/β-catenin complex, closing a negative feedback loop [PMID:24466159]. This braking activity is neutralized when R-spondin ligands clamp into a groove on the RNF43 ectodomain and bridge it to LGR4 in a ternary complex, with LGR4 (not LGR5) supplying high-affinity RSPO recruitment; the protease-associated (PA) domain mediates this R-spondin sensitivity but is dispensable for Wnt suppression itself [PMID:24225776, PMID:23756651, PMID:32527265, PMID:37402772]. The RSPO–LGR–RNF43/ZNRF3 module governs stem-cell Wnt responsiveness and tissue homeostasis: loss of RNF43 (with ZNRF3) expands Wnt signaling to drive intestinal and hepatic tumorigenesis and metabolic reprogramming, and RNF43 controls oligodendrocyte progenitor differentiation through Fzd1 [PMID:26023187, PMID:34129813, PMID:34390652, PMID:35039505]. Inactivating RNF43 mutations raise surface Frizzled and render tumors dependent on paracrine Wnt, conferring sensitivity to Porcupine inhibitors and an acquired requirement for FZD5 [PMID:23847203, PMID:26023187, PMID:27869803, PMID:33067269]. Distinct truncating mutations are not simple null alleles: certain C-terminal truncations retain receptor downregulation but trap Casein kinase 1 at the membrane to block β-catenin turnover, while RING-domain and some extracellular missense mutants hyperactivate Wnt by forming inactive dimers with endogenous RNF43/ZNRF3 [PMID:32965059, PMID:33067269]. Beyond canonical Wnt, RNF43 suppresses noncanonical WNT5A signaling by ubiquitinating VANGL2 and internalizing ROR1/ROR2 [PMID:34702444], and additional substrates including B-RAF have been reported [PMID:38225722].","teleology":[{"year":2008,"claim":"Before its Wnt role was known, RNF43 was established as a bona fide E3 ligase capable of autoubiquitylation, fixing its biochemical identity as a RING enzyme.","evidence":"Cell-free autoubiquitylation assay plus fractionation and yeast two-hybrid interaction screening","pmids":["18313049"],"confidence":"Medium","gaps":["No physiological substrate identified at this stage","Reported ER/nuclear-envelope localization later challenged as antibody artifact"]},{"year":2012,"claim":"Defined RNF43's core function: it ubiquitinates surface Frizzled receptors to route them to lysosomes and dampen Wnt signaling, with reconstitution in mutant cells restoring Wnt sensitivity.","evidence":"Overexpression/reconstitution in HEK293T and HCT116, ubiquitination and lysosomal trafficking assays, conditional mouse intestinal knockout","pmids":["22895187"],"confidence":"High","gaps":["Did not resolve receptor selectivity among FZD members","Adaptor and recruitment mechanism unknown"]},{"year":2013,"claim":"Connected RNF43 loss to cancer dependency, showing inactivating mutations raise surface Frizzled and create selective addiction to upstream Wnt that is druggable with Porcupine inhibitors.","evidence":"Loss-of-function and reconstitution in pancreatic cancer lines, surface-Frizzled measurement, xenografts, LGK974 sensitivity","pmids":["23847203"],"confidence":"High","gaps":["Mechanism distinguishing mutant classes not addressed","Did not identify the specific FZD required"]},{"year":2013,"claim":"Structurally defined how R-spondin inhibits RNF43, showing R-spondin clamps the RNF43 ectodomain and bridges it to LGR4/5 in a ternary complex.","evidence":"X-ray crystallography of RNF43 ectodomain with R-spondin and of the ternary RSPO1-LGR5-RNF43 complex, with disease-mutation mapping","pmids":["24225776","23756651"],"confidence":"High","gaps":["Did not establish how complex formation translates to reduced ligase activity in cells","LGR4 vs LGR5 functional asymmetry not yet quantified"]},{"year":2014,"claim":"Established the regulatory architecture: RNF43 is itself a Wnt/β-catenin transcriptional target, defining a self-limiting negative feedback loop neutralized by RSPO/LGR.","evidence":"Reporter assays with WRE mutagenesis, β-catenin knockdown and ChIP; review synthesis of epistasis and binding data","pmids":["24466159","24532711"],"confidence":"Medium","gaps":["Feedback kinetics in vivo not quantified","Cell-type specificity of WRE usage unexplored"]},{"year":2015,"claim":"Identified Dishevelled as the obligate adaptor recruiting RNF43 to Wnt receptors, and dissected separable extracellular/RING and cytoplasmic determinants for canonical versus noncanonical suppression.","evidence":"DVL knockout, reciprocal co-IP, domain mapping and rescue; domain-deletion/localization and Wnt reporter assays","pmids":["25891077","25825523"],"confidence":"High","gaps":["How DVL DEP/PDZ engagement is regulated remains open","Relative contribution of canonical vs noncanonical arms in vivo unresolved"]},{"year":2015,"claim":"In vivo genetics tied RNF43/ZNRF3 loss to paracrine-Wnt-driven tumor growth, demonstrating Paneth-cell Wnt3 and Porcupine activity as the actionable dependency.","evidence":"Math1 and Wnt3 deletions in Rnf43/Znrf3 DKO mice, Porcupine inhibitor C59 treatment","pmids":["26023187"],"confidence":"High","gaps":["Source-cell dependency may differ in other tissues","Did not address mutant-specific signaling outputs"]},{"year":2016,"claim":"Pinpointed FZD5 as the specific receptor required by RNF43-mutant pancreatic cancer, nominating a precise antibody target.","evidence":"Genome-wide CRISPR screen, anti-FZD5/8 antibodies, in vitro growth, xenografts, patient-derived organoids","pmids":["27869803"],"confidence":"High","gaps":["Molecular basis of FZD5 selectivity not yet defined","Generality across other RNF43-mutant tissues untested"]},{"year":2016,"claim":"Extended the receptor module to physiology, showing the RSPO-LGR-ZNRF3/RNF43 axis sets the hepatic Wnt gradient controlling metabolic liver zonation and regeneration.","evidence":"Liver-specific conditional knockouts, recombinant RSPO1, LGR4/5 loss-of-function, size and regeneration readouts","pmids":["27088858"],"confidence":"High","gaps":["Did not separate RNF43 from ZNRF3 contributions","Downstream metabolic effectors not defined"]},{"year":2020,"claim":"Resolved that distinct mutation classes act through different mechanisms: a phospho-switch licenses ligase activity, C-terminal truncations trap CK1 to drive ligand-independent Wnt, and systematic profiling separated dominant-negative dimerizing mutants from loss-of-function truncations.","evidence":"Serine-triplet mutagenesis with zebrafish/organoid models; CRISPR knock-in of patient truncations with CK1 co-IP and organoid assays; systematic functional assay of 164 patient mutations","pmids":["32934222","32965059","33067269","32527265"],"confidence":"High","gaps":["Reports conflict on whether specific C-terminal truncations are loss-of-function or retain activity (contrast with PMID 32103169)","Kinase responsible for the serine-triplet phospho-switch not identified"]},{"year":2021,"claim":"Broadened the substrate and regulatory landscape: USP42 stabilizes RNF43/ZNRF3 at the membrane, RNF43/ZNRF3 cooperate to restrain hepatic Wnt and lipid metabolism, RNF43 suppresses noncanonical WNT5A signaling via VANGL2/ROR ubiquitination and internalization, and it tunes OPC differentiation through Fzd1.","evidence":"Co-IP/deubiquitination assays; hepatocyte conditional double-KO with scRNA-seq; BioID, ubiquitination and internalization assays in melanoma; conditional Rnf43 KO in OPCs with injury models","pmids":["33786993","34129813","34702444","34390652"],"confidence":"High","gaps":["Tissue-specific balance between canonical and noncanonical roles unclear","Whether noncanonical substrates are targeted in non-melanoma contexts untested"]},{"year":2024,"claim":"Defined the molecular determinant of receptor selectivity and Wnt-stimulated targeting, and added new substrates linking RNF43 to RAS-MAPK signaling.","evidence":"TMD domain-swap endocytosis assays for multiple FZD members; Wnt-stimulated FZD5/8 degradation assays with KO validation; B-RAF co-IP, K499/T491 mutagenesis and combination drug studies","pmids":["38969364","41070826","38225722"],"confidence":"Medium","gaps":["B-RAF as substrate from a single lab without independent confirmation","How Wnt ligand promotes the FZD5-RNF43 interaction mechanistically unresolved"]},{"year":null,"claim":"It remains unresolved which of RNF43's many reported non-Wnt substrates (p53, E-cadherin, PAR2, PD-L1, NDUFS1, EGFR, p85) represent core physiological functions versus context-restricted or unconfirmed activities, and what the validated subcellular localization is given antibody-artifact concerns.","evidence":"Multiple single-lab co-IP/ubiquitination studies, several contradicted or flagged as artifacts","pmids":[],"confidence":"Low","gaps":["E-cadherin substrate claim contradicted by an independent study","Prior nuclear localization shown to be antibody artifact","Several substrates rest on single Co-IP without reciprocal or in vivo validation"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[0,10,24,31]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,24,31]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,20,30]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[7]},{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[7,10]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,1,6,24]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[15,25]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[9,16,21]},{"term_id":"R-HSA-9609507","term_label":"Protein localization","supporting_discovery_ids":[0,30,37]}],"complexes":["RSPO-LGR4/5-RNF43/ZNRF3 ternary complex"],"partners":["FZD5","DVL","LGR4","RSPO1","VANGL2","ROR1","USP42","CSNK1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q68DV7","full_name":"E3 ubiquitin-protein ligase RNF43","aliases":["RING finger protein 43","RING-type E3 ubiquitin transferase RNF43"],"length_aa":783,"mass_kda":85.7,"function":"E3 ubiquitin-protein ligase that acts as a negative regulator of the Wnt signaling pathway by mediating the ubiquitination, endocytosis and subsequent degradation of Wnt receptor complex components Frizzled. Acts on both canonical and non-canonical Wnt signaling pathway (PubMed:18313049, PubMed:22575959, PubMed:22895187). Along with RSPO2 and ZNRF3, constitutes a master switch that governs limb specification (By similarity)","subcellular_location":"Cell membrane; Endoplasmic reticulum membrane; Nucleus envelope","url":"https://www.uniprot.org/uniprotkb/Q68DV7/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/RNF43","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/RNF43","total_profiled":1310},"omim":[{"mim_id":"620946","title":"UBIQUITIN-SPECIFIC PEPTIDASE 42; USP42","url":"https://www.omim.org/entry/620946"},{"mim_id":"618022","title":"HUMEROFEMORAL HYPOPLASIA WITH RADIOTIBIAL RAY DEFICIENCY; HHRRD","url":"https://www.omim.org/entry/618022"},{"mim_id":"618021","title":"TETRAAMELIA SYNDROME 2; TETAMS2","url":"https://www.omim.org/entry/618021"},{"mim_id":"617108","title":"SESSILE SERRATED POLYPOSIS CANCER SYNDROME; SSPCS","url":"https://www.omim.org/entry/617108"},{"mim_id":"612482","title":"RING FINGER PROTEIN 43; RNF43","url":"https://www.omim.org/entry/612482"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"intestine","ntpm":21.4}],"url":"https://www.proteinatlas.org/search/RNF43"},"hgnc":{"alias_symbol":["FLJ20315","RNF124","DKFZp781H0392","URCC"],"prev_symbol":[]},"alphafold":{"accession":"Q68DV7","domains":[{"cath_id":"3.50.30.30","chopping":"44-192","consensus_level":"high","plddt":87.356,"start":44,"end":192},{"cath_id":"3.30.40.10","chopping":"233-249_271-319","consensus_level":"medium","plddt":84.2442,"start":233,"end":319},{"cath_id":"1.20.5","chopping":"194-223","consensus_level":"medium","plddt":81.6147,"start":194,"end":223}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q68DV7","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q68DV7-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q68DV7-F1-predicted_aligned_error_v6.png","plddt_mean":55.5},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=RNF43","jax_strain_url":"https://www.jax.org/strain/search?query=RNF43"},"sequence":{"accession":"Q68DV7","fasta_url":"https://rest.uniprot.org/uniprotkb/Q68DV7.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q68DV7/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q68DV7"}},"corpus_meta":[{"pmid":"22895187","id":"PMC_22895187","title":"Tumour 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Expression of RNF43 in HEK293T cells blocks Wnt responses and targets surface-expressed frizzled receptors to lysosomes; reconstitution of RNF43 in RNF43-mutant HCT116 cells removes their response to exogenous Wnt.\",\n      \"method\": \"Overexpression and reconstitution in cell lines, ubiquitination assays, lysosomal trafficking assays, mouse intestinal epithelium conditional knockout\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal functional assays in multiple cell lines and in vivo mouse KO, widely replicated foundational study\",\n      \"pmids\": [\"22895187\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"RNF43 inhibits Wnt/β-catenin signaling by reducing the membrane level of Frizzled in pancreatic cancer cells, serving as a negative feedback mechanism. Inactivating mutations in RNF43 increase cell-surface Frizzled and render pancreatic cancer cells dependent on autocrine/paracrine Wnt signaling, demonstrated by sensitivity to the Porcupine inhibitor LGK974 selectively in RNF43-mutant lines.\",\n      \"method\": \"Loss-of-function cell culture assays, cell-surface Frizzled measurement, wild-type RNF43 reconstitution, xenograft models, pharmacological Wnt inhibition\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (reconstitution, surface receptor measurement, xenograft) in a single focused study\",\n      \"pmids\": [\"23847203\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Crystal structure of the RNF43 ectodomain in complex with R-spondin 2 (Rspo2 Fu1-Fu2 fragment) reveals that a prominent loop in the Fu1 domain of Rspo2 clamps into a groove on the RNF43 ectodomain surface, forming a ternary complex with LGR4/5. LGR5 does not directly contact RNF43 but increases RSPO1 affinity for RNF43, supporting LGR5 as an engagement receptor and RNF43 as an effector receptor.\",\n      \"method\": \"X-ray crystallography, biophysical binding assays, mutational analysis\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structures of RNF43 ectodomain alone and in complex with R-spondin, validated by biophysical and cellular assays\",\n      \"pmids\": [\"24225776\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Crystal structure of RSPO1 bound simultaneously to both LGR5 and RNF43 ectodomains confirms physical linkage; RSPO1 is sandwiched by LGR5 and RNF43, with its CRD rod module contacting LGR5 and a hairpin inserted into the RNF43 ectodomain groove. Disease mutations map to the RSPO1-RNF43 interface.\",\n      \"method\": \"X-ray crystallography of ternary RSPO1-LGR5-RNF43 complex\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure of ternary complex with disease mutation mapping\",\n      \"pmids\": [\"23756651\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"RNF43 (and ZNRF3) are encoded by Wnt target genes and constitute a negative Wnt feedback loop; the LGR5/R-spondin complex neutralizes RNF43/ZNRF3 activity, thereby amplifying Wnt signaling in stem cells. This module controls removal of Wnt receptors from the stem cell surface.\",\n      \"method\": \"Review integrating genetic epistasis and binding studies (mechanistic synthesis of prior experimental work)\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Strong — review synthesizing replicated experimental findings; cited as mechanistic consensus\",\n      \"pmids\": [\"24532711\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"RNF43 expression is directly regulated by the canonical Wnt/β-catenin pathway via two Wnt-responsive elements (WREs) in intron 2 that associate with the TCF4/β-catenin complex, establishing RNF43 as a direct transcriptional target of TCF4/β-catenin and part of a negative feedback loop.\",\n      \"method\": \"Reporter gene assay with WRE mutagenesis, siRNA knockdown of β-catenin, chromatin immunoprecipitation (ChIP)\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP and reporter assays with mutagenesis in a single lab\",\n      \"pmids\": [\"24466159\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Dishevelled (DVL) acts as a dual-function adaptor that recruits RNF43/ZNRF3 to Wnt receptors. DVL knockout increases cell-surface FZD and LRP6; DVL is required for RNF43/ZNRF3-mediated ubiquitination and degradation of FZD. Physical interaction between DVL and ZNRF3/RNF43 is essential for their Wnt-inhibitory activity, and the DEP domain of DVL is required for FZD binding and downregulation.\",\n      \"method\": \"DVL knockout cells, co-immunoprecipitation, ubiquitination assays, domain fusion rescue experiments\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal co-IP, KO phenotype, domain mapping, and rescue experiments in one focused study\",\n      \"pmids\": [\"25891077\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"RNF43 suppresses both Wnt/β-catenin (canonical) and noncanonical Wnt signaling by distinct mechanisms. Canonical suppression requires interaction between the extracellular PA domain of RNF43 and the CRD of Frizzled, plus the intracellular RING domain. Noncanonical suppression requires the C-terminal cytoplasmic region of RNF43 interacting with the PDZ domain of Dishevelled. Missense mutations in the extracellular domain change RNF43 localization from endosome to ER, abolishing canonical but not noncanonical Wnt suppression.\",\n      \"method\": \"Domain deletion/mutation analysis, co-immunoprecipitation, immunofluorescence localization, Wnt reporter assays\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple domain mutants, localization and functional readouts in one study\",\n      \"pmids\": [\"25825523\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"RNF43 physically interacts with TCF4 in the nucleus and tethers TCF4 to the nuclear membrane, silencing TCF4 transcriptional activity even in the presence of constitutively active β-catenin mutants. This nuclear inhibitory mechanism is disrupted by tumor-associated RNF43 mutations, and RING domain mutation of RNF43 transactivates Wnt target genes in cells and Xenopus embryos.\",\n      \"method\": \"Co-immunoprecipitation, immunofluorescence (nuclear localization), TCF4 transcriptional reporter assays, Xenopus embryo functional assays, RNF43 RING domain mutagenesis\",\n      \"journal\": \"Science signaling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP and functional reporter with Xenopus validation; nuclear localization later questioned by antibody artifact paper (PMID 37023034), lowering confidence\",\n      \"pmids\": [\"26350900\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Paracrine Wnt3 secretion from Paneth cells is an essential driver of Rnf43/Znrf3-double-knockout intestinal tumor growth; removal of Paneth cells by Math1 mutation or deletion of Wnt3 inhibits tumor formation. Treatment with the Porcupine inhibitor C59 strongly inhibits RZ−/− neoplasia growth while sparing adjacent normal crypts.\",\n      \"method\": \"Genetic epistasis (Math1 KO, Wnt3 KO in Rnf43/Znrf3 DKO mice), pharmacological Porcupine inhibitor treatment in vivo\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo genetic epistasis with two independent gene deletions and pharmacological validation\",\n      \"pmids\": [\"26023187\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"RNF43 protein resides in the endoplasmic reticulum and at least partially in the nuclear envelope/inner nuclear membrane. Recombinant RNF43 has autoubiquitylation activity in a cell-free system. RNF43 interacts with HAP95 (a chromatin-associated nuclear envelope protein) by yeast two-hybrid and co-immunoprecipitation, but HAP95 is not a ubiquitination substrate of RNF43.\",\n      \"method\": \"Immunofluorescence, biochemical fractionation, sucrose density gradient, cell-free autoubiquitylation assay, yeast two-hybrid, co-immunoprecipitation\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro autoubiquitylation plus binding partner identification; single lab, and localization findings partially contradicted by later antibody artifact study\",\n      \"pmids\": [\"18313049\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"RNF43 physically interacts with the PSF/p54nrb heterodimer (nuclear RNA-binding proteins); co-expression of PSF relocates RNF43 from the nuclear periphery to the nucleoplasm.\",\n      \"method\": \"Pull-down assay with mass spectrometry identification, co-immunoprecipitation, immunofluorescence\",\n      \"journal\": \"Proteomics\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single pulldown/MS identification with co-IP confirmation; functional consequence not established; localization claims subject to antibody artifact caveat\",\n      \"pmids\": [\"18655028\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"RNF43 interacts with NEDL1 (a p53-activating E3 ligase) and with p53 itself; RNF43 suppresses p53 transcriptional activity in H1299 cells and attenuates UV-induced apoptosis.\",\n      \"method\": \"Yeast two-hybrid screening, co-immunoprecipitation, p53 transcriptional reporter assay, apoptosis assay after UV irradiation\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast two-hybrid confirmed by co-IP, functional readout (reporter + apoptosis); single lab\",\n      \"pmids\": [\"21108931\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"RNF43 promotes growth of colorectal cancer cells in an autocrine manner; the RNF43 protein is secreted and conditioned medium from RNF43-transfected cells enhances NIH3T3 cell growth. Exogenous RNF43 expression confers growth-promoting effects; suppression by siRNA retards colon cancer cell growth.\",\n      \"method\": \"Overexpression in COS7 and NIH3T3 cells, conditioned medium assay, siRNA knockdown, growth assays\",\n      \"journal\": \"International journal of oncology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, single overexpression/knockdown approach; secreted growth-promoting function not confirmed by later mechanistic work, which instead established RNF43 as a tumor suppressor\",\n      \"pmids\": [\"15492824\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"The nucleoprotein (NP) of influenza A virus (IAV) interacts with RNF43 (identified by yeast two-hybrid). IAV infection attenuates RNF43 transcript and protein levels; RNF43-depleted cells show escalated viral replication. RNF43 polyubiquitinates p53, leading to p53 destabilization. NP of IAV targets RNF43 to prevent p53 ubiquitination, causing p53 stabilization and enhanced apoptosis.\",\n      \"method\": \"Yeast two-hybrid, RNF43 depletion (viral replication assay), ubiquitination assay of p53, viral infection models\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, yeast two-hybrid interaction, ubiquitination assay; p53 as RNF43 substrate needs independent confirmation\",\n      \"pmids\": [\"25996295\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"The RSPO-LGR4/5-ZNRF3/RNF43 module controls Wnt/β-catenin-mediated metabolic liver zonation and hepatic growth. Liver-specific ZNRF3/RNF43 loss-of-function expands the hepatic Wnt/β-catenin signaling gradient; RSPO1 increases liver size and improves regeneration in an LGR4/5-dependent manner.\",\n      \"method\": \"Liver-specific conditional knockout mice, recombinant RSPO1 treatment, LGR4/5 LOF, liver size and regeneration readouts\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple genetic models (LOF and GOF) with clear in vivo phenotypic readouts, validated by pharmacological RSPO1\",\n      \"pmids\": [\"27088858\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Genome-wide CRISPR-Cas9 screens in RNF43-mutant pancreatic cancer cells identify a specific requirement for the Wnt receptor FZD5 for proliferation. Antibodies against FZD5 (and FZD8) inhibit growth of RNF43-mutant PDAC in vitro and as xenografts. This specificity could not be explained by FZD5 expression patterns alone.\",\n      \"method\": \"Genome-wide CRISPR-Cas9 screen, recombinant anti-FZD antibodies, in vitro growth assays, xenograft models, patient-derived organoids\",\n      \"journal\": \"Nature medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genome-wide genetic screen with orthogonal antibody validation in vitro and in vivo\",\n      \"pmids\": [\"27869803\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"RNF43 ubiquitinates E-cadherin phosphorylated at tyrosine 797 by c-Src (at lysine 816), leading to E-cadherin degradation, nuclear β-catenin translocation and EMT in lung adenocarcinoma cells. This ubiquitination requires Frizzled 8 (FZD8) interaction with RNF43 but not the PA domain of RNF43.\",\n      \"method\": \"Protein antibody microarray, E3 ligase profiling, immunoprecipitation, immunoblotting, immunofluorescence, shRNA knockdown, xenograft models, site-directed mutagenesis of E-cadherin phosphorylation and ubiquitination sites\",\n      \"journal\": \"BMC cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — site-specific mutagenesis plus in vivo xenograft; single lab; contradicted by another study (PMID 32103169) that could not confirm E-cadherin as RNF43 substrate\",\n      \"pmids\": [\"31286874\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"RNF43 C-terminal truncation mutants (including G659fs), when expressed at endogenous levels via CRISPR knock-in, retain the ability to suppress β-catenin signaling and Wnt-receptor turnover equivalently to wild-type RNF43, and retain DVL-binding. Only N-terminal RNF43 mutations convey Wnt-dependency onto cancer cells. (NOTE: this finding partially contradicts other studies that report C-terminal truncations as loss-of-function.)\",\n      \"method\": \"CRISPR-Cas9 endogenous knock-in/knockout in HEK293T and KM12 cells, β-catenin reporter assay, Wnt-receptor surface flow cytometry, co-immunoprecipitation\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — endogenous CRISPR models with multiple readouts; single lab; contradicted by other CRISPR-based studies\",\n      \"pmids\": [\"32103169\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"RNF43 activity requires phosphorylation at a conserved triplet of serines (phospho-switch). This phospho-regulation is required for zebrafish development and mouse intestinal organoid growth. Cancer-associated mutations that abrogate RNF43 phosphorylation cooperate with active Ras to promote tumorigenesis by abolishing Wnt-inhibitory function of RNF43 while maintaining p53-inhibitory function. Phosphomimetic substitutions of the serine trio restore tumor-suppressive activity of extracellular oncogenic mutants.\",\n      \"method\": \"Site-directed mutagenesis of serine phosphosites, phosphomimetic substitutions, zebrafish developmental assays, mouse intestinal organoids, co-expression with active Ras, Wnt reporter assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — mutagenesis, multiple organism models (zebrafish, mouse organoids), and rescue experiments in one study\",\n      \"pmids\": [\"32934222\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"A class of RNF43 truncating cancer mutations (C-terminal truncations) retains Wnt receptor downregulation activity but interferes with a ubiquitin-independent suppressor role of the RNF43 cytosolic tail involving Casein kinase 1 (CK1) binding and phosphorylation. Truncated RNF43 variants trap CK1 at the plasma membrane, preventing β-catenin turnover and driving ligand-independent Wnt/β-catenin transcription. Gene editing in human colon stem cells shows these truncations cooperate with p53 loss for niche-independent self-renewal.\",\n      \"method\": \"CRISPR gene editing of patient-derived mutations, CK1 co-immunoprecipitation, phosphorylation assays, β-catenin reporter assays, PORCN inhibitor sensitivity assays, human colon organoids\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — endogenous CRISPR mutations, biochemical CK1 interaction, functional organoid assays in one focused study\",\n      \"pmids\": [\"32965059\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Systematic functional assay of 119 missense and 45 truncating patient-derived RNF43 mutations reveals: RING domain missense mutations and a subset of extracellular domain mutations hyperactivate Wnt/β-catenin by forming inactive dimers with endogenous RNF43 or ZNRF3. C-terminal truncation mutants (including G659fs) are loss-of-function at endogenous levels, increase cell-surface Frizzled and Wnt/β-catenin signaling, and render xenografts responsive to PORCN inhibition.\",\n      \"method\": \"Cell-based reporter assays, genome editing, flow cytometry for surface Frizzled, immunofluorescence, patient-derived xenografts and cell lines\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — systematic mutagenesis with multiple orthogonal methods and in vivo validation\",\n      \"pmids\": [\"33067269\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"USP42 deubiquitinase antagonizes R-spondin by protecting ZNRF3/RNF43 from ubiquitin-dependent membrane clearance. USP42 binds to the Dishevelled interacting region (DIR) of ZNRF3 and stalls the R-spondin-LGR4-ZNRF3 ternary complex by deubiquitinating ZNRF3, increasing LRP6 and FZD turnover and inhibiting Wnt signaling.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, deubiquitination assays, Wnt reporter assays, colon cancer cells and mouse small intestinal organoids\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — biochemical interaction and functional rescue; single lab\",\n      \"pmids\": [\"33786993\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ZNRF3 deletion alone promotes hepatocyte proliferation, which becomes limited by compensatory RNF43 upregulation. Concomitant deletion of both ZNRF3 and RNF43 in hepatocytes results in metabolic reprogramming of periportal hepatocytes, clonal expansion, and liver tumor development, demonstrating cooperative roles of the two ligases in restricting WNT/β-catenin activity to balance metabolic function and proliferation.\",\n      \"method\": \"Hepatocyte-specific conditional double knockout mice (ZNRF3 and RNF43), single-cell RNA sequencing, organoid culture, chromatin accessibility analysis\",\n      \"journal\": \"Cell stem cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional KO in vivo with scRNA-seq and organoid validation\",\n      \"pmids\": [\"34129813\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"RNF43 is a negative regulator of noncanonical WNT5A signaling. RNF43 interacts with noncanonical Wnt receptor complex components ROR1, ROR2, VANGL1, and VANGL2 (identified by BioID and immunoprecipitation). RNF43 triggers VANGL2 ubiquitination and proteasomal degradation and induces clathrin-dependent internalization of ROR1 receptor and inhibits ROR2 activation, thereby blocking pro-metastatic WNT5A signaling in melanoma.\",\n      \"method\": \"BioID proximity labeling, co-immunoprecipitation, ubiquitination assays, receptor internalization assays, in vitro and in vivo invasion assays, BRAF/MEK inhibitor resistance models\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — BioID plus co-IP, ubiquitination, internalization assays, and in vivo validation in one study\",\n      \"pmids\": [\"34702444\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Rnf43 (but not Znrf3) is potently activated by Wnt signaling in oligodendrocyte progenitor cells (OPCs) and promotes OPC differentiation specifically in injury contexts by negatively regulating Wnt signal strength at the level of Fzd1 receptor presentation on the OPC cell surface. Inhibition of Fzd1 using UM206 promotes remyelination following demyelinating injury.\",\n      \"method\": \"Conditional Rnf43 knockout in OPCs, neonatal hypoxic injury and adult demyelination models, Fzd1 inhibition, in vivo and ex vivo remyelination assays\",\n      \"journal\": \"Neuron\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional KO with injury models and pharmacological Fzd1 inhibition as mechanistic confirmation\",\n      \"pmids\": [\"34390652\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RNF43_p.G659fs promotes cancer cell growth independent of Wnt signaling. RNF43-G659fs mutant binds the PI3K regulatory subunit p85, leading to increased PI3K signaling through p85 ubiquitination and degradation. PI3K/mTOR inhibitors selectively kill RNF43-G659fs cells in isogenic lines and xenografts, and reduce interferon response gene expression which is reversed by PI3K/mTOR inhibition.\",\n      \"method\": \"Drug repurposing library screen, isogenic cell lines, co-immunoprecipitation (G659fs:p85 interaction), ubiquitination assay, xenograft models, patient-derived organoids, RNA-seq\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — biochemical interaction plus multiple functional models; single lab; novel finding requiring independent replication\",\n      \"pmids\": [\"35676246\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RNF43/ZNRF3 hepatocyte-specific double knockout results in steatohepatitis, accumulation of unsaturated lipids, defective hepatocyte regeneration upon injury, and liver cancer development. Differentiation defects and lipid alterations are, in part, cell-autonomous, demonstrated by hepatocyte-, hepatoblast-, and ductal cell-derived organoids.\",\n      \"method\": \"Hepatocyte-specific conditional Rnf43/Znrf3 double KO mice, organoid cultures from multiple liver cell types, lipidomic analysis\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional KO with multiple orthogonal organoid models and metabolic readouts\",\n      \"pmids\": [\"35039505\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"RNF43 is a negative feedback regulator of PAR2 (protease-activated receptor 2, a GPCR): RNF43 co-associates with PAR2 and promotes its membrane elimination and polyubiquitination, reducing PAR2-induced β-catenin signaling. This degradation is rescued by R-spondin2 in the presence of LGR5.\",\n      \"method\": \"Co-immunoprecipitation, cell-surface biotinylation assay, polyubiquitination assay, β-catenin reporter assays, mouse AOM/DSS colon cancer model with Par2 KO\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP, ubiquitination assay, and in vivo mouse model; single lab\",\n      \"pmids\": [\"36468684\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"RNF43 directly interacts with γH2AX (phosphorylated H2AX) and its loss impairs DNA damage response (DDR) activation in gastric cells, conferring resistance to γ-radiation and chemotherapy by dampening DDR and preventing apoptosis.\",\n      \"method\": \"Co-immunoprecipitation (RNF43:γH2AX), RNF43 depletion, γ-radiation and chemotherapy sensitivity assays, Helicobacter pylori infection in Rnf43ΔEx8 mice\",\n      \"journal\": \"Cellular and molecular gastroenterology and hepatology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct interaction by co-IP with multiple functional readouts in vitro and in vivo; single lab\",\n      \"pmids\": [\"33188943\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"RNF43 preferentially targets FZD1, FZD5, and FZD7 for endocytosis, while the related ZNRF3 preferentially targets FZD6. The RNF43 transmembrane domain (TMD) is a key molecular determinant for FZD5 endocytosis specificity; swapping the TMD between RNF43 and ZNRF3 redirects their FZD preferences.\",\n      \"method\": \"Endocytosis assays comparing RNF43 vs. ZNRF3 for each FZD, TMD domain-swap mutagenesis, flow cytometry for surface FZD levels\",\n      \"journal\": \"Life science alliance\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — domain-swap mutagenesis with functional endocytosis readouts for multiple FZD members\",\n      \"pmids\": [\"38969364\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"RNF43 ubiquitinates B-RAF at K499 to promote proteasome-dependent B-RAF degradation, reducing MEK activity and proliferative ability in cancer cells. Phosphorylation of B-RAF at T491 suppresses this ubiquitination by decreasing the RNF43:B-RAF interaction. MEK and Wnt inhibitors synergistically suppress growth of RNF43-mutated pancreatic cancer cells in vitro and in vivo.\",\n      \"method\": \"Co-immunoprecipitation (RNF43:B-RAF), ubiquitination assay, site-directed mutagenesis (K499, T491), MEK activity assays, xenograft models, combination drug studies\",\n      \"journal\": \"Advanced science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — site-specific mutagenesis and ubiquitination assay with in vivo validation; single lab; novel finding\",\n      \"pmids\": [\"38225722\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ZNRF3 and RNF43 interact with EGFR via their extracellular domains, leading to EGFR ubiquitination and subsequent degradation via the RING E3 ligase domain. Overexpression of ZNRF3 reduces EGFR levels and suppresses cancer cell growth; knockout of ZNRF3/RNF43 upregulates EGFR signaling and promotes tumorigenesis.\",\n      \"method\": \"Biochemical co-immunoprecipitation (extracellular domain interaction), ubiquitination assay, ZNRF3 overexpression, ZNRF3/RNF43 double knockout, in vitro and in vivo tumor growth assays, proteogenomic correlation\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP, ubiquitination assay, KO phenotype, and in vivo models; preprint, not yet peer-reviewed\",\n      \"pmids\": [\"38260423\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"RNF43 interacts with PD-L1 in gastric cancer cells and augments both K48- and K63-linked ubiquitination of PD-L1, reducing PD-L1 surface expression and enhancing T cell antitumor activity.\",\n      \"method\": \"Co-immunoprecipitation (RNF43:PD-L1), ubiquitination assay (K48/K63 linkage-specific), flow cytometry for PD-L1 surface levels, T cell co-culture killing assay\",\n      \"journal\": \"Scandinavian journal of immunology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single co-IP and ubiquitination assay; single lab; limited mechanistic follow-up\",\n      \"pmids\": [\"39007965\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"m6A methylation (mediated by METTL3/IGF2BP2 axis) regulates RNF43 mRNA stability. RNF43 protein directly interacts with and ubiquitinates NDUFS1 (NADH dehydrogenase Fe-S protein 1), promoting its proteasomal degradation and suppressing oxidative phosphorylation in endometrial stromal cells.\",\n      \"method\": \"m6A modification assay, METTL3/IGF2BP2 knockdown, co-immunoprecipitation (RNF43:NDUFS1), ubiquitination assay, NDUFS1 siRNA knockdown, cell viability/migration assays, rat endometriosis model\",\n      \"journal\": \"Journal of cellular physiology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — co-IP and ubiquitination assay for NDUFS1; single lab; novel substrate, limited orthogonal validation\",\n      \"pmids\": [\"38988031\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"The extracellular Protease Associated (PA) domain of RNF43 is not necessary for suppression of Wnt/β-catenin signaling. RNF43ΔPA mutant still inhibits LRP6 phosphorylation, DVL2/DVL3 phosphorylation, and β-catenin-dependent gene expression. RNF43ΔPA is not sensitive to R-spondin1 treatment, confirming that R-spondin inhibits RNF43 function through the PA domain.\",\n      \"method\": \"TetON controlled overexpression, CRISPR/Cas9 RNF43/ZNRF3 double KO rescue, Western blot, TOPflash dual luciferase assay\",\n      \"journal\": \"Cell communication and signaling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CRISPR rescue in defined KO background, multiple signaling readouts; single lab\",\n      \"pmids\": [\"32527265\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Commercial RNF43 antibodies targeting exons 8-9 epitopes yield non-specific signals in immunoblotting, immunofluorescence, and immunohistochemistry; nuclear staining patterns attributed to RNF43 are antibody artifacts. Endogenous RNF43 localization to the nucleus (as reported in earlier studies) is not supported by validated antibody data.\",\n      \"method\": \"CRISPR deletion of RNF43 exons 8-9, immunoblotting, immunofluorescence, immunohistochemistry with knockout-validated negative controls for four commercial antibodies\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CRISPR-validated antibody controls; directly refutes prior nuclear localization claims; single lab\",\n      \"pmids\": [\"37023034\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Wnt ligands specifically induce FZD5/8 endocytosis and degradation in a ZNRF3/RNF43-dependent manner; ZNRF3/RNF43 selectively target FZD5/8 for Wnt-stimulated degradation. DVL proteins promote ligand-independent FZD endocytosis but are dispensable for Wnt-induced FZD5/8 degradation. Wnt promotes the interaction between FZD5 and RNF43.\",\n      \"method\": \"FZD endocytosis assays with Wnt stimulation, ZNRF3/RNF43 knockdown/knockout, DVL knockout, co-immunoprecipitation (FZD5:RNF43), receptor internalization fluorescence assays\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO validation, co-IP, and functional endocytosis assays; single lab\",\n      \"pmids\": [\"41070826\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"LGR4 (but not LGR5) forms a 2:2 complex with RNF43/ZNRF3 that accommodates bivalent RSPO binding, providing high-affinity RSPO recruitment. Co-expression of ZNRF3 with LGR4 dramatically increases monovalent RSPO2 binding affinity, whereas co-expression of ZNRF3 with LGR5 has no effect, explaining why only LGR4 mediates the RSPO-RNF43/ZNRF3 inhibitory complex.\",\n      \"method\": \"Whole-cell binding affinity measurements with monovalent and bivalent RSPO2 furin domains, co-expression of LGR4/5 with ZNRF3 in cells, structural modeling\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — systematic binding measurements in whole cells with controls; single lab\",\n      \"pmids\": [\"37402772\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"RNF43 is a transmembrane RING-type E3 ubiquitin ligase that functions primarily as a negative regulator of Wnt signaling by ubiquitinating Frizzled receptors (preferentially FZD1/5/7) and LRP6 for lysosomal/proteasomal degradation, a process requiring Dishevelled as an adaptor and phosphorylation of a conserved serine triplet; its activity is inhibited when R-spondin ligands recruit LGR4 to form a ternary complex with the RNF43 ectodomain, and is stabilized at the membrane by the deubiquitinase USP42; beyond canonical Wnt, RNF43 also suppresses noncanonical WNT5A signaling by ubiquitinating VANGL2 and internalizing ROR1/ROR2, targets additional substrates including B-RAF and PAR2 for ubiquitin-mediated degradation, and contains a cytosolic tail that sequesters Casein kinase 1 (CK1) to promote β-catenin turnover through a ubiquitin-independent mechanism; loss-of-function mutations in RNF43 increase surface Frizzled abundance, rendering tumors dependent on paracrine Wnt signaling and sensitive to upstream Wnt inhibitors.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"RNF43 is a transmembrane RING-type E3 ubiquitin ligase that operates as a negative-feedback brake on Wnt signaling, limiting the abundance of Wnt receptors at the cell surface [#0, #1]. It ubiquitinates Frizzled receptors and targets them for lysosomal degradation, with member-selective preference for FZD1/5/7 dictated by its transmembrane domain [#0, #30]; receptor engagement is mediated by Dishevelled, which acts as an adaptor recruiting RNF43 to Frizzled/LRP6 and is required for receptor downregulation [#6, #7]. Catalytic activity is gated by phosphorylation of a conserved serine triplet, and cancer mutations that abolish this phospho-switch eliminate Wnt-inhibitory function while sparing other activities [#19]. RNF43 is itself a direct transcriptional target of the TCF4/\\u03b2-catenin complex, closing a negative feedback loop [#5]. This braking activity is neutralized when R-spondin ligands clamp into a groove on the RNF43 ectodomain and bridge it to LGR4 in a ternary complex, with LGR4 (not LGR5) supplying high-affinity RSPO recruitment; the protease-associated (PA) domain mediates this R-spondin sensitivity but is dispensable for Wnt suppression itself [#2, #3, #35, #38]. The RSPO\\u2013LGR\\u2013RNF43/ZNRF3 module governs stem-cell Wnt responsiveness and tissue homeostasis: loss of RNF43 (with ZNRF3) expands Wnt signaling to drive intestinal and hepatic tumorigenesis and metabolic reprogramming, and RNF43 controls oligodendrocyte progenitor differentiation through Fzd1 [#9, #23, #25, #27]. Inactivating RNF43 mutations raise surface Frizzled and render tumors dependent on paracrine Wnt, conferring sensitivity to Porcupine inhibitors and an acquired requirement for FZD5 [#1, #9, #16, #21]. Distinct truncating mutations are not simple null alleles: certain C-terminal truncations retain receptor downregulation but trap Casein kinase 1 at the membrane to block \\u03b2-catenin turnover, while RING-domain and some extracellular missense mutants hyperactivate Wnt by forming inactive dimers with endogenous RNF43/ZNRF3 [#20, #21]. Beyond canonical Wnt, RNF43 suppresses noncanonical WNT5A signaling by ubiquitinating VANGL2 and internalizing ROR1/ROR2 [#24], and additional substrates including B-RAF have been reported [#31].\",\n  \"teleology\": [\n    {\n      \"year\": 2008,\n      \"claim\": \"Before its Wnt role was known, RNF43 was established as a bona fide E3 ligase capable of autoubiquitylation, fixing its biochemical identity as a RING enzyme.\",\n      \"evidence\": \"Cell-free autoubiquitylation assay plus fractionation and yeast two-hybrid interaction screening\",\n      \"pmids\": [\"18313049\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No physiological substrate identified at this stage\", \"Reported ER/nuclear-envelope localization later challenged as antibody artifact\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Defined RNF43's core function: it ubiquitinates surface Frizzled receptors to route them to lysosomes and dampen Wnt signaling, with reconstitution in mutant cells restoring Wnt sensitivity.\",\n      \"evidence\": \"Overexpression/reconstitution in HEK293T and HCT116, ubiquitination and lysosomal trafficking assays, conditional mouse intestinal knockout\",\n      \"pmids\": [\"22895187\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve receptor selectivity among FZD members\", \"Adaptor and recruitment mechanism unknown\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Connected RNF43 loss to cancer dependency, showing inactivating mutations raise surface Frizzled and create selective addiction to upstream Wnt that is druggable with Porcupine inhibitors.\",\n      \"evidence\": \"Loss-of-function and reconstitution in pancreatic cancer lines, surface-Frizzled measurement, xenografts, LGK974 sensitivity\",\n      \"pmids\": [\"23847203\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism distinguishing mutant classes not addressed\", \"Did not identify the specific FZD required\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Structurally defined how R-spondin inhibits RNF43, showing R-spondin clamps the RNF43 ectodomain and bridges it to LGR4/5 in a ternary complex.\",\n      \"evidence\": \"X-ray crystallography of RNF43 ectodomain with R-spondin and of the ternary RSPO1-LGR5-RNF43 complex, with disease-mutation mapping\",\n      \"pmids\": [\"24225776\", \"23756651\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not establish how complex formation translates to reduced ligase activity in cells\", \"LGR4 vs LGR5 functional asymmetry not yet quantified\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Established the regulatory architecture: RNF43 is itself a Wnt/\\u03b2-catenin transcriptional target, defining a self-limiting negative feedback loop neutralized by RSPO/LGR.\",\n      \"evidence\": \"Reporter assays with WRE mutagenesis, \\u03b2-catenin knockdown and ChIP; review synthesis of epistasis and binding data\",\n      \"pmids\": [\"24466159\", \"24532711\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Feedback kinetics in vivo not quantified\", \"Cell-type specificity of WRE usage unexplored\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Identified Dishevelled as the obligate adaptor recruiting RNF43 to Wnt receptors, and dissected separable extracellular/RING and cytoplasmic determinants for canonical versus noncanonical suppression.\",\n      \"evidence\": \"DVL knockout, reciprocal co-IP, domain mapping and rescue; domain-deletion/localization and Wnt reporter assays\",\n      \"pmids\": [\"25891077\", \"25825523\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How DVL DEP/PDZ engagement is regulated remains open\", \"Relative contribution of canonical vs noncanonical arms in vivo unresolved\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"In vivo genetics tied RNF43/ZNRF3 loss to paracrine-Wnt-driven tumor growth, demonstrating Paneth-cell Wnt3 and Porcupine activity as the actionable dependency.\",\n      \"evidence\": \"Math1 and Wnt3 deletions in Rnf43/Znrf3 DKO mice, Porcupine inhibitor C59 treatment\",\n      \"pmids\": [\"26023187\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Source-cell dependency may differ in other tissues\", \"Did not address mutant-specific signaling outputs\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Pinpointed FZD5 as the specific receptor required by RNF43-mutant pancreatic cancer, nominating a precise antibody target.\",\n      \"evidence\": \"Genome-wide CRISPR screen, anti-FZD5/8 antibodies, in vitro growth, xenografts, patient-derived organoids\",\n      \"pmids\": [\"27869803\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis of FZD5 selectivity not yet defined\", \"Generality across other RNF43-mutant tissues untested\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Extended the receptor module to physiology, showing the RSPO-LGR-ZNRF3/RNF43 axis sets the hepatic Wnt gradient controlling metabolic liver zonation and regeneration.\",\n      \"evidence\": \"Liver-specific conditional knockouts, recombinant RSPO1, LGR4/5 loss-of-function, size and regeneration readouts\",\n      \"pmids\": [\"27088858\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not separate RNF43 from ZNRF3 contributions\", \"Downstream metabolic effectors not defined\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Resolved that distinct mutation classes act through different mechanisms: a phospho-switch licenses ligase activity, C-terminal truncations trap CK1 to drive ligand-independent Wnt, and systematic profiling separated dominant-negative dimerizing mutants from loss-of-function truncations.\",\n      \"evidence\": \"Serine-triplet mutagenesis with zebrafish/organoid models; CRISPR knock-in of patient truncations with CK1 co-IP and organoid assays; systematic functional assay of 164 patient mutations\",\n      \"pmids\": [\"32934222\", \"32965059\", \"33067269\", \"32527265\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Reports conflict on whether specific C-terminal truncations are loss-of-function or retain activity (contrast with PMID 32103169)\", \"Kinase responsible for the serine-triplet phospho-switch not identified\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Broadened the substrate and regulatory landscape: USP42 stabilizes RNF43/ZNRF3 at the membrane, RNF43/ZNRF3 cooperate to restrain hepatic Wnt and lipid metabolism, RNF43 suppresses noncanonical WNT5A signaling via VANGL2/ROR ubiquitination and internalization, and it tunes OPC differentiation through Fzd1.\",\n      \"evidence\": \"Co-IP/deubiquitination assays; hepatocyte conditional double-KO with scRNA-seq; BioID, ubiquitination and internalization assays in melanoma; conditional Rnf43 KO in OPCs with injury models\",\n      \"pmids\": [\"33786993\", \"34129813\", \"34702444\", \"34390652\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Tissue-specific balance between canonical and noncanonical roles unclear\", \"Whether noncanonical substrates are targeted in non-melanoma contexts untested\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Defined the molecular determinant of receptor selectivity and Wnt-stimulated targeting, and added new substrates linking RNF43 to RAS-MAPK signaling.\",\n      \"evidence\": \"TMD domain-swap endocytosis assays for multiple FZD members; Wnt-stimulated FZD5/8 degradation assays with KO validation; B-RAF co-IP, K499/T491 mutagenesis and combination drug studies\",\n      \"pmids\": [\"38969364\", \"41070826\", \"38225722\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"B-RAF as substrate from a single lab without independent confirmation\", \"How Wnt ligand promotes the FZD5-RNF43 interaction mechanistically unresolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unresolved which of RNF43's many reported non-Wnt substrates (p53, E-cadherin, PAR2, PD-L1, NDUFS1, EGFR, p85) represent core physiological functions versus context-restricted or unconfirmed activities, and what the validated subcellular localization is given antibody-artifact concerns.\",\n      \"evidence\": \"Multiple single-lab co-IP/ubiquitination studies, several contradicted or flagged as artifacts\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"E-cadherin substrate claim contradicted by an independent study\", \"Prior nuclear localization shown to be antibody artifact\", \"Several substrates rest on single Co-IP without reciprocal or in vivo validation\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [0, 10, 24, 31]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 24, 31]},\n      {\"term_id\": \"GO:0061630\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 20, 30]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [7]},\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [7, 10]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 1, 6, 24]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [15, 25]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [9, 16, 21]},\n      {\"term_id\": \"R-HSA-9609507\", \"supporting_discovery_ids\": [0, 30, 37]}\n    ],\n    \"complexes\": [\n      \"RSPO-LGR4/5-RNF43/ZNRF3 ternary complex\"\n    ],\n    \"partners\": [\n      \"FZD5\",\n      \"DVL\",\n      \"LGR4\",\n      \"RSPO1\",\n      \"VANGL2\",\n      \"ROR1\",\n      \"USP42\",\n      \"CSNK1\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":9,"faith_total":9,"faith_pct":100.0}}