{"gene":"TMEM67","run_date":"2026-06-10T10:51:55","timeline":{"discoveries":[{"year":2006,"finding":"TMEM67 encodes meckelin, a 995-amino acid seven-transmembrane receptor protein. Positional cloning identified pathogenic mutations in TMEM67 in Meckel-Gruber syndrome families, establishing it as the MKS3 gene product.","method":"Positional cloning, mutation analysis, sequence conservation analysis","journal":"Nature genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — positional cloning with multiple independent family mutations, replicated across labs in subsequent studies","pmids":["16415887"],"is_preprint":false},{"year":2009,"finding":"MKS3 (TMEM67) and MKS1 are required for ciliary structure and function; loss-of-function leads to defects in centrosome number, cilia number and length, including multi-ciliated phenotypes, centrosome over-duplication, and functional defects of the connecting cilium in the eye (lack of outer segment formation) and very short sperm flagella in the wpk rat model.","method":"Analysis of wpk rat model (in vivo), patient kidney tissue analysis, stable shRNA knockdown of Mks3 in IMCD3 cells (in vitro), immunofluorescence, electron microscopy","journal":"Human molecular genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (in vivo animal model, patient tissue, shRNA knockdown in cell lines) with defined cellular phenotypes","pmids":["19515853"],"is_preprint":false},{"year":2010,"finding":"In C. elegans, MKS-3 localizes to the distal end of dendrites and the cilium base (not the cilium itself) of ciliated sensory neurons. Genetic analysis shows mks-3 functions in a pathway with other mks genes, and mks-1/mks-3 genetically interact with a separate nphp-1/nphp-4 pathway to influence cilia positioning, orientation, and formation. Combined disruption has cell non-autonomous effects on sensilla.","method":"GFP localization in C. elegans, genetic epistasis analysis (double and triple mutants), chemoreception assays","journal":"Journal of the American Society of Nephrology : JASN","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct localization experiment combined with genetic epistasis across multiple mutant combinations, establishing pathway position","pmids":["20150540"],"is_preprint":false},{"year":2009,"finding":"MKS3/TMEM67 (meckelin) functions in endoplasmic reticulum-associated degradation (ERAD) of misfolded surfactant protein C (SP-C). MKS3 is a membrane glycoprotein predominantly localized to the ER; its ER lumenal domain interacts with mutant SP-C and associated chaperones, while its transmembrane/cytosolic domains interact with the AAA-ATPase p97. Knockdown of MKS3 inhibited degradation of mutant SP-C, and deletion of the TM/cytosolic domains abrogated p97 interaction and caused accumulation of mutant SP-C.","method":"Co-immunoprecipitation, domain deletion constructs, siRNA knockdown, immunofluorescence/subcellular fractionation, Western blotting","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal co-IP with domain mapping, functional knockdown with defined phenotypic readout, localization by fractionation, single lab with multiple orthogonal methods","pmids":["19815549"],"is_preprint":false},{"year":2013,"finding":"In the Tmem67 null (bpck) mouse, canonical Wnt signaling is upregulated in cyst linings and isolated fibroblasts. Analysis of zebrafish tmem67 morphants and the bpck mouse did not support a global loss of planar cell polarity (PCP). Defective cilia loading (not loss of ciliogenesis, basal body docking, or PCP signaling) leads to dysfunctional cilia in MKS3 tissues.","method":"Tmem67 null mouse analysis, zebrafish morpholino knockdown, immunofluorescence for PCP markers, Wnt signaling reporter assays, ciliary loading assays","journal":"Human molecular genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple genetic models and orthogonal assays in a single lab; negative result for PCP is mechanistically informative","pmids":["23393159"],"is_preprint":false},{"year":2013,"finding":"Overexpression of TMEM67 in HEK293 cells activates ERK and JNK signaling pathways (and 4E-BP1 phosphorylation), and these activations are suppressed by pharmacological ERK or JNK inhibitors. In the bpck mouse kidney, phosphorylation of tyrosine-phosphorylated proteins, ERK, and 4E-BP1 is elevated at different postnatal ages.","method":"Overexpression in HEK293 cells, pharmacological inhibition, Western blotting for phospho-ERK/JNK/4E-BP1, bpck mouse kidney analysis","journal":"Cell biology international","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — in vitro overexpression plus in vivo mouse model, single lab, but relies on overexpression rather than clean KO/KD with rescue","pmids":["23456819"],"is_preprint":false},{"year":2015,"finding":"TMEM67 (meckelin) is essential for phosphorylation of the non-canonical Wnt receptor ROR2 upon stimulation with Wnt5a. ROR2 colocalizes and interacts with TMEM67 at the ciliary transition zone (Co-IP). The extracellular N-terminal domain of TMEM67 preferentially binds Wnt5a in an in vitro binding assay. Tmem67 mutant mouse lungs fail to respond to Wnt5a-stimulated epithelial branching morphogenesis, and this pulmonary hypoplasia is rescued by activating RhoA downstream of the Wnt5a-TMEM67-ROR2 axis.","method":"Co-immunoprecipitation of TMEM67 and ROR2, in vitro Wnt5a binding assay with N-terminal domain, Wnt5a-stimulated ROR2 phosphorylation assay, Tmem67 mutant mouse lung culture rescue by RhoA activation, immunofluorescence colocalization","journal":"Disease models & mechanisms","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — multiple orthogonal methods (co-IP, in vitro binding assay, phosphorylation assay, genetic rescue), single lab but strong mechanistic chain","pmids":["26035863"],"is_preprint":false},{"year":2017,"finding":"In zebrafish tmem67 morphants, Wnt signaling (but not Hedgehog signaling) is suppressed. Wild-type human TMEM67 RNA rescues morphant phenotypes, whereas RNA harboring patient mutations (p.Gly132Ala or p.Tyr920ThrfsX40) does not, functionally validating these as pathogenic. The p.Tyr920ThrfsX40 truncation mutation accelerates TMEM67 protein turnover as shown by Western blotting.","method":"Zebrafish morpholino knockdown with mRNA rescue, Western blotting for protein stability, Wnt and Hedgehog signaling assays","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo morpholino model with specific rescue experiments and signaling pathway assays, single lab","pmids":["28860541"],"is_preprint":false},{"year":2019,"finding":"In the Tmem67 knockout mouse cerebellum, loss of TMEM67 leads to aberrantly high canonical Wnt/β-catenin signaling and increased expression of homeobox transcription factor HOXB5. HOXB5 protein occupancy at the β-catenin promoter is significantly increased upon canonical Wnt activation in Tmem67-/- cerebellar neurons, demonstrating that increased canonical Wnt signaling following loss of TMEM67 is directly dependent on HOXB5.","method":"Tmem67 knockout mouse analysis, transcriptome profiling, chromatin immunoprecipitation (ChIP) for HOXB5 at β-catenin promoter, Western blotting, immunofluorescence","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo KO model with transcriptomic profiling and ChIP validation, single lab, multiple orthogonal methods","pmids":["30931988"],"is_preprint":false},{"year":2019,"finding":"TMEM67 is required for regulation of choroid plexus epithelial cell fluid and electrolyte homeostasis in the Wpk rat; TMEM67 point mutation leads to gene dose-dependent hydrocephalus with increased Na+, K+, and Cl- in CSF of severely hydrocephalic animals, while aquaporin 1 and claudin-1 remain normally polarized in all genotypes.","method":"Wpk rat model (homozygous and heterozygous), CSF ion analysis, immunofluorescence for AQP1 and claudin-1 localization, MRI/CT volumetric imaging","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo genetic model with biochemical CSF analysis and protein localization, single lab","pmids":["30705305"],"is_preprint":false},{"year":2021,"finding":"In adult zebrafish tmem67 mutants, absence of a single cilium precedes cystogenesis and mTOR signaling is hyperactivated. mTOR inhibition (via hypomorphic mtor strain or rapamycin) ameliorates renal cysts in both embryonic and adult zebrafish tmem67 mutants and rescues ciliary abnormalities in adult mutants, placing mTOR signaling downstream of tmem67 loss.","method":"TALEN-generated tmem67 zebrafish mutants, 2D/3D imaging, rapamycin treatment, hypomorphic mtor genetic cross, cilia quantification","journal":"Journal of the American Society of Nephrology : JASN","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic model with pharmacological and genetic mTOR inhibition, multiple orthogonal approaches, single lab","pmids":["33574160"],"is_preprint":false},{"year":2021,"finding":"TMEM67 and TMEM237 are unique protein components of the photoreceptor outer segment plasma membrane, as identified by protein correlation profiling using label-free quantitative mass spectrometry comparing OS plasma membrane enriched versus total OS membrane preparations.","method":"Label-free quantitative mass spectrometry (protein correlation profiling), subcellular fractionation of photoreceptor outer segments","journal":"Molecular & cellular proteomics : MCP","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — quantitative MS-based protein correlation profiling with enriched membrane fractions, single study","pmids":["33933680"],"is_preprint":false},{"year":2022,"finding":"TMEM67 is required for the transition zone (TZ) gating function that controls entry of membrane-associated proteins ARL13B and INPP5E into primary cilia. TMEM67-KO cells show impaired ciliogenesis, elongated cilia, and perturbed ciliary localization of ARL13B and INPP5E, but normal recruitment of TZ proteins CEP290, RPGRIP1L, and NPHP5. TMEM67 localization extends beyond the TZ into the cilium itself. Ciliopathy-associated TMEM67 mutants restore ARL13B/INPP5E ciliary localization but fail to rescue aberrant cilium elongation.","method":"CRISPR/Cas9 TMEM67 knockout in hTERT-RPE1 cells, immunofluorescence for TZ and ciliary proteins, exogenous expression of patient variants","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean KO with defined ciliary phenotypes and partial rescue with patient variants, single lab","pmids":["36334440"],"is_preprint":false},{"year":2025,"finding":"TMEM67 contains a cleavage motif in its extracellular domain that is cleaved by the extracellular matrix metalloproteinase ADAMTS9. This cleavage generates two functional forms: a C-terminal portion that localizes to the ciliary transition zone and regulates ciliogenesis, and a non-cleaved full-length form that regulates non-canonical Wnt signaling. A non-cleavable TMEM67 knock-in mouse develops severe ciliopathy phenocopying Tmem67-/- mice but retains normal Wnt signaling, demonstrating that the two functions are uncoupled by cleavage. Patient variants within the cleavage motif disrupt cilia structure and function in mammalian cells and C. elegans.","method":"Identification of ADAMTS9 cleavage motif, in vitro cleavage assays, non-cleavable TMEM67 knock-in mouse model, C. elegans patient variant characterization, mammalian cell culture, Wnt signaling assays, ciliary localization studies","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro cleavage assay, non-cleavable mouse model, C. elegans genetic validation, and Wnt signaling assays constitute multiple rigorous orthogonal methods establishing mechanism; peer-reviewed publication","pmids":["40436881"],"is_preprint":false},{"year":2025,"finding":"FUT8 interacts with TMEM67 and catalyzes its core fucosylation (N-linked glycosylation). Core fucosylation stabilizes TMEM67 by impeding its degradation via the autophagy pathway, ensuring proper localization of TMEM67 to the ciliary transition zone and promoting cilium formation. Fut8-deficient mice exhibit ciliary defects in kidney, brain, and trachea.","method":"Mass spectrometry-based proteomic analysis, co-immunoprecipitation of FUT8 and TMEM67, functional studies in Fut8-deficient mice, autophagy pathway inhibition assays, immunofluorescence for transition zone localization","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — MS identification, co-IP, in vivo KO mouse model with ciliary phenotypes, and biochemical pathway (autophagy) studies constitute multiple orthogonal methods in a single study","pmids":["40728580"],"is_preprint":false},{"year":2024,"finding":"The extracellular cleavage by ADAMTS9 uncouples TMEM67's two functions: the non-cleaved form regulates Wnt signaling while the C-terminal cleavage product mediates ciliogenesis via the transition zone. (Preprint version of the peer-reviewed Nature Communications paper PMID:40436881.)","method":"Identification of ADAMTS9 cleavage motif, non-cleavable TMEM67 mouse model, C. elegans patient variant characterization, Wnt signaling assays","journal":"bioRxiv","confidence":"High","confidence_rationale":"Tier 1 / Strong — same study as PMID:40436881; included as preprint record but superseded by peer-reviewed publication","pmids":["39282264"],"is_preprint":true},{"year":2022,"finding":"TMEM67 protein variants associated with mild cholestasis show significantly decreased protein levels in vitro, but their interaction with MKS1 remains unaffected, establishing that MKS1-TMEM67 protein interaction is preserved even with reduced protein stability caused by these hypomorphic variants.","method":"Western blotting for TMEM67 protein levels, co-immunoprecipitation of TMEM67 and MKS1","journal":"Journal of cellular physiology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, single co-IP for MKS1 interaction, limited mechanistic follow-up","pmids":["35621037"],"is_preprint":false}],"current_model":"TMEM67 (meckelin) is a seven-transmembrane receptor protein that functions at the ciliary transition zone as a gating component controlling entry of membrane-associated proteins (ARL13B, INPP5E) into primary cilia, regulates cilia length and number, and is proteolytically cleaved in its extracellular domain by the metalloproteinase ADAMTS9 to generate two functional forms: a C-terminal fragment that mediates ciliogenesis at the transition zone, and a full-length non-cleaved form that transduces non-canonical Wnt signaling via direct binding to Wnt5a and interaction with the co-receptor ROR2; additionally, TMEM67 is stabilized by FUT8-mediated core fucosylation (which blocks autophagy-mediated degradation), participates in ERAD of misfolded surfactant protein C via interaction with p97, and its loss leads to hyperactivation of canonical Wnt/β-catenin signaling (partly through HOXB5) and mTOR signaling, with genetic epistasis in C. elegans placing mks-3 in the MKS pathway that synergizes with the NPHP pathway for normal ciliogenesis."},"narrative":{"mechanistic_narrative":"TMEM67 (meckelin) is a seven-transmembrane receptor protein that functions at the ciliary transition zone to control ciliogenesis and to transduce non-canonical Wnt signaling, with biallelic mutations causing Meckel-Gruber syndrome (MKS3) [PMID:16415887]. Loss of TMEM67 disrupts centrosome number, cilia number and length, and produces functional cilia defects across tissues; the underlying defect is impaired ciliary cargo loading rather than failure of basal-body docking or planar cell polarity [PMID:19515853, PMID:23393159]. At the transition zone, TMEM67 acts as a gate selectively controlling entry of membrane-associated proteins ARL13B and INPP5E into the cilium while leaving recruitment of core transition-zone proteins intact, and it restrains cilium elongation [PMID:36334440]. A defining feature of TMEM67 is that its extracellular domain is cleaved by the metalloproteinase ADAMTS9, uncoupling its two activities: the C-terminal cleavage product localizes to the transition zone and drives ciliogenesis, whereas the full-length non-cleaved form transduces non-canonical Wnt signaling [PMID:40436881]. In that signaling axis, the TMEM67 N-terminal domain binds Wnt5a and the protein interacts with the co-receptor ROR2 at the transition zone, where it is required for Wnt5a-stimulated ROR2 phosphorylation and downstream RhoA-dependent branching morphogenesis [PMID:26035863]. Conversely, loss of TMEM67 derepresses canonical Wnt/β-catenin signaling in a HOXB5-dependent manner [PMID:30931988] and hyperactivates mTOR signaling, the inhibition of which rescues ciliary and cystic phenotypes [PMID:33574160]. TMEM67 protein abundance and transition-zone localization are controlled post-translationally: FUT8-mediated core fucosylation stabilizes TMEM67 by blocking autophagic degradation [PMID:40728580]. Separately, an ER-localized pool of meckelin participates in ERAD of misfolded surfactant protein C through its lumenal domain binding mutant SP-C and its transmembrane/cytosolic domains recruiting the AAA-ATPase p97 [PMID:19815549].","teleology":[{"year":2006,"claim":"Established the gene's existence and disease relevance: identifying TMEM67 as the MKS3 gene defined meckelin as a seven-transmembrane receptor whose mutation causes Meckel-Gruber syndrome.","evidence":"Positional cloning and mutation analysis in Meckel-Gruber syndrome families","pmids":["16415887"],"confidence":"High","gaps":["Molecular function and subcellular site of action not defined at this stage","No ligand or interaction partner identified"]},{"year":2009,"claim":"Placed TMEM67 in ciliary biology by showing loss-of-function disrupts centrosome and cilia number/length and impairs specialized cilia, answering whether the disease gene acts on cilia.","evidence":"wpk rat model, patient kidney tissue, and shRNA knockdown in IMCD3 cells with IF and EM","pmids":["19515853"],"confidence":"High","gaps":["Did not resolve the molecular step in ciliogenesis that TMEM67 controls","Subciliary localization not established"]},{"year":2009,"claim":"Revealed a distinct ER pool of meckelin functioning in ERAD, showing the protein has activities beyond the cilium by bridging misfolded SP-C to the p97 degradation machinery.","evidence":"Reciprocal Co-IP with domain-deletion mapping, siRNA knockdown, and subcellular fractionation","pmids":["19815549"],"confidence":"High","gaps":["Relationship between ER/ERAD role and ciliary role unresolved","Generality beyond SP-C substrate untested"]},{"year":2010,"claim":"Defined TMEM67's pathway position genetically, establishing it as part of an MKS module that synergizes with the NPHP module for ciliogenesis and localizes to the cilium base.","evidence":"GFP localization and genetic epistasis (double/triple mutants) in C. elegans sensory neurons","pmids":["20150540"],"confidence":"High","gaps":["Mammalian counterpart of the genetic interactions not directly demonstrated","Molecular nature of MKS-NPHP synergy unknown"]},{"year":2013,"claim":"Distinguished the cellular lesion as defective ciliary cargo loading rather than loss of ciliogenesis or PCP, and linked TMEM67 loss to elevated canonical Wnt signaling.","evidence":"Tmem67-null mouse and zebrafish morphants with PCP markers, Wnt reporters, and ciliary loading assays","pmids":["23393159"],"confidence":"Medium","gaps":["Identity of mis-loaded cargo not pinpointed","Mechanism connecting TMEM67 loss to canonical Wnt upregulation unresolved"]},{"year":2013,"claim":"Connected TMEM67 to ERK/JNK and 4E-BP1 signaling, hinting at downstream kinase pathways affected in disease tissue.","evidence":"Overexpression in HEK293 cells with pharmacological inhibitors and phospho-Western blotting, plus bpck mouse kidney","pmids":["23456819"],"confidence":"Medium","gaps":["Relies on overexpression rather than loss-of-function with rescue","Directness of TMEM67 effect on these kinases unestablished"]},{"year":2015,"claim":"Identified TMEM67 as a non-canonical Wnt receptor: it binds Wnt5a via its N-terminal domain and partners with ROR2 to enable Wnt5a-stimulated ROR2 phosphorylation and RhoA-dependent morphogenesis.","evidence":"Co-IP with ROR2, in vitro Wnt5a binding assay, ROR2 phosphorylation assay, and RhoA rescue of mutant mouse lung","pmids":["26035863"],"confidence":"High","gaps":["Single-lab mechanistic chain","How receptor signaling integrates with the ciliary gating role not addressed"]},{"year":2017,"claim":"Functionally validated patient mutations and showed TMEM67 selectively supports Wnt (not Hedgehog) signaling, with a truncating mutation destabilizing the protein.","evidence":"Zebrafish morpholino with wild-type vs mutant mRNA rescue and protein-stability Western blots","pmids":["28860541"],"confidence":"Medium","gaps":["Morpholino-based knockdown specificity limitations","Mechanism of accelerated turnover not defined"]},{"year":2019,"claim":"Defined the mechanism by which TMEM67 loss elevates canonical Wnt: derepressed HOXB5 occupies the β-catenin promoter and is required for the elevated signaling.","evidence":"Tmem67-knockout mouse cerebellum with transcriptome profiling and HOXB5 ChIP at the β-catenin promoter","pmids":["30931988"],"confidence":"Medium","gaps":["How TMEM67 normally restrains HOXB5 is unknown","Tissue generality beyond cerebellum untested"]},{"year":2019,"claim":"Linked TMEM67 to epithelial fluid/electrolyte homeostasis, showing dose-dependent hydrocephalus with altered CSF ion content but preserved AQP1/claudin-1 polarity.","evidence":"Wpk rat genotypes with CSF ion analysis, IF for tight-junction/water channels, and volumetric imaging","pmids":["30705305"],"confidence":"Medium","gaps":["Molecular transport defect causing CSF ion changes unidentified","Causal link to ciliary defect not established"]},{"year":2021,"claim":"Placed mTOR hyperactivation downstream of TMEM67 loss and identified it as a therapeutically tractable driver of cystogenesis.","evidence":"TALEN tmem67 zebrafish mutants with rapamycin and hypomorphic mtor genetic rescue plus cilia quantification","pmids":["33574160"],"confidence":"Medium","gaps":["Direct biochemical link between TMEM67 and mTOR not defined","Relationship to Wnt-axis dysregulation unclear"]},{"year":2021,"claim":"Localized TMEM67 to the photoreceptor outer segment plasma membrane, refining the membrane compartment in which it resides in a specialized cilium.","evidence":"Label-free quantitative MS protein correlation profiling of fractionated outer-segment membranes","pmids":["33933680"],"confidence":"Medium","gaps":["Functional role at the OS plasma membrane not tested","Single proteomic study"]},{"year":2022,"claim":"Resolved the ciliary gating function: TMEM67 is required for entry of ARL13B and INPP5E into cilia and restrains cilium length while leaving core TZ assembly intact.","evidence":"CRISPR TMEM67 knockout in RPE1 cells with IF for TZ/ciliary proteins and patient-variant expression","pmids":["36334440"],"confidence":"Medium","gaps":["Mechanism of selective cargo gating not defined","Single-lab knockout system"]},{"year":2022,"claim":"Showed mild-cholestasis variants reduce TMEM67 protein levels without disrupting the MKS1 interaction, indicating destabilization rather than complex disassembly.","evidence":"Western blotting for protein levels and Co-IP of TMEM67 with MKS1","pmids":["35621037"],"confidence":"Low","gaps":["Single Co-IP without reciprocal validation for the MKS1 interaction","Causal link between reduced stability and cholestasis not established"]},{"year":2025,"claim":"Established the central regulatory logic: ADAMTS9 cleavage of the extracellular domain uncouples TMEM67's ciliary and Wnt functions into a C-terminal ciliogenic fragment and a full-length Wnt-signaling form.","evidence":"ADAMTS9 cleavage-motif mapping, in vitro cleavage assays, a non-cleavable knock-in mouse, C. elegans patient-variant studies, and Wnt assays","pmids":["40436881"],"confidence":"High","gaps":["Spatial/temporal regulation of ADAMTS9-mediated cleavage in vivo not fully defined","How the C-terminal fragment is retained at the TZ unresolved"]},{"year":2025,"claim":"Identified a post-translational stabilization mechanism: FUT8-mediated core fucosylation protects TMEM67 from autophagic degradation, ensuring its transition-zone localization and ciliogenesis.","evidence":"MS proteomics, FUT8-TMEM67 Co-IP, autophagy inhibition assays, and Fut8-deficient mice with ciliary defects","pmids":["40728580"],"confidence":"High","gaps":["Whether fucosylation also modulates the Wnt-signaling form is untested","Glycosylation site(s) on TMEM67 not enumerated"]},{"year":null,"claim":"How TMEM67's transition-zone gating activity, its ADAMTS9-cleavage-regulated dual functions, and its downstream control of canonical Wnt/HOXB5 and mTOR signaling are mechanistically integrated into a single coherent pathway remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of the TMEM67 receptor or its cleavage","Direct biochemical link from TMEM67 to mTOR and to canonical Wnt repression not established","Mechanism of selective ARL13B/INPP5E gating unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[6]},{"term_id":"GO:0048018","term_label":"receptor ligand activity","supporting_discovery_ids":[6]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[3,12]}],"localization":[{"term_id":"GO:0005929","term_label":"cilium","supporting_discovery_ids":[2,12,13]},{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[3]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[11]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[6,8,10]},{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[1,12,13]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[3,14]}],"complexes":[],"partners":["ROR2","WNT5A","P97","ADAMTS9","FUT8","MKS1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q5HYA8","full_name":"Meckelin","aliases":["Meckel syndrome type 3 protein","Transmembrane protein 67"],"length_aa":995,"mass_kda":111.7,"function":"Required for ciliary structure and function. Part of the tectonic-like complex which is required for tissue-specific ciliogenesis and may regulate ciliary membrane composition (By similarity). Involved in centrosome migration to the apical cell surface during early ciliogenesis. Involved in the regulation of cilia length and appropriate number through the control of centrosome duplication. Is a key regulator of stereociliary bundle orientation (By similarity). Required for epithelial cell branching morphology. Essential for endoplasmic reticulum-associated degradation (ERAD) of surfactant protein C (SFTPC). Involved in the negative regulation of canonical Wnt signaling, and activation of the non-canonical cascade stimulated by WNT5A (PubMed:26035863). In non-canonical Wnt signaling, it may act as ROR2 coreceptor (By similarity)","subcellular_location":"Cell membrane; Endoplasmic reticulum membrane; Cell projection, cilium; Cytoplasm, cytoskeleton, cilium basal body","url":"https://www.uniprot.org/uniprotkb/Q5HYA8/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/TMEM67","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":[{"gene":"CANX","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/TMEM67","total_profiled":1310},"omim":[{"mim_id":"617778","title":"THIOREDOXIN DOMAIN-CONTAINING PROTEIN 15; TXNDC15","url":"https://www.omim.org/entry/617778"},{"mim_id":"615586","title":"CENTROSOMAL PROTEIN, 19-KD; CEP19","url":"https://www.omim.org/entry/615586"},{"mim_id":"614423","title":"TRANSMEMBRANE PROTEIN 237; TMEM237","url":"https://www.omim.org/entry/614423"},{"mim_id":"614144","title":"B9 DOMAIN-CONTAINING PROTEIN 1; B9D1","url":"https://www.omim.org/entry/614144"},{"mim_id":"613550","title":"NEPHRONOPHTHISIS 11; NPHP11","url":"https://www.omim.org/entry/613550"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"heart muscle","ntpm":20.8}],"url":"https://www.proteinatlas.org/search/TMEM67"},"hgnc":{"alias_symbol":["MGC26979","JBTS6","NPHP11"],"prev_symbol":["MKS3"]},"alphafold":{"accession":"Q5HYA8","domains":[{"cath_id":"-","chopping":"47-170","consensus_level":"medium","plddt":75.0135,"start":47,"end":170}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q5HYA8","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q5HYA8-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q5HYA8-F1-predicted_aligned_error_v6.png","plddt_mean":84.69},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=TMEM67","jax_strain_url":"https://www.jax.org/strain/search?query=TMEM67"},"sequence":{"accession":"Q5HYA8","fasta_url":"https://rest.uniprot.org/uniprotkb/Q5HYA8.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q5HYA8/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q5HYA8"}},"corpus_meta":[{"pmid":"16415887","id":"PMC_16415887","title":"The transmembrane protein meckelin (MKS3) is mutated in Meckel-Gruber syndrome and the wpk rat.","date":"2006","source":"Nature genetics","url":"https://pubmed.ncbi.nlm.nih.gov/16415887","citation_count":219,"is_preprint":false},{"pmid":"17160906","id":"PMC_17160906","title":"The Meckel-Gruber syndrome gene, MKS3, is mutated in Joubert syndrome.","date":"2006","source":"American journal of human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/17160906","citation_count":188,"is_preprint":false},{"pmid":"19574260","id":"PMC_19574260","title":"Mutations in 3 genes (MKS3, CC2D2A and RPGRIP1L) cause COACH syndrome (Joubert syndrome with congenital hepatic fibrosis).","date":"2009","source":"Journal of medical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/19574260","citation_count":111,"is_preprint":false},{"pmid":"19508969","id":"PMC_19508969","title":"Hypomorphic mutations in meckelin (MKS3/TMEM67) cause nephronophthisis with liver fibrosis (NPHP11).","date":"2009","source":"Journal of medical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/19508969","citation_count":105,"is_preprint":false},{"pmid":"19515853","id":"PMC_19515853","title":"Ciliary and centrosomal defects associated with mutation and depletion of the Meckel syndrome genes MKS1 and MKS3.","date":"2009","source":"Human molecular genetics","url":"https://pubmed.ncbi.nlm.nih.gov/19515853","citation_count":100,"is_preprint":false},{"pmid":"19058225","id":"PMC_19058225","title":"MKS3/TMEM67 mutations are a major cause of COACH Syndrome, a Joubert Syndrome related disorder with liver involvement.","date":"2009","source":"Human mutation","url":"https://pubmed.ncbi.nlm.nih.gov/19058225","citation_count":79,"is_preprint":false},{"pmid":"20232449","id":"PMC_20232449","title":"Novel TMEM67 mutations and genotype-phenotype correlates in meckelin-related ciliopathies.","date":"2010","source":"Human mutation","url":"https://pubmed.ncbi.nlm.nih.gov/20232449","citation_count":73,"is_preprint":false},{"pmid":"17397051","id":"PMC_17397051","title":"Spectrum of MKS1 and MKS3 mutations in Meckel syndrome: a genotype-phenotype correlation. Mutation in brief #960. 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Positional cloning identified pathogenic mutations in TMEM67 in Meckel-Gruber syndrome families, establishing it as the MKS3 gene product.\",\n      \"method\": \"Positional cloning, mutation analysis, sequence conservation analysis\",\n      \"journal\": \"Nature genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — positional cloning with multiple independent family mutations, replicated across labs in subsequent studies\",\n      \"pmids\": [\"16415887\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"MKS3 (TMEM67) and MKS1 are required for ciliary structure and function; loss-of-function leads to defects in centrosome number, cilia number and length, including multi-ciliated phenotypes, centrosome over-duplication, and functional defects of the connecting cilium in the eye (lack of outer segment formation) and very short sperm flagella in the wpk rat model.\",\n      \"method\": \"Analysis of wpk rat model (in vivo), patient kidney tissue analysis, stable shRNA knockdown of Mks3 in IMCD3 cells (in vitro), immunofluorescence, electron microscopy\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (in vivo animal model, patient tissue, shRNA knockdown in cell lines) with defined cellular phenotypes\",\n      \"pmids\": [\"19515853\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"In C. elegans, MKS-3 localizes to the distal end of dendrites and the cilium base (not the cilium itself) of ciliated sensory neurons. Genetic analysis shows mks-3 functions in a pathway with other mks genes, and mks-1/mks-3 genetically interact with a separate nphp-1/nphp-4 pathway to influence cilia positioning, orientation, and formation. Combined disruption has cell non-autonomous effects on sensilla.\",\n      \"method\": \"GFP localization in C. elegans, genetic epistasis analysis (double and triple mutants), chemoreception assays\",\n      \"journal\": \"Journal of the American Society of Nephrology : JASN\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct localization experiment combined with genetic epistasis across multiple mutant combinations, establishing pathway position\",\n      \"pmids\": [\"20150540\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"MKS3/TMEM67 (meckelin) functions in endoplasmic reticulum-associated degradation (ERAD) of misfolded surfactant protein C (SP-C). MKS3 is a membrane glycoprotein predominantly localized to the ER; its ER lumenal domain interacts with mutant SP-C and associated chaperones, while its transmembrane/cytosolic domains interact with the AAA-ATPase p97. Knockdown of MKS3 inhibited degradation of mutant SP-C, and deletion of the TM/cytosolic domains abrogated p97 interaction and caused accumulation of mutant SP-C.\",\n      \"method\": \"Co-immunoprecipitation, domain deletion constructs, siRNA knockdown, immunofluorescence/subcellular fractionation, Western blotting\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal co-IP with domain mapping, functional knockdown with defined phenotypic readout, localization by fractionation, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"19815549\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"In the Tmem67 null (bpck) mouse, canonical Wnt signaling is upregulated in cyst linings and isolated fibroblasts. Analysis of zebrafish tmem67 morphants and the bpck mouse did not support a global loss of planar cell polarity (PCP). Defective cilia loading (not loss of ciliogenesis, basal body docking, or PCP signaling) leads to dysfunctional cilia in MKS3 tissues.\",\n      \"method\": \"Tmem67 null mouse analysis, zebrafish morpholino knockdown, immunofluorescence for PCP markers, Wnt signaling reporter assays, ciliary loading assays\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple genetic models and orthogonal assays in a single lab; negative result for PCP is mechanistically informative\",\n      \"pmids\": [\"23393159\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Overexpression of TMEM67 in HEK293 cells activates ERK and JNK signaling pathways (and 4E-BP1 phosphorylation), and these activations are suppressed by pharmacological ERK or JNK inhibitors. In the bpck mouse kidney, phosphorylation of tyrosine-phosphorylated proteins, ERK, and 4E-BP1 is elevated at different postnatal ages.\",\n      \"method\": \"Overexpression in HEK293 cells, pharmacological inhibition, Western blotting for phospho-ERK/JNK/4E-BP1, bpck mouse kidney analysis\",\n      \"journal\": \"Cell biology international\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — in vitro overexpression plus in vivo mouse model, single lab, but relies on overexpression rather than clean KO/KD with rescue\",\n      \"pmids\": [\"23456819\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"TMEM67 (meckelin) is essential for phosphorylation of the non-canonical Wnt receptor ROR2 upon stimulation with Wnt5a. ROR2 colocalizes and interacts with TMEM67 at the ciliary transition zone (Co-IP). The extracellular N-terminal domain of TMEM67 preferentially binds Wnt5a in an in vitro binding assay. Tmem67 mutant mouse lungs fail to respond to Wnt5a-stimulated epithelial branching morphogenesis, and this pulmonary hypoplasia is rescued by activating RhoA downstream of the Wnt5a-TMEM67-ROR2 axis.\",\n      \"method\": \"Co-immunoprecipitation of TMEM67 and ROR2, in vitro Wnt5a binding assay with N-terminal domain, Wnt5a-stimulated ROR2 phosphorylation assay, Tmem67 mutant mouse lung culture rescue by RhoA activation, immunofluorescence colocalization\",\n      \"journal\": \"Disease models & mechanisms\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — multiple orthogonal methods (co-IP, in vitro binding assay, phosphorylation assay, genetic rescue), single lab but strong mechanistic chain\",\n      \"pmids\": [\"26035863\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"In zebrafish tmem67 morphants, Wnt signaling (but not Hedgehog signaling) is suppressed. Wild-type human TMEM67 RNA rescues morphant phenotypes, whereas RNA harboring patient mutations (p.Gly132Ala or p.Tyr920ThrfsX40) does not, functionally validating these as pathogenic. The p.Tyr920ThrfsX40 truncation mutation accelerates TMEM67 protein turnover as shown by Western blotting.\",\n      \"method\": \"Zebrafish morpholino knockdown with mRNA rescue, Western blotting for protein stability, Wnt and Hedgehog signaling assays\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo morpholino model with specific rescue experiments and signaling pathway assays, single lab\",\n      \"pmids\": [\"28860541\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"In the Tmem67 knockout mouse cerebellum, loss of TMEM67 leads to aberrantly high canonical Wnt/β-catenin signaling and increased expression of homeobox transcription factor HOXB5. HOXB5 protein occupancy at the β-catenin promoter is significantly increased upon canonical Wnt activation in Tmem67-/- cerebellar neurons, demonstrating that increased canonical Wnt signaling following loss of TMEM67 is directly dependent on HOXB5.\",\n      \"method\": \"Tmem67 knockout mouse analysis, transcriptome profiling, chromatin immunoprecipitation (ChIP) for HOXB5 at β-catenin promoter, Western blotting, immunofluorescence\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo KO model with transcriptomic profiling and ChIP validation, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"30931988\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"TMEM67 is required for regulation of choroid plexus epithelial cell fluid and electrolyte homeostasis in the Wpk rat; TMEM67 point mutation leads to gene dose-dependent hydrocephalus with increased Na+, K+, and Cl- in CSF of severely hydrocephalic animals, while aquaporin 1 and claudin-1 remain normally polarized in all genotypes.\",\n      \"method\": \"Wpk rat model (homozygous and heterozygous), CSF ion analysis, immunofluorescence for AQP1 and claudin-1 localization, MRI/CT volumetric imaging\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo genetic model with biochemical CSF analysis and protein localization, single lab\",\n      \"pmids\": [\"30705305\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"In adult zebrafish tmem67 mutants, absence of a single cilium precedes cystogenesis and mTOR signaling is hyperactivated. mTOR inhibition (via hypomorphic mtor strain or rapamycin) ameliorates renal cysts in both embryonic and adult zebrafish tmem67 mutants and rescues ciliary abnormalities in adult mutants, placing mTOR signaling downstream of tmem67 loss.\",\n      \"method\": \"TALEN-generated tmem67 zebrafish mutants, 2D/3D imaging, rapamycin treatment, hypomorphic mtor genetic cross, cilia quantification\",\n      \"journal\": \"Journal of the American Society of Nephrology : JASN\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic model with pharmacological and genetic mTOR inhibition, multiple orthogonal approaches, single lab\",\n      \"pmids\": [\"33574160\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TMEM67 and TMEM237 are unique protein components of the photoreceptor outer segment plasma membrane, as identified by protein correlation profiling using label-free quantitative mass spectrometry comparing OS plasma membrane enriched versus total OS membrane preparations.\",\n      \"method\": \"Label-free quantitative mass spectrometry (protein correlation profiling), subcellular fractionation of photoreceptor outer segments\",\n      \"journal\": \"Molecular & cellular proteomics : MCP\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — quantitative MS-based protein correlation profiling with enriched membrane fractions, single study\",\n      \"pmids\": [\"33933680\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"TMEM67 is required for the transition zone (TZ) gating function that controls entry of membrane-associated proteins ARL13B and INPP5E into primary cilia. TMEM67-KO cells show impaired ciliogenesis, elongated cilia, and perturbed ciliary localization of ARL13B and INPP5E, but normal recruitment of TZ proteins CEP290, RPGRIP1L, and NPHP5. TMEM67 localization extends beyond the TZ into the cilium itself. Ciliopathy-associated TMEM67 mutants restore ARL13B/INPP5E ciliary localization but fail to rescue aberrant cilium elongation.\",\n      \"method\": \"CRISPR/Cas9 TMEM67 knockout in hTERT-RPE1 cells, immunofluorescence for TZ and ciliary proteins, exogenous expression of patient variants\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KO with defined ciliary phenotypes and partial rescue with patient variants, single lab\",\n      \"pmids\": [\"36334440\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TMEM67 contains a cleavage motif in its extracellular domain that is cleaved by the extracellular matrix metalloproteinase ADAMTS9. This cleavage generates two functional forms: a C-terminal portion that localizes to the ciliary transition zone and regulates ciliogenesis, and a non-cleaved full-length form that regulates non-canonical Wnt signaling. A non-cleavable TMEM67 knock-in mouse develops severe ciliopathy phenocopying Tmem67-/- mice but retains normal Wnt signaling, demonstrating that the two functions are uncoupled by cleavage. Patient variants within the cleavage motif disrupt cilia structure and function in mammalian cells and C. elegans.\",\n      \"method\": \"Identification of ADAMTS9 cleavage motif, in vitro cleavage assays, non-cleavable TMEM67 knock-in mouse model, C. elegans patient variant characterization, mammalian cell culture, Wnt signaling assays, ciliary localization studies\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro cleavage assay, non-cleavable mouse model, C. elegans genetic validation, and Wnt signaling assays constitute multiple rigorous orthogonal methods establishing mechanism; peer-reviewed publication\",\n      \"pmids\": [\"40436881\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"FUT8 interacts with TMEM67 and catalyzes its core fucosylation (N-linked glycosylation). Core fucosylation stabilizes TMEM67 by impeding its degradation via the autophagy pathway, ensuring proper localization of TMEM67 to the ciliary transition zone and promoting cilium formation. Fut8-deficient mice exhibit ciliary defects in kidney, brain, and trachea.\",\n      \"method\": \"Mass spectrometry-based proteomic analysis, co-immunoprecipitation of FUT8 and TMEM67, functional studies in Fut8-deficient mice, autophagy pathway inhibition assays, immunofluorescence for transition zone localization\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — MS identification, co-IP, in vivo KO mouse model with ciliary phenotypes, and biochemical pathway (autophagy) studies constitute multiple orthogonal methods in a single study\",\n      \"pmids\": [\"40728580\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"The extracellular cleavage by ADAMTS9 uncouples TMEM67's two functions: the non-cleaved form regulates Wnt signaling while the C-terminal cleavage product mediates ciliogenesis via the transition zone. (Preprint version of the peer-reviewed Nature Communications paper PMID:40436881.)\",\n      \"method\": \"Identification of ADAMTS9 cleavage motif, non-cleavable TMEM67 mouse model, C. elegans patient variant characterization, Wnt signaling assays\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — same study as PMID:40436881; included as preprint record but superseded by peer-reviewed publication\",\n      \"pmids\": [\"39282264\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"TMEM67 protein variants associated with mild cholestasis show significantly decreased protein levels in vitro, but their interaction with MKS1 remains unaffected, establishing that MKS1-TMEM67 protein interaction is preserved even with reduced protein stability caused by these hypomorphic variants.\",\n      \"method\": \"Western blotting for TMEM67 protein levels, co-immunoprecipitation of TMEM67 and MKS1\",\n      \"journal\": \"Journal of cellular physiology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, single co-IP for MKS1 interaction, limited mechanistic follow-up\",\n      \"pmids\": [\"35621037\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"TMEM67 (meckelin) is a seven-transmembrane receptor protein that functions at the ciliary transition zone as a gating component controlling entry of membrane-associated proteins (ARL13B, INPP5E) into primary cilia, regulates cilia length and number, and is proteolytically cleaved in its extracellular domain by the metalloproteinase ADAMTS9 to generate two functional forms: a C-terminal fragment that mediates ciliogenesis at the transition zone, and a full-length non-cleaved form that transduces non-canonical Wnt signaling via direct binding to Wnt5a and interaction with the co-receptor ROR2; additionally, TMEM67 is stabilized by FUT8-mediated core fucosylation (which blocks autophagy-mediated degradation), participates in ERAD of misfolded surfactant protein C via interaction with p97, and its loss leads to hyperactivation of canonical Wnt/β-catenin signaling (partly through HOXB5) and mTOR signaling, with genetic epistasis in C. elegans placing mks-3 in the MKS pathway that synergizes with the NPHP pathway for normal ciliogenesis.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TMEM67 (meckelin) is a seven-transmembrane receptor protein that functions at the ciliary transition zone to control ciliogenesis and to transduce non-canonical Wnt signaling, with biallelic mutations causing Meckel-Gruber syndrome (MKS3) [#0]. Loss of TMEM67 disrupts centrosome number, cilia number and length, and produces functional cilia defects across tissues; the underlying defect is impaired ciliary cargo loading rather than failure of basal-body docking or planar cell polarity [#1, #4]. At the transition zone, TMEM67 acts as a gate selectively controlling entry of membrane-associated proteins ARL13B and INPP5E into the cilium while leaving recruitment of core transition-zone proteins intact, and it restrains cilium elongation [#12]. A defining feature of TMEM67 is that its extracellular domain is cleaved by the metalloproteinase ADAMTS9, uncoupling its two activities: the C-terminal cleavage product localizes to the transition zone and drives ciliogenesis, whereas the full-length non-cleaved form transduces non-canonical Wnt signaling [#13]. In that signaling axis, the TMEM67 N-terminal domain binds Wnt5a and the protein interacts with the co-receptor ROR2 at the transition zone, where it is required for Wnt5a-stimulated ROR2 phosphorylation and downstream RhoA-dependent branching morphogenesis [#6]. Conversely, loss of TMEM67 derepresses canonical Wnt/\\u03b2-catenin signaling in a HOXB5-dependent manner [#8] and hyperactivates mTOR signaling, the inhibition of which rescues ciliary and cystic phenotypes [#10]. TMEM67 protein abundance and transition-zone localization are controlled post-translationally: FUT8-mediated core fucosylation stabilizes TMEM67 by blocking autophagic degradation [#14]. Separately, an ER-localized pool of meckelin participates in ERAD of misfolded surfactant protein C through its lumenal domain binding mutant SP-C and its transmembrane/cytosolic domains recruiting the AAA-ATPase p97 [#3].\"\n,\n  \"teleology\": [\n    {\n      \"year\": 2006,\n      \"claim\": \"Established the gene's existence and disease relevance: identifying TMEM67 as the MKS3 gene defined meckelin as a seven-transmembrane receptor whose mutation causes Meckel-Gruber syndrome.\",\n      \"evidence\": \"Positional cloning and mutation analysis in Meckel-Gruber syndrome families\",\n      \"pmids\": [\"16415887\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular function and subcellular site of action not defined at this stage\", \"No ligand or interaction partner identified\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Placed TMEM67 in ciliary biology by showing loss-of-function disrupts centrosome and cilia number/length and impairs specialized cilia, answering whether the disease gene acts on cilia.\",\n      \"evidence\": \"wpk rat model, patient kidney tissue, and shRNA knockdown in IMCD3 cells with IF and EM\",\n      \"pmids\": [\"19515853\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve the molecular step in ciliogenesis that TMEM67 controls\", \"Subciliary localization not established\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Revealed a distinct ER pool of meckelin functioning in ERAD, showing the protein has activities beyond the cilium by bridging misfolded SP-C to the p97 degradation machinery.\",\n      \"evidence\": \"Reciprocal Co-IP with domain-deletion mapping, siRNA knockdown, and subcellular fractionation\",\n      \"pmids\": [\"19815549\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relationship between ER/ERAD role and ciliary role unresolved\", \"Generality beyond SP-C substrate untested\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Defined TMEM67's pathway position genetically, establishing it as part of an MKS module that synergizes with the NPHP module for ciliogenesis and localizes to the cilium base.\",\n      \"evidence\": \"GFP localization and genetic epistasis (double/triple mutants) in C. elegans sensory neurons\",\n      \"pmids\": [\"20150540\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mammalian counterpart of the genetic interactions not directly demonstrated\", \"Molecular nature of MKS-NPHP synergy unknown\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Distinguished the cellular lesion as defective ciliary cargo loading rather than loss of ciliogenesis or PCP, and linked TMEM67 loss to elevated canonical Wnt signaling.\",\n      \"evidence\": \"Tmem67-null mouse and zebrafish morphants with PCP markers, Wnt reporters, and ciliary loading assays\",\n      \"pmids\": [\"23393159\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Identity of mis-loaded cargo not pinpointed\", \"Mechanism connecting TMEM67 loss to canonical Wnt upregulation unresolved\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Connected TMEM67 to ERK/JNK and 4E-BP1 signaling, hinting at downstream kinase pathways affected in disease tissue.\",\n      \"evidence\": \"Overexpression in HEK293 cells with pharmacological inhibitors and phospho-Western blotting, plus bpck mouse kidney\",\n      \"pmids\": [\"23456819\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Relies on overexpression rather than loss-of-function with rescue\", \"Directness of TMEM67 effect on these kinases unestablished\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Identified TMEM67 as a non-canonical Wnt receptor: it binds Wnt5a via its N-terminal domain and partners with ROR2 to enable Wnt5a-stimulated ROR2 phosphorylation and RhoA-dependent morphogenesis.\",\n      \"evidence\": \"Co-IP with ROR2, in vitro Wnt5a binding assay, ROR2 phosphorylation assay, and RhoA rescue of mutant mouse lung\",\n      \"pmids\": [\"26035863\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Single-lab mechanistic chain\", \"How receptor signaling integrates with the ciliary gating role not addressed\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Functionally validated patient mutations and showed TMEM67 selectively supports Wnt (not Hedgehog) signaling, with a truncating mutation destabilizing the protein.\",\n      \"evidence\": \"Zebrafish morpholino with wild-type vs mutant mRNA rescue and protein-stability Western blots\",\n      \"pmids\": [\"28860541\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Morpholino-based knockdown specificity limitations\", \"Mechanism of accelerated turnover not defined\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Defined the mechanism by which TMEM67 loss elevates canonical Wnt: derepressed HOXB5 occupies the \\u03b2-catenin promoter and is required for the elevated signaling.\",\n      \"evidence\": \"Tmem67-knockout mouse cerebellum with transcriptome profiling and HOXB5 ChIP at the \\u03b2-catenin promoter\",\n      \"pmids\": [\"30931988\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How TMEM67 normally restrains HOXB5 is unknown\", \"Tissue generality beyond cerebellum untested\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Linked TMEM67 to epithelial fluid/electrolyte homeostasis, showing dose-dependent hydrocephalus with altered CSF ion content but preserved AQP1/claudin-1 polarity.\",\n      \"evidence\": \"Wpk rat genotypes with CSF ion analysis, IF for tight-junction/water channels, and volumetric imaging\",\n      \"pmids\": [\"30705305\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular transport defect causing CSF ion changes unidentified\", \"Causal link to ciliary defect not established\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Placed mTOR hyperactivation downstream of TMEM67 loss and identified it as a therapeutically tractable driver of cystogenesis.\",\n      \"evidence\": \"TALEN tmem67 zebrafish mutants with rapamycin and hypomorphic mtor genetic rescue plus cilia quantification\",\n      \"pmids\": [\"33574160\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct biochemical link between TMEM67 and mTOR not defined\", \"Relationship to Wnt-axis dysregulation unclear\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Localized TMEM67 to the photoreceptor outer segment plasma membrane, refining the membrane compartment in which it resides in a specialized cilium.\",\n      \"evidence\": \"Label-free quantitative MS protein correlation profiling of fractionated outer-segment membranes\",\n      \"pmids\": [\"33933680\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional role at the OS plasma membrane not tested\", \"Single proteomic study\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Resolved the ciliary gating function: TMEM67 is required for entry of ARL13B and INPP5E into cilia and restrains cilium length while leaving core TZ assembly intact.\",\n      \"evidence\": \"CRISPR TMEM67 knockout in RPE1 cells with IF for TZ/ciliary proteins and patient-variant expression\",\n      \"pmids\": [\"36334440\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of selective cargo gating not defined\", \"Single-lab knockout system\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Showed mild-cholestasis variants reduce TMEM67 protein levels without disrupting the MKS1 interaction, indicating destabilization rather than complex disassembly.\",\n      \"evidence\": \"Western blotting for protein levels and Co-IP of TMEM67 with MKS1\",\n      \"pmids\": [\"35621037\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single Co-IP without reciprocal validation for the MKS1 interaction\", \"Causal link between reduced stability and cholestasis not established\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Established the central regulatory logic: ADAMTS9 cleavage of the extracellular domain uncouples TMEM67's ciliary and Wnt functions into a C-terminal ciliogenic fragment and a full-length Wnt-signaling form.\",\n      \"evidence\": \"ADAMTS9 cleavage-motif mapping, in vitro cleavage assays, a non-cleavable knock-in mouse, C. elegans patient-variant studies, and Wnt assays\",\n      \"pmids\": [\"40436881\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Spatial/temporal regulation of ADAMTS9-mediated cleavage in vivo not fully defined\", \"How the C-terminal fragment is retained at the TZ unresolved\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Identified a post-translational stabilization mechanism: FUT8-mediated core fucosylation protects TMEM67 from autophagic degradation, ensuring its transition-zone localization and ciliogenesis.\",\n      \"evidence\": \"MS proteomics, FUT8-TMEM67 Co-IP, autophagy inhibition assays, and Fut8-deficient mice with ciliary defects\",\n      \"pmids\": [\"40728580\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether fucosylation also modulates the Wnt-signaling form is untested\", \"Glycosylation site(s) on TMEM67 not enumerated\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How TMEM67's transition-zone gating activity, its ADAMTS9-cleavage-regulated dual functions, and its downstream control of canonical Wnt/HOXB5 and mTOR signaling are mechanistically integrated into a single coherent pathway remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of the TMEM67 receptor or its cleavage\", \"Direct biochemical link from TMEM67 to mTOR and to canonical Wnt repression not established\", \"Mechanism of selective ARL13B/INPP5E gating unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [6]},\n      {\"term_id\": \"GO:0048018\", \"supporting_discovery_ids\": [6]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [3, 12]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005929\", \"supporting_discovery_ids\": [2, 12, 13]},\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [3]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [11]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0007275\", \"supporting_discovery_ids\": [6]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [6, 8, 10]},\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [1, 12, 13]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [3, 14]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"ROR2\", \"Wnt5a\", \"p97\", \"ADAMTS9\", \"FUT8\", \"MKS1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}