{"gene":"EPB41L3","run_date":"2026-06-09T23:54:43","timeline":{"discoveries":[{"year":2002,"finding":"DAL-1/4.1B (EPB41L3) directly associates with the tumor suppressor TSLC1 (CADM1) at cell-cell contact sites, and this complex interacts with the actin filament cytoskeleton; the complex formation is dependent on the integrity of the actin cytoskeleton.","method":"Co-immunoprecipitation, colocalization imaging, actin disruption experiments","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal co-IP and colocalization with functional validation (cytoskeleton disruption), single lab but multiple orthogonal methods","pmids":["12234973"],"is_preprint":false},{"year":2010,"finding":"Crystal structure of the DAL-1/4.1B FERM domain in complex with the TSLC1 cytoplasmic domain reveals that binding occurs through a hydrophobic pocket in the C-lobe of the FERM domain; Tyr406 and Thr408 of TSLC1 form the critical contacts, confirmed by surface plasmon resonance.","method":"X-ray crystallography, surface plasmon resonance","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure plus orthogonal biophysical validation (SPR), rigorous mechanistic detail","pmids":["21131357"],"is_preprint":false},{"year":2000,"finding":"Protein 4.1B is localized specifically at the plasma membrane in regions of cell-cell contact; multiple splice isoforms exist with functionally relevant heterogeneity in the spectrin-actin binding and NuMA binding domains; only muscle isoforms possess a functional spectrin-actin binding domain.","method":"Immunofluorescence, Western blot, cDNA characterization, in situ hybridization","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct localization by immunofluorescence with functional domain analysis, replicated across tissue types, multiple orthogonal methods in one study","pmids":["10652311"],"is_preprint":false},{"year":2003,"finding":"Protein 4.1B binds to the conserved intracellular GNP motifs of both Caspr/paranodin (at paranodes) and Caspr2 (at juxtaparanodes) of myelinated axons, as demonstrated by co-immunoprecipitation from brain homogenates and progressive accumulation during postnatal development.","method":"Co-immunoprecipitation from brain homogenate, in vitro binding assay, immunohistochemistry","journal":"The European journal of neuroscience","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal co-IP from native tissue plus in vitro binding assay, single lab with multiple orthogonal methods","pmids":["12542678"],"is_preprint":false},{"year":2010,"finding":"Protein 4.1B is required for Caspr2 accumulation at the juxtaparanodal region (JXP) and for Kv1 channel clustering there; the interaction of Caspr with 4.1B is necessary for generating an efficient membrane barrier at the paranodal junction; Caspr2 and Kv1 channels are not clustered at JXP in 4.1B-null mice.","method":"Transgenic rescue experiments (Caspr-d4.1 and Caspr2-d4.1 mutants in null mice), immunofluorescence, 4.1B-null mouse analysis","journal":"The Journal of neuroscience","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis using transgenic rescue in null mice, multiple readouts, independently confirmed in related papers (PMID:21966409, 21632923)","pmids":["20164332"],"is_preprint":false},{"year":2004,"finding":"DAL-1/4.1B interacts with protein arginine N-methyltransferase 3 (PRMT3) via the C-terminal catalytic core domain of PRMT3 and the FERM domain of DAL-1/4.1B; DAL-1/4.1B is not a PRMT3 substrate but inhibits PRMT3-mediated methylation of GAR-containing substrates in vitro and in cells.","method":"Yeast two-hybrid, co-immunoprecipitation, in vitro binding assay (GST pulldown), in vitro methylation assay, induced expression in MCF-7 cells","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — yeast 2-hybrid discovery confirmed by co-IP and in vitro methylation assay, enzymatic function tested with mutagenesis-level controls","pmids":["15334060"],"is_preprint":false},{"year":2002,"finding":"DAL-1/4.1B interacts with 14-3-3 isoforms (β, γ, η) via residues Pro244–Leu280 within its FERM domain; this interaction is specific to DAL-1/4.1B and is not shared by merlin, ezrin, or radixin.","method":"Yeast two-hybrid, GST affinity chromatography, co-immunoprecipitation","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 2 / Moderate — yeast 2-hybrid confirmed by orthogonal in vitro and in vivo co-IP, binding domain mapped, specificity tested across family members","pmids":["11996670"],"is_preprint":false},{"year":2004,"finding":"Disruption of 14-3-3 binding to DAL-1/4.1B (via F359Y mutation) does not impair DAL-1 growth suppressor function in meningioma cells, indicating 14-3-3 binding is not required for growth suppression.","method":"Site-directed mutagenesis, GST affinity chromatography, co-immunoprecipitation, clonogenic assay, thymidine incorporation","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — mutagenesis with multiple functional readouts demonstrating a specific negative mechanistic result, single lab","pmids":["15116094"],"is_preprint":false},{"year":2005,"finding":"The U2 domain of protein 4.1B, when localized to the plasma membrane, is necessary and sufficient for meningioma growth suppression; deletion of U2 domain eliminates growth suppressor function; artificial membrane targeting of U2 alone recapitulates full-length DAL-1 suppression.","method":"Truncation/deletion constructs, clonogenic assay, thymidine incorporation, caspase-3 activation, membrane localization signal fusion","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 2 / Moderate — domain dissection with multiple functional assays, gain-of-function rescue with membrane-targeted U2 domain, single lab","pmids":["15688033"],"is_preprint":false},{"year":2006,"finding":"Protein 4.1B/DAL-1 growth suppression in meningioma cells requires the sequential activation of Src, Rac1, and JNK; inhibition of Rac1 or JNK abrogates 4.1B-mediated growth suppression and cyclin A regulation; this pathway requires the U2 domain localized to the plasma membrane.","method":"Genetic and pharmacologic inhibition of Src, Rac1, JNK; dominant-negative constructs; clonogenic assay; JNK activity assay; cyclin A analysis","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 2 / Moderate — genetic epistasis with pharmacologic validation and multiple pathway readouts, single lab with orthogonal approaches","pmids":["16707455"],"is_preprint":false},{"year":2005,"finding":"DAL-1/4.1B modulates PRMT5 activity in a substrate-specific manner: it inhibits PRMT5-mediated methylation of Sm proteins but enhances methylation of myelin basic protein.","method":"Yeast two-hybrid, in vitro methylation assay, cell-based methylation assay","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 1-2 / Weak — in vitro enzymatic assay with substrate specificity, single lab, limited orthogonal validation","pmids":["15737618"],"is_preprint":false},{"year":2006,"finding":"DAL-1/4.1B-induced apoptosis in MCF-7 breast cancer cells is primarily mediated through activation of caspase-8; inhibition of caspase-8 activation blocks DAL-1/4.1B-induced cell death; protein methylation cooperates in this apoptotic pathway.","method":"Flow cytometry, caspase activation assays, methylation inhibitor (AdOX) treatment","journal":"Molecular cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — flow cytometry and caspase assays with pharmacologic inhibition, single lab, single study","pmids":["16420693"],"is_preprint":false},{"year":2005,"finding":"Protein 4.1B interacts selectively with the β8 integrin cytoplasmic tail via its C-terminal domain; this interaction was identified in cultured astrocytes and brain tissue.","method":"Yeast two-hybrid, co-immunoprecipitation, colocalization by immunofluorescence","journal":"Proceedings of the National Academy of Sciences","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — co-IP and colocalization in native tissue plus yeast 2-hybrid, single lab, limited functional follow-up in this paper","pmids":["16157875"],"is_preprint":false},{"year":2009,"finding":"Protein 4.1B acts as an intracellular effector of SynCAM1 to recruit NMDA-type receptors to SynCAM1 adhesion sites; 4.1B in conjunction with SynCAM1 increases NMDAR-mediated mEPSC frequency and presynaptic contact area; manipulation of 4.1B levels in hippocampal neurons specifically affects NMDAR-mediated activity and localization.","method":"Co-immunoprecipitation, COS7 cell reconstitution assay, HEK293/neuron co-culture electrophysiology, hippocampal neuron knockdown/overexpression","journal":"Molecular and cellular neurosciences","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reconstitution in heterologous cells plus functional electrophysiology in neurons, multiple orthogonal methods, single lab","pmids":["19796685"],"is_preprint":false},{"year":2009,"finding":"A 200 kDa isoform of protein 4.1B is specifically associated with the Golgi apparatus; depletion of this Golgi-specific 4.1B by siRNA disrupts Golgi structure and prevents Na+/K+-ATPase, ZO-1, and ZO-2 from migrating to the plasma membrane.","method":"siRNA knockdown, Brefeldin A treatment, immunofluorescence, fractionation, Western blot","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 / Moderate — direct localization with functional consequence (KD showing failure of membrane protein targeting), Brefeldin A confirmation, multiple orthogonal methods, single lab","pmids":["19299464"],"is_preprint":false},{"year":2011,"finding":"4.1B-null mice show mislocalization of Caspr at paranodes, destabilization of paranodal axoglial septate junctions in both PNS and CNS, and progressive disruption of paranodal ultrastructure; the 4.1B locus generates multiple splice isoforms differentially in PNS vs. CNS.","method":"4.1B-null mouse generation, immunofluorescence, electron microscopy, RT-PCR for splice isoforms","journal":"The Journal of neuroscience","confidence":"High","confidence_rationale":"Tier 2 / Strong — KO mouse with ultrastructural and molecular readouts, independently replicated across multiple 4.1B KO papers","pmids":["21632923"],"is_preprint":false},{"year":2011,"finding":"4.1B KO mice show loss of proper molecular compartmentalization at the axon initial segment region: the Caspr+ para-AIS barrier is disrupted, preventing proper segregation and compartmentalization of Kv1 channels.","method":"4.1B KO mouse immunofluorescence, confocal analysis of AIS compartments","journal":"BMC biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO mouse with defined molecular phenotype, single lab but consistent with other 4.1B KO papers","pmids":["21958379"],"is_preprint":false},{"year":2011,"finding":"In 4.1B KO mice, Caspr/paranodin distribution and levels are decreased at paranodal regions, Caspr2/TAG-1/Kv1.1 enrichment is absent at juxtaparanodes, βII spectrin enrichment along the axolemma is decreased, axon calibers are reduced, and electrophysiological recordings show neuronal hyperexcitability; these defects are neuron-autonomous.","method":"4.1B KO mouse, immunofluorescence, immuno-EM, electrophysiology, myelinating co-culture with WT Schwann cells and 4.1B-null neurons","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 2 / Strong — KO mouse with ultrastructural, electrophysiological, and cell-autonomous validation, multiple orthogonal approaches, replicated across 4.1B KO papers","pmids":["21966409"],"is_preprint":false},{"year":2012,"finding":"4.1B is expressed subjacent to the axon membrane in all myelinated axon domains except nodes; in 4.1B-deficient mice, juxtaparanodal Kv1/Caspr2 clustering is substantially disrupted in both PNS and CNS; loss of 4.1B in axons is associated with reduced internodal Necl-1, Necl-2, and α-2 spectrin; and 4.1B unexpectedly regulates myelin sheath thickness.","method":"Immunofluorescence, immuno-EM, 4.1B KO mouse, myelinating co-culture (neuron-autonomous test), nerve conduction velocity measurement","journal":"Glia","confidence":"High","confidence_rationale":"Tier 2 / Strong — KO mouse with ultrastructural analysis and cell-autonomous myelinating coculture validation, multiple molecular and functional readouts","pmids":["23109359"],"is_preprint":false},{"year":2013,"finding":"The 130 kDa isoform of protein 4.1B, localized predominantly at the plasma membrane, is required for cell adhesion, spreading, and migration in mouse embryonic fibroblasts; loss of 130 kDa 4.1B causes failure to form actin stress fibers; rescue with 130 kDa but not 60 kDa 4.1B restores spreading and stress fiber formation.","method":"4.1B KO MEF cells, re-expression of specific isoforms, immunofluorescence, adhesion/spreading/migration assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — isoform-specific rescue in KO cells with multiple functional assays, single lab with orthogonal methods","pmids":["24381168"],"is_preprint":false},{"year":2001,"finding":"DAL-1 suppresses cell proliferation in meningioma cells but not schwannoma cells; DAL-1 interacts with ERM proteins and βII-spectrin but not with the merlin-interactor SCHIP-1.","method":"Cell proliferation assays, co-immunoprecipitation","journal":"Neurobiology of disease","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — co-IP with proliferation assays, cell-type specificity tested, single lab","pmids":["11300722"],"is_preprint":false},{"year":2002,"finding":"Expression of DAL-1 in MCF-7 breast cancer cells suppresses cell growth partly through induction of apoptosis, and increases cell attachment to extracellular matrix substrates.","method":"Constitutive and inducible expression in MCF-7 cells, proliferation assays, apoptosis assays, attachment assays","journal":"International journal of cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — loss-of-function/gain-of-function with multiple phenotypic readouts, single lab","pmids":["12115567"],"is_preprint":false},{"year":2007,"finding":"Knockdown of 4.1B in non-metastatic sarcoma cells by RNAi causes loss of actin stress fibers and doubles cell migration speed; forced expression of 4.1B in metastatic cells halves migration speed and suppresses chemotaxis, implicating 4.1B in regulation of actin cytoskeleton and cell motility.","method":"RNAi knockdown, overexpression, F-actin staining, cell migration/chemotaxis assays","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — bidirectional manipulation (KD and OE) with matched phenotypic readouts, single lab","pmids":["17264155"],"is_preprint":false},{"year":2007,"finding":"4.1B-deficient mice display increased susceptibility to aggressive, spontaneous prostate carcinomas with reduced apoptosis; downregulation of 4.1B in poorly metastatic prostate cancer cells increases their metastatic propensity in an orthotopic model.","method":"4.1B KO mouse, orthotopic prostate cancer model, histopathology, apoptosis assay","journal":"Proceedings of the National Academy of Sciences","confidence":"High","confidence_rationale":"Tier 2 / Moderate — in vivo KO mouse model plus orthotopic transplant model with defined readouts, single lab with orthogonal in vivo approaches","pmids":["17640904"],"is_preprint":false},{"year":2005,"finding":"4.1B expression is dramatically upregulated in mammary epithelial cells during pregnancy; 4.1B-null mice show increased mammary epithelial cell proliferation during pregnancy; 4.1B induces G1 cell cycle arrest with decreased cyclin A, reduced Rb phosphorylation, and reduced erbB2 phosphorylation in mammary epithelial cell lines.","method":"4.1B KO mouse mammary gland analysis, in vitro cell cycle analysis (FACS), Western blot for cyclin A, Rb, erbB2 phosphorylation, immunohistochemistry","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 2 / Moderate — KO mouse in vivo plus mechanistic in vitro cell cycle analysis, multiple molecular readouts, single lab","pmids":["16007173"],"is_preprint":false},{"year":2010,"finding":"EPB41L3 (4.1B) functions as a tumor suppressor in ovarian cancer; reexpression in 3D spheroid models causes growth suppression and induces apoptosis; chromosome 18 transfer including EPB41L3 induces neoplastic suppression in TOV21G ovarian cancer cells.","method":"Microcell-mediated chromosome transfer, gene expression microarray, immunoblot, immunohistochemistry, 3D spheroid growth assay, electron microscopy, inducible expression","journal":"Neoplasia","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional complementation by chromosome transfer plus inducible re-expression with apoptosis readout, single lab","pmids":["20651987"],"is_preprint":false},{"year":2007,"finding":"p55 (membrane-associated guanylate kinase), protein 4.1B, and sodium bicarbonate cotransporter 1 (NBC1) form a molecular complex at the basolateral membrane of renal S1-S2 proximal tubules, as shown by co-immunoprecipitation and GST pulldown.","method":"Co-immunoprecipitation with anti-p55 and anti-4.1B antibodies, GST pulldown with NBC1 and 4.1B regions, immunohistochemistry","journal":"The journal of histochemistry and cytochemistry","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — reciprocal co-IP and GST pulldown identifying a ternary complex, single lab, limited functional follow-up","pmids":["17712176"],"is_preprint":false},{"year":2011,"finding":"β8 integrin and Band 4.1B cooperatively regulate cardiac morphogenesis; double-null embryos for β8 integrin and 4.1B show cardiovascular defects and death by E11.5, with defective cardiac outflow tract development and reduced smooth muscle α-actin in neural crest-derived cells.","method":"Double-null mouse genetics, embryo phenotyping, immunofluorescence for SMAα-actin","journal":"Developmental dynamics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis (double KO) with defined developmental phenotype, single lab","pmids":["21181944"],"is_preprint":false},{"year":2016,"finding":"4.1B is required for proper targeting of Caspr2 early during myelination; lateral stability of paranodal Caspr (assessed by FRAP) is not altered in 4.1B KO mice, indicating 4.1B is not required for paranodal junction assembly/stability but is needed for juxtaparanodal Caspr2 targeting.","method":"4.1B KO mouse, myelinating DRG/Schwann cell co-culture, adenoviral Caspr-GFP expression, FRAP, immunofluorescence","journal":"Glia","confidence":"High","confidence_rationale":"Tier 2 / Moderate — KO with live FRAP and adenoviral rescue, multiple orthogonal methods, single lab","pmids":["26840208"],"is_preprint":false},{"year":2018,"finding":"Combined ablation of 4.1B and Whirlin (Whrn) in double-mutant mice leads to axonal transport defects in cerebellar Purkinje neuron myelinated axons, manifested by axonal swellings containing cytoskeletal components and vesicles, and progressive impairment of locomotor performance.","method":"Single and double KO mouse, immunoblot, immunofluorescence, gait analysis","journal":"Journal of neuroscience research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic interaction (double KO) with ultrastructural and behavioral readouts, single lab","pmids":["30447021"],"is_preprint":false},{"year":2019,"finding":"The FERM domain of protein 4.1B interacts with EGFR through the initial 13 amino acids (P13) of the intracellular juxtamembrane segment; this binding inhibits EGFR dimerization and autophosphorylation and suppresses downstream EGFR/MAPK/ERK1/2 and PI3K/AKT signaling, inhibiting gastric cancer cell proliferation.","method":"Co-immunoprecipitation, immunofluorescence colocalization, domain mapping, EGFR phosphorylation/dimerization assays, KO MEF cells, gastric cancer cell overexpression/knockdown","journal":"Cell communication and signaling","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — co-IP and domain mapping with downstream signaling readouts, single lab","pmids":["31492173"],"is_preprint":false},{"year":2015,"finding":"DAL-1 directly binds E-cadherin and regulates its promoter, attenuating EMT in lung cancer; HSPA5 (GRP78) was identified as a direct DAL-1 binding protein by co-immunoprecipitation.","method":"Co-immunoprecipitation, Western blot, EMT marker analysis, promoter analysis, overexpression and knockdown","journal":"Journal of experimental & clinical cancer research","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — single co-IP identifying binding partner, EMT marker panel, single lab; promoter binding not definitively demonstrated by ChIP","pmids":["25609022"],"is_preprint":false},{"year":2017,"finding":"DAL-1 suppresses HSPA5 expression at mRNA and protein levels and inhibits the PI3K/Akt/Mdm2 signaling pathway through downregulation of HSPA5; DAL-1 and HSPA5 proteins co-localize in the cytoplasm and nucleus.","method":"Co-immunoprecipitation, immunofluorescence colocalization, Western blot, qRT-PCR, migration/invasion assay","journal":"Oncology reports","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — co-localization and co-IP with pathway analysis, single lab, limited mechanistic depth","pmids":["29048640"],"is_preprint":false},{"year":2016,"finding":"EPB41L3 suppresses esophageal squamous cell carcinoma cell invasion and migration; overexpression of EPB41L3 downregulates MMP2 and MMP9 expression and p-AKT levels.","method":"Overexpression and siRNA knockdown, wound healing and Transwell assays, Western blot for MMP2, MMP9, p-AKT","journal":"Cell biochemistry and function","confidence":"Medium","confidence_rationale":"Tier 3 / Weak — bidirectional manipulation with functional and pathway readouts, single lab, limited mechanistic depth","pmids":["26916087"],"is_preprint":false},{"year":2021,"finding":"DAL-1/4.1B promotes exosome uptake in lung cancer cells via upregulation of heparan sulfate proteoglycan 2 (HSPG2); heparin and heparinase counteract the uptake enhancement by DAL-1/4.1B; HSPG2 expression correlates positively with DAL-1/4.1B levels.","method":"siRNA knockdown, overexpression, vesicle uptake inhibitors/heparinase treatment, Western blot, qRT-PCR","journal":"Molecular and cellular biochemistry","confidence":"Low","confidence_rationale":"Tier 3 / Weak — pharmacologic inhibition and knockdown, single lab, indirect pathway evidence","pmids":["34657240"],"is_preprint":false},{"year":2004,"finding":"Protein 4.1B isoforms in kidney and brain arise from two alternative promoters (exons 1A and 1B) coupled to differential alternative splicing at exon 2; exon 1B transcripts encode larger 4.1B isoforms with N-terminal extension; differentiation of PC12 cells causes translocation of large 4.1B isoforms into the nucleus.","method":"Promoter mapping, RT-PCR, Western blot, PC12 differentiation with subcellular fractionation/immunofluorescence","journal":"Biochimica et biophysica acta","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — transcriptional and protein-level characterization with cell-biology follow-up, single lab, multiple orthogonal methods","pmids":["15488987"],"is_preprint":false},{"year":2024,"finding":"Biallelic loss-of-function variants in EPB41L3 cause a human autosomal recessive developmental disorder (EADD) with seizures, hypotonia, and delayed myelination; patient fibroblasts show ablation of 4.1B protein; Epb41l3-deficient mouse oligodendroglia show reduced myelin gene expression, reduced branching, and increased apoptosis.","method":"Exome sequencing, Western blot, qRT-PCR of patient fibroblasts, NMD inhibition assay, mouse oligodendroglia KO culture with gene expression and morphological analysis","journal":"Brain","confidence":"High","confidence_rationale":"Tier 2 / Moderate — human genetics plus functional validation in patient cells and mouse oligodendroglia, multiple orthogonal methods, multi-center study","pmids":["39292993"],"is_preprint":false},{"year":2016,"finding":"Knockout of 4.1B in SV40T-immortalized mouse embryonic fibroblasts is sufficient to trigger malignant transformation, enabling tumor formation in nude mice; loss of 4.1B activates ERK, AKT, the p16INK4A-pRb pathway, and Wnt/β-catenin nuclear accumulation; 4.1B interacts with E-cadherin in MEF cells.","method":"4.1B KO iMEF cells, tumor formation in nude mice, Western blot for ERK/AKT/Wnt pathway, co-immunoprecipitation (4.1B-E-cadherin), immunofluorescence for β-catenin","journal":"Molecular carcinogenesis","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — in vivo transformation assay with pathway analysis and co-IP, single lab","pmids":["27312663"],"is_preprint":false},{"year":2022,"finding":"EPB41L3 overexpression in cervical cancer cells inhibits PI3K and AKT phosphorylation, leading to apoptosis; overexpression suppresses tumorigenicity in nude mice.","method":"Lentiviral overexpression, Western blot for PI3K/AKT phosphorylation, apoptosis assay, xenograft mouse model","journal":"Acta biochimica Polonica","confidence":"Low","confidence_rationale":"Tier 3 / Weak — overexpression with signaling readouts, single lab, limited mechanistic depth","pmids":["35569139"],"is_preprint":false}],"current_model":"EPB41L3 (protein 4.1B/DAL-1) is a FERM domain-containing membrane-cytoskeleton adaptor that links the plasma membrane to the actin-spectrin cytoskeleton at cell-cell contacts; it directly binds TSLC1/CADM1 (via a hydrophobic pocket in its FERM C-lobe, crystal structure resolved), Caspr and Caspr2 at paranodal/juxtaparanodal axonal domains (required for Kv1 channel clustering and paranodal barrier integrity in myelinated axons), β8 integrin (via its C-terminal domain), EGFR (via its FERM domain, inhibiting EGFR dimerization and downstream MAPK/PI3K signaling), PRMT3 and PRMT5 (inhibiting or modulating their methyltransferase activity in a substrate-specific manner), 14-3-3 proteins (though this interaction is not required for growth suppression), ERM proteins and βII-spectrin; its tumor suppressor functions in meningioma, lung, breast, prostate, ovarian, and other cancers are mediated through the U2 domain at the plasma membrane, activating Src-Rac1-JNK signaling, inducing G1 cell cycle arrest (decreasing cyclin A and Rb phosphorylation), and suppressing EMT and cell motility through regulation of actin stress fiber organization; biallelic loss-of-function variants in humans cause an autosomal recessive neurodevelopmental syndrome with seizures and myelination defects, and 4.1B-null mice display juxtaparanodal disorganization, axonal hyperexcitability, and increased susceptibility to prostate carcinoma."},"narrative":{"mechanistic_narrative":"EPB41L3 (protein 4.1B/DAL-1) is a FERM-domain membrane-cytoskeleton adaptor that organizes specialized plasma-membrane microdomains at cell-cell contacts and along myelinated axons, and acts as a tumor suppressor across multiple epithelial lineages [PMID:12234973, PMID:15688033, PMID:21966409, PMID:17640904]. It is localized at sites of cell-cell contact and links transmembrane partners to the actin-spectrin cytoskeleton; complex formation with the adhesion molecule TSLC1/CADM1 depends on an intact actin network, and crystallography places this interaction in a hydrophobic pocket of the FERM C-lobe contacting TSLC1 residues Tyr406/Thr408 [PMID:12234973, PMID:21131357]. In the nervous system, 4.1B binds the GNP motifs of Caspr at paranodes and Caspr2 at juxtaparanodes, where it is required to target Caspr2 and cluster Kv1 channels and to generate the paranodal membrane barrier; 4.1B-null mice show paranodal/juxtaparanodal disorganization, reduced βII-spectrin enrichment, axonal hyperexcitability, and altered myelin sheath thickness [PMID:12542678, PMID:20164332, PMID:21966409, PMID:23109359, PMID:26840208]. Through its plasma-membrane-localized U2 domain, 4.1B suppresses growth by activating sequential Src-Rac1-JNK signaling, inducing G1 arrest with reduced cyclin A and Rb phosphorylation, and it restrains EGFR by binding the receptor juxtamembrane segment to block dimerization and downstream MAPK and PI3K/AKT signaling [PMID:15688033, PMID:16707455, PMID:16007173, PMID:31492173]. 4.1B also controls actin stress-fiber organization, adhesion, and motility in an isoform-specific manner, with the 130 kDa plasma-membrane isoform required for spreading and stress-fiber formation [PMID:24381168, PMID:17264155]. Loss of 4.1B promotes transformation and tumor progression in vivo, including increased susceptibility to prostate carcinoma and enhanced metastatic propensity [PMID:17640904, PMID:27312663]. Biallelic loss-of-function variants in EPB41L3 cause an autosomal recessive developmental disorder with seizures, hypotonia, and delayed myelination [PMID:39292993].","teleology":[{"year":2000,"claim":"Established that 4.1B is positioned at cell-cell contact sites and that splice isoforms diverge functionally in their cytoskeletal-binding capacity, framing it as a membrane adaptor with isoform-specific roles.","evidence":"Immunofluorescence, cDNA characterization, and domain analysis across tissues","pmids":["10652311"],"confidence":"High","gaps":["Did not identify the transmembrane binding partner at contacts","Functional consequence of isoform heterogeneity untested"]},{"year":2002,"claim":"Answered what 4.1B binds at cell-cell contacts by showing a direct, actin-dependent complex with the tumor suppressor TSLC1/CADM1, linking adhesion to the cytoskeleton.","evidence":"Co-IP, colocalization, and actin-disruption experiments","pmids":["12234973"],"confidence":"High","gaps":["Structural basis of binding not resolved","Did not establish how the complex suppresses growth"]},{"year":2010,"claim":"Resolved the atomic basis of the TSLC1 interaction, mapping it to a hydrophobic FERM C-lobe pocket and defining the critical TSLC1 contact residues.","evidence":"X-ray crystallography of the FERM–TSLC1 complex with SPR validation","pmids":["21131357"],"confidence":"High","gaps":["Structure of other partner complexes (Caspr, EGFR, integrin) not solved","In vivo relevance of specific contacts not tested"]},{"year":2003,"claim":"Extended 4.1B function to myelinated axons by showing direct binding to the GNP motifs of Caspr and Caspr2, implicating it in axonal domain organization.","evidence":"Co-IP from brain homogenate and in vitro binding assays","pmids":["12542678"],"confidence":"High","gaps":["Did not test whether binding is required for axonal compartmentalization in vivo"]},{"year":2004,"claim":"Identified 4.1B as a regulator of arginine methyltransferases, binding PRMT3 without being a substrate and inhibiting its methylation of GAR substrates.","evidence":"Yeast two-hybrid, co-IP, in vitro and cellular methylation assays","pmids":["15334060"],"confidence":"High","gaps":["Biological consequence of PRMT3 inhibition not linked to a phenotype","Methylation substrates relevant in vivo unknown"]},{"year":2005,"claim":"Defined the U2 domain at the plasma membrane as necessary and sufficient for meningioma growth suppression, localizing the tumor-suppressor activity to a discrete module.","evidence":"Truncation/deletion constructs with clonogenic, proliferation, and apoptosis readouts and membrane-targeting fusions","pmids":["15688033"],"confidence":"High","gaps":["Downstream effectors of U2 not yet identified","Mechanism of membrane requirement unresolved at this stage"]},{"year":2006,"claim":"Linked the U2/membrane-dependent growth suppression to a defined signaling cascade, showing sequential Src-Rac1-JNK activation drives cyclin A regulation.","evidence":"Genetic and pharmacologic inhibition, dominant-negatives, JNK activity and cyclin A assays","pmids":["16707455"],"confidence":"High","gaps":["How U2 at the membrane initiates Src activation is unknown","Generality of the pathway across cancer types untested"]},{"year":2010,"claim":"Demonstrated genetically that 4.1B is required in vivo for juxtaparanodal Caspr2 accumulation, Kv1 clustering, and the paranodal membrane barrier.","evidence":"Transgenic 4.1-binding-deficient rescue constructs in 4.1B-null mice with immunofluorescence","pmids":["20164332"],"confidence":"High","gaps":["Did not distinguish targeting from stabilization","Effect on conduction not directly measured here"]},{"year":2011,"claim":"Multiple 4.1B-null mouse analyses established that loss disrupts paranodal septate junctions, juxtaparanodal Kv1/Caspr2 clustering, βII-spectrin enrichment, axon caliber, and causes neuronal hyperexcitability cell-autonomously.","evidence":"4.1B-null mice with immunofluorescence, immuno-EM, electrophysiology, and myelinating co-cultures","pmids":["21632923","21958379","21966409"],"confidence":"High","gaps":["Molecular mechanism connecting 4.1B loss to myelin/caliber changes incomplete"]},{"year":2013,"claim":"Resolved isoform specificity in adhesion/motility by showing the 130 kDa plasma-membrane isoform, not the 60 kDa form, is required for stress-fiber formation, spreading, and migration.","evidence":"Isoform-specific rescue in 4.1B-KO MEFs with adhesion/spreading/migration assays","pmids":["24381168"],"confidence":"High","gaps":["Direct cytoskeletal effector engaged by the 130 kDa isoform not defined"]},{"year":2019,"claim":"Defined a direct molecular mechanism for receptor-level tumor suppression: 4.1B FERM binds the EGFR juxtamembrane segment to block dimerization, autophosphorylation, and MAPK/PI3K signaling.","evidence":"Co-IP, domain mapping, EGFR 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\"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal co-IP and colocalization with functional validation (cytoskeleton disruption), single lab but multiple orthogonal methods\",\n      \"pmids\": [\"12234973\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Crystal structure of the DAL-1/4.1B FERM domain in complex with the TSLC1 cytoplasmic domain reveals that binding occurs through a hydrophobic pocket in the C-lobe of the FERM domain; Tyr406 and Thr408 of TSLC1 form the critical contacts, confirmed by surface plasmon resonance.\",\n      \"method\": \"X-ray crystallography, surface plasmon resonance\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure plus orthogonal biophysical validation (SPR), rigorous mechanistic detail\",\n      \"pmids\": [\"21131357\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Protein 4.1B is localized specifically at the plasma membrane in regions of cell-cell contact; multiple splice isoforms exist with functionally relevant heterogeneity in the spectrin-actin binding and NuMA binding domains; only muscle isoforms possess a functional spectrin-actin binding domain.\",\n      \"method\": \"Immunofluorescence, Western blot, cDNA characterization, in situ hybridization\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct localization by immunofluorescence with functional domain analysis, replicated across tissue types, multiple orthogonal methods in one study\",\n      \"pmids\": [\"10652311\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Protein 4.1B binds to the conserved intracellular GNP motifs of both Caspr/paranodin (at paranodes) and Caspr2 (at juxtaparanodes) of myelinated axons, as demonstrated by co-immunoprecipitation from brain homogenates and progressive accumulation during postnatal development.\",\n      \"method\": \"Co-immunoprecipitation from brain homogenate, in vitro binding assay, immunohistochemistry\",\n      \"journal\": \"The European journal of neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal co-IP from native tissue plus in vitro binding assay, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"12542678\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Protein 4.1B is required for Caspr2 accumulation at the juxtaparanodal region (JXP) and for Kv1 channel clustering there; the interaction of Caspr with 4.1B is necessary for generating an efficient membrane barrier at the paranodal junction; Caspr2 and Kv1 channels are not clustered at JXP in 4.1B-null mice.\",\n      \"method\": \"Transgenic rescue experiments (Caspr-d4.1 and Caspr2-d4.1 mutants in null mice), immunofluorescence, 4.1B-null mouse analysis\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis using transgenic rescue in null mice, multiple readouts, independently confirmed in related papers (PMID:21966409, 21632923)\",\n      \"pmids\": [\"20164332\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"DAL-1/4.1B interacts with protein arginine N-methyltransferase 3 (PRMT3) via the C-terminal catalytic core domain of PRMT3 and the FERM domain of DAL-1/4.1B; DAL-1/4.1B is not a PRMT3 substrate but inhibits PRMT3-mediated methylation of GAR-containing substrates in vitro and in cells.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, in vitro binding assay (GST pulldown), in vitro methylation assay, induced expression in MCF-7 cells\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — yeast 2-hybrid discovery confirmed by co-IP and in vitro methylation assay, enzymatic function tested with mutagenesis-level controls\",\n      \"pmids\": [\"15334060\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"DAL-1/4.1B interacts with 14-3-3 isoforms (β, γ, η) via residues Pro244–Leu280 within its FERM domain; this interaction is specific to DAL-1/4.1B and is not shared by merlin, ezrin, or radixin.\",\n      \"method\": \"Yeast two-hybrid, GST affinity chromatography, co-immunoprecipitation\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast 2-hybrid confirmed by orthogonal in vitro and in vivo co-IP, binding domain mapped, specificity tested across family members\",\n      \"pmids\": [\"11996670\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Disruption of 14-3-3 binding to DAL-1/4.1B (via F359Y mutation) does not impair DAL-1 growth suppressor function in meningioma cells, indicating 14-3-3 binding is not required for growth suppression.\",\n      \"method\": \"Site-directed mutagenesis, GST affinity chromatography, co-immunoprecipitation, clonogenic assay, thymidine incorporation\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — mutagenesis with multiple functional readouts demonstrating a specific negative mechanistic result, single lab\",\n      \"pmids\": [\"15116094\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"The U2 domain of protein 4.1B, when localized to the plasma membrane, is necessary and sufficient for meningioma growth suppression; deletion of U2 domain eliminates growth suppressor function; artificial membrane targeting of U2 alone recapitulates full-length DAL-1 suppression.\",\n      \"method\": \"Truncation/deletion constructs, clonogenic assay, thymidine incorporation, caspase-3 activation, membrane localization signal fusion\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain dissection with multiple functional assays, gain-of-function rescue with membrane-targeted U2 domain, single lab\",\n      \"pmids\": [\"15688033\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Protein 4.1B/DAL-1 growth suppression in meningioma cells requires the sequential activation of Src, Rac1, and JNK; inhibition of Rac1 or JNK abrogates 4.1B-mediated growth suppression and cyclin A regulation; this pathway requires the U2 domain localized to the plasma membrane.\",\n      \"method\": \"Genetic and pharmacologic inhibition of Src, Rac1, JNK; dominant-negative constructs; clonogenic assay; JNK activity assay; cyclin A analysis\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis with pharmacologic validation and multiple pathway readouts, single lab with orthogonal approaches\",\n      \"pmids\": [\"16707455\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"DAL-1/4.1B modulates PRMT5 activity in a substrate-specific manner: it inhibits PRMT5-mediated methylation of Sm proteins but enhances methylation of myelin basic protein.\",\n      \"method\": \"Yeast two-hybrid, in vitro methylation assay, cell-based methylation assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 / Weak — in vitro enzymatic assay with substrate specificity, single lab, limited orthogonal validation\",\n      \"pmids\": [\"15737618\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"DAL-1/4.1B-induced apoptosis in MCF-7 breast cancer cells is primarily mediated through activation of caspase-8; inhibition of caspase-8 activation blocks DAL-1/4.1B-induced cell death; protein methylation cooperates in this apoptotic pathway.\",\n      \"method\": \"Flow cytometry, caspase activation assays, methylation inhibitor (AdOX) treatment\",\n      \"journal\": \"Molecular cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — flow cytometry and caspase assays with pharmacologic inhibition, single lab, single study\",\n      \"pmids\": [\"16420693\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Protein 4.1B interacts selectively with the β8 integrin cytoplasmic tail via its C-terminal domain; this interaction was identified in cultured astrocytes and brain tissue.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, colocalization by immunofluorescence\",\n      \"journal\": \"Proceedings of the National Academy of Sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — co-IP and colocalization in native tissue plus yeast 2-hybrid, single lab, limited functional follow-up in this paper\",\n      \"pmids\": [\"16157875\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Protein 4.1B acts as an intracellular effector of SynCAM1 to recruit NMDA-type receptors to SynCAM1 adhesion sites; 4.1B in conjunction with SynCAM1 increases NMDAR-mediated mEPSC frequency and presynaptic contact area; manipulation of 4.1B levels in hippocampal neurons specifically affects NMDAR-mediated activity and localization.\",\n      \"method\": \"Co-immunoprecipitation, COS7 cell reconstitution assay, HEK293/neuron co-culture electrophysiology, hippocampal neuron knockdown/overexpression\",\n      \"journal\": \"Molecular and cellular neurosciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reconstitution in heterologous cells plus functional electrophysiology in neurons, multiple orthogonal methods, single lab\",\n      \"pmids\": [\"19796685\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"A 200 kDa isoform of protein 4.1B is specifically associated with the Golgi apparatus; depletion of this Golgi-specific 4.1B by siRNA disrupts Golgi structure and prevents Na+/K+-ATPase, ZO-1, and ZO-2 from migrating to the plasma membrane.\",\n      \"method\": \"siRNA knockdown, Brefeldin A treatment, immunofluorescence, fractionation, Western blot\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization with functional consequence (KD showing failure of membrane protein targeting), Brefeldin A confirmation, multiple orthogonal methods, single lab\",\n      \"pmids\": [\"19299464\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"4.1B-null mice show mislocalization of Caspr at paranodes, destabilization of paranodal axoglial septate junctions in both PNS and CNS, and progressive disruption of paranodal ultrastructure; the 4.1B locus generates multiple splice isoforms differentially in PNS vs. CNS.\",\n      \"method\": \"4.1B-null mouse generation, immunofluorescence, electron microscopy, RT-PCR for splice isoforms\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — KO mouse with ultrastructural and molecular readouts, independently replicated across multiple 4.1B KO papers\",\n      \"pmids\": [\"21632923\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"4.1B KO mice show loss of proper molecular compartmentalization at the axon initial segment region: the Caspr+ para-AIS barrier is disrupted, preventing proper segregation and compartmentalization of Kv1 channels.\",\n      \"method\": \"4.1B KO mouse immunofluorescence, confocal analysis of AIS compartments\",\n      \"journal\": \"BMC biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO mouse with defined molecular phenotype, single lab but consistent with other 4.1B KO papers\",\n      \"pmids\": [\"21958379\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"In 4.1B KO mice, Caspr/paranodin distribution and levels are decreased at paranodal regions, Caspr2/TAG-1/Kv1.1 enrichment is absent at juxtaparanodes, βII spectrin enrichment along the axolemma is decreased, axon calibers are reduced, and electrophysiological recordings show neuronal hyperexcitability; these defects are neuron-autonomous.\",\n      \"method\": \"4.1B KO mouse, immunofluorescence, immuno-EM, electrophysiology, myelinating co-culture with WT Schwann cells and 4.1B-null neurons\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — KO mouse with ultrastructural, electrophysiological, and cell-autonomous validation, multiple orthogonal approaches, replicated across 4.1B KO papers\",\n      \"pmids\": [\"21966409\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"4.1B is expressed subjacent to the axon membrane in all myelinated axon domains except nodes; in 4.1B-deficient mice, juxtaparanodal Kv1/Caspr2 clustering is substantially disrupted in both PNS and CNS; loss of 4.1B in axons is associated with reduced internodal Necl-1, Necl-2, and α-2 spectrin; and 4.1B unexpectedly regulates myelin sheath thickness.\",\n      \"method\": \"Immunofluorescence, immuno-EM, 4.1B KO mouse, myelinating co-culture (neuron-autonomous test), nerve conduction velocity measurement\",\n      \"journal\": \"Glia\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — KO mouse with ultrastructural analysis and cell-autonomous myelinating coculture validation, multiple molecular and functional readouts\",\n      \"pmids\": [\"23109359\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"The 130 kDa isoform of protein 4.1B, localized predominantly at the plasma membrane, is required for cell adhesion, spreading, and migration in mouse embryonic fibroblasts; loss of 130 kDa 4.1B causes failure to form actin stress fibers; rescue with 130 kDa but not 60 kDa 4.1B restores spreading and stress fiber formation.\",\n      \"method\": \"4.1B KO MEF cells, re-expression of specific isoforms, immunofluorescence, adhesion/spreading/migration assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — isoform-specific rescue in KO cells with multiple functional assays, single lab with orthogonal methods\",\n      \"pmids\": [\"24381168\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"DAL-1 suppresses cell proliferation in meningioma cells but not schwannoma cells; DAL-1 interacts with ERM proteins and βII-spectrin but not with the merlin-interactor SCHIP-1.\",\n      \"method\": \"Cell proliferation assays, co-immunoprecipitation\",\n      \"journal\": \"Neurobiology of disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — co-IP with proliferation assays, cell-type specificity tested, single lab\",\n      \"pmids\": [\"11300722\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Expression of DAL-1 in MCF-7 breast cancer cells suppresses cell growth partly through induction of apoptosis, and increases cell attachment to extracellular matrix substrates.\",\n      \"method\": \"Constitutive and inducible expression in MCF-7 cells, proliferation assays, apoptosis assays, attachment assays\",\n      \"journal\": \"International journal of cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — loss-of-function/gain-of-function with multiple phenotypic readouts, single lab\",\n      \"pmids\": [\"12115567\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Knockdown of 4.1B in non-metastatic sarcoma cells by RNAi causes loss of actin stress fibers and doubles cell migration speed; forced expression of 4.1B in metastatic cells halves migration speed and suppresses chemotaxis, implicating 4.1B in regulation of actin cytoskeleton and cell motility.\",\n      \"method\": \"RNAi knockdown, overexpression, F-actin staining, cell migration/chemotaxis assays\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — bidirectional manipulation (KD and OE) with matched phenotypic readouts, single lab\",\n      \"pmids\": [\"17264155\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"4.1B-deficient mice display increased susceptibility to aggressive, spontaneous prostate carcinomas with reduced apoptosis; downregulation of 4.1B in poorly metastatic prostate cancer cells increases their metastatic propensity in an orthotopic model.\",\n      \"method\": \"4.1B KO mouse, orthotopic prostate cancer model, histopathology, apoptosis assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo KO mouse model plus orthotopic transplant model with defined readouts, single lab with orthogonal in vivo approaches\",\n      \"pmids\": [\"17640904\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"4.1B expression is dramatically upregulated in mammary epithelial cells during pregnancy; 4.1B-null mice show increased mammary epithelial cell proliferation during pregnancy; 4.1B induces G1 cell cycle arrest with decreased cyclin A, reduced Rb phosphorylation, and reduced erbB2 phosphorylation in mammary epithelial cell lines.\",\n      \"method\": \"4.1B KO mouse mammary gland analysis, in vitro cell cycle analysis (FACS), Western blot for cyclin A, Rb, erbB2 phosphorylation, immunohistochemistry\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO mouse in vivo plus mechanistic in vitro cell cycle analysis, multiple molecular readouts, single lab\",\n      \"pmids\": [\"16007173\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"EPB41L3 (4.1B) functions as a tumor suppressor in ovarian cancer; reexpression in 3D spheroid models causes growth suppression and induces apoptosis; chromosome 18 transfer including EPB41L3 induces neoplastic suppression in TOV21G ovarian cancer cells.\",\n      \"method\": \"Microcell-mediated chromosome transfer, gene expression microarray, immunoblot, immunohistochemistry, 3D spheroid growth assay, electron microscopy, inducible expression\",\n      \"journal\": \"Neoplasia\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional complementation by chromosome transfer plus inducible re-expression with apoptosis readout, single lab\",\n      \"pmids\": [\"20651987\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"p55 (membrane-associated guanylate kinase), protein 4.1B, and sodium bicarbonate cotransporter 1 (NBC1) form a molecular complex at the basolateral membrane of renal S1-S2 proximal tubules, as shown by co-immunoprecipitation and GST pulldown.\",\n      \"method\": \"Co-immunoprecipitation with anti-p55 and anti-4.1B antibodies, GST pulldown with NBC1 and 4.1B regions, immunohistochemistry\",\n      \"journal\": \"The journal of histochemistry and cytochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — reciprocal co-IP and GST pulldown identifying a ternary complex, single lab, limited functional follow-up\",\n      \"pmids\": [\"17712176\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"β8 integrin and Band 4.1B cooperatively regulate cardiac morphogenesis; double-null embryos for β8 integrin and 4.1B show cardiovascular defects and death by E11.5, with defective cardiac outflow tract development and reduced smooth muscle α-actin in neural crest-derived cells.\",\n      \"method\": \"Double-null mouse genetics, embryo phenotyping, immunofluorescence for SMAα-actin\",\n      \"journal\": \"Developmental dynamics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis (double KO) with defined developmental phenotype, single lab\",\n      \"pmids\": [\"21181944\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"4.1B is required for proper targeting of Caspr2 early during myelination; lateral stability of paranodal Caspr (assessed by FRAP) is not altered in 4.1B KO mice, indicating 4.1B is not required for paranodal junction assembly/stability but is needed for juxtaparanodal Caspr2 targeting.\",\n      \"method\": \"4.1B KO mouse, myelinating DRG/Schwann cell co-culture, adenoviral Caspr-GFP expression, FRAP, immunofluorescence\",\n      \"journal\": \"Glia\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO with live FRAP and adenoviral rescue, multiple orthogonal methods, single lab\",\n      \"pmids\": [\"26840208\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Combined ablation of 4.1B and Whirlin (Whrn) in double-mutant mice leads to axonal transport defects in cerebellar Purkinje neuron myelinated axons, manifested by axonal swellings containing cytoskeletal components and vesicles, and progressive impairment of locomotor performance.\",\n      \"method\": \"Single and double KO mouse, immunoblot, immunofluorescence, gait analysis\",\n      \"journal\": \"Journal of neuroscience research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic interaction (double KO) with ultrastructural and behavioral readouts, single lab\",\n      \"pmids\": [\"30447021\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"The FERM domain of protein 4.1B interacts with EGFR through the initial 13 amino acids (P13) of the intracellular juxtamembrane segment; this binding inhibits EGFR dimerization and autophosphorylation and suppresses downstream EGFR/MAPK/ERK1/2 and PI3K/AKT signaling, inhibiting gastric cancer cell proliferation.\",\n      \"method\": \"Co-immunoprecipitation, immunofluorescence colocalization, domain mapping, EGFR phosphorylation/dimerization assays, KO MEF cells, gastric cancer cell overexpression/knockdown\",\n      \"journal\": \"Cell communication and signaling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — co-IP and domain mapping with downstream signaling readouts, single lab\",\n      \"pmids\": [\"31492173\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"DAL-1 directly binds E-cadherin and regulates its promoter, attenuating EMT in lung cancer; HSPA5 (GRP78) was identified as a direct DAL-1 binding protein by co-immunoprecipitation.\",\n      \"method\": \"Co-immunoprecipitation, Western blot, EMT marker analysis, promoter analysis, overexpression and knockdown\",\n      \"journal\": \"Journal of experimental & clinical cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — single co-IP identifying binding partner, EMT marker panel, single lab; promoter binding not definitively demonstrated by ChIP\",\n      \"pmids\": [\"25609022\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"DAL-1 suppresses HSPA5 expression at mRNA and protein levels and inhibits the PI3K/Akt/Mdm2 signaling pathway through downregulation of HSPA5; DAL-1 and HSPA5 proteins co-localize in the cytoplasm and nucleus.\",\n      \"method\": \"Co-immunoprecipitation, immunofluorescence colocalization, Western blot, qRT-PCR, migration/invasion assay\",\n      \"journal\": \"Oncology reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — co-localization and co-IP with pathway analysis, single lab, limited mechanistic depth\",\n      \"pmids\": [\"29048640\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"EPB41L3 suppresses esophageal squamous cell carcinoma cell invasion and migration; overexpression of EPB41L3 downregulates MMP2 and MMP9 expression and p-AKT levels.\",\n      \"method\": \"Overexpression and siRNA knockdown, wound healing and Transwell assays, Western blot for MMP2, MMP9, p-AKT\",\n      \"journal\": \"Cell biochemistry and function\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Weak — bidirectional manipulation with functional and pathway readouts, single lab, limited mechanistic depth\",\n      \"pmids\": [\"26916087\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"DAL-1/4.1B promotes exosome uptake in lung cancer cells via upregulation of heparan sulfate proteoglycan 2 (HSPG2); heparin and heparinase counteract the uptake enhancement by DAL-1/4.1B; HSPG2 expression correlates positively with DAL-1/4.1B levels.\",\n      \"method\": \"siRNA knockdown, overexpression, vesicle uptake inhibitors/heparinase treatment, Western blot, qRT-PCR\",\n      \"journal\": \"Molecular and cellular biochemistry\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — pharmacologic inhibition and knockdown, single lab, indirect pathway evidence\",\n      \"pmids\": [\"34657240\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Protein 4.1B isoforms in kidney and brain arise from two alternative promoters (exons 1A and 1B) coupled to differential alternative splicing at exon 2; exon 1B transcripts encode larger 4.1B isoforms with N-terminal extension; differentiation of PC12 cells causes translocation of large 4.1B isoforms into the nucleus.\",\n      \"method\": \"Promoter mapping, RT-PCR, Western blot, PC12 differentiation with subcellular fractionation/immunofluorescence\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — transcriptional and protein-level characterization with cell-biology follow-up, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"15488987\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Biallelic loss-of-function variants in EPB41L3 cause a human autosomal recessive developmental disorder (EADD) with seizures, hypotonia, and delayed myelination; patient fibroblasts show ablation of 4.1B protein; Epb41l3-deficient mouse oligodendroglia show reduced myelin gene expression, reduced branching, and increased apoptosis.\",\n      \"method\": \"Exome sequencing, Western blot, qRT-PCR of patient fibroblasts, NMD inhibition assay, mouse oligodendroglia KO culture with gene expression and morphological analysis\",\n      \"journal\": \"Brain\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — human genetics plus functional validation in patient cells and mouse oligodendroglia, multiple orthogonal methods, multi-center study\",\n      \"pmids\": [\"39292993\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Knockout of 4.1B in SV40T-immortalized mouse embryonic fibroblasts is sufficient to trigger malignant transformation, enabling tumor formation in nude mice; loss of 4.1B activates ERK, AKT, the p16INK4A-pRb pathway, and Wnt/β-catenin nuclear accumulation; 4.1B interacts with E-cadherin in MEF cells.\",\n      \"method\": \"4.1B KO iMEF cells, tumor formation in nude mice, Western blot for ERK/AKT/Wnt pathway, co-immunoprecipitation (4.1B-E-cadherin), immunofluorescence for β-catenin\",\n      \"journal\": \"Molecular carcinogenesis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — in vivo transformation assay with pathway analysis and co-IP, single lab\",\n      \"pmids\": [\"27312663\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"EPB41L3 overexpression in cervical cancer cells inhibits PI3K and AKT phosphorylation, leading to apoptosis; overexpression suppresses tumorigenicity in nude mice.\",\n      \"method\": \"Lentiviral overexpression, Western blot for PI3K/AKT phosphorylation, apoptosis assay, xenograft mouse model\",\n      \"journal\": \"Acta biochimica Polonica\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — overexpression with signaling readouts, single lab, limited mechanistic depth\",\n      \"pmids\": [\"35569139\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"EPB41L3 (protein 4.1B/DAL-1) is a FERM domain-containing membrane-cytoskeleton adaptor that links the plasma membrane to the actin-spectrin cytoskeleton at cell-cell contacts; it directly binds TSLC1/CADM1 (via a hydrophobic pocket in its FERM C-lobe, crystal structure resolved), Caspr and Caspr2 at paranodal/juxtaparanodal axonal domains (required for Kv1 channel clustering and paranodal barrier integrity in myelinated axons), β8 integrin (via its C-terminal domain), EGFR (via its FERM domain, inhibiting EGFR dimerization and downstream MAPK/PI3K signaling), PRMT3 and PRMT5 (inhibiting or modulating their methyltransferase activity in a substrate-specific manner), 14-3-3 proteins (though this interaction is not required for growth suppression), ERM proteins and βII-spectrin; its tumor suppressor functions in meningioma, lung, breast, prostate, ovarian, and other cancers are mediated through the U2 domain at the plasma membrane, activating Src-Rac1-JNK signaling, inducing G1 cell cycle arrest (decreasing cyclin A and Rb phosphorylation), and suppressing EMT and cell motility through regulation of actin stress fiber organization; biallelic loss-of-function variants in humans cause an autosomal recessive neurodevelopmental syndrome with seizures and myelination defects, and 4.1B-null mice display juxtaparanodal disorganization, axonal hyperexcitability, and increased susceptibility to prostate carcinoma.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"EPB41L3 (protein 4.1B/DAL-1) is a FERM-domain membrane-cytoskeleton adaptor that organizes specialized plasma-membrane microdomains at cell-cell contacts and along myelinated axons, and acts as a tumor suppressor across multiple epithelial lineages [#0, #8, #17, #23]. It is localized at sites of cell-cell contact and links transmembrane partners to the actin-spectrin cytoskeleton; complex formation with the adhesion molecule TSLC1/CADM1 depends on an intact actin network, and crystallography places this interaction in a hydrophobic pocket of the FERM C-lobe contacting TSLC1 residues Tyr406/Thr408 [#0, #1]. In the nervous system, 4.1B binds the GNP motifs of Caspr at paranodes and Caspr2 at juxtaparanodes, where it is required to target Caspr2 and cluster Kv1 channels and to generate the paranodal membrane barrier; 4.1B-null mice show paranodal/juxtaparanodal disorganization, reduced βII-spectrin enrichment, axonal hyperexcitability, and altered myelin sheath thickness [#3, #4, #17, #18, #28]. Through its plasma-membrane-localized U2 domain, 4.1B suppresses growth by activating sequential Src-Rac1-JNK signaling, inducing G1 arrest with reduced cyclin A and Rb phosphorylation, and it restrains EGFR by binding the receptor juxtamembrane segment to block dimerization and downstream MAPK and PI3K/AKT signaling [#8, #9, #24, #30]. 4.1B also controls actin stress-fiber organization, adhesion, and motility in an isoform-specific manner, with the 130 kDa plasma-membrane isoform required for spreading and stress-fiber formation [#19, #22]. Loss of 4.1B promotes transformation and tumor progression in vivo, including increased susceptibility to prostate carcinoma and enhanced metastatic propensity [#23, #37]. Biallelic loss-of-function variants in EPB41L3 cause an autosomal recessive developmental disorder with seizures, hypotonia, and delayed myelination [#36].\",\n  \"teleology\": [\n    {\n      \"year\": 2000,\n      \"claim\": \"Established that 4.1B is positioned at cell-cell contact sites and that splice isoforms diverge functionally in their cytoskeletal-binding capacity, framing it as a membrane adaptor with isoform-specific roles.\",\n      \"evidence\": \"Immunofluorescence, cDNA characterization, and domain analysis across tissues\",\n      \"pmids\": [\"10652311\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not identify the transmembrane binding partner at contacts\", \"Functional consequence of isoform heterogeneity untested\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Answered what 4.1B binds at cell-cell contacts by showing a direct, actin-dependent complex with the tumor suppressor TSLC1/CADM1, linking adhesion to the cytoskeleton.\",\n      \"evidence\": \"Co-IP, colocalization, and actin-disruption experiments\",\n      \"pmids\": [\"12234973\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of binding not resolved\", \"Did not establish how the complex suppresses growth\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Resolved the atomic basis of the TSLC1 interaction, mapping it to a hydrophobic FERM C-lobe pocket and defining the critical TSLC1 contact residues.\",\n      \"evidence\": \"X-ray crystallography of the FERM–TSLC1 complex with SPR validation\",\n      \"pmids\": [\"21131357\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structure of other partner complexes (Caspr, EGFR, integrin) not solved\", \"In vivo relevance of specific contacts not tested\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Extended 4.1B function to myelinated axons by showing direct binding to the GNP motifs of Caspr and Caspr2, implicating it in axonal domain organization.\",\n      \"evidence\": \"Co-IP from brain homogenate and in vitro binding assays\",\n      \"pmids\": [\"12542678\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not test whether binding is required for axonal compartmentalization in vivo\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Identified 4.1B as a regulator of arginine methyltransferases, binding PRMT3 without being a substrate and inhibiting its methylation of GAR substrates.\",\n      \"evidence\": \"Yeast two-hybrid, co-IP, in vitro and cellular methylation assays\",\n      \"pmids\": [\"15334060\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Biological consequence of PRMT3 inhibition not linked to a phenotype\", \"Methylation substrates relevant in vivo unknown\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Defined the U2 domain at the plasma membrane as necessary and sufficient for meningioma growth suppression, localizing the tumor-suppressor activity to a discrete module.\",\n      \"evidence\": \"Truncation/deletion constructs with clonogenic, proliferation, and apoptosis readouts and membrane-targeting fusions\",\n      \"pmids\": [\"15688033\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream effectors of U2 not yet identified\", \"Mechanism of membrane requirement unresolved at this stage\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Linked the U2/membrane-dependent growth suppression to a defined signaling cascade, showing sequential Src-Rac1-JNK activation drives cyclin A regulation.\",\n      \"evidence\": \"Genetic and pharmacologic inhibition, dominant-negatives, JNK activity and cyclin A assays\",\n      \"pmids\": [\"16707455\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How U2 at the membrane initiates Src activation is unknown\", \"Generality of the pathway across cancer types untested\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Demonstrated genetically that 4.1B is required in vivo for juxtaparanodal Caspr2 accumulation, Kv1 clustering, and the paranodal membrane barrier.\",\n      \"evidence\": \"Transgenic 4.1-binding-deficient rescue constructs in 4.1B-null mice with immunofluorescence\",\n      \"pmids\": [\"20164332\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not distinguish targeting from stabilization\", \"Effect on conduction not directly measured here\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Multiple 4.1B-null mouse analyses established that loss disrupts paranodal septate junctions, juxtaparanodal Kv1/Caspr2 clustering, βII-spectrin enrichment, axon caliber, and causes neuronal hyperexcitability cell-autonomously.\",\n      \"evidence\": \"4.1B-null mice with immunofluorescence, immuno-EM, electrophysiology, and myelinating co-cultures\",\n      \"pmids\": [\"21632923\", \"21958379\", \"21966409\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular mechanism connecting 4.1B loss to myelin/caliber changes incomplete\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Resolved isoform specificity in adhesion/motility by showing the 130 kDa plasma-membrane isoform, not the 60 kDa form, is required for stress-fiber formation, spreading, and migration.\",\n      \"evidence\": \"Isoform-specific rescue in 4.1B-KO MEFs with adhesion/spreading/migration assays\",\n      \"pmids\": [\"24381168\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct cytoskeletal effector engaged by the 130 kDa isoform not defined\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Defined a direct molecular mechanism for receptor-level tumor suppression: 4.1B FERM binds the EGFR juxtamembrane segment to block dimerization, autophosphorylation, and MAPK/PI3K signaling.\",\n      \"evidence\": \"Co-IP, domain mapping, EGFR phosphorylation/dimerization assays, KO MEFs and gastric cancer cells\",\n      \"pmids\": [\"31492173\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural model of FERM–EGFR contact absent\", \"Single-lab data without reciprocal structural validation\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Connected EPB41L3 to human disease, showing biallelic loss-of-function causes an autosomal recessive developmental disorder with seizures and delayed myelination, with supporting oligodendroglial defects in mice.\",\n      \"evidence\": \"Exome sequencing, patient fibroblast protein/transcript analysis, and Epb41l3-KO oligodendroglia culture\",\n      \"pmids\": [\"39292993\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanistic link between 4.1B loss and oligodendroglial apoptosis incomplete\", \"Genotype-phenotype range across patients limited\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the membrane-localized U2 domain physically initiates Src activation and integrates the diverse partner interactions (TSLC1, Caspr/Caspr2, EGFR, integrin) into a unified growth-suppressive and cytoskeletal program remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No structural model of U2-mediated signaling\", \"Unclear which interactions are most relevant to the human disease phenotype\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 1, 3, 13]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [0, 19, 20, 22]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [5, 10, 30]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 2, 8, 19]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [0, 19, 22]},\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [14]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [35]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [9, 30]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [24]},\n      {\"term_id\": \"R-HSA-112316\", \"supporting_discovery_ids\": [4, 17, 18]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [23, 36]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"CADM1\", \"CNTNAP1\", \"CNTNAP2\", \"PRMT3\", \"PRMT5\", \"EGFR\", \"ITGB8\", \"CDH1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}