{"gene":"EPB41L3","run_date":"2026-04-28T17:46:03","timeline":{"discoveries":[{"year":2002,"finding":"EPB41L3 (DAL-1/4.1B) directly associates with the tumor suppressor TSLC1 (CADM1) at cell-cell contact sites and links to the actin cytoskeleton; redistribution of both proteins to membrane ruffles suggests involvement in cell motility accompanying actin rearrangement.","method":"Co-immunoprecipitation, co-localization, actin disruption assays, in vivo metastasis model","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 1-2 — reciprocal Co-IP, actin cytoskeleton functional validation, and in vivo metastasis assay; replicated in structural study (PMID 21131357)","pmids":["12234973"],"is_preprint":false},{"year":2010,"finding":"Crystal structure of the DAL-1 FERM domain in complex with the TSLC1 cytoplasmic domain revealed that DAL-1 binds TSLC1 through conserved residues in a hydrophobic pocket in the C-lobe of the FERM domain, with TSLC1 Tyr406 and Thr408 forming the most critical contacts, as confirmed by surface plasmon resonance.","method":"X-ray crystallography, surface plasmon resonance, mutagenesis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — atomic-resolution crystal structure with mutagenesis and SPR validation in a single study","pmids":["21131357"],"is_preprint":false},{"year":2004,"finding":"The FERM domain of DAL-1/4.1B interacts with protein arginine N-methyltransferase 3 (PRMT3) via the C-terminal catalytic core domain of PRMT3; DAL-1/4.1B is not itself a PRMT3 substrate but inhibits PRMT3-mediated methylation of GAR-containing substrates both in vitro and in cells.","method":"Yeast two-hybrid, co-immunoprecipitation, in vitro GST binding, in vitro methylation assay, inducible overexpression in MCF-7 cells","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 1-2 — multiple orthogonal methods (Y2H, Co-IP, in vitro binding, enzymatic assay) in a single study","pmids":["15334060"],"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 while enhancing methylation of myelin basic protein, demonstrating bidirectional regulation of PRMT5.","method":"Yeast two-hybrid, in vitro methylation assay, co-immunoprecipitation","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 1-2 — in vitro enzymatic assay with multiple substrates; single lab follow-up to PRMT3 study","pmids":["15737618"],"is_preprint":false},{"year":2002,"finding":"The FERM domain of DAL-1/Protein 4.1B specifically binds 14-3-3 isoforms β, γ, and η (but not other 4.1 family members such as merlin, ezrin, or radixin); the binding site was mapped to residues Pro244–Leu280 within the FERM domain.","method":"Yeast two-hybrid, GST affinity chromatography, co-immunoprecipitation","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 2 — reciprocal Co-IP and in vitro binding with domain mapping; single lab","pmids":["11996670"],"is_preprint":false},{"year":2004,"finding":"14-3-3 binding to Protein 4.1B (via the F359Y mutation that abolishes 14-3-3 binding without affecting other interactors) is dispensable for Protein 4.1B growth suppression in meningioma cells.","method":"Site-directed mutagenesis, GST affinity chromatography, co-immunoprecipitation, clonogenic assay, thymidine incorporation","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 1-2 — mutagenesis with functional rescue experiments; single lab","pmids":["15116094"],"is_preprint":false},{"year":2005,"finding":"The U2 domain of Protein 4.1B, when targeted to the plasma membrane, is necessary and sufficient for meningioma growth suppression; deletion of the U2 domain abolishes growth suppression and caspase-3 activation.","method":"Truncation/deletion mutants, clonogenic assay, thymidine incorporation, caspase-3 activation assay, membrane targeting construct","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 — domain dissection with multiple functional readouts; single lab","pmids":["15688033"],"is_preprint":false},{"year":2006,"finding":"Protein 4.1B/DAL-1 expression in meningioma cells activates JNK through sequential activation of Src and Rac1; inhibition of Rac1 or JNK abrogates 4.1B-mediated growth suppression and cyclin A regulation; this function requires the U2 domain at the plasma membrane.","method":"Pharmacologic inhibitors, dominant-negative constructs, JNK activity assays, colony formation, cyclin A western blot","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2 — genetic/pharmacologic epistasis with multiple pathway readouts; single lab","pmids":["16707455"],"is_preprint":false},{"year":2006,"finding":"DAL-1/4.1B-induced apoptosis in MCF-7 cells is primarily mediated through caspase-8 activation; inhibition of caspase-8 blocks DAL-1/4.1B-induced cell death; protein methylation cooperates with DAL-1/4.1B in this process.","method":"Flow cytometry (apoptosis), caspase activation assays, methylation inhibitor (AdOX), inducible expression system","journal":"Molecular cancer","confidence":"Medium","confidence_rationale":"Tier 2 — pharmacologic and genetic dissection of apoptosis pathway; single lab","pmids":["16420693"],"is_preprint":false},{"year":2003,"finding":"Protein 4.1B associates with both Caspr/paranodin (at paranodes) and Caspr2 (at juxtaparanodes) of myelinated axons through their conserved GNP motifs; these interactions were validated by co-immunoprecipitation from brain homogenates.","method":"In vitro binding assays, co-immunoprecipitation from brain homogenates, immunofluorescence in developing CNS/PNS","journal":"The European journal of neuroscience","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP from tissue plus domain mapping; replicated by multiple subsequent KO studies","pmids":["12542678"],"is_preprint":false},{"year":2010,"finding":"4.1B interaction with Caspr (via the 4.1-binding sequence) is required for generating an effective membrane barrier at the paranodal junction, as Caspr lacking the 4.1-binding site fails to exclude Kv1 channels from paranodes; 4.1B interaction with Caspr2 is required for Caspr2 and Kv1 channel accumulation at the juxtaparanodal membrane, demonstrated in 4.1B-null mice.","method":"Transgenic mice expressing Caspr/Caspr2 lacking 4.1-binding sequence, 4.1B-null mice, immunofluorescence, Kv1 channel localization assays","journal":"The Journal of neuroscience","confidence":"High","confidence_rationale":"Tier 2 — genetic epistasis using domain-specific transgene rescue in null mice; two complementary transgenic lines","pmids":["20164332"],"is_preprint":false},{"year":2011,"finding":"In 4.1B-null mice, Caspr localization is disrupted at paranodes and paranodal axoglial septate junctions (AGSJs) are destabilized in both PNS and CNS, and juxtaparanodal clustering of Caspr2/TAG-1/Kv1 channels is lost; βII spectrin enrichment along the axolemma is reduced.","method":"4.1B-null mice (independent generation), immunofluorescence, ultrastructural EM, electrophysiology","journal":"The Journal of neuroscience","confidence":"High","confidence_rationale":"Tier 2 — loss-of-function in vivo with ultrastructural and electrophysiological readouts; independently replicated by two labs (PMIDs 21632923 and 21966409)","pmids":["21632923","21966409"],"is_preprint":false},{"year":2012,"finding":"4.1B is expressed by neurons subjacent to the axon membrane in all myelinated axon domains except nodes; its loss reduces internodal Necl-1, Necl-2, and α2-spectrin levels, and causes hypermyelination and increased Schmidt-Lanterman incisures; juxtaparanodal clustering defects are neuron-autonomous as shown by myelinating cocultures.","method":"4.1B-null mice, immunofluorescence, immuno-EM, myelinating cocultures (neuron-autonomous test), nerve conduction velocity measurement","journal":"Glia","confidence":"High","confidence_rationale":"Tier 2 — cell-autonomous rescue cocultures and multiple ultrastructural readouts; independent lab confirmation","pmids":["23109359"],"is_preprint":false},{"year":2011,"finding":"Protein 4.1B is necessary to form the Caspr+ para-AIS barrier at the first myelin attachment site of motor neurons, ensuring compartmentalization of Kv1 channels and segregation of AIS, para-AIS, and juxtapara-AIS compartments.","method":"4.1B knockout mice, immunofluorescence for AIS and nodal markers","journal":"BMC biology","confidence":"Medium","confidence_rationale":"Tier 2 — KO mice with defined regional phenotype; single lab","pmids":["21958379"],"is_preprint":false},{"year":2009,"finding":"Protein 4.1B acts as an intracellular effector of SynCAM1 to recruit NMDA-type receptors (NMDARs) to SynCAM1 adhesion sites; manipulation of 4.1B expression specifically affects NMDAR-mediated activity and localization in hippocampal neurons, whereas SynCAM1-4.1N interaction specifically recruits AMPARs.","method":"COS7 cell adhesion assay, HEK293-neuron coculture assay, siRNA knockdown and overexpression in hippocampal neurons, electrophysiology (mEPSC recording)","journal":"Molecular and cellular neurosciences","confidence":"Medium","confidence_rationale":"Tier 2 — multiple cell-based assays with electrophysiological validation; single lab","pmids":["19796685"],"is_preprint":false},{"year":2005,"finding":"The C-terminal domain of Band 4.1B (a region conserved across 4.1 family members) interacts selectively with the β8 integrin cytoplasmic tail; 4.1B colocalizes with αvβ8 in cultured astrocytes and brain.","method":"Yeast two-hybrid, in vitro binding assay, co-immunoprecipitation, immunofluorescence colocalization","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 — multiple binding assays with colocalization; single lab","pmids":["16157875"],"is_preprint":false},{"year":2011,"finding":"αvβ8 integrin and Band 4.1B cooperatively regulate cardiac morphogenesis: ~60% of β8/4.1B double-null mouse embryos display cardiovascular defects and die by E11.5 due to defective cardiac outflow tract development with reduced SMA-actin expression in neural crest-derived cells.","method":"Double-null mouse genetics, embryo phenotyping, immunostaining for SMA-actin","journal":"Developmental dynamics","confidence":"Medium","confidence_rationale":"Tier 2 — genetic epistasis in double-KO mice with defined developmental phenotype; single lab","pmids":["21181944"],"is_preprint":false},{"year":2009,"finding":"A 200 kDa isoform of protein 4.1B localizes to the Golgi apparatus; siRNA depletion disrupts Golgi structure and prevents Na+/K+-ATPase, ZO-1, and ZO-2 from trafficking to the plasma membrane.","method":"siRNA knockdown, Brefeldin A treatment, immunofluorescence, subcellular fractionation","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 — siRNA KD with organelle and trafficking phenotype; single lab","pmids":["19299464"],"is_preprint":false},{"year":2007,"finding":"Protein 4.1B suppresses prostate cancer progression and metastasis in vivo: down-regulation of 4.1B increased metastatic propensity in an orthotopic prostate cancer model, and 4.1B-deficient mice showed increased susceptibility to spontaneous aggressive prostate carcinomas; enhanced malignancy was associated with reduced apoptosis.","method":"shRNA knockdown in orthotopic mouse model, 4.1B-null mouse spontaneous tumor study, apoptosis assays","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 — two independent in vivo genetic approaches (knockdown + KO mice) with consistent phenotype","pmids":["17640904"],"is_preprint":false},{"year":2007,"finding":"RNAi-mediated knockdown of 4.1B in non-metastatic sarcoma cells reduced actin stress fibers and doubled migration speed; re-expression of 4.1B in metastatic cells halved migration speed and suppressed chemotaxis, establishing 4.1B as a regulator of actin organization and cell motility.","method":"RNAi knockdown, exogenous re-expression, wound-healing migration assay, chemotaxis assay, F-actin staining","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 — bidirectional genetic manipulation with quantitative motility readouts; single lab","pmids":["17264155"],"is_preprint":false},{"year":2013,"finding":"The 130-kDa plasma membrane isoform of protein 4.1B (but not the 60-kDa nuclear isoform) is required for cell adhesion, spreading, migration, and actin stress fiber formation in mouse embryonic fibroblasts; these defects are rescued by re-expression of the 130-kDa isoform.","method":"4.1B KO MEFs, isoform-selective re-expression, adhesion/spreading/migration assays, F-actin staining","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — isoform-specific rescue with multiple cellular phenotypes; single lab","pmids":["24381168"],"is_preprint":false},{"year":2005,"finding":"4.1B inhibits mammary epithelial cell proliferation by inducing G1 cell cycle arrest with decreased cyclin A expression, reduced Rb phosphorylation, and reduced erbB2 phosphorylation, independently of MAPK, JNK, or Akt; 4.1B-null mice show increased mammary epithelial proliferation during pregnancy.","method":"4.1B-null mice (mammary gland proliferation analysis), cell cycle analysis, western blot for cyclin A/Rb/erbB2 phosphorylation","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 — in vivo KO phenotype plus in vitro mechanistic pathway analysis; single lab","pmids":["16007173"],"is_preprint":false},{"year":2001,"finding":"DAL-1 interacts with ERM proteins and βII-spectrin (similar to merlin) but not SCHIP-1 (a merlin-specific interactor); DAL-1 suppresses cell proliferation in meningioma cells but not in schwannoma cells, indicating cell-type-specific growth regulation.","method":"Co-immunoprecipitation, proliferation assay (cell counting)","journal":"Neurobiology of disease","confidence":"Medium","confidence_rationale":"Tier 3 — single Co-IP plus proliferation assay; single lab but consistent with broader 4.1B literature","pmids":["11300722"],"is_preprint":false},{"year":2002,"finding":"Re-expression of DAL-1 in MCF-7 breast cancer cells suppresses growth (partly via apoptosis induction) and increases cell attachment to multiple extracellular matrices.","method":"Constitutive and inducible DAL-1 expression, cell growth assays, apoptosis assays, adhesion assays to ECM substrates","journal":"International journal of cancer","confidence":"Medium","confidence_rationale":"Tier 2 — inducible system with multiple functional readouts; single lab","pmids":["12115567"],"is_preprint":false},{"year":2007,"finding":"Protein 4.1B interacts with p55 and sodium bicarbonate cotransporter 1 (NBC1) in the basolateral membrane of renal S1-S2 proximal tubules; GST pull-down and co-immunoprecipitation from kidney lysates confirmed a trimeric complex (NBC1-4.1B-p55).","method":"Co-immunoprecipitation from kidney lysates, GST pull-down, immunohistochemistry co-localization","journal":"The journal of histochemistry and cytochemistry","confidence":"Medium","confidence_rationale":"Tier 2 — reciprocal Co-IP plus in vitro pull-down identifying a trimeric complex; single lab","pmids":["17712176"],"is_preprint":false},{"year":2019,"finding":"The FERM domain of 4.1B interacts with the first 13 amino acids (P13) of the EGFR intracellular juxtamembrane segment; 4.1B binding inhibits EGFR dimerization and autophosphorylation, thereby suppressing EGFR/MAPK/ERK1/2 and PI3K/AKT signaling and inhibiting cancer cell proliferation.","method":"Co-immunoprecipitation, immunofluorescence colocalization, domain-mapping constructs, EGFR phosphorylation/dimerization assays, proliferation assays in GC cell lines and MEFs","journal":"Cell communication and signaling","confidence":"Medium","confidence_rationale":"Tier 2 — domain mapping with functional consequence on receptor signaling; single lab","pmids":["31492173"],"is_preprint":false},{"year":2015,"finding":"DAL-1 directly binds HSPA5 (GRP78/BiP), regulates its expression, and thereby suppresses EMT markers (E-cadherin, β-catenin, Vimentin, N-cadherin) and inhibits lung cancer cell migration and invasion; additionally, DAL-1 binds E-cadherin promoter to regulate its transcription.","method":"Co-immunoprecipitation, chromatin immunoprecipitation (direct E-cadherin promoter binding), EMT marker western blot, migration/invasion assays","journal":"Journal of experimental & clinical cancer research","confidence":"Medium","confidence_rationale":"Tier 2 — CoIP for protein interaction plus ChIP for transcriptional regulation; single lab","pmids":["25609022"],"is_preprint":false},{"year":2021,"finding":"DAL-1/4.1B promotes exosome uptake by lung cancer cells by upregulating HSPG2 (heparan sulfate proteoglycan 2); HSPG2 knockdown abolishes the enhanced exosome uptake caused by DAL-1/4.1B overexpression.","method":"Overexpression/knockdown of DAL-1/4.1B, HSPG2 siRNA, vesicle uptake inhibitor panel, fluorescent exosome uptake assay","journal":"Molecular and cellular biochemistry","confidence":"Low","confidence_rationale":"Tier 3 — single lab, single approach, no direct binding shown between 4.1B and HSPG2","pmids":["34657240"],"is_preprint":false},{"year":2016,"finding":"EPB41L3 overexpression suppresses ESCC cell invasion and migration and downregulates MMP2 and MMP9 expression; knockdown has the opposite effect.","method":"Overexpression plasmid and siRNA knockdown, transwell invasion/wound-healing assays, western blot for MMP2/MMP9/p-AKT","journal":"Cell biochemistry and function","confidence":"Low","confidence_rationale":"Tier 3 — single lab, no direct mechanistic link between 4.1B and MMP regulation established","pmids":["26916087"],"is_preprint":false},{"year":2000,"finding":"Protein 4.1B is localized specifically at the plasma membrane in regions of cell-cell contact; it contains functional spectrin-actin binding and NuMA-binding domains subject to regulated alternative splicing (spectrin-actin domain present only in muscle isoforms); focal high expression occurs in select neuronal populations including Purkinje cells and hippocampal pyramidal neurons.","method":"In situ hybridization, immunofluorescence, Western blot, alternative splicing characterization","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — multiple complementary methods establishing localization and domain structure; foundational characterization paper","pmids":["10652311"],"is_preprint":false},{"year":2010,"finding":"Microcell-mediated chromosome 18 transfer suppressed ovarian cancer neoplasia; EPB41L3 re-expression in 3D spheroids caused growth suppression and induced apoptosis, identifying EPB41L3 as the chromosome 18 tumor suppressor gene responsible for neoplastic suppression.","method":"Microcell-mediated chromosome transfer, gene expression microarray, EPB41L3 reconstitution in 3D spheroid model, transmission/scanning electron microscopy, apoptosis assays","journal":"Neoplasia","confidence":"Medium","confidence_rationale":"Tier 2 — functional complementation approach with 3D reconstitution and apoptosis readout; single lab","pmids":["20651987"],"is_preprint":false},{"year":2016,"finding":"4.1B knockout in SV40T-immortalized MEFs is sufficient for malignant transformation: 4.1B-null iMEFs form tumors in nude mice (desmoid-type), display nuclear β-catenin accumulation, E-cadherin downregulation, and ERK/AKT activation; 4.1B interacts with E-cadherin in MEF cells.","method":"4.1B-null iMEF transformation assay, xenograft in nude mice, western blot pathway analysis, co-immunoprecipitation (4.1B–E-cadherin)","journal":"Molecular carcinogenesis","confidence":"Medium","confidence_rationale":"Tier 2 — in vivo transformation assay with mechanistic pathway analysis; single lab","pmids":["27312663"],"is_preprint":false},{"year":2011,"finding":"miR-223, induced by the transcription factor Twist, posttranscriptionally downregulates EPB41L3 expression by directly targeting its 3'-UTR, thereby promoting gastric cancer cell migration and invasion.","method":"Luciferase reporter assay (3'-UTR targeting), qRT-PCR, western blot, migration/invasion assays, miR-223 transfection","journal":"Molecular cancer research : MCR","confidence":"Medium","confidence_rationale":"Tier 2 — direct 3'-UTR luciferase validation plus functional migration assay; replicated in lung cancer context (PMID 25881295)","pmids":["21628394"],"is_preprint":false}],"current_model":"EPB41L3 (protein 4.1B/DAL-1) is a FERM-domain membrane-cytoskeletal adaptor that links transmembrane proteins—including TSLC1/CADM1, Caspr, Caspr2, αvβ8 integrin, EGFR, NBC1, and SynCAM1—to the actin-spectrin cytoskeleton at sites of cell-cell contact and specialized axonal domains; it suppresses tumor cell growth, motility, and metastasis through multiple mechanisms including inhibition of EGFR dimerization/autophosphorylation (suppressing EGFR/MAPK and PI3K/AKT), activation of Src-Rac1-JNK signaling in a membrane-localized U2-domain-dependent manner, induction of caspase-8-mediated apoptosis, modulation of PRMT3/PRMT5 arginine methyltransferase activity, and maintenance of E-cadherin expression to oppose EMT; in neurons it is essential for clustering Caspr/Caspr2 at paranodal and juxtaparanodal axonal domains and organizing Kv1 channel compartmentalization around nodes of Ranvier."},"narrative":{"teleology":[{"year":2000,"claim":"Establishing the basic identity of 4.1B as a plasma-membrane adaptor at cell–cell contacts with tissue-specific alternative splicing resolved what domain architecture underlies this new 4.1 family member.","evidence":"In situ hybridization, immunofluorescence, and splicing analysis in multiple tissues","pmids":["10652311"],"confidence":"Medium","gaps":["No functional consequence of alternative splicing shown","No binding partners identified at this stage"]},{"year":2001,"claim":"Demonstrating that DAL-1 interacts with ERM proteins and βII-spectrin and suppresses meningioma cell growth established it as a candidate tumor suppressor linked to the cortical cytoskeleton.","evidence":"Co-immunoprecipitation and proliferation assays in meningioma and schwannoma cells","pmids":["11996670","11300722"],"confidence":"Medium","gaps":["Mechanism of growth suppression unknown","Cell-type specificity unexplained"]},{"year":2002,"claim":"Identification of direct TSLC1/CADM1 binding and co-redistribution to membrane ruffles linked 4.1B to a known tumor suppressor pathway and implicated it in actin-dependent cell motility and metastasis suppression.","evidence":"Reciprocal co-immunoprecipitation, actin disruption, and in vivo metastasis model","pmids":["12234973","12115567"],"confidence":"High","gaps":["Structural basis of TSLC1 interaction not yet resolved","Downstream signaling from the 4.1B–TSLC1 complex unknown"]},{"year":2003,"claim":"Discovery that 4.1B binds Caspr at paranodes and Caspr2 at juxtaparanodes through conserved GNP motifs established a neuronal role entirely distinct from tumor suppression.","evidence":"In vitro binding and co-immunoprecipitation from brain homogenates with immunofluorescence in developing CNS/PNS","pmids":["12542678"],"confidence":"High","gaps":["In vivo requirement not yet tested by knockout","Whether 4.1B is sufficient or redundant with other 4.1 proteins at nodes unknown"]},{"year":2004,"claim":"Showing that the FERM domain inhibits PRMT3 methyltransferase activity without being a substrate itself revealed an unexpected enzymatic regulatory function and linked 4.1B to protein arginine methylation.","evidence":"Yeast two-hybrid, co-IP, GST binding, and in vitro methylation assays in MCF-7 cells","pmids":["15334060"],"confidence":"High","gaps":["Physiological relevance of PRMT3 inhibition to tumor suppression not demonstrated","Whether endogenous methylation targets change in 4.1B-expressing cells unclear"]},{"year":2005,"claim":"Domain dissection identified the U2 domain as the minimal growth-suppressive unit when membrane-targeted, and showed 4.1B induces G1 arrest with decreased cyclin A and Rb phosphorylation in mammary epithelium, separating the mechanism from 14-3-3 binding.","evidence":"Deletion mutants with clonogenic/caspase-3 assays; 4.1B-null mouse mammary gland analysis; 14-3-3-binding-defective mutant retaining growth suppression","pmids":["15688033","16007173","15116094","15737618","16157875"],"confidence":"Medium","gaps":["U2 domain binding partners mediating growth suppression not identified","How membrane localization activates U2-dependent signaling unknown"]},{"year":2006,"claim":"Epistasis experiments placing Src→Rac1→JNK downstream of membrane-localized 4.1B, and identification of caspase-8 as the apoptotic effector, defined two parallel pathways through which 4.1B suppresses growth.","evidence":"Pharmacologic/dominant-negative inhibition of Rac1/JNK in meningioma cells; caspase-8 inhibition in MCF-7 cells","pmids":["16707455","16420693"],"confidence":"Medium","gaps":["How U2 domain activates Src not known","Whether JNK and caspase-8 pathways converge or act in different cell types unclear"]},{"year":2007,"claim":"In vivo loss-of-function in prostate cancer models proved 4.1B is a bona fide metastasis suppressor, while bidirectional manipulation in sarcoma cells showed it directly controls actin stress fibers and migration speed.","evidence":"shRNA orthotopic prostate model, 4.1B-null spontaneous tumor study, RNAi/re-expression migration assays in sarcoma cells","pmids":["17640904","17264155"],"confidence":"High","gaps":["Whether metastasis suppression depends on TSLC1, Src–Rac1–JNK, or another pathway not resolved","Actin-organizing mechanism at the molecular level not defined"]},{"year":2009,"claim":"Discovery that a 200 kDa Golgi-localized isoform is required for trafficking of Na⁺/K⁺-ATPase and ZO-1/ZO-2 to the plasma membrane, and that 4.1B acts as an intracellular effector of SynCAM1 to recruit NMDARs, expanded the functional repertoire beyond cortical cytoskeletal scaffolding.","evidence":"siRNA depletion with Golgi and trafficking phenotypes; SynCAM1–4.1B coculture assay with electrophysiology in hippocampal neurons","pmids":["19299464","19796685"],"confidence":"Medium","gaps":["Golgi isoform domain requirements not mapped","Whether NMDAR recruitment occurs in vivo not confirmed"]},{"year":2010,"claim":"Transgenic rescue in 4.1B-null mice proved the 4.1-binding motifs of Caspr and Caspr2 are individually required for paranodal barrier function and juxtaparanodal Kv1 channel clustering, establishing 4.1B as an essential axonal domain organizer.","evidence":"Caspr/Caspr2 transgenes lacking 4.1-binding sequence in 4.1B-null mice with Kv1 channel localization","pmids":["20164332"],"confidence":"High","gaps":["Whether 4.1B directly connects Caspr/Caspr2 to spectrin in vivo not biochemically shown","Functional consequences for nerve conduction not fully characterized at this point"]},{"year":2011,"claim":"Independent 4.1B-null mouse studies confirmed paranodal AGSJ destabilization, loss of juxtaparanodal Caspr2/Kv1, reduced βII-spectrin, and extended the phenotype to the para-AIS compartment, consolidating 4.1B as the primary cytoskeletal anchor at multiple axonal domains; the crystal structure of FERM–TSLC1 defined the atomic basis of the key tumor-suppressive interaction.","evidence":"Two independent 4.1B-null lines with ultrastructural EM and electrophysiology; X-ray crystallography with SPR and mutagenesis","pmids":["21632923","21966409","21958379","21131357"],"confidence":"High","gaps":["Why 4.1G partially compensates in CNS but not PNS remains unclear","Structural basis for U2 domain signaling still unknown"]},{"year":2012,"claim":"Showing that 4.1B is expressed by neurons in all myelinated domains except nodes, and that juxtaparanodal defects are neuron-autonomous, resolved the cell-of-origin question and revealed additional roles in controlling myelin thickness and Schmidt-Lanterman incisures.","evidence":"4.1B-null mice, immuno-EM, myelinating neuron-Schwann cell cocultures","pmids":["23109359"],"confidence":"High","gaps":["How 4.1B loss leads to hypermyelination is mechanistically unexplained","Whether internodal Necl loss is cause or consequence of myelin defects unclear"]},{"year":2016,"claim":"Demonstrating that 4.1B loss in SV40T-immortalized MEFs is sufficient for malignant transformation with E-cadherin loss, nuclear β-catenin, and ERK/AKT activation—and that 4.1B physically interacts with E-cadherin—unified tumor suppression with adherens junction maintenance.","evidence":"4.1B-null iMEF xenograft, co-immunoprecipitation of 4.1B–E-cadherin, pathway western blots","pmids":["27312663"],"confidence":"Medium","gaps":["Whether 4.1B stabilizes E-cadherin at the membrane or regulates its transcription not separated","Relative contributions of EGFR, E-cadherin, and Src–Rac1 pathways to transformation not ranked"]},{"year":2019,"claim":"Mapping the FERM domain interaction to the first 13 residues of the EGFR juxtamembrane segment and showing this inhibits receptor dimerization and autophosphorylation provided a direct mechanism for 4.1B suppression of MAPK/ERK and PI3K/AKT signaling.","evidence":"Co-IP, domain-mapping constructs, EGFR dimerization/phosphorylation assays in gastric cancer lines and MEFs","pmids":["31492173"],"confidence":"Medium","gaps":["Whether EGFR inhibition accounts for tumor suppression in vivo not tested","Structural model of FERM–EGFR complex not available"]},{"year":null,"claim":"Major open questions include the structural basis of U2-domain-mediated Src activation, how 4.1B loss causes hypermyelination, the relative contribution of each signaling axis (EGFR, E-cadherin/β-catenin, Src–Rac1–JNK, caspase-8) to tumor suppression in different tissues, and whether the Golgi-trafficking and synaptic NMDAR-recruitment roles are connected to the canonical membrane-cytoskeletal scaffold function.","evidence":"","pmids":[],"confidence":"Low","gaps":["No structural model for U2 domain or its effector interface","In vivo hierarchy among tumor-suppressive pathways not established","Golgi isoform biology largely unexplored beyond initial siRNA study"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,9,14,15,24,25]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[0,19,20,22,29]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[2,3,25]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,15,20,24,25,29]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[0,19,20,29]},{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[17]}],"pathway":[{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[8,18,30]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[7,25,31]},{"term_id":"R-HSA-112316","term_label":"Neuronal System","supporting_discovery_ids":[9,10,11,14]},{"term_id":"R-HSA-1500931","term_label":"Cell-Cell communication","supporting_discovery_ids":[0,15,31]}],"complexes":[],"partners":["CADM1","CNTNAP1","CNTNAP2","EGFR","PRMT3","PRMT5","SLC4A4"],"other_free_text":[]},"mechanistic_narrative":"EPB41L3 (protein 4.1B/DAL-1) is a FERM-domain-containing membrane-cytoskeletal adaptor that functions as a tumor suppressor and an organizer of specialized axonal membrane domains. Its FERM domain directly engages transmembrane partners—including TSLC1/CADM1, EGFR, and Caspr/Caspr2—while its C-terminal domain binds β8 integrin and its U2 domain, when membrane-localized, activates a Src–Rac1–JNK growth-suppressive cascade; it inhibits EGFR dimerization/autophosphorylation to dampen MAPK/ERK and PI3K/AKT signaling, induces caspase-8-mediated apoptosis, and maintains E-cadherin expression to oppose epithelial–mesenchymal transition [PMID:31492173, PMID:16420693, PMID:27312663, PMID:16707455]. In myelinated neurons, 4.1B is essential for anchoring Caspr at paranodes and Caspr2/Kv1 channels at juxtaparanodes, thereby establishing the membrane barriers that compartmentalize ion channels around nodes of Ranvier, as demonstrated by loss of these domains in 4.1B-null mice [PMID:20164332, PMID:21632923]. A 200 kDa isoform localizes to the Golgi apparatus and is required for trafficking of Na⁺/K⁺-ATPase and tight-junction proteins to the plasma membrane [PMID:19299464]."},"prefetch_data":{"uniprot":{"accession":"Q9Y2J2","full_name":"Band 4.1-like protein 3","aliases":["4.1B","Differentially expressed in adenocarcinoma of the lung protein 1","DAL-1","Erythrocyte membrane protein band 4.1-like 3"],"length_aa":1087,"mass_kda":120.7,"function":"Tumor suppressor that inhibits cell proliferation and promotes apoptosis. 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 \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — reciprocal Co-IP, actin cytoskeleton functional validation, and in vivo metastasis assay; replicated in structural study (PMID 21131357)\",\n      \"pmids\": [\"12234973\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Crystal structure of the DAL-1 FERM domain in complex with the TSLC1 cytoplasmic domain revealed that DAL-1 binds TSLC1 through conserved residues in a hydrophobic pocket in the C-lobe of the FERM domain, with TSLC1 Tyr406 and Thr408 forming the most critical contacts, as confirmed by surface plasmon resonance.\",\n      \"method\": \"X-ray crystallography, surface plasmon resonance, mutagenesis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — atomic-resolution crystal structure with mutagenesis and SPR validation in a single study\",\n      \"pmids\": [\"21131357\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"The FERM domain of DAL-1/4.1B interacts with protein arginine N-methyltransferase 3 (PRMT3) via the C-terminal catalytic core domain of PRMT3; DAL-1/4.1B is not itself a PRMT3 substrate but inhibits PRMT3-mediated methylation of GAR-containing substrates both in vitro and in cells.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, in vitro GST binding, in vitro methylation assay, inducible overexpression in MCF-7 cells\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — multiple orthogonal methods (Y2H, Co-IP, in vitro binding, enzymatic assay) in a single study\",\n      \"pmids\": [\"15334060\"],\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 while enhancing methylation of myelin basic protein, demonstrating bidirectional regulation of PRMT5.\",\n      \"method\": \"Yeast two-hybrid, in vitro methylation assay, co-immunoprecipitation\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro enzymatic assay with multiple substrates; single lab follow-up to PRMT3 study\",\n      \"pmids\": [\"15737618\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"The FERM domain of DAL-1/Protein 4.1B specifically binds 14-3-3 isoforms β, γ, and η (but not other 4.1 family members such as merlin, ezrin, or radixin); the binding site was mapped to residues Pro244–Leu280 within the FERM domain.\",\n      \"method\": \"Yeast two-hybrid, GST affinity chromatography, co-immunoprecipitation\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP and in vitro binding with domain mapping; single lab\",\n      \"pmids\": [\"11996670\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"14-3-3 binding to Protein 4.1B (via the F359Y mutation that abolishes 14-3-3 binding without affecting other interactors) is dispensable for Protein 4.1B growth suppression in meningioma cells.\",\n      \"method\": \"Site-directed mutagenesis, GST affinity chromatography, co-immunoprecipitation, clonogenic assay, thymidine incorporation\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 — mutagenesis with functional rescue experiments; single lab\",\n      \"pmids\": [\"15116094\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"The U2 domain of Protein 4.1B, when targeted to the plasma membrane, is necessary and sufficient for meningioma growth suppression; deletion of the U2 domain abolishes growth suppression and caspase-3 activation.\",\n      \"method\": \"Truncation/deletion mutants, clonogenic assay, thymidine incorporation, caspase-3 activation assay, membrane targeting construct\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — domain dissection with multiple functional readouts; single lab\",\n      \"pmids\": [\"15688033\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Protein 4.1B/DAL-1 expression in meningioma cells activates JNK through sequential activation of Src and Rac1; inhibition of Rac1 or JNK abrogates 4.1B-mediated growth suppression and cyclin A regulation; this function requires the U2 domain at the plasma membrane.\",\n      \"method\": \"Pharmacologic inhibitors, dominant-negative constructs, JNK activity assays, colony formation, cyclin A western blot\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic/pharmacologic epistasis with multiple pathway readouts; single lab\",\n      \"pmids\": [\"16707455\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"DAL-1/4.1B-induced apoptosis in MCF-7 cells is primarily mediated through caspase-8 activation; inhibition of caspase-8 blocks DAL-1/4.1B-induced cell death; protein methylation cooperates with DAL-1/4.1B in this process.\",\n      \"method\": \"Flow cytometry (apoptosis), caspase activation assays, methylation inhibitor (AdOX), inducible expression system\",\n      \"journal\": \"Molecular cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — pharmacologic and genetic dissection of apoptosis pathway; single lab\",\n      \"pmids\": [\"16420693\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Protein 4.1B associates with both Caspr/paranodin (at paranodes) and Caspr2 (at juxtaparanodes) of myelinated axons through their conserved GNP motifs; these interactions were validated by co-immunoprecipitation from brain homogenates.\",\n      \"method\": \"In vitro binding assays, co-immunoprecipitation from brain homogenates, immunofluorescence in developing CNS/PNS\",\n      \"journal\": \"The European journal of neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP from tissue plus domain mapping; replicated by multiple subsequent KO studies\",\n      \"pmids\": [\"12542678\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"4.1B interaction with Caspr (via the 4.1-binding sequence) is required for generating an effective membrane barrier at the paranodal junction, as Caspr lacking the 4.1-binding site fails to exclude Kv1 channels from paranodes; 4.1B interaction with Caspr2 is required for Caspr2 and Kv1 channel accumulation at the juxtaparanodal membrane, demonstrated in 4.1B-null mice.\",\n      \"method\": \"Transgenic mice expressing Caspr/Caspr2 lacking 4.1-binding sequence, 4.1B-null mice, immunofluorescence, Kv1 channel localization assays\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis using domain-specific transgene rescue in null mice; two complementary transgenic lines\",\n      \"pmids\": [\"20164332\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"In 4.1B-null mice, Caspr localization is disrupted at paranodes and paranodal axoglial septate junctions (AGSJs) are destabilized in both PNS and CNS, and juxtaparanodal clustering of Caspr2/TAG-1/Kv1 channels is lost; βII spectrin enrichment along the axolemma is reduced.\",\n      \"method\": \"4.1B-null mice (independent generation), immunofluorescence, ultrastructural EM, electrophysiology\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — loss-of-function in vivo with ultrastructural and electrophysiological readouts; independently replicated by two labs (PMIDs 21632923 and 21966409)\",\n      \"pmids\": [\"21632923\", \"21966409\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"4.1B is expressed by neurons subjacent to the axon membrane in all myelinated axon domains except nodes; its loss reduces internodal Necl-1, Necl-2, and α2-spectrin levels, and causes hypermyelination and increased Schmidt-Lanterman incisures; juxtaparanodal clustering defects are neuron-autonomous as shown by myelinating cocultures.\",\n      \"method\": \"4.1B-null mice, immunofluorescence, immuno-EM, myelinating cocultures (neuron-autonomous test), nerve conduction velocity measurement\",\n      \"journal\": \"Glia\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — cell-autonomous rescue cocultures and multiple ultrastructural readouts; independent lab confirmation\",\n      \"pmids\": [\"23109359\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Protein 4.1B is necessary to form the Caspr+ para-AIS barrier at the first myelin attachment site of motor neurons, ensuring compartmentalization of Kv1 channels and segregation of AIS, para-AIS, and juxtapara-AIS compartments.\",\n      \"method\": \"4.1B knockout mice, immunofluorescence for AIS and nodal markers\",\n      \"journal\": \"BMC biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — KO mice with defined regional phenotype; single lab\",\n      \"pmids\": [\"21958379\"],\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 (NMDARs) to SynCAM1 adhesion sites; manipulation of 4.1B expression specifically affects NMDAR-mediated activity and localization in hippocampal neurons, whereas SynCAM1-4.1N interaction specifically recruits AMPARs.\",\n      \"method\": \"COS7 cell adhesion assay, HEK293-neuron coculture assay, siRNA knockdown and overexpression in hippocampal neurons, electrophysiology (mEPSC recording)\",\n      \"journal\": \"Molecular and cellular neurosciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple cell-based assays with electrophysiological validation; single lab\",\n      \"pmids\": [\"19796685\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"The C-terminal domain of Band 4.1B (a region conserved across 4.1 family members) interacts selectively with the β8 integrin cytoplasmic tail; 4.1B colocalizes with αvβ8 in cultured astrocytes and brain.\",\n      \"method\": \"Yeast two-hybrid, in vitro binding assay, co-immunoprecipitation, immunofluorescence colocalization\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple binding assays with colocalization; single lab\",\n      \"pmids\": [\"16157875\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"αvβ8 integrin and Band 4.1B cooperatively regulate cardiac morphogenesis: ~60% of β8/4.1B double-null mouse embryos display cardiovascular defects and die by E11.5 due to defective cardiac outflow tract development with reduced SMA-actin expression in neural crest-derived cells.\",\n      \"method\": \"Double-null mouse genetics, embryo phenotyping, immunostaining for SMA-actin\",\n      \"journal\": \"Developmental dynamics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis in double-KO mice with defined developmental phenotype; single lab\",\n      \"pmids\": [\"21181944\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"A 200 kDa isoform of protein 4.1B localizes to the Golgi apparatus; siRNA depletion disrupts Golgi structure and prevents Na+/K+-ATPase, ZO-1, and ZO-2 from trafficking to the plasma membrane.\",\n      \"method\": \"siRNA knockdown, Brefeldin A treatment, immunofluorescence, subcellular fractionation\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — siRNA KD with organelle and trafficking phenotype; single lab\",\n      \"pmids\": [\"19299464\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Protein 4.1B suppresses prostate cancer progression and metastasis in vivo: down-regulation of 4.1B increased metastatic propensity in an orthotopic prostate cancer model, and 4.1B-deficient mice showed increased susceptibility to spontaneous aggressive prostate carcinomas; enhanced malignancy was associated with reduced apoptosis.\",\n      \"method\": \"shRNA knockdown in orthotopic mouse model, 4.1B-null mouse spontaneous tumor study, apoptosis assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — two independent in vivo genetic approaches (knockdown + KO mice) with consistent phenotype\",\n      \"pmids\": [\"17640904\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"RNAi-mediated knockdown of 4.1B in non-metastatic sarcoma cells reduced actin stress fibers and doubled migration speed; re-expression of 4.1B in metastatic cells halved migration speed and suppressed chemotaxis, establishing 4.1B as a regulator of actin organization and cell motility.\",\n      \"method\": \"RNAi knockdown, exogenous re-expression, wound-healing migration assay, chemotaxis assay, F-actin staining\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — bidirectional genetic manipulation with quantitative motility readouts; single lab\",\n      \"pmids\": [\"17264155\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"The 130-kDa plasma membrane isoform of protein 4.1B (but not the 60-kDa nuclear isoform) is required for cell adhesion, spreading, migration, and actin stress fiber formation in mouse embryonic fibroblasts; these defects are rescued by re-expression of the 130-kDa isoform.\",\n      \"method\": \"4.1B KO MEFs, isoform-selective re-expression, adhesion/spreading/migration assays, F-actin staining\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — isoform-specific rescue with multiple cellular phenotypes; single lab\",\n      \"pmids\": [\"24381168\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"4.1B inhibits mammary epithelial cell proliferation by inducing G1 cell cycle arrest with decreased cyclin A expression, reduced Rb phosphorylation, and reduced erbB2 phosphorylation, independently of MAPK, JNK, or Akt; 4.1B-null mice show increased mammary epithelial proliferation during pregnancy.\",\n      \"method\": \"4.1B-null mice (mammary gland proliferation analysis), cell cycle analysis, western blot for cyclin A/Rb/erbB2 phosphorylation\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vivo KO phenotype plus in vitro mechanistic pathway analysis; single lab\",\n      \"pmids\": [\"16007173\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"DAL-1 interacts with ERM proteins and βII-spectrin (similar to merlin) but not SCHIP-1 (a merlin-specific interactor); DAL-1 suppresses cell proliferation in meningioma cells but not in schwannoma cells, indicating cell-type-specific growth regulation.\",\n      \"method\": \"Co-immunoprecipitation, proliferation assay (cell counting)\",\n      \"journal\": \"Neurobiology of disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — single Co-IP plus proliferation assay; single lab but consistent with broader 4.1B literature\",\n      \"pmids\": [\"11300722\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Re-expression of DAL-1 in MCF-7 breast cancer cells suppresses growth (partly via apoptosis induction) and increases cell attachment to multiple extracellular matrices.\",\n      \"method\": \"Constitutive and inducible DAL-1 expression, cell growth assays, apoptosis assays, adhesion assays to ECM substrates\",\n      \"journal\": \"International journal of cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — inducible system with multiple functional readouts; single lab\",\n      \"pmids\": [\"12115567\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Protein 4.1B interacts with p55 and sodium bicarbonate cotransporter 1 (NBC1) in the basolateral membrane of renal S1-S2 proximal tubules; GST pull-down and co-immunoprecipitation from kidney lysates confirmed a trimeric complex (NBC1-4.1B-p55).\",\n      \"method\": \"Co-immunoprecipitation from kidney lysates, GST pull-down, immunohistochemistry co-localization\",\n      \"journal\": \"The journal of histochemistry and cytochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP plus in vitro pull-down identifying a trimeric complex; single lab\",\n      \"pmids\": [\"17712176\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"The FERM domain of 4.1B interacts with the first 13 amino acids (P13) of the EGFR intracellular juxtamembrane segment; 4.1B binding inhibits EGFR dimerization and autophosphorylation, thereby suppressing EGFR/MAPK/ERK1/2 and PI3K/AKT signaling and inhibiting cancer cell proliferation.\",\n      \"method\": \"Co-immunoprecipitation, immunofluorescence colocalization, domain-mapping constructs, EGFR phosphorylation/dimerization assays, proliferation assays in GC cell lines and MEFs\",\n      \"journal\": \"Cell communication and signaling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — domain mapping with functional consequence on receptor signaling; single lab\",\n      \"pmids\": [\"31492173\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"DAL-1 directly binds HSPA5 (GRP78/BiP), regulates its expression, and thereby suppresses EMT markers (E-cadherin, β-catenin, Vimentin, N-cadherin) and inhibits lung cancer cell migration and invasion; additionally, DAL-1 binds E-cadherin promoter to regulate its transcription.\",\n      \"method\": \"Co-immunoprecipitation, chromatin immunoprecipitation (direct E-cadherin promoter binding), EMT marker western blot, migration/invasion assays\",\n      \"journal\": \"Journal of experimental & clinical cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — CoIP for protein interaction plus ChIP for transcriptional regulation; single lab\",\n      \"pmids\": [\"25609022\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"DAL-1/4.1B promotes exosome uptake by lung cancer cells by upregulating HSPG2 (heparan sulfate proteoglycan 2); HSPG2 knockdown abolishes the enhanced exosome uptake caused by DAL-1/4.1B overexpression.\",\n      \"method\": \"Overexpression/knockdown of DAL-1/4.1B, HSPG2 siRNA, vesicle uptake inhibitor panel, fluorescent exosome uptake assay\",\n      \"journal\": \"Molecular and cellular biochemistry\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single lab, single approach, no direct binding shown between 4.1B and HSPG2\",\n      \"pmids\": [\"34657240\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"EPB41L3 overexpression suppresses ESCC cell invasion and migration and downregulates MMP2 and MMP9 expression; knockdown has the opposite effect.\",\n      \"method\": \"Overexpression plasmid and siRNA knockdown, transwell invasion/wound-healing assays, western blot for MMP2/MMP9/p-AKT\",\n      \"journal\": \"Cell biochemistry and function\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single lab, no direct mechanistic link between 4.1B and MMP regulation established\",\n      \"pmids\": [\"26916087\"],\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; it contains functional spectrin-actin binding and NuMA-binding domains subject to regulated alternative splicing (spectrin-actin domain present only in muscle isoforms); focal high expression occurs in select neuronal populations including Purkinje cells and hippocampal pyramidal neurons.\",\n      \"method\": \"In situ hybridization, immunofluorescence, Western blot, alternative splicing characterization\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple complementary methods establishing localization and domain structure; foundational characterization paper\",\n      \"pmids\": [\"10652311\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Microcell-mediated chromosome 18 transfer suppressed ovarian cancer neoplasia; EPB41L3 re-expression in 3D spheroids caused growth suppression and induced apoptosis, identifying EPB41L3 as the chromosome 18 tumor suppressor gene responsible for neoplastic suppression.\",\n      \"method\": \"Microcell-mediated chromosome transfer, gene expression microarray, EPB41L3 reconstitution in 3D spheroid model, transmission/scanning electron microscopy, apoptosis assays\",\n      \"journal\": \"Neoplasia\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — functional complementation approach with 3D reconstitution and apoptosis readout; single lab\",\n      \"pmids\": [\"20651987\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"4.1B knockout in SV40T-immortalized MEFs is sufficient for malignant transformation: 4.1B-null iMEFs form tumors in nude mice (desmoid-type), display nuclear β-catenin accumulation, E-cadherin downregulation, and ERK/AKT activation; 4.1B interacts with E-cadherin in MEF cells.\",\n      \"method\": \"4.1B-null iMEF transformation assay, xenograft in nude mice, western blot pathway analysis, co-immunoprecipitation (4.1B–E-cadherin)\",\n      \"journal\": \"Molecular carcinogenesis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vivo transformation assay with mechanistic pathway analysis; single lab\",\n      \"pmids\": [\"27312663\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"miR-223, induced by the transcription factor Twist, posttranscriptionally downregulates EPB41L3 expression by directly targeting its 3'-UTR, thereby promoting gastric cancer cell migration and invasion.\",\n      \"method\": \"Luciferase reporter assay (3'-UTR targeting), qRT-PCR, western blot, migration/invasion assays, miR-223 transfection\",\n      \"journal\": \"Molecular cancer research : MCR\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct 3'-UTR luciferase validation plus functional migration assay; replicated in lung cancer context (PMID 25881295)\",\n      \"pmids\": [\"21628394\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"EPB41L3 (protein 4.1B/DAL-1) is a FERM-domain membrane-cytoskeletal adaptor that links transmembrane proteins—including TSLC1/CADM1, Caspr, Caspr2, αvβ8 integrin, EGFR, NBC1, and SynCAM1—to the actin-spectrin cytoskeleton at sites of cell-cell contact and specialized axonal domains; it suppresses tumor cell growth, motility, and metastasis through multiple mechanisms including inhibition of EGFR dimerization/autophosphorylation (suppressing EGFR/MAPK and PI3K/AKT), activation of Src-Rac1-JNK signaling in a membrane-localized U2-domain-dependent manner, induction of caspase-8-mediated apoptosis, modulation of PRMT3/PRMT5 arginine methyltransferase activity, and maintenance of E-cadherin expression to oppose EMT; in neurons it is essential for clustering Caspr/Caspr2 at paranodal and juxtaparanodal axonal domains and organizing Kv1 channel compartmentalization around nodes of Ranvier.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"EPB41L3 (protein 4.1B/DAL-1) is a FERM-domain-containing membrane-cytoskeletal adaptor that functions as a tumor suppressor and an organizer of specialized axonal membrane domains. Its FERM domain directly engages transmembrane partners—including TSLC1/CADM1, EGFR, and Caspr/Caspr2—while its C-terminal domain binds β8 integrin and its U2 domain, when membrane-localized, activates a Src–Rac1–JNK growth-suppressive cascade; it inhibits EGFR dimerization/autophosphorylation to dampen MAPK/ERK and PI3K/AKT signaling, induces caspase-8-mediated apoptosis, and maintains E-cadherin expression to oppose epithelial–mesenchymal transition [PMID:31492173, PMID:16420693, PMID:27312663, PMID:16707455]. In myelinated neurons, 4.1B is essential for anchoring Caspr at paranodes and Caspr2/Kv1 channels at juxtaparanodes, thereby establishing the membrane barriers that compartmentalize ion channels around nodes of Ranvier, as demonstrated by loss of these domains in 4.1B-null mice [PMID:20164332, PMID:21632923]. A 200 kDa isoform localizes to the Golgi apparatus and is required for trafficking of Na⁺/K⁺-ATPase and tight-junction proteins to the plasma membrane [PMID:19299464].\",\n  \"teleology\": [\n    {\n      \"year\": 2000,\n      \"claim\": \"Establishing the basic identity of 4.1B as a plasma-membrane adaptor at cell–cell contacts with tissue-specific alternative splicing resolved what domain architecture underlies this new 4.1 family member.\",\n      \"evidence\": \"In situ hybridization, immunofluorescence, and splicing analysis in multiple tissues\",\n      \"pmids\": [\"10652311\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No functional consequence of alternative splicing shown\", \"No binding partners identified at this stage\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Demonstrating that DAL-1 interacts with ERM proteins and βII-spectrin and suppresses meningioma cell growth established it as a candidate tumor suppressor linked to the cortical cytoskeleton.\",\n      \"evidence\": \"Co-immunoprecipitation and proliferation assays in meningioma and schwannoma cells\",\n      \"pmids\": [\"11996670\", \"11300722\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of growth suppression unknown\", \"Cell-type specificity unexplained\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Identification of direct TSLC1/CADM1 binding and co-redistribution to membrane ruffles linked 4.1B to a known tumor suppressor pathway and implicated it in actin-dependent cell motility and metastasis suppression.\",\n      \"evidence\": \"Reciprocal co-immunoprecipitation, actin disruption, and in vivo metastasis model\",\n      \"pmids\": [\"12234973\", \"12115567\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of TSLC1 interaction not yet resolved\", \"Downstream signaling from the 4.1B–TSLC1 complex unknown\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Discovery that 4.1B binds Caspr at paranodes and Caspr2 at juxtaparanodes through conserved GNP motifs established a neuronal role entirely distinct from tumor suppression.\",\n      \"evidence\": \"In vitro binding and co-immunoprecipitation from brain homogenates with immunofluorescence in developing CNS/PNS\",\n      \"pmids\": [\"12542678\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo requirement not yet tested by knockout\", \"Whether 4.1B is sufficient or redundant with other 4.1 proteins at nodes unknown\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Showing that the FERM domain inhibits PRMT3 methyltransferase activity without being a substrate itself revealed an unexpected enzymatic regulatory function and linked 4.1B to protein arginine methylation.\",\n      \"evidence\": \"Yeast two-hybrid, co-IP, GST binding, and in vitro methylation assays in MCF-7 cells\",\n      \"pmids\": [\"15334060\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological relevance of PRMT3 inhibition to tumor suppression not demonstrated\", \"Whether endogenous methylation targets change in 4.1B-expressing cells unclear\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Domain dissection identified the U2 domain as the minimal growth-suppressive unit when membrane-targeted, and showed 4.1B induces G1 arrest with decreased cyclin A and Rb phosphorylation in mammary epithelium, separating the mechanism from 14-3-3 binding.\",\n      \"evidence\": \"Deletion mutants with clonogenic/caspase-3 assays; 4.1B-null mouse mammary gland analysis; 14-3-3-binding-defective mutant retaining growth suppression\",\n      \"pmids\": [\"15688033\", \"16007173\", \"15116094\", \"15737618\", \"16157875\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"U2 domain binding partners mediating growth suppression not identified\", \"How membrane localization activates U2-dependent signaling unknown\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Epistasis experiments placing Src→Rac1→JNK downstream of membrane-localized 4.1B, and identification of caspase-8 as the apoptotic effector, defined two parallel pathways through which 4.1B suppresses growth.\",\n      \"evidence\": \"Pharmacologic/dominant-negative inhibition of Rac1/JNK in meningioma cells; caspase-8 inhibition in MCF-7 cells\",\n      \"pmids\": [\"16707455\", \"16420693\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How U2 domain activates Src not known\", \"Whether JNK and caspase-8 pathways converge or act in different cell types unclear\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"In vivo loss-of-function in prostate cancer models proved 4.1B is a bona fide metastasis suppressor, while bidirectional manipulation in sarcoma cells showed it directly controls actin stress fibers and migration speed.\",\n      \"evidence\": \"shRNA orthotopic prostate model, 4.1B-null spontaneous tumor study, RNAi/re-expression migration assays in sarcoma cells\",\n      \"pmids\": [\"17640904\", \"17264155\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether metastasis suppression depends on TSLC1, Src–Rac1–JNK, or another pathway not resolved\", \"Actin-organizing mechanism at the molecular level not defined\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Discovery that a 200 kDa Golgi-localized isoform is required for trafficking of Na⁺/K⁺-ATPase and ZO-1/ZO-2 to the plasma membrane, and that 4.1B acts as an intracellular effector of SynCAM1 to recruit NMDARs, expanded the functional repertoire beyond cortical cytoskeletal scaffolding.\",\n      \"evidence\": \"siRNA depletion with Golgi and trafficking phenotypes; SynCAM1–4.1B coculture assay with electrophysiology in hippocampal neurons\",\n      \"pmids\": [\"19299464\", \"19796685\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Golgi isoform domain requirements not mapped\", \"Whether NMDAR recruitment occurs in vivo not confirmed\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Transgenic rescue in 4.1B-null mice proved the 4.1-binding motifs of Caspr and Caspr2 are individually required for paranodal barrier function and juxtaparanodal Kv1 channel clustering, establishing 4.1B as an essential axonal domain organizer.\",\n      \"evidence\": \"Caspr/Caspr2 transgenes lacking 4.1-binding sequence in 4.1B-null mice with Kv1 channel localization\",\n      \"pmids\": [\"20164332\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether 4.1B directly connects Caspr/Caspr2 to spectrin in vivo not biochemically shown\", \"Functional consequences for nerve conduction not fully characterized at this point\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Independent 4.1B-null mouse studies confirmed paranodal AGSJ destabilization, loss of juxtaparanodal Caspr2/Kv1, reduced βII-spectrin, and extended the phenotype to the para-AIS compartment, consolidating 4.1B as the primary cytoskeletal anchor at multiple axonal domains; the crystal structure of FERM–TSLC1 defined the atomic basis of the key tumor-suppressive interaction.\",\n      \"evidence\": \"Two independent 4.1B-null lines with ultrastructural EM and electrophysiology; X-ray crystallography with SPR and mutagenesis\",\n      \"pmids\": [\"21632923\", \"21966409\", \"21958379\", \"21131357\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Why 4.1G partially compensates in CNS but not PNS remains unclear\", \"Structural basis for U2 domain signaling still unknown\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Showing that 4.1B is expressed by neurons in all myelinated domains except nodes, and that juxtaparanodal defects are neuron-autonomous, resolved the cell-of-origin question and revealed additional roles in controlling myelin thickness and Schmidt-Lanterman incisures.\",\n      \"evidence\": \"4.1B-null mice, immuno-EM, myelinating neuron-Schwann cell cocultures\",\n      \"pmids\": [\"23109359\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How 4.1B loss leads to hypermyelination is mechanistically unexplained\", \"Whether internodal Necl loss is cause or consequence of myelin defects unclear\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Demonstrating that 4.1B loss in SV40T-immortalized MEFs is sufficient for malignant transformation with E-cadherin loss, nuclear β-catenin, and ERK/AKT activation—and that 4.1B physically interacts with E-cadherin—unified tumor suppression with adherens junction maintenance.\",\n      \"evidence\": \"4.1B-null iMEF xenograft, co-immunoprecipitation of 4.1B–E-cadherin, pathway western blots\",\n      \"pmids\": [\"27312663\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether 4.1B stabilizes E-cadherin at the membrane or regulates its transcription not separated\", \"Relative contributions of EGFR, E-cadherin, and Src–Rac1 pathways to transformation not ranked\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Mapping the FERM domain interaction to the first 13 residues of the EGFR juxtamembrane segment and showing this inhibits receptor dimerization and autophosphorylation provided a direct mechanism for 4.1B suppression of MAPK/ERK and PI3K/AKT signaling.\",\n      \"evidence\": \"Co-IP, domain-mapping constructs, EGFR dimerization/phosphorylation assays in gastric cancer lines and MEFs\",\n      \"pmids\": [\"31492173\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether EGFR inhibition accounts for tumor suppression in vivo not tested\", \"Structural model of FERM–EGFR complex not available\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Major open questions include the structural basis of U2-domain-mediated Src activation, how 4.1B loss causes hypermyelination, the relative contribution of each signaling axis (EGFR, E-cadherin/β-catenin, Src–Rac1–JNK, caspase-8) to tumor suppression in different tissues, and whether the Golgi-trafficking and synaptic NMDAR-recruitment roles are connected to the canonical membrane-cytoskeletal scaffold function.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No structural model for U2 domain or its effector interface\", \"In vivo hierarchy among tumor-suppressive pathways not established\", \"Golgi isoform biology largely unexplored beyond initial siRNA study\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 9, 14, 15, 24, 25]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [0, 19, 20, 22, 29]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [2, 3, 25]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 15, 20, 24, 25, 29]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [0, 19, 20, 29]},\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [17]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [8, 18, 30]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [7, 25, 31]},\n      {\"term_id\": \"R-HSA-112316\", \"supporting_discovery_ids\": [9, 10, 11, 14]},\n      {\"term_id\": \"R-HSA-1500931\", \"supporting_discovery_ids\": [0, 15, 31]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"CADM1\",\n      \"CNTNAP1\",\n      \"CNTNAP2\",\n      \"EGFR\",\n      \"PRMT3\",\n      \"PRMT5\",\n      \"SLC4A4\",\n      \"CADM1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}