{"gene":"EFNB1","run_date":"2026-06-09T23:54:42","timeline":{"discoveries":[{"year":1995,"finding":"LERK-2 (EFNB1) is a membrane-bound ligand that signals through three different Eph-related receptor tyrosine kinases: Cek5, Cek10, and Elk, but fails to interact functionally with Cek9. Preliminary mutagenesis suggested a negative regulatory role for the LERK-2 cytoplasmic domain in LERK-2 signaling.","method":"Chimeric receptor constructs (Eph ectodomain + TrkB cytoplasmic domain) expressed in NIH 3T3 cells; focus formation assays; binding assays; mutagenesis","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with chimeric receptors, binding assays, and mutagenesis; receptor selectivity demonstrated across multiple receptors","pmids":["7621826"],"is_preprint":false},{"year":1996,"finding":"Membrane-bound LERK-2 (EFNB1) binds HEK2 (an EPH-family receptor) and signals through it, inducing receptor phosphorylation and cell-cell adhesion/aggregate formation. Soluble LERK-2 can also signal through HEK2. Co-expression of HEK2 and LERK-2 in the same cell reduces kinase activity and autophosphorylation compared to juxtacrine stimulation.","method":"Expression of HEK2 in 32D cells; coincubation assays; receptor phosphorylation assays; cell aggregation assays; inhibition with soluble LERK-2","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — direct receptor phosphorylation assay and cell adhesion functional readout with multiple orthogonal approaches in a single study","pmids":["8798744"],"is_preprint":false},{"year":1996,"finding":"ELK receptor and its ligand LERK-2 (EFNB1) are co-expressed on human renal microvascular endothelial cells (HRMEC). LERK-2 (but not LERK-1/B61) promotes in vitro assembly of HRMEC into capillary-like structures, demonstrating receptor-ligand specificity in endothelial morphogenesis.","method":"In vitro capillary-like assembly assay comparing HRMEC and HUVEC responses to LERK-1 vs LERK-2; immunohistochemistry of developing kidney","journal":"Kidney international. Supplement","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional in vitro assembly assay with receptor-ligand specificity comparison, single lab","pmids":["8941926"],"is_preprint":false},{"year":2004,"finding":"Heterozygous loss-of-function mutations in EFNB1 cause craniofrontonasal syndrome (CFNS). The more severe female phenotype is proposed to result from patchwork loss of ephrin-B1 due to X-inactivation, disturbing tissue boundary formation at the developing coronal suture. EFNB1 is X-inactivated but X-inactivation is not markedly skewed in blood or cranial periosteum of CFNS females.","method":"Mutation analysis (sequencing); X-inactivation analysis in patient tissues; in situ expression analysis in mouse embryos","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (sequencing, X-inactivation analysis, in situ expression); replicated by multiple subsequent studies","pmids":["15166289"],"is_preprint":false},{"year":2005,"finding":"The majority of CFNS-causing EFNB1 mutations (26/33) are located in exons 2 and 3 encoding the extracellular ephrin domain. Frameshift mutations in the last 25 codons disrupt intracellular binding sites for Grb4 and PDZ-effector proteins involved in reverse signaling, demonstrating that ephrin-B1 reverse signaling is mechanistically important.","method":"DNA sequencing of EFNB1 in CFNS patients; mutation spectrum analysis","journal":"Human mutation","confidence":"Medium","confidence_rationale":"Tier 3 / Strong — sequencing-based mutation mapping across large patient cohort identifies functional domain requirements, but no direct biochemical assay of reverse signaling","pmids":["15959873"],"is_preprint":false},{"year":2007,"finding":"EFNB1 missense mutations in CFNS patients disrupt Eph receptor-ephrin-B1 interactions at the ligand-receptor dimerization interface (G-H loop). The P54L mutation prevents Eph-receptor-mediated cell cluster formation and abolishes intracellular ephrin-B1 Tyr324 and Tyr329 phosphorylation. The T111I mutation reduces Tyr324/Tyr329 phosphorylation. Splice-site mutation c.406+2T>C causes intron retention or cryptic splice site activation. Frameshift c.614_615delCT escapes nonsense-mediated decay but does not produce soluble ephrin-B1 protein.","method":"RT-PCR; Western blot; time-lapse microscopy; cell culture transfection of NIH 3T3 cells with site-directed mutants; phosphorylation analysis","journal":"BMC medical genetics","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — multiple orthogonal methods (Western blot, phosphorylation assay, time-lapse microscopy, RT-PCR) on patient-derived and engineered mutations; functional consequences directly measured","pmids":["20565770"],"is_preprint":false},{"year":2007,"finding":"EFNB1 mutations can result in differential degradation by the nonsense-mediated mRNA decay (NMD) pathway: splice-site mutation c.407-2A>T and frameshift c.377_384delTCAAGAAG show severe transcript depletion, while c.614_615delCT (premature termination codon near 3' end of penultimate exon) escapes NMD, consistent with the '50-55 bp' rule. Clonal expansion of patient fibroblasts confirmed cellular mosaicism for wild-type vs. mutant EFNB1-expressing cells.","method":"RT-PCR on fibroblast cultures from CFNS females; quantitative mRNA analysis; clonal expansion of patient-derived cells","journal":"European journal of human genetics : EJHG","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct mRNA quantification in patient fibroblasts with clonal expansion; single lab, multiple mutations analyzed","pmids":["18043713"],"is_preprint":false},{"year":2011,"finding":"Ephrin-B1 (EFNB1) reverse signaling reduces RhoA activation and vascular smooth muscle cell (VSMC) contractility in vitro. In mesenteric arteries from smooth muscle-specific Efnb1 knockout mice, RhoA activity and myosin light chain phosphorylation were increased. Knockdown of GRIP1 (associated with Efnb1 intracellular tail) partially eliminated Efnb1's effect on VSMC contractility. Efnb1 KO mice on high-salt diet showed heightened blood pressure increments.","method":"Conditional Efnb1 knockout mice; siRNA knockdown of Grip1; RhoA activity assay; myosin light chain phosphorylation assay; ex vivo artery analysis; blood pressure measurement","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (KO mice, ex vivo assay, siRNA, phosphorylation assay, in vivo BP measurement) in a single study establishing reverse signaling through GRIP1 controlling RhoA/VSMC contractility","pmids":["22393061"],"is_preprint":false},{"year":2011,"finding":"Double conditional knockout of both Efnb1 and Efnb2 in T cells compromised IL-6 signaling, specifically abating STAT3 phosphorylation upon IL-6 stimulation, contributing to defects in T cell development, homeostatic expansion, and Th1/Th17 differentiation.","method":"loxP-mediated conditional double knockout mice; STAT3 phosphorylation assay; competitive bone marrow reconstitution; in vitro T cell differentiation assays; LCMV infection model","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean KO with defined cellular phenotype and downstream signaling readout; note that single Efnb1 KO showed no phenotype (redundancy), so dual KO required","pmids":["21976681"],"is_preprint":false},{"year":2011,"finding":"Single T cell-specific EFNB1 knockout mice showed no significant defects in T cell development, activation, proliferation, or differentiation into Th1, Th2, Th17 or Treg cells, indicating functional redundancy with other ephrin family members in T cells.","method":"T cell-specific EFNB1 conditional knockout; flow cytometry of thymus/spleen populations; competitive bone marrow reconstitution; T cell differentiation assays","journal":"BMC immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — clean KO with defined negative result across multiple T cell readouts; single lab","pmids":["22182253"],"is_preprint":false},{"year":2011,"finding":"In Xenopus tissue, cells overexpressing ephrinB1 cluster and sort out from wild-type cells, providing in vitro mechanistic evidence that cellular interference (due to mosaic EFNB1 expression) drives the CFNS phenotype in heterozygous females.","method":"In vitro cell sorting/clustering experiments in Xenopus tissue with overexpression of ephrinB1","journal":"Clinical genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct experimental demonstration of cell sorting in a model system; supports the cellular interference mechanism proposed from human genetics","pmids":["23614707"],"is_preprint":false},{"year":2011,"finding":"Duplication of EFNB1 produces ~1.6-fold more EFNB1 transcript from the duplicated X chromosome. In a mouse model carrying targeted human EFNB1 cDNA, abnormal cell sorting occurs in the cranial region, demonstrating that cellular mosaicism for different levels of ephrin-B1 (not just presence/absence) causes craniofacial abnormalities.","method":"Array-CGH; quantitative mRNA analysis; mouse model with targeted EFNB1 cDNA; cell sorting analysis in cranial region","journal":"Human mutation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mouse model with direct cell sorting observation; mRNA quantification confirming dosage imbalance; single lab","pmids":["21542058"],"is_preprint":false},{"year":2013,"finding":"Males mosaic for EFNB1 mutations are more severely affected than constitutionally hemizygous males. A 5' UTR variant mutating the stop codon of a small upstream open reading frame (uORF) was demonstrated using a dual-luciferase reporter construct to exacerbate interference with translation of the wild-type EFNB1 protein.","method":"Denaturing HPLC; massively parallel sequencing; MLPA; Pyrosequencing; dual-luciferase reporter assay for 5' UTR uORF translation interference","journal":"Human molecular genetics","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — functional luciferase assay directly demonstrating translational regulation by uORF; multiple orthogonal detection methods for mosaicism; replicated mechanism across 6 cases","pmids":["23335590"],"is_preprint":false},{"year":2013,"finding":"EFNB1 forms a novel complex with EGFR in head and neck squamous cell carcinoma cells. Cetuximab (anti-EGFR) treatment drives a shift in EGFR dimerization partners toward EFNB1, and EFNB1 phosphorylation and downstream ERK signaling persist in the presence of cetuximab. EFNB1 attenuation slowed tumor growth and increased survival in a murine HNSCC model.","method":"Co-immunoprecipitation; signaling assays; EFNB1 knockdown; murine tumor model","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP demonstrating EGFR-EFNB1 complex; in vivo tumor model; single lab, mechanistic follow-up partially detailed in abstract","pmids":["23811940"],"is_preprint":false},{"year":2015,"finding":"Deletion mutation of Efnb1 and Efnb2 intracellular tails revealed critical regions in controlling T cell chemotaxis toward CXCL12. T cells from dKO mice were compromised in migration to arthritic paws in vivo and in chemotaxis in vitro.","method":"Double conditional KO mice; intracellular tail deletion mutants; in vitro chemotaxis assay (CXCL12); in vivo migration to arthritic paws","journal":"Arthritis & rheumatology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — domain-specific deletion mutants identifying intracellular tail requirement for T cell chemotaxis; in vitro and in vivo confirmation; single lab","pmids":["25779027"],"is_preprint":false},{"year":2018,"finding":"A novel uATG variant in the 5' UTR of EFNB1 (c.-411) creates a new upstream ATG that significantly reduces EFNB1 protein translation without affecting mRNA levels, as demonstrated by dual-luciferase reporter assays.","method":"Dual-luciferase reporter assay; mRNA quantification","journal":"Molecular syndromology","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — direct functional reporter assay demonstrating translational repression by uATG; single lab, single method","pmids":["30976278"],"is_preprint":false},{"year":2018,"finding":"EfnB1 in osteogenic lineage cells supports hematopoietic stem/progenitor cell (HSPC) maintenance. EFNB1 reverse signaling in stromal cells is required for activation of the HSPC-promoting factor CXCL12. Activation of EPHB2 on CD34+ HSPCs by EFNB1-Fc stimulation enhanced myeloid/erythroid colony formation, while blocking EPHB1 or EPHB2 inhibited LTC-IC maintenance.","method":"Conditional EfnB1 knockout in osteogenic lineage (Osterix-Cre); LTC-IC assays; colony formation assays; EFNB1-Fc stimulation; functional blocking antibodies against EPHB1/EPHB2; CXCL12 activation assays","journal":"Experimental hematology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — conditional KO with defined cellular phenotype, functional blocking experiments, and reverse signaling readout (CXCL12 activation); single lab","pmids":["30326247"],"is_preprint":false},{"year":2020,"finding":"Upon stimulation by EFNB1 (ligand), EPHB2 receptor is ubiquitinated by the E3 ubiquitin ligase RNF186 at Lys892. This ubiquitination event recruits MAP1LC3B to trigger autophagy in colonic epithelial cells, contributing to intestinal homeostasis.","method":"Co-IP; site-directed mutagenesis (Lys892Arg EPHB2 mutant); EPHB2 ubiquitination assay; autophagy assays (MAP1LC3B recruitment); rnf186 and ephb2 knockout mice; DSS-induced colitis model; EFNB1-Fc recombinant protein treatment","journal":"Autophagy","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — site-directed mutagenesis identifying specific ubiquitination site, multiple orthogonal methods (Co-IP, ubiquitination assay, KO mice, in vivo model), functional consequence demonstrated","pmids":["33280498"],"is_preprint":false},{"year":2020,"finding":"EFNB1 and BDNF-dependent neuroprotection against excitotoxicity requires de novo formation of 'survival complexes' consisting of NMDA receptor, EFNB1's receptor EPHB2, and presenilin 1. Absence of presenilin 1 reduces survival complex formation and abolishes neuroprotection. Familial Alzheimer disease presenilin 1 mutants decrease factor-stimulated survival complex formation and neuroprotection.","method":"Co-immunoprecipitation; EPHB2- and NMDA receptor-derived peptides disrupting complex formation; survival assays; electrophysiology (NMDA receptor-mediated EPSCs); analysis of post-mortem Alzheimer brain samples","journal":"Brain communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP demonstrating complex formation, peptide disruption experiments, multiple readouts; single lab","pmids":["33005890"],"is_preprint":false},{"year":2023,"finding":"EFNB1-EPHB2 interaction activates downstream Wnt/β-catenin and FAK signaling pathways to promote gastric cancer cell invasion and migration. Fc-EFNB1 treatment increased invasion/migration abilities and induced high EPHB2 expression. EPHB2 knockdown completely abolished ephrin ligand-induced effects.","method":"Patient-derived xenografts; PCR array; gain/loss-of-function experiments; Fc-EFNB1 treatment; EPHB2 knockdown; signaling analysis (Wnt/β-catenin, FAK)","journal":"International journal of biological macromolecules","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function with defined signaling readouts and rescue; single lab","pmids":["38114003"],"is_preprint":false},{"year":2024,"finding":"Tumor cell-expressed EPHB1 and platelet-expressed EFNB1 mediate contact-dependent activation of platelets via EFNB1 reverse signaling-mediated AKT signaling activation. Activated platelets then release 5-HT (serotonin) which further enhances tumor growth in the liver metastatic niche of pancreatic ductal adenocarcinoma.","method":"mCherry metastatic niche-labeling system; platelet depletion in liver metastasis mouse model; gain/loss-of-function of EPHB1; recombinant protein treatment; Tph1-knockout mice; tumor-platelet adhesion assays; AKT signaling analysis","journal":"Cancer communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (KO mice, gain/loss-of-function, recombinant protein) establishing EFNB1 reverse signaling through AKT; single lab","pmids":["39648610"],"is_preprint":false},{"year":2025,"finding":"EFNB1 physically associates with EGFR in cells, and this complex formation depends on EFNB1's PDZ-binding motif (PBM). EGFR directly phosphorylates tyrosine residues within EFNB1's PBM, which disrupts the EFNB1-EGFR complex. The EFNB1-EGFR association is required for EFNB1-dependent cell adhesion to fibronectin.","method":"Proximity labeling proteomics (BioID); Co-IP; in vitro kinase assay demonstrating direct EGFR phosphorylation of EFNB1; PBM mutants; cell adhesion assay to fibronectin","journal":"Molecular & cellular proteomics : MCP","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro kinase reconstitution (direct phosphorylation), proximity proteomics, Co-IP, mutagenesis of PBM, functional adhesion readout; multiple orthogonal methods in single study","pmids":["40619000"],"is_preprint":false},{"year":2025,"finding":"Dengue virus NS1 protein binds EFNB1 at the endothelial cell surface to trigger endothelial barrier dysfunction. Phosphorylation of EFNB1 is necessary for NS1-induced barrier dysfunction. EFNB1-Fc fusion proteins act as decoys to block NS1-induced barrier dysfunction in vitro and in vivo.","method":"Comparative mass spectrometry (NS1 interactome); biochemical and computational interface mapping of EFNB1-NS1 complex; phosphorylation assays; barrier dysfunction assays in vitro and in vivo with EFNB1-Fc decoys","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (MS interactome, biochemical complex mapping, phosphorylation assay, functional in vivo decoy experiment); preprint, not peer-reviewed","pmids":["bio_10.1101_2025.11.19.689067"],"is_preprint":true}],"current_model":"EFNB1 (ephrin-B1) is a transmembrane ligand for Eph receptor tyrosine kinases that mediates bidirectional signaling: in forward signaling it activates Eph receptors (including EphB2, ELK/EphB4, and others) to promote cell-cell adhesion, endothelial morphogenesis, tissue boundary formation, and autophagy (via RNF186-mediated ubiquitination of EPHB2 at Lys892 recruiting MAP1LC3B); in reverse signaling through its intracellular domain (involving PDZ-binding motif interactions with GRIP1 and other effectors, and phosphorylation of Tyr324/Tyr329), it regulates RhoA activity and vascular smooth muscle contractility, T cell chemotaxis, CXCL12 production in stromal cells, and neuroprotection via NMDA receptor/presenilin-1 survival complexes; additionally, EFNB1 associates with EGFR (dependent on its PBM) and is directly phosphorylated by EGFR within the PBM to control cell adhesion to fibronectin, and its mosaic loss due to X-inactivation in heterozygous females causes craniofrontonasal syndrome through cellular interference and abnormal tissue boundary formation."},"narrative":{"mechanistic_narrative":"EFNB1 (ephrin-B1) is a membrane-bound ligand for Eph-family receptor tyrosine kinases that mediates bidirectional cell-cell signaling to control tissue boundary formation, endothelial morphogenesis, and cellular adhesion [PMID:7621826, PMID:8941926]. In forward signaling it binds and activates multiple Eph receptors — including EphB2/HEK2, ELK, and others — inducing receptor autophosphorylation and driving cell-cell adhesion and aggregate formation, with juxtacrine presentation being more potent than soluble ligand [PMID:7621826, PMID:8798744]. Through EphB2 specifically, EFNB1 engagement triggers RNF186-mediated ubiquitination of EPHB2 at Lys892, which recruits MAP1LC3B to initiate autophagy and maintain intestinal epithelial homeostasis [PMID:33280498], and activates downstream Wnt/β-catenin and FAK signaling [PMID:38114003]. EFNB1 reverse signaling through its intracellular tail and PDZ-binding motif acts via GRIP1 to suppress RhoA activation and vascular smooth muscle contractility [PMID:22393061], promotes CXCL12 production by osteogenic stromal cells to support hematopoietic stem/progenitor maintenance [PMID:30326247], and directs T cell chemotaxis toward CXCL12 [PMID:25779027]. EFNB1 also forms a PDZ-binding-motif-dependent complex with EGFR, and direct EGFR phosphorylation of tyrosines within this motif disrupts the complex and controls EFNB1-dependent cell adhesion to fibronectin [PMID:40619000, PMID:23811940]. Heterozygous loss-of-function mutations in EFNB1 cause craniofrontonasal syndrome, where mosaic ephrin-B1 expression arising from X-inactivation produces aberrant cell sorting and disrupted coronal-suture boundary formation [PMID:15166289, PMID:23614707].","teleology":[{"year":1995,"claim":"Establishing that the membrane-bound ligand EFNB1 functionally engages a defined subset of Eph receptors answered which receptors transduce its signal and revealed receptor selectivity.","evidence":"Chimeric Eph-TrkB receptor constructs in NIH 3T3 cells with focus-formation and binding assays","pmids":["7621826"],"confidence":"High","gaps":["Cytoplasmic-domain regulatory role only preliminarily mapped","Downstream effectors of receptor activation not identified"]},{"year":1996,"claim":"Demonstrating that EFNB1 binding induces Eph receptor phosphorylation and cell aggregation, and that co-expression dampens kinase activity, defined the forward-signaling output and the importance of juxtacrine presentation.","evidence":"HEK2 expression in 32D cells; receptor phosphorylation and cell aggregation assays; soluble-ligand competition","pmids":["8798744"],"confidence":"High","gaps":["Mechanism of cis-inhibition by co-expression not resolved","Physiological cell context not addressed"]},{"year":1996,"claim":"Showing ligand-specific promotion of endothelial capillary-like assembly tied EFNB1-Eph signaling to a tissue morphogenetic process.","evidence":"In vitro capillary-assembly assays in renal microvascular endothelial cells comparing LERK-1 vs LERK-2","pmids":["8941926"],"confidence":"Medium","gaps":["Single-lab in vitro assay","Molecular mediators of morphogenesis not defined"]},{"year":2004,"claim":"Linking heterozygous EFNB1 loss-of-function to craniofrontonasal syndrome and the paradoxically severe female phenotype established the X-inactivation/mosaic-interference disease model.","evidence":"Mutation sequencing, X-inactivation analysis in patient tissues, and mouse embryo in situ expression","pmids":["15166289"],"confidence":"High","gaps":["Direct mechanistic test of cellular interference not yet performed in this study","X-inactivation skewing not detected as explanation"]},{"year":2005,"claim":"Mapping the CFNS mutation spectrum to the extracellular ephrin domain and the reverse-signaling intracellular sites established that both ligand-receptor binding and reverse signaling are mechanistically required.","evidence":"DNA sequencing and mutation-spectrum analysis across a CFNS cohort","pmids":["15959873"],"confidence":"Medium","gaps":["No direct biochemical assay of reverse signaling in this study","Genotype-phenotype correlation incomplete"]},{"year":2007,"claim":"Demonstrating that specific CFNS missense mutations disrupt receptor binding and abolish intracellular Tyr324/Tyr329 phosphorylation, and that some alleles escape nonsense-mediated decay, linked mutation class to molecular consequence.","evidence":"Site-directed mutants in NIH 3T3 cells with Western blot, phosphorylation assays, time-lapse microscopy, RT-PCR; patient-fibroblast clonal expansion","pmids":["20565770","18043713"],"confidence":"High","gaps":["Downstream signaling consequences of lost Tyr phosphorylation not traced","Quantitative threshold of mosaicism for phenotype unknown"]},{"year":2011,"claim":"Showing that EFNB1 reverse signaling suppresses RhoA and smooth muscle contractility through GRIP1 defined a concrete reverse-signaling effector pathway with physiological output.","evidence":"Smooth-muscle-specific Efnb1 KO mice, Grip1 siRNA, RhoA and MLC phosphorylation assays, ex vivo arteries, blood pressure measurement","pmids":["22393061"],"confidence":"High","gaps":["Direct GRIP1-RhoA biochemical link not fully resolved","Relevance to human vascular disease untested"]},{"year":2011,"claim":"Defining EFNB1/EFNB2 roles in T cell IL-6/STAT3 signaling and the redundancy revealed by single versus double knockouts clarified its immunological function.","evidence":"Conditional single and double KO mice; STAT3 phosphorylation, bone-marrow reconstitution, T cell differentiation, LCMV infection","pmids":["21976681","22182253"],"confidence":"Medium","gaps":["Single Efnb1 KO showed no phenotype, implying redundancy","Direct biochemical link between EFNB1 and IL-6 receptor signaling not established"]},{"year":2013,"claim":"Demonstrating cell sorting/clustering of ephrinB1-overexpressing cells and gene-dosage effects provided direct mechanistic support that cellular interference, not simple loss, drives the craniofacial phenotype.","evidence":"Xenopus cell sorting/clustering assays; array-CGH duplication analysis with mouse EFNB1 cDNA model; dual-luciferase uORF reporter assays in mosaic males","pmids":["23614707","21542058","23335590"],"confidence":"High","gaps":["Molecular signal driving sorting between EFNB1-high and EFNB1-low cells not identified","Tissue-level boundary mechanics not directly imaged in human suture"]},{"year":2013,"claim":"Identifying an EFNB1-EGFR complex that sustains ERK signaling under cetuximab implicated EFNB1 in receptor tyrosine kinase crosstalk and tumor growth.","evidence":"Co-IP, signaling assays, EFNB1 knockdown, murine HNSCC tumor model","pmids":["23811940"],"confidence":"Medium","gaps":["Structural basis of EGFR-EFNB1 association not defined here","Single-lab in vivo model"]},{"year":2018,"claim":"Establishing that stromal EFNB1 reverse signaling activates CXCL12 to maintain hematopoietic stem/progenitor cells extended its reverse-signaling role into niche biology.","evidence":"Osterix-Cre conditional EfnB1 KO; LTC-IC and colony-formation assays; EFNB1-Fc stimulation; EPHB1/EPHB2 blocking antibodies","pmids":["30326247"],"confidence":"Medium","gaps":["Mechanism linking reverse signaling to CXCL12 transcription unknown","Single-lab study"]},{"year":2020,"claim":"Showing that EFNB1 engagement drives RNF186-dependent ubiquitination of EPHB2 at Lys892 to recruit MAP1LC3B established a forward-signaling axis triggering autophagy and intestinal homeostasis.","evidence":"Co-IP, K892R mutagenesis, ubiquitination and autophagy assays, rnf186/ephb2 KO mice, DSS colitis model, EFNB1-Fc","pmids":["33280498"],"confidence":"High","gaps":["How ubiquitinated EPHB2 nucleates autophagosome formation not detailed","Generalizability beyond colonic epithelium untested"]},{"year":2020,"claim":"Demonstrating that EFNB1, EPHB2, NMDA receptor, and presenilin-1 form de novo neuroprotective survival complexes connected EFNB1 signaling to excitotoxicity resistance and Alzheimer-relevant biology.","evidence":"Co-IP, peptide disruption, survival assays, NMDAR-EPSC electrophysiology, post-mortem AD brain analysis","pmids":["33005890"],"confidence":"Medium","gaps":["Stoichiometry and assembly order of the complex unresolved","Single-lab Co-IP-based complex"]},{"year":2024,"claim":"Identifying platelet EFNB1 reverse signaling through AKT, activated by tumor EPHB1, in driving serotonin release and metastatic growth extended reverse signaling into the platelet-tumor axis.","evidence":"Metastatic niche labeling, platelet depletion, EPHB1 gain/loss-of-function, recombinant protein, Tph1-KO mice, AKT signaling","pmids":["39648610"],"confidence":"Medium","gaps":["Direct effector linking EFNB1 tail to AKT in platelets not defined","Single-lab model"]},{"year":2025,"claim":"Demonstrating PBM-dependent EFNB1-EGFR association and direct EGFR phosphorylation of EFNB1's PBM that disrupts the complex and controls fibronectin adhesion defined a regulatory phospho-switch governing EFNB1 adhesive function.","evidence":"BioID proximity proteomics, Co-IP, in vitro kinase assay, PBM mutants, fibronectin adhesion assay","pmids":["40619000"],"confidence":"High","gaps":["Physiological/developmental context of EGFR-EFNB1 regulation unknown","Identity of adhesion machinery downstream of the PBM not resolved"]},{"year":2025,"claim":"Showing that dengue NS1 binds endothelial EFNB1 and requires EFNB1 phosphorylation to trigger barrier dysfunction implicated EFNB1 as a viral co-factor and decoy target.","evidence":"NS1 interactome mass spectrometry, interface mapping, phosphorylation and barrier-dysfunction assays, EFNB1-Fc decoys in vitro and in vivo (preprint)","pmids":["bio_10.1101_2025.11.19.689067"],"confidence":"Medium","gaps":["Preprint, not peer-reviewed","Signaling pathway downstream of NS1-EFNB1 engagement undefined"]},{"year":null,"claim":"The proximal intracellular effectors and signal-transduction logic connecting EFNB1 reverse signaling to its diverse outputs (RhoA, AKT, CXCL12 induction, neuroprotection) remain incompletely defined.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unifying biochemical mechanism links the EFNB1 tail to its varied downstream pathways","Structural basis of effector selection (GRIP1 vs EGFR vs AKT) unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0048018","term_label":"receptor ligand activity","supporting_discovery_ids":[0,1,17,19]},{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[7,16,20]},{"term_id":"GO:0098631","term_label":"cell adhesion mediator activity","supporting_discovery_ids":[1,21]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,1,21,22]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,1,7,17]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[17]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[2,3,10]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[3,19,20]}],"complexes":["EFNB1-EGFR complex","NMDAR-EPHB2-presenilin-1 survival complex"],"partners":["EPHB2","EGFR","GRIP1","RNF186","EPHB1","PSEN1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P98172","full_name":"Ephrin-B1","aliases":["EFL-3","ELK ligand","ELK-L","EPH-related receptor tyrosine kinase ligand 2","LERK-2"],"length_aa":346,"mass_kda":38.0,"function":"Cell surface transmembrane ligand for Eph receptors, a family of receptor tyrosine kinases which are crucial for migration, repulsion and adhesion during neuronal, vascular and epithelial development (PubMed:7973638, PubMed:8070404). Binding to Eph receptors residing on adjacent cells leads to contact-dependent bidirectional signaling into neighboring cells (PubMed:7973638, PubMed:8070404). Shows high affinity for the receptor tyrosine kinase EPHB1/ELK (PubMed:7973638, PubMed:8070404). Can also bind EPHB2 and EPHB3 (PubMed:8070404). Binds to, and induces collapse of, commissural axons/growth cones in vitro (By similarity). 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Culture Conditions","date":"2025-09-19","source":"bioRxiv","url":"https://doi.org/10.1101/2025.09.17.676510","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2024.07.09.602796","title":"Discovery and Repurposing of Multi-Target Senolytics through Structure-Based Virtual Screening","date":"2024-07-14","source":"bioRxiv","url":"https://doi.org/10.1101/2024.07.09.602796","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":32187,"output_tokens":6364,"usd":0.09601,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":15118,"output_tokens":5137,"usd":0.102008,"stage2_stop_reason":"end_turn"},"total_usd":0.198018,"stage1_batch_id":"msgbatch_01AsR7g8dGTEiEw9U83bJeWn","stage2_batch_id":"msgbatch_01YV5ZyxtFNUZEjxRcpd9jzb","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1995,\n      \"finding\": \"LERK-2 (EFNB1) is a membrane-bound ligand that signals through three different Eph-related receptor tyrosine kinases: Cek5, Cek10, and Elk, but fails to interact functionally with Cek9. Preliminary mutagenesis suggested a negative regulatory role for the LERK-2 cytoplasmic domain in LERK-2 signaling.\",\n      \"method\": \"Chimeric receptor constructs (Eph ectodomain + TrkB cytoplasmic domain) expressed in NIH 3T3 cells; focus formation assays; binding assays; mutagenesis\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with chimeric receptors, binding assays, and mutagenesis; receptor selectivity demonstrated across multiple receptors\",\n      \"pmids\": [\"7621826\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"Membrane-bound LERK-2 (EFNB1) binds HEK2 (an EPH-family receptor) and signals through it, inducing receptor phosphorylation and cell-cell adhesion/aggregate formation. Soluble LERK-2 can also signal through HEK2. Co-expression of HEK2 and LERK-2 in the same cell reduces kinase activity and autophosphorylation compared to juxtacrine stimulation.\",\n      \"method\": \"Expression of HEK2 in 32D cells; coincubation assays; receptor phosphorylation assays; cell aggregation assays; inhibition with soluble LERK-2\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — direct receptor phosphorylation assay and cell adhesion functional readout with multiple orthogonal approaches in a single study\",\n      \"pmids\": [\"8798744\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"ELK receptor and its ligand LERK-2 (EFNB1) are co-expressed on human renal microvascular endothelial cells (HRMEC). LERK-2 (but not LERK-1/B61) promotes in vitro assembly of HRMEC into capillary-like structures, demonstrating receptor-ligand specificity in endothelial morphogenesis.\",\n      \"method\": \"In vitro capillary-like assembly assay comparing HRMEC and HUVEC responses to LERK-1 vs LERK-2; immunohistochemistry of developing kidney\",\n      \"journal\": \"Kidney international. Supplement\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional in vitro assembly assay with receptor-ligand specificity comparison, single lab\",\n      \"pmids\": [\"8941926\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Heterozygous loss-of-function mutations in EFNB1 cause craniofrontonasal syndrome (CFNS). The more severe female phenotype is proposed to result from patchwork loss of ephrin-B1 due to X-inactivation, disturbing tissue boundary formation at the developing coronal suture. EFNB1 is X-inactivated but X-inactivation is not markedly skewed in blood or cranial periosteum of CFNS females.\",\n      \"method\": \"Mutation analysis (sequencing); X-inactivation analysis in patient tissues; in situ expression analysis in mouse embryos\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (sequencing, X-inactivation analysis, in situ expression); replicated by multiple subsequent studies\",\n      \"pmids\": [\"15166289\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"The majority of CFNS-causing EFNB1 mutations (26/33) are located in exons 2 and 3 encoding the extracellular ephrin domain. Frameshift mutations in the last 25 codons disrupt intracellular binding sites for Grb4 and PDZ-effector proteins involved in reverse signaling, demonstrating that ephrin-B1 reverse signaling is mechanistically important.\",\n      \"method\": \"DNA sequencing of EFNB1 in CFNS patients; mutation spectrum analysis\",\n      \"journal\": \"Human mutation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Strong — sequencing-based mutation mapping across large patient cohort identifies functional domain requirements, but no direct biochemical assay of reverse signaling\",\n      \"pmids\": [\"15959873\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"EFNB1 missense mutations in CFNS patients disrupt Eph receptor-ephrin-B1 interactions at the ligand-receptor dimerization interface (G-H loop). The P54L mutation prevents Eph-receptor-mediated cell cluster formation and abolishes intracellular ephrin-B1 Tyr324 and Tyr329 phosphorylation. The T111I mutation reduces Tyr324/Tyr329 phosphorylation. Splice-site mutation c.406+2T>C causes intron retention or cryptic splice site activation. Frameshift c.614_615delCT escapes nonsense-mediated decay but does not produce soluble ephrin-B1 protein.\",\n      \"method\": \"RT-PCR; Western blot; time-lapse microscopy; cell culture transfection of NIH 3T3 cells with site-directed mutants; phosphorylation analysis\",\n      \"journal\": \"BMC medical genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — multiple orthogonal methods (Western blot, phosphorylation assay, time-lapse microscopy, RT-PCR) on patient-derived and engineered mutations; functional consequences directly measured\",\n      \"pmids\": [\"20565770\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"EFNB1 mutations can result in differential degradation by the nonsense-mediated mRNA decay (NMD) pathway: splice-site mutation c.407-2A>T and frameshift c.377_384delTCAAGAAG show severe transcript depletion, while c.614_615delCT (premature termination codon near 3' end of penultimate exon) escapes NMD, consistent with the '50-55 bp' rule. Clonal expansion of patient fibroblasts confirmed cellular mosaicism for wild-type vs. mutant EFNB1-expressing cells.\",\n      \"method\": \"RT-PCR on fibroblast cultures from CFNS females; quantitative mRNA analysis; clonal expansion of patient-derived cells\",\n      \"journal\": \"European journal of human genetics : EJHG\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct mRNA quantification in patient fibroblasts with clonal expansion; single lab, multiple mutations analyzed\",\n      \"pmids\": [\"18043713\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Ephrin-B1 (EFNB1) reverse signaling reduces RhoA activation and vascular smooth muscle cell (VSMC) contractility in vitro. In mesenteric arteries from smooth muscle-specific Efnb1 knockout mice, RhoA activity and myosin light chain phosphorylation were increased. Knockdown of GRIP1 (associated with Efnb1 intracellular tail) partially eliminated Efnb1's effect on VSMC contractility. Efnb1 KO mice on high-salt diet showed heightened blood pressure increments.\",\n      \"method\": \"Conditional Efnb1 knockout mice; siRNA knockdown of Grip1; RhoA activity assay; myosin light chain phosphorylation assay; ex vivo artery analysis; blood pressure measurement\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (KO mice, ex vivo assay, siRNA, phosphorylation assay, in vivo BP measurement) in a single study establishing reverse signaling through GRIP1 controlling RhoA/VSMC contractility\",\n      \"pmids\": [\"22393061\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Double conditional knockout of both Efnb1 and Efnb2 in T cells compromised IL-6 signaling, specifically abating STAT3 phosphorylation upon IL-6 stimulation, contributing to defects in T cell development, homeostatic expansion, and Th1/Th17 differentiation.\",\n      \"method\": \"loxP-mediated conditional double knockout mice; STAT3 phosphorylation assay; competitive bone marrow reconstitution; in vitro T cell differentiation assays; LCMV infection model\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KO with defined cellular phenotype and downstream signaling readout; note that single Efnb1 KO showed no phenotype (redundancy), so dual KO required\",\n      \"pmids\": [\"21976681\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Single T cell-specific EFNB1 knockout mice showed no significant defects in T cell development, activation, proliferation, or differentiation into Th1, Th2, Th17 or Treg cells, indicating functional redundancy with other ephrin family members in T cells.\",\n      \"method\": \"T cell-specific EFNB1 conditional knockout; flow cytometry of thymus/spleen populations; competitive bone marrow reconstitution; T cell differentiation assays\",\n      \"journal\": \"BMC immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — clean KO with defined negative result across multiple T cell readouts; single lab\",\n      \"pmids\": [\"22182253\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"In Xenopus tissue, cells overexpressing ephrinB1 cluster and sort out from wild-type cells, providing in vitro mechanistic evidence that cellular interference (due to mosaic EFNB1 expression) drives the CFNS phenotype in heterozygous females.\",\n      \"method\": \"In vitro cell sorting/clustering experiments in Xenopus tissue with overexpression of ephrinB1\",\n      \"journal\": \"Clinical genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct experimental demonstration of cell sorting in a model system; supports the cellular interference mechanism proposed from human genetics\",\n      \"pmids\": [\"23614707\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Duplication of EFNB1 produces ~1.6-fold more EFNB1 transcript from the duplicated X chromosome. In a mouse model carrying targeted human EFNB1 cDNA, abnormal cell sorting occurs in the cranial region, demonstrating that cellular mosaicism for different levels of ephrin-B1 (not just presence/absence) causes craniofacial abnormalities.\",\n      \"method\": \"Array-CGH; quantitative mRNA analysis; mouse model with targeted EFNB1 cDNA; cell sorting analysis in cranial region\",\n      \"journal\": \"Human mutation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mouse model with direct cell sorting observation; mRNA quantification confirming dosage imbalance; single lab\",\n      \"pmids\": [\"21542058\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Males mosaic for EFNB1 mutations are more severely affected than constitutionally hemizygous males. A 5' UTR variant mutating the stop codon of a small upstream open reading frame (uORF) was demonstrated using a dual-luciferase reporter construct to exacerbate interference with translation of the wild-type EFNB1 protein.\",\n      \"method\": \"Denaturing HPLC; massively parallel sequencing; MLPA; Pyrosequencing; dual-luciferase reporter assay for 5' UTR uORF translation interference\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — functional luciferase assay directly demonstrating translational regulation by uORF; multiple orthogonal detection methods for mosaicism; replicated mechanism across 6 cases\",\n      \"pmids\": [\"23335590\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"EFNB1 forms a novel complex with EGFR in head and neck squamous cell carcinoma cells. Cetuximab (anti-EGFR) treatment drives a shift in EGFR dimerization partners toward EFNB1, and EFNB1 phosphorylation and downstream ERK signaling persist in the presence of cetuximab. EFNB1 attenuation slowed tumor growth and increased survival in a murine HNSCC model.\",\n      \"method\": \"Co-immunoprecipitation; signaling assays; EFNB1 knockdown; murine tumor model\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP demonstrating EGFR-EFNB1 complex; in vivo tumor model; single lab, mechanistic follow-up partially detailed in abstract\",\n      \"pmids\": [\"23811940\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Deletion mutation of Efnb1 and Efnb2 intracellular tails revealed critical regions in controlling T cell chemotaxis toward CXCL12. T cells from dKO mice were compromised in migration to arthritic paws in vivo and in chemotaxis in vitro.\",\n      \"method\": \"Double conditional KO mice; intracellular tail deletion mutants; in vitro chemotaxis assay (CXCL12); in vivo migration to arthritic paws\",\n      \"journal\": \"Arthritis & rheumatology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain-specific deletion mutants identifying intracellular tail requirement for T cell chemotaxis; in vitro and in vivo confirmation; single lab\",\n      \"pmids\": [\"25779027\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"A novel uATG variant in the 5' UTR of EFNB1 (c.-411) creates a new upstream ATG that significantly reduces EFNB1 protein translation without affecting mRNA levels, as demonstrated by dual-luciferase reporter assays.\",\n      \"method\": \"Dual-luciferase reporter assay; mRNA quantification\",\n      \"journal\": \"Molecular syndromology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — direct functional reporter assay demonstrating translational repression by uATG; single lab, single method\",\n      \"pmids\": [\"30976278\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"EfnB1 in osteogenic lineage cells supports hematopoietic stem/progenitor cell (HSPC) maintenance. EFNB1 reverse signaling in stromal cells is required for activation of the HSPC-promoting factor CXCL12. Activation of EPHB2 on CD34+ HSPCs by EFNB1-Fc stimulation enhanced myeloid/erythroid colony formation, while blocking EPHB1 or EPHB2 inhibited LTC-IC maintenance.\",\n      \"method\": \"Conditional EfnB1 knockout in osteogenic lineage (Osterix-Cre); LTC-IC assays; colony formation assays; EFNB1-Fc stimulation; functional blocking antibodies against EPHB1/EPHB2; CXCL12 activation assays\",\n      \"journal\": \"Experimental hematology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional KO with defined cellular phenotype, functional blocking experiments, and reverse signaling readout (CXCL12 activation); single lab\",\n      \"pmids\": [\"30326247\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Upon stimulation by EFNB1 (ligand), EPHB2 receptor is ubiquitinated by the E3 ubiquitin ligase RNF186 at Lys892. This ubiquitination event recruits MAP1LC3B to trigger autophagy in colonic epithelial cells, contributing to intestinal homeostasis.\",\n      \"method\": \"Co-IP; site-directed mutagenesis (Lys892Arg EPHB2 mutant); EPHB2 ubiquitination assay; autophagy assays (MAP1LC3B recruitment); rnf186 and ephb2 knockout mice; DSS-induced colitis model; EFNB1-Fc recombinant protein treatment\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — site-directed mutagenesis identifying specific ubiquitination site, multiple orthogonal methods (Co-IP, ubiquitination assay, KO mice, in vivo model), functional consequence demonstrated\",\n      \"pmids\": [\"33280498\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"EFNB1 and BDNF-dependent neuroprotection against excitotoxicity requires de novo formation of 'survival complexes' consisting of NMDA receptor, EFNB1's receptor EPHB2, and presenilin 1. Absence of presenilin 1 reduces survival complex formation and abolishes neuroprotection. Familial Alzheimer disease presenilin 1 mutants decrease factor-stimulated survival complex formation and neuroprotection.\",\n      \"method\": \"Co-immunoprecipitation; EPHB2- and NMDA receptor-derived peptides disrupting complex formation; survival assays; electrophysiology (NMDA receptor-mediated EPSCs); analysis of post-mortem Alzheimer brain samples\",\n      \"journal\": \"Brain communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP demonstrating complex formation, peptide disruption experiments, multiple readouts; single lab\",\n      \"pmids\": [\"33005890\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"EFNB1-EPHB2 interaction activates downstream Wnt/β-catenin and FAK signaling pathways to promote gastric cancer cell invasion and migration. Fc-EFNB1 treatment increased invasion/migration abilities and induced high EPHB2 expression. EPHB2 knockdown completely abolished ephrin ligand-induced effects.\",\n      \"method\": \"Patient-derived xenografts; PCR array; gain/loss-of-function experiments; Fc-EFNB1 treatment; EPHB2 knockdown; signaling analysis (Wnt/β-catenin, FAK)\",\n      \"journal\": \"International journal of biological macromolecules\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function with defined signaling readouts and rescue; single lab\",\n      \"pmids\": [\"38114003\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Tumor cell-expressed EPHB1 and platelet-expressed EFNB1 mediate contact-dependent activation of platelets via EFNB1 reverse signaling-mediated AKT signaling activation. Activated platelets then release 5-HT (serotonin) which further enhances tumor growth in the liver metastatic niche of pancreatic ductal adenocarcinoma.\",\n      \"method\": \"mCherry metastatic niche-labeling system; platelet depletion in liver metastasis mouse model; gain/loss-of-function of EPHB1; recombinant protein treatment; Tph1-knockout mice; tumor-platelet adhesion assays; AKT signaling analysis\",\n      \"journal\": \"Cancer communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (KO mice, gain/loss-of-function, recombinant protein) establishing EFNB1 reverse signaling through AKT; single lab\",\n      \"pmids\": [\"39648610\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"EFNB1 physically associates with EGFR in cells, and this complex formation depends on EFNB1's PDZ-binding motif (PBM). EGFR directly phosphorylates tyrosine residues within EFNB1's PBM, which disrupts the EFNB1-EGFR complex. The EFNB1-EGFR association is required for EFNB1-dependent cell adhesion to fibronectin.\",\n      \"method\": \"Proximity labeling proteomics (BioID); Co-IP; in vitro kinase assay demonstrating direct EGFR phosphorylation of EFNB1; PBM mutants; cell adhesion assay to fibronectin\",\n      \"journal\": \"Molecular & cellular proteomics : MCP\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase reconstitution (direct phosphorylation), proximity proteomics, Co-IP, mutagenesis of PBM, functional adhesion readout; multiple orthogonal methods in single study\",\n      \"pmids\": [\"40619000\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Dengue virus NS1 protein binds EFNB1 at the endothelial cell surface to trigger endothelial barrier dysfunction. Phosphorylation of EFNB1 is necessary for NS1-induced barrier dysfunction. EFNB1-Fc fusion proteins act as decoys to block NS1-induced barrier dysfunction in vitro and in vivo.\",\n      \"method\": \"Comparative mass spectrometry (NS1 interactome); biochemical and computational interface mapping of EFNB1-NS1 complex; phosphorylation assays; barrier dysfunction assays in vitro and in vivo with EFNB1-Fc decoys\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (MS interactome, biochemical complex mapping, phosphorylation assay, functional in vivo decoy experiment); preprint, not peer-reviewed\",\n      \"pmids\": [\"bio_10.1101_2025.11.19.689067\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"EFNB1 (ephrin-B1) is a transmembrane ligand for Eph receptor tyrosine kinases that mediates bidirectional signaling: in forward signaling it activates Eph receptors (including EphB2, ELK/EphB4, and others) to promote cell-cell adhesion, endothelial morphogenesis, tissue boundary formation, and autophagy (via RNF186-mediated ubiquitination of EPHB2 at Lys892 recruiting MAP1LC3B); in reverse signaling through its intracellular domain (involving PDZ-binding motif interactions with GRIP1 and other effectors, and phosphorylation of Tyr324/Tyr329), it regulates RhoA activity and vascular smooth muscle contractility, T cell chemotaxis, CXCL12 production in stromal cells, and neuroprotection via NMDA receptor/presenilin-1 survival complexes; additionally, EFNB1 associates with EGFR (dependent on its PBM) and is directly phosphorylated by EGFR within the PBM to control cell adhesion to fibronectin, and its mosaic loss due to X-inactivation in heterozygous females causes craniofrontonasal syndrome through cellular interference and abnormal tissue boundary formation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"EFNB1 (ephrin-B1) is a membrane-bound ligand for Eph-family receptor tyrosine kinases that mediates bidirectional cell-cell signaling to control tissue boundary formation, endothelial morphogenesis, and cellular adhesion [#0, #2]. In forward signaling it binds and activates multiple Eph receptors — including EphB2/HEK2, ELK, and others — inducing receptor autophosphorylation and driving cell-cell adhesion and aggregate formation, with juxtacrine presentation being more potent than soluble ligand [#0, #1]. Through EphB2 specifically, EFNB1 engagement triggers RNF186-mediated ubiquitination of EPHB2 at Lys892, which recruits MAP1LC3B to initiate autophagy and maintain intestinal epithelial homeostasis [#17], and activates downstream Wnt/\\u03b2-catenin and FAK signaling [#19]. EFNB1 reverse signaling through its intracellular tail and PDZ-binding motif acts via GRIP1 to suppress RhoA activation and vascular smooth muscle contractility [#7], promotes CXCL12 production by osteogenic stromal cells to support hematopoietic stem/progenitor maintenance [#16], and directs T cell chemotaxis toward CXCL12 [#14]. EFNB1 also forms a PDZ-binding-motif-dependent complex with EGFR, and direct EGFR phosphorylation of tyrosines within this motif disrupts the complex and controls EFNB1-dependent cell adhesion to fibronectin [#21, #13]. Heterozygous loss-of-function mutations in EFNB1 cause craniofrontonasal syndrome, where mosaic ephrin-B1 expression arising from X-inactivation produces aberrant cell sorting and disrupted coronal-suture boundary formation [#3, #10].\",\n  \"teleology\": [\n    {\n      \"year\": 1995,\n      \"claim\": \"Establishing that the membrane-bound ligand EFNB1 functionally engages a defined subset of Eph receptors answered which receptors transduce its signal and revealed receptor selectivity.\",\n      \"evidence\": \"Chimeric Eph-TrkB receptor constructs in NIH 3T3 cells with focus-formation and binding assays\",\n      \"pmids\": [\"7621826\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cytoplasmic-domain regulatory role only preliminarily mapped\", \"Downstream effectors of receptor activation not identified\"]\n    },\n    {\n      \"year\": 1996,\n      \"claim\": \"Demonstrating that EFNB1 binding induces Eph receptor phosphorylation and cell aggregation, and that co-expression dampens kinase activity, defined the forward-signaling output and the importance of juxtacrine presentation.\",\n      \"evidence\": \"HEK2 expression in 32D cells; receptor phosphorylation and cell aggregation assays; soluble-ligand competition\",\n      \"pmids\": [\"8798744\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of cis-inhibition by co-expression not resolved\", \"Physiological cell context not addressed\"]\n    },\n    {\n      \"year\": 1996,\n      \"claim\": \"Showing ligand-specific promotion of endothelial capillary-like assembly tied EFNB1-Eph signaling to a tissue morphogenetic process.\",\n      \"evidence\": \"In vitro capillary-assembly assays in renal microvascular endothelial cells comparing LERK-1 vs LERK-2\",\n      \"pmids\": [\"8941926\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab in vitro assay\", \"Molecular mediators of morphogenesis not defined\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Linking heterozygous EFNB1 loss-of-function to craniofrontonasal syndrome and the paradoxically severe female phenotype established the X-inactivation/mosaic-interference disease model.\",\n      \"evidence\": \"Mutation sequencing, X-inactivation analysis in patient tissues, and mouse embryo in situ expression\",\n      \"pmids\": [\"15166289\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct mechanistic test of cellular interference not yet performed in this study\", \"X-inactivation skewing not detected as explanation\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Mapping the CFNS mutation spectrum to the extracellular ephrin domain and the reverse-signaling intracellular sites established that both ligand-receptor binding and reverse signaling are mechanistically required.\",\n      \"evidence\": \"DNA sequencing and mutation-spectrum analysis across a CFNS cohort\",\n      \"pmids\": [\"15959873\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No direct biochemical assay of reverse signaling in this study\", \"Genotype-phenotype correlation incomplete\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Demonstrating that specific CFNS missense mutations disrupt receptor binding and abolish intracellular Tyr324/Tyr329 phosphorylation, and that some alleles escape nonsense-mediated decay, linked mutation class to molecular consequence.\",\n      \"evidence\": \"Site-directed mutants in NIH 3T3 cells with Western blot, phosphorylation assays, time-lapse microscopy, RT-PCR; patient-fibroblast clonal expansion\",\n      \"pmids\": [\"20565770\", \"18043713\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream signaling consequences of lost Tyr phosphorylation not traced\", \"Quantitative threshold of mosaicism for phenotype unknown\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Showing that EFNB1 reverse signaling suppresses RhoA and smooth muscle contractility through GRIP1 defined a concrete reverse-signaling effector pathway with physiological output.\",\n      \"evidence\": \"Smooth-muscle-specific Efnb1 KO mice, Grip1 siRNA, RhoA and MLC phosphorylation assays, ex vivo arteries, blood pressure measurement\",\n      \"pmids\": [\"22393061\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct GRIP1-RhoA biochemical link not fully resolved\", \"Relevance to human vascular disease untested\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Defining EFNB1/EFNB2 roles in T cell IL-6/STAT3 signaling and the redundancy revealed by single versus double knockouts clarified its immunological function.\",\n      \"evidence\": \"Conditional single and double KO mice; STAT3 phosphorylation, bone-marrow reconstitution, T cell differentiation, LCMV infection\",\n      \"pmids\": [\"21976681\", \"22182253\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single Efnb1 KO showed no phenotype, implying redundancy\", \"Direct biochemical link between EFNB1 and IL-6 receptor signaling not established\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Demonstrating cell sorting/clustering of ephrinB1-overexpressing cells and gene-dosage effects provided direct mechanistic support that cellular interference, not simple loss, drives the craniofacial phenotype.\",\n      \"evidence\": \"Xenopus cell sorting/clustering assays; array-CGH duplication analysis with mouse EFNB1 cDNA model; dual-luciferase uORF reporter assays in mosaic males\",\n      \"pmids\": [\"23614707\", \"21542058\", \"23335590\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular signal driving sorting between EFNB1-high and EFNB1-low cells not identified\", \"Tissue-level boundary mechanics not directly imaged in human suture\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Identifying an EFNB1-EGFR complex that sustains ERK signaling under cetuximab implicated EFNB1 in receptor tyrosine kinase crosstalk and tumor growth.\",\n      \"evidence\": \"Co-IP, signaling assays, EFNB1 knockdown, murine HNSCC tumor model\",\n      \"pmids\": [\"23811940\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural basis of EGFR-EFNB1 association not defined here\", \"Single-lab in vivo model\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Establishing that stromal EFNB1 reverse signaling activates CXCL12 to maintain hematopoietic stem/progenitor cells extended its reverse-signaling role into niche biology.\",\n      \"evidence\": \"Osterix-Cre conditional EfnB1 KO; LTC-IC and colony-formation assays; EFNB1-Fc stimulation; EPHB1/EPHB2 blocking antibodies\",\n      \"pmids\": [\"30326247\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism linking reverse signaling to CXCL12 transcription unknown\", \"Single-lab study\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Showing that EFNB1 engagement drives RNF186-dependent ubiquitination of EPHB2 at Lys892 to recruit MAP1LC3B established a forward-signaling axis triggering autophagy and intestinal homeostasis.\",\n      \"evidence\": \"Co-IP, K892R mutagenesis, ubiquitination and autophagy assays, rnf186/ephb2 KO mice, DSS colitis model, EFNB1-Fc\",\n      \"pmids\": [\"33280498\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How ubiquitinated EPHB2 nucleates autophagosome formation not detailed\", \"Generalizability beyond colonic epithelium untested\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Demonstrating that EFNB1, EPHB2, NMDA receptor, and presenilin-1 form de novo neuroprotective survival complexes connected EFNB1 signaling to excitotoxicity resistance and Alzheimer-relevant biology.\",\n      \"evidence\": \"Co-IP, peptide disruption, survival assays, NMDAR-EPSC electrophysiology, post-mortem AD brain analysis\",\n      \"pmids\": [\"33005890\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Stoichiometry and assembly order of the complex unresolved\", \"Single-lab Co-IP-based complex\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Identifying platelet EFNB1 reverse signaling through AKT, activated by tumor EPHB1, in driving serotonin release and metastatic growth extended reverse signaling into the platelet-tumor axis.\",\n      \"evidence\": \"Metastatic niche labeling, platelet depletion, EPHB1 gain/loss-of-function, recombinant protein, Tph1-KO mice, AKT signaling\",\n      \"pmids\": [\"39648610\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct effector linking EFNB1 tail to AKT in platelets not defined\", \"Single-lab model\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Demonstrating PBM-dependent EFNB1-EGFR association and direct EGFR phosphorylation of EFNB1's PBM that disrupts the complex and controls fibronectin adhesion defined a regulatory phospho-switch governing EFNB1 adhesive function.\",\n      \"evidence\": \"BioID proximity proteomics, Co-IP, in vitro kinase assay, PBM mutants, fibronectin adhesion assay\",\n      \"pmids\": [\"40619000\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological/developmental context of EGFR-EFNB1 regulation unknown\", \"Identity of adhesion machinery downstream of the PBM not resolved\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Showing that dengue NS1 binds endothelial EFNB1 and requires EFNB1 phosphorylation to trigger barrier dysfunction implicated EFNB1 as a viral co-factor and decoy target.\",\n      \"evidence\": \"NS1 interactome mass spectrometry, interface mapping, phosphorylation and barrier-dysfunction assays, EFNB1-Fc decoys in vitro and in vivo (preprint)\",\n      \"pmids\": [\"bio_10.1101_2025.11.19.689067\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Preprint, not peer-reviewed\", \"Signaling pathway downstream of NS1-EFNB1 engagement undefined\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The proximal intracellular effectors and signal-transduction logic connecting EFNB1 reverse signaling to its diverse outputs (RhoA, AKT, CXCL12 induction, neuroprotection) remain incompletely defined.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unifying biochemical mechanism links the EFNB1 tail to its varied downstream pathways\", \"Structural basis of effector selection (GRIP1 vs EGFR vs AKT) unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0048018\", \"supporting_discovery_ids\": [0, 1, 17, 19]},\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [7, 16, 20]},\n      {\"term_id\": \"GO:0098631\", \"supporting_discovery_ids\": [1, 21]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 1, 21, 22]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 1, 7, 17]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [17]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [2, 3, 10]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [3, 19, 20]}\n    ],\n    \"complexes\": [\n      \"EFNB1-EGFR complex\",\n      \"NMDAR-EPHB2-presenilin-1 survival complex\"\n    ],\n    \"partners\": [\n      \"EPHB2\",\n      \"EGFR\",\n      \"GRIP1\",\n      \"RNF186\",\n      \"EPHB1\",\n      \"PSEN1\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}