{"gene":"EPHB2","run_date":"2026-06-09T23:54:43","timeline":{"discoveries":[{"year":2001,"finding":"Crystal structure of the autoinhibited, unphosphorylated EphB2 kinase domain (with juxtamembrane region) at 1.9 Å resolution reveals that the juxtamembrane segment adopts a helical conformation that distorts the small lobe of the kinase domain and blocks the activation segment, providing structural autoinhibition. Phosphorylation of conserved juxtamembrane tyrosines relieves this autoinhibition by disrupting the JM-kinase interface and liberating phosphotyrosine sites for SH2-domain binding.","method":"X-ray crystallography + site-directed mutagenesis","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure at 1.9 Å combined with mutagenesis validation in a single rigorous study","pmids":["11572780"],"is_preprint":false},{"year":1998,"finding":"Crystal structure of the EphB2 ligand-binding domain at 2.9 Å resolution reveals a jellyroll beta-sandwich of 11 antiparallel beta-strands. Structure-based mutagenesis identified an extended loop critical for ligand binding and subclass specificity, localizing the ligand-binding surface to the concave beta-sandwich near positions where missense mutations cause signaling defects.","method":"X-ray crystallography + structure-based mutagenesis","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure combined with mutagenesis in a single focused study","pmids":["9853759"],"is_preprint":false},{"year":2004,"finding":"EphB2 and ephrin-A5 (a cross-subclass pair) bind each other with high affinity, triggering receptor clustering, autophosphorylation, and downstream signaling. Ephrin-A5 induces EphB2-mediated growth cone collapse and neurite retraction. Crystal structure reveals a heterodimeric complex architecturally distinct from the tetrameric EphB2-ephrinB2 structure, providing a molecular basis for cross-subclass signaling.","method":"X-ray crystallography, cell-based signaling assays (receptor clustering, autophosphorylation), growth cone collapse assay","journal":"Nature neuroscience","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure plus multiple orthogonal functional assays in one study","pmids":["15107857"],"is_preprint":false},{"year":1998,"finding":"Phosphorylated tyrosine 611 in the juxtamembrane region of EphB2 is a major autophosphorylation site and is required for interaction with the SH2 domain of Src kinase. Tyrosines 605 and 611 are important for EphB2 kinase activity. EphB2 and Src physically associate (co-immunoprecipitation), and activated EphB2 induces increased tyrosine phosphorylation of Src.","method":"Yeast two-hybrid, site-directed mutagenesis, co-immunoprecipitation, phosphopeptide mapping","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — multiple orthogonal methods (Y2H + mutagenesis + Co-IP + phosphopeptide mapping) in one study","pmids":["9632142"],"is_preprint":false},{"year":2001,"finding":"Abl and Arg non-receptor tyrosine kinases associate with EphB2 through multiple distinct interactions: (1) their SH2 domains bind phosphorylated juxtamembrane tyrosines of EphB2; (2) a phosphorylation-independent interaction involves C-terminal sequences of Abl/Arg; (3) a third interaction requires EphB2 phosphorylation but not direct SH2 binding. Activated EphB2 phosphorylates Abl and Arg, and ephrin-B1 stimulation decreases endogenous Abl kinase activity.","method":"Yeast two-hybrid, co-immunoprecipitation, kinase activity assay, ephrin-B1 stimulation of endogenous EphB2","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP plus multiple interaction modes validated, single lab","pmids":["11494128"],"is_preprint":false},{"year":2001,"finding":"EphB2 null mice exhibit reduced NMDA receptor-mediated synaptic current and reduced synaptically localized NR1 subunit (immunogold) in dentate granule neurons. Mice lacking only the intracellular kinase domain retain wild-type LTP, indicating that kinase-independent EphB2 functions mediate certain synaptic roles, whereas the extracellular/transmembrane domain is required for NMDA receptor regulation.","method":"EphB2 knockout mouse, electrophysiology (LTP, NMDA currents), immunogold localization","journal":"Neuron","confidence":"High","confidence_rationale":"Tier 2 / Strong — KO mouse with electrophysiology + immunogold, replicated by companion paper (PMID 11754835)","pmids":["11754836"],"is_preprint":false},{"year":2001,"finding":"EphB2 kinase signaling is not required for protein synthesis-dependent LTP or for two forms of synaptic depression in hippocampal slices; a C-terminally truncated EphB2 (lacking kinase domain) rescues the EphB2 null phenotype, demonstrating kinase-independent regulation of synaptic plasticity. Eph receptor stimulation in cultured neurons modulates signaling pathways implicated in synaptic plasticity, suggesting cross-talk with NMDA receptor-activated pathways.","method":"EphB2 knockout mouse, targeted knock-in of truncated EphB2, LTP/LTD electrophysiology in hippocampal slices","journal":"Neuron","confidence":"High","confidence_rationale":"Tier 2 / Strong — knock-in rescue experiment with defined readout, replicated by companion paper (PMID 11754836)","pmids":["11754835"],"is_preprint":false},{"year":2010,"finding":"Amyloid-β oligomers bind to the fibronectin repeats domain of EphB2 and trigger EphB2 degradation via the proteasome. EphB2 knockdown by shRNA reduces NMDA receptor currents and impairs LTP in dentate gyrus; increasing EphB2 expression in dentate gyrus of hAPP transgenic mice reverses NMDA receptor-dependent LTP deficits and memory impairments.","method":"Biochemical binding assay (Aβ oligomers + EphB2), lentiviral shRNA knockdown and overexpression in vivo, electrophysiology, behavioral memory tests","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct binding demonstrated, bidirectional genetic manipulation with multiple functional readouts","pmids":["21113149"],"is_preprint":false},{"year":2011,"finding":"The serine protease neuropsin cleaves EphB2 in the amygdala upon stress, causing dissociation of EphB2 from the NR1 subunit of the NMDA receptor and promoting membrane turnover of EphB2 receptors. Dynamic EphB2-NR1 interaction enhances NMDA receptor current, induces Fkbp5 gene expression, and increases anxiety-like behaviour. Neuropsin-deficient mice lack EphB2 cleavage and show attenuated anxiety.","method":"Neuropsin knockout mice, intra-amygdala injection of neuropsin/antibodies/shRNA, EphB2-NR1 co-immunoprecipitation, NMDA receptor current recordings, behavioural assays","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods in KO and rescue experiments with defined mechanistic pathway","pmids":["21508957"],"is_preprint":false},{"year":2007,"finding":"EphB2 receptor ectodomain is cleaved after residue 543; the remaining membrane fragment is then cleaved by presenilin-dependent γ-secretase after residue 569, releasing an intracellular peptide (EphB2/CTF2). Ephrin-B ligand-induced processing requires endocytosis and is sensitive to proteasomal (not metalloproteinase) inhibitors. Calcium influx/NMDA-induced processing is sensitive to GM6001 and ADAM10 inhibitors and occurs at the plasma membrane without endocytosis. Ligand stimulation also induces ubiquitination of EphB2.","method":"Pharmacological inhibitors, endocytosis blockade, biochemical cleavage mapping, ubiquitination assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal pharmacological and biochemical methods mapping distinct cleavage pathways","pmids":["17428795"],"is_preprint":false},{"year":2009,"finding":"The γ-secretase-derived intracellular peptide EphB2/CTF2 retains tyrosine kinase activity and directly phosphorylates NMDA receptor subunits (NR1, NR2) in cell lines and primary neuronal cultures, independently of Src kinases. A kinase-dead EphB2/CTF2 mutant lacks this activity. EphB2/CTF2 promotes cell-surface expression of NMDA receptors.","method":"In vitro kinase assay, Src inhibitors, kinase-dead mutant, cell-surface expression assay (primary neurons and cell lines)","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro kinase reconstitution plus mutagenesis and Src-inhibitor controls, single lab","pmids":["19661068"],"is_preprint":false},{"year":2002,"finding":"EphB2 recruits p120RasGAP via juxtamembrane (JM) binding sites, leading to down-regulation of the Ras-Erk MAPK pathway and neurite retraction. Elimination of RasGAP binding sites with addition of Grb2 binding sites within the kinase domain activates Erk MAPK upon ephrin-B1 stimulation. This confirms that EphB2-mediated neurite retraction correlates with Ras-Erk MAPK down-regulation mediated through RasGAP recruitment.","method":"Receptor mutagenesis/re-engineering, phospho-Erk immunoblotting, neurite retraction functional assay in NG108 neurons","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — systematic mutagenesis with structure-function correlation and functional readout","pmids":["12486127"],"is_preprint":false},{"year":2008,"finding":"MMP-2 and MMP-9 cleave EphB2 in its ectodomain upon ephrin-B2 binding in vitro and in vivo. MMP-specific inhibition or cleavage-resistant EphB2 ectodomain mutations prevent EphB2-mediated cell-cell repulsion in HEK293 cells and block ephrin-B1-induced growth cone withdrawal in hippocampal neurons. Expression of cleavable but not non-cleavable EphB2 rescues ephrin-B1-induced growth cone collapse in EphB-deficient neurons.","method":"MMP inhibitors, cleavage-resistant mutagenesis, cell repulsion assay, growth cone collapse assay in EphB-deficient neurons, in vitro and in vivo cleavage assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — mutagenesis plus rescue in KO neurons plus pharmacological inhibitors, multiple orthogonal methods","pmids":["18713744"],"is_preprint":false},{"year":2009,"finding":"EphB2 regulates intestinal progenitor cell migration and proliferation through two independent signaling pathways: kinase-independent, PI3K-mediated control of cell positioning/migration; and tyrosine kinase-dependent regulation of proliferation through an Abl-cyclin D1 pathway. These pathways dissociate during colon carcinoma progression, allowing continued proliferation with invasive growth.","method":"Genetic dissection with EphB2 mutants (kinase-dead, truncated), PI3K inhibitors, Abl inhibitors, cyclin D1 measurement, intestinal cell positioning assays","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 / Moderate — clean genetic dissection with multiple mutants, pharmacological validation, clear pathway placement","pmids":["19914164"],"is_preprint":false},{"year":2005,"finding":"Upon EphB2 activation, R-Ras associates with EphB2 receptor and becomes highly phosphorylated. siRNA depletion of R-Ras abrogates EphB2 effects on glioma cell adhesion, proliferation, and invasion. Anti-proliferative responses correlate with suppressed MAPK activity, identifying EphB2/R-Ras as a functionally relevant signaling axis.","method":"Co-immunoprecipitation, siRNA knockdown, invasion/adhesion/proliferation assays, MAPK activity assay","journal":"The American journal of pathology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus siRNA rescue with functional readout, single lab","pmids":["16049340"],"is_preprint":false},{"year":2008,"finding":"FGFR1 activation prevents EphB2-mediated cell segregation and repulsion by: (1) increasing basal EphB2 phosphorylation through down-regulation of the leukocyte common antigen-related (LAR) tyrosine phosphatase that dephosphorylates EphB2; and (2) inhibiting further EphB2 phosphorylation upon ephrinB1 stimulation via MAPK pathway-induced Sprouty genes. EphB2 normally activates the MAPK pathway, which feeds back positively to promote EphB2 activation.","method":"Phosphorylation analysis, LAR phosphatase knockdown, MAPK inhibitors, Sprouty expression, cell segregation/repulsion assays","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pharmacological and genetic dissection in cell-based assays, single lab","pmids":["19047466"],"is_preprint":false},{"year":2014,"finding":"EphB2 clustering determines repulsive signaling strength: small clusters (trimers/tetramers) are sufficient to induce cell collapse, with dimers being inactive. Extended EphB2 arrays are dispensable. The ratio of multimers to dimers within a cluster population determines collapse response strength. The C-terminal modules of EphB2 have negative regulatory effects on ephrin-induced clustering.","method":"Chemical dimerizers, fluorescence anisotropy in live cells, quantitative imaging, collapse response assays","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — quantitative live-cell imaging with chemical genetics, single lab","pmids":["24469634"],"is_preprint":false},{"year":2016,"finding":"Rac-dependent F-actin enrichment at sites of EphB2 internalization (not during vesicle trafficking) mediates trans-endocytosis. Systematic Rho GTPase depletion identified Rac subfamily and Tiam2 (Rac-specific GEF) as key components of EphB2 trans-endocytosis from opposing ephrinB+ cells, and this pathway is required for EphB2-stimulated contact repulsion.","method":"Live imaging, systematic siRNA screen of Rho GTPases, Tiam2 knockdown, trans-endocytosis assay, cell repulsion assay","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — systematic genetic screen plus functional validation, single lab","pmids":["27597758"],"is_preprint":false},{"year":2012,"finding":"EphB2 forward signaling mediates cortical growth cone collapse via Pak and Nck: kinase-active EphB2 binds Pak, promotes growth cone repulsion via Pak kinase activity, Pak-Nck binding, RhoA signaling and endocytosis. This occurs independently of Rac/Cdc42-GTP production after ephrinB treatment of cortical neurons.","method":"Co-immunoprecipitation (EphB2-Pak), dominant-negative constructs, endocytosis blockade, growth cone collapse assay in cortical neurons","journal":"Molecular and cellular neurosciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus functional rescue experiments with pathway inhibitors, single lab","pmids":["23147113"],"is_preprint":false},{"year":2017,"finding":"Optogenetic (OptoEphB2) photoactivation of EphB2 in dendrites of hippocampal neurons induces rapid actin polymerization and dynamic dendritic filopodial growth. Rac1 and Cdc42 inhibition does not abolish this effect; instead, Abl2/Arg kinase is required as a necessary effector for OptoEphB2-induced filopodia growth in dendrites.","method":"Optogenetic EphB2 activation, Rac1/Cdc42 inhibitors, Arg kinase knockdown/inhibition, live-cell actin imaging","journal":"Biology open","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — optogenetic spatial control plus pharmacological/genetic dissection, single lab","pmids":["29158322"],"is_preprint":false},{"year":2015,"finding":"EphB2 activation (by ephrinB1/Fc or overexpression plus ligand) induces tau dephosphorylation at multiple AD-associated sites through PI3K/Akt-mediated inhibition of GSK-3β. PI3K inhibition or GSK-3β upregulation abolishes this effect. Deletion of the tyrosine kinase domain eliminates EphB2 stimulation-induced GSK-3β inhibition and tau dephosphorylation, demonstrating kinase-dependence.","method":"Kinase-dead mutant, PI3K inhibitors, GSK-3β overexpression, phospho-tau immunoblotting in cell lines, primary neurons, and transgenic mice","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis plus pharmacological rescue, in vitro and in vivo, single lab","pmids":["26119563"],"is_preprint":false},{"year":2015,"finding":"The protective effect of EphB2 against Aβ-induced depletion of surface NMDA receptors depends on EphB2's PDZ-binding motif and the presence of neuronal activity, but not on its kinase activity. The GluA2 subunit of AMPA receptors associates with EphB2's PDZ-binding motif via PDZ domain-containing proteins and promotes NMDA receptor retention at the membrane.","method":"Pharmacological treatments, primary neuronal cultures expressing wild-type or PDZ-motif/kinase-dead EphB2 mutants, biochemical co-association assays","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — structure-function mutagenesis with defined cellular readout, single lab","pmids":["26589795"],"is_preprint":false},{"year":2018,"finding":"Photoactivation of OptoEphB2 (kinase-active) during fear conditioning (but not minutes afterward) enhances long-term fear memory consolidation and activates CREB in lateral amygdala neurons. Kinase-dead OptoEphB2 lacks these effects. EphB2lacZ/lacZ mice lacking forward signaling show impaired long-term auditory fear conditioning memory, rescued by optoEphB2 activation.","method":"Optogenetics, kinase-dead mutant, EphB2lacZ/lacZ knockout mice, CREB phosphorylation, behavioral fear conditioning","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — optogenetic and genetic dissection with defined molecular and behavioral readouts, single lab","pmids":["29768201"],"is_preprint":false},{"year":2011,"finding":"EphB2 tyrosine kinase forward signaling (catalytic activity is essential) controls migration of nestin-positive neural progenitor cells from the dentate notch neuroepithelium to populate the lateral suprapyramidal blade (LSB) of the dentate gyrus. Ephrin-B1 acts as the ligand for EphB2 in this context. Loss of EphB2 forward signaling dramatically reduces Reelin immunoreactivity in a compartment above the developing LSB.","method":"EphB2 kinase-dead and truncation mutants in mice, BrdU/nestin labeling, Reelin immunostaining, ephrin-B1 knockout comparison","journal":"The Journal of neuroscience","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple genetic alleles with defined cellular phenotype, single lab","pmids":["21832177"],"is_preprint":false},{"year":2000,"finding":"EphB2 is expressed in K+-secreting dark cells of the inner ear epithelium and is required for proper efferent axon midline guidance and for endolymph homeostasis. PDZ domain-containing proteins bind the C-terminus of EphB2 and can also recognize cytoplasmic tails of anion exchangers and aquaporins, suggesting EphB2 regulates ionic homeostasis through macromolecular complexes with membrane channels.","method":"EphB2 knockout mouse (circling behavior, vestibular phenotype analysis), PDZ binding assay, ultrastructural analysis","journal":"Neuron","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO mouse with defined phenotype plus biochemical PDZ interaction, single lab","pmids":["10839360"],"is_preprint":false},{"year":2006,"finding":"Bidirectional signaling by EphB2 (forward) and ephrin-B2 (reverse) is required for proper K+ secretion and ionic homeostasis in vestibular endolymph. Mutations disrupting EphB2 forward signaling or ephrin-B2 reverse signaling decrease K+ concentration and endolymphatic potential, demonstrating that B-subclass Eph/ephrin signaling controls epithelial ion transport function.","method":"Targeted mutations (cytoplasmic domain) of EphB2 and ephrin-B2 in mice, direct electrophysiological measurement of endolymphatic potential and K+ concentration in live animals","journal":"Hearing research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct measurement in vivo with bidirectional genetic controls, single lab","pmids":["17158005"],"is_preprint":false},{"year":2019,"finding":"EPHB2 carried on small extracellular vesicles (exosomes) induces tumor angiogenesis by transporting the transmembrane receptor to non-adjacent endothelial cells, stimulating ephrin-B reverse signaling and STAT3 phosphorylation. A STAT3 inhibitor greatly reduces SEV-induced angiogenesis.","method":"Proteomic analysis of SEVs, EPHB2 knockdown in SEVs, in vitro and in vivo angiogenesis assays, STAT3 phosphorylation analysis, STAT3 inhibitor treatment","journal":"JCI insight","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional knockdown with defined signaling readout, single lab, multiple assays","pmids":["31661464"],"is_preprint":false},{"year":2020,"finding":"RNF186, an E3 ubiquitin ligase, ubiquitinates EPHB2 at Lys892 upon stimulation by ligand EFNB1. Ubiquitinated EPHB2 recruits MAP1LC3B to activate autophagy in colonic epithelial cells. rnf186 and ephb2 knockout mice show more severe DSS-induced colitis due to autophagy defects in colon epithelial cells.","method":"Co-immunoprecipitation, ubiquitination assay (K892R mutant), autophagy markers (LC3B), KO mice + DSS colitis model","journal":"Autophagy","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ubiquitination site mutagenesis plus KO mouse model with defined phenotype, single lab","pmids":["33280498"],"is_preprint":false},{"year":2021,"finding":"EPHB2 drives liver cancer stemness and sorafenib resistance through activation of the SRC/AKT/GSK3β/β-catenin signaling cascade. EPHB2 expression is regulated by TCF1 via promoter activation, forming a positive Wnt/β-catenin feedback loop. EPHB2 knockout attenuates tumor development and rAAV-8-shEPHB2 sensitizes HCC cells to sorafenib.","method":"EPHB2 knockout (endogenous), RNA-seq, signaling pathway analysis, xenograft and immunocompetent mouse models, rAAV-mediated shRNA knockdown","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO plus in vivo knockdown with defined signaling cascade, single lab","pmids":["33903122"],"is_preprint":false},{"year":2023,"finding":"EphB2, acting as a downstream effector of Notch signaling in hepatocytes, is sufficient to induce cell-autonomous inflammation during NASH progression. Knockdown of Ephb2 in hepatocytes ameliorates inflammation and fibrosis in a mouse NASH model. EphB2-expressing hepatocytes are specifically demarcated as a profibrotic/inflammatory subpopulation.","method":"Single-nucleus transcriptomics/epigenomics, Notch pathway analysis, hepatocyte-specific Ephb2 knockdown in NASH mouse model, inflammation/fibrosis readouts","journal":"Science translational medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — hepatocyte-specific knockdown with defined mechanistic pathway placement, replicated in mouse and human data","pmids":["36753562"],"is_preprint":false},{"year":2018,"finding":"EphB2 is upregulated and activated in hepatic stellate cells (HSCs) during fibrogenesis. EphB2-deficient HSCs show impaired proliferation and suboptimal differentiation into fibrogenic myofibroblasts in vitro, identifying HSC activation as a cellular mechanism through which EphB2 promotes liver fibrosis.","method":"EphB2 knockout mice, in vitro HSC activation, fibrosis markers, apoptosis assays","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO mouse with in vitro mechanistic follow-up in HSCs, single lab","pmids":["29416088"],"is_preprint":false},{"year":2014,"finding":"Tissue factor (TF)/coagulation factor VIIa (FVIIa) cleaves EphB2 ectodomain at a key arginine residue (identified by Edman sequencing and LC-MS/MS). This cleavage is independent of PAR2 signaling and potentiates cell repulsion mediated by the EphB2 ligand ephrin-B1, identifying TF/FVIIa as a novel protease controlling Eph-mediated cell segregation.","method":"N-terminal Edman sequencing, LC-MS/MS, PAR2 inhibition, cell repulsion assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MS-based cleavage site identification plus functional cell repulsion assay, single lab","pmids":["25281742"],"is_preprint":false},{"year":2018,"finding":"A human missense mutation p.R745C in the EphB2 kinase domain impairs EphB2 autophosphorylation but not ephrin ligand-induced EphB2 clustering. This results in defects in platelet aggregation, αIIbβ3 activation, granule secretion, and thrombus formation. EPHB2 positively regulates GPVI platelet signaling (Lyn, Syk, FcRγ phosphorylation) and Src activation downstream of PAR4.","method":"Whole-exome sequencing, patient platelets, overexpression in RBL-2H3 cells, platelet aggregation/flow assays, phosphorylation analysis","journal":"Blood","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — human mutation with defined kinase-activity defect, validated in patient cells and heterologous expression, multiple functional readouts","pmids":["30213874"],"is_preprint":false},{"year":2014,"finding":"EphB2 co-associates with and activates focal adhesion kinase (FAK) in glioblastoma neurosphere cells, facilitating focal adhesion formation, cytoskeletal changes, and cell migration. EphB2 expression stimulates invasion and inhibits proliferation in vitro and in vivo.","method":"Co-immunoprecipitation (EphB2-FAK), EphB2 overexpression/silencing, FAK phosphorylation analysis, migration/invasion/proliferation assays, xenograft model","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus genetic gain/loss of function with functional readout, single lab","pmids":["22310282"],"is_preprint":false},{"year":2017,"finding":"EphB2 signaling induces mitochondrial translocation of Sirt3. EphrinB2-Fc induces nuclear translocation of Nrf-2 via c-Src phosphorylation, and Sirt3 expression is regulated by Nrf-2. EphB2/Sirt3 signaling increases MnSOD activity (by deacetylation at Lys68) to reduce mitochondrial ROS and suppress MSC senescence.","method":"EphB2 knockdown, Sirt3 knockdown, subcellular fractionation, Nrf-2/Src phosphorylation analysis, MnSOD deacetylation/activity assay, ROS measurement","journal":"Free radical biology & medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic knockdown with defined mechanistic chain, single lab, multiple orthogonal readouts","pmids":["28687409"],"is_preprint":false},{"year":2024,"finding":"Ephrin-B2 acts on mouse and human DRG neurons via EphB2 to induce nociceptor plasticity and hyperalgesic priming through MNK-eIF4E signaling. Sensory neuron-specific EphB2 knockout (Pirt-Cre) abrogates ephrin-B2-induced mechanical hypersensitivity and enhanced Ca2+ responses to PGE2. MNK1 knockout or MNK inhibitor (eFT508) blocks acute and priming responses.","method":"Conditional EphB2 knockout (Pirt-Cre), MNK1 knockout, MNK inhibitor, Ca2+ imaging in cultured DRG neurons (mouse and human), behavioral pain assays","journal":"Pharmacological research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — neuron-specific KO plus pharmacological validation with functional readout in two species, single lab","pmids":["38925462"],"is_preprint":false},{"year":2017,"finding":"EphB2 receptor repulsion trans-endocytosis requires Eps15R and clathrin. Eps15R (but not Eps15) knockdown significantly reduces EphB2-mediated cell repulsion. A novel DPFxxLDPF motif in Eps15R binds directly to the clathrin terminal domain in vitro; wild-type Eps15R but not the clathrin-binding mutant rescues cell repulsion.","method":"siRNA knockdown, in vitro clathrin-binding assay, rescue experiments in co-culture cell repulsion assay","journal":"Traffic","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro binding plus genetic rescue in cell repulsion assay, single lab","pmids":["28972287"],"is_preprint":false},{"year":2016,"finding":"EphB2 activation in the medial prefrontal cortex (mPFC) via EphrinB1-Fc produces stress-resistant and antidepressant-like effects. EphB2 shRNA knockdown in the mPFC increases stress susceptibility. These effects are associated with changes in cofilin phosphorylation, membrane AMPA receptor trafficking, and spine remodeling.","method":"Lentiviral shRNA knockdown, EphrinB1-Fc intra-mPFC injection, behavioral assays, cofilin phosphorylation, AMPAR surface trafficking, spine analysis","journal":"Neuropsychopharmacology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — bidirectional genetic manipulation with defined molecular and behavioral readouts, single lab","pmids":["27103064"],"is_preprint":false},{"year":2021,"finding":"Loss of EPHB2 in prostate cancer cells increases lipogenic DGAT1, DGAT2, and PLIN2 and decreases lipolytic ATGL and PEDF, leading to lipid droplet accumulation in cytoplasmic and nuclear compartments. A DGAT1-specific inhibitor abrogates LD accumulation and proliferative effects induced by EPHB2 loss, mechanistically linking EPHB2 to lipid homeostasis via DGAT1/ATGL regulation.","method":"EPHB2 siRNA knockdown, DGAT1 inhibitor, lipid droplet quantification, proliferation assays, protein expression analysis","journal":"Laboratory investigation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — knockdown plus pharmacological rescue with defined molecular targets, single lab","pmids":["33824421"],"is_preprint":false},{"year":1995,"finding":"DRT (EPHB2/EPHT3) encodes a receptor-type tyrosine kinase of the EPH family. Its 11 kb transcript is preferentially expressed in fetal brain and is upregulated upon retinoic acid-induced neuronal differentiation of NTera-2 cells, indicating a role in neurogenesis. The gene was mapped to human chromosome 1p35-1p36.1.","method":"cDNA library screening with anti-phosphotyrosine antibody, 5' RACE, RT-PCR, chromosomal mapping by somatic cell hybrid panels and FISH, retinoic acid differentiation assay","journal":"Human molecular genetics","confidence":"Low","confidence_rationale":"Tier 3 / Weak — initial cloning and expression characterization without mechanistic functional assays","pmids":["8589679"],"is_preprint":false},{"year":2014,"finding":"EphB2 signaling in B cells promotes proliferation, TNF-α secretion, and IgG production in human naive B cells. EphB2 knockdown attenuates B-cell activation via reduction of Src-p65 (NF-κB) and Notch1 signaling pathways.","method":"EphB2 siRNA in human primary B cells, proliferation assay, ELISA (cytokines/Ig), Src/p65/Notch1 pathway analysis","journal":"FASEB journal","confidence":"Low","confidence_rationale":"Tier 3 / Weak — siRNA knockdown with pathway measurement, single lab, limited mechanistic depth","pmids":["24803541"],"is_preprint":false}],"current_model":"EphB2 is a transmembrane receptor tyrosine kinase that is autoinhibited by its juxtamembrane segment (which distorts the kinase activation loop) and activated by phosphorylation of conserved juxtamembrane tyrosines (Y605/Y611), which also create docking sites for SH2-domain proteins including Src, Abl/Arg, and RasGAP; it binds both ephrin-B ligands (canonical) and ephrin-A5 (cross-subclass) to form distinct signaling complexes, signals bidirectionally with ephrins to control cell repulsion via MMP-mediated ectodomain cleavage, Rac/Tiam2-dependent trans-endocytosis, and a Nck/Pak/RhoA pathway for growth cone collapse; in neurons it regulates NMDA receptor surface expression and function both through kinase-dependent mechanisms (direct NR1/NR2 phosphorylation via the γ-secretase-released CTF2 fragment, and PI3K/Akt/GSK-3β-mediated tau dephosphorylation) and kinase-independent mechanisms (PDZ-motif-mediated GluA2/NMDAR retention); its activity is modulated by neuropsin cleavage, ADAM10/MMP ectodomain shedding, presenilin/γ-secretase processing, RNF186-mediated ubiquitination at K892 (triggering autophagy), and amyloid-β-induced proteasomal degradation; and in non-neural contexts EphB2 drives intestinal progenitor proliferation (via Abl-cyclin D1) and migration (via PI3K) through distinct pathways, promotes liver fibrosis via hepatic stellate cell activation, regulates platelet GPVI/GPCR signaling through its kinase domain, and in cancer contexts activates SRC/AKT/GSK3β/β-catenin and R-Ras/MAPK cascades to control invasion, stemness, and drug resistance."},"narrative":{"mechanistic_narrative":"EphB2 is a transmembrane receptor tyrosine kinase that governs cell repulsion, axon guidance, synaptic function, and tissue patterning through ephrin-driven bidirectional signaling [PMID:15107857, PMID:18713744]. Its catalytic activity is held in check by an autoinhibitory juxtamembrane segment that adopts a helical conformation distorting the kinase small lobe and blocking the activation segment; autophosphorylation of conserved juxtamembrane tyrosines (notably Y605/Y611) relieves this autoinhibition and creates phosphotyrosine docking sites for SH2-domain effectors including Src and the Abl/Arg non-receptor kinases [PMID:11572780, PMID:9632142, PMID:11494128]. Ligand engagement occurs through a jellyroll beta-sandwich ligand-binding domain that binds ephrin-B ligands and, across subclass, ephrin-A5, with clustering above the dimer threshold determining repulsive signaling strength [PMID:9853759, PMID:15107857, PMID:24469634]. Downstream, EphB2 controls cytoskeletal collapse and contact repulsion through multiple effector arms: recruitment of p120RasGAP to downregulate Ras-Erk, a Pak/Nck/RhoA pathway for growth cone collapse, Rac/Tiam2-dependent and Eps15R/clathrin-mediated trans-endocytosis, and MMP-2/MMP-9 ectodomain cleavage required for repulsion [PMID:12486127, PMID:18713744, PMID:27597758, PMID:23147113, PMID:28972287]. EphB2 is sequentially processed by ectodomain sheddases and presenilin/γ-secretase to release an intracellular CTF2 fragment that retains kinase activity and directly phosphorylates NMDA receptor subunits to promote their surface expression [PMID:17428795, PMID:19661068]. In neurons EphB2 regulates NMDA receptor-mediated currents and synaptic plasticity through both kinase-dependent and kinase-independent (including PDZ-motif-mediated) mechanisms, and amyloid-β oligomers bind its fibronectin repeats to trigger proteasomal degradation, linking EphB2 to memory deficits [PMID:11754836, PMID:11754835, PMID:21113149, PMID:26589795]. Beyond the nervous system, EphB2 partitions intestinal progenitor proliferation (Abl-cyclin D1) from migration (PI3K), drives cancer stemness and drug resistance via SRC/AKT/GSK3β/β-catenin and R-Ras/MAPK cascades, promotes liver fibrosis and NASH inflammation, and supports platelet GPVI/PAR signaling through its kinase domain, where a human p.R745C mutation impairing autophosphorylation causes a platelet function disorder [PMID:19914164, PMID:16049340, PMID:33903122, PMID:36753562, PMID:29416088, PMID:30213874].","teleology":[{"year":1995,"claim":"Establishing EPHB2 as an EPH-family receptor tyrosine kinase enriched in fetal brain and induced during neuronal differentiation framed it as a candidate regulator of neurogenesis.","evidence":"cDNA cloning, expression profiling, and chromosomal mapping in differentiating NTera-2 cells","pmids":["8589679"],"confidence":"Low","gaps":["Initial cloning lacks any mechanistic functional assay","No ligand or downstream pathway defined at this stage"]},{"year":1998,"claim":"Solving the ligand-binding domain structure and identifying Y611 as the SH2 docking site for Src defined how EphB2 recognizes ephrins and how its phosphorylated juxtamembrane region recruits effectors.","evidence":"X-ray crystallography of the ligand-binding domain plus Y2H, mutagenesis, Co-IP and phosphopeptide mapping for Src association","pmids":["9853759","9632142"],"confidence":"High","gaps":["Did not resolve the autoinhibited conformation of the kinase domain","Full repertoire of SH2 effectors not yet enumerated"]},{"year":2000,"claim":"EphB2 knockout phenotypes in the inner ear revealed a role in efferent axon guidance and epithelial ion homeostasis through PDZ-mediated complexes with membrane channels, extending its function beyond axon repulsion.","evidence":"EphB2 knockout mouse phenotyping plus PDZ-binding assays","pmids":["10839360"],"confidence":"Medium","gaps":["Direct channel partners within the macromolecular complex not biochemically confirmed in vivo","Forward versus reverse signaling contributions not separated here"]},{"year":2001,"claim":"Crystallography of the autoinhibited kinase domain and dissection of Abl/Arg binding established the structural switch underlying activation and the breadth of non-receptor kinase effectors, while knockout/knock-in mice showed EphB2 regulates NMDA receptor synaptic function partly independently of its kinase domain.","evidence":"1.9 Å crystal structure with mutagenesis; Y2H and Co-IP for Abl/Arg; EphB2-null and truncated knock-in mice with electrophysiology and immunogold","pmids":["11572780","11494128","11754836","11754835"],"confidence":"High","gaps":["Molecular basis of kinase-independent NMDA receptor regulation left undefined","How distinct Abl/Arg interaction modes are coordinated in vivo unresolved"]},{"year":2002,"claim":"Re-engineering juxtamembrane docking sites showed that RasGAP recruitment downregulates Ras-Erk to drive neurite retraction, providing a defined effector pathway linking EphB2 phosphorylation to cytoskeletal collapse.","evidence":"Receptor mutagenesis with phospho-Erk immunoblotting and neurite retraction assays in NG108 neurons","pmids":["12486127"],"confidence":"High","gaps":["Relative contribution of RasGAP versus other repulsive arms not quantified","In vivo relevance of this pathway not tested"]},{"year":2004,"claim":"Demonstrating high-affinity cross-subclass binding of EphB2 to ephrin-A5 with a distinct complex architecture revised the rule that EphB and ephrin-A pairs do not interact, broadening EphB2's ligand repertoire and signaling outputs.","evidence":"Crystallography plus receptor clustering, autophosphorylation, and growth cone collapse assays","pmids":["15107857"],"confidence":"High","gaps":["Physiological contexts where ephrin-A5/EphB2 signaling operates not established","Whether cross-subclass complexes assemble in vivo unaddressed"]},{"year":2005,"claim":"Identifying R-Ras as an EphB2-associated effector controlling glioma adhesion, proliferation, and invasion linked EphB2 to a tumor-relevant MAPK-suppressing axis.","evidence":"Co-IP, siRNA depletion, and adhesion/invasion/proliferation assays with MAPK readout","pmids":["16049340"],"confidence":"Medium","gaps":["Single-lab Co-IP without reciprocal structural mapping","Direct versus indirect EphB2-R-Ras association not resolved"]},{"year":2007,"claim":"Mapping sequential ectodomain shedding and presenilin/γ-secretase cleavage that releases EphB2/CTF2 revealed regulated intramembrane proteolysis as a mechanism converting receptor activation into an intracellular signaling fragment.","evidence":"Pharmacological inhibitors, endocytosis blockade, biochemical cleavage mapping, and ubiquitination assays","pmids":["17428795"],"confidence":"High","gaps":["Function of the released CTF2 fragment not yet demonstrated here","Identity of the initial ectodomain sheddase only partially defined"]},{"year":2008,"claim":"Showing MMP-2/MMP-9 ectodomain cleavage is required for repulsion, and that FGFR1 suppresses EphB2 via LAR phosphatase and Sprouty feedback, established proteolytic and cross-receptor control of EphB2 segregation activity.","evidence":"MMP inhibitors and cleavage-resistant mutants with rescue in EphB-deficient neurons; LAR knockdown, MAPK inhibitors, and Sprouty analysis in segregation assays","pmids":["18713744","19047466"],"confidence":"Medium","gaps":["Quantitative integration of cleavage with clustering thresholds not addressed","FGFR1 cross-talk validated in cell-based assays only"]},{"year":2009,"claim":"Demonstrating that EphB2/CTF2 directly phosphorylates NR1/NR2 independently of Src, and that EphB2 segregates intestinal proliferation (Abl-cyclin D1) from migration (PI3K), defined parallel kinase-dependent and kinase-independent output modules whose uncoupling enables cancer.","evidence":"In vitro kinase assays with kinase-dead mutants and Src inhibitors; genetic dissection of intestinal progenitors with pathway inhibitors","pmids":["19661068","19914164"],"confidence":"High","gaps":["Substrate selectivity of CTF2 beyond NMDA subunits not mapped","Mechanism dissociating proliferation and migration pathways in carcinoma unresolved"]},{"year":2010,"claim":"Identifying amyloid-β oligomer binding to EphB2 fibronectin repeats with consequent proteasomal degradation, and rescue of LTP/memory by EphB2 restoration, placed EphB2 mechanistically in Alzheimer's disease synaptic dysfunction.","evidence":"Biochemical Aβ binding, in vivo shRNA/overexpression, electrophysiology, and behavioral memory tests in hAPP mice","pmids":["21113149"],"confidence":"High","gaps":["Degradation machinery downstream of Aβ binding not fully defined","Whether degradation acts on full-length receptor or processed fragments unclear"]},{"year":2011,"claim":"Neuropsin-mediated cleavage controlling EphB2-NR1 dissociation in the amygdala, and EphB2 kinase-dependent control of dentate progenitor migration, showed proteolytic and forward-signaling regulation of EphB2 in behavior and neurodevelopment.","evidence":"Neuropsin-KO mice with EphB2-NR1 Co-IP, NMDA currents, and anxiety assays; EphB2 kinase-dead/truncation alleles with BrdU/nestin and Reelin staining","pmids":["21508957","21832177"],"confidence":"High","gaps":["Generality of neuropsin cleavage outside the amygdala not established","Reelin link to progenitor migration mechanistically indirect"]},{"year":2014,"claim":"Defining clustering-threshold-dependent repulsion, TF/FVIIa ectodomain cleavage potentiating repulsion, and EphB2-FAK coupling in glioblastoma refined the quantitative and proteolytic control of EphB2 signaling strength and migration.","evidence":"Chemical dimerizers and fluorescence anisotropy; Edman/LC-MS cleavage mapping; Co-IP and gain/loss-of-function with FAK phosphorylation","pmids":["24469634","25281742","22310282"],"confidence":"Medium","gaps":["In vivo relevance of cluster-size thresholds not tested","TF/FVIIa cleavage characterized in single-lab cell assays"]},{"year":2016,"claim":"Identifying Rac/Tiam2-dependent F-actin enrichment driving trans-endocytosis and antidepressant-like mPFC effects of EphB2 activation linked specific GTPase machinery and synaptic remodeling to EphB2 forward signaling outcomes.","evidence":"siRNA Rho-GTPase screen, Tiam2 knockdown, and trans-endocytosis/repulsion assays; mPFC shRNA/EphrinB1-Fc with cofilin, AMPAR, and behavioral readouts","pmids":["27597758","27103064"],"confidence":"Medium","gaps":["How Rac is locally activated at internalization sites unresolved","mPFC effects mechanistically correlative beyond cofilin/AMPAR changes"]},{"year":2017,"claim":"Demonstrating Eps15R/clathrin dependence of trans-endocytosis, Abl2/Arg-dependent dendritic filopodia growth, and an EphB2/Sirt3 antioxidant axis expanded the effector network connecting EphB2 to endocytic, cytoskeletal, and redox outcomes.","evidence":"siRNA and in vitro clathrin-binding rescue; optogenetic EphB2 with Arg knockdown and actin imaging; knockdown with Nrf-2/Src and MnSOD deacetylation assays","pmids":["28972287","29158322","28687409"],"confidence":"Medium","gaps":["Each pathway validated in single labs and distinct cell systems","Integration of endocytic and actin-nucleating arms not unified"]},{"year":2018,"claim":"A human kinase-domain mutation (p.R745C) impairing autophosphorylation but not clustering established EphB2 as a positive regulator of platelet GPVI/PAR signaling and the basis of a human platelet function disorder, while optogenetic studies placed EphB2 forward signaling in fear memory consolidation and HSC-driven liver fibrosis.","evidence":"Whole-exome sequencing with patient platelets and heterologous expression; optogenetics with kinase-dead and lacZ-null mice and CREB readout; EphB2-KO mice with in vitro HSC activation","pmids":["30213874","29768201","29416088"],"confidence":"High","gaps":["Direct platelet substrates of EphB2 not defined","Whether HSC fibrosis effect is cell-autonomous unresolved"]},{"year":2019,"claim":"Showing exosome-delivered EphB2 stimulates ephrin-B reverse signaling and STAT3 phosphorylation in endothelial cells revealed a non-cell-autonomous, vesicle-mediated route for EphB2 to drive tumor angiogenesis.","evidence":"SEV proteomics, EphB2 knockdown in SEVs, angiogenesis assays, and STAT3 inhibitor treatment","pmids":["31661464"],"confidence":"Medium","gaps":["Mechanism of EphB2 loading into exosomes not defined","Direct endothelial receptor mediating reverse signaling not identified"]},{"year":2020,"claim":"Identifying RNF186-mediated ubiquitination of EphB2 at K892 triggering LC3B-dependent autophagy in colonic epithelium connected EphB2 to a post-translational autophagy switch protective against colitis.","evidence":"Co-IP, K892R ubiquitination mutant, LC3B autophagy markers, and rnf186/ephb2 KO mice in DSS colitis","pmids":["33280498"],"confidence":"Medium","gaps":["How ubiquitinated EphB2 recruits the autophagy machinery mechanistically unresolved","Single-lab finding without reciprocal validation"]},{"year":2021,"claim":"Defining a TCF1-EphB2-SRC/AKT/GSK3β/β-catenin positive feedback loop driving liver cancer stemness and sorafenib resistance, and an EphB2-DGAT1/ATGL lipid-homeostasis link in prostate cancer, established opposing context-dependent roles of EphB2 in malignancy.","evidence":"EPHB2 KO and rAAV-shRNA with RNA-seq and xenograft models; siRNA knockdown with DGAT1 inhibitor and lipid droplet quantification","pmids":["33903122","33824421"],"confidence":"Medium","gaps":["Reconciliation of tumor-promoting versus tumor-suppressive roles across cancers unresolved","Direct kinase substrates within these cascades not identified"]},{"year":2023,"claim":"Placing EphB2 as a Notch-downstream effector marking a profibrotic/inflammatory hepatocyte subpopulation showed it is sufficient to drive cell-autonomous inflammation in NASH progression.","evidence":"Single-nucleus multi-omics and hepatocyte-specific Ephb2 knockdown in a mouse NASH model with human data","pmids":["36753562"],"confidence":"Medium","gaps":["Downstream inflammatory effectors of hepatocyte EphB2 not fully defined","Ligand driving hepatocyte EphB2 activation unidentified"]},{"year":2024,"claim":"Demonstrating ephrin-B2/EphB2 signaling through MNK-eIF4E to induce nociceptor plasticity and hyperalgesic priming in mouse and human DRG neurons defined a translational-control mechanism for EphB2 in pain.","evidence":"Sensory-neuron-specific EphB2 KO, MNK1 KO, MNK inhibitor, Ca2+ imaging, and behavioral pain assays in two species","pmids":["38925462"],"confidence":"Medium","gaps":["How EphB2 forward signaling couples to MNK-eIF4E not biochemically mapped","Whether receptor proteolysis participates in this pathway untested"]},{"year":null,"claim":"How EphB2's multiple effector arms, proteolytic processing events, and context-specific oncogenic versus tumor-suppressive outputs are coordinated at the level of a single activated receptor remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model linking clustering state to choice of effector pathway","Determinants of opposing roles across tumor types not established","Comprehensive substrate map of full-length EphB2 and CTF2 lacking"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[3,9,10,20,32]},{"term_id":"GO:0001618","term_label":"virus receptor activity","supporting_discovery_ids":[1,2]},{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[2,12,25]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[11,21,24]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[9,16,12]},{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[17,26,36]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,2,11,13]},{"term_id":"R-HSA-112316","term_label":"Neuronal System","supporting_discovery_ids":[5,7,8,18,22]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[9,12,27,31]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[28,29,33,38]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[13,23,24,25]}],"complexes":[],"partners":["EFNB1","EFNB2","EFNA5","SRC","ABL1","RASA1","PAK1","PTK2"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P29323","full_name":"Ephrin type-B receptor 2","aliases":["Developmentally-regulated Eph-related tyrosine kinase","ELK-related tyrosine kinase","EPH tyrosine kinase 3","EPH-like kinase 5","EK5","hEK5","Renal carcinoma antigen NY-REN-47","Tyrosine-protein kinase TYRO5","Tyrosine-protein kinase receptor EPH-3"],"length_aa":1055,"mass_kda":117.5,"function":"Receptor tyrosine kinase which binds promiscuously transmembrane ephrin-B family ligands residing on adjacent cells, leading to contact-dependent bidirectional signaling into neighboring cells. The signaling pathway downstream of the receptor is referred to as forward signaling while the signaling pathway downstream of the ephrin ligand is referred to as reverse signaling. Functions in axon guidance during development. Involved in the guidance of commissural axons, that form a major interhemispheric connection between the 2 temporal lobes of the cerebral cortex. Also involved in guidance of contralateral inner ear efferent growth cones at the midline and of retinal ganglion cell axons to the optic disk. In addition to axon guidance, also regulates dendritic spines development and maturation and stimulates the formation of excitatory synapses. Upon activation by EFNB1, abolishes the ARHGEF15-mediated negative regulation on excitatory synapse formation. Controls other aspects of development including angiogenesis, palate development and in inner ear development through regulation of endolymph production. Forward and reverse signaling through the EFNB2/EPHB2 complex regulate movement and adhesion of cells that tubularize the urethra and septate the cloaca. May function as a tumor suppressor. May be involved in the regulation of platelet activation and blood coagulation (PubMed:30213874)","subcellular_location":"Cell membrane; Cell projection, axon; Cell projection, dendrite","url":"https://www.uniprot.org/uniprotkb/P29323/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/EPHB2","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/EPHB2","total_profiled":1310},"omim":[{"mim_id":"618462","title":"BLEEDING DISORDER, PLATELET-TYPE, 22; BDPLT22","url":"https://www.omim.org/entry/618462"},{"mim_id":"614150","title":"PROTEIN KINASE DOMAIN-CONTAINING PROTEIN, CYTOPLASMIC; PKDCC","url":"https://www.omim.org/entry/614150"},{"mim_id":"613199","title":"TAO KINASE 2; TAOK2","url":"https://www.omim.org/entry/613199"},{"mim_id":"610456","title":"STERILE ALPHA MOTIF DOMAIN-CONTAINING PROTEIN 9; SAMD9","url":"https://www.omim.org/entry/610456"},{"mim_id":"608504","title":"RHO GUANINE NUCLEOTIDE EXCHANGE FACTOR 15; ARHGEF15","url":"https://www.omim.org/entry/608504"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Plasma membrane","reliability":"Supported"},{"location":"Nucleoplasm","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"intestine","ntpm":21.9}],"url":"https://www.proteinatlas.org/search/EPHB2"},"hgnc":{"alias_symbol":["Hek5","Tyro5"],"prev_symbol":["DRT","ERK","EPHT3"]},"alphafold":{"accession":"P29323","domains":[{"cath_id":"2.60.120.260","chopping":"20-196","consensus_level":"high","plddt":88.7093,"start":20,"end":196},{"cath_id":"2.60.40.10","chopping":"329-431","consensus_level":"medium","plddt":90.835,"start":329,"end":431},{"cath_id":"2.60.40.10","chopping":"443-533","consensus_level":"medium","plddt":89.3146,"start":443,"end":533},{"cath_id":"3.30.200.20","chopping":"617-702","consensus_level":"medium","plddt":88.101,"start":617,"end":702},{"cath_id":"1.10.510.10","chopping":"703-771_788-893","consensus_level":"medium","plddt":89.2427,"start":703,"end":893},{"cath_id":"1.10.150.50","chopping":"919-977","consensus_level":"high","plddt":84.9041,"start":919,"end":977}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P29323","model_url":"https://alphafold.ebi.ac.uk/files/AF-P29323-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P29323-F1-predicted_aligned_error_v6.png","plddt_mean":80.0},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=EPHB2","jax_strain_url":"https://www.jax.org/strain/search?query=EPHB2"},"sequence":{"accession":"P29323","fasta_url":"https://rest.uniprot.org/uniprotkb/P29323.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P29323/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P29323"}},"corpus_meta":[{"pmid":"15107857","id":"PMC_15107857","title":"Repelling 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Phosphorylation of conserved juxtamembrane tyrosines relieves this autoinhibition by disrupting the JM-kinase interface and liberating phosphotyrosine sites for SH2-domain binding.\",\n      \"method\": \"X-ray crystallography + site-directed mutagenesis\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure at 1.9 Å combined with mutagenesis validation in a single rigorous study\",\n      \"pmids\": [\"11572780\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Crystal structure of the EphB2 ligand-binding domain at 2.9 Å resolution reveals a jellyroll beta-sandwich of 11 antiparallel beta-strands. Structure-based mutagenesis identified an extended loop critical for ligand binding and subclass specificity, localizing the ligand-binding surface to the concave beta-sandwich near positions where missense mutations cause signaling defects.\",\n      \"method\": \"X-ray crystallography + structure-based mutagenesis\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure combined with mutagenesis in a single focused study\",\n      \"pmids\": [\"9853759\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"EphB2 and ephrin-A5 (a cross-subclass pair) bind each other with high affinity, triggering receptor clustering, autophosphorylation, and downstream signaling. Ephrin-A5 induces EphB2-mediated growth cone collapse and neurite retraction. Crystal structure reveals a heterodimeric complex architecturally distinct from the tetrameric EphB2-ephrinB2 structure, providing a molecular basis for cross-subclass signaling.\",\n      \"method\": \"X-ray crystallography, cell-based signaling assays (receptor clustering, autophosphorylation), growth cone collapse assay\",\n      \"journal\": \"Nature neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure plus multiple orthogonal functional assays in one study\",\n      \"pmids\": [\"15107857\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Phosphorylated tyrosine 611 in the juxtamembrane region of EphB2 is a major autophosphorylation site and is required for interaction with the SH2 domain of Src kinase. Tyrosines 605 and 611 are important for EphB2 kinase activity. EphB2 and Src physically associate (co-immunoprecipitation), and activated EphB2 induces increased tyrosine phosphorylation of Src.\",\n      \"method\": \"Yeast two-hybrid, site-directed mutagenesis, co-immunoprecipitation, phosphopeptide mapping\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — multiple orthogonal methods (Y2H + mutagenesis + Co-IP + phosphopeptide mapping) in one study\",\n      \"pmids\": [\"9632142\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Abl and Arg non-receptor tyrosine kinases associate with EphB2 through multiple distinct interactions: (1) their SH2 domains bind phosphorylated juxtamembrane tyrosines of EphB2; (2) a phosphorylation-independent interaction involves C-terminal sequences of Abl/Arg; (3) a third interaction requires EphB2 phosphorylation but not direct SH2 binding. Activated EphB2 phosphorylates Abl and Arg, and ephrin-B1 stimulation decreases endogenous Abl kinase activity.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, kinase activity assay, ephrin-B1 stimulation of endogenous EphB2\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP plus multiple interaction modes validated, single lab\",\n      \"pmids\": [\"11494128\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"EphB2 null mice exhibit reduced NMDA receptor-mediated synaptic current and reduced synaptically localized NR1 subunit (immunogold) in dentate granule neurons. Mice lacking only the intracellular kinase domain retain wild-type LTP, indicating that kinase-independent EphB2 functions mediate certain synaptic roles, whereas the extracellular/transmembrane domain is required for NMDA receptor regulation.\",\n      \"method\": \"EphB2 knockout mouse, electrophysiology (LTP, NMDA currents), immunogold localization\",\n      \"journal\": \"Neuron\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — KO mouse with electrophysiology + immunogold, replicated by companion paper (PMID 11754835)\",\n      \"pmids\": [\"11754836\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"EphB2 kinase signaling is not required for protein synthesis-dependent LTP or for two forms of synaptic depression in hippocampal slices; a C-terminally truncated EphB2 (lacking kinase domain) rescues the EphB2 null phenotype, demonstrating kinase-independent regulation of synaptic plasticity. Eph receptor stimulation in cultured neurons modulates signaling pathways implicated in synaptic plasticity, suggesting cross-talk with NMDA receptor-activated pathways.\",\n      \"method\": \"EphB2 knockout mouse, targeted knock-in of truncated EphB2, LTP/LTD electrophysiology in hippocampal slices\",\n      \"journal\": \"Neuron\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — knock-in rescue experiment with defined readout, replicated by companion paper (PMID 11754836)\",\n      \"pmids\": [\"11754835\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Amyloid-β oligomers bind to the fibronectin repeats domain of EphB2 and trigger EphB2 degradation via the proteasome. EphB2 knockdown by shRNA reduces NMDA receptor currents and impairs LTP in dentate gyrus; increasing EphB2 expression in dentate gyrus of hAPP transgenic mice reverses NMDA receptor-dependent LTP deficits and memory impairments.\",\n      \"method\": \"Biochemical binding assay (Aβ oligomers + EphB2), lentiviral shRNA knockdown and overexpression in vivo, electrophysiology, behavioral memory tests\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct binding demonstrated, bidirectional genetic manipulation with multiple functional readouts\",\n      \"pmids\": [\"21113149\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"The serine protease neuropsin cleaves EphB2 in the amygdala upon stress, causing dissociation of EphB2 from the NR1 subunit of the NMDA receptor and promoting membrane turnover of EphB2 receptors. Dynamic EphB2-NR1 interaction enhances NMDA receptor current, induces Fkbp5 gene expression, and increases anxiety-like behaviour. Neuropsin-deficient mice lack EphB2 cleavage and show attenuated anxiety.\",\n      \"method\": \"Neuropsin knockout mice, intra-amygdala injection of neuropsin/antibodies/shRNA, EphB2-NR1 co-immunoprecipitation, NMDA receptor current recordings, behavioural assays\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods in KO and rescue experiments with defined mechanistic pathway\",\n      \"pmids\": [\"21508957\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"EphB2 receptor ectodomain is cleaved after residue 543; the remaining membrane fragment is then cleaved by presenilin-dependent γ-secretase after residue 569, releasing an intracellular peptide (EphB2/CTF2). Ephrin-B ligand-induced processing requires endocytosis and is sensitive to proteasomal (not metalloproteinase) inhibitors. Calcium influx/NMDA-induced processing is sensitive to GM6001 and ADAM10 inhibitors and occurs at the plasma membrane without endocytosis. Ligand stimulation also induces ubiquitination of EphB2.\",\n      \"method\": \"Pharmacological inhibitors, endocytosis blockade, biochemical cleavage mapping, ubiquitination assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal pharmacological and biochemical methods mapping distinct cleavage pathways\",\n      \"pmids\": [\"17428795\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"The γ-secretase-derived intracellular peptide EphB2/CTF2 retains tyrosine kinase activity and directly phosphorylates NMDA receptor subunits (NR1, NR2) in cell lines and primary neuronal cultures, independently of Src kinases. A kinase-dead EphB2/CTF2 mutant lacks this activity. EphB2/CTF2 promotes cell-surface expression of NMDA receptors.\",\n      \"method\": \"In vitro kinase assay, Src inhibitors, kinase-dead mutant, cell-surface expression assay (primary neurons and cell lines)\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase reconstitution plus mutagenesis and Src-inhibitor controls, single lab\",\n      \"pmids\": [\"19661068\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"EphB2 recruits p120RasGAP via juxtamembrane (JM) binding sites, leading to down-regulation of the Ras-Erk MAPK pathway and neurite retraction. Elimination of RasGAP binding sites with addition of Grb2 binding sites within the kinase domain activates Erk MAPK upon ephrin-B1 stimulation. This confirms that EphB2-mediated neurite retraction correlates with Ras-Erk MAPK down-regulation mediated through RasGAP recruitment.\",\n      \"method\": \"Receptor mutagenesis/re-engineering, phospho-Erk immunoblotting, neurite retraction functional assay in NG108 neurons\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — systematic mutagenesis with structure-function correlation and functional readout\",\n      \"pmids\": [\"12486127\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"MMP-2 and MMP-9 cleave EphB2 in its ectodomain upon ephrin-B2 binding in vitro and in vivo. MMP-specific inhibition or cleavage-resistant EphB2 ectodomain mutations prevent EphB2-mediated cell-cell repulsion in HEK293 cells and block ephrin-B1-induced growth cone withdrawal in hippocampal neurons. Expression of cleavable but not non-cleavable EphB2 rescues ephrin-B1-induced growth cone collapse in EphB-deficient neurons.\",\n      \"method\": \"MMP inhibitors, cleavage-resistant mutagenesis, cell repulsion assay, growth cone collapse assay in EphB-deficient neurons, in vitro and in vivo cleavage assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — mutagenesis plus rescue in KO neurons plus pharmacological inhibitors, multiple orthogonal methods\",\n      \"pmids\": [\"18713744\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"EphB2 regulates intestinal progenitor cell migration and proliferation through two independent signaling pathways: kinase-independent, PI3K-mediated control of cell positioning/migration; and tyrosine kinase-dependent regulation of proliferation through an Abl-cyclin D1 pathway. These pathways dissociate during colon carcinoma progression, allowing continued proliferation with invasive growth.\",\n      \"method\": \"Genetic dissection with EphB2 mutants (kinase-dead, truncated), PI3K inhibitors, Abl inhibitors, cyclin D1 measurement, intestinal cell positioning assays\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean genetic dissection with multiple mutants, pharmacological validation, clear pathway placement\",\n      \"pmids\": [\"19914164\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Upon EphB2 activation, R-Ras associates with EphB2 receptor and becomes highly phosphorylated. siRNA depletion of R-Ras abrogates EphB2 effects on glioma cell adhesion, proliferation, and invasion. Anti-proliferative responses correlate with suppressed MAPK activity, identifying EphB2/R-Ras as a functionally relevant signaling axis.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, invasion/adhesion/proliferation assays, MAPK activity assay\",\n      \"journal\": \"The American journal of pathology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus siRNA rescue with functional readout, single lab\",\n      \"pmids\": [\"16049340\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"FGFR1 activation prevents EphB2-mediated cell segregation and repulsion by: (1) increasing basal EphB2 phosphorylation through down-regulation of the leukocyte common antigen-related (LAR) tyrosine phosphatase that dephosphorylates EphB2; and (2) inhibiting further EphB2 phosphorylation upon ephrinB1 stimulation via MAPK pathway-induced Sprouty genes. EphB2 normally activates the MAPK pathway, which feeds back positively to promote EphB2 activation.\",\n      \"method\": \"Phosphorylation analysis, LAR phosphatase knockdown, MAPK inhibitors, Sprouty expression, cell segregation/repulsion assays\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pharmacological and genetic dissection in cell-based assays, single lab\",\n      \"pmids\": [\"19047466\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"EphB2 clustering determines repulsive signaling strength: small clusters (trimers/tetramers) are sufficient to induce cell collapse, with dimers being inactive. Extended EphB2 arrays are dispensable. The ratio of multimers to dimers within a cluster population determines collapse response strength. The C-terminal modules of EphB2 have negative regulatory effects on ephrin-induced clustering.\",\n      \"method\": \"Chemical dimerizers, fluorescence anisotropy in live cells, quantitative imaging, collapse response assays\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — quantitative live-cell imaging with chemical genetics, single lab\",\n      \"pmids\": [\"24469634\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Rac-dependent F-actin enrichment at sites of EphB2 internalization (not during vesicle trafficking) mediates trans-endocytosis. Systematic Rho GTPase depletion identified Rac subfamily and Tiam2 (Rac-specific GEF) as key components of EphB2 trans-endocytosis from opposing ephrinB+ cells, and this pathway is required for EphB2-stimulated contact repulsion.\",\n      \"method\": \"Live imaging, systematic siRNA screen of Rho GTPases, Tiam2 knockdown, trans-endocytosis assay, cell repulsion assay\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — systematic genetic screen plus functional validation, single lab\",\n      \"pmids\": [\"27597758\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"EphB2 forward signaling mediates cortical growth cone collapse via Pak and Nck: kinase-active EphB2 binds Pak, promotes growth cone repulsion via Pak kinase activity, Pak-Nck binding, RhoA signaling and endocytosis. This occurs independently of Rac/Cdc42-GTP production after ephrinB treatment of cortical neurons.\",\n      \"method\": \"Co-immunoprecipitation (EphB2-Pak), dominant-negative constructs, endocytosis blockade, growth cone collapse assay in cortical neurons\",\n      \"journal\": \"Molecular and cellular neurosciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus functional rescue experiments with pathway inhibitors, single lab\",\n      \"pmids\": [\"23147113\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Optogenetic (OptoEphB2) photoactivation of EphB2 in dendrites of hippocampal neurons induces rapid actin polymerization and dynamic dendritic filopodial growth. Rac1 and Cdc42 inhibition does not abolish this effect; instead, Abl2/Arg kinase is required as a necessary effector for OptoEphB2-induced filopodia growth in dendrites.\",\n      \"method\": \"Optogenetic EphB2 activation, Rac1/Cdc42 inhibitors, Arg kinase knockdown/inhibition, live-cell actin imaging\",\n      \"journal\": \"Biology open\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — optogenetic spatial control plus pharmacological/genetic dissection, single lab\",\n      \"pmids\": [\"29158322\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"EphB2 activation (by ephrinB1/Fc or overexpression plus ligand) induces tau dephosphorylation at multiple AD-associated sites through PI3K/Akt-mediated inhibition of GSK-3β. PI3K inhibition or GSK-3β upregulation abolishes this effect. Deletion of the tyrosine kinase domain eliminates EphB2 stimulation-induced GSK-3β inhibition and tau dephosphorylation, demonstrating kinase-dependence.\",\n      \"method\": \"Kinase-dead mutant, PI3K inhibitors, GSK-3β overexpression, phospho-tau immunoblotting in cell lines, primary neurons, and transgenic mice\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis plus pharmacological rescue, in vitro and in vivo, single lab\",\n      \"pmids\": [\"26119563\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"The protective effect of EphB2 against Aβ-induced depletion of surface NMDA receptors depends on EphB2's PDZ-binding motif and the presence of neuronal activity, but not on its kinase activity. The GluA2 subunit of AMPA receptors associates with EphB2's PDZ-binding motif via PDZ domain-containing proteins and promotes NMDA receptor retention at the membrane.\",\n      \"method\": \"Pharmacological treatments, primary neuronal cultures expressing wild-type or PDZ-motif/kinase-dead EphB2 mutants, biochemical co-association assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — structure-function mutagenesis with defined cellular readout, single lab\",\n      \"pmids\": [\"26589795\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Photoactivation of OptoEphB2 (kinase-active) during fear conditioning (but not minutes afterward) enhances long-term fear memory consolidation and activates CREB in lateral amygdala neurons. Kinase-dead OptoEphB2 lacks these effects. EphB2lacZ/lacZ mice lacking forward signaling show impaired long-term auditory fear conditioning memory, rescued by optoEphB2 activation.\",\n      \"method\": \"Optogenetics, kinase-dead mutant, EphB2lacZ/lacZ knockout mice, CREB phosphorylation, behavioral fear conditioning\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — optogenetic and genetic dissection with defined molecular and behavioral readouts, single lab\",\n      \"pmids\": [\"29768201\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"EphB2 tyrosine kinase forward signaling (catalytic activity is essential) controls migration of nestin-positive neural progenitor cells from the dentate notch neuroepithelium to populate the lateral suprapyramidal blade (LSB) of the dentate gyrus. Ephrin-B1 acts as the ligand for EphB2 in this context. Loss of EphB2 forward signaling dramatically reduces Reelin immunoreactivity in a compartment above the developing LSB.\",\n      \"method\": \"EphB2 kinase-dead and truncation mutants in mice, BrdU/nestin labeling, Reelin immunostaining, ephrin-B1 knockout comparison\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple genetic alleles with defined cellular phenotype, single lab\",\n      \"pmids\": [\"21832177\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"EphB2 is expressed in K+-secreting dark cells of the inner ear epithelium and is required for proper efferent axon midline guidance and for endolymph homeostasis. PDZ domain-containing proteins bind the C-terminus of EphB2 and can also recognize cytoplasmic tails of anion exchangers and aquaporins, suggesting EphB2 regulates ionic homeostasis through macromolecular complexes with membrane channels.\",\n      \"method\": \"EphB2 knockout mouse (circling behavior, vestibular phenotype analysis), PDZ binding assay, ultrastructural analysis\",\n      \"journal\": \"Neuron\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO mouse with defined phenotype plus biochemical PDZ interaction, single lab\",\n      \"pmids\": [\"10839360\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Bidirectional signaling by EphB2 (forward) and ephrin-B2 (reverse) is required for proper K+ secretion and ionic homeostasis in vestibular endolymph. Mutations disrupting EphB2 forward signaling or ephrin-B2 reverse signaling decrease K+ concentration and endolymphatic potential, demonstrating that B-subclass Eph/ephrin signaling controls epithelial ion transport function.\",\n      \"method\": \"Targeted mutations (cytoplasmic domain) of EphB2 and ephrin-B2 in mice, direct electrophysiological measurement of endolymphatic potential and K+ concentration in live animals\",\n      \"journal\": \"Hearing research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct measurement in vivo with bidirectional genetic controls, single lab\",\n      \"pmids\": [\"17158005\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"EPHB2 carried on small extracellular vesicles (exosomes) induces tumor angiogenesis by transporting the transmembrane receptor to non-adjacent endothelial cells, stimulating ephrin-B reverse signaling and STAT3 phosphorylation. A STAT3 inhibitor greatly reduces SEV-induced angiogenesis.\",\n      \"method\": \"Proteomic analysis of SEVs, EPHB2 knockdown in SEVs, in vitro and in vivo angiogenesis assays, STAT3 phosphorylation analysis, STAT3 inhibitor treatment\",\n      \"journal\": \"JCI insight\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional knockdown with defined signaling readout, single lab, multiple assays\",\n      \"pmids\": [\"31661464\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"RNF186, an E3 ubiquitin ligase, ubiquitinates EPHB2 at Lys892 upon stimulation by ligand EFNB1. Ubiquitinated EPHB2 recruits MAP1LC3B to activate autophagy in colonic epithelial cells. rnf186 and ephb2 knockout mice show more severe DSS-induced colitis due to autophagy defects in colon epithelial cells.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay (K892R mutant), autophagy markers (LC3B), KO mice + DSS colitis model\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ubiquitination site mutagenesis plus KO mouse model with defined phenotype, single lab\",\n      \"pmids\": [\"33280498\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"EPHB2 drives liver cancer stemness and sorafenib resistance through activation of the SRC/AKT/GSK3β/β-catenin signaling cascade. EPHB2 expression is regulated by TCF1 via promoter activation, forming a positive Wnt/β-catenin feedback loop. EPHB2 knockout attenuates tumor development and rAAV-8-shEPHB2 sensitizes HCC cells to sorafenib.\",\n      \"method\": \"EPHB2 knockout (endogenous), RNA-seq, signaling pathway analysis, xenograft and immunocompetent mouse models, rAAV-mediated shRNA knockdown\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO plus in vivo knockdown with defined signaling cascade, single lab\",\n      \"pmids\": [\"33903122\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"EphB2, acting as a downstream effector of Notch signaling in hepatocytes, is sufficient to induce cell-autonomous inflammation during NASH progression. Knockdown of Ephb2 in hepatocytes ameliorates inflammation and fibrosis in a mouse NASH model. EphB2-expressing hepatocytes are specifically demarcated as a profibrotic/inflammatory subpopulation.\",\n      \"method\": \"Single-nucleus transcriptomics/epigenomics, Notch pathway analysis, hepatocyte-specific Ephb2 knockdown in NASH mouse model, inflammation/fibrosis readouts\",\n      \"journal\": \"Science translational medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — hepatocyte-specific knockdown with defined mechanistic pathway placement, replicated in mouse and human data\",\n      \"pmids\": [\"36753562\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"EphB2 is upregulated and activated in hepatic stellate cells (HSCs) during fibrogenesis. EphB2-deficient HSCs show impaired proliferation and suboptimal differentiation into fibrogenic myofibroblasts in vitro, identifying HSC activation as a cellular mechanism through which EphB2 promotes liver fibrosis.\",\n      \"method\": \"EphB2 knockout mice, in vitro HSC activation, fibrosis markers, apoptosis assays\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO mouse with in vitro mechanistic follow-up in HSCs, single lab\",\n      \"pmids\": [\"29416088\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Tissue factor (TF)/coagulation factor VIIa (FVIIa) cleaves EphB2 ectodomain at a key arginine residue (identified by Edman sequencing and LC-MS/MS). This cleavage is independent of PAR2 signaling and potentiates cell repulsion mediated by the EphB2 ligand ephrin-B1, identifying TF/FVIIa as a novel protease controlling Eph-mediated cell segregation.\",\n      \"method\": \"N-terminal Edman sequencing, LC-MS/MS, PAR2 inhibition, cell repulsion assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MS-based cleavage site identification plus functional cell repulsion assay, single lab\",\n      \"pmids\": [\"25281742\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"A human missense mutation p.R745C in the EphB2 kinase domain impairs EphB2 autophosphorylation but not ephrin ligand-induced EphB2 clustering. This results in defects in platelet aggregation, αIIbβ3 activation, granule secretion, and thrombus formation. EPHB2 positively regulates GPVI platelet signaling (Lyn, Syk, FcRγ phosphorylation) and Src activation downstream of PAR4.\",\n      \"method\": \"Whole-exome sequencing, patient platelets, overexpression in RBL-2H3 cells, platelet aggregation/flow assays, phosphorylation analysis\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — human mutation with defined kinase-activity defect, validated in patient cells and heterologous expression, multiple functional readouts\",\n      \"pmids\": [\"30213874\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"EphB2 co-associates with and activates focal adhesion kinase (FAK) in glioblastoma neurosphere cells, facilitating focal adhesion formation, cytoskeletal changes, and cell migration. EphB2 expression stimulates invasion and inhibits proliferation in vitro and in vivo.\",\n      \"method\": \"Co-immunoprecipitation (EphB2-FAK), EphB2 overexpression/silencing, FAK phosphorylation analysis, migration/invasion/proliferation assays, xenograft model\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus genetic gain/loss of function with functional readout, single lab\",\n      \"pmids\": [\"22310282\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"EphB2 signaling induces mitochondrial translocation of Sirt3. EphrinB2-Fc induces nuclear translocation of Nrf-2 via c-Src phosphorylation, and Sirt3 expression is regulated by Nrf-2. EphB2/Sirt3 signaling increases MnSOD activity (by deacetylation at Lys68) to reduce mitochondrial ROS and suppress MSC senescence.\",\n      \"method\": \"EphB2 knockdown, Sirt3 knockdown, subcellular fractionation, Nrf-2/Src phosphorylation analysis, MnSOD deacetylation/activity assay, ROS measurement\",\n      \"journal\": \"Free radical biology & medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic knockdown with defined mechanistic chain, single lab, multiple orthogonal readouts\",\n      \"pmids\": [\"28687409\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Ephrin-B2 acts on mouse and human DRG neurons via EphB2 to induce nociceptor plasticity and hyperalgesic priming through MNK-eIF4E signaling. Sensory neuron-specific EphB2 knockout (Pirt-Cre) abrogates ephrin-B2-induced mechanical hypersensitivity and enhanced Ca2+ responses to PGE2. MNK1 knockout or MNK inhibitor (eFT508) blocks acute and priming responses.\",\n      \"method\": \"Conditional EphB2 knockout (Pirt-Cre), MNK1 knockout, MNK inhibitor, Ca2+ imaging in cultured DRG neurons (mouse and human), behavioral pain assays\",\n      \"journal\": \"Pharmacological research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — neuron-specific KO plus pharmacological validation with functional readout in two species, single lab\",\n      \"pmids\": [\"38925462\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"EphB2 receptor repulsion trans-endocytosis requires Eps15R and clathrin. Eps15R (but not Eps15) knockdown significantly reduces EphB2-mediated cell repulsion. A novel DPFxxLDPF motif in Eps15R binds directly to the clathrin terminal domain in vitro; wild-type Eps15R but not the clathrin-binding mutant rescues cell repulsion.\",\n      \"method\": \"siRNA knockdown, in vitro clathrin-binding assay, rescue experiments in co-culture cell repulsion assay\",\n      \"journal\": \"Traffic\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro binding plus genetic rescue in cell repulsion assay, single lab\",\n      \"pmids\": [\"28972287\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"EphB2 activation in the medial prefrontal cortex (mPFC) via EphrinB1-Fc produces stress-resistant and antidepressant-like effects. EphB2 shRNA knockdown in the mPFC increases stress susceptibility. These effects are associated with changes in cofilin phosphorylation, membrane AMPA receptor trafficking, and spine remodeling.\",\n      \"method\": \"Lentiviral shRNA knockdown, EphrinB1-Fc intra-mPFC injection, behavioral assays, cofilin phosphorylation, AMPAR surface trafficking, spine analysis\",\n      \"journal\": \"Neuropsychopharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — bidirectional genetic manipulation with defined molecular and behavioral readouts, single lab\",\n      \"pmids\": [\"27103064\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Loss of EPHB2 in prostate cancer cells increases lipogenic DGAT1, DGAT2, and PLIN2 and decreases lipolytic ATGL and PEDF, leading to lipid droplet accumulation in cytoplasmic and nuclear compartments. A DGAT1-specific inhibitor abrogates LD accumulation and proliferative effects induced by EPHB2 loss, mechanistically linking EPHB2 to lipid homeostasis via DGAT1/ATGL regulation.\",\n      \"method\": \"EPHB2 siRNA knockdown, DGAT1 inhibitor, lipid droplet quantification, proliferation assays, protein expression analysis\",\n      \"journal\": \"Laboratory investigation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — knockdown plus pharmacological rescue with defined molecular targets, single lab\",\n      \"pmids\": [\"33824421\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"DRT (EPHB2/EPHT3) encodes a receptor-type tyrosine kinase of the EPH family. Its 11 kb transcript is preferentially expressed in fetal brain and is upregulated upon retinoic acid-induced neuronal differentiation of NTera-2 cells, indicating a role in neurogenesis. The gene was mapped to human chromosome 1p35-1p36.1.\",\n      \"method\": \"cDNA library screening with anti-phosphotyrosine antibody, 5' RACE, RT-PCR, chromosomal mapping by somatic cell hybrid panels and FISH, retinoic acid differentiation assay\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — initial cloning and expression characterization without mechanistic functional assays\",\n      \"pmids\": [\"8589679\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"EphB2 signaling in B cells promotes proliferation, TNF-α secretion, and IgG production in human naive B cells. EphB2 knockdown attenuates B-cell activation via reduction of Src-p65 (NF-κB) and Notch1 signaling pathways.\",\n      \"method\": \"EphB2 siRNA in human primary B cells, proliferation assay, ELISA (cytokines/Ig), Src/p65/Notch1 pathway analysis\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — siRNA knockdown with pathway measurement, single lab, limited mechanistic depth\",\n      \"pmids\": [\"24803541\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"EphB2 is a transmembrane receptor tyrosine kinase that is autoinhibited by its juxtamembrane segment (which distorts the kinase activation loop) and activated by phosphorylation of conserved juxtamembrane tyrosines (Y605/Y611), which also create docking sites for SH2-domain proteins including Src, Abl/Arg, and RasGAP; it binds both ephrin-B ligands (canonical) and ephrin-A5 (cross-subclass) to form distinct signaling complexes, signals bidirectionally with ephrins to control cell repulsion via MMP-mediated ectodomain cleavage, Rac/Tiam2-dependent trans-endocytosis, and a Nck/Pak/RhoA pathway for growth cone collapse; in neurons it regulates NMDA receptor surface expression and function both through kinase-dependent mechanisms (direct NR1/NR2 phosphorylation via the γ-secretase-released CTF2 fragment, and PI3K/Akt/GSK-3β-mediated tau dephosphorylation) and kinase-independent mechanisms (PDZ-motif-mediated GluA2/NMDAR retention); its activity is modulated by neuropsin cleavage, ADAM10/MMP ectodomain shedding, presenilin/γ-secretase processing, RNF186-mediated ubiquitination at K892 (triggering autophagy), and amyloid-β-induced proteasomal degradation; and in non-neural contexts EphB2 drives intestinal progenitor proliferation (via Abl-cyclin D1) and migration (via PI3K) through distinct pathways, promotes liver fibrosis via hepatic stellate cell activation, regulates platelet GPVI/GPCR signaling through its kinase domain, and in cancer contexts activates SRC/AKT/GSK3β/β-catenin and R-Ras/MAPK cascades to control invasion, stemness, and drug resistance.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"EphB2 is a transmembrane receptor tyrosine kinase that governs cell repulsion, axon guidance, synaptic function, and tissue patterning through ephrin-driven bidirectional signaling [#2, #12]. Its catalytic activity is held in check by an autoinhibitory juxtamembrane segment that adopts a helical conformation distorting the kinase small lobe and blocking the activation segment; autophosphorylation of conserved juxtamembrane tyrosines (notably Y605/Y611) relieves this autoinhibition and creates phosphotyrosine docking sites for SH2-domain effectors including Src and the Abl/Arg non-receptor kinases [#0, #3, #4]. Ligand engagement occurs through a jellyroll beta-sandwich ligand-binding domain that binds ephrin-B ligands and, across subclass, ephrin-A5, with clustering above the dimer threshold determining repulsive signaling strength [#1, #2, #16]. Downstream, EphB2 controls cytoskeletal collapse and contact repulsion through multiple effector arms: recruitment of p120RasGAP to downregulate Ras-Erk, a Pak/Nck/RhoA pathway for growth cone collapse, Rac/Tiam2-dependent and Eps15R/clathrin-mediated trans-endocytosis, and MMP-2/MMP-9 ectodomain cleavage required for repulsion [#11, #12, #17, #18, #36]. EphB2 is sequentially processed by ectodomain sheddases and presenilin/γ-secretase to release an intracellular CTF2 fragment that retains kinase activity and directly phosphorylates NMDA receptor subunits to promote their surface expression [#9, #10]. In neurons EphB2 regulates NMDA receptor-mediated currents and synaptic plasticity through both kinase-dependent and kinase-independent (including PDZ-motif-mediated) mechanisms, and amyloid-β oligomers bind its fibronectin repeats to trigger proteasomal degradation, linking EphB2 to memory deficits [#5, #6, #7, #21]. Beyond the nervous system, EphB2 partitions intestinal progenitor proliferation (Abl-cyclin D1) from migration (PI3K), drives cancer stemness and drug resistance via SRC/AKT/GSK3β/β-catenin and R-Ras/MAPK cascades, promotes liver fibrosis and NASH inflammation, and supports platelet GPVI/PAR signaling through its kinase domain, where a human p.R745C mutation impairing autophosphorylation causes a platelet function disorder [#13, #14, #28, #29, #30, #32].\",\n  \"teleology\": [\n    {\n      \"year\": 1995,\n      \"claim\": \"Establishing EPHB2 as an EPH-family receptor tyrosine kinase enriched in fetal brain and induced during neuronal differentiation framed it as a candidate regulator of neurogenesis.\",\n      \"evidence\": \"cDNA cloning, expression profiling, and chromosomal mapping in differentiating NTera-2 cells\",\n      \"pmids\": [\"8589679\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"Initial cloning lacks any mechanistic functional assay\",\n        \"No ligand or downstream pathway defined at this stage\"\n      ]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Solving the ligand-binding domain structure and identifying Y611 as the SH2 docking site for Src defined how EphB2 recognizes ephrins and how its phosphorylated juxtamembrane region recruits effectors.\",\n      \"evidence\": \"X-ray crystallography of the ligand-binding domain plus Y2H, mutagenesis, Co-IP and phosphopeptide mapping for Src association\",\n      \"pmids\": [\"9853759\", \"9632142\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Did not resolve the autoinhibited conformation of the kinase domain\",\n        \"Full repertoire of SH2 effectors not yet enumerated\"\n      ]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"EphB2 knockout phenotypes in the inner ear revealed a role in efferent axon guidance and epithelial ion homeostasis through PDZ-mediated complexes with membrane channels, extending its function beyond axon repulsion.\",\n      \"evidence\": \"EphB2 knockout mouse phenotyping plus PDZ-binding assays\",\n      \"pmids\": [\"10839360\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Direct channel partners within the macromolecular complex not biochemically confirmed in vivo\",\n        \"Forward versus reverse signaling contributions not separated here\"\n      ]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Crystallography of the autoinhibited kinase domain and dissection of Abl/Arg binding established the structural switch underlying activation and the breadth of non-receptor kinase effectors, while knockout/knock-in mice showed EphB2 regulates NMDA receptor synaptic function partly independently of its kinase domain.\",\n      \"evidence\": \"1.9 Å crystal structure with mutagenesis; Y2H and Co-IP for Abl/Arg; EphB2-null and truncated knock-in mice with electrophysiology and immunogold\",\n      \"pmids\": [\"11572780\", \"11494128\", \"11754836\", \"11754835\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Molecular basis of kinase-independent NMDA receptor regulation left undefined\",\n        \"How distinct Abl/Arg interaction modes are coordinated in vivo unresolved\"\n      ]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Re-engineering juxtamembrane docking sites showed that RasGAP recruitment downregulates Ras-Erk to drive neurite retraction, providing a defined effector pathway linking EphB2 phosphorylation to cytoskeletal collapse.\",\n      \"evidence\": \"Receptor mutagenesis with phospho-Erk immunoblotting and neurite retraction assays in NG108 neurons\",\n      \"pmids\": [\"12486127\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Relative contribution of RasGAP versus other repulsive arms not quantified\",\n        \"In vivo relevance of this pathway not tested\"\n      ]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Demonstrating high-affinity cross-subclass binding of EphB2 to ephrin-A5 with a distinct complex architecture revised the rule that EphB and ephrin-A pairs do not interact, broadening EphB2's ligand repertoire and signaling outputs.\",\n      \"evidence\": \"Crystallography plus receptor clustering, autophosphorylation, and growth cone collapse assays\",\n      \"pmids\": [\"15107857\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Physiological contexts where ephrin-A5/EphB2 signaling operates not established\",\n        \"Whether cross-subclass complexes assemble in vivo unaddressed\"\n      ]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Identifying R-Ras as an EphB2-associated effector controlling glioma adhesion, proliferation, and invasion linked EphB2 to a tumor-relevant MAPK-suppressing axis.\",\n      \"evidence\": \"Co-IP, siRNA depletion, and adhesion/invasion/proliferation assays with MAPK readout\",\n      \"pmids\": [\"16049340\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Single-lab Co-IP without reciprocal structural mapping\",\n        \"Direct versus indirect EphB2-R-Ras association not resolved\"\n      ]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Mapping sequential ectodomain shedding and presenilin/γ-secretase cleavage that releases EphB2/CTF2 revealed regulated intramembrane proteolysis as a mechanism converting receptor activation into an intracellular signaling fragment.\",\n      \"evidence\": \"Pharmacological inhibitors, endocytosis blockade, biochemical cleavage mapping, and ubiquitination assays\",\n      \"pmids\": [\"17428795\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Function of the released CTF2 fragment not yet demonstrated here\",\n        \"Identity of the initial ectodomain sheddase only partially defined\"\n      ]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Showing MMP-2/MMP-9 ectodomain cleavage is required for repulsion, and that FGFR1 suppresses EphB2 via LAR phosphatase and Sprouty feedback, established proteolytic and cross-receptor control of EphB2 segregation activity.\",\n      \"evidence\": \"MMP inhibitors and cleavage-resistant mutants with rescue in EphB-deficient neurons; LAR knockdown, MAPK inhibitors, and Sprouty analysis in segregation assays\",\n      \"pmids\": [\"18713744\", \"19047466\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Quantitative integration of cleavage with clustering thresholds not addressed\",\n        \"FGFR1 cross-talk validated in cell-based assays only\"\n      ]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Demonstrating that EphB2/CTF2 directly phosphorylates NR1/NR2 independently of Src, and that EphB2 segregates intestinal proliferation (Abl-cyclin D1) from migration (PI3K), defined parallel kinase-dependent and kinase-independent output modules whose uncoupling enables cancer.\",\n      \"evidence\": \"In vitro kinase assays with kinase-dead mutants and Src inhibitors; genetic dissection of intestinal progenitors with pathway inhibitors\",\n      \"pmids\": [\"19661068\", \"19914164\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Substrate selectivity of CTF2 beyond NMDA subunits not mapped\",\n        \"Mechanism dissociating proliferation and migration pathways in carcinoma unresolved\"\n      ]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Identifying amyloid-β oligomer binding to EphB2 fibronectin repeats with consequent proteasomal degradation, and rescue of LTP/memory by EphB2 restoration, placed EphB2 mechanistically in Alzheimer's disease synaptic dysfunction.\",\n      \"evidence\": \"Biochemical Aβ binding, in vivo shRNA/overexpression, electrophysiology, and behavioral memory tests in hAPP mice\",\n      \"pmids\": [\"21113149\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Degradation machinery downstream of Aβ binding not fully defined\",\n        \"Whether degradation acts on full-length receptor or processed fragments unclear\"\n      ]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Neuropsin-mediated cleavage controlling EphB2-NR1 dissociation in the amygdala, and EphB2 kinase-dependent control of dentate progenitor migration, showed proteolytic and forward-signaling regulation of EphB2 in behavior and neurodevelopment.\",\n      \"evidence\": \"Neuropsin-KO mice with EphB2-NR1 Co-IP, NMDA currents, and anxiety assays; EphB2 kinase-dead/truncation alleles with BrdU/nestin and Reelin staining\",\n      \"pmids\": [\"21508957\", \"21832177\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Generality of neuropsin cleavage outside the amygdala not established\",\n        \"Reelin link to progenitor migration mechanistically indirect\"\n      ]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Defining clustering-threshold-dependent repulsion, TF/FVIIa ectodomain cleavage potentiating repulsion, and EphB2-FAK coupling in glioblastoma refined the quantitative and proteolytic control of EphB2 signaling strength and migration.\",\n      \"evidence\": \"Chemical dimerizers and fluorescence anisotropy; Edman/LC-MS cleavage mapping; Co-IP and gain/loss-of-function with FAK phosphorylation\",\n      \"pmids\": [\"24469634\", \"25281742\", \"22310282\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"In vivo relevance of cluster-size thresholds not tested\",\n        \"TF/FVIIa cleavage characterized in single-lab cell assays\"\n      ]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Identifying Rac/Tiam2-dependent F-actin enrichment driving trans-endocytosis and antidepressant-like mPFC effects of EphB2 activation linked specific GTPase machinery and synaptic remodeling to EphB2 forward signaling outcomes.\",\n      \"evidence\": \"siRNA Rho-GTPase screen, Tiam2 knockdown, and trans-endocytosis/repulsion assays; mPFC shRNA/EphrinB1-Fc with cofilin, AMPAR, and behavioral readouts\",\n      \"pmids\": [\"27597758\", \"27103064\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"How Rac is locally activated at internalization sites unresolved\",\n        \"mPFC effects mechanistically correlative beyond cofilin/AMPAR changes\"\n      ]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Demonstrating Eps15R/clathrin dependence of trans-endocytosis, Abl2/Arg-dependent dendritic filopodia growth, and an EphB2/Sirt3 antioxidant axis expanded the effector network connecting EphB2 to endocytic, cytoskeletal, and redox outcomes.\",\n      \"evidence\": \"siRNA and in vitro clathrin-binding rescue; optogenetic EphB2 with Arg knockdown and actin imaging; knockdown with Nrf-2/Src and MnSOD deacetylation assays\",\n      \"pmids\": [\"28972287\", \"29158322\", \"28687409\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Each pathway validated in single labs and distinct cell systems\",\n        \"Integration of endocytic and actin-nucleating arms not unified\"\n      ]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"A human kinase-domain mutation (p.R745C) impairing autophosphorylation but not clustering established EphB2 as a positive regulator of platelet GPVI/PAR signaling and the basis of a human platelet function disorder, while optogenetic studies placed EphB2 forward signaling in fear memory consolidation and HSC-driven liver fibrosis.\",\n      \"evidence\": \"Whole-exome sequencing with patient platelets and heterologous expression; optogenetics with kinase-dead and lacZ-null mice and CREB readout; EphB2-KO mice with in vitro HSC activation\",\n      \"pmids\": [\"30213874\", \"29768201\", \"29416088\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Direct platelet substrates of EphB2 not defined\",\n        \"Whether HSC fibrosis effect is cell-autonomous unresolved\"\n      ]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Showing exosome-delivered EphB2 stimulates ephrin-B reverse signaling and STAT3 phosphorylation in endothelial cells revealed a non-cell-autonomous, vesicle-mediated route for EphB2 to drive tumor angiogenesis.\",\n      \"evidence\": \"SEV proteomics, EphB2 knockdown in SEVs, angiogenesis assays, and STAT3 inhibitor treatment\",\n      \"pmids\": [\"31661464\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Mechanism of EphB2 loading into exosomes not defined\",\n        \"Direct endothelial receptor mediating reverse signaling not identified\"\n      ]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Identifying RNF186-mediated ubiquitination of EphB2 at K892 triggering LC3B-dependent autophagy in colonic epithelium connected EphB2 to a post-translational autophagy switch protective against colitis.\",\n      \"evidence\": \"Co-IP, K892R ubiquitination mutant, LC3B autophagy markers, and rnf186/ephb2 KO mice in DSS colitis\",\n      \"pmids\": [\"33280498\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"How ubiquitinated EphB2 recruits the autophagy machinery mechanistically unresolved\",\n        \"Single-lab finding without reciprocal validation\"\n      ]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Defining a TCF1-EphB2-SRC/AKT/GSK3β/β-catenin positive feedback loop driving liver cancer stemness and sorafenib resistance, and an EphB2-DGAT1/ATGL lipid-homeostasis link in prostate cancer, established opposing context-dependent roles of EphB2 in malignancy.\",\n      \"evidence\": \"EPHB2 KO and rAAV-shRNA with RNA-seq and xenograft models; siRNA knockdown with DGAT1 inhibitor and lipid droplet quantification\",\n      \"pmids\": [\"33903122\", \"33824421\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Reconciliation of tumor-promoting versus tumor-suppressive roles across cancers unresolved\",\n        \"Direct kinase substrates within these cascades not identified\"\n      ]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Placing EphB2 as a Notch-downstream effector marking a profibrotic/inflammatory hepatocyte subpopulation showed it is sufficient to drive cell-autonomous inflammation in NASH progression.\",\n      \"evidence\": \"Single-nucleus multi-omics and hepatocyte-specific Ephb2 knockdown in a mouse NASH model with human data\",\n      \"pmids\": [\"36753562\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Downstream inflammatory effectors of hepatocyte EphB2 not fully defined\",\n        \"Ligand driving hepatocyte EphB2 activation unidentified\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Demonstrating ephrin-B2/EphB2 signaling through MNK-eIF4E to induce nociceptor plasticity and hyperalgesic priming in mouse and human DRG neurons defined a translational-control mechanism for EphB2 in pain.\",\n      \"evidence\": \"Sensory-neuron-specific EphB2 KO, MNK1 KO, MNK inhibitor, Ca2+ imaging, and behavioral pain assays in two species\",\n      \"pmids\": [\"38925462\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"How EphB2 forward signaling couples to MNK-eIF4E not biochemically mapped\",\n        \"Whether receptor proteolysis participates in this pathway untested\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How EphB2's multiple effector arms, proteolytic processing events, and context-specific oncogenic versus tumor-suppressive outputs are coordinated at the level of a single activated receptor remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"No unified model linking clustering state to choice of effector pathway\",\n        \"Determinants of opposing roles across tumor types not established\",\n        \"Comprehensive substrate map of full-length EphB2 and CTF2 lacking\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [3, 9, 10, 20, 32]},\n      {\"term_id\": \"GO:0001618\", \"supporting_discovery_ids\": [1, 2]},\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [2, 12, 25]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [11, 21, 24]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [9, 16, 12]},\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [17, 26, 36]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 2, 11, 13]},\n      {\"term_id\": \"R-HSA-112316\", \"supporting_discovery_ids\": [5, 7, 8, 18, 22]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [9, 12, 27, 31]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [28, 29, 33, 38]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [13, 23, 24, 25]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"EFNB1\", \"EFNB2\", \"EFNA5\", \"SRC\", \"ABL1\", \"RASA1\", \"PAK1\", \"PTK2\"],\n    \"other_free_text\": []\n  }\n}\n```","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}