{"gene":"EPHB3","run_date":"2026-06-09T23:54:43","timeline":{"discoveries":[{"year":1998,"finding":"EphB3 (HEK2) interacts via its C-terminal PDZ-binding motif with the PDZ domain of the Ras-binding protein AF6; this interaction with full-length AF6 (but not the isolated PDZ domain) depends on EphB3 kinase activity. Endogenous AF6 is phosphorylated by EphB3 and EphB2 in a ligand-dependent fashion in NIH 3T3 and NG108 cells.","method":"Yeast two-hybrid, mutational analysis, immunoprecipitation, in-cell phosphorylation assays","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal interaction confirmed by yeast two-hybrid and co-IP, kinase-dependence shown by mutant, replicated in multiple cell lines","pmids":["9707552"],"is_preprint":false},{"year":1998,"finding":"Tyrosine-614 (Y614) is the major autophosphorylation site of EphB3 (HEK2) and functions as a multi-docking site for SH2-domain-containing proteins rasGAP (via its N-terminal SH2 domain), Crk, and Fyn; a Y614F substitution abolishes binding of all three.","method":"In vitro binding assays, immunoprecipitation, yeast two-hybrid, site-directed mutagenesis (Y614F)","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro assay combined with site-directed mutagenesis and co-IP; multiple orthogonal methods in single study","pmids":["9674711"],"is_preprint":false},{"year":1998,"finding":"Ephrin-B3 is a high-affinity ligand for EphB3 (Kd ~1 nM) with selectivity among the transmembrane ephrin-B family; it is expressed at the dorsal and ventral midline of the neural tube (floor plate).","method":"Receptor-binding affinity assays, in situ hybridization","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct binding measurement with defined Kd, anatomical localization by ISH; single lab","pmids":["9484836"],"is_preprint":false},{"year":2002,"finding":"Human RYK associates with EphB2 and EphB3, requiring both extracellular and cytoplasmic domains of RYK; this association is not dependent on Eph receptor activation. Unlike murine Ryk, human RYK is not phosphorylated by EphB3.","method":"Co-immunoprecipitation, in vitro binding assays","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal co-IP and domain-deletion analysis; single lab","pmids":["11956217"],"is_preprint":false},{"year":2004,"finding":"EphB3 kinase activity is required for inhibition of integrin-mediated cell adhesion and induction of cell rounding, but directional cell migration inhibition is kinase-independent. Both wild-type and kinase-dead EphB3 reduce Rac1 and Cdc42 activity upon ephrin-B1 stimulation, with a relative increase in RhoA signaling mediating migration inhibition.","method":"Stable transfection of WT vs. kinase-dead EphB3, adhesion assays, migration assays, GTPase activity assays, pharmacological inhibition, integrin-activating antibodies","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple orthogonal functional assays with kinase-dead mutant, GTPase measurements, and pharmacological rescue in same study","pmids":["15536074"],"is_preprint":false},{"year":2006,"finding":"EphB3 expressed by recruited macrophages at the optic nerve injury site supports adult retinal ganglion cell (RGC) axon re-extension and sprouting after injury; loss of EphB3 greatly decreases axon plasticity without affecting intrinsic axon growth potential. Injured RGC axons express EphrinB3 and bind recombinant EphB3 protein in vivo.","method":"In vivo optic nerve injury in EphB3 heterozygous and null mice, mRNA expression analysis, recombinant protein injection, in vitro axon outgrowth and turning assays","journal":"The Journal of neuroscience","confidence":"High","confidence_rationale":"Tier 2 / Strong — loss-of-function genetic model with specific phenotypic readout, in vivo protein-binding, in vitro functional assays, multiple orthogonal methods","pmids":["16554460"],"is_preprint":false},{"year":2009,"finding":"EphB3 overexpression in HT-29 colorectal cancer cells promotes mesenchymal-to-epithelial transition (MET): reorganizes cortical actin, increases E-cadherin/ZO-1/plakoglobin, decreases fibronectin and nuclear β-catenin, inactivates CrkL-Rac1, increases Ca2+-dependent cell-cell adhesion, and suppresses tumor growth in xenografts.","method":"Stable transfection, morphological analysis, immunofluorescence, transwell migration, soft agar assay, xenograft, GTPase assays","journal":"Carcinogenesis","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple functional readouts with defined molecular markers; single lab","pmids":["19483190"],"is_preprint":false},{"year":2010,"finding":"EphB3 signaling suppresses neural stem/progenitor cell (NSPC) proliferation in the subventricular zone in a p53-dependent manner. EphB3 activation by ephrinB3-Fc increases p53 expression; pharmacological inhibition or siRNA knockdown of p53 attenuates ephrinB3-Fc-mediated growth suppression. In the absence of ligand, EphB3 induces cell death.","method":"EphB3-/- and ephrinB3-/- knockout mice, BrdU incorporation, Ki67 immunostaining, lateral ventricle ephrinB3-Fc infusion, siRNA knockdown of p53, pharmacological p53 inhibition, cultured NSPCs","journal":"Stem cells","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO with specific phenotype, epistasis via p53 knockdown/inhibition, multiple orthogonal readouts","pmids":["20496368"],"is_preprint":false},{"year":2011,"finding":"In NSCLC cells, EphB3 promotes cell growth and migration in a kinase-independent manner when overexpressed; silencing EphB3 reduces DNA synthesis, activates caspase-8-mediated apoptosis, and increases focal adhesion accumulation to suppress migration.","method":"Stable overexpression and siRNA knockdown in NSCLC cell lines, proliferation/migration assays, xenograft models, apoptosis assays, focal adhesion staining","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss- and gain-of-function with defined molecular readouts; kinase-independence established by kinase-dead constructs in related study context; single lab","pmids":["21266352"],"is_preprint":false},{"year":2012,"finding":"Kinase-activated EphB3 in NSCLC assembles a ternary signaling complex comprising RACK1, PP2A, and Akt, leading to reduced Akt phosphorylation and inhibition of cell migration. RACK1 mediates assembly of this complex in response to EphB3 activation.","method":"Co-immunoprecipitation, forced EphB3 kinase activation, in vitro migration assay, in vivo metastasis seeding, identification of RACK1 as EphB3-binding protein","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — novel binding partner identified by co-IP, ternary complex assembled, functional consequence (Akt dephosphorylation, migration inhibition) demonstrated in vitro and in vivo","pmids":["22314363"],"is_preprint":false},{"year":2012,"finding":"EphB2 and EphB3 forward signaling (not reverse signaling) are required for palatal mesenchyme proliferation and palate development; foetuses with cytoplasmic-truncated EphB2 (capable of reverse but not forward signaling) plus EphB3-null develop cleft palate due to reduced palatal shelf proliferation.","method":"Genetic epistasis using kinase-dead/truncated EphB2 knockin and EphB3-/- compound mutant mice, palatal shelf culture, BrdU proliferation assays","journal":"Mechanisms of development","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis with allelic series distinguishing forward vs. reverse signaling; specific cellular phenotype (reduced proliferation) quantified","pmids":["19032981"],"is_preprint":false},{"year":2014,"finding":"SNAIL1 silences EPHB3 by competitively displacing the stem cell factor ASCL2 from an E-box motif in the EPHB3 promoter/enhancer, then recruiting corepressor complexes containing HDACs and the histone demethylase LSD1, collapsing active chromatin. SNAIL2, but not ZEB1, also represses EPHB3 reporter constructs.","method":"Reporter assays, ChIP for ASCL2/p300/TCF7L2/LSD1/HDACs, endogenous locus analysis, xenotransplant models with/without sustained EPHB3 expression","journal":"Molecular oncology","confidence":"High","confidence_rationale":"Tier 2 / Strong — ChIP demonstrating displacement of activators and recruitment of repressors, functional rescue by EPHB3 re-expression, multiple orthogonal methods","pmids":["25277775"],"is_preprint":false},{"year":2014,"finding":"A transcriptional enhancer at the EPHB3 locus integrates input from ASCL2, Wnt/β-catenin, MAP kinase, and Notch signaling; low Notch activity causes enhancer decommissioning and EPHB3 silencing in colorectal carcinoma. Restoring Notch activity re-establishes enhancer function and EPHB3 expression.","method":"Enhancer reporter assays, ChIP, Notch pathway manipulation, gene expression analysis in cell lines and tumors","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — functional enhancer mapping, ChIP for pathway effectors, rescue by Notch activation; multiple orthogonal methods","pmids":["24707046"],"is_preprint":false},{"year":2014,"finding":"EphB3 functions as a dependence receptor in neurons: in the absence of ephrinB3 ligand, EphB3 is cleaved by caspases and induces cell death; ephrinB3 stimulation blocks this cleavage and prevents apoptosis. Infusion of clustered ephrinB3-Fc reduces cortical infarct and cell death after controlled cortical impact injury via EphB3-dependent mechanism.","method":"EphB3-/- and ephrinB3-/- mice, controlled cortical impact injury, TUNEL staining, ephrinB3-Fc infusion, caspase cleavage assays","journal":"Cell death & disease","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO distinguishes EphB3-dependent from EphB3-independent effects; caspase cleavage demonstrated; ephrinB3-Fc rescue only in EphB3-expressing mice","pmids":["24810043"],"is_preprint":false},{"year":2015,"finding":"EphB3 mediates oligodendrocyte cell death in the injured spinal cord through a dependence receptor mechanism; ephrinB3 ligand administration promotes oligodendrocyte survival both in vivo and in vitro under pro-apoptotic conditions. Genetic ablation of EphB3 increases myelin basic protein expression and improves locomotor function after SCI.","method":"EphB3-/- mice, contusive spinal cord injury, oligodendrocyte survival assays, ephrinB3 ligand administration, locomotor behavioral assessment","journal":"Cell death & disease","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO with behavioral and molecular rescue, ligand rescue in vitro and in vivo, multiple readouts","pmids":["26469970"],"is_preprint":false},{"year":2016,"finding":"TCF7L1 represses EPHB3 expression in colorectal cancer cells; loss of TCF7L1 activates EPHB3 as a CTNNB1/TCF target gene. Knockdown of EPHB3 partially restores growth and normal cell cycle progression in TCF7L1-null CRC cells.","method":"TCF7L1 knockdown/knockout in CRC cells, gene expression analysis, EPHB3 siRNA epistasis, xenograft tumor models, colony formation assays","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — epistasis by double knockdown, xenograft validation; single lab","pmids":["27333864"],"is_preprint":false},{"year":2016,"finding":"EphB3 stimulates migration and metastasis in papillary thyroid cancer in a kinase-dependent manner through activation of Vav2, a Rho GEF, leading to increased Rac1 activity and decreased RhoA activity. EphB3 knockdown suppresses Vav2 and reverses this GTPase balance.","method":"siRNA knockdown, EphB3 overexpression, ephrin-B1/B2-Fc stimulation, EphB3-Fc blocking, Rho GTPase activity assays, in vivo metastasis model","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — kinase-dependence shown by kinase blockade, GTPase activity measured, Vav2 identified as downstream effector; single lab","pmids":["27986811"],"is_preprint":false},{"year":2016,"finding":"The Mule E3 ubiquitin ligase targets EphB3 for proteasomal and lysosomal degradation in the intestinal stem cell niche, controlling EphB3 protein levels and thus crypt-villus cell positioning.","method":"In vivo mouse genetics (Mule conditional KO), western blotting, proteasome/lysosome inhibitor treatment","journal":"Cell stem cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo genetic model with biochemical confirmation of degradation pathway; single lab","pmids":["27184401"],"is_preprint":false},{"year":2016,"finding":"EphB3 receptors in the juxtamembrane region undergo autophosphorylation in trans (not in cis), demonstrated using isoform-specific covalent inhibitors; a unique cysteine in the EphB3 hinge region (absent in other human kinases) allows covalent inhibitor targeting. Co-crystal structures confirmed covalent linkage between EphB3 and quinazoline inhibitors.","method":"Specific irreversible inhibitor synthesis, kinase assays, co-crystal structures, 'clickable' inhibitor for proteome-wide target engagement","journal":"Journal of the American Chemical Society","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structures confirming covalent binding, in vitro kinase assay with specific inhibitor, trans-autophosphorylation mechanism validated; multiple orthogonal methods","pmids":["27478969"],"is_preprint":false},{"year":2016,"finding":"EphB3 signaling in the hippocampus after traumatic brain injury promotes synaptic loss and reduces d-serine levels; EphB3-/- mice show preserved long-term potentiation, improved hippocampal-dependent learning, and attenuated synapse loss and d-serine reduction compared to wild-type injured mice.","method":"EphB3-/- mice, controlled cortical impact injury, LTP electrophysiology, synaptic protein quantification, d-serine measurement, behavioral learning assays","journal":"Neurobiology of disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO with electrophysiological and molecular readouts; single lab","pmids":["27317833"],"is_preprint":false},{"year":2018,"finding":"EphB3 functions as a pro-apoptotic dependence receptor in cortical endothelial cells after TBI; in the absence of EphB3, endothelial cell survival is increased, blood-brain barrier permeability is reduced, and astrocyte-EC membrane interactions are enhanced after controlled cortical impact.","method":"EphB3-/- mice, CCI injury, BBB permeability assays, endothelial cell survival quantification, immunofluorescence of astrocyte-EC contacts","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO with specific vascular phenotype; single lab","pmids":["29311672"],"is_preprint":false},{"year":2018,"finding":"EphB3 suppresses glioma invasion, migration, and proliferation by downregulating the EGFR-PI3K/AKT signaling pathway; EphB3 knockdown upregulates EGFR, p-PI3K, and p-AKT, while overexpression downregulates these proteins.","method":"siRNA knockdown and overexpression in U87MG and U251 glioma cell lines, western blotting, invasion/migration/proliferation assays","journal":"Brain research","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — bidirectional modulation with consistent molecular pathway readout; single lab, no in vivo epistasis","pmids":["38341169"],"is_preprint":false},{"year":2019,"finding":"In cetuximab-resistant colorectal cancer, EGFR and EphB3 physically interact (co-immunoprecipitation), and this binding increases upon resistance acquisition, leading to STAT3 activation; inhibition of EphB3 decreases STAT3 activity.","method":"Co-immunoprecipitation, western blotting for STAT3 phosphorylation, RTK array, siRNA knockdown, cetuximab-resistant cell line model","journal":"Theranostics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP demonstrates physical EGFR-EphB3 interaction, STAT3 signaling measured after EphB3 inhibition; single lab","pmids":["31149041"],"is_preprint":false},{"year":2020,"finding":"EphB3 interacts with initiator caspases (caspase-8 or caspase-9) and the adaptor protein Dral/FHL-2 to mediate its cleavage and activate dependence receptor cell death in oligodendrocytes after brain injury; blocking this pathway with ephrinB3-Fc improves oligodendrocyte survival, myelin sparing, axonal conductance, and behavioral recovery in mice and conserved mechanism found in human TBI patients.","method":"Co-immunoprecipitation, caspase cleavage assays, EphB3-/- mice, CCI injury, ephrinB3-Fc administration, behavioral/electrophysiological readouts, human TBI tissue analysis","journal":"Brain communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — protein interaction (co-IP with FHL-2 and caspases), genetic KO rescue, ligand rescue, conserved in human samples; multiple orthogonal methods","pmids":["33305261"],"is_preprint":false},{"year":2022,"finding":"YTHDF2 binds to m6A sites in the 3'UTR of EPHB3 mRNA to decrease its stability and reduce EPHB3 protein levels, thereby activating the PI3K/Akt and NF-κB signaling pathways and promoting temozolomide resistance in glioblastoma.","method":"RNA immunoprecipitation, FISH, dual-luciferase reporter, siRNA knockdown of YTHDF2, RNA sequencing, rescue experiments with PI3K/Akt and NF-κB inhibitors","journal":"Clinical & translational immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — RNA-IP demonstrates direct m6A-YTHDF2-EPHB3 interaction, luciferase reporter validates 3'UTR binding, pathway epistasis confirmed by inhibitors; single lab","pmids":["35582627"],"is_preprint":false},{"year":2024,"finding":"EphB3 acts as a negative regulator of osteogenic differentiation in mesenchymal stromal/stem cells; absence of EphB3 signaling reduces expression of BMP pathway inhibitors, increases Bmp7 expression, increases osteoprogenitor and preosteoblast proportions, and reduces osteoclast numbers, preventing bone loss in ovariectomy and glucocorticoid-induced osteoporosis mouse models.","method":"EphB3-/- and EphB2-/- mice, in vitro MSC differentiation assays, qRT-PCR, histological staining, ovariectomy and dexamethasone bone loss models, bone volume/density measurement","journal":"Journal of bone and mineral research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO with mechanistic pathway (BMP signaling) and in vivo bone loss phenotype; single lab","pmids":["38739682"],"is_preprint":false},{"year":2025,"finding":"Astrocytic EphB3 receptors regulate synaptic d-serine availability at CA3-CA1 hippocampal synapses: stimulation of EphB3 with exogenous ephrinB3 increases d-serine and NMDAR activity, while inhibition of endogenous EphB3 impairs NMDAR-dependent LTP. Astrocyte-specific knockdown of EphB3 causes hippocampal plasticity deficits and novel object recognition memory impairment that can be rescued by exogenous d-serine.","method":"Acute hippocampal slice electrophysiology, exogenous ephrinB3 application, EphB3 inhibition, astrocyte-specific EphB3 knockdown, d-serine measurement, LTP recording, behavioral memory testing","journal":"Progress in neurobiology","confidence":"High","confidence_rationale":"Tier 2 / Strong — cell-type-specific KD with LTP/memory phenotype, d-serine rescue, multiple orthogonal methods in single study","pmids":["40081519"],"is_preprint":false},{"year":2025,"finding":"MHV68 gH/gL glycoprotein complex directly interacts with EphB3 (and EphA4), enabling viral entry; ectopic expression of EphB3 enables MHV68 infection of otherwise non-permissive human B cells, demonstrating EphB3 functions as an entry receptor for this gammaherpesvirus.","method":"Direct binding assays, ectopic EphB3 expression in non-permissive B cells, soluble decoy receptor competition, mutagenesis informed by structural predictions, neutralizing antibody adsorption","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct binding demonstrated, functional receptor role confirmed by ectopic expression enabling infection; preprint, not yet peer-reviewed","pmids":["bio_10.1101_2025.06.05.657996"],"is_preprint":true}],"current_model":"EphB3 is a receptor tyrosine kinase whose kinase activity mediates phosphorylation-dependent assembly of downstream signaling complexes (including RACK1-PP2A-Akt and SH2-domain adaptors rasGAP/Crk/Fyn at Y614), while kinase-independent mechanisms allow it to inhibit directional cell migration via Rho GTPase rebalancing; it functions as a dependence receptor that induces caspase/FHL-2-mediated apoptosis in the absence of its primary ligand ephrinB3 and is suppressed by ephrinB3 binding, regulates intestinal epithelial compartmentalization and MET, controls neural stem cell proliferation through p53, regulates synaptic NMDAR function via astrocytic d-serine release, and is transcriptionally controlled by ASCL2/Wnt/Notch/SNAIL1-mediated enhancer mechanisms and post-translationally by Mule-dependent proteasomal/lysosomal degradation."},"narrative":{"mechanistic_narrative":"EphB3 is an ephrin-B-family receptor tyrosine kinase that converts ephrinB3 engagement into bidirectional control of cell adhesion, migration, proliferation, and survival across epithelial, neural, and skeletal tissues [PMID:15536074, PMID:20496368, PMID:24810043]. Upon ligand binding it autophosphorylates in trans at juxtamembrane tyrosines, with Tyr-614 serving as the principal docking site that recruits the SH2 adaptors rasGAP, Crk, and Fyn, while its C-terminal PDZ motif binds the Ras effector AF6 in a kinase-dependent manner [PMID:9674711, PMID:9707552, PMID:27478969]. Active EphB3 nucleates downstream signaling complexes—assembling a RACK1–PP2A–Akt module that dampens Akt phosphorylation and migration in lung cancer [PMID:22314363], and rebalancing Rho-family GTPases by lowering Rac1/Cdc42 and raising RhoA to inhibit directional migration; this migratory restraint is kinase-independent whereas inhibition of integrin adhesion requires kinase activity [PMID:15536074]. In epithelia EphB3 drives a mesenchymal-to-epithelial transition with restored E-cadherin and cell–cell adhesion and suppressed tumor growth, consistent with a role in intestinal compartmentalization that is set by Mule-dependent degradation of EphB3 protein [PMID:19483190, PMID:27184401]. In the absence of ephrinB3, EphB3 behaves as a dependence receptor: it is cleaved by initiator caspases via the adaptor Dral/FHL-2 to trigger apoptosis in neurons, oligodendrocytes, and endothelial cells, and ligand binding blocks this death program—a mechanism conserved in human injury tissue [PMID:24810043, PMID:33305261, PMID:29311672]. EphB3 also restrains neural stem/progenitor proliferation through p53 [PMID:20496368], and astrocytic EphB3 controls synaptic d-serine availability and NMDAR-dependent plasticity and memory [PMID:40081519]. EPHB3 expression is tightly controlled transcriptionally—activated by an ASCL2/Wnt/Notch enhancer and silenced by SNAIL1-directed recruitment of LSD1/HDAC corepressors and by TCF7L1—and post-transcriptionally by YTHDF2-mediated m6A decay of its mRNA [PMID:24707046, PMID:25277775, PMID:27333864, PMID:35582627].","teleology":[{"year":1998,"claim":"Establishing how ligand-activated EphB3 transmits a signal: identification of its autophosphorylation site and the adaptors it recruits defined the receptor's proximal signaling output.","evidence":"Site-directed mutagenesis (Y614F), in vitro binding, yeast two-hybrid and co-IP identifying rasGAP/Crk/Fyn at Y614 and kinase-dependent AF6 binding via the PDZ motif","pmids":["9674711","9707552"],"confidence":"High","gaps":["Functional consequences of each adaptor downstream of Y614 not dissected","Whether AF6 phosphorylation alters its Ras-effector function not resolved"]},{"year":1998,"claim":"Defining the cognate ligand: ephrinB3 was shown to be a high-affinity, selective EphB3 ligand with midline expression, anchoring EphB3 in neural-tube patterning.","evidence":"Receptor-binding affinity assays (Kd ~1 nM) and in situ hybridization","pmids":["9484836"],"confidence":"Medium","gaps":["In vivo developmental phenotype of this ligand-receptor pair not addressed here","Single-lab affinity measurement"]},{"year":2004,"claim":"Separating kinase-dependent from kinase-independent EphB3 outputs clarified that adhesion control needs catalytic activity but migration inhibition does not, mediated by GTPase rebalancing toward RhoA.","evidence":"WT vs kinase-dead stable lines, adhesion/migration assays, GTPase activity assays, pharmacological rescue","pmids":["15536074"],"confidence":"High","gaps":["Molecular link between kinase-dead EphB3 and RhoA activation not identified","Effector GEF/GAP responsible not pinned down"]},{"year":2010,"claim":"Linking EphB3 to growth control: it suppresses neural stem/progenitor proliferation through p53 and induces death without ligand, foreshadowing its dependence-receptor behavior.","evidence":"EphB3-/- and ephrinB3-/- mice, BrdU/Ki67, ephrinB3-Fc infusion, p53 siRNA/inhibition epistasis","pmids":["20496368"],"confidence":"High","gaps":["Mechanism connecting EphB3 to p53 induction unresolved","Cleavage machinery for ligand-free death not yet identified at this stage"]},{"year":2012,"claim":"Identifying RACK1 as an EphB3 partner showed how kinase-active receptor assembles a phosphatase-containing complex to suppress Akt and migration.","evidence":"Co-IP, forced kinase activation, in vitro migration and in vivo metastasis assays in NSCLC","pmids":["22314363"],"confidence":"High","gaps":["How RACK1 is recruited to active EphB3 not detailed","Reconciliation with kinase-independent migration effects not addressed"]},{"year":2012,"claim":"Genetic epistasis distinguished forward from reverse signaling, establishing that EphB3 forward signaling drives palatal mesenchyme proliferation in development.","evidence":"Compound EphB2 truncation knockin / EphB3-null mice, palatal shelf culture, BrdU assays","pmids":["19032981"],"confidence":"High","gaps":["Downstream proliferative effectors in palate not defined","EphB2/EphB3 redundancy boundaries not fully mapped"]},{"year":2014,"claim":"Defining EPHB3 transcriptional control: an ASCL2/Wnt/Notch enhancer activates the gene while SNAIL1 displaces ASCL2 and recruits LSD1/HDAC corepressors to silence it, explaining loss of EPHB3 in colorectal carcinoma.","evidence":"Enhancer/promoter reporter assays, ChIP for activators and repressors, Notch manipulation, xenografts with sustained EPHB3","pmids":["24707046","25277775"],"confidence":"High","gaps":["Hierarchy among Wnt/Notch/MAPK inputs at the enhancer not fully ordered","In vivo relevance in human tumors beyond cell models limited"]},{"year":2014,"claim":"Establishing EphB3 as a dependence receptor: ligand-free EphB3 is caspase-cleaved to drive death, and ephrinB3 blocks cleavage to protect cells after brain injury.","evidence":"EphB3-/- and ephrinB3-/- mice, controlled cortical impact, TUNEL, caspase cleavage assays, ephrinB3-Fc rescue","pmids":["24810043"],"confidence":"High","gaps":["Identity of the cleavage adaptor not yet known at this point","Threshold of ligand needed to suppress death not quantified"]},{"year":2016,"claim":"Multiple layers of EphB3 regulation and context-dependent signaling were defined: Mule-mediated degradation sets protein levels in the intestinal niche, TCF7L1 represses EPHB3 transcriptionally, and EphB3 can drive or suppress migration via Vav2/Rho GTPase rebalancing depending on context.","evidence":"Mule conditional KO with degradation-pathway inhibitors; TCF7L1 knockdown with EPHB3 epistasis; Vav2/GTPase activity assays in thyroid cancer","pmids":["27184401","27333864","27986811"],"confidence":"Medium","gaps":["Reconciliation of pro- vs anti-migratory roles across tissues not unified","Single-lab findings for each regulatory axis"]},{"year":2016,"claim":"Mechanistic and structural definition of EphB3 catalysis: it autophosphorylates in trans, and a unique hinge cysteine enables covalent inhibitor targeting confirmed by co-crystal structures.","evidence":"Isoform-specific irreversible inhibitors, kinase assays, co-crystal structures, clickable probe","pmids":["27478969"],"confidence":"High","gaps":["Cellular consequences of selective EphB3 inhibition in disease not tested here","Whether trans-autophosphorylation requires specific receptor clustering geometry unresolved"]},{"year":2020,"claim":"Completing the dependence-receptor mechanism: EphB3 recruits initiator caspases and the adaptor Dral/FHL-2 to execute cleavage-dependent death, with the pathway conserved in human TBI tissue.","evidence":"Co-IP with FHL-2/caspases, caspase cleavage assays, EphB3-/- mice, ephrinB3-Fc rescue, human tissue analysis","pmids":["33305261"],"confidence":"High","gaps":["Structural basis of FHL-2/caspase recruitment to EphB3 not solved","Whether the same adaptor operates in non-CNS cell types not established"]},{"year":2022,"claim":"Post-transcriptional control was extended: YTHDF2-mediated m6A decay of EPHB3 mRNA lowers protein levels and activates PI3K/Akt and NF-κB to promote glioblastoma drug resistance.","evidence":"RNA-IP, FISH, dual-luciferase 3'UTR reporter, YTHDF2 knockdown, pathway-inhibitor rescue","pmids":["35582627"],"confidence":"Medium","gaps":["Whether EphB3 protein loss alone, versus other YTHDF2 targets, drives the pathway activation not isolated","Single-lab study"]},{"year":2025,"claim":"Defining a non-canonical CNS function: astrocytic EphB3 controls synaptic d-serine availability to set NMDAR-dependent plasticity and memory, with d-serine rescuing loss-of-function deficits.","evidence":"Astrocyte-specific EphB3 knockdown, slice LTP electrophysiology, d-serine measurement and rescue, novel object recognition","pmids":["40081519"],"confidence":"High","gaps":["Signaling link from EphB3 to d-serine synthesis/release not delineated","Relationship to EphB3's neuronal dependence-receptor role not integrated"]},{"year":null,"claim":"How EphB3 switches between kinase-dependent signaling, kinase-independent migration control, and ligand-free dependence-receptor death within a single cell, and what determines its opposing pro- versus anti-tumor outputs across tissues, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model reconciling context-specific migratory and survival outputs","Structural basis for the death-versus-signaling switch unknown","In vivo therapeutic targeting consequences untested"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,1,4,18]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[1,18]},{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[4,13,16]},{"term_id":"GO:0001618","term_label":"virus receptor activity","supporting_discovery_ids":[27]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[4,13,18]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[1,4,9,16]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[13,14,20,23]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[10,7,25]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[11,12,15]}],"complexes":["RACK1-PP2A-Akt complex","EphB3-FHL-2/Dral-caspase death complex"],"partners":["EFNB3","AF6","RASA1","CRK","FYN","RACK1","FHL2","EGFR"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P54753","full_name":"Ephrin type-B receptor 3","aliases":["EPH-like tyrosine kinase 2","EPH-like kinase 2","Embryonic kinase 2","EK2","hEK2","Tyrosine-protein kinase TYRO6"],"length_aa":998,"mass_kda":110.3,"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. Generally has an overlapping and redundant function with EPHB2. Like EPHB2, functions in axon guidance during development regulating for instance the neurons forming the corpus callosum and the anterior commissure, 2 major interhemispheric connections between the temporal lobes of the cerebral cortex. In addition to its role in axon guidance also plays an important redundant role with other ephrin-B receptors in development and maturation of dendritic spines and the formation of excitatory synapses. Controls other aspects of development through regulation of cell migration and positioning. This includes angiogenesis, palate development and thymic epithelium development for instance. Forward and reverse signaling through the EFNB2/EPHB3 complex also regulate migration and adhesion of cells that tubularize the urethra and septate the cloaca. 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Endogenous AF6 is phosphorylated by EphB3 and EphB2 in a ligand-dependent fashion in NIH 3T3 and NG108 cells.\",\n      \"method\": \"Yeast two-hybrid, mutational analysis, immunoprecipitation, in-cell phosphorylation assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal interaction confirmed by yeast two-hybrid and co-IP, kinase-dependence shown by mutant, replicated in multiple cell lines\",\n      \"pmids\": [\"9707552\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Tyrosine-614 (Y614) is the major autophosphorylation site of EphB3 (HEK2) and functions as a multi-docking site for SH2-domain-containing proteins rasGAP (via its N-terminal SH2 domain), Crk, and Fyn; a Y614F substitution abolishes binding of all three.\",\n      \"method\": \"In vitro binding assays, immunoprecipitation, yeast two-hybrid, site-directed mutagenesis (Y614F)\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro assay combined with site-directed mutagenesis and co-IP; multiple orthogonal methods in single study\",\n      \"pmids\": [\"9674711\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Ephrin-B3 is a high-affinity ligand for EphB3 (Kd ~1 nM) with selectivity among the transmembrane ephrin-B family; it is expressed at the dorsal and ventral midline of the neural tube (floor plate).\",\n      \"method\": \"Receptor-binding affinity assays, in situ hybridization\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct binding measurement with defined Kd, anatomical localization by ISH; single lab\",\n      \"pmids\": [\"9484836\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Human RYK associates with EphB2 and EphB3, requiring both extracellular and cytoplasmic domains of RYK; this association is not dependent on Eph receptor activation. Unlike murine Ryk, human RYK is not phosphorylated by EphB3.\",\n      \"method\": \"Co-immunoprecipitation, in vitro binding assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal co-IP and domain-deletion analysis; single lab\",\n      \"pmids\": [\"11956217\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"EphB3 kinase activity is required for inhibition of integrin-mediated cell adhesion and induction of cell rounding, but directional cell migration inhibition is kinase-independent. Both wild-type and kinase-dead EphB3 reduce Rac1 and Cdc42 activity upon ephrin-B1 stimulation, with a relative increase in RhoA signaling mediating migration inhibition.\",\n      \"method\": \"Stable transfection of WT vs. kinase-dead EphB3, adhesion assays, migration assays, GTPase activity assays, pharmacological inhibition, integrin-activating antibodies\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple orthogonal functional assays with kinase-dead mutant, GTPase measurements, and pharmacological rescue in same study\",\n      \"pmids\": [\"15536074\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"EphB3 expressed by recruited macrophages at the optic nerve injury site supports adult retinal ganglion cell (RGC) axon re-extension and sprouting after injury; loss of EphB3 greatly decreases axon plasticity without affecting intrinsic axon growth potential. Injured RGC axons express EphrinB3 and bind recombinant EphB3 protein in vivo.\",\n      \"method\": \"In vivo optic nerve injury in EphB3 heterozygous and null mice, mRNA expression analysis, recombinant protein injection, in vitro axon outgrowth and turning assays\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — loss-of-function genetic model with specific phenotypic readout, in vivo protein-binding, in vitro functional assays, multiple orthogonal methods\",\n      \"pmids\": [\"16554460\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"EphB3 overexpression in HT-29 colorectal cancer cells promotes mesenchymal-to-epithelial transition (MET): reorganizes cortical actin, increases E-cadherin/ZO-1/plakoglobin, decreases fibronectin and nuclear β-catenin, inactivates CrkL-Rac1, increases Ca2+-dependent cell-cell adhesion, and suppresses tumor growth in xenografts.\",\n      \"method\": \"Stable transfection, morphological analysis, immunofluorescence, transwell migration, soft agar assay, xenograft, GTPase assays\",\n      \"journal\": \"Carcinogenesis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple functional readouts with defined molecular markers; single lab\",\n      \"pmids\": [\"19483190\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"EphB3 signaling suppresses neural stem/progenitor cell (NSPC) proliferation in the subventricular zone in a p53-dependent manner. EphB3 activation by ephrinB3-Fc increases p53 expression; pharmacological inhibition or siRNA knockdown of p53 attenuates ephrinB3-Fc-mediated growth suppression. In the absence of ligand, EphB3 induces cell death.\",\n      \"method\": \"EphB3-/- and ephrinB3-/- knockout mice, BrdU incorporation, Ki67 immunostaining, lateral ventricle ephrinB3-Fc infusion, siRNA knockdown of p53, pharmacological p53 inhibition, cultured NSPCs\",\n      \"journal\": \"Stem cells\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO with specific phenotype, epistasis via p53 knockdown/inhibition, multiple orthogonal readouts\",\n      \"pmids\": [\"20496368\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"In NSCLC cells, EphB3 promotes cell growth and migration in a kinase-independent manner when overexpressed; silencing EphB3 reduces DNA synthesis, activates caspase-8-mediated apoptosis, and increases focal adhesion accumulation to suppress migration.\",\n      \"method\": \"Stable overexpression and siRNA knockdown in NSCLC cell lines, proliferation/migration assays, xenograft models, apoptosis assays, focal adhesion staining\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss- and gain-of-function with defined molecular readouts; kinase-independence established by kinase-dead constructs in related study context; single lab\",\n      \"pmids\": [\"21266352\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Kinase-activated EphB3 in NSCLC assembles a ternary signaling complex comprising RACK1, PP2A, and Akt, leading to reduced Akt phosphorylation and inhibition of cell migration. RACK1 mediates assembly of this complex in response to EphB3 activation.\",\n      \"method\": \"Co-immunoprecipitation, forced EphB3 kinase activation, in vitro migration assay, in vivo metastasis seeding, identification of RACK1 as EphB3-binding protein\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — novel binding partner identified by co-IP, ternary complex assembled, functional consequence (Akt dephosphorylation, migration inhibition) demonstrated in vitro and in vivo\",\n      \"pmids\": [\"22314363\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"EphB2 and EphB3 forward signaling (not reverse signaling) are required for palatal mesenchyme proliferation and palate development; foetuses with cytoplasmic-truncated EphB2 (capable of reverse but not forward signaling) plus EphB3-null develop cleft palate due to reduced palatal shelf proliferation.\",\n      \"method\": \"Genetic epistasis using kinase-dead/truncated EphB2 knockin and EphB3-/- compound mutant mice, palatal shelf culture, BrdU proliferation assays\",\n      \"journal\": \"Mechanisms of development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis with allelic series distinguishing forward vs. reverse signaling; specific cellular phenotype (reduced proliferation) quantified\",\n      \"pmids\": [\"19032981\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"SNAIL1 silences EPHB3 by competitively displacing the stem cell factor ASCL2 from an E-box motif in the EPHB3 promoter/enhancer, then recruiting corepressor complexes containing HDACs and the histone demethylase LSD1, collapsing active chromatin. SNAIL2, but not ZEB1, also represses EPHB3 reporter constructs.\",\n      \"method\": \"Reporter assays, ChIP for ASCL2/p300/TCF7L2/LSD1/HDACs, endogenous locus analysis, xenotransplant models with/without sustained EPHB3 expression\",\n      \"journal\": \"Molecular oncology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — ChIP demonstrating displacement of activators and recruitment of repressors, functional rescue by EPHB3 re-expression, multiple orthogonal methods\",\n      \"pmids\": [\"25277775\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"A transcriptional enhancer at the EPHB3 locus integrates input from ASCL2, Wnt/β-catenin, MAP kinase, and Notch signaling; low Notch activity causes enhancer decommissioning and EPHB3 silencing in colorectal carcinoma. Restoring Notch activity re-establishes enhancer function and EPHB3 expression.\",\n      \"method\": \"Enhancer reporter assays, ChIP, Notch pathway manipulation, gene expression analysis in cell lines and tumors\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — functional enhancer mapping, ChIP for pathway effectors, rescue by Notch activation; multiple orthogonal methods\",\n      \"pmids\": [\"24707046\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"EphB3 functions as a dependence receptor in neurons: in the absence of ephrinB3 ligand, EphB3 is cleaved by caspases and induces cell death; ephrinB3 stimulation blocks this cleavage and prevents apoptosis. Infusion of clustered ephrinB3-Fc reduces cortical infarct and cell death after controlled cortical impact injury via EphB3-dependent mechanism.\",\n      \"method\": \"EphB3-/- and ephrinB3-/- mice, controlled cortical impact injury, TUNEL staining, ephrinB3-Fc infusion, caspase cleavage assays\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO distinguishes EphB3-dependent from EphB3-independent effects; caspase cleavage demonstrated; ephrinB3-Fc rescue only in EphB3-expressing mice\",\n      \"pmids\": [\"24810043\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"EphB3 mediates oligodendrocyte cell death in the injured spinal cord through a dependence receptor mechanism; ephrinB3 ligand administration promotes oligodendrocyte survival both in vivo and in vitro under pro-apoptotic conditions. Genetic ablation of EphB3 increases myelin basic protein expression and improves locomotor function after SCI.\",\n      \"method\": \"EphB3-/- mice, contusive spinal cord injury, oligodendrocyte survival assays, ephrinB3 ligand administration, locomotor behavioral assessment\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO with behavioral and molecular rescue, ligand rescue in vitro and in vivo, multiple readouts\",\n      \"pmids\": [\"26469970\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"TCF7L1 represses EPHB3 expression in colorectal cancer cells; loss of TCF7L1 activates EPHB3 as a CTNNB1/TCF target gene. Knockdown of EPHB3 partially restores growth and normal cell cycle progression in TCF7L1-null CRC cells.\",\n      \"method\": \"TCF7L1 knockdown/knockout in CRC cells, gene expression analysis, EPHB3 siRNA epistasis, xenograft tumor models, colony formation assays\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — epistasis by double knockdown, xenograft validation; single lab\",\n      \"pmids\": [\"27333864\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"EphB3 stimulates migration and metastasis in papillary thyroid cancer in a kinase-dependent manner through activation of Vav2, a Rho GEF, leading to increased Rac1 activity and decreased RhoA activity. EphB3 knockdown suppresses Vav2 and reverses this GTPase balance.\",\n      \"method\": \"siRNA knockdown, EphB3 overexpression, ephrin-B1/B2-Fc stimulation, EphB3-Fc blocking, Rho GTPase activity assays, in vivo metastasis model\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — kinase-dependence shown by kinase blockade, GTPase activity measured, Vav2 identified as downstream effector; single lab\",\n      \"pmids\": [\"27986811\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"The Mule E3 ubiquitin ligase targets EphB3 for proteasomal and lysosomal degradation in the intestinal stem cell niche, controlling EphB3 protein levels and thus crypt-villus cell positioning.\",\n      \"method\": \"In vivo mouse genetics (Mule conditional KO), western blotting, proteasome/lysosome inhibitor treatment\",\n      \"journal\": \"Cell stem cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo genetic model with biochemical confirmation of degradation pathway; single lab\",\n      \"pmids\": [\"27184401\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"EphB3 receptors in the juxtamembrane region undergo autophosphorylation in trans (not in cis), demonstrated using isoform-specific covalent inhibitors; a unique cysteine in the EphB3 hinge region (absent in other human kinases) allows covalent inhibitor targeting. Co-crystal structures confirmed covalent linkage between EphB3 and quinazoline inhibitors.\",\n      \"method\": \"Specific irreversible inhibitor synthesis, kinase assays, co-crystal structures, 'clickable' inhibitor for proteome-wide target engagement\",\n      \"journal\": \"Journal of the American Chemical Society\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structures confirming covalent binding, in vitro kinase assay with specific inhibitor, trans-autophosphorylation mechanism validated; multiple orthogonal methods\",\n      \"pmids\": [\"27478969\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"EphB3 signaling in the hippocampus after traumatic brain injury promotes synaptic loss and reduces d-serine levels; EphB3-/- mice show preserved long-term potentiation, improved hippocampal-dependent learning, and attenuated synapse loss and d-serine reduction compared to wild-type injured mice.\",\n      \"method\": \"EphB3-/- mice, controlled cortical impact injury, LTP electrophysiology, synaptic protein quantification, d-serine measurement, behavioral learning assays\",\n      \"journal\": \"Neurobiology of disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO with electrophysiological and molecular readouts; single lab\",\n      \"pmids\": [\"27317833\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"EphB3 functions as a pro-apoptotic dependence receptor in cortical endothelial cells after TBI; in the absence of EphB3, endothelial cell survival is increased, blood-brain barrier permeability is reduced, and astrocyte-EC membrane interactions are enhanced after controlled cortical impact.\",\n      \"method\": \"EphB3-/- mice, CCI injury, BBB permeability assays, endothelial cell survival quantification, immunofluorescence of astrocyte-EC contacts\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO with specific vascular phenotype; single lab\",\n      \"pmids\": [\"29311672\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"EphB3 suppresses glioma invasion, migration, and proliferation by downregulating the EGFR-PI3K/AKT signaling pathway; EphB3 knockdown upregulates EGFR, p-PI3K, and p-AKT, while overexpression downregulates these proteins.\",\n      \"method\": \"siRNA knockdown and overexpression in U87MG and U251 glioma cell lines, western blotting, invasion/migration/proliferation assays\",\n      \"journal\": \"Brain research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — bidirectional modulation with consistent molecular pathway readout; single lab, no in vivo epistasis\",\n      \"pmids\": [\"38341169\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"In cetuximab-resistant colorectal cancer, EGFR and EphB3 physically interact (co-immunoprecipitation), and this binding increases upon resistance acquisition, leading to STAT3 activation; inhibition of EphB3 decreases STAT3 activity.\",\n      \"method\": \"Co-immunoprecipitation, western blotting for STAT3 phosphorylation, RTK array, siRNA knockdown, cetuximab-resistant cell line model\",\n      \"journal\": \"Theranostics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP demonstrates physical EGFR-EphB3 interaction, STAT3 signaling measured after EphB3 inhibition; single lab\",\n      \"pmids\": [\"31149041\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"EphB3 interacts with initiator caspases (caspase-8 or caspase-9) and the adaptor protein Dral/FHL-2 to mediate its cleavage and activate dependence receptor cell death in oligodendrocytes after brain injury; blocking this pathway with ephrinB3-Fc improves oligodendrocyte survival, myelin sparing, axonal conductance, and behavioral recovery in mice and conserved mechanism found in human TBI patients.\",\n      \"method\": \"Co-immunoprecipitation, caspase cleavage assays, EphB3-/- mice, CCI injury, ephrinB3-Fc administration, behavioral/electrophysiological readouts, human TBI tissue analysis\",\n      \"journal\": \"Brain communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — protein interaction (co-IP with FHL-2 and caspases), genetic KO rescue, ligand rescue, conserved in human samples; multiple orthogonal methods\",\n      \"pmids\": [\"33305261\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"YTHDF2 binds to m6A sites in the 3'UTR of EPHB3 mRNA to decrease its stability and reduce EPHB3 protein levels, thereby activating the PI3K/Akt and NF-κB signaling pathways and promoting temozolomide resistance in glioblastoma.\",\n      \"method\": \"RNA immunoprecipitation, FISH, dual-luciferase reporter, siRNA knockdown of YTHDF2, RNA sequencing, rescue experiments with PI3K/Akt and NF-κB inhibitors\",\n      \"journal\": \"Clinical & translational immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — RNA-IP demonstrates direct m6A-YTHDF2-EPHB3 interaction, luciferase reporter validates 3'UTR binding, pathway epistasis confirmed by inhibitors; single lab\",\n      \"pmids\": [\"35582627\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"EphB3 acts as a negative regulator of osteogenic differentiation in mesenchymal stromal/stem cells; absence of EphB3 signaling reduces expression of BMP pathway inhibitors, increases Bmp7 expression, increases osteoprogenitor and preosteoblast proportions, and reduces osteoclast numbers, preventing bone loss in ovariectomy and glucocorticoid-induced osteoporosis mouse models.\",\n      \"method\": \"EphB3-/- and EphB2-/- mice, in vitro MSC differentiation assays, qRT-PCR, histological staining, ovariectomy and dexamethasone bone loss models, bone volume/density measurement\",\n      \"journal\": \"Journal of bone and mineral research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO with mechanistic pathway (BMP signaling) and in vivo bone loss phenotype; single lab\",\n      \"pmids\": [\"38739682\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Astrocytic EphB3 receptors regulate synaptic d-serine availability at CA3-CA1 hippocampal synapses: stimulation of EphB3 with exogenous ephrinB3 increases d-serine and NMDAR activity, while inhibition of endogenous EphB3 impairs NMDAR-dependent LTP. Astrocyte-specific knockdown of EphB3 causes hippocampal plasticity deficits and novel object recognition memory impairment that can be rescued by exogenous d-serine.\",\n      \"method\": \"Acute hippocampal slice electrophysiology, exogenous ephrinB3 application, EphB3 inhibition, astrocyte-specific EphB3 knockdown, d-serine measurement, LTP recording, behavioral memory testing\",\n      \"journal\": \"Progress in neurobiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — cell-type-specific KD with LTP/memory phenotype, d-serine rescue, multiple orthogonal methods in single study\",\n      \"pmids\": [\"40081519\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"MHV68 gH/gL glycoprotein complex directly interacts with EphB3 (and EphA4), enabling viral entry; ectopic expression of EphB3 enables MHV68 infection of otherwise non-permissive human B cells, demonstrating EphB3 functions as an entry receptor for this gammaherpesvirus.\",\n      \"method\": \"Direct binding assays, ectopic EphB3 expression in non-permissive B cells, soluble decoy receptor competition, mutagenesis informed by structural predictions, neutralizing antibody adsorption\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct binding demonstrated, functional receptor role confirmed by ectopic expression enabling infection; preprint, not yet peer-reviewed\",\n      \"pmids\": [\"bio_10.1101_2025.06.05.657996\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"EphB3 is a receptor tyrosine kinase whose kinase activity mediates phosphorylation-dependent assembly of downstream signaling complexes (including RACK1-PP2A-Akt and SH2-domain adaptors rasGAP/Crk/Fyn at Y614), while kinase-independent mechanisms allow it to inhibit directional cell migration via Rho GTPase rebalancing; it functions as a dependence receptor that induces caspase/FHL-2-mediated apoptosis in the absence of its primary ligand ephrinB3 and is suppressed by ephrinB3 binding, regulates intestinal epithelial compartmentalization and MET, controls neural stem cell proliferation through p53, regulates synaptic NMDAR function via astrocytic d-serine release, and is transcriptionally controlled by ASCL2/Wnt/Notch/SNAIL1-mediated enhancer mechanisms and post-translationally by Mule-dependent proteasomal/lysosomal degradation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"EphB3 is an ephrin-B-family receptor tyrosine kinase that converts ephrinB3 engagement into bidirectional control of cell adhesion, migration, proliferation, and survival across epithelial, neural, and skeletal tissues [#4, #7, #13]. Upon ligand binding it autophosphorylates in trans at juxtamembrane tyrosines, with Tyr-614 serving as the principal docking site that recruits the SH2 adaptors rasGAP, Crk, and Fyn, while its C-terminal PDZ motif binds the Ras effector AF6 in a kinase-dependent manner [#1, #0, #18]. Active EphB3 nucleates downstream signaling complexes—assembling a RACK1–PP2A–Akt module that dampens Akt phosphorylation and migration in lung cancer [#9], and rebalancing Rho-family GTPases by lowering Rac1/Cdc42 and raising RhoA to inhibit directional migration; this migratory restraint is kinase-independent whereas inhibition of integrin adhesion requires kinase activity [#4]. In epithelia EphB3 drives a mesenchymal-to-epithelial transition with restored E-cadherin and cell–cell adhesion and suppressed tumor growth, consistent with a role in intestinal compartmentalization that is set by Mule-dependent degradation of EphB3 protein [#6, #17]. In the absence of ephrinB3, EphB3 behaves as a dependence receptor: it is cleaved by initiator caspases via the adaptor Dral/FHL-2 to trigger apoptosis in neurons, oligodendrocytes, and endothelial cells, and ligand binding blocks this death program—a mechanism conserved in human injury tissue [#13, #23, #20]. EphB3 also restrains neural stem/progenitor proliferation through p53 [#7], and astrocytic EphB3 controls synaptic d-serine availability and NMDAR-dependent plasticity and memory [#26]. EPHB3 expression is tightly controlled transcriptionally—activated by an ASCL2/Wnt/Notch enhancer and silenced by SNAIL1-directed recruitment of LSD1/HDAC corepressors and by TCF7L1—and post-transcriptionally by YTHDF2-mediated m6A decay of its mRNA [#12, #11, #15, #24].\",\n  \"teleology\": [\n    {\n      \"year\": 1998,\n      \"claim\": \"Establishing how ligand-activated EphB3 transmits a signal: identification of its autophosphorylation site and the adaptors it recruits defined the receptor's proximal signaling output.\",\n      \"evidence\": \"Site-directed mutagenesis (Y614F), in vitro binding, yeast two-hybrid and co-IP identifying rasGAP/Crk/Fyn at Y614 and kinase-dependent AF6 binding via the PDZ motif\",\n      \"pmids\": [\"9674711\", \"9707552\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequences of each adaptor downstream of Y614 not dissected\", \"Whether AF6 phosphorylation alters its Ras-effector function not resolved\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Defining the cognate ligand: ephrinB3 was shown to be a high-affinity, selective EphB3 ligand with midline expression, anchoring EphB3 in neural-tube patterning.\",\n      \"evidence\": \"Receptor-binding affinity assays (Kd ~1 nM) and in situ hybridization\",\n      \"pmids\": [\"9484836\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vivo developmental phenotype of this ligand-receptor pair not addressed here\", \"Single-lab affinity measurement\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Separating kinase-dependent from kinase-independent EphB3 outputs clarified that adhesion control needs catalytic activity but migration inhibition does not, mediated by GTPase rebalancing toward RhoA.\",\n      \"evidence\": \"WT vs kinase-dead stable lines, adhesion/migration assays, GTPase activity assays, pharmacological rescue\",\n      \"pmids\": [\"15536074\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular link between kinase-dead EphB3 and RhoA activation not identified\", \"Effector GEF/GAP responsible not pinned down\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Linking EphB3 to growth control: it suppresses neural stem/progenitor proliferation through p53 and induces death without ligand, foreshadowing its dependence-receptor behavior.\",\n      \"evidence\": \"EphB3-/- and ephrinB3-/- mice, BrdU/Ki67, ephrinB3-Fc infusion, p53 siRNA/inhibition epistasis\",\n      \"pmids\": [\"20496368\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism connecting EphB3 to p53 induction unresolved\", \"Cleavage machinery for ligand-free death not yet identified at this stage\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Identifying RACK1 as an EphB3 partner showed how kinase-active receptor assembles a phosphatase-containing complex to suppress Akt and migration.\",\n      \"evidence\": \"Co-IP, forced kinase activation, in vitro migration and in vivo metastasis assays in NSCLC\",\n      \"pmids\": [\"22314363\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How RACK1 is recruited to active EphB3 not detailed\", \"Reconciliation with kinase-independent migration effects not addressed\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Genetic epistasis distinguished forward from reverse signaling, establishing that EphB3 forward signaling drives palatal mesenchyme proliferation in development.\",\n      \"evidence\": \"Compound EphB2 truncation knockin / EphB3-null mice, palatal shelf culture, BrdU assays\",\n      \"pmids\": [\"19032981\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream proliferative effectors in palate not defined\", \"EphB2/EphB3 redundancy boundaries not fully mapped\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Defining EPHB3 transcriptional control: an ASCL2/Wnt/Notch enhancer activates the gene while SNAIL1 displaces ASCL2 and recruits LSD1/HDAC corepressors to silence it, explaining loss of EPHB3 in colorectal carcinoma.\",\n      \"evidence\": \"Enhancer/promoter reporter assays, ChIP for activators and repressors, Notch manipulation, xenografts with sustained EPHB3\",\n      \"pmids\": [\"24707046\", \"25277775\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Hierarchy among Wnt/Notch/MAPK inputs at the enhancer not fully ordered\", \"In vivo relevance in human tumors beyond cell models limited\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Establishing EphB3 as a dependence receptor: ligand-free EphB3 is caspase-cleaved to drive death, and ephrinB3 blocks cleavage to protect cells after brain injury.\",\n      \"evidence\": \"EphB3-/- and ephrinB3-/- mice, controlled cortical impact, TUNEL, caspase cleavage assays, ephrinB3-Fc rescue\",\n      \"pmids\": [\"24810043\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the cleavage adaptor not yet known at this point\", \"Threshold of ligand needed to suppress death not quantified\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Multiple layers of EphB3 regulation and context-dependent signaling were defined: Mule-mediated degradation sets protein levels in the intestinal niche, TCF7L1 represses EPHB3 transcriptionally, and EphB3 can drive or suppress migration via Vav2/Rho GTPase rebalancing depending on context.\",\n      \"evidence\": \"Mule conditional KO with degradation-pathway inhibitors; TCF7L1 knockdown with EPHB3 epistasis; Vav2/GTPase activity assays in thyroid cancer\",\n      \"pmids\": [\"27184401\", \"27333864\", \"27986811\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Reconciliation of pro- vs anti-migratory roles across tissues not unified\", \"Single-lab findings for each regulatory axis\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Mechanistic and structural definition of EphB3 catalysis: it autophosphorylates in trans, and a unique hinge cysteine enables covalent inhibitor targeting confirmed by co-crystal structures.\",\n      \"evidence\": \"Isoform-specific irreversible inhibitors, kinase assays, co-crystal structures, clickable probe\",\n      \"pmids\": [\"27478969\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cellular consequences of selective EphB3 inhibition in disease not tested here\", \"Whether trans-autophosphorylation requires specific receptor clustering geometry unresolved\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Completing the dependence-receptor mechanism: EphB3 recruits initiator caspases and the adaptor Dral/FHL-2 to execute cleavage-dependent death, with the pathway conserved in human TBI tissue.\",\n      \"evidence\": \"Co-IP with FHL-2/caspases, caspase cleavage assays, EphB3-/- mice, ephrinB3-Fc rescue, human tissue analysis\",\n      \"pmids\": [\"33305261\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of FHL-2/caspase recruitment to EphB3 not solved\", \"Whether the same adaptor operates in non-CNS cell types not established\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Post-transcriptional control was extended: YTHDF2-mediated m6A decay of EPHB3 mRNA lowers protein levels and activates PI3K/Akt and NF-κB to promote glioblastoma drug resistance.\",\n      \"evidence\": \"RNA-IP, FISH, dual-luciferase 3'UTR reporter, YTHDF2 knockdown, pathway-inhibitor rescue\",\n      \"pmids\": [\"35582627\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether EphB3 protein loss alone, versus other YTHDF2 targets, drives the pathway activation not isolated\", \"Single-lab study\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Defining a non-canonical CNS function: astrocytic EphB3 controls synaptic d-serine availability to set NMDAR-dependent plasticity and memory, with d-serine rescuing loss-of-function deficits.\",\n      \"evidence\": \"Astrocyte-specific EphB3 knockdown, slice LTP electrophysiology, d-serine measurement and rescue, novel object recognition\",\n      \"pmids\": [\"40081519\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Signaling link from EphB3 to d-serine synthesis/release not delineated\", \"Relationship to EphB3's neuronal dependence-receptor role not integrated\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How EphB3 switches between kinase-dependent signaling, kinase-independent migration control, and ligand-free dependence-receptor death within a single cell, and what determines its opposing pro- versus anti-tumor outputs across tissues, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified model reconciling context-specific migratory and survival outputs\", \"Structural basis for the death-versus-signaling switch unknown\", \"In vivo therapeutic targeting consequences untested\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 1, 4, 18]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [1, 18]},\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [4, 13, 16]},\n      {\"term_id\": \"GO:0001618\", \"supporting_discovery_ids\": [27]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [4, 13, 18]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [1, 4, 9, 16]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [13, 14, 20, 23]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [10, 7, 25]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [11, 12, 15]}\n    ],\n    \"complexes\": [\n      \"RACK1-PP2A-Akt complex\",\n      \"EphB3-FHL-2/Dral-caspase death complex\"\n    ],\n    \"partners\": [\n      \"EFNB3\",\n      \"AF6\",\n      \"RASA1\",\n      \"CRK\",\n      \"FYN\",\n      \"RACK1\",\n      \"FHL2\",\n      \"EGFR\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}