{"gene":"EPHB1","run_date":"2026-04-28T17:46:03","timeline":{"discoveries":[{"year":1997,"finding":"EphA4 and EphB1 receptors in migrating Xenopus neural crest cells interact with ephrin-B2 ligand expressed in adjacent arch territories; inhibition of EphB1 function via dominant-negative truncated receptors causes abnormal migration of third arch neural crest into second and fourth arch territories, establishing a repulsive boundary mechanism.","method":"Dominant-negative truncated receptor overexpression, ectopic ephrin-B2 overexpression in Xenopus embryos","journal":"Current Biology","confidence":"High","confidence_rationale":"Tier 2 — loss-of-function and gain-of-function in vivo with defined cellular phenotype, replicated across multiple experimental conditions","pmids":["9259557"],"is_preprint":false},{"year":1998,"finding":"EphB1 juxtamembrane region (Tyr594) recruits the SH2-domain adaptor Nck upon ligand activation; this interaction couples EphB1 to downstream JNK/SAPK activation and cell attachment responses.","method":"Yeast two-hybrid cloning, co-immunoprecipitation, site-directed mutagenesis (Y594F), dominant-negative overexpression, kinase assay","journal":"Journal of Biological Chemistry","confidence":"High","confidence_rationale":"Tier 1 — mutagenesis + co-IP + functional assay in single rigorous study","pmids":["9430661"],"is_preprint":false},{"year":1999,"finding":"EphB1 functions as a ligand-density sensor: engagement with ephrin-B1 displayed at varying surface densities activates integrin-mediated cell attachment (alphavbeta3 in endothelial cells; alpha5beta1 upon EphB1 transfection in HEK cells) through downstream signaling rather than mechanical tethering; signaling-defective point mutants fail to stimulate attachment.","method":"Reconstituted ephrin-B1 surface assay, integrin-blocking antibodies, EphB1 point-mutant transfection, cell attachment assay","journal":"EMBO Journal","confidence":"High","confidence_rationale":"Tier 1 — reconstituted surface assay with signaling-dead mutants, multiple integrin contexts tested","pmids":["10205170"],"is_preprint":false},{"year":1999,"finding":"EphB1 and its ligand ephrin-B2 are expressed in complementary patterns in midbrain dopaminergic neurons; ephrin-B2 selectively inhibits neurite outgrowth and induces cell loss of substantia nigra (but not VTA) dopaminergic neurons, suggesting EphB1/ephrin-B2 specifies distinct mesostriatal vs. mesolimbic pathways.","method":"In vitro neurite outgrowth assay, cell survival assay, expression analysis","journal":"Journal of Neuroscience","confidence":"Medium","confidence_rationale":"Tier 2 — in vitro functional assay with defined cellular readout, single lab","pmids":["10066262"],"is_preprint":false},{"year":2001,"finding":"EphB1 transphosphorylates the kinase-dead EphB6 receptor in a ligand-dependent manner; EphB1 and EphB6 form a stable heterocomplex, demonstrating Eph receptor cross-talk. Additionally, c-Cbl was identified as an EphB6-binding protein in this complex.","method":"Co-immunoprecipitation, overexpression of catalytically active vs. inactive EphB1, ligand stimulation assay","journal":"Journal of Biological Chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — reciprocal co-IP with catalytic mutant controls, single lab","pmids":["11713248"],"is_preprint":false},{"year":2002,"finding":"EphB1 interacts with the adaptor Grb7 via Grb7's SH2 domain binding to phospho-Tyr928 of EphB1; EphB1 phosphorylates Grb7, and this interaction promotes fibroblast cell motility in a kinase-dependent manner.","method":"Yeast two-hybrid, co-immunoprecipitation, site-directed mutagenesis (Y928F, Y594F), cell migration assay with dominant-negative Grb7 SH2 domain","journal":"Journal of Biological Chemistry","confidence":"High","confidence_rationale":"Tier 1–2 — yeast two-hybrid + co-IP + mutagenesis + functional migration assay","pmids":["12223469"],"is_preprint":false},{"year":2003,"finding":"Ephrin-B2 at the optic chiasm midline selectively repels EphB1-expressing retinal ganglion cell axons from ventrotemporal retina; EphB1 null mice show dramatically reduced ipsilateral retinal projection, establishing EphB1 as the key receptor mediating retinal axon divergence at the chiasm.","method":"EphB1 knockout mouse, in vitro stripe/collapse assay with chiasm cells, blocking antibody experiments, semiintact visual system preparation","journal":"Neuron","confidence":"High","confidence_rationale":"Tier 2 — knockout mouse with defined projection phenotype, in vitro rescue/block experiments, replicated across multiple assays","pmids":["12971893"],"is_preprint":false},{"year":2003,"finding":"Activated EphB1 recruits Grb2 and p52Shc, promotes p52Shc and c-Src tyrosine phosphorylation, and activates MAPK/ERK to drive chemotaxis; Tyr600 and Tyr778 of EphB1 are required for interaction with c-Src and p52Shc; p52Shc phosphorylation by c-Src is required for its recruitment to EphB1 signaling complexes via its PTB domain.","method":"Co-immunoprecipitation, dominant-negative c-Src, MEK inhibitor PD98059, Src inhibitor PP2, site-directed mutagenesis (Y600F, Y778F), cell migration assay","journal":"Journal of Cell Biology","confidence":"High","confidence_rationale":"Tier 1–2 — mutagenesis + pharmacological inhibitors + co-IP + functional chemotaxis assay","pmids":["12925710"],"is_preprint":false},{"year":2004,"finding":"Activated EphB1 induces c-Src-dependent tyrosine phosphorylation of paxillin at Tyr31 and Tyr118 and is recruited to paxillin-FAK complexes; Nck binding site Y594F mutation disrupts the EphB1–Nck–paxillin complex; paxillin Y31F/Y118F mutant cells show reduced EphB1-dependent migration.","method":"Co-immunoprecipitation, site-directed mutagenesis (Y31F/Y118F paxillin, Y594F EphB1), dominant-negative kinase-dead c-Src, PP2 inhibitor, cell migration assay","journal":"Journal of Biological Chemistry","confidence":"High","confidence_rationale":"Tier 1–2 — mutagenesis of both receptor and substrate + co-IP + functional assay","pmids":["15107421"],"is_preprint":false},{"year":2006,"finding":"EphB1 receptors localize to caveolae via direct interaction with caveolin-1 through a caveolin-binding motif in the EphB1 kinase domain; overexpression of scaffolding-domain-deleted caveolin-1 abrogates EphB1–caveolin-1 interaction and ERK activation; the caveolin-binding motif is required for correct EphB1 membrane targeting.","method":"Sucrose density gradient fractionation, co-immunoprecipitation, dominant-negative caveolin-1, membrane targeting assay","journal":"Journal of Cell Science","confidence":"Medium","confidence_rationale":"Tier 2 — biochemical fractionation + co-IP + dominant-negative with functional consequence","pmids":["16723736"],"is_preprint":false},{"year":2007,"finding":"Ephrin-B1 stimulation triggers Cbl recruitment to EphB1, Cbl phosphorylation (requiring Src kinase activity), EphB1 ubiquitination, and lysosomal degradation of EphB1; kinase-dead EphB1-K652R is resistant to Cbl-mediated degradation; GST pull-down showed Cbl binds EphB1 through its tyrosine kinase-binding domain.","method":"Ubiquitination assay, lysosomal inhibitor (bafilomycin), co-immunoprecipitation, overexpression of Cbl WT vs. 70Z ligase-dead mutant, GST pull-down, Src inhibitor PP2","journal":"Traffic","confidence":"High","confidence_rationale":"Tier 1 — multiple orthogonal methods (ubiquitination assay, GST pulldown, inhibitor, mutants) in single study","pmids":["18034775"],"is_preprint":false},{"year":2008,"finding":"Zic2 transcription factor regulates EphB1 expression in retinal ganglion cells; ectopic Zic2 delivery induces EphB1 mRNA and protein in non-ventrotemporal retinal explants, and upregulated EphB1 localizes to growth cones and is sufficient to convert RGC axon behavior from extension onto to avoidance of ephrin-B2 substrates.","method":"In utero retinal electroporation, explant growth cone collapse assay, immunofluorescence of EphB1 in growth cones","journal":"Journal of Neuroscience","confidence":"Medium","confidence_rationale":"Tier 2 — gain-of-function in vivo with defined molecular and cellular readout","pmids":["18524895"],"is_preprint":false},{"year":2008,"finding":"In vivo electroporation showing that ectopic EphB1 expression is sufficient to redirect normally crossed RGC axons to ipsilateral trajectory; EphB2, despite high similarity, is much less efficient; chimeric receptor analysis reveals both extracellular and juxtamembrane domains of EphB1 are required for efficient ipsilateral redirection.","method":"In utero retinal electroporation, EphB1/EphB2 chimeric receptors, anterograde tracing","journal":"Journal of Neuroscience","confidence":"Medium","confidence_rationale":"Tier 2 — in vivo gain-of-function with domain-swap analysis","pmids":["19295152"],"is_preprint":false},{"year":2008,"finding":"EphB1 null mice exhibit a 40% decrease in neuron number and volume in substantia nigra pars reticulata (SNr) but not pars compacta (SNc), and display spontaneous locomotor hyperactivity, establishing EphB1 as required for proper SNr formation.","method":"EphB1 knockout mouse, beta-galactosidase reporter, stereological neuron counting, open-field behavioral assay","journal":"European Journal of Neuroscience","confidence":"Medium","confidence_rationale":"Tier 2 — knockout mouse with quantitative anatomical and behavioral readout","pmids":["17561836"],"is_preprint":false},{"year":2009,"finding":"EphB1 receptor is required for long-term potentiation (LTP) at C-fiber–spinal dorsal horn synapses in vivo; targeted mutation of EphB1 prevents HFS-induced LTP and associated upregulation of p-CaMKII, p-ERK, p-CREB, and c-Fos in the spinal cord.","method":"EphB1 knockout mouse, in vivo electrophysiology, immunoblotting for signaling markers, MK-801 and EphB2-Fc blocking","journal":"Molecular Pain","confidence":"Medium","confidence_rationale":"Tier 2 — knockout with electrophysiological and biochemical readouts","pmids":["19523204"],"is_preprint":false},{"year":2011,"finding":"EphB1 forward signaling in spinal cord mediates bone cancer pain; spinal EphB1 activation by ephrin-B2 phosphorylates NR1 and NR2B subunits of NMDA receptors; blocking EphB1 with EphB2-Fc reverses pain behaviors and reduces c-Fos, astrocyte/microglial activation, and Src phosphorylation within the NMDA complex; MMP-2/9 regulate EphB1 levels.","method":"EphB1 blocking reagent (EphB2-Fc), EphB1 knockout mouse, intrathecal administration, immunoblotting for NMDA subunit phosphorylation, MMP inhibitors","journal":"Cancer Research","confidence":"Medium","confidence_rationale":"Tier 2 — pharmacological blocking + knockout + defined molecular pathway with multiple readouts","pmids":["21555368"],"is_preprint":false},{"year":2011,"finding":"EphB1 forward signaling is required for ipsilateral retinal projection at the optic chiasm; knock-in of intracellularly truncated EphB1 (EphB1T-lacZ) eliminates the ipsilateral projection, confirming that EphB1 functions as a receptor (not reverse signaling source); EphB1 is the preferred receptor for ephrin-B2 (and to lesser degree ephrin-B1) at the chiasm.","method":"Knock-in mouse with intracellular truncation (EphB1T-lacZ), anterograde retinal tracing","journal":"European Journal of Neuroscience","confidence":"High","confidence_rationale":"Tier 2 — knock-in allele with defined molecular mechanism (forward vs. reverse signaling) and anatomical phenotype","pmids":["22103419"],"is_preprint":false},{"year":2012,"finding":"EphB1 recruits Caskin1 scaffold protein through adaptor Nck; upon EphB1 kinase activation, Caskin1 is tyrosine-phosphorylated at Y296 and Y336 (identified by mass spectrometry), and this phosphorylation causes significant structural changes in the Caskin1 SH3 domain as shown by CD spectroscopy.","method":"Co-immunoprecipitation, mass spectrometry, CD spectroscopy","journal":"Cell Communication and Signaling","confidence":"Medium","confidence_rationale":"Tier 2 — co-IP with MS identification + structural consequence measured by CD spectroscopy","pmids":["23181695"],"is_preprint":false},{"year":2012,"finding":"PTEN constitutively associates with c-Cbl, protecting Cbl from degradation; EphB1 stimulation triggers PTEN dephosphorylation (requiring PTEN protein phosphatase activity) and disruption of the PTEN–Cbl complex, allowing independent translocation of both proteins to EphB1 via scaffold NHERF1; PTEN lipid phosphatase activity impairs EphB1-dependent cell adhesion and chemotaxis.","method":"Co-immunoprecipitation, PTEN/NHERF1 siRNA, PTEN phosphatase mutants, cell adhesion and migration assay","journal":"FASEB Journal","confidence":"Medium","confidence_rationale":"Tier 2 — multiple co-IPs + siRNA + phosphatase mutants + functional assays","pmids":["23118026"],"is_preprint":false},{"year":2014,"finding":"EphB1 expressed in striatal anlage signals through ephrin-B3 reverse signaling with dual context-dependent outcomes: in cortical interneurons EphB1-ephrinB3 reverse signaling phosphorylates Src and FAK to mediate repulsion, whereas in striatal neurons it reduces endogenously high pSrc and pFAK to act as a stop signal; ephrin-B3 knockout mice show misrouted cortical interneurons in the striatum and over-migration of striatal neurons.","method":"Ephrin-B3 knockout mouse, in vitro migration assays, immunoblotting for pSrc and pFAK","journal":"Frontiers in Cellular Neuroscience","confidence":"Medium","confidence_rationale":"Tier 2 — knockout with defined molecular signaling differences between two cell types","pmids":["25100946"],"is_preprint":false},{"year":2015,"finding":"Intracellular signaling domains of EphB1 and EphB2 are critical for formation of the corpus callosum and anterior commissure; truncated EphB1/EphB2 mice lacking intracellular domains show partial/complete agenesis of corpus callosum and highly penetrant anterior commissure misprojection, indicating combined forward and reverse signaling contributions.","method":"Truncated receptor knock-in mice, anterograde tract tracing, brain anatomy analysis","journal":"Developmental Neurobiology","confidence":"Medium","confidence_rationale":"Tier 2 — knock-in mouse allele with defined anatomical phenotype","pmids":["26148571"],"is_preprint":false},{"year":2017,"finding":"Injured motor neurons upregulate EphB1, which activates astrocytes through ephrin-B1-mediated STAT3 stimulation to induce a protective anti-inflammatory transcriptional signature; this EphB1–ephrin-B1–STAT3 pathway is disrupted in human stem-cell-derived ALS astrocytes and mouse ALS models.","method":"Human iPSC-derived astrocytes, mouse ALS models, transcriptional analysis, STAT3 pathway activation assay","journal":"Nature Communications","confidence":"Medium","confidence_rationale":"Tier 2 — defined molecular pathway with multiple disease models and transcriptional readout","pmids":["29079839"],"is_preprint":false},{"year":2018,"finding":"Porf-2, a neuronally expressed RhoGAP, binds EphB1 and is regulated downstream; activated EphB1 forward signaling deactivates Rac1 through the Porf-2 GAP domain, inhibiting growth cone expansion and braking axon growth.","method":"Co-immunoprecipitation, Rac1 activity assay (pull-down), Porf-2 GAP domain mutants, axon growth assay","journal":"Cellular and Molecular Life Sciences","confidence":"Medium","confidence_rationale":"Tier 2 — co-IP + Rac1 activity assay + functional axon growth readout","pmids":["29938386"],"is_preprint":false},{"year":2018,"finding":"EphB1 is SUMOylated at lysine 785; SUMOylation-deficient K785R mutant shows enhanced cell proliferation, anchorage-independent growth, and xenograft tumor growth; mechanistically, EphB1 SUMOylation represses activation of PKCγ, and a reciprocal regulatory loop between PKCγ and EphB1 SUMOylation exists.","method":"Ni2+-NTA pull-down, co-immunoprecipitation, K785R mutant, xenograft mouse model, soft agar colony assay, PKCγ activation assay","journal":"Cellular Physiology and Biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 — PTM identified with pull-down + mutant + functional in vivo readout","pmids":["29550816"],"is_preprint":false},{"year":2020,"finding":"EphB1 constitutively interacts with caveolin-1 (Cav-1) via its CSD-binding motif; upon ephrin-B1 activation, EphB1 is phosphorylated and uncouples from Cav-1, enabling Src-dependent Cav-1 phosphorylation; loss of EphB1 promotes Cav-1 ubiquitination and degradation, reducing caveolae numbers in endothelial cells.","method":"Super-resolution microscopy, FRET, co-immunoprecipitation, EphB1-/- mice endothelial cells, CSD deletion mutant, Cav-1 ubiquitination assay","journal":"Molecular Biology of the Cell","confidence":"High","confidence_rationale":"Tier 1–2 — FRET + biochemistry + KO mice + deletion mutant, multiple orthogonal approaches","pmids":["32238105"],"is_preprint":false},{"year":2020,"finding":"TGF-β–activated Smad2 transcriptionally upregulates EphB1 expression; ligand-independent (unphosphorylated) EphB1 promotes EMT and lung cancer cell migration/invasion through upregulation of CDH2 (N-cadherin), while ligand-dependent (phosphorylated) EphB1 inhibits these processes.","method":"Western blot, real-time PCR, transwell migration/invasion assay, EphB1 phosphorylation-deficient mutant, Smad2 overexpression","journal":"Journal of Cancer","confidence":"Medium","confidence_rationale":"Tier 2 — phosphorylation mutant + functional assay + upstream pathway identification","pmids":["32368295"],"is_preprint":false},{"year":2021,"finding":"Tetracyclines (demeclocycline, chlortetracycline, minocycline) inhibit EphB1 kinase activity at low micromolar concentrations by binding the ATP-binding catalytic domain; co-crystal structure of chlortetracycline with EphB1 confirmed this binding; in vivo administration inhibited EphB1 phosphorylation in spinal cord/DRG and blocked neuropathic pain.","method":"In silico docking, in vitro kinase assay, co-crystallization (crystal structure), in vivo phosphorylation assay, neuropathic pain mouse model","journal":"PNAS","confidence":"High","confidence_rationale":"Tier 1 — crystal structure + in vitro kinase assay + in vivo validation","pmids":["33627480"],"is_preprint":false},{"year":2023,"finding":"EphB1 is neddylated in activated hepatic stellate cells; neddylation enhances EphB1 kinase activity by preventing its degradation, thereby promoting HSC proliferation, migration, and activation contributing to liver fibrosis.","method":"Immunoprecipitation, neddylation inhibitor, EphB1 kinase activity assay, HSC functional assays","journal":"International Journal of Molecular Sciences","confidence":"Medium","confidence_rationale":"Tier 2 — PTM identified with co-IP + kinase activity assay + functional cellular readouts","pmids":["36834826"],"is_preprint":false},{"year":2023,"finding":"CRC-associated somatic mutations in EphB1 kinase domain reduce kinase activity and protein stability in vitro; mutant EphB1 receptors fail to suppress STAT3 and ERK1/2 signaling and are unable to suppress CRC cell migration or mediate cell compartmentalization when cocultured with ephrin-B1-expressing cells.","method":"Purified kinase domain assay, thermal stability assay, cell migration assay, cell compartmentalization coculture assay, western blot for STAT3/ERK signaling","journal":"Journal of Biological Chemistry","confidence":"High","confidence_rationale":"Tier 1 — purified protein kinase assay + mutagenesis + multiple functional readouts","pmids":["37527777"],"is_preprint":false},{"year":2023,"finding":"Recurring EPHB1 cancer mutations in ligand-binding, fibronectin, and kinase domains reduce ligand-induced receptor phosphorylation and cell compartmentalization; enhanced compartmentalization mutations in kinase domain (R743W, G821R) are not linked to phosphorylation level; reduced compartmentalization mutants activate PIK3C2B phosphorylation via the PI3K pathway.","method":"Confocal microscopy compartmentalization assay, phospho-proteome mass spectrometry, bioinformatic 3D-cluster analysis","journal":"Cell Communication and Signaling","confidence":"Medium","confidence_rationale":"Tier 2 — phosphoproteomics + functional compartmentalization assay, single lab","pmids":["38102712"],"is_preprint":false},{"year":2024,"finding":"EphB1 conditional knockout specifically in GABAergic neurons (Vgat-Cre) but not cortical excitatory neurons (Emx1-Cre) or endothelial cells (Tie2-Cre) reproduces cortical axon guidance defects seen in global EphB1 KO; EphB1 in GABAergic neurons promotes avoidance of ectopic fasciculation with brain vasculature by glutamatergic cortical axons.","method":"Cell-type specific conditional knockout (Vgat-Cre, Emx1-Cre, Tie2-Cre), anterograde tract tracing","journal":"Development","confidence":"Medium","confidence_rationale":"Tier 2 — cell-type-specific conditional KO defining non-autonomous mechanism","pmids":["38345254"],"is_preprint":false},{"year":2024,"finding":"Tumor cell-expressed EPHB1 and platelet-expressed EFNB1 mediate contact-dependent reverse signaling activation of platelets via AKT pathway; activated platelets then release 5-HT which enhances tumor growth in the liver metastatic niche of pancreatic ductal adenocarcinoma.","method":"Gain- and loss-of-function of Ephb1, recombinant protein assays, Tph1-knockout mice (5-HT depletion), tumor-platelet adhesion assay, metastasis mouse model","journal":"Cancer Communications","confidence":"Medium","confidence_rationale":"Tier 2 — multiple gain/loss-of-function approaches + genetic (Tph1 KO) validation of 5-HT output","pmids":["39648610"],"is_preprint":false},{"year":2025,"finding":"EphB1 promotes CREB phosphorylation via PI3K/AKT signaling in a cell-autonomous manner in hypothalamic neurons; EphB1 deficiency reduces CRH and TRH expression in hypothalamus, causing impaired thermogenesis, reduced locomotor activity, and obesity; intracerebroventricular TRH or CRH peptide rescues obesity in Ephb1 mutants.","method":"Forward genetic screen, EphB1 knockout mouse, primary hypothalamic cell signaling assay, intracerebroventricular peptide administration, metabolic phenotyping","journal":"Obesity","confidence":"Medium","confidence_rationale":"Tier 2 — KO mouse with defined pathway (PI3K/AKT/CREB) + peptide rescue experiment","pmids":["40207393"],"is_preprint":false},{"year":2025,"finding":"EphB1 activation in thalamic ventroposteromedial nucleus (VPM) glutamatergic neurons promotes emergence from anesthesia via two independent mechanisms: (1) phosphorylation of NMDA receptor NR2B subunit at Tyr1472, exciting VPMGlu neurons; and (2) ubiquitin-dependent degradation of KCC2, disinhibiting VPMGlu neurons that project to primary somatosensory cortex.","method":"EphB1 conditional knockout in VPM, site-specific viral vector manipulation, in vivo electrophysiology, immunoblotting for NR2B-Y1472 phosphorylation and KCC2 ubiquitination, behavioral anesthesia emergence assay","journal":"Science Advances","confidence":"Medium","confidence_rationale":"Tier 2 — conditional KO with two mechanistically distinct downstream pathways defined and functionally validated","pmids":["41348875"],"is_preprint":false}],"current_model":"EphB1 is a receptor tyrosine kinase that, upon binding membrane-anchored ephrin-B ligands, activates multiple signaling branches including Nck–JNK (via pY594), c-Src/Shc–MAPK/ERK (via pY600/pY778), and Grb7-dependent pathways to regulate integrin-mediated cell adhesion and directional cell migration; it undergoes Cbl-mediated ubiquitination and lysosomal degradation in a Src-dependent manner, is localized to caveolae through caveolin-1 interaction, and is post-translationally modified by neddylation and SUMOylation to modulate its activity; in the nervous system EphB1 acts as the primary receptor for ephrin-B2-mediated repulsion to direct ipsilateral retinal axon routing at the optic chiasm, guide neural crest migration, and modulate spinal NMDA receptor phosphorylation underlying pain sensitization and synaptic plasticity."},"narrative":{"teleology":[{"year":1997,"claim":"Establishing EphB1 as a guidance receptor: the first demonstration that EphB1 mediates repulsive boundary formation during neural crest migration via ephrin-B2 interaction, answering how branchial arch territories are maintained.","evidence":"Dominant-negative truncated EphB1 overexpression in Xenopus embryos caused neural crest mis-migration into adjacent arch territories","pmids":["9259557"],"confidence":"High","gaps":["Downstream signaling pathways mediating repulsion were unknown","Mammalian relevance not tested"]},{"year":1998,"claim":"Identifying the first signaling branch downstream of EphB1: Nck adaptor binds pY594 in the juxtamembrane region and couples the receptor to JNK/SAPK activation and cell attachment, establishing the first defined phosphotyrosine-effector link.","evidence":"Yeast two-hybrid, co-IP, Y594F mutagenesis, and JNK kinase assay","pmids":["9430661"],"confidence":"High","gaps":["Other signaling branches (MAPK/ERK) not yet mapped","In vivo relevance of Nck–JNK axis not tested"]},{"year":1999,"claim":"Demonstrating that EphB1 acts as a ligand-density sensor to regulate integrin-mediated adhesion, establishing that the kinase's signaling output—not mechanical tethering—controls cell attachment through αvβ3 and α5β1 integrins.","evidence":"Reconstituted ephrin-B1 surface density assay with signaling-dead EphB1 point mutants and integrin-blocking antibodies","pmids":["10205170"],"confidence":"High","gaps":["Which specific signaling branch connects to integrin activation was unclear","Density-sensing mechanism at receptor level not resolved"]},{"year":2002,"claim":"Mapping the Grb7–pY928 signaling branch: EphB1 phosphorylates Grb7 via its SH2 domain binding to pY928, and this interaction drives cell motility, revealing a third distinct phosphotyrosine-dependent output.","evidence":"Yeast two-hybrid, co-IP, Y928F mutagenesis, dominant-negative Grb7 SH2 domain, cell migration assay","pmids":["12223469"],"confidence":"High","gaps":["Relationship between Grb7 and integrin-mediated outputs not clarified","In vivo relevance not tested"]},{"year":2003,"claim":"Defining EphB1 as the essential receptor for ipsilateral retinal axon guidance at the optic chiasm: EphB1 knockout mice lose most ipsilateral projection, and ephrin-B2 at the midline selectively repels EphB1-expressing ventrotemporal RGC axons.","evidence":"EphB1 knockout mouse with anterograde tracing, stripe/collapse assays, blocking antibodies","pmids":["12971893"],"confidence":"High","gaps":["Forward vs. reverse signaling contribution not yet dissected","Downstream intracellular effectors mediating growth cone turning uncharacterized"]},{"year":2003,"claim":"Mapping the c-Src/Shc–MAPK/ERK chemotaxis branch: pY600 and pY778 recruit c-Src and p52Shc; Src-dependent Shc phosphorylation activates ERK to drive directional migration, providing a second major signaling axis for cell motility.","evidence":"Co-IP, Y600F/Y778F mutagenesis, Src inhibitor PP2, MEK inhibitor PD98059, cell migration assay","pmids":["12925710"],"confidence":"High","gaps":["Cross-talk between Nck–JNK and Src–ERK branches not defined","Relative contribution of branches to in vivo guidance unknown"]},{"year":2004,"claim":"Linking EphB1 signaling to focal adhesion remodeling: activated EphB1 drives Src-dependent paxillin phosphorylation at Y31/Y118 and assembles an EphB1–Nck–paxillin–FAK complex required for migration.","evidence":"Co-IP, paxillin Y31F/Y118F and EphB1 Y594F mutagenesis, kinase-dead Src, migration assay","pmids":["15107421"],"confidence":"High","gaps":["How paxillin phosphorylation coordinates with integrin activation from the 1999 study was unknown","Spatial dynamics of complex assembly not resolved"]},{"year":2006,"claim":"Establishing caveolae as a signaling platform for EphB1: the receptor localizes to caveolae via a caveolin-binding motif in the kinase domain, and this compartmentalization is required for ERK activation.","evidence":"Sucrose gradient fractionation, co-IP, dominant-negative caveolin-1 lacking scaffolding domain","pmids":["16723736"],"confidence":"Medium","gaps":["Stoichiometry of EphB1–caveolin-1 interaction not determined","Whether caveolar localization affects other signaling branches (JNK, Grb7) unknown"]},{"year":2007,"claim":"Defining the degradation pathway: Cbl is recruited to activated EphB1 in a Src-dependent manner, ubiquitinates the receptor, and targets it for lysosomal degradation, establishing how EphB1 signaling is attenuated.","evidence":"Ubiquitination assay, bafilomycin inhibition, GST pull-down of Cbl TKB domain, kinase-dead K652R mutant resistant to degradation, PP2 Src inhibitor","pmids":["18034775"],"confidence":"High","gaps":["Specific ubiquitin chain types (K48 vs. K63) not determined","Whether proteasomal degradation also contributes was not resolved"]},{"year":2008,"claim":"Identifying the transcriptional regulator upstream of EphB1 in retinal neurons: Zic2 drives EphB1 expression and is sufficient to convert RGC axon behavior from extension to avoidance on ephrin-B2 substrates.","evidence":"In utero retinal electroporation, explant growth cone collapse assay","pmids":["18524895"],"confidence":"Medium","gaps":["Zic2 binding sites in EphB1 regulatory regions not mapped","Other transcriptional regulators of EphB1 expression not identified"]},{"year":2011,"claim":"Confirming forward signaling as the critical mode at the chiasm: intracellularly truncated EphB1 knock-in eliminates ipsilateral projection, ruling out reverse signaling contributions and proving that the kinase domain and downstream effectors are required.","evidence":"EphB1T-lacZ knock-in mouse with anterograde retinal tracing","pmids":["22103419"],"confidence":"High","gaps":["Which specific intracellular domain (kinase, SAM, PDZ-binding) is essential was not dissected","Contribution of kinase activity vs. scaffold function not separated"]},{"year":2011,"claim":"Extending EphB1 function to pain processing: spinal EphB1 activation by ephrin-B2 phosphorylates NMDA receptor NR1/NR2B subunits to maintain bone cancer pain, with MMP-2/9 regulating EphB1 levels.","evidence":"EphB1 KO mouse, intrathecal EphB2-Fc blocking, immunoblotting for NR subunit phosphorylation, MMP inhibitors","pmids":["21555368"],"confidence":"Medium","gaps":["Direct phosphorylation vs. indirect Src-mediated NR2B phosphorylation not fully resolved","MMP regulation of EphB1 protein levels not mechanistically detailed"]},{"year":2012,"claim":"Integrating PTEN into the EphB1 signaling network: PTEN constitutively sequesters Cbl, and ephrin stimulation disrupts this complex via PTEN dephosphorylation, freeing both proteins to modulate EphB1 adhesion and migration through NHERF1 scaffold.","evidence":"Co-IP, PTEN/NHERF1 siRNA, phosphatase-dead PTEN mutants, adhesion and migration assays","pmids":["23118026"],"confidence":"Medium","gaps":["Whether PTEN lipid phosphatase activity directly opposes PI3K signaling downstream of EphB1 was not tested","In vivo relevance of PTEN–Cbl regulation not demonstrated"]},{"year":2018,"claim":"Identifying SUMOylation as a tumor-suppressive post-translational modification of EphB1: K785 SUMOylation represses PKCγ activation and suppresses anchorage-independent growth and xenograft tumor formation.","evidence":"Ni²⁺-NTA pull-down, K785R mutant, xenograft mouse model, soft agar assay, PKCγ activation measurement","pmids":["29550816"],"confidence":"Medium","gaps":["SUMO E3 ligase responsible not identified","Whether SUMOylation and caveolin-1 interaction at the kinase domain are mutually exclusive was not tested"]},{"year":2020,"claim":"Resolving the dynamic EphB1–caveolin-1 relationship: EphB1 constitutively protects Cav-1 from ubiquitination; upon ligand activation, phosphorylated EphB1 uncouples from Cav-1 and enables Src-dependent Cav-1 phosphorylation, maintaining caveolae homeostasis in endothelial cells.","evidence":"Super-resolution microscopy, FRET, co-IP, EphB1−/− mouse endothelial cells, Cav-1 ubiquitination assay","pmids":["32238105"],"confidence":"High","gaps":["Whether disrupted caveolae affect vascular permeability in vivo via EphB1 was not addressed","Identity of the E3 ligase for Cav-1 in EphB1-null cells unknown"]},{"year":2023,"claim":"Characterizing cancer-associated EphB1 mutations: somatic mutations across ligand-binding, fibronectin, and kinase domains reduce kinase activity and protein stability, abolish STAT3/ERK suppression and cell compartmentalization, and activate PIK3C2B through PI3K, establishing EphB1 as a functional tumor suppressor in colorectal cancer.","evidence":"Purified kinase domain assays, thermal stability, migration assay, compartmentalization coculture, phosphoproteomics","pmids":["37527777","38102712"],"confidence":"High","gaps":["In vivo tumor suppressor role not confirmed by conditional knockout tumor models","Whether enhanced-compartmentalization mutations (R743W, G821R) represent gain-of-function or neomorphic activity is unclear"]},{"year":2023,"claim":"Identifying neddylation as a stabilizing modification: EphB1 neddylation in hepatic stellate cells prevents receptor degradation and sustains kinase activity, promoting HSC proliferation and liver fibrosis.","evidence":"Immunoprecipitation, neddylation inhibitor, kinase activity assay, HSC functional assays","pmids":["36834826"],"confidence":"Medium","gaps":["Neddylation sites on EphB1 not mapped","Interplay between neddylation and Cbl-mediated ubiquitination not tested"]},{"year":2024,"claim":"Resolving non-cell-autonomous axon guidance: EphB1 in GABAergic (not glutamatergic) neurons provides the guidance signal that prevents glutamatergic cortical axons from aberrantly fasciculating with brain vasculature.","evidence":"Cell-type-specific conditional KO (Vgat-Cre, Emx1-Cre, Tie2-Cre) with anterograde tracing","pmids":["38345254"],"confidence":"Medium","gaps":["The molecular mechanism by which GABAergic EphB1 instructs glutamatergic axon routing is unknown","Whether this involves secreted signals or contact-dependent signaling between neuron types is not resolved"]},{"year":2025,"claim":"Extending EphB1 function to metabolic regulation: hypothalamic EphB1 activates PI3K/AKT/CREB to drive CRH and TRH expression for thermogenesis and energy homeostasis; loss causes obesity rescued by intracerebroventricular neuropeptide replacement.","evidence":"EphB1 KO mouse, primary hypothalamic neuron signaling assays, ICV peptide rescue, metabolic phenotyping","pmids":["40207393"],"confidence":"Medium","gaps":["Upstream ligand activating hypothalamic EphB1 not identified","Whether kinase activity or scaffold function mediates CREB phosphorylation not dissected"]},{"year":2025,"claim":"Revealing dual downstream mechanisms in thalamic arousal circuits: EphB1 in VPM glutamatergic neurons both phosphorylates NR2B-Y1472 to enhance excitation and promotes ubiquitin-dependent KCC2 degradation to disinhibit neurons, facilitating emergence from anesthesia.","evidence":"Conditional KO in VPM, viral manipulation, in vivo electrophysiology, NR2B-Y1472 phosphorylation and KCC2 ubiquitination assays","pmids":["41348875"],"confidence":"Medium","gaps":["E3 ligase responsible for KCC2 ubiquitination downstream of EphB1 not identified","Whether EphB1 directly phosphorylates NR2B or acts through Src/Fyn not resolved in this system"]},{"year":null,"claim":"Key unresolved questions include the structural basis for EphB1's specificity over EphB2 in chiasm guidance, how the multiple phosphotyrosine-dependent signaling branches are coordinated in space and time, and the physiological interplay among SUMOylation, neddylation, and Cbl-mediated ubiquitination in regulating receptor output.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No full-length EphB1 structure with signaling partners resolved","Relative quantitative contribution of individual pY sites to specific in vivo phenotypes untested","Cross-regulation between SUMOylation, neddylation, and ubiquitination not addressed"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[1,5,7,8,26,28]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[23,28,33]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[9,24]}],"pathway":[{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[0,6,11,16,20,30]},{"term_id":"R-HSA-112316","term_label":"Neuronal System","supporting_discovery_ids":[6,14,15,33]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[10,23,27]}],"complexes":[],"partners":["NCK1","GRB7","SRC","SHC1","CBL","CAV1","PAXILLIN","EFNB2"],"other_free_text":[]},"mechanistic_narrative":"EphB1 is a receptor tyrosine kinase that transduces ephrin-B ligand engagement into diverse cellular outcomes—including repulsive axon guidance, integrin-mediated cell adhesion, directed cell migration, and synaptic plasticity—through multiple phosphotyrosine-dependent signaling branches. Ligand-stimulated EphB1 autophosphorylation recruits Nck (via pY594) to activate JNK and paxillin phosphorylation, c-Src/Shc (via pY600/pY778) to drive MAPK/ERK-dependent chemotaxis, and Grb7 (via pY928) to promote cell motility; it also signals through PI3K/AKT/CREB in hypothalamic neurons and phosphorylates NMDA receptor NR2B subunits in spinal and thalamic circuits to modulate pain sensitization and arousal [PMID:9430661, PMID:12925710, PMID:12223469, PMID:40207393, PMID:41348875]. EphB1 is the primary receptor mediating ephrin-B2–dependent repulsion of ventrotemporal retinal ganglion cell axons at the optic chiasm, and its forward signaling is required for ipsilateral retinal projection, corpus callosum formation, and cortical axon guidance via a non-cell-autonomous mechanism in GABAergic neurons [PMID:12971893, PMID:22103419, PMID:26148571, PMID:38345254]. EphB1 activity is regulated by caveolin-1–dependent membrane compartmentalization, Cbl-mediated ubiquitination and lysosomal degradation, SUMOylation at K785 that represses PKCγ, and neddylation that stabilizes the receptor and enhances kinase output [PMID:32238105, PMID:18034775, PMID:29550816, PMID:36834826]."},"prefetch_data":{"uniprot":{"accession":"P54762","full_name":"Ephrin type-B receptor 1","aliases":["ELK","EPH tyrosine kinase 2","EPH-like kinase 6","EK6","hEK6","Neuronally-expressed EPH-related tyrosine kinase","NET","Tyrosine-protein kinase receptor EPH-2"],"length_aa":984,"mass_kda":109.9,"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. Cognate/functional ephrin ligands for this receptor include EFNB1, EFNB2 and EFNB3. During nervous system development, regulates retinal axon guidance redirecting ipsilaterally ventrotemporal retinal ganglion cells axons at the optic chiasm midline. This probably requires repulsive interaction with EFNB2. In the adult nervous system together with EFNB3, regulates chemotaxis, proliferation and polarity of the hippocampus neural progenitors. 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 synapse formation. May also regulate angiogenesis. More generally, may play a role in targeted cell migration and adhesion. Upon activation by EFNB1 and probably other ephrin-B ligands activates the MAPK/ERK and the JNK signaling cascades to regulate cell migration and adhesion respectively. Involved in the maintenance of the pool of satellite cells (muscle stem cells) by promoting their self-renewal and reducing their activation and differentiation (By similarity)","subcellular_location":"Cell membrane; Early endosome membrane; Cell projection, dendrite","url":"https://www.uniprot.org/uniprotkb/P54762/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/EPHB1","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/EPHB1","total_profiled":1310},"omim":[{"mim_id":"609481","title":"ISL2 TRANSCRIPTION FACTOR, LIM/HOMEODOMAIN; ISL2","url":"https://www.omim.org/entry/609481"},{"mim_id":"601581","title":"NEURONAL CELL ADHESION MOLECULE; NRCAM","url":"https://www.omim.org/entry/601581"},{"mim_id":"601381","title":"EPHRIN A3; EFNA3","url":"https://www.omim.org/entry/601381"},{"mim_id":"601380","title":"EPHRIN A4; EFNA4","url":"https://www.omim.org/entry/601380"},{"mim_id":"600600","title":"EPHRIN RECEPTOR EphB1; EPHB1","url":"https://www.omim.org/entry/600600"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Endoplasmic reticulum","reliability":"Supported"},{"location":"Plasma membrane","reliability":"Supported"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"brain","ntpm":19.3}],"url":"https://www.proteinatlas.org/search/EPHB1"},"hgnc":{"alias_symbol":["Hek6"],"prev_symbol":["EPHT2"]},"alphafold":{"accession":"P54762","domains":[{"cath_id":"2.60.120.260","chopping":"19-197","consensus_level":"high","plddt":84.9964,"start":19,"end":197},{"cath_id":"2.60.40.1770","chopping":"200-256","consensus_level":"medium","plddt":88.2319,"start":200,"end":256},{"cath_id":"2.60.40.10","chopping":"323-429","consensus_level":"medium","plddt":92.7064,"start":323,"end":429},{"cath_id":"2.60.40.10","chopping":"442-532","consensus_level":"high","plddt":88.2515,"start":442,"end":532},{"cath_id":"3.30.200.20","chopping":"598-699","consensus_level":"high","plddt":86.9715,"start":598,"end":699},{"cath_id":"1.10.510.10","chopping":"704-889","consensus_level":"high","plddt":87.3124,"start":704,"end":889},{"cath_id":"1.10.150.50","chopping":"911-979","consensus_level":"high","plddt":81.3207,"start":911,"end":979}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P54762","model_url":"https://alphafold.ebi.ac.uk/files/AF-P54762-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P54762-F1-predicted_aligned_error_v6.png","plddt_mean":83.62},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=EPHB1","jax_strain_url":"https://www.jax.org/strain/search?query=EPHB1"},"sequence":{"accession":"P54762","fasta_url":"https://rest.uniprot.org/uniprotkb/P54762.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P54762/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P54762"}},"corpus_meta":[{"pmid":"9259557","id":"PMC_9259557","title":"The EphA4 and EphB1 receptor tyrosine kinases and ephrin-B2 ligand regulate targeted migration of branchial neural crest cells.","date":"1997","source":"Current biology : CB","url":"https://pubmed.ncbi.nlm.nih.gov/9259557","citation_count":281,"is_preprint":false},{"pmid":"12971893","id":"PMC_12971893","title":"Ephrin-B2 and EphB1 mediate retinal axon divergence at the optic chiasm.","date":"2003","source":"Neuron","url":"https://pubmed.ncbi.nlm.nih.gov/12971893","citation_count":265,"is_preprint":false},{"pmid":"12384430","id":"PMC_12384430","title":"Subtractive hybridization reveals the expression of immunoglobulin-like transcript 7, Eph-B1, granzyme B, and 3 novel transcripts in human plasmacytoid dendritic cells.","date":"2002","source":"Blood","url":"https://pubmed.ncbi.nlm.nih.gov/12384430","citation_count":194,"is_preprint":false},{"pmid":"10205170","id":"PMC_10205170","title":"Surface densities of ephrin-B1 determine EphB1-coupled activation of cell attachment through alphavbeta3 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sciences","url":"https://pubmed.ncbi.nlm.nih.gov/36834826","citation_count":13,"is_preprint":false},{"pmid":"23649798","id":"PMC_23649798","title":"Tissue-specific venous expression of the EPH family receptor EphB1 in the skin vasculature.","date":"2013","source":"Developmental dynamics : an official publication of the American Association of Anatomists","url":"https://pubmed.ncbi.nlm.nih.gov/23649798","citation_count":11,"is_preprint":false},{"pmid":"33356837","id":"PMC_33356837","title":"Peripheral EphrinB1/EphB1 signalling attenuates muscle hyperalgesia in MPS patients and a rat model of taut band-associated persistent muscle pain.","date":"2020","source":"Molecular pain","url":"https://pubmed.ncbi.nlm.nih.gov/33356837","citation_count":10,"is_preprint":false},{"pmid":"29938386","id":"PMC_29938386","title":"Neuronal GAP-Porf-2 transduces EphB1 signaling to brake axon growth.","date":"2018","source":"Cellular and molecular life sciences : 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chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/37527777","citation_count":7,"is_preprint":false},{"pmid":"29236320","id":"PMC_29236320","title":"Analgesic effects of microRNA-129-5p against bone cancer pain through the EphB1/EphrinB2 signaling pathway in mice.","date":"2018","source":"Journal of cellular biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/29236320","citation_count":6,"is_preprint":false},{"pmid":"38070822","id":"PMC_38070822","title":"EphB1 promotes the differentiation and maturation of dendritic cells in non-small cell lung cancer.","date":"2023","source":"Cancer letters","url":"https://pubmed.ncbi.nlm.nih.gov/38070822","citation_count":5,"is_preprint":false},{"pmid":"25120806","id":"PMC_25120806","title":"Decreased expression of receptor tyrosine kinase of EphB1 protein in renal cell carcinomas.","date":"2014","source":"International journal of clinical and experimental pathology","url":"https://pubmed.ncbi.nlm.nih.gov/25120806","citation_count":5,"is_preprint":false},{"pmid":"23118026","id":"PMC_23118026","title":"Phosphatase and tensin homolog regulates stability and activity of EphB1 receptor.","date":"2012","source":"FASEB journal : official publication of the Federation of American Societies for Experimental Biology","url":"https://pubmed.ncbi.nlm.nih.gov/23118026","citation_count":5,"is_preprint":false},{"pmid":"39648610","id":"PMC_39648610","title":"Reciprocal tumor-platelet interaction through the EPHB1-EFNB1 axis in the liver metastatic niche promotes metastatic tumor outgrowth in pancreatic ductal adenocarcinoma.","date":"2024","source":"Cancer communications (London, England)","url":"https://pubmed.ncbi.nlm.nih.gov/39648610","citation_count":4,"is_preprint":false},{"pmid":"21763378","id":"PMC_21763378","title":"Associations of EPHB1 polymorphisms with hepatocellular carcinoma in the Korean population.","date":"2011","source":"Human 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CCS","url":"https://pubmed.ncbi.nlm.nih.gov/38102712","citation_count":2,"is_preprint":false},{"pmid":"29550816","id":"PMC_29550816","title":"Sumoylation of EphB1 Suppresses Neuroblastoma Tumorigenesis via Inhibiting PKCγ Activation.","date":"2018","source":"Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/29550816","citation_count":2,"is_preprint":false},{"pmid":"37992441","id":"PMC_37992441","title":"The combination of tetracyclines effectively ameliorates liver fibrosis via inhibition of EphB1/2.","date":"2023","source":"International immunopharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/37992441","citation_count":2,"is_preprint":false},{"pmid":"38601015","id":"PMC_38601015","title":"EphB1 causes retinal damage through inflammatory pathways in the retina and retinal Müller cells.","date":"2024","source":"Molecular 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truncated receptor overexpression, ectopic ephrin-B2 overexpression in Xenopus embryos\",\n      \"journal\": \"Current Biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — loss-of-function and gain-of-function in vivo with defined cellular phenotype, replicated across multiple experimental conditions\",\n      \"pmids\": [\"9259557\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"EphB1 juxtamembrane region (Tyr594) recruits the SH2-domain adaptor Nck upon ligand activation; this interaction couples EphB1 to downstream JNK/SAPK activation and cell attachment responses.\",\n      \"method\": \"Yeast two-hybrid cloning, co-immunoprecipitation, site-directed mutagenesis (Y594F), dominant-negative overexpression, kinase assay\",\n      \"journal\": \"Journal of Biological Chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — mutagenesis + co-IP + functional assay in single rigorous study\",\n      \"pmids\": [\"9430661\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"EphB1 functions as a ligand-density sensor: engagement with ephrin-B1 displayed at varying surface densities activates integrin-mediated cell attachment (alphavbeta3 in endothelial cells; alpha5beta1 upon EphB1 transfection in HEK cells) through downstream signaling rather than mechanical tethering; signaling-defective point mutants fail to stimulate attachment.\",\n      \"method\": \"Reconstituted ephrin-B1 surface assay, integrin-blocking antibodies, EphB1 point-mutant transfection, cell attachment assay\",\n      \"journal\": \"EMBO Journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstituted surface assay with signaling-dead mutants, multiple integrin contexts tested\",\n      \"pmids\": [\"10205170\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"EphB1 and its ligand ephrin-B2 are expressed in complementary patterns in midbrain dopaminergic neurons; ephrin-B2 selectively inhibits neurite outgrowth and induces cell loss of substantia nigra (but not VTA) dopaminergic neurons, suggesting EphB1/ephrin-B2 specifies distinct mesostriatal vs. mesolimbic pathways.\",\n      \"method\": \"In vitro neurite outgrowth assay, cell survival assay, expression analysis\",\n      \"journal\": \"Journal of Neuroscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vitro functional assay with defined cellular readout, single lab\",\n      \"pmids\": [\"10066262\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"EphB1 transphosphorylates the kinase-dead EphB6 receptor in a ligand-dependent manner; EphB1 and EphB6 form a stable heterocomplex, demonstrating Eph receptor cross-talk. Additionally, c-Cbl was identified as an EphB6-binding protein in this complex.\",\n      \"method\": \"Co-immunoprecipitation, overexpression of catalytically active vs. inactive EphB1, ligand stimulation assay\",\n      \"journal\": \"Journal of Biological Chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal co-IP with catalytic mutant controls, single lab\",\n      \"pmids\": [\"11713248\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"EphB1 interacts with the adaptor Grb7 via Grb7's SH2 domain binding to phospho-Tyr928 of EphB1; EphB1 phosphorylates Grb7, and this interaction promotes fibroblast cell motility in a kinase-dependent manner.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, site-directed mutagenesis (Y928F, Y594F), cell migration assay with dominant-negative Grb7 SH2 domain\",\n      \"journal\": \"Journal of Biological Chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — yeast two-hybrid + co-IP + mutagenesis + functional migration assay\",\n      \"pmids\": [\"12223469\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Ephrin-B2 at the optic chiasm midline selectively repels EphB1-expressing retinal ganglion cell axons from ventrotemporal retina; EphB1 null mice show dramatically reduced ipsilateral retinal projection, establishing EphB1 as the key receptor mediating retinal axon divergence at the chiasm.\",\n      \"method\": \"EphB1 knockout mouse, in vitro stripe/collapse assay with chiasm cells, blocking antibody experiments, semiintact visual system preparation\",\n      \"journal\": \"Neuron\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — knockout mouse with defined projection phenotype, in vitro rescue/block experiments, replicated across multiple assays\",\n      \"pmids\": [\"12971893\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Activated EphB1 recruits Grb2 and p52Shc, promotes p52Shc and c-Src tyrosine phosphorylation, and activates MAPK/ERK to drive chemotaxis; Tyr600 and Tyr778 of EphB1 are required for interaction with c-Src and p52Shc; p52Shc phosphorylation by c-Src is required for its recruitment to EphB1 signaling complexes via its PTB domain.\",\n      \"method\": \"Co-immunoprecipitation, dominant-negative c-Src, MEK inhibitor PD98059, Src inhibitor PP2, site-directed mutagenesis (Y600F, Y778F), cell migration assay\",\n      \"journal\": \"Journal of Cell Biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — mutagenesis + pharmacological inhibitors + co-IP + functional chemotaxis assay\",\n      \"pmids\": [\"12925710\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Activated EphB1 induces c-Src-dependent tyrosine phosphorylation of paxillin at Tyr31 and Tyr118 and is recruited to paxillin-FAK complexes; Nck binding site Y594F mutation disrupts the EphB1–Nck–paxillin complex; paxillin Y31F/Y118F mutant cells show reduced EphB1-dependent migration.\",\n      \"method\": \"Co-immunoprecipitation, site-directed mutagenesis (Y31F/Y118F paxillin, Y594F EphB1), dominant-negative kinase-dead c-Src, PP2 inhibitor, cell migration assay\",\n      \"journal\": \"Journal of Biological Chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — mutagenesis of both receptor and substrate + co-IP + functional assay\",\n      \"pmids\": [\"15107421\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"EphB1 receptors localize to caveolae via direct interaction with caveolin-1 through a caveolin-binding motif in the EphB1 kinase domain; overexpression of scaffolding-domain-deleted caveolin-1 abrogates EphB1–caveolin-1 interaction and ERK activation; the caveolin-binding motif is required for correct EphB1 membrane targeting.\",\n      \"method\": \"Sucrose density gradient fractionation, co-immunoprecipitation, dominant-negative caveolin-1, membrane targeting assay\",\n      \"journal\": \"Journal of Cell Science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — biochemical fractionation + co-IP + dominant-negative with functional consequence\",\n      \"pmids\": [\"16723736\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Ephrin-B1 stimulation triggers Cbl recruitment to EphB1, Cbl phosphorylation (requiring Src kinase activity), EphB1 ubiquitination, and lysosomal degradation of EphB1; kinase-dead EphB1-K652R is resistant to Cbl-mediated degradation; GST pull-down showed Cbl binds EphB1 through its tyrosine kinase-binding domain.\",\n      \"method\": \"Ubiquitination assay, lysosomal inhibitor (bafilomycin), co-immunoprecipitation, overexpression of Cbl WT vs. 70Z ligase-dead mutant, GST pull-down, Src inhibitor PP2\",\n      \"journal\": \"Traffic\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — multiple orthogonal methods (ubiquitination assay, GST pulldown, inhibitor, mutants) in single study\",\n      \"pmids\": [\"18034775\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Zic2 transcription factor regulates EphB1 expression in retinal ganglion cells; ectopic Zic2 delivery induces EphB1 mRNA and protein in non-ventrotemporal retinal explants, and upregulated EphB1 localizes to growth cones and is sufficient to convert RGC axon behavior from extension onto to avoidance of ephrin-B2 substrates.\",\n      \"method\": \"In utero retinal electroporation, explant growth cone collapse assay, immunofluorescence of EphB1 in growth cones\",\n      \"journal\": \"Journal of Neuroscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — gain-of-function in vivo with defined molecular and cellular readout\",\n      \"pmids\": [\"18524895\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"In vivo electroporation showing that ectopic EphB1 expression is sufficient to redirect normally crossed RGC axons to ipsilateral trajectory; EphB2, despite high similarity, is much less efficient; chimeric receptor analysis reveals both extracellular and juxtamembrane domains of EphB1 are required for efficient ipsilateral redirection.\",\n      \"method\": \"In utero retinal electroporation, EphB1/EphB2 chimeric receptors, anterograde tracing\",\n      \"journal\": \"Journal of Neuroscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vivo gain-of-function with domain-swap analysis\",\n      \"pmids\": [\"19295152\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"EphB1 null mice exhibit a 40% decrease in neuron number and volume in substantia nigra pars reticulata (SNr) but not pars compacta (SNc), and display spontaneous locomotor hyperactivity, establishing EphB1 as required for proper SNr formation.\",\n      \"method\": \"EphB1 knockout mouse, beta-galactosidase reporter, stereological neuron counting, open-field behavioral assay\",\n      \"journal\": \"European Journal of Neuroscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — knockout mouse with quantitative anatomical and behavioral readout\",\n      \"pmids\": [\"17561836\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"EphB1 receptor is required for long-term potentiation (LTP) at C-fiber–spinal dorsal horn synapses in vivo; targeted mutation of EphB1 prevents HFS-induced LTP and associated upregulation of p-CaMKII, p-ERK, p-CREB, and c-Fos in the spinal cord.\",\n      \"method\": \"EphB1 knockout mouse, in vivo electrophysiology, immunoblotting for signaling markers, MK-801 and EphB2-Fc blocking\",\n      \"journal\": \"Molecular Pain\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — knockout with electrophysiological and biochemical readouts\",\n      \"pmids\": [\"19523204\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"EphB1 forward signaling in spinal cord mediates bone cancer pain; spinal EphB1 activation by ephrin-B2 phosphorylates NR1 and NR2B subunits of NMDA receptors; blocking EphB1 with EphB2-Fc reverses pain behaviors and reduces c-Fos, astrocyte/microglial activation, and Src phosphorylation within the NMDA complex; MMP-2/9 regulate EphB1 levels.\",\n      \"method\": \"EphB1 blocking reagent (EphB2-Fc), EphB1 knockout mouse, intrathecal administration, immunoblotting for NMDA subunit phosphorylation, MMP inhibitors\",\n      \"journal\": \"Cancer Research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — pharmacological blocking + knockout + defined molecular pathway with multiple readouts\",\n      \"pmids\": [\"21555368\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"EphB1 forward signaling is required for ipsilateral retinal projection at the optic chiasm; knock-in of intracellularly truncated EphB1 (EphB1T-lacZ) eliminates the ipsilateral projection, confirming that EphB1 functions as a receptor (not reverse signaling source); EphB1 is the preferred receptor for ephrin-B2 (and to lesser degree ephrin-B1) at the chiasm.\",\n      \"method\": \"Knock-in mouse with intracellular truncation (EphB1T-lacZ), anterograde retinal tracing\",\n      \"journal\": \"European Journal of Neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — knock-in allele with defined molecular mechanism (forward vs. reverse signaling) and anatomical phenotype\",\n      \"pmids\": [\"22103419\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"EphB1 recruits Caskin1 scaffold protein through adaptor Nck; upon EphB1 kinase activation, Caskin1 is tyrosine-phosphorylated at Y296 and Y336 (identified by mass spectrometry), and this phosphorylation causes significant structural changes in the Caskin1 SH3 domain as shown by CD spectroscopy.\",\n      \"method\": \"Co-immunoprecipitation, mass spectrometry, CD spectroscopy\",\n      \"journal\": \"Cell Communication and Signaling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — co-IP with MS identification + structural consequence measured by CD spectroscopy\",\n      \"pmids\": [\"23181695\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"PTEN constitutively associates with c-Cbl, protecting Cbl from degradation; EphB1 stimulation triggers PTEN dephosphorylation (requiring PTEN protein phosphatase activity) and disruption of the PTEN–Cbl complex, allowing independent translocation of both proteins to EphB1 via scaffold NHERF1; PTEN lipid phosphatase activity impairs EphB1-dependent cell adhesion and chemotaxis.\",\n      \"method\": \"Co-immunoprecipitation, PTEN/NHERF1 siRNA, PTEN phosphatase mutants, cell adhesion and migration assay\",\n      \"journal\": \"FASEB Journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple co-IPs + siRNA + phosphatase mutants + functional assays\",\n      \"pmids\": [\"23118026\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"EphB1 expressed in striatal anlage signals through ephrin-B3 reverse signaling with dual context-dependent outcomes: in cortical interneurons EphB1-ephrinB3 reverse signaling phosphorylates Src and FAK to mediate repulsion, whereas in striatal neurons it reduces endogenously high pSrc and pFAK to act as a stop signal; ephrin-B3 knockout mice show misrouted cortical interneurons in the striatum and over-migration of striatal neurons.\",\n      \"method\": \"Ephrin-B3 knockout mouse, in vitro migration assays, immunoblotting for pSrc and pFAK\",\n      \"journal\": \"Frontiers in Cellular Neuroscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — knockout with defined molecular signaling differences between two cell types\",\n      \"pmids\": [\"25100946\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Intracellular signaling domains of EphB1 and EphB2 are critical for formation of the corpus callosum and anterior commissure; truncated EphB1/EphB2 mice lacking intracellular domains show partial/complete agenesis of corpus callosum and highly penetrant anterior commissure misprojection, indicating combined forward and reverse signaling contributions.\",\n      \"method\": \"Truncated receptor knock-in mice, anterograde tract tracing, brain anatomy analysis\",\n      \"journal\": \"Developmental Neurobiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — knock-in mouse allele with defined anatomical phenotype\",\n      \"pmids\": [\"26148571\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Injured motor neurons upregulate EphB1, which activates astrocytes through ephrin-B1-mediated STAT3 stimulation to induce a protective anti-inflammatory transcriptional signature; this EphB1–ephrin-B1–STAT3 pathway is disrupted in human stem-cell-derived ALS astrocytes and mouse ALS models.\",\n      \"method\": \"Human iPSC-derived astrocytes, mouse ALS models, transcriptional analysis, STAT3 pathway activation assay\",\n      \"journal\": \"Nature Communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — defined molecular pathway with multiple disease models and transcriptional readout\",\n      \"pmids\": [\"29079839\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Porf-2, a neuronally expressed RhoGAP, binds EphB1 and is regulated downstream; activated EphB1 forward signaling deactivates Rac1 through the Porf-2 GAP domain, inhibiting growth cone expansion and braking axon growth.\",\n      \"method\": \"Co-immunoprecipitation, Rac1 activity assay (pull-down), Porf-2 GAP domain mutants, axon growth assay\",\n      \"journal\": \"Cellular and Molecular Life Sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — co-IP + Rac1 activity assay + functional axon growth readout\",\n      \"pmids\": [\"29938386\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"EphB1 is SUMOylated at lysine 785; SUMOylation-deficient K785R mutant shows enhanced cell proliferation, anchorage-independent growth, and xenograft tumor growth; mechanistically, EphB1 SUMOylation represses activation of PKCγ, and a reciprocal regulatory loop between PKCγ and EphB1 SUMOylation exists.\",\n      \"method\": \"Ni2+-NTA pull-down, co-immunoprecipitation, K785R mutant, xenograft mouse model, soft agar colony assay, PKCγ activation assay\",\n      \"journal\": \"Cellular Physiology and Biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — PTM identified with pull-down + mutant + functional in vivo readout\",\n      \"pmids\": [\"29550816\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"EphB1 constitutively interacts with caveolin-1 (Cav-1) via its CSD-binding motif; upon ephrin-B1 activation, EphB1 is phosphorylated and uncouples from Cav-1, enabling Src-dependent Cav-1 phosphorylation; loss of EphB1 promotes Cav-1 ubiquitination and degradation, reducing caveolae numbers in endothelial cells.\",\n      \"method\": \"Super-resolution microscopy, FRET, co-immunoprecipitation, EphB1-/- mice endothelial cells, CSD deletion mutant, Cav-1 ubiquitination assay\",\n      \"journal\": \"Molecular Biology of the Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — FRET + biochemistry + KO mice + deletion mutant, multiple orthogonal approaches\",\n      \"pmids\": [\"32238105\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"TGF-β–activated Smad2 transcriptionally upregulates EphB1 expression; ligand-independent (unphosphorylated) EphB1 promotes EMT and lung cancer cell migration/invasion through upregulation of CDH2 (N-cadherin), while ligand-dependent (phosphorylated) EphB1 inhibits these processes.\",\n      \"method\": \"Western blot, real-time PCR, transwell migration/invasion assay, EphB1 phosphorylation-deficient mutant, Smad2 overexpression\",\n      \"journal\": \"Journal of Cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — phosphorylation mutant + functional assay + upstream pathway identification\",\n      \"pmids\": [\"32368295\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Tetracyclines (demeclocycline, chlortetracycline, minocycline) inhibit EphB1 kinase activity at low micromolar concentrations by binding the ATP-binding catalytic domain; co-crystal structure of chlortetracycline with EphB1 confirmed this binding; in vivo administration inhibited EphB1 phosphorylation in spinal cord/DRG and blocked neuropathic pain.\",\n      \"method\": \"In silico docking, in vitro kinase assay, co-crystallization (crystal structure), in vivo phosphorylation assay, neuropathic pain mouse model\",\n      \"journal\": \"PNAS\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure + in vitro kinase assay + in vivo validation\",\n      \"pmids\": [\"33627480\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"EphB1 is neddylated in activated hepatic stellate cells; neddylation enhances EphB1 kinase activity by preventing its degradation, thereby promoting HSC proliferation, migration, and activation contributing to liver fibrosis.\",\n      \"method\": \"Immunoprecipitation, neddylation inhibitor, EphB1 kinase activity assay, HSC functional assays\",\n      \"journal\": \"International Journal of Molecular Sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — PTM identified with co-IP + kinase activity assay + functional cellular readouts\",\n      \"pmids\": [\"36834826\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"CRC-associated somatic mutations in EphB1 kinase domain reduce kinase activity and protein stability in vitro; mutant EphB1 receptors fail to suppress STAT3 and ERK1/2 signaling and are unable to suppress CRC cell migration or mediate cell compartmentalization when cocultured with ephrin-B1-expressing cells.\",\n      \"method\": \"Purified kinase domain assay, thermal stability assay, cell migration assay, cell compartmentalization coculture assay, western blot for STAT3/ERK signaling\",\n      \"journal\": \"Journal of Biological Chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — purified protein kinase assay + mutagenesis + multiple functional readouts\",\n      \"pmids\": [\"37527777\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Recurring EPHB1 cancer mutations in ligand-binding, fibronectin, and kinase domains reduce ligand-induced receptor phosphorylation and cell compartmentalization; enhanced compartmentalization mutations in kinase domain (R743W, G821R) are not linked to phosphorylation level; reduced compartmentalization mutants activate PIK3C2B phosphorylation via the PI3K pathway.\",\n      \"method\": \"Confocal microscopy compartmentalization assay, phospho-proteome mass spectrometry, bioinformatic 3D-cluster analysis\",\n      \"journal\": \"Cell Communication and Signaling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — phosphoproteomics + functional compartmentalization assay, single lab\",\n      \"pmids\": [\"38102712\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"EphB1 conditional knockout specifically in GABAergic neurons (Vgat-Cre) but not cortical excitatory neurons (Emx1-Cre) or endothelial cells (Tie2-Cre) reproduces cortical axon guidance defects seen in global EphB1 KO; EphB1 in GABAergic neurons promotes avoidance of ectopic fasciculation with brain vasculature by glutamatergic cortical axons.\",\n      \"method\": \"Cell-type specific conditional knockout (Vgat-Cre, Emx1-Cre, Tie2-Cre), anterograde tract tracing\",\n      \"journal\": \"Development\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — cell-type-specific conditional KO defining non-autonomous mechanism\",\n      \"pmids\": [\"38345254\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Tumor cell-expressed EPHB1 and platelet-expressed EFNB1 mediate contact-dependent reverse signaling activation of platelets via AKT pathway; activated platelets then release 5-HT which enhances tumor growth in the liver metastatic niche of pancreatic ductal adenocarcinoma.\",\n      \"method\": \"Gain- and loss-of-function of Ephb1, recombinant protein assays, Tph1-knockout mice (5-HT depletion), tumor-platelet adhesion assay, metastasis mouse model\",\n      \"journal\": \"Cancer Communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple gain/loss-of-function approaches + genetic (Tph1 KO) validation of 5-HT output\",\n      \"pmids\": [\"39648610\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"EphB1 promotes CREB phosphorylation via PI3K/AKT signaling in a cell-autonomous manner in hypothalamic neurons; EphB1 deficiency reduces CRH and TRH expression in hypothalamus, causing impaired thermogenesis, reduced locomotor activity, and obesity; intracerebroventricular TRH or CRH peptide rescues obesity in Ephb1 mutants.\",\n      \"method\": \"Forward genetic screen, EphB1 knockout mouse, primary hypothalamic cell signaling assay, intracerebroventricular peptide administration, metabolic phenotyping\",\n      \"journal\": \"Obesity\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — KO mouse with defined pathway (PI3K/AKT/CREB) + peptide rescue experiment\",\n      \"pmids\": [\"40207393\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"EphB1 activation in thalamic ventroposteromedial nucleus (VPM) glutamatergic neurons promotes emergence from anesthesia via two independent mechanisms: (1) phosphorylation of NMDA receptor NR2B subunit at Tyr1472, exciting VPMGlu neurons; and (2) ubiquitin-dependent degradation of KCC2, disinhibiting VPMGlu neurons that project to primary somatosensory cortex.\",\n      \"method\": \"EphB1 conditional knockout in VPM, site-specific viral vector manipulation, in vivo electrophysiology, immunoblotting for NR2B-Y1472 phosphorylation and KCC2 ubiquitination, behavioral anesthesia emergence assay\",\n      \"journal\": \"Science Advances\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — conditional KO with two mechanistically distinct downstream pathways defined and functionally validated\",\n      \"pmids\": [\"41348875\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"EphB1 is a receptor tyrosine kinase that, upon binding membrane-anchored ephrin-B ligands, activates multiple signaling branches including Nck–JNK (via pY594), c-Src/Shc–MAPK/ERK (via pY600/pY778), and Grb7-dependent pathways to regulate integrin-mediated cell adhesion and directional cell migration; it undergoes Cbl-mediated ubiquitination and lysosomal degradation in a Src-dependent manner, is localized to caveolae through caveolin-1 interaction, and is post-translationally modified by neddylation and SUMOylation to modulate its activity; in the nervous system EphB1 acts as the primary receptor for ephrin-B2-mediated repulsion to direct ipsilateral retinal axon routing at the optic chiasm, guide neural crest migration, and modulate spinal NMDA receptor phosphorylation underlying pain sensitization and synaptic plasticity.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"EphB1 is a receptor tyrosine kinase that transduces ephrin-B ligand engagement into diverse cellular outcomes—including repulsive axon guidance, integrin-mediated cell adhesion, directed cell migration, and synaptic plasticity—through multiple phosphotyrosine-dependent signaling branches. Ligand-stimulated EphB1 autophosphorylation recruits Nck (via pY594) to activate JNK and paxillin phosphorylation, c-Src/Shc (via pY600/pY778) to drive MAPK/ERK-dependent chemotaxis, and Grb7 (via pY928) to promote cell motility; it also signals through PI3K/AKT/CREB in hypothalamic neurons and phosphorylates NMDA receptor NR2B subunits in spinal and thalamic circuits to modulate pain sensitization and arousal [PMID:9430661, PMID:12925710, PMID:12223469, PMID:40207393, PMID:41348875]. EphB1 is the primary receptor mediating ephrin-B2–dependent repulsion of ventrotemporal retinal ganglion cell axons at the optic chiasm, and its forward signaling is required for ipsilateral retinal projection, corpus callosum formation, and cortical axon guidance via a non-cell-autonomous mechanism in GABAergic neurons [PMID:12971893, PMID:22103419, PMID:26148571, PMID:38345254]. EphB1 activity is regulated by caveolin-1–dependent membrane compartmentalization, Cbl-mediated ubiquitination and lysosomal degradation, SUMOylation at K785 that represses PKCγ, and neddylation that stabilizes the receptor and enhances kinase output [PMID:32238105, PMID:18034775, PMID:29550816, PMID:36834826].\",\n  \"teleology\": [\n    {\n      \"year\": 1997,\n      \"claim\": \"Establishing EphB1 as a guidance receptor: the first demonstration that EphB1 mediates repulsive boundary formation during neural crest migration via ephrin-B2 interaction, answering how branchial arch territories are maintained.\",\n      \"evidence\": \"Dominant-negative truncated EphB1 overexpression in Xenopus embryos caused neural crest mis-migration into adjacent arch territories\",\n      \"pmids\": [\"9259557\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream signaling pathways mediating repulsion were unknown\", \"Mammalian relevance not tested\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Identifying the first signaling branch downstream of EphB1: Nck adaptor binds pY594 in the juxtamembrane region and couples the receptor to JNK/SAPK activation and cell attachment, establishing the first defined phosphotyrosine-effector link.\",\n      \"evidence\": \"Yeast two-hybrid, co-IP, Y594F mutagenesis, and JNK kinase assay\",\n      \"pmids\": [\"9430661\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Other signaling branches (MAPK/ERK) not yet mapped\", \"In vivo relevance of Nck–JNK axis not tested\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Demonstrating that EphB1 acts as a ligand-density sensor to regulate integrin-mediated adhesion, establishing that the kinase's signaling output—not mechanical tethering—controls cell attachment through αvβ3 and α5β1 integrins.\",\n      \"evidence\": \"Reconstituted ephrin-B1 surface density assay with signaling-dead EphB1 point mutants and integrin-blocking antibodies\",\n      \"pmids\": [\"10205170\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Which specific signaling branch connects to integrin activation was unclear\", \"Density-sensing mechanism at receptor level not resolved\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Mapping the Grb7–pY928 signaling branch: EphB1 phosphorylates Grb7 via its SH2 domain binding to pY928, and this interaction drives cell motility, revealing a third distinct phosphotyrosine-dependent output.\",\n      \"evidence\": \"Yeast two-hybrid, co-IP, Y928F mutagenesis, dominant-negative Grb7 SH2 domain, cell migration assay\",\n      \"pmids\": [\"12223469\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relationship between Grb7 and integrin-mediated outputs not clarified\", \"In vivo relevance not tested\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Defining EphB1 as the essential receptor for ipsilateral retinal axon guidance at the optic chiasm: EphB1 knockout mice lose most ipsilateral projection, and ephrin-B2 at the midline selectively repels EphB1-expressing ventrotemporal RGC axons.\",\n      \"evidence\": \"EphB1 knockout mouse with anterograde tracing, stripe/collapse assays, blocking antibodies\",\n      \"pmids\": [\"12971893\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Forward vs. reverse signaling contribution not yet dissected\", \"Downstream intracellular effectors mediating growth cone turning uncharacterized\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Mapping the c-Src/Shc–MAPK/ERK chemotaxis branch: pY600 and pY778 recruit c-Src and p52Shc; Src-dependent Shc phosphorylation activates ERK to drive directional migration, providing a second major signaling axis for cell motility.\",\n      \"evidence\": \"Co-IP, Y600F/Y778F mutagenesis, Src inhibitor PP2, MEK inhibitor PD98059, cell migration assay\",\n      \"pmids\": [\"12925710\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cross-talk between Nck–JNK and Src–ERK branches not defined\", \"Relative contribution of branches to in vivo guidance unknown\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Linking EphB1 signaling to focal adhesion remodeling: activated EphB1 drives Src-dependent paxillin phosphorylation at Y31/Y118 and assembles an EphB1–Nck–paxillin–FAK complex required for migration.\",\n      \"evidence\": \"Co-IP, paxillin Y31F/Y118F and EphB1 Y594F mutagenesis, kinase-dead Src, migration assay\",\n      \"pmids\": [\"15107421\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How paxillin phosphorylation coordinates with integrin activation from the 1999 study was unknown\", \"Spatial dynamics of complex assembly not resolved\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Establishing caveolae as a signaling platform for EphB1: the receptor localizes to caveolae via a caveolin-binding motif in the kinase domain, and this compartmentalization is required for ERK activation.\",\n      \"evidence\": \"Sucrose gradient fractionation, co-IP, dominant-negative caveolin-1 lacking scaffolding domain\",\n      \"pmids\": [\"16723736\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Stoichiometry of EphB1–caveolin-1 interaction not determined\", \"Whether caveolar localization affects other signaling branches (JNK, Grb7) unknown\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Defining the degradation pathway: Cbl is recruited to activated EphB1 in a Src-dependent manner, ubiquitinates the receptor, and targets it for lysosomal degradation, establishing how EphB1 signaling is attenuated.\",\n      \"evidence\": \"Ubiquitination assay, bafilomycin inhibition, GST pull-down of Cbl TKB domain, kinase-dead K652R mutant resistant to degradation, PP2 Src inhibitor\",\n      \"pmids\": [\"18034775\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Specific ubiquitin chain types (K48 vs. K63) not determined\", \"Whether proteasomal degradation also contributes was not resolved\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Identifying the transcriptional regulator upstream of EphB1 in retinal neurons: Zic2 drives EphB1 expression and is sufficient to convert RGC axon behavior from extension to avoidance on ephrin-B2 substrates.\",\n      \"evidence\": \"In utero retinal electroporation, explant growth cone collapse assay\",\n      \"pmids\": [\"18524895\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Zic2 binding sites in EphB1 regulatory regions not mapped\", \"Other transcriptional regulators of EphB1 expression not identified\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Confirming forward signaling as the critical mode at the chiasm: intracellularly truncated EphB1 knock-in eliminates ipsilateral projection, ruling out reverse signaling contributions and proving that the kinase domain and downstream effectors are required.\",\n      \"evidence\": \"EphB1T-lacZ knock-in mouse with anterograde retinal tracing\",\n      \"pmids\": [\"22103419\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Which specific intracellular domain (kinase, SAM, PDZ-binding) is essential was not dissected\", \"Contribution of kinase activity vs. scaffold function not separated\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Extending EphB1 function to pain processing: spinal EphB1 activation by ephrin-B2 phosphorylates NMDA receptor NR1/NR2B subunits to maintain bone cancer pain, with MMP-2/9 regulating EphB1 levels.\",\n      \"evidence\": \"EphB1 KO mouse, intrathecal EphB2-Fc blocking, immunoblotting for NR subunit phosphorylation, MMP inhibitors\",\n      \"pmids\": [\"21555368\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct phosphorylation vs. indirect Src-mediated NR2B phosphorylation not fully resolved\", \"MMP regulation of EphB1 protein levels not mechanistically detailed\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Integrating PTEN into the EphB1 signaling network: PTEN constitutively sequesters Cbl, and ephrin stimulation disrupts this complex via PTEN dephosphorylation, freeing both proteins to modulate EphB1 adhesion and migration through NHERF1 scaffold.\",\n      \"evidence\": \"Co-IP, PTEN/NHERF1 siRNA, phosphatase-dead PTEN mutants, adhesion and migration assays\",\n      \"pmids\": [\"23118026\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether PTEN lipid phosphatase activity directly opposes PI3K signaling downstream of EphB1 was not tested\", \"In vivo relevance of PTEN–Cbl regulation not demonstrated\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Identifying SUMOylation as a tumor-suppressive post-translational modification of EphB1: K785 SUMOylation represses PKCγ activation and suppresses anchorage-independent growth and xenograft tumor formation.\",\n      \"evidence\": \"Ni²⁺-NTA pull-down, K785R mutant, xenograft mouse model, soft agar assay, PKCγ activation measurement\",\n      \"pmids\": [\"29550816\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"SUMO E3 ligase responsible not identified\", \"Whether SUMOylation and caveolin-1 interaction at the kinase domain are mutually exclusive was not tested\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Resolving the dynamic EphB1–caveolin-1 relationship: EphB1 constitutively protects Cav-1 from ubiquitination; upon ligand activation, phosphorylated EphB1 uncouples from Cav-1 and enables Src-dependent Cav-1 phosphorylation, maintaining caveolae homeostasis in endothelial cells.\",\n      \"evidence\": \"Super-resolution microscopy, FRET, co-IP, EphB1−/− mouse endothelial cells, Cav-1 ubiquitination assay\",\n      \"pmids\": [\"32238105\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether disrupted caveolae affect vascular permeability in vivo via EphB1 was not addressed\", \"Identity of the E3 ligase for Cav-1 in EphB1-null cells unknown\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Characterizing cancer-associated EphB1 mutations: somatic mutations across ligand-binding, fibronectin, and kinase domains reduce kinase activity and protein stability, abolish STAT3/ERK suppression and cell compartmentalization, and activate PIK3C2B through PI3K, establishing EphB1 as a functional tumor suppressor in colorectal cancer.\",\n      \"evidence\": \"Purified kinase domain assays, thermal stability, migration assay, compartmentalization coculture, phosphoproteomics\",\n      \"pmids\": [\"37527777\", \"38102712\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo tumor suppressor role not confirmed by conditional knockout tumor models\", \"Whether enhanced-compartmentalization mutations (R743W, G821R) represent gain-of-function or neomorphic activity is unclear\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identifying neddylation as a stabilizing modification: EphB1 neddylation in hepatic stellate cells prevents receptor degradation and sustains kinase activity, promoting HSC proliferation and liver fibrosis.\",\n      \"evidence\": \"Immunoprecipitation, neddylation inhibitor, kinase activity assay, HSC functional assays\",\n      \"pmids\": [\"36834826\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Neddylation sites on EphB1 not mapped\", \"Interplay between neddylation and Cbl-mediated ubiquitination not tested\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Resolving non-cell-autonomous axon guidance: EphB1 in GABAergic (not glutamatergic) neurons provides the guidance signal that prevents glutamatergic cortical axons from aberrantly fasciculating with brain vasculature.\",\n      \"evidence\": \"Cell-type-specific conditional KO (Vgat-Cre, Emx1-Cre, Tie2-Cre) with anterograde tracing\",\n      \"pmids\": [\"38345254\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"The molecular mechanism by which GABAergic EphB1 instructs glutamatergic axon routing is unknown\", \"Whether this involves secreted signals or contact-dependent signaling between neuron types is not resolved\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Extending EphB1 function to metabolic regulation: hypothalamic EphB1 activates PI3K/AKT/CREB to drive CRH and TRH expression for thermogenesis and energy homeostasis; loss causes obesity rescued by intracerebroventricular neuropeptide replacement.\",\n      \"evidence\": \"EphB1 KO mouse, primary hypothalamic neuron signaling assays, ICV peptide rescue, metabolic phenotyping\",\n      \"pmids\": [\"40207393\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Upstream ligand activating hypothalamic EphB1 not identified\", \"Whether kinase activity or scaffold function mediates CREB phosphorylation not dissected\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Revealing dual downstream mechanisms in thalamic arousal circuits: EphB1 in VPM glutamatergic neurons both phosphorylates NR2B-Y1472 to enhance excitation and promotes ubiquitin-dependent KCC2 degradation to disinhibit neurons, facilitating emergence from anesthesia.\",\n      \"evidence\": \"Conditional KO in VPM, viral manipulation, in vivo electrophysiology, NR2B-Y1472 phosphorylation and KCC2 ubiquitination assays\",\n      \"pmids\": [\"41348875\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"E3 ligase responsible for KCC2 ubiquitination downstream of EphB1 not identified\", \"Whether EphB1 directly phosphorylates NR2B or acts through Src/Fyn not resolved in this system\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the structural basis for EphB1's specificity over EphB2 in chiasm guidance, how the multiple phosphotyrosine-dependent signaling branches are coordinated in space and time, and the physiological interplay among SUMOylation, neddylation, and Cbl-mediated ubiquitination in regulating receptor output.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No full-length EphB1 structure with signaling partners resolved\", \"Relative quantitative contribution of individual pY sites to specific in vivo phenotypes untested\", \"Cross-regulation between SUMOylation, neddylation, and ubiquitination not addressed\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [1, 5, 7, 8, 26, 28]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [23, 28, 33]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [9, 24]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0162582\", \"supporting_discovery_ids\": [1, 5, 7, 8, 15, 32, 33]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [0, 6, 11, 16, 20, 30]},\n      {\"term_id\": \"R-HSA-112316\", \"supporting_discovery_ids\": [6, 14, 15, 33]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [10, 23, 27]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"NCK1\",\n      \"GRB7\",\n      \"SRC\",\n      \"SHC1\",\n      \"CBL\",\n      \"CAV1\",\n      \"PAXILLIN\",\n      \"EFNB2\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```\n\nWait, I need to fix the pathway term_ids. I used \"GO:0162582\" which is wrong. Let me correct:\n\n```json\n{\n  \"mechanistic_narrative\": \"EphB1 is a receptor tyrosine kinase that transduces ephrin-B ligand engagement into diverse cellular outcomes—including repulsive axon guidance, integrin-mediated cell adhesion, directed cell migration, and synaptic plasticity—through multiple phosphotyrosine-dependent signaling branches. Ligand-stimulated EphB1 autophosphorylation recruits Nck (via pY594) to activate JNK and paxillin phosphorylation, c-Src/Shc (via pY600/pY778) to drive MAPK/ERK-dependent chemotaxis, and Grb7 (via pY928) to promote cell motility; it also signals through PI3K/AKT/CREB in hypothalamic neurons and phosphorylates NMDA receptor NR2B subunits in spinal and thalamic circuits to modulate pain sensitization and arousal [PMID:9430661, PMID:12925710, PMID:12223469, PMID:40207393, PMID:41348875]. EphB1 is the primary receptor mediating ephrin-B2–dependent repulsion of ventrotemporal retinal ganglion cell axons at the optic chiasm, and its forward signaling is required for ipsilateral retinal projection, corpus callosum formation, and cortical axon guidance via a non-cell-autonomous mechanism in GABAergic neurons [PMID:12971893, PMID:22103419, PMID:26148571, PMID:38345254]. EphB1 activity is regulated by caveolin-1–dependent membrane compartmentalization, Cbl-mediated ubiquitination and lysosomal degradation, SUMOylation at K785 that represses PKCγ, and neddylation that stabilizes the receptor and enhances kinase output [PMID:32238105, PMID:18034775, PMID:29550816, PMID:36834826].\",\n  \"teleology\": [\n    {\n      \"year\": 1997,\n      \"claim\": \"Establishing EphB1 as a guidance receptor: the first demonstration that EphB1 mediates repulsive boundary formation during neural crest migration via ephrin-B2 interaction, answering how branchial arch territories are maintained.\",\n      \"evidence\": \"Dominant-negative truncated EphB1 overexpression in Xenopus embryos caused neural crest mis-migration into adjacent arch territories\",\n      \"pmids\": [\"9259557\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream signaling pathways mediating repulsion were unknown\", \"Mammalian relevance not tested\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Identifying the first signaling branch downstream of EphB1: Nck adaptor binds pY594 in the juxtamembrane region and couples the receptor to JNK/SAPK activation and cell attachment, establishing the first defined phosphotyrosine-effector link.\",\n      \"evidence\": \"Yeast two-hybrid, co-IP, Y594F mutagenesis, and JNK kinase assay\",\n      \"pmids\": [\"9430661\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Other signaling branches (MAPK/ERK) not yet mapped\", \"In vivo relevance of Nck–JNK axis not tested\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Demonstrating that EphB1 acts as a ligand-density sensor to regulate integrin-mediated adhesion, establishing that the kinase's signaling output—not mechanical tethering—controls cell attachment through αvβ3 and α5β1 integrins.\",\n      \"evidence\": \"Reconstituted ephrin-B1 surface density assay with signaling-dead EphB1 point mutants and integrin-blocking antibodies\",\n      \"pmids\": [\"10205170\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Which specific signaling branch connects to integrin activation was unclear\", \"Density-sensing mechanism at receptor level not resolved\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Mapping the Grb7–pY928 signaling branch: EphB1 phosphorylates Grb7 via its SH2 domain binding to pY928, and this interaction drives cell motility, revealing a third distinct phosphotyrosine-dependent output.\",\n      \"evidence\": \"Yeast two-hybrid, co-IP, Y928F mutagenesis, dominant-negative Grb7 SH2 domain, cell migration assay\",\n      \"pmids\": [\"12223469\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relationship between Grb7 and integrin-mediated outputs not clarified\", \"In vivo relevance not tested\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Defining EphB1 as the essential receptor for ipsilateral retinal axon guidance at the optic chiasm: EphB1 knockout mice lose most ipsilateral projection, and ephrin-B2 at the midline selectively repels EphB1-expressing ventrotemporal RGC axons.\",\n      \"evidence\": \"EphB1 knockout mouse with anterograde tracing, stripe/collapse assays, blocking antibodies\",\n      \"pmids\": [\"12971893\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Forward vs. reverse signaling contribution not yet dissected\", \"Downstream intracellular effectors mediating growth cone turning uncharacterized\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Mapping the c-Src/Shc–MAPK/ERK chemotaxis branch: pY600 and pY778 recruit c-Src and p52Shc; Src-dependent Shc phosphorylation activates ERK to drive directional migration, providing a second major signaling axis for cell motility.\",\n      \"evidence\": \"Co-IP, Y600F/Y778F mutagenesis, Src inhibitor PP2, MEK inhibitor PD98059, cell migration assay\",\n      \"pmids\": [\"12925710\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cross-talk between Nck–JNK and Src–ERK branches not defined\", \"Relative contribution of branches to in vivo guidance unknown\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Linking EphB1 signaling to focal adhesion remodeling: activated EphB1 drives Src-dependent paxillin phosphorylation at Y31/Y118 and assembles an EphB1–Nck–paxillin–FAK complex required for migration.\",\n      \"evidence\": \"Co-IP, paxillin Y31F/Y118F and EphB1 Y594F mutagenesis, kinase-dead Src, migration assay\",\n      \"pmids\": [\"15107421\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How paxillin phosphorylation coordinates with integrin activation was unclear\", \"Spatial dynamics of complex assembly not resolved\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Establishing caveolae as a signaling platform for EphB1: the receptor localizes to caveolae via a caveolin-binding motif in the kinase domain, and this compartmentalization is required for ERK activation.\",\n      \"evidence\": \"Sucrose gradient fractionation, co-IP, dominant-negative caveolin-1 lacking scaffolding domain\",\n      \"pmids\": [\"16723736\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Stoichiometry of EphB1–caveolin-1 interaction not determined\", \"Whether caveolar localization affects other signaling branches (JNK, Grb7) unknown\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Defining the degradation pathway: Cbl is recruited to activated EphB1 in a Src-dependent manner, ubiquitinates the receptor, and targets it for lysosomal degradation, establishing how EphB1 signaling is attenuated.\",\n      \"evidence\": \"Ubiquitination assay, bafilomycin inhibition, GST pull-down of Cbl TKB domain, kinase-dead K652R mutant, PP2 Src inhibitor\",\n      \"pmids\": [\"18034775\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Specific ubiquitin chain types (K48 vs. K63) not determined\", \"Whether proteasomal degradation also contributes was not resolved\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Identifying the transcriptional regulator upstream of EphB1 in retinal neurons: Zic2 drives EphB1 expression and is sufficient to convert RGC axon behavior from extension to avoidance on ephrin-B2 substrates.\",\n      \"evidence\": \"In utero retinal electroporation, explant growth cone collapse assay\",\n      \"pmids\": [\"18524895\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Zic2 binding sites in EphB1 regulatory regions not mapped\", \"Other transcriptional regulators of EphB1 expression not identified\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Confirming forward signaling as the critical mode at the chiasm: intracellularly truncated EphB1 knock-in eliminates ipsilateral projection, ruling out reverse signaling contributions and proving that the kinase domain and downstream effectors are required.\",\n      \"evidence\": \"EphB1T-lacZ knock-in mouse with anterograde retinal tracing\",\n      \"pmids\": [\"22103419\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Which specific intracellular domain (kinase, SAM, PDZ-binding) is essential was not dissected\", \"Contribution of kinase activity vs. scaffold function not separated\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Extending EphB1 function to pain processing: spinal EphB1 activation by ephrin-B2 phosphorylates NMDA receptor NR1/NR2B subunits to maintain bone cancer pain, with MMP-2/9 regulating EphB1 levels.\",\n      \"evidence\": \"EphB1 KO mouse, intrathecal EphB2-Fc blocking, immunoblotting for NR subunit phosphorylation, MMP inhibitors\",\n      \"pmids\": [\"21555368\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct phosphorylation vs. indirect Src-mediated NR2B phosphorylation not fully resolved\", \"MMP regulation of EphB1 protein levels not mechanistically detailed\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Integrating PTEN into the EphB1 signaling network: PTEN constitutively sequesters Cbl, and ephrin stimulation disrupts this complex via PTEN dephosphorylation, freeing both proteins to modulate EphB1 adhesion and migration through NHERF1 scaffold.\",\n      \"evidence\": \"Co-IP, PTEN/NHERF1 siRNA, phosphatase-dead PTEN mutants, adhesion and migration assays\",\n      \"pmids\": [\"23118026\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether PTEN lipid phosphatase activity directly opposes PI3K signaling downstream of EphB1 was not tested\", \"In vivo relevance of PTEN–Cbl regulation not demonstrated\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Identifying SUMOylation as a tumor-suppressive post-translational modification of EphB1: K785 SUMOylation represses PKCγ activation and suppresses anchorage-independent growth and xenograft tumor formation.\",\n      \"evidence\": \"Ni²⁺-NTA pull-down, K785R mutant, xenograft mouse model, soft agar assay, PKCγ activation measurement\",\n      \"pmids\": [\"29550816\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"SUMO E3 ligase responsible not identified\", \"Whether SUMOylation and caveolin-1 interaction at the kinase domain are mutually exclusive was not tested\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Resolving the dynamic EphB1–caveolin-1 relationship: EphB1 constitutively protects Cav-1 from ubiquitination; upon ligand activation, phosphorylated EphB1 uncouples from Cav-1 and enables Src-dependent Cav-1 phosphorylation, maintaining caveolae homeostasis in endothelial cells.\",\n      \"evidence\": \"Super-resolution microscopy, FRET, co-IP, EphB1−/− mouse endothelial cells, Cav-1 ubiquitination assay\",\n      \"pmids\": [\"32238105\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether disrupted caveolae affect vascular permeability in vivo via EphB1 was not addressed\", \"Identity of the E3 ligase for Cav-1 in EphB1-null cells unknown\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Characterizing cancer-associated EphB1 mutations: somatic mutations across ligand-binding, fibronectin, and kinase domains reduce kinase activity and protein stability, abolish STAT3/ERK suppression and cell compartmentalization, and activate PIK3C2B through PI3K, establishing EphB1 as a functional tumor suppressor in colorectal cancer.\",\n      \"evidence\": \"Purified kinase domain assays, thermal stability, migration assay, compartmentalization coculture, phosphoproteomics\",\n      \"pmids\": [\"37527777\", \"38102712\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo tumor suppressor role not confirmed by conditional knockout tumor models\", \"Whether enhanced-compartmentalization mutations (R743W, G821R) represent gain-of-function or neomorphic activity is unclear\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identifying neddylation as a stabilizing modification: EphB1 neddylation in hepatic stellate cells prevents receptor degradation and sustains kinase activity, promoting HSC proliferation and liver fibrosis.\",\n      \"evidence\": \"Immunoprecipitation, neddylation inhibitor, kinase activity assay, HSC functional assays\",\n      \"pmids\": [\"36834826\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Neddylation sites on EphB1 not mapped\", \"Interplay between neddylation and Cbl-mediated ubiquitination not tested\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Resolving non-cell-autonomous axon guidance: EphB1 in GABAergic (not glutamatergic) neurons provides the guidance signal that prevents glutamatergic cortical axons from aberrantly fasciculating with brain vasculature.\",\n      \"evidence\": \"Cell-type-specific conditional KO (Vgat-Cre, Emx1-Cre, Tie2-Cre) with anterograde tracing\",\n      \"pmids\": [\"38345254\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"The molecular mechanism by which GABAergic EphB1 instructs glutamatergic axon routing is unknown\", \"Whether this involves secreted signals or contact-dependent signaling between neuron types is not resolved\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Extending EphB1 function to metabolic regulation: hypothalamic EphB1 activates PI3K/AKT/CREB to drive CRH and TRH expression for thermogenesis and energy homeostasis; loss causes obesity rescued by intracerebroventricular neuropeptide replacement.\",\n      \"evidence\": \"EphB1 KO mouse, primary hypothalamic neuron signaling assays, ICV peptide rescue, metabolic phenotyping\",\n      \"pmids\": [\"40207393\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Upstream ligand activating hypothalamic EphB1 not identified\", \"Whether kinase activity or scaffold function mediates CREB phosphorylation not dissected\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Revealing dual downstream mechanisms in thalamic arousal circuits: EphB1 in VPM glutamatergic neurons both phosphorylates NR2B-Y1472 to enhance excitation and promotes ubiquitin-dependent KCC2 degradation to disinhibit neurons, facilitating emergence from anesthesia.\",\n      \"evidence\": \"Conditional KO in VPM, viral manipulation, in vivo electrophysiology, NR2B-Y1472 phosphorylation and KCC2 ubiquitination assays\",\n      \"pmids\": [\"41348875\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"E3 ligase responsible for KCC2 ubiquitination downstream of EphB1 not identified\", \"Whether EphB1 directly phosphorylates NR2B or acts through Src/Fyn not resolved in this system\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the structural basis for EphB1's specificity over EphB2 in chiasm guidance, how the multiple phosphotyrosine-dependent signaling branches are coordinated in space and time, and the physiological interplay among SUMOylation, neddylation, and Cbl-mediated ubiquitination in regulating receptor output.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No full-length EphB1 structure with signaling partners resolved\", \"Relative quantitative contribution of individual pY sites to specific in vivo phenotypes untested\", \"Cross-regulation between SUMOylation, neddylation, and ubiquitination not addressed\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [1, 5, 7, 8, 26, 28]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [9, 24]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [1, 5, 7, 8, 15, 32, 33]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [0, 6, 11, 16, 20, 30]},\n      {\"term_id\": \"R-HSA-112316\", \"supporting_discovery_ids\": [6, 14, 15, 33]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [10, 23, 27]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"NCK1\",\n      \"GRB7\",\n      \"SRC\",\n      \"SHC1\",\n      \"CBL\",\n      \"CAV1\",\n      \"PXN\",\n      \"EFNB2\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}