{"gene":"EPHB6","run_date":"2026-06-09T23:54:43","timeline":{"discoveries":[{"year":2001,"finding":"EphB6, despite lacking intrinsic kinase activity, undergoes ligand-induced tyrosine phosphorylation via transphosphorylation by the catalytically active EphB1 receptor. EphB1 and EphB6 form a stable heterocomplex, and EphB1-induced EphB6 phosphorylation is ligand-dependent and requires EphB1 catalytic activity. Additionally, the proto-oncogene c-Cbl was identified as an EphB6-binding protein; EphB6-Cbl association is constitutive and requires a functional phosphotyrosine binding domain on Cbl.","method":"Co-immunoprecipitation, overexpression of catalytically active/inactive EphB1, ligand stimulation with ephrin-B1, kinase assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP and functional mutagenesis (kinase-dead EphB1 control), single lab with multiple orthogonal methods","pmids":["11713248"],"is_preprint":false},{"year":2001,"finding":"Cross-linking of EphB6 in Jurkat T cells triggers general protein tyrosine phosphorylation and associates with Cbl. EphB6 cross-linking results in Cbl dephosphorylation and dissociation of Cbl from SHP-1, leading to altered lymphokine secretion, proliferation inhibition, and Fas-mediated apoptosis.","method":"Co-cross-linking with anti-EphB6 antibody and anti-CD3, Co-immunoprecipitation, apoptosis assays","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — Co-IP and functional cellular assays, single lab, single study","pmids":["11466354"],"is_preprint":false},{"year":2002,"finding":"EphB6 crosslinking by anti-EphB6 mAb or ephrin-B2 in the presence of suboptimal TCR stimulation co-stimulates T cell proliferation and cytokine secretion. Within two minutes of anti-CD3/anti-CD28 stimulation, EphB6 aggregates and co-localizes with TCR at membrane rafts, and this is followed by p38 MAPK activation.","method":"Anti-EphB6 crosslinking, confocal microscopy for co-localization with TCR, cytokine ELISA, proliferation assays, p38 MAPK activation assays","journal":"The Journal of clinical investigation","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — direct imaging of co-localization plus functional readouts, single lab","pmids":["12393850"],"is_preprint":false},{"year":2003,"finding":"EphB6 overexpression in Jurkat T cells followed by ephrin-B1 stimulation selectively inhibits TCR-mediated JNK activation but not the MAPK pathway. The mechanism involves suppression of TCR-induced Rac1 GTPase activation upstream of JNK. EphB6 also blocks anti-CD3-induced IL-2 secretion and CD25 expression in a ligand-dependent manner. Dominant-negative EphB6 reverses these inhibitory effects. In murine thymocytes, ephrin-B1 stimulation inhibits TCR-mediated apoptosis and CD25 upregulation.","method":"Overexpression of wild-type and dominant-negative EphB6, Rac1 activation assay (GST-PAK pull-down), JNK kinase assay, IL-2 ELISA, flow cytometry for CD25","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods including dominant-negative rescue, kinase assays, and Rac1 pull-down in single study","pmids":["12517763"],"is_preprint":false},{"year":2004,"finding":"EphB6 migrates to aggregated TCRs and lipid rafts after TCR activation in T cells. In EphB6-deficient mice, ZAP-70 activation, LAT phosphorylation, PLCγ1 association with SLP-76, and p44/42 MAPK activation are diminished upon TCR stimulation, demonstrating EphB6 is required for full TCR downstream signaling.","method":"EphB6 knockout mice, biochemical fractionation to isolate rafts, immunoblotting for ZAP-70, LAT, PLCγ1/SLP-76 co-IP, MAPK activation assays, in vivo T cell functional assays","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO model with multiple downstream signaling readouts and in vivo validation, replicated across multiple assays","pmids":["15599401"],"is_preprint":false},{"year":2000,"finding":"Ephrin-B2 is a high-affinity ligand for EphB6. EphB6-Fc fusion protein binds ephrin-B2 but not ephrin-B1 on transfected COS cells in a saturable manner. The ligand identified on Reh pro-B cells is a transmembrane ephrin (not GPI-anchored, as PIPLC treatment did not abrogate binding).","method":"EphB6-Fc fusion protein binding assay, COS cell transfection with ephrin-B1 or ephrin-B2, phosphatidylinositol-specific phospholipase C treatment, saturation binding analysis","journal":"FEBS letters","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct in vitro binding reconstitution with fusion protein, saturable binding, negative control with ephrin-B1, confirmed in multiple cell types","pmids":["10648835"],"is_preprint":false},{"year":2002,"finding":"Ephrin-B2 is the unique ligand for EphB6 among eight known ephrins, as demonstrated by differential binding of ephrin-Fc proteins to EphB6-deficient versus wild-type thymocytes. EphB6 is the dominant ephrin-B2-binding receptor on adult thymocytes.","method":"EphB6 knockout mice, ephrin-Fc binding assays on thymocytes","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO mouse model with direct binding assay, replicates ligand specificity finding from PMID 10648835","pmids":["12379224"],"is_preprint":false},{"year":2005,"finding":"EphB6 exerts biphasic effects on cell adhesion and migration: low concentrations of ephrin-B2 promote adhesion and migration, while high concentrations induce repulsion and inhibit migration. A truncated EphB6 lacking the cytoplasmic domain shows only positive (monophasic) effects, indicating the cytoplasmic domain mediates negative effects. EphB6 is constitutively associated with the Src family kinase Fyn, and high ephrin-B2 concentrations induce EphB6 tyrosine phosphorylation through Src family kinase activity as the molecular switch.","method":"Cell adhesion and migration assays with varying ligand concentrations, cytoplasmic domain deletion mutants, co-immunoprecipitation with Fyn, Src family kinase inhibitors, tyrosine phosphorylation assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — domain deletion mutants, kinase inhibitors, and Co-IP identifying Fyn interaction; multiple orthogonal approaches in single study","pmids":["15955811"],"is_preprint":false},{"year":2010,"finding":"EphB6 suppresses breast cancer cell invasiveness through a mechanism requiring EphB4-mediated transphosphorylation of EphB6, followed by EphB6-c-Cbl interaction and phosphorylation of the Abl kinase. Cbl silencing suppresses Abl phosphorylation, cell adhesion, and morphological changes, and blocks EphB6's ability to inhibit invasiveness. EphB4 also acts in an EphB6-independent manner to enhance invasiveness, suggesting cancer invasiveness is defined by the balance in the EphB6-EphB4 system.","method":"Restoration of EphB6 expression in invasive breast cancer cells, Co-immunoprecipitation of EphB6 with c-Cbl, siRNA knockdown of Cbl, Abl phosphorylation assays, invasion assays","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 2 / Moderate — Co-IP, siRNA rescue experiments, multiple functional readouts in single study","pmids":["20086179"],"is_preprint":false},{"year":2011,"finding":"EPHB6 interacts with EPHB2 and with EPHA2 (the first demonstrated A-type/B-type Eph receptor interaction), but does not interact with another kinase-deficient receptor EPHA10. The tumor-invasiveness-suppressing activity of EPHB6 is mediated partly by sequestering kinase-sufficient oncogenic EPH receptors.","method":"Co-immunoprecipitation in mammalian breast carcinoma cell lines","journal":"Cancer genomics & proteomics","confidence":"Medium","confidence_rationale":"Tier 3 / Weak — single Co-IP experiment per interaction pair, single lab","pmids":["21737611"],"is_preprint":false},{"year":2014,"finding":"EphB6 interacts with EphA2 and suppresses EphA2-mediated anoikis resistance in MCF7 breast cancer cells by decreasing serine 897 phosphorylation of EphA2 and suppressing EphA2-Ephexin4 interaction and RhoG activation. Knockdown of EphB6 promotes anoikis resistance.","method":"Co-immunoprecipitation, phosphorylation assays, siRNA knockdown of EphB6, RhoG activation assay, anoikis assay","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — Co-IP plus functional knockdown with defined pathway placement, single lab","pmids":["25239188"],"is_preprint":false},{"year":2012,"finding":"EPHB6 and its ephrin-B ligands are expressed on vascular smooth muscle cells (VSMC). In castrated male Ephb6-KO mice, small arteries show increased contractility, RhoA activation, and constitutive myosin light chain phosphorylation ex vivo. Cross-linking ephrin-Bs (reverse signaling) but not EphB6 (forward signaling) reduces VSMC contractions, mediated by the adaptor protein GRIP1. Male Ephb6-KO mice show decreased 24-hour urine catecholamines as a compensatory mechanism.","method":"EphB6 knockout mice, ex vivo arterial contractility assays, RhoA activation assay, myosin light chain phosphorylation immunoblot, cross-linking assays, blood pressure measurement, catecholamine measurement","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — KO mouse model with multiple ex vivo and in vivo readouts, mechanistic pathway identification via GRIP1 and RhoA","pmids":["22223652"],"is_preprint":false},{"year":2019,"finding":"EPHB6 controls catecholamine biosynthesis in adrenal gland chromaffin cells (AGCCs) by upregulating tyrosine hydroxylase transcription. Ephb6-KO male mice have reduced epinephrine content due to decreased tyrosine hydroxylase. The signaling pathway from EPHB6 to tyrosine hydroxylase involves RAC1, MKK7, JNK, c-Jun, AP1, and EGR1. This effect operates via reverse signaling through ephrin-B1. ERK1/2, p38 MAPK, and ELK1 signaling were not affected by EPHB6 deletion.","method":"EphB6 knockout mice, RT-qPCR, immunoblotting, gene microarray, signaling pathway inhibitors, HPLC catecholamine measurement","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — KO model with multiple orthogonal methods (qPCR, immunoblot, microarray) and defined pathway components","pmids":["30824540"],"is_preprint":false},{"year":2018,"finding":"In male Ephb6-KO adrenal gland chromaffin cells, acetylcholine-triggered Ca2+ influx is reduced due to augmented large conductance calcium-activated potassium (BKCa) channel currents in a testosterone-dependent manner. Blocking BKCa channels in KO cells corrects the reduced Ca2+ influx. This phenotype depends on the non-genomic effect of testosterone and is absent in female or castrated KO mice.","method":"Patch-clamp electrophysiology, Ca2+ imaging, BKCa channel pharmacological blockade, EphB6 knockout mice, castration experiments","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 1 / Moderate — electrophysiology with pharmacological rescue, KO model, sex-hormone dependence established with castration controls","pmids":["29339804"],"is_preprint":false},{"year":2020,"finding":"In male Ephb6-KO adrenal gland chromaffin cells, catecholamine secretion via exocytosis is compromised and is accompanied by reduced cortical F-actin disassembly. Reverse signaling from EPHB6 to ephrin-B1 and a 7-amino acid segment in the EFNB1 intracellular tail are essential for this regulation. Downstream, RHOA and FYN-ABL-MICAL-1 pathways mediate signaling from EFNB1 to defective F-actin disassembly. This phenotype is testosterone-dependent and absent in female KO mice.","method":"Amperometry recordings of catecholamine secretion, F-actin staining/quantification, EphB6 knockout mice, ephrin-B1 tail deletion mutants, pathway inhibition assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — amperometry, domain mutagenesis of ephrin-B1, KO model with defined pathway (RHOA, FYN-ABL-MICAL-1)","pmids":["32321761"],"is_preprint":false},{"year":2018,"finding":"EPHB6 interacts with the GRB2 adapter protein and promotes expansion of tumor-initiating cells (TICs) via activation of the RAS-ERK pathway, elevating OCT4 expression. Suppression of ERK or OCT4 blocks EPHB6-induced pro-proliferative responses. EPHB6 also partially suppresses epithelial-mesenchymal transition in TNBC cells.","method":"Co-immunoprecipitation of EPHB6 with GRB2, ERK pathway inhibitor experiments, OCT4 knockdown, xenograft models, flow cytometry for TIC populations","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus pathway inhibitor rescue, xenograft validation, multiple orthogonal approaches","pmids":["29700392"],"is_preprint":false},{"year":2016,"finding":"EPHB6 induces mitochondrial network fragmentation in triple-negative breast cancer cells by activating the ERK-DRP1 pathway, increasing organelle fission. This mitochondrial fragmentation renders cancer cells more susceptible to DR5-mediated apoptosis. DRP1 activity is essential for the EPHB6-mediated pro-apoptotic response.","method":"Mitochondrial morphology imaging, ERK activation assays, DRP1 phosphorylation assays, DRP1 inhibitor/knockdown experiments, DR5 agonist apoptosis assays","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — imaging of mitochondrial fragmentation, pathway inhibition, functional apoptosis assays; single lab","pmids":["27788485"],"is_preprint":false},{"year":2014,"finding":"Ligand-induced (ephrin-B2) downregulation of EphB6 occurs through internalization via clathrin-coated pits followed by lysosomal degradation, dependent on Rab5 GTPase activity. EphB6 interacts constitutively with the Hsp90 chaperone, and ligand-induced downregulation is preceded by rapid EphB6-Hsp90 dissociation. Hsp90 inhibition alone induces EphB6 degradation, mimicking ligand-induced downregulation.","method":"Dominant-negative Rab5 mutant expression, clathrin inhibitors, lysosomal pathway inhibitors, Co-immunoprecipitation of EphB6 with Hsp90, Hsp90 inhibitor (geldanamycin) treatment, receptor degradation assays","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — dominant-negative mutant plus pharmacological inhibitors plus Co-IP, multiple orthogonal approaches, single lab","pmids":["25152371"],"is_preprint":false},{"year":2021,"finding":"The intracellular regions of EphB6 comprising the juxtamembrane (JM) region, pseudokinase, and SAM domains display high flexibility in solution and a propensity for inter-domain interactions. Tyrosine residues in the JM region of EphB6 are substrates for EphB4 kinase and, when phosphorylated, bind SH2 domains of Abl, Src, and Vav3. EphB6 and EphA10 pseudokinase domains can bind ATP and ATP-competitive small molecules.","method":"Small-angle X-ray scattering (SAXS), cross-linking mass spectrometry, in vitro EphB4 kinase assay, SH2 domain pull-down assays, ATP binding assays","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1 / Moderate — SAXS structural data, in vitro kinase assay, pull-down with specific SH2 domains; multiple orthogonal Tier 1 methods","pmids":["34431498"],"is_preprint":false},{"year":2021,"finding":"Crystal structure of the EphB6 extracellular domain (ECD) was determined by X-ray crystallography, revealing the overall architecture including ligand-binding domain (LBD) and fibronectin III domain (FN3). EphB6-LBD/FN3 interactions are similar to those observed in unliganded A-class Eph receptors, suggesting general importance. Unique structural features of EphB6 likely reflect its requirement for co-receptors due to lack of intrinsic kinase activity.","method":"X-ray crystallography of EphB6-ECD","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure determination with structural validation","pmids":["33770085"],"is_preprint":false},{"year":2023,"finding":"EPHB6 modulates EGFR signaling to enhance proliferation of cancer cells and tumor development, as identified by genetic screening and BioID proteomics integrated with machine learning. Further experiments confirmed the EPHB6-EGFR crosstalk.","method":"Genome-wide genetic interaction screen, BioID proximity proteomics, machine learning integration, functional validation experiments","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — BioID proteomics plus functional validation, multiple complementary approaches, single lab","pmids":["37392382"],"is_preprint":false},{"year":2009,"finding":"Expression of EPHB6 in A549 lung adenocarcinoma cells leads to phosphorylation of ERK (MAP kinase). siRNA-based knockdown of EPHB6 reverses ERK phosphorylation. However, EPHB6-induced ERK phosphorylation is uncoupled from activation of the Elk-1 transcriptional factor.","method":"EPHB6 overexpression, siRNA knockdown, ERK phosphorylation immunoblotting, Elk-1 reporter assays","journal":"International journal of oncology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — overexpression and siRNA knockdown with consistent result, single lab, single study","pmids":["19513565"],"is_preprint":false},{"year":2020,"finding":"CLDN1 upregulates cellular EPHB6 levels and enhances its activation, resulting in suppression of ERK1/2 signaling and downstream inhibition of SLUG expression, forming a CLDN1-EPHB6-ERK1/2-SLUG feedback axis that represses metastasis, drug resistance, and cancer stemness in lung adenocarcinoma.","method":"Immunoblotting, immunoprecipitation, migration assays, sphere assay, xenograft experiments, siRNA knockdown","journal":"Theranostics","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — immunoprecipitation and functional assays defining pathway, single lab","pmids":["32754286"],"is_preprint":false},{"year":2019,"finding":"EPHB6 mutation-induced paclitaxel resistance is mediated by interaction with EPHA2, which promotes JNK-mediated cadherin 11 (CDH11) expression. EPHB6-mutated cells acquire cell adhesion-mediated drug resistance via CDH11 expression and RhoA/FAK activation. Targeted inhibition of EPHA2 or CDH11 reverses paclitaxel resistance.","method":"Co-immunoprecipitation of EPHB6 with EPHA2, CDH11 expression assays, JNK phosphorylation assays, RhoA/FAK activation assays, pharmacological inhibitor experiments, drug resistance assays","journal":"Experimental & molecular medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — Co-IP, pathway inhibitors, functional drug resistance assays; single lab","pmids":["31160603"],"is_preprint":false},{"year":2024,"finding":"EphB6 interacts with ephrin-B1 in trans on neighboring cells, leading to formation of large co-clusters at the plasma membrane. These co-clusters show decreased propensity for endocytosis. Co-clustering promotes formation of double-membrane tubular structures between cells, stabilizes cell-cell adhesion, and reduces invasive behavior of cancer cells.","method":"Lattice light-sheet microscopy, 3D structured illumination microscopy, cryo-electron tomography, endocytosis assays, cell invasion assays","journal":"Communications biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — multiple high-resolution structural imaging modalities (cryo-ET, LLSM, SIM) plus functional invasion assays","pmids":["38627519"],"is_preprint":false},{"year":2023,"finding":"EphB6 deficiency in mice promotes tumor growth in a colorectal cancer xenograft model by increasing GABA in the tumor microenvironment through intestinal neurons. The mechanism involves increased expression of synaptosomal-associated protein 25 (SNAP25) in the intestinal myenteric plexus, mediating GABA release. Botulinum toxin A injection into the rectum of EphB6-deficient mice eliminated the pro-tumorigenic effect, confirming the neuronal dependency.","method":"EphB6-deficient mouse xenograft model, GABA measurement, SNAP25 immunoblotting, botulinum toxin A neuronal inhibition, gut microbiota controls","journal":"Carcinogenesis","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — KO mouse model with pharmacological rescue (botulinum toxin) and defined mediator (SNAP25/GABA), single lab","pmids":["37294054"],"is_preprint":false},{"year":2021,"finding":"EphB6 promotes survival of disseminated dormant cancer cells (DDCCs) in the lung through activation of the TFEB-lysosomal axis. Direct co-culture of DDCCs with lung epithelial cells induces EphB6 expression and lysosomal accumulation. EphB6 contributes to TFEB transcriptional activity and lysosome formation. EphB6 signaling also promotes proliferation of surrounding lung parenchymal cells in vivo.","method":"Lung organotypic co-culture system, in vivo dissemination assays, TFEB reporter assays, lysosome staining, EphB6 knockdown, xenograft models","journal":"Cancers","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — in vitro and in vivo functional assays with defined TFEB pathway, single lab","pmids":["33802447"],"is_preprint":false},{"year":2026,"finding":"SIAH2 promotes K48-linked polyubiquitination and proteasomal degradation of EPHB6 in hepatocellular carcinoma cells. Loss of EPHB6 through SIAH2-mediated degradation promotes filopodia formation and cell invasion/migration via upregulation of RHOF.","method":"Co-immunoprecipitation of SIAH2 with EPHB6, ubiquitination assays (K48-linkage specific), EPHB6 knockdown/overexpression, RHOF expression assays, filopodia quantification, invasion/migration assays","journal":"Cell & bioscience","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — Co-IP, ubiquitination assay, functional rescue experiments; single lab, single study","pmids":["42036676"],"is_preprint":false},{"year":2024,"finding":"EPHB6 interacts with CBX7 in cervical cancer C33A cells, and EPHB6 inhibits cell proliferation, invasion, migration, and cell cycle acceleration by binding to CBX7.","method":"Co-immunoprecipitation, HDOCK molecular docking prediction, functional assays (CCK-8, EdU, wound healing, transwell), flow cytometry for cell cycle","journal":"Cell biochemistry and biophysics","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP with computational docking support, functional assays without detailed mechanistic dissection, single lab","pmids":["39322790"],"is_preprint":false},{"year":2017,"finding":"Enzalutamide-mediated androgen receptor (AR) activation binds to an androgen-response-element (ARE) on the EPHB6 promoter to decrease EPHB6 expression, which then activates JNK phosphorylation and increases MMP9 expression to promote CRPC cell invasion. JNK inhibitor SP600125 blocks Enzalutamide-increased CRPC cell invasion.","method":"ARE binding assay/ChIP, EPHB6 promoter reporter assay, JNK phosphorylation immunoblot, JNK inhibitor treatment, invasion assays","journal":"Cancer letters","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — promoter binding assay and pharmacological rescue with defined pathway components, single lab","pmids":["28826721"],"is_preprint":false},{"year":2023,"finding":"Overexpression of EphB6 via in utero electroporation results in clumping of cortical neurons (soma spacing defect), as does overexpression of ephrin-B2. When both EphB6 and ephrin-B2 are co-overexpressed in cortical neurons, the soma clumping phenotypes disappear, demonstrating mutual inhibition requiring interaction of their specific domains.","method":"In utero electroporation, cortical neuron imaging, domain-specific interaction analysis","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — in vivo overexpression with quantitative phenotypic readout and domain specificity analysis, single lab","pmids":["37149633"],"is_preprint":false}],"current_model":"EPHB6 is an intrinsically kinase-dead (pseudokinase) Eph receptor that signals via transphosphorylation by catalytically active partners (EphB1, EphB4, Src/Fyn), recruits adaptor proteins (c-Cbl, GRB2, Hsp90) through its phosphorylated juxtamembrane region, and modulates multiple downstream pathways (JNK/Rac1, ERK-DRP1, RAS-ERK, RhoA) to regulate T cell activation, cell adhesion and migration, vascular smooth muscle contractility, adrenal catecholamine biosynthesis and secretion, and cancer cell invasiveness and drug sensitivity; it also engages in trans co-clustering with ephrin-B1 to stabilize cell-cell adhesion and suppress cancer invasion, and is degraded via clathrin/Rab5-dependent lysosomal targeting following ligand-induced Hsp90 dissociation or via SIAH2-mediated K48-ubiquitination."},"narrative":{"mechanistic_narrative":"EPHB6 is an intrinsically kinase-deficient (pseudokinase) Eph receptor that binds ephrin-B2 as its high-affinity ligand and signals not through its own catalytic activity but by being transphosphorylated by catalytically active partners and by acting as a scaffold for adaptor recruitment [PMID:10648835, PMID:11713248]. Its juxtamembrane tyrosines are substrates for active EphB-family and Src-family kinases—EphB1, EphB4, and the constitutively associated Fyn—and once phosphorylated they recruit SH2-domain proteins including Abl, Src, and Vav3, while constitutive recruitment of c-Cbl and Hsp90 occurs through the cytoplasmic region [PMID:11713248, PMID:15955811, PMID:34431498, PMID:20086179, PMID:25152371]. Structurally, the extracellular domain adopts an architecture resembling unliganded A-class Eph receptors and the intracellular pseudokinase and SAM modules are highly flexible, retaining residual ATP binding despite catalytic deficiency [PMID:33770085, PMID:34431498]. In T cells EPHB6 partitions to TCR-containing lipid rafts and is required for full proximal TCR signaling (ZAP-70, LAT, PLCγ1-SLP-76, MAPK), exerting ligand- and concentration-dependent modulation of Rac1/JNK, IL-2 secretion, proliferation, and apoptosis [PMID:15599401, PMID:12393850, PMID:12517763]. Through ephrin-B reverse signaling and adaptor coupling, EPHB6 controls vascular smooth muscle contractility via RhoA and GRIP1 and governs adrenal chromaffin-cell catecholamine biosynthesis (RAC1-MKK7-JNK-c-Jun/AP1-EGR1 driving tyrosine hydroxylase) and testosterone-dependent secretion (BKCa-regulated Ca2+ influx and RHOA/FYN-ABL-MICAL-1-dependent cortical F-actin disassembly) [PMID:22223652, PMID:30824540, PMID:29339804, PMID:32321761]. In cancer, EPHB6 generally suppresses invasiveness—by transphosphorylation-dependent c-Cbl/Abl signaling, by sequestering and restraining kinase-active oncogenic Eph receptors such as EphA2, and by trans co-clustering with ephrin-B1 to stabilize cell-cell adhesion—but also drives context-specific pro-tumorigenic outputs through GRB2-RAS-ERK-OCT4, ERK-DRP1 mitochondrial fission, EGFR crosstalk, and a TFEB-lysosomal survival program [PMID:20086179, PMID:21737611, PMID:25239188, PMID:38627519, PMID:29700392, PMID:27788485, PMID:37392382, PMID:33802447]. Receptor levels are set post-translationally by ligand-induced clathrin/Rab5-dependent lysosomal degradation following Hsp90 dissociation and by SIAH2-mediated K48-linked ubiquitination [PMID:25152371, PMID:42036676].","teleology":[{"year":2000,"claim":"Establishing which ephrin engages EPHB6 was the prerequisite for any signaling model; the work defined ephrin-B2 as its specific high-affinity ligand.","evidence":"EphB6-Fc binding to ephrin-transfected COS and Reh cells with PIPLC and saturation controls","pmids":["10648835"],"confidence":"High","gaps":["Did not address downstream signaling consequences of binding","In vitro fusion-protein binding may not reflect full-length receptor avidity"]},{"year":2001,"claim":"Because EPHB6 lacks intrinsic kinase activity, how it is activated was unresolved; transphosphorylation by catalytically active EphB1 within a stable heterocomplex, plus constitutive c-Cbl binding, defined the pseudokinase signaling logic.","evidence":"Co-IP with catalytically active/inactive EphB1, ligand stimulation, kinase assays","pmids":["11713248"],"confidence":"High","gaps":["Which juxtamembrane tyrosines are phosphorylated not mapped here","Functional consequence of Cbl binding not determined"]},{"year":2001,"claim":"Whether EPHB6 has cell-autonomous immune function was unknown; cross-linking in T cells showed it couples to Cbl/SHP-1 dynamics and controls proliferation and apoptosis.","evidence":"Anti-EphB6/anti-CD3 co-cross-linking, Co-IP, apoptosis assays in Jurkat cells","pmids":["11466354"],"confidence":"Medium","gaps":["Single cell line and single study","Mechanism linking Cbl dephosphorylation to apoptosis not dissected"]},{"year":2002,"claim":"The directionality of EPHB6's T-cell role was clarified by showing it co-localizes with the TCR at membrane rafts and co-stimulates proliferation via p38, and by confirming ephrin-B2 as its unique thymocyte ligand.","evidence":"Confocal co-localization, cytokine/proliferation assays, p38 assays; ephrin-Fc binding on KO thymocytes","pmids":["12393850","12379224"],"confidence":"Medium","gaps":["Adaptor linkage between EPHB6 and p38 not defined","Co-stimulation context requires suboptimal TCR signal"]},{"year":2003,"claim":"To resolve which signaling arm EPHB6 modulates in T cells, ligand stimulation was shown to selectively inhibit TCR-induced Rac1-JNK (not MAPK) and IL-2/CD25, with dominant-negative rescue.","evidence":"WT and dominant-negative overexpression, Rac1 PAK pull-down, JNK assay, IL-2 ELISA, flow cytometry","pmids":["12517763"],"confidence":"High","gaps":["Direct biochemical link from EPHB6 to Rac1 regulation not shown","Overexpression context"]},{"year":2004,"claim":"Genetic loss-of-function defined EPHB6 as required (not merely modulatory) for full proximal TCR signaling, anchoring its physiological immune role.","evidence":"EphB6-KO mice, raft fractionation, ZAP-70/LAT/PLCγ1-SLP-76/MAPK readouts, in vivo T-cell assays","pmids":["15599401"],"confidence":"High","gaps":["Molecular mechanism of how a kinase-dead receptor enhances ZAP-70/LAT not resolved"]},{"year":2005,"claim":"The dual positive/negative behavior of EPHB6 was explained by a concentration-dependent switch in which constitutive Fyn association and Src-family-driven tyrosine phosphorylation convert adhesion-promoting into repulsive signaling.","evidence":"Adhesion/migration assays at varying ligand dose, cytoplasmic deletion mutants, Fyn Co-IP, SFK inhibitors","pmids":["15955811"],"confidence":"High","gaps":["Identity of phosphosites driving the switch not mapped","Downstream effectors of repulsion not defined"]},{"year":2009,"claim":"Whether EPHB6 engages ERK in epithelial cancers and whether that drives transcription was tested, showing ERK phosphorylation uncoupled from Elk-1 activation.","evidence":"Overexpression/siRNA in A549 cells, ERK immunoblot, Elk-1 reporter","pmids":["19513565"],"confidence":"Medium","gaps":["Mechanism of ERK-Elk1 uncoupling unexplained","Single cell line"]},{"year":2010,"claim":"The mechanism of EPHB6's tumor-suppressive activity was placed downstream of EphB4 transphosphorylation, requiring c-Cbl-dependent Abl phosphorylation to inhibit invasiveness.","evidence":"EPHB6 restoration in breast cancer cells, c-Cbl Co-IP, Cbl siRNA rescue, Abl phosphorylation and invasion assays","pmids":["20086179"],"confidence":"High","gaps":["Quantitative balance of EphB6/EphB4 in vivo not established"]},{"year":2011,"claim":"A sequestration model for tumor suppression was supported by demonstrating EPHB6 heterointeractions with EphB2 and, unprecedentedly, with the A-class EphA2.","evidence":"Co-IP across breast carcinoma cell lines","pmids":["21737611"],"confidence":"Medium","gaps":["Single Co-IP per pair without reciprocal/functional validation","Stoichiometry of sequestration unknown"]},{"year":2014,"claim":"Receptor turnover and the EPHB6-EphA2 axis were mechanistically defined: ligand-induced Hsp90 dissociation triggers clathrin/Rab5 lysosomal degradation, and EPHB6 restrains EphA2 S897 phosphorylation/Ephexin4-RhoG to suppress anoikis resistance.","evidence":"Dominant-negative Rab5, clathrin/lysosome inhibitors, Hsp90 Co-IP and geldanamycin; EphA2 phospho/RhoG/anoikis assays","pmids":["25152371","25239188"],"confidence":"Medium","gaps":["E3 ligase for lysosomal route not identified here","Hsp90 binding site on EPHB6 unmapped"]},{"year":2012,"claim":"Beyond immunity, EPHB6 was assigned a cardiovascular role: KO arteries show heightened contractility via RhoA/myosin light chain, with ephrin-B reverse signaling acting through GRIP1.","evidence":"EphB6-KO mice, ex vivo contractility, RhoA assay, MLC phospho-immunoblot, cross-linking, BP/catecholamine measurement","pmids":["22223652"],"confidence":"High","gaps":["Sex/testosterone dependence first hinted but not mechanistically resolved here"]},{"year":2018,"claim":"EPHB6's adrenal function was opened by linking it to chromaffin-cell excitability: KO augments BKCa currents and reduces ACh-triggered Ca2+ influx in a testosterone-dependent manner.","evidence":"Patch-clamp, Ca2+ imaging, BKCa blockade, KO mice with castration controls","pmids":["29339804"],"confidence":"High","gaps":["How EPHB6 regulates BKCa channels molecularly not defined","Mechanism of testosterone dependence unresolved"]},{"year":2019,"claim":"The biosynthetic arm of catecholamine control was defined: EPHB6 reverse signaling through ephrin-B1 drives tyrosine hydroxylase transcription via RAC1-MKK7-JNK-c-Jun/AP1-EGR1.","evidence":"KO mice, RT-qPCR, immunoblot, microarray, pathway inhibitors, HPLC catecholamines","pmids":["30824540"],"confidence":"High","gaps":["Direct transcription factor occupancy at TH promoter not shown"]},{"year":2020,"claim":"The secretory arm was completed by showing EPHB6-to-ephrin-B1 reverse signaling regulates exocytosis through RHOA and FYN-ABL-MICAL-1-dependent cortical F-actin disassembly, with a defined 7-residue ephrin-B1 tail motif.","evidence":"Amperometry, F-actin quantification, ephrin-B1 tail deletion mutants, pathway inhibition, KO mice","pmids":["32321761"],"confidence":"High","gaps":["How testosterone gates this pathway not molecularly resolved"]},{"year":2016,"claim":"A pro-apoptotic cancer output was identified whereby EPHB6 activates ERK-DRP1 to fragment mitochondria, sensitizing TNBC cells to DR5-mediated apoptosis.","evidence":"Mitochondrial imaging, ERK/DRP1 phospho assays, DRP1 inhibitor/knockdown, DR5 agonist apoptosis","pmids":["27788485"],"confidence":"Medium","gaps":["Link from EPHB6 to ERK activation upstream not biochemically defined","Single lab"]},{"year":2018,"claim":"A context-dependent pro-tumorigenic function was shown via GRB2 recruitment activating RAS-ERK-OCT4 to expand tumor-initiating cells, revealing EPHB6's dual cancer roles.","evidence":"GRB2 Co-IP, ERK inhibitor and OCT4 knockdown, xenografts, TIC flow cytometry","pmids":["29700392"],"confidence":"Medium","gaps":["Reconciliation with tumor-suppressive roles context-dependent and unresolved"]},{"year":2017,"claim":"Transcriptional regulation of EPHB6 in prostate cancer was defined: AR binds an ARE to repress EPHB6, derepressing JNK-MMP9-driven invasion.","evidence":"ChIP/ARE binding, promoter reporter, JNK phospho, JNK inhibitor, invasion assays","pmids":["28826721"],"confidence":"Medium","gaps":["Single lab; direct EPHB6-to-JNK biochemical link not shown"]},{"year":2019,"claim":"A drug-resistance mechanism was mapped: mutated EPHB6 engages EphA2 to drive JNK-CDH11 and RhoA/FAK, conferring adhesion-mediated paclitaxel resistance reversible by targeting EphA2 or CDH11.","evidence":"EphA2 Co-IP, CDH11/JNK/RhoA-FAK assays, inhibitors, resistance assays","pmids":["31160603"],"confidence":"Medium","gaps":["Specific EPHB6 mutations and their structural effect not resolved","Single lab"]},{"year":2020,"claim":"An upstream regulator-EPHB6-effector axis in lung cancer was defined whereby CLDN1 enhances EPHB6 to suppress ERK1/2-SLUG, repressing metastasis and stemness.","evidence":"IP, migration/sphere/xenograft assays, siRNA knockdown","pmids":["32754286"],"confidence":"Medium","gaps":["How CLDN1 enhances EPHB6 activation mechanistically unclear"]},{"year":2021,"claim":"Structural understanding of the kinase-dead receptor was advanced: the ECD resembles unliganded A-class Ephs, and the flexible intracellular JM/pseudokinase/SAM region presents EphB4-phosphorylated tyrosines that bind Abl/Src/Vav3 SH2 domains, with residual ATP binding.","evidence":"X-ray crystallography of ECD; SAXS, XL-MS, in vitro EphB4 kinase, SH2 pull-downs, ATP binding","pmids":["33770085","34431498"],"confidence":"High","gaps":["Full-length receptor structure absent","Functional role of residual ATP binding in cells untested"]},{"year":2021,"claim":"A pro-survival role in metastatic dormancy was identified through EPHB6 activation of the TFEB-lysosomal axis in disseminated dormant cancer cells.","evidence":"Lung organotypic co-culture, in vivo dissemination, TFEB reporter, lysosome staining, knockdown, xenograft","pmids":["33802447"],"confidence":"Medium","gaps":["Signaling link from EPHB6 to TFEB not biochemically defined"]},{"year":2023,"claim":"Unbiased screening extended EPHB6 partners to EGFR, showing crosstalk that enhances cancer-cell proliferation and tumor development; separately, EPHB6 deficiency was shown to promote colorectal tumor growth via intestinal-neuron GABA/SNAP25.","evidence":"Genetic screen + BioID + machine learning with validation; KO xenograft, GABA/SNAP25 assays, botulinum toxin rescue","pmids":["37392382","37294054"],"confidence":"Medium","gaps":["Direct EPHB6-EGFR biochemical interface not defined","Neuronal-tumor axis from single lab"]},{"year":2023,"claim":"A developmental neuronal role was shown in which EPHB6 and ephrin-B2 overexpression each cause cortical soma clumping that mutually cancels, indicating domain-specific cis/trans interaction controlling neuronal spacing.","evidence":"In utero electroporation, cortical neuron imaging, domain-specific interaction analysis","pmids":["37149633"],"confidence":"Medium","gaps":["Loss-of-function neuronal phenotype not shown","Downstream effectors unmapped"]},{"year":2024,"claim":"High-resolution imaging defined the adhesive output: EPHB6 trans co-clustering with ephrin-B1 forms endocytosis-resistant clusters and double-membrane tubules that stabilize cell-cell contacts and reduce invasion.","evidence":"Lattice light-sheet, 3D-SIM, cryo-electron tomography, endocytosis and invasion assays","pmids":["38627519"],"confidence":"High","gaps":["Molecular composition of tubular structures not fully resolved"]},{"year":2026,"claim":"Post-translational control was extended by showing SIAH2 drives K48-ubiquitination and proteasomal degradation of EPHB6, derepressing RHOF-driven filopodia and invasion in hepatocellular carcinoma.","evidence":"SIAH2-EPHB6 Co-IP, K48-linkage ubiquitination assay, knockdown/overexpression, RHOF and filopodia/invasion assays","pmids":["42036676"],"confidence":"Medium","gaps":["SIAH2 recognition motif on EPHB6 not mapped","Single lab, single study"]},{"year":null,"claim":"How EPHB6's many context-specific outputs (tumor suppression vs promotion, immune, vascular, adrenal, neuronal) are selected by partner availability, phosphosite occupancy, and tissue context remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model reconciling opposing cancer roles","Endogenous heterocomplex stoichiometry across tissues unknown","Full-length receptor signaling structure undetermined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,8,18]},{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[5,24,7]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,7,15,18]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[9,10]},{"term_id":"GO:0140657","term_label":"ATP-dependent activity","supporting_discovery_ids":[18]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[2,4,24]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[17,24]},{"term_id":"GO:0005764","term_label":"lysosome","supporting_discovery_ids":[17,26]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,7,3,11]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[2,3,4]},{"term_id":"R-HSA-1500931","term_label":"Cell-Cell communication","supporting_discovery_ids":[7,24,30]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[8,15,23,27]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[17,27]}],"complexes":[],"partners":["EFNB2","EFNB1","EPHB1","EPHB4","EPHA2","FYN","CBL","GRB2"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O15197","full_name":"Ephrin type-B receptor 6","aliases":["HEP","Tyrosine-protein kinase-defective receptor EPH-6"],"length_aa":1021,"mass_kda":110.7,"function":"Kinase-defective receptor for members of the ephrin-B family. Binds to ephrin-B1 and ephrin-B2. Modulates cell adhesion and migration by exerting both positive and negative effects upon stimulation with ephrin-B2. Inhibits JNK activation, T-cell receptor-induced IL-2 secretion and CD25 expression upon stimulation with ephrin-B2","subcellular_location":"Secreted","url":"https://www.uniprot.org/uniprotkb/O15197/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/EPHB6","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/EPHB6","total_profiled":1310},"omim":[{"mim_id":"602757","title":"EPHRIN RECEPTOR EphB6; EPHB6","url":"https://www.omim.org/entry/602757"},{"mim_id":"300035","title":"EPHRIN B1; EFNB1","url":"https://www.omim.org/entry/300035"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Uncertain","locations":[{"location":"Nuclear speckles","reliability":"Uncertain"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"brain","ntpm":69.8},{"tissue":"skin 1","ntpm":79.7}],"url":"https://www.proteinatlas.org/search/EPHB6"},"hgnc":{"alias_symbol":["HEP"],"prev_symbol":[]},"alphafold":{"accession":"O15197","domains":[{"cath_id":"2.60.120.260","chopping":"33-166_195-232","consensus_level":"high","plddt":85.1317,"start":33,"end":232},{"cath_id":"2.60.40.1770","chopping":"234-272_280-302","consensus_level":"medium","plddt":81.835,"start":234,"end":302},{"cath_id":"2.60.40.10","chopping":"374-414_424-484","consensus_level":"medium","plddt":87.8439,"start":374,"end":484},{"cath_id":"2.60.40.10","chopping":"496-579","consensus_level":"high","plddt":89.8535,"start":496,"end":579},{"cath_id":"3.30.200.20","chopping":"649-751","consensus_level":"high","plddt":86.7389,"start":649,"end":751},{"cath_id":"1.10.510.10","chopping":"752-929","consensus_level":"medium","plddt":88.1249,"start":752,"end":929},{"cath_id":"1.10.150.50","chopping":"944-1021","consensus_level":"medium","plddt":77.4015,"start":944,"end":1021}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O15197","model_url":"https://alphafold.ebi.ac.uk/files/AF-O15197-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O15197-F1-predicted_aligned_error_v6.png","plddt_mean":80.25},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=EPHB6","jax_strain_url":"https://www.jax.org/strain/search?query=EPHB6"},"sequence":{"accession":"O15197","fasta_url":"https://rest.uniprot.org/uniprotkb/O15197.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O15197/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O15197"}},"corpus_meta":[{"pmid":"32819434","id":"PMC_32819434","title":"The 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controls catecholamine biosynthesis by up-regulating tyrosine hydroxylase transcription in adrenal gland chromaffin cells.","date":"2019","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/30824540","citation_count":9,"is_preprint":false},{"pmid":"29339804","id":"PMC_29339804","title":"EPHB6 and testosterone in concert regulate epinephrine release by adrenal gland chromaffin cells.","date":"2018","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/29339804","citation_count":9,"is_preprint":false},{"pmid":"20952760","id":"PMC_20952760","title":"Tyrosine kinase-deficient EphB6 receptor-dependent alterations in proteomic profiles of invasive breast carcinoma cells as determined by difference gel electrophoresis.","date":"2010","source":"Cancer genomics & proteomics","url":"https://pubmed.ncbi.nlm.nih.gov/20952760","citation_count":8,"is_preprint":false},{"pmid":"37441529","id":"PMC_37441529","title":"Oat beta-glucan reduces colitis by 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EphB1 and EphB6 form a stable heterocomplex, and EphB1-induced EphB6 phosphorylation is ligand-dependent and requires EphB1 catalytic activity. Additionally, the proto-oncogene c-Cbl was identified as an EphB6-binding protein; EphB6-Cbl association is constitutive and requires a functional phosphotyrosine binding domain on Cbl.\",\n      \"method\": \"Co-immunoprecipitation, overexpression of catalytically active/inactive EphB1, ligand stimulation with ephrin-B1, kinase assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP and functional mutagenesis (kinase-dead EphB1 control), single lab with multiple orthogonal methods\",\n      \"pmids\": [\"11713248\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Cross-linking of EphB6 in Jurkat T cells triggers general protein tyrosine phosphorylation and associates with Cbl. EphB6 cross-linking results in Cbl dephosphorylation and dissociation of Cbl from SHP-1, leading to altered lymphokine secretion, proliferation inhibition, and Fas-mediated apoptosis.\",\n      \"method\": \"Co-cross-linking with anti-EphB6 antibody and anti-CD3, Co-immunoprecipitation, apoptosis assays\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — Co-IP and functional cellular assays, single lab, single study\",\n      \"pmids\": [\"11466354\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"EphB6 crosslinking by anti-EphB6 mAb or ephrin-B2 in the presence of suboptimal TCR stimulation co-stimulates T cell proliferation and cytokine secretion. Within two minutes of anti-CD3/anti-CD28 stimulation, EphB6 aggregates and co-localizes with TCR at membrane rafts, and this is followed by p38 MAPK activation.\",\n      \"method\": \"Anti-EphB6 crosslinking, confocal microscopy for co-localization with TCR, cytokine ELISA, proliferation assays, p38 MAPK activation assays\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — direct imaging of co-localization plus functional readouts, single lab\",\n      \"pmids\": [\"12393850\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"EphB6 overexpression in Jurkat T cells followed by ephrin-B1 stimulation selectively inhibits TCR-mediated JNK activation but not the MAPK pathway. The mechanism involves suppression of TCR-induced Rac1 GTPase activation upstream of JNK. EphB6 also blocks anti-CD3-induced IL-2 secretion and CD25 expression in a ligand-dependent manner. Dominant-negative EphB6 reverses these inhibitory effects. In murine thymocytes, ephrin-B1 stimulation inhibits TCR-mediated apoptosis and CD25 upregulation.\",\n      \"method\": \"Overexpression of wild-type and dominant-negative EphB6, Rac1 activation assay (GST-PAK pull-down), JNK kinase assay, IL-2 ELISA, flow cytometry for CD25\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods including dominant-negative rescue, kinase assays, and Rac1 pull-down in single study\",\n      \"pmids\": [\"12517763\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"EphB6 migrates to aggregated TCRs and lipid rafts after TCR activation in T cells. In EphB6-deficient mice, ZAP-70 activation, LAT phosphorylation, PLCγ1 association with SLP-76, and p44/42 MAPK activation are diminished upon TCR stimulation, demonstrating EphB6 is required for full TCR downstream signaling.\",\n      \"method\": \"EphB6 knockout mice, biochemical fractionation to isolate rafts, immunoblotting for ZAP-70, LAT, PLCγ1/SLP-76 co-IP, MAPK activation assays, in vivo T cell functional assays\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO model with multiple downstream signaling readouts and in vivo validation, replicated across multiple assays\",\n      \"pmids\": [\"15599401\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Ephrin-B2 is a high-affinity ligand for EphB6. EphB6-Fc fusion protein binds ephrin-B2 but not ephrin-B1 on transfected COS cells in a saturable manner. The ligand identified on Reh pro-B cells is a transmembrane ephrin (not GPI-anchored, as PIPLC treatment did not abrogate binding).\",\n      \"method\": \"EphB6-Fc fusion protein binding assay, COS cell transfection with ephrin-B1 or ephrin-B2, phosphatidylinositol-specific phospholipase C treatment, saturation binding analysis\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct in vitro binding reconstitution with fusion protein, saturable binding, negative control with ephrin-B1, confirmed in multiple cell types\",\n      \"pmids\": [\"10648835\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Ephrin-B2 is the unique ligand for EphB6 among eight known ephrins, as demonstrated by differential binding of ephrin-Fc proteins to EphB6-deficient versus wild-type thymocytes. EphB6 is the dominant ephrin-B2-binding receptor on adult thymocytes.\",\n      \"method\": \"EphB6 knockout mice, ephrin-Fc binding assays on thymocytes\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO mouse model with direct binding assay, replicates ligand specificity finding from PMID 10648835\",\n      \"pmids\": [\"12379224\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"EphB6 exerts biphasic effects on cell adhesion and migration: low concentrations of ephrin-B2 promote adhesion and migration, while high concentrations induce repulsion and inhibit migration. A truncated EphB6 lacking the cytoplasmic domain shows only positive (monophasic) effects, indicating the cytoplasmic domain mediates negative effects. EphB6 is constitutively associated with the Src family kinase Fyn, and high ephrin-B2 concentrations induce EphB6 tyrosine phosphorylation through Src family kinase activity as the molecular switch.\",\n      \"method\": \"Cell adhesion and migration assays with varying ligand concentrations, cytoplasmic domain deletion mutants, co-immunoprecipitation with Fyn, Src family kinase inhibitors, tyrosine phosphorylation assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain deletion mutants, kinase inhibitors, and Co-IP identifying Fyn interaction; multiple orthogonal approaches in single study\",\n      \"pmids\": [\"15955811\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"EphB6 suppresses breast cancer cell invasiveness through a mechanism requiring EphB4-mediated transphosphorylation of EphB6, followed by EphB6-c-Cbl interaction and phosphorylation of the Abl kinase. Cbl silencing suppresses Abl phosphorylation, cell adhesion, and morphological changes, and blocks EphB6's ability to inhibit invasiveness. EphB4 also acts in an EphB6-independent manner to enhance invasiveness, suggesting cancer invasiveness is defined by the balance in the EphB6-EphB4 system.\",\n      \"method\": \"Restoration of EphB6 expression in invasive breast cancer cells, Co-immunoprecipitation of EphB6 with c-Cbl, siRNA knockdown of Cbl, Abl phosphorylation assays, invasion assays\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, siRNA rescue experiments, multiple functional readouts in single study\",\n      \"pmids\": [\"20086179\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"EPHB6 interacts with EPHB2 and with EPHA2 (the first demonstrated A-type/B-type Eph receptor interaction), but does not interact with another kinase-deficient receptor EPHA10. The tumor-invasiveness-suppressing activity of EPHB6 is mediated partly by sequestering kinase-sufficient oncogenic EPH receptors.\",\n      \"method\": \"Co-immunoprecipitation in mammalian breast carcinoma cell lines\",\n      \"journal\": \"Cancer genomics & proteomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP experiment per interaction pair, single lab\",\n      \"pmids\": [\"21737611\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"EphB6 interacts with EphA2 and suppresses EphA2-mediated anoikis resistance in MCF7 breast cancer cells by decreasing serine 897 phosphorylation of EphA2 and suppressing EphA2-Ephexin4 interaction and RhoG activation. Knockdown of EphB6 promotes anoikis resistance.\",\n      \"method\": \"Co-immunoprecipitation, phosphorylation assays, siRNA knockdown of EphB6, RhoG activation assay, anoikis assay\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — Co-IP plus functional knockdown with defined pathway placement, single lab\",\n      \"pmids\": [\"25239188\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"EPHB6 and its ephrin-B ligands are expressed on vascular smooth muscle cells (VSMC). In castrated male Ephb6-KO mice, small arteries show increased contractility, RhoA activation, and constitutive myosin light chain phosphorylation ex vivo. Cross-linking ephrin-Bs (reverse signaling) but not EphB6 (forward signaling) reduces VSMC contractions, mediated by the adaptor protein GRIP1. Male Ephb6-KO mice show decreased 24-hour urine catecholamines as a compensatory mechanism.\",\n      \"method\": \"EphB6 knockout mice, ex vivo arterial contractility assays, RhoA activation assay, myosin light chain phosphorylation immunoblot, cross-linking assays, blood pressure measurement, catecholamine measurement\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO mouse model with multiple ex vivo and in vivo readouts, mechanistic pathway identification via GRIP1 and RhoA\",\n      \"pmids\": [\"22223652\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"EPHB6 controls catecholamine biosynthesis in adrenal gland chromaffin cells (AGCCs) by upregulating tyrosine hydroxylase transcription. Ephb6-KO male mice have reduced epinephrine content due to decreased tyrosine hydroxylase. The signaling pathway from EPHB6 to tyrosine hydroxylase involves RAC1, MKK7, JNK, c-Jun, AP1, and EGR1. This effect operates via reverse signaling through ephrin-B1. ERK1/2, p38 MAPK, and ELK1 signaling were not affected by EPHB6 deletion.\",\n      \"method\": \"EphB6 knockout mice, RT-qPCR, immunoblotting, gene microarray, signaling pathway inhibitors, HPLC catecholamine measurement\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO model with multiple orthogonal methods (qPCR, immunoblot, microarray) and defined pathway components\",\n      \"pmids\": [\"30824540\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"In male Ephb6-KO adrenal gland chromaffin cells, acetylcholine-triggered Ca2+ influx is reduced due to augmented large conductance calcium-activated potassium (BKCa) channel currents in a testosterone-dependent manner. Blocking BKCa channels in KO cells corrects the reduced Ca2+ influx. This phenotype depends on the non-genomic effect of testosterone and is absent in female or castrated KO mice.\",\n      \"method\": \"Patch-clamp electrophysiology, Ca2+ imaging, BKCa channel pharmacological blockade, EphB6 knockout mice, castration experiments\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — electrophysiology with pharmacological rescue, KO model, sex-hormone dependence established with castration controls\",\n      \"pmids\": [\"29339804\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"In male Ephb6-KO adrenal gland chromaffin cells, catecholamine secretion via exocytosis is compromised and is accompanied by reduced cortical F-actin disassembly. Reverse signaling from EPHB6 to ephrin-B1 and a 7-amino acid segment in the EFNB1 intracellular tail are essential for this regulation. Downstream, RHOA and FYN-ABL-MICAL-1 pathways mediate signaling from EFNB1 to defective F-actin disassembly. This phenotype is testosterone-dependent and absent in female KO mice.\",\n      \"method\": \"Amperometry recordings of catecholamine secretion, F-actin staining/quantification, EphB6 knockout mice, ephrin-B1 tail deletion mutants, pathway inhibition assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — amperometry, domain mutagenesis of ephrin-B1, KO model with defined pathway (RHOA, FYN-ABL-MICAL-1)\",\n      \"pmids\": [\"32321761\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"EPHB6 interacts with the GRB2 adapter protein and promotes expansion of tumor-initiating cells (TICs) via activation of the RAS-ERK pathway, elevating OCT4 expression. Suppression of ERK or OCT4 blocks EPHB6-induced pro-proliferative responses. EPHB6 also partially suppresses epithelial-mesenchymal transition in TNBC cells.\",\n      \"method\": \"Co-immunoprecipitation of EPHB6 with GRB2, ERK pathway inhibitor experiments, OCT4 knockdown, xenograft models, flow cytometry for TIC populations\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus pathway inhibitor rescue, xenograft validation, multiple orthogonal approaches\",\n      \"pmids\": [\"29700392\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"EPHB6 induces mitochondrial network fragmentation in triple-negative breast cancer cells by activating the ERK-DRP1 pathway, increasing organelle fission. This mitochondrial fragmentation renders cancer cells more susceptible to DR5-mediated apoptosis. DRP1 activity is essential for the EPHB6-mediated pro-apoptotic response.\",\n      \"method\": \"Mitochondrial morphology imaging, ERK activation assays, DRP1 phosphorylation assays, DRP1 inhibitor/knockdown experiments, DR5 agonist apoptosis assays\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — imaging of mitochondrial fragmentation, pathway inhibition, functional apoptosis assays; single lab\",\n      \"pmids\": [\"27788485\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Ligand-induced (ephrin-B2) downregulation of EphB6 occurs through internalization via clathrin-coated pits followed by lysosomal degradation, dependent on Rab5 GTPase activity. EphB6 interacts constitutively with the Hsp90 chaperone, and ligand-induced downregulation is preceded by rapid EphB6-Hsp90 dissociation. Hsp90 inhibition alone induces EphB6 degradation, mimicking ligand-induced downregulation.\",\n      \"method\": \"Dominant-negative Rab5 mutant expression, clathrin inhibitors, lysosomal pathway inhibitors, Co-immunoprecipitation of EphB6 with Hsp90, Hsp90 inhibitor (geldanamycin) treatment, receptor degradation assays\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — dominant-negative mutant plus pharmacological inhibitors plus Co-IP, multiple orthogonal approaches, single lab\",\n      \"pmids\": [\"25152371\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"The intracellular regions of EphB6 comprising the juxtamembrane (JM) region, pseudokinase, and SAM domains display high flexibility in solution and a propensity for inter-domain interactions. Tyrosine residues in the JM region of EphB6 are substrates for EphB4 kinase and, when phosphorylated, bind SH2 domains of Abl, Src, and Vav3. EphB6 and EphA10 pseudokinase domains can bind ATP and ATP-competitive small molecules.\",\n      \"method\": \"Small-angle X-ray scattering (SAXS), cross-linking mass spectrometry, in vitro EphB4 kinase assay, SH2 domain pull-down assays, ATP binding assays\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — SAXS structural data, in vitro kinase assay, pull-down with specific SH2 domains; multiple orthogonal Tier 1 methods\",\n      \"pmids\": [\"34431498\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Crystal structure of the EphB6 extracellular domain (ECD) was determined by X-ray crystallography, revealing the overall architecture including ligand-binding domain (LBD) and fibronectin III domain (FN3). EphB6-LBD/FN3 interactions are similar to those observed in unliganded A-class Eph receptors, suggesting general importance. Unique structural features of EphB6 likely reflect its requirement for co-receptors due to lack of intrinsic kinase activity.\",\n      \"method\": \"X-ray crystallography of EphB6-ECD\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure determination with structural validation\",\n      \"pmids\": [\"33770085\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"EPHB6 modulates EGFR signaling to enhance proliferation of cancer cells and tumor development, as identified by genetic screening and BioID proteomics integrated with machine learning. Further experiments confirmed the EPHB6-EGFR crosstalk.\",\n      \"method\": \"Genome-wide genetic interaction screen, BioID proximity proteomics, machine learning integration, functional validation experiments\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — BioID proteomics plus functional validation, multiple complementary approaches, single lab\",\n      \"pmids\": [\"37392382\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Expression of EPHB6 in A549 lung adenocarcinoma cells leads to phosphorylation of ERK (MAP kinase). siRNA-based knockdown of EPHB6 reverses ERK phosphorylation. However, EPHB6-induced ERK phosphorylation is uncoupled from activation of the Elk-1 transcriptional factor.\",\n      \"method\": \"EPHB6 overexpression, siRNA knockdown, ERK phosphorylation immunoblotting, Elk-1 reporter assays\",\n      \"journal\": \"International journal of oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — overexpression and siRNA knockdown with consistent result, single lab, single study\",\n      \"pmids\": [\"19513565\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"CLDN1 upregulates cellular EPHB6 levels and enhances its activation, resulting in suppression of ERK1/2 signaling and downstream inhibition of SLUG expression, forming a CLDN1-EPHB6-ERK1/2-SLUG feedback axis that represses metastasis, drug resistance, and cancer stemness in lung adenocarcinoma.\",\n      \"method\": \"Immunoblotting, immunoprecipitation, migration assays, sphere assay, xenograft experiments, siRNA knockdown\",\n      \"journal\": \"Theranostics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — immunoprecipitation and functional assays defining pathway, single lab\",\n      \"pmids\": [\"32754286\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"EPHB6 mutation-induced paclitaxel resistance is mediated by interaction with EPHA2, which promotes JNK-mediated cadherin 11 (CDH11) expression. EPHB6-mutated cells acquire cell adhesion-mediated drug resistance via CDH11 expression and RhoA/FAK activation. Targeted inhibition of EPHA2 or CDH11 reverses paclitaxel resistance.\",\n      \"method\": \"Co-immunoprecipitation of EPHB6 with EPHA2, CDH11 expression assays, JNK phosphorylation assays, RhoA/FAK activation assays, pharmacological inhibitor experiments, drug resistance assays\",\n      \"journal\": \"Experimental & molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — Co-IP, pathway inhibitors, functional drug resistance assays; single lab\",\n      \"pmids\": [\"31160603\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"EphB6 interacts with ephrin-B1 in trans on neighboring cells, leading to formation of large co-clusters at the plasma membrane. These co-clusters show decreased propensity for endocytosis. Co-clustering promotes formation of double-membrane tubular structures between cells, stabilizes cell-cell adhesion, and reduces invasive behavior of cancer cells.\",\n      \"method\": \"Lattice light-sheet microscopy, 3D structured illumination microscopy, cryo-electron tomography, endocytosis assays, cell invasion assays\",\n      \"journal\": \"Communications biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — multiple high-resolution structural imaging modalities (cryo-ET, LLSM, SIM) plus functional invasion assays\",\n      \"pmids\": [\"38627519\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"EphB6 deficiency in mice promotes tumor growth in a colorectal cancer xenograft model by increasing GABA in the tumor microenvironment through intestinal neurons. The mechanism involves increased expression of synaptosomal-associated protein 25 (SNAP25) in the intestinal myenteric plexus, mediating GABA release. Botulinum toxin A injection into the rectum of EphB6-deficient mice eliminated the pro-tumorigenic effect, confirming the neuronal dependency.\",\n      \"method\": \"EphB6-deficient mouse xenograft model, GABA measurement, SNAP25 immunoblotting, botulinum toxin A neuronal inhibition, gut microbiota controls\",\n      \"journal\": \"Carcinogenesis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — KO mouse model with pharmacological rescue (botulinum toxin) and defined mediator (SNAP25/GABA), single lab\",\n      \"pmids\": [\"37294054\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"EphB6 promotes survival of disseminated dormant cancer cells (DDCCs) in the lung through activation of the TFEB-lysosomal axis. Direct co-culture of DDCCs with lung epithelial cells induces EphB6 expression and lysosomal accumulation. EphB6 contributes to TFEB transcriptional activity and lysosome formation. EphB6 signaling also promotes proliferation of surrounding lung parenchymal cells in vivo.\",\n      \"method\": \"Lung organotypic co-culture system, in vivo dissemination assays, TFEB reporter assays, lysosome staining, EphB6 knockdown, xenograft models\",\n      \"journal\": \"Cancers\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — in vitro and in vivo functional assays with defined TFEB pathway, single lab\",\n      \"pmids\": [\"33802447\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"SIAH2 promotes K48-linked polyubiquitination and proteasomal degradation of EPHB6 in hepatocellular carcinoma cells. Loss of EPHB6 through SIAH2-mediated degradation promotes filopodia formation and cell invasion/migration via upregulation of RHOF.\",\n      \"method\": \"Co-immunoprecipitation of SIAH2 with EPHB6, ubiquitination assays (K48-linkage specific), EPHB6 knockdown/overexpression, RHOF expression assays, filopodia quantification, invasion/migration assays\",\n      \"journal\": \"Cell & bioscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — Co-IP, ubiquitination assay, functional rescue experiments; single lab, single study\",\n      \"pmids\": [\"42036676\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"EPHB6 interacts with CBX7 in cervical cancer C33A cells, and EPHB6 inhibits cell proliferation, invasion, migration, and cell cycle acceleration by binding to CBX7.\",\n      \"method\": \"Co-immunoprecipitation, HDOCK molecular docking prediction, functional assays (CCK-8, EdU, wound healing, transwell), flow cytometry for cell cycle\",\n      \"journal\": \"Cell biochemistry and biophysics\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP with computational docking support, functional assays without detailed mechanistic dissection, single lab\",\n      \"pmids\": [\"39322790\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Enzalutamide-mediated androgen receptor (AR) activation binds to an androgen-response-element (ARE) on the EPHB6 promoter to decrease EPHB6 expression, which then activates JNK phosphorylation and increases MMP9 expression to promote CRPC cell invasion. JNK inhibitor SP600125 blocks Enzalutamide-increased CRPC cell invasion.\",\n      \"method\": \"ARE binding assay/ChIP, EPHB6 promoter reporter assay, JNK phosphorylation immunoblot, JNK inhibitor treatment, invasion assays\",\n      \"journal\": \"Cancer letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — promoter binding assay and pharmacological rescue with defined pathway components, single lab\",\n      \"pmids\": [\"28826721\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Overexpression of EphB6 via in utero electroporation results in clumping of cortical neurons (soma spacing defect), as does overexpression of ephrin-B2. When both EphB6 and ephrin-B2 are co-overexpressed in cortical neurons, the soma clumping phenotypes disappear, demonstrating mutual inhibition requiring interaction of their specific domains.\",\n      \"method\": \"In utero electroporation, cortical neuron imaging, domain-specific interaction analysis\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — in vivo overexpression with quantitative phenotypic readout and domain specificity analysis, single lab\",\n      \"pmids\": [\"37149633\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"EPHB6 is an intrinsically kinase-dead (pseudokinase) Eph receptor that signals via transphosphorylation by catalytically active partners (EphB1, EphB4, Src/Fyn), recruits adaptor proteins (c-Cbl, GRB2, Hsp90) through its phosphorylated juxtamembrane region, and modulates multiple downstream pathways (JNK/Rac1, ERK-DRP1, RAS-ERK, RhoA) to regulate T cell activation, cell adhesion and migration, vascular smooth muscle contractility, adrenal catecholamine biosynthesis and secretion, and cancer cell invasiveness and drug sensitivity; it also engages in trans co-clustering with ephrin-B1 to stabilize cell-cell adhesion and suppress cancer invasion, and is degraded via clathrin/Rab5-dependent lysosomal targeting following ligand-induced Hsp90 dissociation or via SIAH2-mediated K48-ubiquitination.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"EPHB6 is an intrinsically kinase-deficient (pseudokinase) Eph receptor that binds ephrin-B2 as its high-affinity ligand and signals not through its own catalytic activity but by being transphosphorylated by catalytically active partners and by acting as a scaffold for adaptor recruitment [#5, #0]. Its juxtamembrane tyrosines are substrates for active EphB-family and Src-family kinases—EphB1, EphB4, and the constitutively associated Fyn—and once phosphorylated they recruit SH2-domain proteins including Abl, Src, and Vav3, while constitutive recruitment of c-Cbl and Hsp90 occurs through the cytoplasmic region [#0, #7, #18, #8, #17]. Structurally, the extracellular domain adopts an architecture resembling unliganded A-class Eph receptors and the intracellular pseudokinase and SAM modules are highly flexible, retaining residual ATP binding despite catalytic deficiency [#19, #18]. In T cells EPHB6 partitions to TCR-containing lipid rafts and is required for full proximal TCR signaling (ZAP-70, LAT, PLCγ1-SLP-76, MAPK), exerting ligand- and concentration-dependent modulation of Rac1/JNK, IL-2 secretion, proliferation, and apoptosis [#4, #2, #3]. Through ephrin-B reverse signaling and adaptor coupling, EPHB6 controls vascular smooth muscle contractility via RhoA and GRIP1 and governs adrenal chromaffin-cell catecholamine biosynthesis (RAC1-MKK7-JNK-c-Jun/AP1-EGR1 driving tyrosine hydroxylase) and testosterone-dependent secretion (BKCa-regulated Ca2+ influx and RHOA/FYN-ABL-MICAL-1-dependent cortical F-actin disassembly) [#11, #12, #13, #14]. In cancer, EPHB6 generally suppresses invasiveness—by transphosphorylation-dependent c-Cbl/Abl signaling, by sequestering and restraining kinase-active oncogenic Eph receptors such as EphA2, and by trans co-clustering with ephrin-B1 to stabilize cell-cell adhesion—but also drives context-specific pro-tumorigenic outputs through GRB2-RAS-ERK-OCT4, ERK-DRP1 mitochondrial fission, EGFR crosstalk, and a TFEB-lysosomal survival program [#8, #9, #10, #24, #15, #16, #20, #26]. Receptor levels are set post-translationally by ligand-induced clathrin/Rab5-dependent lysosomal degradation following Hsp90 dissociation and by SIAH2-mediated K48-linked ubiquitination [#17, #27].\",\n  \"teleology\": [\n    {\n      \"year\": 2000,\n      \"claim\": \"Establishing which ephrin engages EPHB6 was the prerequisite for any signaling model; the work defined ephrin-B2 as its specific high-affinity ligand.\",\n      \"evidence\": \"EphB6-Fc binding to ephrin-transfected COS and Reh cells with PIPLC and saturation controls\",\n      \"pmids\": [\"10648835\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not address downstream signaling consequences of binding\", \"In vitro fusion-protein binding may not reflect full-length receptor avidity\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Because EPHB6 lacks intrinsic kinase activity, how it is activated was unresolved; transphosphorylation by catalytically active EphB1 within a stable heterocomplex, plus constitutive c-Cbl binding, defined the pseudokinase signaling logic.\",\n      \"evidence\": \"Co-IP with catalytically active/inactive EphB1, ligand stimulation, kinase assays\",\n      \"pmids\": [\"11713248\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Which juxtamembrane tyrosines are phosphorylated not mapped here\", \"Functional consequence of Cbl binding not determined\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Whether EPHB6 has cell-autonomous immune function was unknown; cross-linking in T cells showed it couples to Cbl/SHP-1 dynamics and controls proliferation and apoptosis.\",\n      \"evidence\": \"Anti-EphB6/anti-CD3 co-cross-linking, Co-IP, apoptosis assays in Jurkat cells\",\n      \"pmids\": [\"11466354\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single cell line and single study\", \"Mechanism linking Cbl dephosphorylation to apoptosis not dissected\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"The directionality of EPHB6's T-cell role was clarified by showing it co-localizes with the TCR at membrane rafts and co-stimulates proliferation via p38, and by confirming ephrin-B2 as its unique thymocyte ligand.\",\n      \"evidence\": \"Confocal co-localization, cytokine/proliferation assays, p38 assays; ephrin-Fc binding on KO thymocytes\",\n      \"pmids\": [\"12393850\", \"12379224\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Adaptor linkage between EPHB6 and p38 not defined\", \"Co-stimulation context requires suboptimal TCR signal\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"To resolve which signaling arm EPHB6 modulates in T cells, ligand stimulation was shown to selectively inhibit TCR-induced Rac1-JNK (not MAPK) and IL-2/CD25, with dominant-negative rescue.\",\n      \"evidence\": \"WT and dominant-negative overexpression, Rac1 PAK pull-down, JNK assay, IL-2 ELISA, flow cytometry\",\n      \"pmids\": [\"12517763\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct biochemical link from EPHB6 to Rac1 regulation not shown\", \"Overexpression context\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Genetic loss-of-function defined EPHB6 as required (not merely modulatory) for full proximal TCR signaling, anchoring its physiological immune role.\",\n      \"evidence\": \"EphB6-KO mice, raft fractionation, ZAP-70/LAT/PLCγ1-SLP-76/MAPK readouts, in vivo T-cell assays\",\n      \"pmids\": [\"15599401\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular mechanism of how a kinase-dead receptor enhances ZAP-70/LAT not resolved\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"The dual positive/negative behavior of EPHB6 was explained by a concentration-dependent switch in which constitutive Fyn association and Src-family-driven tyrosine phosphorylation convert adhesion-promoting into repulsive signaling.\",\n      \"evidence\": \"Adhesion/migration assays at varying ligand dose, cytoplasmic deletion mutants, Fyn Co-IP, SFK inhibitors\",\n      \"pmids\": [\"15955811\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of phosphosites driving the switch not mapped\", \"Downstream effectors of repulsion not defined\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Whether EPHB6 engages ERK in epithelial cancers and whether that drives transcription was tested, showing ERK phosphorylation uncoupled from Elk-1 activation.\",\n      \"evidence\": \"Overexpression/siRNA in A549 cells, ERK immunoblot, Elk-1 reporter\",\n      \"pmids\": [\"19513565\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of ERK-Elk1 uncoupling unexplained\", \"Single cell line\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"The mechanism of EPHB6's tumor-suppressive activity was placed downstream of EphB4 transphosphorylation, requiring c-Cbl-dependent Abl phosphorylation to inhibit invasiveness.\",\n      \"evidence\": \"EPHB6 restoration in breast cancer cells, c-Cbl Co-IP, Cbl siRNA rescue, Abl phosphorylation and invasion assays\",\n      \"pmids\": [\"20086179\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Quantitative balance of EphB6/EphB4 in vivo not established\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"A sequestration model for tumor suppression was supported by demonstrating EPHB6 heterointeractions with EphB2 and, unprecedentedly, with the A-class EphA2.\",\n      \"evidence\": \"Co-IP across breast carcinoma cell lines\",\n      \"pmids\": [\"21737611\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single Co-IP per pair without reciprocal/functional validation\", \"Stoichiometry of sequestration unknown\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Receptor turnover and the EPHB6-EphA2 axis were mechanistically defined: ligand-induced Hsp90 dissociation triggers clathrin/Rab5 lysosomal degradation, and EPHB6 restrains EphA2 S897 phosphorylation/Ephexin4-RhoG to suppress anoikis resistance.\",\n      \"evidence\": \"Dominant-negative Rab5, clathrin/lysosome inhibitors, Hsp90 Co-IP and geldanamycin; EphA2 phospho/RhoG/anoikis assays\",\n      \"pmids\": [\"25152371\", \"25239188\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"E3 ligase for lysosomal route not identified here\", \"Hsp90 binding site on EPHB6 unmapped\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Beyond immunity, EPHB6 was assigned a cardiovascular role: KO arteries show heightened contractility via RhoA/myosin light chain, with ephrin-B reverse signaling acting through GRIP1.\",\n      \"evidence\": \"EphB6-KO mice, ex vivo contractility, RhoA assay, MLC phospho-immunoblot, cross-linking, BP/catecholamine measurement\",\n      \"pmids\": [\"22223652\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Sex/testosterone dependence first hinted but not mechanistically resolved here\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"EPHB6's adrenal function was opened by linking it to chromaffin-cell excitability: KO augments BKCa currents and reduces ACh-triggered Ca2+ influx in a testosterone-dependent manner.\",\n      \"evidence\": \"Patch-clamp, Ca2+ imaging, BKCa blockade, KO mice with castration controls\",\n      \"pmids\": [\"29339804\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How EPHB6 regulates BKCa channels molecularly not defined\", \"Mechanism of testosterone dependence unresolved\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"The biosynthetic arm of catecholamine control was defined: EPHB6 reverse signaling through ephrin-B1 drives tyrosine hydroxylase transcription via RAC1-MKK7-JNK-c-Jun/AP1-EGR1.\",\n      \"evidence\": \"KO mice, RT-qPCR, immunoblot, microarray, pathway inhibitors, HPLC catecholamines\",\n      \"pmids\": [\"30824540\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct transcription factor occupancy at TH promoter not shown\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"The secretory arm was completed by showing EPHB6-to-ephrin-B1 reverse signaling regulates exocytosis through RHOA and FYN-ABL-MICAL-1-dependent cortical F-actin disassembly, with a defined 7-residue ephrin-B1 tail motif.\",\n      \"evidence\": \"Amperometry, F-actin quantification, ephrin-B1 tail deletion mutants, pathway inhibition, KO mice\",\n      \"pmids\": [\"32321761\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How testosterone gates this pathway not molecularly resolved\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"A pro-apoptotic cancer output was identified whereby EPHB6 activates ERK-DRP1 to fragment mitochondria, sensitizing TNBC cells to DR5-mediated apoptosis.\",\n      \"evidence\": \"Mitochondrial imaging, ERK/DRP1 phospho assays, DRP1 inhibitor/knockdown, DR5 agonist apoptosis\",\n      \"pmids\": [\"27788485\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Link from EPHB6 to ERK activation upstream not biochemically defined\", \"Single lab\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"A context-dependent pro-tumorigenic function was shown via GRB2 recruitment activating RAS-ERK-OCT4 to expand tumor-initiating cells, revealing EPHB6's dual cancer roles.\",\n      \"evidence\": \"GRB2 Co-IP, ERK inhibitor and OCT4 knockdown, xenografts, TIC flow cytometry\",\n      \"pmids\": [\"29700392\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Reconciliation with tumor-suppressive roles context-dependent and unresolved\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Transcriptional regulation of EPHB6 in prostate cancer was defined: AR binds an ARE to repress EPHB6, derepressing JNK-MMP9-driven invasion.\",\n      \"evidence\": \"ChIP/ARE binding, promoter reporter, JNK phospho, JNK inhibitor, invasion assays\",\n      \"pmids\": [\"28826721\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab; direct EPHB6-to-JNK biochemical link not shown\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"A drug-resistance mechanism was mapped: mutated EPHB6 engages EphA2 to drive JNK-CDH11 and RhoA/FAK, conferring adhesion-mediated paclitaxel resistance reversible by targeting EphA2 or CDH11.\",\n      \"evidence\": \"EphA2 Co-IP, CDH11/JNK/RhoA-FAK assays, inhibitors, resistance assays\",\n      \"pmids\": [\"31160603\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Specific EPHB6 mutations and their structural effect not resolved\", \"Single lab\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"An upstream regulator-EPHB6-effector axis in lung cancer was defined whereby CLDN1 enhances EPHB6 to suppress ERK1/2-SLUG, repressing metastasis and stemness.\",\n      \"evidence\": \"IP, migration/sphere/xenograft assays, siRNA knockdown\",\n      \"pmids\": [\"32754286\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How CLDN1 enhances EPHB6 activation mechanistically unclear\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Structural understanding of the kinase-dead receptor was advanced: the ECD resembles unliganded A-class Ephs, and the flexible intracellular JM/pseudokinase/SAM region presents EphB4-phosphorylated tyrosines that bind Abl/Src/Vav3 SH2 domains, with residual ATP binding.\",\n      \"evidence\": \"X-ray crystallography of ECD; SAXS, XL-MS, in vitro EphB4 kinase, SH2 pull-downs, ATP binding\",\n      \"pmids\": [\"33770085\", \"34431498\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full-length receptor structure absent\", \"Functional role of residual ATP binding in cells untested\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"A pro-survival role in metastatic dormancy was identified through EPHB6 activation of the TFEB-lysosomal axis in disseminated dormant cancer cells.\",\n      \"evidence\": \"Lung organotypic co-culture, in vivo dissemination, TFEB reporter, lysosome staining, knockdown, xenograft\",\n      \"pmids\": [\"33802447\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Signaling link from EPHB6 to TFEB not biochemically defined\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Unbiased screening extended EPHB6 partners to EGFR, showing crosstalk that enhances cancer-cell proliferation and tumor development; separately, EPHB6 deficiency was shown to promote colorectal tumor growth via intestinal-neuron GABA/SNAP25.\",\n      \"evidence\": \"Genetic screen + BioID + machine learning with validation; KO xenograft, GABA/SNAP25 assays, botulinum toxin rescue\",\n      \"pmids\": [\"37392382\", \"37294054\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct EPHB6-EGFR biochemical interface not defined\", \"Neuronal-tumor axis from single lab\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"A developmental neuronal role was shown in which EPHB6 and ephrin-B2 overexpression each cause cortical soma clumping that mutually cancels, indicating domain-specific cis/trans interaction controlling neuronal spacing.\",\n      \"evidence\": \"In utero electroporation, cortical neuron imaging, domain-specific interaction analysis\",\n      \"pmids\": [\"37149633\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Loss-of-function neuronal phenotype not shown\", \"Downstream effectors unmapped\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"High-resolution imaging defined the adhesive output: EPHB6 trans co-clustering with ephrin-B1 forms endocytosis-resistant clusters and double-membrane tubules that stabilize cell-cell contacts and reduce invasion.\",\n      \"evidence\": \"Lattice light-sheet, 3D-SIM, cryo-electron tomography, endocytosis and invasion assays\",\n      \"pmids\": [\"38627519\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular composition of tubular structures not fully resolved\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Post-translational control was extended by showing SIAH2 drives K48-ubiquitination and proteasomal degradation of EPHB6, derepressing RHOF-driven filopodia and invasion in hepatocellular carcinoma.\",\n      \"evidence\": \"SIAH2-EPHB6 Co-IP, K48-linkage ubiquitination assay, knockdown/overexpression, RHOF and filopodia/invasion assays\",\n      \"pmids\": [\"42036676\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"SIAH2 recognition motif on EPHB6 not mapped\", \"Single lab, single study\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How EPHB6's many context-specific outputs (tumor suppression vs promotion, immune, vascular, adrenal, neuronal) are selected by partner availability, phosphosite occupancy, and tissue context remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified model reconciling opposing cancer roles\", \"Endogenous heterocomplex stoichiometry across tissues unknown\", \"Full-length receptor signaling structure undetermined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 8, 18]},\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [5, 24, 7]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 7, 15, 18]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [9, 10]},\n      {\"term_id\": \"GO:0140657\", \"supporting_discovery_ids\": [18]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [2, 4, 24]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [17, 24]},\n      {\"term_id\": \"GO:0005764\", \"supporting_discovery_ids\": [17, 26]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 7, 3, 11]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [2, 3, 4]},\n      {\"term_id\": \"R-HSA-1500931\", \"supporting_discovery_ids\": [7, 24, 30]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [8, 15, 23, 27]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [17, 27]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"EFNB2\", \"EFNB1\", \"EPHB1\", \"EPHB4\", \"EPHA2\", \"FYN\", \"CBL\", \"GRB2\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}