{"gene":"EPHA3","run_date":"2026-06-09T23:54:43","timeline":{"discoveries":[{"year":1998,"finding":"EphA3 extracellular region contains distinct functional subdomains: ligand binding is restricted to the N-terminal region encoded by exon III, while an independent C-terminal domain mediates receptor dimerization/oligomerization. A two-step activation mechanism was proposed involving sequential ligand binding and ligand-independent receptor-receptor oligomerization.","method":"BIAcore kinetic binding analysis, mRNA injection into zebrafish embryos, receptor transphosphorylation analysis using recombinant subdomain proteins","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple orthogonal methods (BIAcore, functional zebrafish assay, transphosphorylation) in a single rigorous study clearly mapping two distinct extracellular subdomains","pmids":["9685371"],"is_preprint":false},{"year":1995,"finding":"EphA3 (hek) binds GPI-anchored membrane proteins (ephrin-A ligands) with high affinity (Kd ~2×10^8 M^-1); two related ephrin ligands were isolated from a human T-lymphoma line that bind both EphA3 and the related ELK receptor tyrosine kinase.","method":"Expression cloning using soluble EphA3-Fc fusion protein as affinity probe; binding affinity measured by Fc-fusion pulldown","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct biochemical isolation of ligands using soluble receptor fusion protein, affinity constants determined, replicated across multiple ligands","pmids":["7838529"],"is_preprint":false},{"year":2002,"finding":"Ephrin-A5 stimulation of EphA3 causes rapid RhoA activation, leading to actin/myosin cytoskeleton reorganization, membrane blebbing, retraction of cellular protrusions, and cell detachment. Receptor clustering (polymeric ephrin-A5) is required. The adaptor CrkII associates with tyrosine-phosphorylated EphA3 in vitro and is recruited in vivo upon ephrin-A5 stimulation; expression of SH3-domain-mutated CrkII abolishes cell rounding, blebbing, and detachment.","method":"Ephrin-A5 stimulation assays, RhoA activation assay, co-immunoprecipitation of CrkII with phosphorylated EphA3, dominant-negative CrkII expression, morphological assays in EphA3-expressing 293T and melanoma cells","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, dominant-negative rescue, RhoA activation assay, and morphological readouts in multiple cell lines; multiple orthogonal methods in one study","pmids":["11870224"],"is_preprint":false},{"year":2003,"finding":"Three distinct surface areas of EphA3 are essential for ephrin-A5 binding and assembly of signaling-competent oligomeric receptor-ligand complexes: two map to interfaces identified in the EphB2-ephrin-B2 crystal structure, and a third falls outside the structurally characterized interaction domains.","method":"Random mutagenesis of EphA3 in yeast expression library, selection for impaired ephrin-A5 binding, functional characterization of point mutants","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — systematic mutagenesis screen with functional validation of multiple mutants, defining molecular determinants of ligand binding","pmids":["14660665"],"is_preprint":false},{"year":2006,"finding":"EphA3 forms a cis-interaction with ephrinA5 (on the same cell surface) that is independent of the established ligand-binding domain of EphA3. This cis-complex abolishes ephrinA5-induced tyrosine phosphorylation of EphA3 and renders retinal axons insensitive to ephrinA in trans, thereby converting uniform EphA3 expression into a functional gradient.","method":"Truncated EphA3 constructs, single-amino acid point mutants of ephrinA5, FRET (fluorescence resonance energy transfer) imaging, tyrosine phosphorylation assays, retinal axon sensitivity assays","journal":"Nature neuroscience","confidence":"High","confidence_rationale":"Tier 1 / Strong — FRET directly demonstrates physical cis-interaction; mutagenesis and functional axon assays confirm mechanistic consequence; multiple orthogonal methods","pmids":["16491080"],"is_preprint":false},{"year":2006,"finding":"EphA3 signaling is required for atrioventricular endocardial cushion development and epithelial-to-mesenchymal transformation (EMT) in the developing heart. EphA3 knockout mice show hypoplastic endocardial cushions and reduced mesenchymal cell migration from cushion explants; EphA3 and its ligand ephrin-A1 are expressed in adjacent cells in the developing endocardial cushions.","method":"EphA3 null mouse generation, cardiac morphological analysis, endocardial cushion explant migration assay, expression analysis by in situ/immunostaining","journal":"Developmental biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean knockout with specific morphological and cellular phenotypes, ligand-receptor co-expression in adjacent cells confirmed, replicated across multiple readouts","pmids":["17046737"],"is_preprint":false},{"year":2008,"finding":"The unphosphorylated juxtamembrane (JM) region of EphA3 inhibits kinase activity through an autoinhibitory mechanism: two tyrosines in the JM region are sites of autophosphorylation and interact with the kinase active site to modulate catalysis. Crystal structures of multiple states (with/without JM segment, wild-type and mutants) revealed a conserved pathway of coupled residues connecting the JM segment to the activation loop and catalytic residues.","method":"X-ray crystallography of EphA3 kinase domain with and without juxtamembrane segment; site-specific mutagenesis of JM tyrosines; structural comparison of multiple states","journal":"Structure","confidence":"High","confidence_rationale":"Tier 1 / Strong — high-resolution crystal structures plus mutagenesis, multiple states characterized, clear mechanistic model established","pmids":["18547520"],"is_preprint":false},{"year":2009,"finding":"Adaptor protein Nck1 interacts with activated EphA3 through binding of the Nck1 SH2 domain to phosphotyrosine Y602 of EphA3. This interaction mediates EphA3-induced inhibition of cell migration and process retraction; dominant-negative Nck1 mutants lacking functional SH2 or SH3 domains partially alleviate the EphA3-mediated inhibitory effects.","method":"Yeast two-hybrid screen using EphA3 cytoplasmic domain as bait; GST pulldown; co-immunoprecipitation; Y602 point mutagenesis; dominant-negative Nck1 rescue experiments; cell migration and process retraction assays","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — yeast 2-hybrid confirmed by GST pulldown and Co-IP; mutagenesis defines exact binding site (Y602); functional rescue with dominant-negative validates mechanistic role","pmids":["19505147"],"is_preprint":false},{"year":2010,"finding":"EphA3-mediated inhibition of cell migration, neurite outgrowth, and growth cone collapse requires collaboration between phosphotyrosine residues Y602 and Y779 in the cytoplasmic domain. Mutation of either residue alone partially decreases EphA3-induced responses; full abrogation requires mutation of both Y602 and Y779 together.","method":"Individual tyrosine-to-phenylalanine mutagenesis of EphA3 cytoplasmic domain residues; cell migration assay (293A cells); neurite outgrowth assay (NG108-15 cells); growth cone collapse assay (hippocampal neurons)","journal":"Cell research","confidence":"High","confidence_rationale":"Tier 2 / Strong — systematic mutagenesis with three independent functional assays in distinct cell types; collaborative model validated across multiple readouts","pmids":["20697431"],"is_preprint":false},{"year":2012,"finding":"Somatic cancer mutations in EphA3 impair kinase activity and/or ephrin ligand binding and/or decrease receptor cell surface localization through multiple distinct mechanisms, consistent with EphA3 having ephrin- and kinase-dependent tumor-suppressing activities disrupted by cancer mutations.","method":"Functional analysis of cancer-derived EphA3 point mutants: in vitro kinase assays, ephrin binding assays, cell surface biotinylation/flow cytometry","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple orthogonal biochemical assays (kinase activity, ligand binding, surface localization) applied systematically to a panel of cancer mutations","pmids":["22242939"],"is_preprint":false},{"year":2012,"finding":"At least two cancer-associated EphA3 somatic mutations function as dominant inhibitors (dominant negative) of wild-type EphA3 protein. Wild-type EphA3 re-expression in lung cancer lines increases apoptosis by suppressing AKT activation in vitro and inhibits tumor xenograft growth in vivo; dominant-negative mutations override these tumor-suppressive effects in trans.","method":"Immunoprecipitation, western blotting, kinase assays; AKT phosphorylation analysis; BrdU proliferation and apoptosis assays; xenograft tumor growth assay","journal":"Journal of the National Cancer Institute","confidence":"High","confidence_rationale":"Tier 2 / Strong — kinase assays plus in vitro signaling plus in vivo xenograft; dominant-negative mechanism validated across multiple methods","pmids":["22829656"],"is_preprint":false},{"year":2013,"finding":"EphA3 maintains glioblastoma tumor-initiating cells in a less differentiated state by modulating MAPK signaling. EphA3 knockdown or depletion of EphA3-positive cells reduces tumorigenic potential.","method":"EphA3 knockdown (siRNA/shRNA), tumor sphere formation, MAPK signaling analysis, xenograft mouse model, radiolabeled EphA3 antibody depletion of EphA3+ cells","journal":"Cancer cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean KD with specific cellular phenotype and MAPK pathway readout, validated in vivo; single lab but multiple methods","pmids":["23410976"],"is_preprint":false},{"year":2005,"finding":"The anti-EphA3 monoclonal antibody IIIA4 binds a conformation-specific epitope within the ephrin-binding domain adjacent to the ephrin-A5 heterotetramerization site. Preclustered IIIA4 triggers EphA3 activation and cytoskeletal contraction similarly to ephrin-A5. Unclustered IIIA4 and ephrin-A5 Fc in combination initiate greatly enhanced EphA3 signaling through allosteric permissive conformational effects.","method":"BIAcore binding analysis, immunoblot for EphA3 phosphorylation, confocal microscopy, wild-type and binding-site mutant EphA3 analysis, cell contraction assays","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 2 / Strong — BIAcore, mutagenesis, phosphorylation assays, and morphological readouts with both wild-type and binding-impaired mutants; multiple orthogonal approaches","pmids":["16061656"],"is_preprint":false},{"year":2015,"finding":"EphA3 forms dimers in the absence of ligand binding on the surface of live cells, and these unliganded dimers are stabilized by interactions involving the EphA3 SAM (sterile α-motif) domain, establishing a new role for the SAM domain in promoting Eph receptor lateral interactions.","method":"Quantitative FRET-based dimerization assay on live cells, deletion mutants removing the SAM domain, comparison of wild-type vs. SAM-deleted EphA3","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — FRET on live cells with domain deletion mutants, directly demonstrating SAM-dependent dimerization; rigorous single-lab study with clear mechanistic conclusion","pmids":["26232493"],"is_preprint":false},{"year":2019,"finding":"Presenilin-1 (PS1)/γ-secretase cleaves EphA3 independently of ligand binding to generate an intracellular domain (ICD) fragment. EphA3 ICD promotes axon elongation by binding to non-muscle myosin IIA (NMIIA) and increasing its phosphorylation at Ser1943, promoting NMIIA filament disassembly and cytoskeletal rearrangement. This PS/EphA3-dependent cleavage also inhibits RhoA signaling to promote axon growth.","method":"Genetic rescue experiments in PS1/γ-secretase- and EphA3-deficient hippocampal neurons; proteomic analysis of EphA3 ICD binding partners; NMIIA phosphorylation assays; pharmacological NMII inhibition; RhoA signaling assays; live imaging","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1 / Strong — proteomic identification of NMIIA as binding partner, biochemical phosphorylation assay, genetic rescue with ICD fragment, pharmacological validation; multiple orthogonal methods","pmids":["31577226"],"is_preprint":false},{"year":2009,"finding":"Stimulation of EphA3 with ephrin-A5 inhibits cell migration and process outgrowth; these effects are partially mediated through the Nck1 SH2-SH3 adaptor protein binding phosphoY602. Dominant-negative Nck1 (lacking functional SH2 or SH3) partially rescues the inhibitory effects, indicating Nck1 is a downstream effector of EphA3-mediated cytoskeletal responses.","method":"Cell migration assays, process retraction assay, dominant-negative Nck1 expression, co-immunoprecipitation","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — functional rescue with domain-specific dominant-negatives plus co-IP, multiple cell-based readouts; described above but merits citation","pmids":["19505147"],"is_preprint":false},{"year":2004,"finding":"EphA3 stimulation by ephrin-A5 in Jurkat and RMS cells causes loss of cell adhesion to fibronectin and recruitment of CrkII to EphA3. EphA3 expression is transcriptionally upregulated by CD28 co-stimulation and by IGF-1/IGF-1R signaling; anti-IGF-1R blocking antibodies suppress this upregulation.","method":"Receptor array screening, ephrin-A5 stimulation assays, Co-IP of CrkII with EphA3, western blotting, anti-IGF-1R blocking antibody experiments","journal":"Experimental cell research","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — single lab, Co-IP of CrkII plus functional adhesion assays; pathway regulation confirmed by blocking antibody","pmids":["14697337"],"is_preprint":false},{"year":2008,"finding":"EphA3 stimulation with ephrin-A5 increases EphA3 phosphorylation and Rho GTPase activity in rhabdomyosarcoma cells, resulting in suppression of cell adhesion to fibronectin and decreased migration toward SDF-1. EphA3 siRNA knockdown increases SDF-1-mediated motility, confirming a suppressive role in cell motility.","method":"Ephrin-A5 stimulation, phosphorylation assays, Rho GTPase activity assay, fibronectin adhesion assay, Transwell migration assay, siRNA knockdown","journal":"Journal of cellular biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean KD with motility phenotype, Rho GTPase assay links receptor to cytoskeletal signaling; single lab","pmids":["18814179"],"is_preprint":false},{"year":2018,"finding":"Neurocan (a perineuronal net proteoglycan) binds NCAM at the EphA3-binding site within the immunoglobulin-2 domain of NCAM, inhibiting NCAM/EphA3 association, membrane clustering of NCAM/EphA3, EphA3 kinase activation, and ephrin-A5-induced growth cone collapse in cortical interneuron axons.","method":"Co-immunoprecipitation of neurocan with NCAM, binding site mapping using NCAM-Ig domain mutants, EphA3 kinase activation assays, NCAM/EphA3 clustering assay by imaging, growth cone collapse assay in cortical neurons","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP with binding site mapping, kinase activation assay, imaging-based clustering, functional growth cone collapse assay; multiple orthogonal methods in single study","pmids":["29670169"],"is_preprint":false},{"year":1999,"finding":"EphA3 expression in hematopoietic tumor cell lines is regulated by DNA methylation of CpG sites downstream of the basal promoter; these methylation sites are absent in normal tissues but present in leukemia patient samples, indicating epigenetic regulation of EphA3 transcription.","method":"Promoter deletion/reporter assays; Southern blot with methylation-sensitive restriction enzymes; bisulfite sequencing of genomic DNA from cell lines and clinical leukemia samples","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 / Strong — bisulfite sequencing plus methylation-sensitive restriction analysis plus promoter reporter assays, validated in clinical samples","pmids":["10498621"],"is_preprint":false},{"year":2014,"finding":"EphA3 is highly expressed on bone marrow-derived mesenchymal/stromal cells (CD90+/Sca1+) and myeloid cells within the tumor microenvironment. Activation of EphA3+ mesenchymal/stromal cells with an agonistic EphA3 antibody leads to cell contraction, cell-cell segregation, and apoptosis, disrupting newly formed tumor stroma and microvasculature and inhibiting tumor growth in vivo.","method":"EphA3 agonist antibody treatment of isolated stromal cells, cell contraction/segregation assays, apoptosis assays, in vivo mouse xenograft model with α-EphA3 antibody treatment","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — defined cellular responses (contraction, segregation, apoptosis) in isolated stromal cells with in vivo validation; single lab","pmids":["25125683"],"is_preprint":false},{"year":2014,"finding":"HIF-1α controls EphA3 expression on human endometrial multipotent mesenchymal stromal cells (eMSCs) under hypoxic conditions. EphA3 silencing significantly inhibits the ability of transplanted eMSCs to support neovascularisation in immunocompromised mice, placing EphA3 downstream of HIF-1α in stromal cell-mediated vascular support.","method":"HIF-1α-EphA3 expression correlation, EphA3 siRNA knockdown in eMSCs, in vivo neovascularization assay in NSG mice","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA KD with defined in vivo vascular phenotype; HIF-1α link established by expression analysis without direct HIF-binding site mutagenesis; single lab","pmids":["25420155"],"is_preprint":false},{"year":2016,"finding":"EPHA3 overexpression in small cell lung cancer cells decreases chemoresistance by increasing apoptosis and inducing G0/G1 arrest, accompanied by reduced phosphorylation of the PI3K/BMX/STAT3 signaling pathway. Conversely, EPHA3 knockdown generates a resistant phenotype with increased PI3K/BMX/STAT3 phosphorylation; PI3K inhibitor (LY294002) and BMX inhibitor (LFM-A13) both reverse the EPHA3-deficiency-induced resistance.","method":"EPHA3 overexpression and siRNA knockdown in SCLC cells; PI3K/BMX/STAT3 phosphorylation western blots; apoptosis and cell cycle assays; pharmacological inhibitor rescue; in vivo xenograft","journal":"Tumour biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — bidirectional genetic perturbation with pharmacological rescue confirming pathway placement; single lab","pmids":["27101199"],"is_preprint":false},{"year":2021,"finding":"EPHA3 regulates PTEN expression epigenetically in radioresistant head and neck cancer cells via two mechanisms downstream of EPHA3/c-Myc signaling: DNMT1-mediated DNA methylation and EZH2-mediated histone H3K27 trimethylation of the PTEN promoter.","method":"Western blotting for DNMT1, EZH2, H3K27me3, and PTEN in radioresistant vs. sensitive cells; EPHA3 knockdown/overexpression; correlation of EPHA3 levels with DNMT1/EZH2/H3K27me3/PTEN","journal":"Biomolecules","confidence":"Low","confidence_rationale":"Tier 3 / Weak — correlative western blots with limited direct mechanistic validation of EPHA3-to-DNMT1/EZH2 pathway; single lab, single method per step","pmids":["33919657"],"is_preprint":false},{"year":2018,"finding":"Androgen receptor (AR) induces EPHA3 expression in prostate cancer cells by forming a transcription factor complex with SP1 that binds SP1 binding sites (−295 to −261) in the EPHA3 core promoter region, as required for androgen-stimulated EPHA3 transcription.","method":"Luciferase reporter assays with EPHA3 promoter deletion constructs; AR overexpression and siRNA knockdown; SP1 inhibitor (mithramycin A) and siSP1 treatment; co-immunoprecipitation and chromatin immunoprecipitation (ChIP) of AR/SP1 complex at EPHA3 promoter","journal":"Oncology reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — ChIP directly demonstrates AR/SP1 occupancy at EPHA3 promoter; luciferase reporters with deletion mapping; Co-IP of AR-SP1 complex; bidirectional genetic perturbation; multiple orthogonal methods","pmids":["29917167"],"is_preprint":false},{"year":2003,"finding":"EphA3(-/-) null mice do not show defects in motor axon guidance to axial muscle targets, contrary to prior prediction based on EphA3 expression in the medial motor column; retrograde labeling studies confirm motor axons reach correct targets without EphA3.","method":"EphA3 null mouse generation; retrograde axonal labeling of motor projections in embryos and adults","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean genetic knockout with direct anatomical readout (retrograde labeling); well-controlled negative result","pmids":["14585969"],"is_preprint":false}],"current_model":"EphA3 is a receptor tyrosine kinase that is activated by clustering upon ephrin-A ligand binding through a two-step mechanism involving an N-terminal ligand-binding domain and a separate C-terminal dimerization domain; the unphosphorylated juxtamembrane region autoinhibits kinase activity, which is relieved by autophosphorylation at two juxtamembrane tyrosines; downstream signaling proceeds through phosphotyrosine docking sites Y602 and Y779 recruiting adaptors including CrkII and Nck1 to activate RhoA and suppress cell adhesion and migration; EphA3 can also be cleaved by presenilin/γ-secretase to generate an ICD that promotes axon elongation via NMIIA phosphorylation; the SAM domain stabilizes ligand-independent receptor dimers on the cell surface; a cis-interaction with ephrinA5 silences receptor activation to shape functional gradients; and EphA3 expression is regulated by DNA methylation, HIF-1α, androgen receptor/SP1, and by perineuronal net protein neurocan inhibiting NCAM/EphA3 complex formation."},"narrative":{"mechanistic_narrative":"EphA3 is a receptor tyrosine kinase that converts ephrin-A ligand engagement into cytoskeletal remodeling, controlling cell adhesion, migration, axon guidance, and tissue morphogenesis [PMID:11870224, PMID:18814179]. It binds GPI-anchored ephrin-A ligands with high affinity through an N-terminal ligand-binding region encoded by exon III, while a separate C-terminal domain drives ligand-independent receptor oligomerization, defining a two-step activation mechanism [PMID:9685371, PMID:7838529]; assembly of signaling-competent oligomers depends on three distinct surface interfaces of the receptor [PMID:14660665], and unliganded receptor dimers are additionally stabilized by the cytoplasmic SAM domain [PMID:26232493]. Kinase activity is held in check by the unphosphorylated juxtamembrane segment, which engages the active site until autophosphorylation of two juxtamembrane tyrosines relieves autoinhibition through a coupled network of residues linking the juxtamembrane region to the activation loop [PMID:18547520]. Activated EphA3 recruits the adaptors CrkII and Nck1 — the latter via its SH2 domain binding phosphotyrosine Y602 — and signals through Y602 together with Y779 to activate RhoA and suppress adhesion to fibronectin, cell migration, neurite outgrowth, and growth cone collapse [PMID:11870224, PMID:19505147, PMID:20697431, PMID:18814179]. A cis-interaction with ephrinA5 on the same cell surface, independent of the canonical ligand-binding domain, silences receptor phosphorylation and converts uniform EphA3 expression into a functional sensitivity gradient in retinal axons [PMID:16491080], and presenilin-1/γ-secretase cleavage of EphA3 generates an intracellular domain that binds non-muscle myosin IIA and increases its Ser1943 phosphorylation to promote axon elongation while inhibiting RhoA [PMID:31577226]. EphA3 is required for atrioventricular endocardial cushion development and epithelial-to-mesenchymal transformation in the heart [PMID:17046737]. In cancer, EphA3 has kinase- and ephrin-dependent tumor-suppressing activity that is disrupted by somatic mutations impairing kinase activity, ligand binding, or surface localization — some acting as dominant negatives — and wild-type re-expression promotes apoptosis by suppressing AKT [PMID:22242939, PMID:22829656, PMID:27101199]. Its expression is controlled by DNA methylation, HIF-1α, and an androgen receptor/SP1 complex binding the core promoter [PMID:10498621, PMID:25420155, PMID:29917167], and at the cell surface neurocan blocks NCAM/EphA3 complex formation and clustering to inhibit ephrin-A5-induced growth cone collapse [PMID:29670169].","teleology":[{"year":1995,"claim":"Establishing the ligands of an orphan-like receptor was prerequisite to any mechanistic model; this work isolated high-affinity GPI-anchored ephrin-A ligands for EphA3.","evidence":"Expression cloning using soluble EphA3-Fc as an affinity probe in a human T-lymphoma line","pmids":["7838529"],"confidence":"High","gaps":["Did not resolve how ligand binding triggers receptor activation","Ligand specificity overlapped with the related ELK receptor"]},{"year":1998,"claim":"How a single ligand-binding event produces an active receptor was unclear; mapping distinct ligand-binding and oligomerization subdomains established a two-step activation mechanism.","evidence":"BIAcore kinetics, zebrafish mRNA injection, and transphosphorylation of recombinant subdomains","pmids":["9685371"],"confidence":"High","gaps":["Atomic-level interfaces not yet defined","Did not address downstream effectors"]},{"year":2002,"claim":"The downstream consequence of activation was undefined; ephrin-A5 clustering was shown to activate RhoA and recruit CrkII to drive cytoskeletal collapse and cell detachment.","evidence":"RhoA activation assays, CrkII Co-IP, dominant-negative CrkII rescue, and morphological assays in 293T and melanoma cells","pmids":["11870224"],"confidence":"High","gaps":["Did not map the receptor docking site for CrkII","Link between RhoA and specific cytoskeletal machinery unresolved"]},{"year":2003,"claim":"The structural determinants of ligand-driven oligomerization were unknown; a mutagenesis screen identified three surface areas required for signaling-competent complex assembly, one outside known interaction domains.","evidence":"Random mutagenesis in yeast with selection for impaired ephrin-A5 binding and functional validation","pmids":["14660665"],"confidence":"High","gaps":["Function of the third, structurally uncharacterized interface not mechanistically explained"]},{"year":2003,"claim":"EphA3 expression predicted a role in motor axon guidance; a clean knockout showed motor axons reach correct targets without EphA3, refining the receptor's in vivo guidance role.","evidence":"EphA3 null mice with retrograde axonal labeling of motor projections","pmids":["14585969"],"confidence":"High","gaps":["Did not exclude redundancy with other Eph receptors","Other guidance contexts left untested"]},{"year":2006,"claim":"How uniform receptor expression yields graded responsiveness was unexplained; a cis-interaction with ephrinA5 was shown to silence receptor phosphorylation and convert expression into a functional gradient.","evidence":"FRET imaging, ephrinA5 point mutants, phosphorylation assays, and retinal axon sensitivity assays","pmids":["16491080"],"confidence":"High","gaps":["Structural basis of the cis-interface not defined","Extent of cis-silencing in non-retinal tissues unknown"]},{"year":2006,"claim":"A developmental requirement for EphA3 was undefined; knockout mice revealed it is required for endocardial cushion development and EMT in the heart.","evidence":"EphA3 null mice with cardiac morphology, cushion explant migration assays, and ligand co-expression analysis","pmids":["17046737"],"confidence":"High","gaps":["Downstream signaling driving cushion EMT not dissected","Cell-autonomy of the requirement not established"]},{"year":2008,"claim":"How kinase activity is restrained was unknown; crystallography revealed juxtamembrane autoinhibition relieved by autophosphorylation through a coupled residue network.","evidence":"X-ray crystallography of multiple kinase-domain states plus juxtamembrane tyrosine mutagenesis","pmids":["18547520"],"confidence":"High","gaps":["Did not connect autoinhibition release to specific downstream substrates","Kinetics of the activation transition in cells unresolved"]},{"year":2009,"claim":"The receptor effector linking activation to migration suppression was unidentified; Nck1 was shown to bind phosphoY602 via its SH2 domain to mediate inhibition of migration and process retraction.","evidence":"Yeast two-hybrid, GST pulldown, Co-IP, Y602 mutagenesis, and dominant-negative Nck1 rescue with migration assays","pmids":["19505147"],"confidence":"High","gaps":["Only partial rescue indicates additional effectors","Connection between Nck1 and RhoA not directly traced"]},{"year":2010,"claim":"Whether a single docking site sufficed for cytoskeletal output was untested; Y602 and Y779 were shown to act collaboratively, with full effect requiring both.","evidence":"Tyrosine-to-phenylalanine mutagenesis with migration, neurite outgrowth, and growth cone collapse assays across cell types","pmids":["20697431"],"confidence":"High","gaps":["Effectors binding Y779 not identified","Mechanism of cooperativity between the two sites unresolved"]},{"year":2012,"claim":"The disease relevance of EphA3 mutations was unclear; cancer-derived mutations were shown to impair kinase activity, ligand binding, or surface localization, with some acting as dominant negatives over wild-type tumor-suppressive signaling.","evidence":"Biochemical kinase, binding, and surface-localization assays on mutant panels plus AKT signaling, apoptosis, and xenograft assays","pmids":["22242939","22829656"],"confidence":"High","gaps":["Tumor-suppressor mechanism beyond AKT suppression incompletely defined","Context dependence of suppressor vs oncogenic roles unresolved"]},{"year":2014,"claim":"How EphA3 contributes to tumors via the microenvironment was unknown; agonist antibody activation of EphA3+ stromal cells was shown to drive contraction, segregation, and apoptosis, disrupting tumor stroma and vasculature.","evidence":"EphA3 agonist antibody on isolated stromal cells with contraction/apoptosis assays and in vivo xenograft treatment","pmids":["25125683"],"confidence":"Medium","gaps":["Single lab","Molecular signaling underlying stromal apoptosis not detailed"]},{"year":2015,"claim":"Whether receptor dimers exist before ligand binding was untested; live-cell FRET showed unliganded dimers stabilized by the SAM domain.","evidence":"Quantitative FRET dimerization assay with SAM-deletion mutants on live cells","pmids":["26232493"],"confidence":"High","gaps":["Functional consequence of pre-formed dimers for signaling output not established","SAM-mediated interface not structurally resolved"]},{"year":2018,"claim":"A surface mechanism restraining EphA3 clustering was unknown; neurocan was shown to block NCAM/EphA3 complex formation by binding the EphA3-binding site on NCAM Ig2, inhibiting kinase activation and growth cone collapse.","evidence":"Co-IP, NCAM Ig-domain binding-site mapping, clustering imaging, kinase activation, and growth cone collapse assays","pmids":["29670169"],"confidence":"High","gaps":["Whether neurocan regulation operates outside cortical interneurons untested"]},{"year":2018,"claim":"Transcriptional control of EphA3 was incompletely mapped; an androgen-receptor/SP1 complex was shown to bind the core promoter and drive androgen-stimulated expression.","evidence":"Promoter luciferase deletion, AR/SP1 perturbation, Co-IP, and ChIP at the EPHA3 promoter","pmids":["29917167"],"confidence":"High","gaps":["Downstream consequence of AR-driven EphA3 in prostate cancer not functionally tied to signaling"]},{"year":2019,"claim":"A ligand-independent signaling output of EphA3 was undefined; presenilin/γ-secretase cleavage was shown to release an ICD that binds NMIIA, raises its Ser1943 phosphorylation, and promotes axon elongation while inhibiting RhoA.","evidence":"Genetic rescue in PS1/EphA3-deficient neurons, ICD interactome proteomics, NMIIA phosphorylation assays, and pharmacological NMII inhibition","pmids":["31577226"],"confidence":"High","gaps":["Trigger for cleavage in vivo not defined","Balance between full-length signaling and ICD function unresolved"]},{"year":null,"claim":"How the receptor's context-dependent switch between tumor-suppressive full-length signaling, microenvironmental effects, and ligand-independent ICD functions is coordinated at the molecular level remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model integrating full-length kinase signaling, cis-silencing, and ICD output","Effectors downstream of Y779 not identified","Mechanistic basis of EZH2/DNMT1-mediated PTEN regulation remains correlative"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[6,9]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,6]},{"term_id":"GO:0001618","term_label":"virus receptor activity","supporting_discovery_ids":[1,3]},{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[2,7,8]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[7]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[4,9,13]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[2,7,8]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[4,5,14]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[9,10,22]}],"complexes":["NCAM/EphA3 complex"],"partners":["EFNA5","CRK","NCK1","MYH9","NCAM1","PSEN1","EFNA1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P29320","full_name":"Ephrin type-A receptor 3","aliases":["EPH-like kinase 4","EK4","hEK4","HEK","Human embryo kinase","Tyrosine-protein kinase TYRO4","Tyrosine-protein kinase receptor ETK1","Eph-like tyrosine kinase 1"],"length_aa":983,"mass_kda":110.1,"function":"Receptor tyrosine kinase which binds promiscuously membrane-bound ephrin family ligands residing on adjacent cells, leading to contact-dependent bidirectional signaling into neighboring cells. The signaling pathway downstream of the receptor is referred to as forward signaling while the signaling pathway downstream of the ephrin ligand is referred to as reverse signaling (PubMed:11870224, PubMed:12794130). Highly promiscuous for ephrin-A ligands it binds preferentially EFNA5 (By similarity). Upon activation by EFNA5 regulates cell-cell adhesion, cytoskeletal organization and cell migration (PubMed:11870224). Also activated by EFNA1, inhibiting epithelial-to-mesenchymal transition of cardiac cells and playing a role in heart development (PubMed:12794130). Involved in the retinotectal mapping of neurons. May also control the segregation but not the guidance of motor and sensory axons during neuromuscular circuit development (By similarity)","subcellular_location":"Secreted","url":"https://www.uniprot.org/uniprotkb/P29320/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/EPHA3","classification":"Not Classified","n_dependent_lines":5,"n_total_lines":1208,"dependency_fraction":0.0041390728476821195},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/EPHA3","total_profiled":1310},"omim":[{"mim_id":"610328","title":"RUN AND FYVE DOMAINS-CONTAINING PROTEIN 2; RUFY2","url":"https://www.omim.org/entry/610328"},{"mim_id":"610327","title":"RUN AND FYVE DOMAINS-CONTAINING PROTEIN 1; RUFY1","url":"https://www.omim.org/entry/610327"},{"mim_id":"608892","title":"CHROMODOMAIN HELICASE DNA-BINDING PROTEIN 7; CHD7","url":"https://www.omim.org/entry/608892"},{"mim_id":"602192","title":"A DISINTEGRIN AND METALLOPROTEINASE DOMAIN 10; ADAM10","url":"https://www.omim.org/entry/602192"},{"mim_id":"602190","title":"EPHRIN RECEPTOR EphA7; EPHA7","url":"https://www.omim.org/entry/602190"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nuclear membrane","reliability":"Supported"},{"location":"Plasma membrane","reliability":"Supported"},{"location":"Cytosol","reliability":"Supported"},{"location":"Nucleoplasm","reliability":"Additional"},{"location":"Actin filaments","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"prostate","ntpm":29.5}],"url":"https://www.proteinatlas.org/search/EPHA3"},"hgnc":{"alias_symbol":["HEK","HEK4"],"prev_symbol":["ETK","ETK1","TYRO4"]},"alphafold":{"accession":"P29320","domains":[{"cath_id":"2.60.120.260","chopping":"28-199","consensus_level":"high","plddt":88.7751,"start":28,"end":199},{"cath_id":"2.60.40.1770","chopping":"204-259","consensus_level":"medium","plddt":90.5427,"start":204,"end":259},{"cath_id":"2.60.40.10","chopping":"329-430","consensus_level":"high","plddt":91.0727,"start":329,"end":430},{"cath_id":"2.60.40.10","chopping":"442-527","consensus_level":"high","plddt":90.0335,"start":442,"end":527},{"cath_id":"3.30.200.20","chopping":"598-699","consensus_level":"high","plddt":84.7519,"start":598,"end":699},{"cath_id":"1.10.510.10","chopping":"706-887","consensus_level":"high","plddt":86.9299,"start":706,"end":887},{"cath_id":"1.10.150.50","chopping":"915-976","consensus_level":"medium","plddt":52.0382,"start":915,"end":976}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P29320","model_url":"https://alphafold.ebi.ac.uk/files/AF-P29320-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P29320-F1-predicted_aligned_error_v6.png","plddt_mean":80.75},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=EPHA3","jax_strain_url":"https://www.jax.org/strain/search?query=EPHA3"},"sequence":{"accession":"P29320","fasta_url":"https://rest.uniprot.org/uniprotkb/P29320.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P29320/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P29320"}},"corpus_meta":[{"pmid":"8987735","id":"PMC_8987735","title":"Protein kinase C activation regulates human serotonin transporters in HEK-293 cells via altered cell surface expression.","date":"1997","source":"The Journal of neuroscience : the official journal of the Society for Neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/8987735","citation_count":307,"is_preprint":false},{"pmid":"23410976","id":"PMC_23410976","title":"EphA3 maintains tumorigenicity and is a therapeutic target in glioblastoma multiforme.","date":"2013","source":"Cancer cell","url":"https://pubmed.ncbi.nlm.nih.gov/23410976","citation_count":175,"is_preprint":false},{"pmid":"16491080","id":"PMC_16491080","title":"Silencing of EphA3 through a cis interaction with ephrinA5.","date":"2006","source":"Nature neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/16491080","citation_count":155,"is_preprint":false},{"pmid":"11870224","id":"PMC_11870224","title":"Ephrin-A5 induces rounding, blebbing and de-adhesion of EphA3-expressing 293T and melanoma cells by CrkII and Rho-mediated signalling.","date":"2002","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/11870224","citation_count":149,"is_preprint":false},{"pmid":"9556622","id":"PMC_9556622","title":"Comparison of Na-K-Cl cotransporters. NKCC1, NKCC2, and the HEK cell Na-L-Cl cotransporter.","date":"1998","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/9556622","citation_count":110,"is_preprint":false},{"pmid":"30176744","id":"PMC_30176744","title":"Nose-to-brain delivery of temozolomide-loaded PLGA nanoparticles functionalized with anti-EPHA3 for glioblastoma targeting.","date":"2018","source":"Drug delivery","url":"https://pubmed.ncbi.nlm.nih.gov/30176744","citation_count":92,"is_preprint":false},{"pmid":"9685371","id":"PMC_9685371","title":"Distinct subdomains of the EphA3 receptor mediate ligand binding and receptor dimerization.","date":"1998","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/9685371","citation_count":86,"is_preprint":false},{"pmid":"7838529","id":"PMC_7838529","title":"Ligands for the receptor tyrosine kinases hek and elk: isolation of cDNAs encoding a family of proteins.","date":"1995","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/7838529","citation_count":83,"is_preprint":false},{"pmid":"14660665","id":"PMC_14660665","title":"Dissecting the EphA3/Ephrin-A5 interactions using a novel functional mutagenesis screen.","date":"2003","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/14660665","citation_count":77,"is_preprint":false},{"pmid":"25125683","id":"PMC_25125683","title":"Targeting EphA3 inhibits cancer growth by disrupting the tumor stromal microenvironment.","date":"2014","source":"Cancer research","url":"https://pubmed.ncbi.nlm.nih.gov/25125683","citation_count":67,"is_preprint":false},{"pmid":"15569306","id":"PMC_15569306","title":"D1 dopamine receptor signaling involves caveolin-2 in HEK-293 cells.","date":"2004","source":"Kidney international","url":"https://pubmed.ncbi.nlm.nih.gov/15569306","citation_count":66,"is_preprint":false},{"pmid":"16061656","id":"PMC_16061656","title":"Concurrent binding of anti-EphA3 antibody and ephrin-A5 amplifies EphA3 signaling and downstream responses: potential as EphA3-specific tumor-targeting reagents.","date":"2005","source":"Cancer research","url":"https://pubmed.ncbi.nlm.nih.gov/16061656","citation_count":65,"is_preprint":false},{"pmid":"10498621","id":"PMC_10498621","title":"Cloning and characterization of EphA3 (Hek) gene promoter: DNA methylation regulates expression in hematopoietic tumor cells.","date":"1999","source":"Blood","url":"https://pubmed.ncbi.nlm.nih.gov/10498621","citation_count":65,"is_preprint":false},{"pmid":"8397371","id":"PMC_8397371","title":"PCR mediated detection of a new human receptor-tyrosine-kinase, HEK 2.","date":"1993","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/8397371","citation_count":64,"is_preprint":false},{"pmid":"16941478","id":"PMC_16941478","title":"Somatic mutations of GUCY2F, EPHA3, and NTRK3 in human cancers.","date":"2006","source":"Human mutation","url":"https://pubmed.ncbi.nlm.nih.gov/16941478","citation_count":63,"is_preprint":false},{"pmid":"17046737","id":"PMC_17046737","title":"A critical role for the EphA3 receptor tyrosine kinase in heart development.","date":"2006","source":"Developmental biology","url":"https://pubmed.ncbi.nlm.nih.gov/17046737","citation_count":60,"is_preprint":false},{"pmid":"12970074","id":"PMC_12970074","title":"Protein kinase C-mediated Ca2+ entry in HEK 293 cells transiently expressing human TRPV4.","date":"2003","source":"British journal of pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/12970074","citation_count":59,"is_preprint":false},{"pmid":"33417456","id":"PMC_33417456","title":"Intranasal Delivery of Temozolomide-Conjugated Gold Nanoparticles Functionalized with Anti-EphA3 for Glioblastoma Targeting.","date":"2021","source":"Molecular pharmaceutics","url":"https://pubmed.ncbi.nlm.nih.gov/33417456","citation_count":57,"is_preprint":false},{"pmid":"22242939","id":"PMC_22242939","title":"Cancer somatic mutations disrupt functions of the EphA3 receptor tyrosine kinase through multiple mechanisms.","date":"2012","source":"Biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/22242939","citation_count":54,"is_preprint":false},{"pmid":"22829656","id":"PMC_22829656","title":"Effects of cancer-associated EPHA3 mutations on lung cancer.","date":"2012","source":"Journal of the National Cancer Institute","url":"https://pubmed.ncbi.nlm.nih.gov/22829656","citation_count":53,"is_preprint":false},{"pmid":"18547520","id":"PMC_18547520","title":"Autoregulation by the juxtamembrane region of the human ephrin receptor tyrosine kinase A3 (EphA3).","date":"2008","source":"Structure (London, England : 1993)","url":"https://pubmed.ncbi.nlm.nih.gov/18547520","citation_count":53,"is_preprint":false},{"pmid":"25391995","id":"PMC_25391995","title":"EphA3 biology and cancer.","date":"2014","source":"Growth factors (Chur, Switzerland)","url":"https://pubmed.ncbi.nlm.nih.gov/25391995","citation_count":52,"is_preprint":false},{"pmid":"7615078","id":"PMC_7615078","title":"G protein beta gamma subunit activates Ras, Raf, and MAP kinase in HEK 293 cells.","date":"1995","source":"FEBS letters","url":"https://pubmed.ncbi.nlm.nih.gov/7615078","citation_count":51,"is_preprint":false},{"pmid":"24696141","id":"PMC_24696141","title":"PACAP-induced ERK activation in HEK cells expressing PAC1 receptors involves both receptor internalization and PKC signaling.","date":"2014","source":"American journal of physiology. Cell physiology","url":"https://pubmed.ncbi.nlm.nih.gov/24696141","citation_count":48,"is_preprint":false},{"pmid":"13129920","id":"PMC_13129920","title":"Expression of Drosophila trehalose-phosphate synthase in HEK-293 cells increases hypoxia tolerance.","date":"2003","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/13129920","citation_count":47,"is_preprint":false},{"pmid":"15979128","id":"PMC_15979128","title":"Endogenous channels in HEK cells and potential roles in HCN ionic current measurements.","date":"2005","source":"Progress in biophysics and molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/15979128","citation_count":45,"is_preprint":false},{"pmid":"22350700","id":"PMC_22350700","title":"Aberrant expression of EphA3 in gastric carcinoma: correlation with tumor angiogenesis and survival.","date":"2012","source":"Journal of gastroenterology","url":"https://pubmed.ncbi.nlm.nih.gov/22350700","citation_count":44,"is_preprint":false},{"pmid":"26232493","id":"PMC_26232493","title":"Unliganded EphA3 dimerization promoted by the SAM domain.","date":"2015","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/26232493","citation_count":44,"is_preprint":false},{"pmid":"25847125","id":"PMC_25847125","title":"DNA damage and S phase arrest induced by Ochratoxin A in human embryonic kidney cells (HEK 293).","date":"2014","source":"Mutation research","url":"https://pubmed.ncbi.nlm.nih.gov/25847125","citation_count":43,"is_preprint":false},{"pmid":"29888905","id":"PMC_29888905","title":"N-Glycosylation of Extracellular Vesicles from HEK-293 and Glioma Cell Lines.","date":"2018","source":"Analytical chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/29888905","citation_count":42,"is_preprint":false},{"pmid":"14697337","id":"PMC_14697337","title":"EphA3 is induced by CD28 and IGF-1 and regulates cell adhesion.","date":"2004","source":"Experimental cell research","url":"https://pubmed.ncbi.nlm.nih.gov/14697337","citation_count":40,"is_preprint":false},{"pmid":"16083359","id":"PMC_16083359","title":"Upregulation of EphA3 receptor after spinal cord injury.","date":"2005","source":"Journal of neurotrauma","url":"https://pubmed.ncbi.nlm.nih.gov/16083359","citation_count":38,"is_preprint":false},{"pmid":"15294877","id":"PMC_15294877","title":"Functional expression and cellular distribution of diastrophic dysplasia sulfate transporter (DTDST) gene mutations in HEK cells.","date":"2004","source":"Human molecular genetics","url":"https://pubmed.ncbi.nlm.nih.gov/15294877","citation_count":38,"is_preprint":false},{"pmid":"27101199","id":"PMC_27101199","title":"EPHA3 regulates the multidrug resistance of small cell lung cancer via the PI3K/BMX/STAT3 signaling pathway.","date":"2016","source":"Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine","url":"https://pubmed.ncbi.nlm.nih.gov/27101199","citation_count":37,"is_preprint":false},{"pmid":"23892143","id":"PMC_23892143","title":"Human respiratory syncytial virus N, P and M protein interactions in HEK-293T cells.","date":"2013","source":"Virus research","url":"https://pubmed.ncbi.nlm.nih.gov/23892143","citation_count":36,"is_preprint":false},{"pmid":"27991764","id":"PMC_27991764","title":"High-Throughput Lipidomic and Transcriptomic Analysis To Compare SP2/0, CHO, and HEK-293 Mammalian Cell Lines.","date":"2017","source":"Analytical chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/27991764","citation_count":35,"is_preprint":false},{"pmid":"21464992","id":"PMC_21464992","title":"Phosphorylation and transport in the Na-K-2Cl cotransporters, NKCC1 and NKCC2A, compared in HEK-293 cells.","date":"2011","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/21464992","citation_count":35,"is_preprint":false},{"pmid":"18814179","id":"PMC_18814179","title":"The EphA3 receptor is expressed in a subset of rhabdomyosarcoma cell lines and suppresses cell adhesion and migration.","date":"2008","source":"Journal of cellular biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/18814179","citation_count":32,"is_preprint":false},{"pmid":"29670169","id":"PMC_29670169","title":"Perineuronal Net Protein Neurocan Inhibits NCAM/EphA3 Repellent Signaling in GABAergic Interneurons.","date":"2018","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/29670169","citation_count":31,"is_preprint":false},{"pmid":"32621117","id":"PMC_32621117","title":"Critical role of EphA3 in cancer and current state of EphA3 drug therapeutics.","date":"2020","source":"Molecular biology reports","url":"https://pubmed.ncbi.nlm.nih.gov/32621117","citation_count":30,"is_preprint":false},{"pmid":"17241243","id":"PMC_17241243","title":"The Hek outer membrane protein of Escherichia coli is an auto-aggregating adhesin and invasin.","date":"2007","source":"FEMS microbiology letters","url":"https://pubmed.ncbi.nlm.nih.gov/17241243","citation_count":30,"is_preprint":false},{"pmid":"27922598","id":"PMC_27922598","title":"EphA3 as a target for antibody immunotherapy in acute lymphoblastic leukemia.","date":"2016","source":"Leukemia","url":"https://pubmed.ncbi.nlm.nih.gov/27922598","citation_count":29,"is_preprint":false},{"pmid":"21454190","id":"PMC_21454190","title":"Copy number variations of EphA3 are associated with multiple types of hematologic malignancies.","date":"2011","source":"Clinical lymphoma, myeloma & leukemia","url":"https://pubmed.ncbi.nlm.nih.gov/21454190","citation_count":29,"is_preprint":false},{"pmid":"32180717","id":"PMC_32180717","title":"ZnO Quantum Dots Induced Oxidative Stress and Apoptosis in HeLa and HEK-293T Cell Lines.","date":"2020","source":"Frontiers in pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/32180717","citation_count":29,"is_preprint":false},{"pmid":"35567642","id":"PMC_35567642","title":"Endogenous ion channels expressed in human embryonic kidney (HEK-293) cells.","date":"2022","source":"Pflugers Archiv : European journal of physiology","url":"https://pubmed.ncbi.nlm.nih.gov/35567642","citation_count":28,"is_preprint":false},{"pmid":"30562956","id":"PMC_30562956","title":"EphA3 Pay-Loaded Antibody Therapeutics for the Treatment of Glioblastoma.","date":"2018","source":"Cancers","url":"https://pubmed.ncbi.nlm.nih.gov/30562956","citation_count":28,"is_preprint":false},{"pmid":"28198671","id":"PMC_28198671","title":"Transcriptome analysis reveals dynamic changes in coxsackievirus A16 infected HEK 293T cells.","date":"2017","source":"BMC genomics","url":"https://pubmed.ncbi.nlm.nih.gov/28198671","citation_count":28,"is_preprint":false},{"pmid":"31358407","id":"PMC_31358407","title":"Characterization of influenza H1N1 Gag virus-like particles and extracellular vesicles co-produced in HEK-293SF.","date":"2019","source":"Vaccine","url":"https://pubmed.ncbi.nlm.nih.gov/31358407","citation_count":27,"is_preprint":false},{"pmid":"19505147","id":"PMC_19505147","title":"Regulation of process retraction and cell migration by EphA3 is mediated by the adaptor protein Nck1.","date":"2009","source":"Biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/19505147","citation_count":26,"is_preprint":false},{"pmid":"19649722","id":"PMC_19649722","title":"The time course of Akt and ERK activation on XIAP expression in HEK 293 cell line.","date":"2009","source":"Molecular biology reports","url":"https://pubmed.ncbi.nlm.nih.gov/19649722","citation_count":24,"is_preprint":false},{"pmid":"27313717","id":"PMC_27313717","title":"Chelerythrine chloride induces apoptosis in renal cancer HEK-293 and SW-839 cell lines.","date":"2016","source":"Oncology letters","url":"https://pubmed.ncbi.nlm.nih.gov/27313717","citation_count":23,"is_preprint":false},{"pmid":"22780212","id":"PMC_22780212","title":"EPHA3 as a novel therapeutic target in the hematological malignancies.","date":"2012","source":"Expert review of hematology","url":"https://pubmed.ncbi.nlm.nih.gov/22780212","citation_count":22,"is_preprint":false},{"pmid":"25683738","id":"PMC_25683738","title":"Transcriptional and post-transcriptional limitations of high-yielding, PEI-mediated transient transfection with CHO and HEK-293E cells.","date":"2015","source":"Biotechnology progress","url":"https://pubmed.ncbi.nlm.nih.gov/25683738","citation_count":22,"is_preprint":false},{"pmid":"20697431","id":"PMC_20697431","title":"EphA3 functions are regulated by collaborating phosphotyrosine residues.","date":"2010","source":"Cell research","url":"https://pubmed.ncbi.nlm.nih.gov/20697431","citation_count":21,"is_preprint":false},{"pmid":"36742140","id":"PMC_36742140","title":"Effect of trans-sodium crocetinate on contrast-induced cytotoxicity in HEK-293 cells.","date":"2023","source":"Iranian journal of basic medical sciences","url":"https://pubmed.ncbi.nlm.nih.gov/36742140","citation_count":21,"is_preprint":false},{"pmid":"25231727","id":"PMC_25231727","title":"EphA3, induced by PC-1/PrLZ, contributes to the malignant progression of prostate cancer.","date":"2014","source":"Oncology reports","url":"https://pubmed.ncbi.nlm.nih.gov/25231727","citation_count":20,"is_preprint":false},{"pmid":"35774066","id":"PMC_35774066","title":"Production of Lentiviral Vectors Using a HEK-293 Producer Cell Line and Advanced Perfusion Processing.","date":"2022","source":"Frontiers in bioengineering and biotechnology","url":"https://pubmed.ncbi.nlm.nih.gov/35774066","citation_count":20,"is_preprint":false},{"pmid":"37435968","id":"PMC_37435968","title":"Peptide ligands for the affinity purification of adeno-associated viruses from HEK 293 cell lysates.","date":"2023","source":"Biotechnology and bioengineering","url":"https://pubmed.ncbi.nlm.nih.gov/37435968","citation_count":20,"is_preprint":false},{"pmid":"20592104","id":"PMC_20592104","title":"Biotin regulates the expression of holocarboxylase synthetase in the miR-539 pathway in HEK-293 cells.","date":"2010","source":"The Journal of nutrition","url":"https://pubmed.ncbi.nlm.nih.gov/20592104","citation_count":20,"is_preprint":false},{"pmid":"17097906","id":"PMC_17097906","title":"Transcriptional profiling of batch and fed-batch protein-free 293-HEK cultures.","date":"2006","source":"Metabolic engineering","url":"https://pubmed.ncbi.nlm.nih.gov/17097906","citation_count":20,"is_preprint":false},{"pmid":"37445652","id":"PMC_37445652","title":"LRRK2 Knockout Confers Resistance in HEK-293 Cells to Rotenone-Induced Oxidative Stress, Mitochondrial Damage, and Apoptosis.","date":"2023","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/37445652","citation_count":20,"is_preprint":false},{"pmid":"30346389","id":"PMC_30346389","title":"Rapid Isolation of the Mitoribosome from HEK Cells.","date":"2018","source":"Journal of visualized experiments : JoVE","url":"https://pubmed.ncbi.nlm.nih.gov/30346389","citation_count":20,"is_preprint":false},{"pmid":"33945505","id":"PMC_33945505","title":"Antibody-mediated depletion of CCR10+EphA3+ cells ameliorates fibrosis in IPF.","date":"2021","source":"JCI insight","url":"https://pubmed.ncbi.nlm.nih.gov/33945505","citation_count":19,"is_preprint":false},{"pmid":"14585969","id":"PMC_14585969","title":"EphA3 null mutants do not demonstrate motor axon guidance defects.","date":"2003","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/14585969","citation_count":19,"is_preprint":false},{"pmid":"31262977","id":"PMC_31262977","title":"EPHA3 enhances macrophage autophagy and apoptosis by disrupting the mTOR signaling pathway in mice with endometriosis.","date":"2019","source":"Bioscience reports","url":"https://pubmed.ncbi.nlm.nih.gov/31262977","citation_count":18,"is_preprint":false},{"pmid":"31821898","id":"PMC_31821898","title":"Mitochondrial mRNA fragments are circularized in a human HEK cell line.","date":"2019","source":"Mitochondrion","url":"https://pubmed.ncbi.nlm.nih.gov/31821898","citation_count":18,"is_preprint":false},{"pmid":"39111833","id":"PMC_39111833","title":"EphA3-targeted chimeric antigen receptor T cells are effective in glioma and generate curative memory T cell responses.","date":"2024","source":"Journal for immunotherapy of cancer","url":"https://pubmed.ncbi.nlm.nih.gov/39111833","citation_count":17,"is_preprint":false},{"pmid":"30066881","id":"PMC_30066881","title":"EphA3 contributes to tumor growth and angiogenesis in human gastric cancer cells.","date":"2018","source":"Oncology reports","url":"https://pubmed.ncbi.nlm.nih.gov/30066881","citation_count":17,"is_preprint":false},{"pmid":"27721017","id":"PMC_27721017","title":"EphA3 promotes malignant transformation of colorectal epithelial cells by upregulating oncogenic pathways.","date":"2016","source":"Cancer letters","url":"https://pubmed.ncbi.nlm.nih.gov/27721017","citation_count":17,"is_preprint":false},{"pmid":"23324396","id":"PMC_23324396","title":"A high-content cellular senescence screen identifies candidate tumor suppressors, including EPHA3.","date":"2013","source":"Cell cycle (Georgetown, Tex.)","url":"https://pubmed.ncbi.nlm.nih.gov/23324396","citation_count":17,"is_preprint":false},{"pmid":"28415715","id":"PMC_28415715","title":"Epha3 acts as proangiogenic factor in multiple myeloma.","date":"2017","source":"Oncotarget","url":"https://pubmed.ncbi.nlm.nih.gov/28415715","citation_count":16,"is_preprint":false},{"pmid":"29660208","id":"PMC_29660208","title":"MiR-340 suppresses the metastasis by targeting EphA3 in cervical cancer.","date":"2018","source":"Cell biology international","url":"https://pubmed.ncbi.nlm.nih.gov/29660208","citation_count":16,"is_preprint":false},{"pmid":"33919657","id":"PMC_33919657","title":"EPHA3 Contributes to Epigenetic Suppression of PTEN in Radioresistant Head and Neck Cancer.","date":"2021","source":"Biomolecules","url":"https://pubmed.ncbi.nlm.nih.gov/33919657","citation_count":15,"is_preprint":false},{"pmid":"25978371","id":"PMC_25978371","title":"Antitumor Effects of Oncolytic Adenovirus-Carrying siRNA Targeting Potential Oncogene EphA3.","date":"2015","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/25978371","citation_count":15,"is_preprint":false},{"pmid":"9458884","id":"PMC_9458884","title":"IL-1 beta alters the expression of the receptor tyrosine kinase gene r-EphA3 in neonatal rat cardiomyocytes.","date":"1998","source":"The American journal of physiology","url":"https://pubmed.ncbi.nlm.nih.gov/9458884","citation_count":15,"is_preprint":false},{"pmid":"34446852","id":"PMC_34446852","title":"Signaling profiles in HEK 293T cells co-expressing GLP-1 and GIP receptors.","date":"2021","source":"Acta pharmacologica Sinica","url":"https://pubmed.ncbi.nlm.nih.gov/34446852","citation_count":15,"is_preprint":false},{"pmid":"31577226","id":"PMC_31577226","title":"Presenilin/γ-secretase-dependent EphA3 processing mediates axon elongation through non-muscle myosin IIA.","date":"2019","source":"eLife","url":"https://pubmed.ncbi.nlm.nih.gov/31577226","citation_count":14,"is_preprint":false},{"pmid":"39111832","id":"PMC_39111832","title":"EphA3 CAR T cells are effective against glioblastoma in preclinical models.","date":"2024","source":"Journal for immunotherapy of cancer","url":"https://pubmed.ncbi.nlm.nih.gov/39111832","citation_count":14,"is_preprint":false},{"pmid":"33493920","id":"PMC_33493920","title":"Designing and evaluation of MERS-CoV siRNAs in HEK-293 cell line.","date":"2020","source":"Journal of infection and public health","url":"https://pubmed.ncbi.nlm.nih.gov/33493920","citation_count":14,"is_preprint":false},{"pmid":"18674612","id":"PMC_18674612","title":"TRP-2 expression protects HEK cells from dopamine- and hydroquinone-induced toxicity.","date":"2008","source":"Free radical biology & medicine","url":"https://pubmed.ncbi.nlm.nih.gov/18674612","citation_count":14,"is_preprint":false},{"pmid":"25420155","id":"PMC_25420155","title":"Hypoxia-controlled EphA3 marks a human endometrium-derived multipotent mesenchymal stromal cell that supports vascular growth.","date":"2014","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/25420155","citation_count":14,"is_preprint":false},{"pmid":"29917167","id":"PMC_29917167","title":"Androgen receptor induces EPHA3 expression by interacting with transcription factor SP1.","date":"2018","source":"Oncology reports","url":"https://pubmed.ncbi.nlm.nih.gov/29917167","citation_count":13,"is_preprint":false},{"pmid":"30483759","id":"PMC_30483759","title":"EphA3 inhibits migration and invasion of esophageal cancer cells by activating the mesenchymal‑epithelial transition process.","date":"2018","source":"International journal of oncology","url":"https://pubmed.ncbi.nlm.nih.gov/30483759","citation_count":13,"is_preprint":false},{"pmid":"36449077","id":"PMC_36449077","title":"Apolipoprotein L1 (APOL1) cation current in HEK-293 cells and in human podocytes.","date":"2022","source":"Pflugers Archiv : European journal of physiology","url":"https://pubmed.ncbi.nlm.nih.gov/36449077","citation_count":13,"is_preprint":false},{"pmid":"17479408","id":"PMC_17479408","title":"JWA gene is involved in cadmium-induced growth inhibition and apoptosis in HEK-293T cells.","date":"2007","source":"Journal of toxicology and environmental health. Part A","url":"https://pubmed.ncbi.nlm.nih.gov/17479408","citation_count":13,"is_preprint":false},{"pmid":"19820835","id":"PMC_19820835","title":"Infection kinetics of human adenovirus serotype 41 in HEK 293 cells.","date":"2009","source":"Memorias do Instituto Oswaldo Cruz","url":"https://pubmed.ncbi.nlm.nih.gov/19820835","citation_count":13,"is_preprint":false},{"pmid":"29653204","id":"PMC_29653204","title":"EphA3 maintains radioresistance in head and neck cancers through epithelial mesenchymal transition.","date":"2018","source":"Cellular signalling","url":"https://pubmed.ncbi.nlm.nih.gov/29653204","citation_count":12,"is_preprint":false},{"pmid":"28721629","id":"PMC_28721629","title":"EphA3 targeting reduces in vitro adhesion and invasion and in vivo growth and angiogenesis of multiple myeloma cells.","date":"2017","source":"Cellular oncology (Dordrecht, Netherlands)","url":"https://pubmed.ncbi.nlm.nih.gov/28721629","citation_count":12,"is_preprint":false},{"pmid":"21864196","id":"PMC_21864196","title":"Reporter gene HEK 293 cells and WNT/Frizzled fusion proteins as tools to study WNT signaling pathways.","date":"2011","source":"Biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/21864196","citation_count":12,"is_preprint":false},{"pmid":"11029633","id":"PMC_11029633","title":"Graded expression of EphA3 in the retina and ephrin-A2 in the superior colliculus during initial development of coarse topography in the wallaby retinocollicular projection.","date":"2000","source":"The European journal of neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/11029633","citation_count":11,"is_preprint":false},{"pmid":"25713296","id":"PMC_25713296","title":"The putative tumor suppressor gene EphA3 fails to demonstrate a crucial role in murine lung tumorigenesis or morphogenesis.","date":"2015","source":"Disease models & mechanisms","url":"https://pubmed.ncbi.nlm.nih.gov/25713296","citation_count":11,"is_preprint":false},{"pmid":"25413948","id":"PMC_25413948","title":"A high affinity recombinant antibody to the human EphA3 receptor with enhanced ADCC activity.","date":"2014","source":"Growth factors (Chur, Switzerland)","url":"https://pubmed.ncbi.nlm.nih.gov/25413948","citation_count":11,"is_preprint":false},{"pmid":"37118847","id":"PMC_37118847","title":"Circular RNA EPHA3 suppresses progression and metastasis in prostate cancer through the miR-513a-3p/BMP2 axis.","date":"2023","source":"Journal of translational medicine","url":"https://pubmed.ncbi.nlm.nih.gov/37118847","citation_count":10,"is_preprint":false},{"pmid":"37760615","id":"PMC_37760615","title":"Inhibition of EphA3 Expression in Tumour Stromal Cells Suppresses Tumour Growth and Progression.","date":"2023","source":"Cancers","url":"https://pubmed.ncbi.nlm.nih.gov/37760615","citation_count":10,"is_preprint":false},{"pmid":"28169277","id":"PMC_28169277","title":"Investigation of the role of tyrosine kinase receptor EPHA3 in colorectal cancer.","date":"2017","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/28169277","citation_count":10,"is_preprint":false},{"pmid":"37434266","id":"PMC_37434266","title":"The Ephrin tyrosine kinase a3 (EphA3) is a novel mediator of RAGE-prompted motility of breast cancer cells.","date":"2023","source":"Journal of experimental & clinical cancer research : CR","url":"https://pubmed.ncbi.nlm.nih.gov/37434266","citation_count":10,"is_preprint":false},{"pmid":"31433759","id":"PMC_31433759","title":"Sam68 Promotes the Progression of Human Breast Cancer through inducing Activation of EphA3.","date":"2020","source":"Current cancer drug targets","url":"https://pubmed.ncbi.nlm.nih.gov/31433759","citation_count":10,"is_preprint":false},{"pmid":"29091272","id":"PMC_29091272","title":"Expression and Purification of Protein Complexes Suitable for Structural Studies Using Mammalian HEK 293F Cells.","date":"2017","source":"Current protocols in protein science","url":"https://pubmed.ncbi.nlm.nih.gov/29091272","citation_count":10,"is_preprint":false},{"pmid":"35510320","id":"PMC_35510320","title":"Thapsigargin blocks electromagnetic field-elicited intracellular Ca2+ increase in HEK 293 cells.","date":"2022","source":"Physiological reports","url":"https://pubmed.ncbi.nlm.nih.gov/35510320","citation_count":10,"is_preprint":false},{"pmid":"8188238","id":"PMC_8188238","title":"Localization of a human receptor tyrosine kinase (ETK1) to chromosome region 3p11.2.","date":"1994","source":"Genomics","url":"https://pubmed.ncbi.nlm.nih.gov/8188238","citation_count":9,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":49735,"output_tokens":7029,"usd":0.12732,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":16238,"output_tokens":5017,"usd":0.103307,"stage2_stop_reason":"end_turn"},"total_usd":0.230627,"stage1_batch_id":"msgbatch_01AsR7g8dGTEiEw9U83bJeWn","stage2_batch_id":"msgbatch_01YV5ZyxtFNUZEjxRcpd9jzb","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1998,\n      \"finding\": \"EphA3 extracellular region contains distinct functional subdomains: ligand binding is restricted to the N-terminal region encoded by exon III, while an independent C-terminal domain mediates receptor dimerization/oligomerization. A two-step activation mechanism was proposed involving sequential ligand binding and ligand-independent receptor-receptor oligomerization.\",\n      \"method\": \"BIAcore kinetic binding analysis, mRNA injection into zebrafish embryos, receptor transphosphorylation analysis using recombinant subdomain proteins\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple orthogonal methods (BIAcore, functional zebrafish assay, transphosphorylation) in a single rigorous study clearly mapping two distinct extracellular subdomains\",\n      \"pmids\": [\"9685371\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"EphA3 (hek) binds GPI-anchored membrane proteins (ephrin-A ligands) with high affinity (Kd ~2×10^8 M^-1); two related ephrin ligands were isolated from a human T-lymphoma line that bind both EphA3 and the related ELK receptor tyrosine kinase.\",\n      \"method\": \"Expression cloning using soluble EphA3-Fc fusion protein as affinity probe; binding affinity measured by Fc-fusion pulldown\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct biochemical isolation of ligands using soluble receptor fusion protein, affinity constants determined, replicated across multiple ligands\",\n      \"pmids\": [\"7838529\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Ephrin-A5 stimulation of EphA3 causes rapid RhoA activation, leading to actin/myosin cytoskeleton reorganization, membrane blebbing, retraction of cellular protrusions, and cell detachment. Receptor clustering (polymeric ephrin-A5) is required. The adaptor CrkII associates with tyrosine-phosphorylated EphA3 in vitro and is recruited in vivo upon ephrin-A5 stimulation; expression of SH3-domain-mutated CrkII abolishes cell rounding, blebbing, and detachment.\",\n      \"method\": \"Ephrin-A5 stimulation assays, RhoA activation assay, co-immunoprecipitation of CrkII with phosphorylated EphA3, dominant-negative CrkII expression, morphological assays in EphA3-expressing 293T and melanoma cells\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, dominant-negative rescue, RhoA activation assay, and morphological readouts in multiple cell lines; multiple orthogonal methods in one study\",\n      \"pmids\": [\"11870224\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Three distinct surface areas of EphA3 are essential for ephrin-A5 binding and assembly of signaling-competent oligomeric receptor-ligand complexes: two map to interfaces identified in the EphB2-ephrin-B2 crystal structure, and a third falls outside the structurally characterized interaction domains.\",\n      \"method\": \"Random mutagenesis of EphA3 in yeast expression library, selection for impaired ephrin-A5 binding, functional characterization of point mutants\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — systematic mutagenesis screen with functional validation of multiple mutants, defining molecular determinants of ligand binding\",\n      \"pmids\": [\"14660665\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"EphA3 forms a cis-interaction with ephrinA5 (on the same cell surface) that is independent of the established ligand-binding domain of EphA3. This cis-complex abolishes ephrinA5-induced tyrosine phosphorylation of EphA3 and renders retinal axons insensitive to ephrinA in trans, thereby converting uniform EphA3 expression into a functional gradient.\",\n      \"method\": \"Truncated EphA3 constructs, single-amino acid point mutants of ephrinA5, FRET (fluorescence resonance energy transfer) imaging, tyrosine phosphorylation assays, retinal axon sensitivity assays\",\n      \"journal\": \"Nature neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — FRET directly demonstrates physical cis-interaction; mutagenesis and functional axon assays confirm mechanistic consequence; multiple orthogonal methods\",\n      \"pmids\": [\"16491080\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"EphA3 signaling is required for atrioventricular endocardial cushion development and epithelial-to-mesenchymal transformation (EMT) in the developing heart. EphA3 knockout mice show hypoplastic endocardial cushions and reduced mesenchymal cell migration from cushion explants; EphA3 and its ligand ephrin-A1 are expressed in adjacent cells in the developing endocardial cushions.\",\n      \"method\": \"EphA3 null mouse generation, cardiac morphological analysis, endocardial cushion explant migration assay, expression analysis by in situ/immunostaining\",\n      \"journal\": \"Developmental biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean knockout with specific morphological and cellular phenotypes, ligand-receptor co-expression in adjacent cells confirmed, replicated across multiple readouts\",\n      \"pmids\": [\"17046737\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"The unphosphorylated juxtamembrane (JM) region of EphA3 inhibits kinase activity through an autoinhibitory mechanism: two tyrosines in the JM region are sites of autophosphorylation and interact with the kinase active site to modulate catalysis. Crystal structures of multiple states (with/without JM segment, wild-type and mutants) revealed a conserved pathway of coupled residues connecting the JM segment to the activation loop and catalytic residues.\",\n      \"method\": \"X-ray crystallography of EphA3 kinase domain with and without juxtamembrane segment; site-specific mutagenesis of JM tyrosines; structural comparison of multiple states\",\n      \"journal\": \"Structure\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — high-resolution crystal structures plus mutagenesis, multiple states characterized, clear mechanistic model established\",\n      \"pmids\": [\"18547520\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Adaptor protein Nck1 interacts with activated EphA3 through binding of the Nck1 SH2 domain to phosphotyrosine Y602 of EphA3. This interaction mediates EphA3-induced inhibition of cell migration and process retraction; dominant-negative Nck1 mutants lacking functional SH2 or SH3 domains partially alleviate the EphA3-mediated inhibitory effects.\",\n      \"method\": \"Yeast two-hybrid screen using EphA3 cytoplasmic domain as bait; GST pulldown; co-immunoprecipitation; Y602 point mutagenesis; dominant-negative Nck1 rescue experiments; cell migration and process retraction assays\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — yeast 2-hybrid confirmed by GST pulldown and Co-IP; mutagenesis defines exact binding site (Y602); functional rescue with dominant-negative validates mechanistic role\",\n      \"pmids\": [\"19505147\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"EphA3-mediated inhibition of cell migration, neurite outgrowth, and growth cone collapse requires collaboration between phosphotyrosine residues Y602 and Y779 in the cytoplasmic domain. Mutation of either residue alone partially decreases EphA3-induced responses; full abrogation requires mutation of both Y602 and Y779 together.\",\n      \"method\": \"Individual tyrosine-to-phenylalanine mutagenesis of EphA3 cytoplasmic domain residues; cell migration assay (293A cells); neurite outgrowth assay (NG108-15 cells); growth cone collapse assay (hippocampal neurons)\",\n      \"journal\": \"Cell research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — systematic mutagenesis with three independent functional assays in distinct cell types; collaborative model validated across multiple readouts\",\n      \"pmids\": [\"20697431\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Somatic cancer mutations in EphA3 impair kinase activity and/or ephrin ligand binding and/or decrease receptor cell surface localization through multiple distinct mechanisms, consistent with EphA3 having ephrin- and kinase-dependent tumor-suppressing activities disrupted by cancer mutations.\",\n      \"method\": \"Functional analysis of cancer-derived EphA3 point mutants: in vitro kinase assays, ephrin binding assays, cell surface biotinylation/flow cytometry\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple orthogonal biochemical assays (kinase activity, ligand binding, surface localization) applied systematically to a panel of cancer mutations\",\n      \"pmids\": [\"22242939\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"At least two cancer-associated EphA3 somatic mutations function as dominant inhibitors (dominant negative) of wild-type EphA3 protein. Wild-type EphA3 re-expression in lung cancer lines increases apoptosis by suppressing AKT activation in vitro and inhibits tumor xenograft growth in vivo; dominant-negative mutations override these tumor-suppressive effects in trans.\",\n      \"method\": \"Immunoprecipitation, western blotting, kinase assays; AKT phosphorylation analysis; BrdU proliferation and apoptosis assays; xenograft tumor growth assay\",\n      \"journal\": \"Journal of the National Cancer Institute\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — kinase assays plus in vitro signaling plus in vivo xenograft; dominant-negative mechanism validated across multiple methods\",\n      \"pmids\": [\"22829656\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"EphA3 maintains glioblastoma tumor-initiating cells in a less differentiated state by modulating MAPK signaling. EphA3 knockdown or depletion of EphA3-positive cells reduces tumorigenic potential.\",\n      \"method\": \"EphA3 knockdown (siRNA/shRNA), tumor sphere formation, MAPK signaling analysis, xenograft mouse model, radiolabeled EphA3 antibody depletion of EphA3+ cells\",\n      \"journal\": \"Cancer cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KD with specific cellular phenotype and MAPK pathway readout, validated in vivo; single lab but multiple methods\",\n      \"pmids\": [\"23410976\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"The anti-EphA3 monoclonal antibody IIIA4 binds a conformation-specific epitope within the ephrin-binding domain adjacent to the ephrin-A5 heterotetramerization site. Preclustered IIIA4 triggers EphA3 activation and cytoskeletal contraction similarly to ephrin-A5. Unclustered IIIA4 and ephrin-A5 Fc in combination initiate greatly enhanced EphA3 signaling through allosteric permissive conformational effects.\",\n      \"method\": \"BIAcore binding analysis, immunoblot for EphA3 phosphorylation, confocal microscopy, wild-type and binding-site mutant EphA3 analysis, cell contraction assays\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — BIAcore, mutagenesis, phosphorylation assays, and morphological readouts with both wild-type and binding-impaired mutants; multiple orthogonal approaches\",\n      \"pmids\": [\"16061656\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"EphA3 forms dimers in the absence of ligand binding on the surface of live cells, and these unliganded dimers are stabilized by interactions involving the EphA3 SAM (sterile α-motif) domain, establishing a new role for the SAM domain in promoting Eph receptor lateral interactions.\",\n      \"method\": \"Quantitative FRET-based dimerization assay on live cells, deletion mutants removing the SAM domain, comparison of wild-type vs. SAM-deleted EphA3\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — FRET on live cells with domain deletion mutants, directly demonstrating SAM-dependent dimerization; rigorous single-lab study with clear mechanistic conclusion\",\n      \"pmids\": [\"26232493\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Presenilin-1 (PS1)/γ-secretase cleaves EphA3 independently of ligand binding to generate an intracellular domain (ICD) fragment. EphA3 ICD promotes axon elongation by binding to non-muscle myosin IIA (NMIIA) and increasing its phosphorylation at Ser1943, promoting NMIIA filament disassembly and cytoskeletal rearrangement. This PS/EphA3-dependent cleavage also inhibits RhoA signaling to promote axon growth.\",\n      \"method\": \"Genetic rescue experiments in PS1/γ-secretase- and EphA3-deficient hippocampal neurons; proteomic analysis of EphA3 ICD binding partners; NMIIA phosphorylation assays; pharmacological NMII inhibition; RhoA signaling assays; live imaging\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — proteomic identification of NMIIA as binding partner, biochemical phosphorylation assay, genetic rescue with ICD fragment, pharmacological validation; multiple orthogonal methods\",\n      \"pmids\": [\"31577226\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Stimulation of EphA3 with ephrin-A5 inhibits cell migration and process outgrowth; these effects are partially mediated through the Nck1 SH2-SH3 adaptor protein binding phosphoY602. Dominant-negative Nck1 (lacking functional SH2 or SH3) partially rescues the inhibitory effects, indicating Nck1 is a downstream effector of EphA3-mediated cytoskeletal responses.\",\n      \"method\": \"Cell migration assays, process retraction assay, dominant-negative Nck1 expression, co-immunoprecipitation\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — functional rescue with domain-specific dominant-negatives plus co-IP, multiple cell-based readouts; described above but merits citation\",\n      \"pmids\": [\"19505147\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"EphA3 stimulation by ephrin-A5 in Jurkat and RMS cells causes loss of cell adhesion to fibronectin and recruitment of CrkII to EphA3. EphA3 expression is transcriptionally upregulated by CD28 co-stimulation and by IGF-1/IGF-1R signaling; anti-IGF-1R blocking antibodies suppress this upregulation.\",\n      \"method\": \"Receptor array screening, ephrin-A5 stimulation assays, Co-IP of CrkII with EphA3, western blotting, anti-IGF-1R blocking antibody experiments\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — single lab, Co-IP of CrkII plus functional adhesion assays; pathway regulation confirmed by blocking antibody\",\n      \"pmids\": [\"14697337\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"EphA3 stimulation with ephrin-A5 increases EphA3 phosphorylation and Rho GTPase activity in rhabdomyosarcoma cells, resulting in suppression of cell adhesion to fibronectin and decreased migration toward SDF-1. EphA3 siRNA knockdown increases SDF-1-mediated motility, confirming a suppressive role in cell motility.\",\n      \"method\": \"Ephrin-A5 stimulation, phosphorylation assays, Rho GTPase activity assay, fibronectin adhesion assay, Transwell migration assay, siRNA knockdown\",\n      \"journal\": \"Journal of cellular biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KD with motility phenotype, Rho GTPase assay links receptor to cytoskeletal signaling; single lab\",\n      \"pmids\": [\"18814179\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Neurocan (a perineuronal net proteoglycan) binds NCAM at the EphA3-binding site within the immunoglobulin-2 domain of NCAM, inhibiting NCAM/EphA3 association, membrane clustering of NCAM/EphA3, EphA3 kinase activation, and ephrin-A5-induced growth cone collapse in cortical interneuron axons.\",\n      \"method\": \"Co-immunoprecipitation of neurocan with NCAM, binding site mapping using NCAM-Ig domain mutants, EphA3 kinase activation assays, NCAM/EphA3 clustering assay by imaging, growth cone collapse assay in cortical neurons\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP with binding site mapping, kinase activation assay, imaging-based clustering, functional growth cone collapse assay; multiple orthogonal methods in single study\",\n      \"pmids\": [\"29670169\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"EphA3 expression in hematopoietic tumor cell lines is regulated by DNA methylation of CpG sites downstream of the basal promoter; these methylation sites are absent in normal tissues but present in leukemia patient samples, indicating epigenetic regulation of EphA3 transcription.\",\n      \"method\": \"Promoter deletion/reporter assays; Southern blot with methylation-sensitive restriction enzymes; bisulfite sequencing of genomic DNA from cell lines and clinical leukemia samples\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — bisulfite sequencing plus methylation-sensitive restriction analysis plus promoter reporter assays, validated in clinical samples\",\n      \"pmids\": [\"10498621\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"EphA3 is highly expressed on bone marrow-derived mesenchymal/stromal cells (CD90+/Sca1+) and myeloid cells within the tumor microenvironment. Activation of EphA3+ mesenchymal/stromal cells with an agonistic EphA3 antibody leads to cell contraction, cell-cell segregation, and apoptosis, disrupting newly formed tumor stroma and microvasculature and inhibiting tumor growth in vivo.\",\n      \"method\": \"EphA3 agonist antibody treatment of isolated stromal cells, cell contraction/segregation assays, apoptosis assays, in vivo mouse xenograft model with α-EphA3 antibody treatment\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — defined cellular responses (contraction, segregation, apoptosis) in isolated stromal cells with in vivo validation; single lab\",\n      \"pmids\": [\"25125683\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"HIF-1α controls EphA3 expression on human endometrial multipotent mesenchymal stromal cells (eMSCs) under hypoxic conditions. EphA3 silencing significantly inhibits the ability of transplanted eMSCs to support neovascularisation in immunocompromised mice, placing EphA3 downstream of HIF-1α in stromal cell-mediated vascular support.\",\n      \"method\": \"HIF-1α-EphA3 expression correlation, EphA3 siRNA knockdown in eMSCs, in vivo neovascularization assay in NSG mice\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA KD with defined in vivo vascular phenotype; HIF-1α link established by expression analysis without direct HIF-binding site mutagenesis; single lab\",\n      \"pmids\": [\"25420155\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"EPHA3 overexpression in small cell lung cancer cells decreases chemoresistance by increasing apoptosis and inducing G0/G1 arrest, accompanied by reduced phosphorylation of the PI3K/BMX/STAT3 signaling pathway. Conversely, EPHA3 knockdown generates a resistant phenotype with increased PI3K/BMX/STAT3 phosphorylation; PI3K inhibitor (LY294002) and BMX inhibitor (LFM-A13) both reverse the EPHA3-deficiency-induced resistance.\",\n      \"method\": \"EPHA3 overexpression and siRNA knockdown in SCLC cells; PI3K/BMX/STAT3 phosphorylation western blots; apoptosis and cell cycle assays; pharmacological inhibitor rescue; in vivo xenograft\",\n      \"journal\": \"Tumour biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — bidirectional genetic perturbation with pharmacological rescue confirming pathway placement; single lab\",\n      \"pmids\": [\"27101199\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"EPHA3 regulates PTEN expression epigenetically in radioresistant head and neck cancer cells via two mechanisms downstream of EPHA3/c-Myc signaling: DNMT1-mediated DNA methylation and EZH2-mediated histone H3K27 trimethylation of the PTEN promoter.\",\n      \"method\": \"Western blotting for DNMT1, EZH2, H3K27me3, and PTEN in radioresistant vs. sensitive cells; EPHA3 knockdown/overexpression; correlation of EPHA3 levels with DNMT1/EZH2/H3K27me3/PTEN\",\n      \"journal\": \"Biomolecules\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — correlative western blots with limited direct mechanistic validation of EPHA3-to-DNMT1/EZH2 pathway; single lab, single method per step\",\n      \"pmids\": [\"33919657\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Androgen receptor (AR) induces EPHA3 expression in prostate cancer cells by forming a transcription factor complex with SP1 that binds SP1 binding sites (−295 to −261) in the EPHA3 core promoter region, as required for androgen-stimulated EPHA3 transcription.\",\n      \"method\": \"Luciferase reporter assays with EPHA3 promoter deletion constructs; AR overexpression and siRNA knockdown; SP1 inhibitor (mithramycin A) and siSP1 treatment; co-immunoprecipitation and chromatin immunoprecipitation (ChIP) of AR/SP1 complex at EPHA3 promoter\",\n      \"journal\": \"Oncology reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — ChIP directly demonstrates AR/SP1 occupancy at EPHA3 promoter; luciferase reporters with deletion mapping; Co-IP of AR-SP1 complex; bidirectional genetic perturbation; multiple orthogonal methods\",\n      \"pmids\": [\"29917167\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"EphA3(-/-) null mice do not show defects in motor axon guidance to axial muscle targets, contrary to prior prediction based on EphA3 expression in the medial motor column; retrograde labeling studies confirm motor axons reach correct targets without EphA3.\",\n      \"method\": \"EphA3 null mouse generation; retrograde axonal labeling of motor projections in embryos and adults\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean genetic knockout with direct anatomical readout (retrograde labeling); well-controlled negative result\",\n      \"pmids\": [\"14585969\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"EphA3 is a receptor tyrosine kinase that is activated by clustering upon ephrin-A ligand binding through a two-step mechanism involving an N-terminal ligand-binding domain and a separate C-terminal dimerization domain; the unphosphorylated juxtamembrane region autoinhibits kinase activity, which is relieved by autophosphorylation at two juxtamembrane tyrosines; downstream signaling proceeds through phosphotyrosine docking sites Y602 and Y779 recruiting adaptors including CrkII and Nck1 to activate RhoA and suppress cell adhesion and migration; EphA3 can also be cleaved by presenilin/γ-secretase to generate an ICD that promotes axon elongation via NMIIA phosphorylation; the SAM domain stabilizes ligand-independent receptor dimers on the cell surface; a cis-interaction with ephrinA5 silences receptor activation to shape functional gradients; and EphA3 expression is regulated by DNA methylation, HIF-1α, androgen receptor/SP1, and by perineuronal net protein neurocan inhibiting NCAM/EphA3 complex formation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"EphA3 is a receptor tyrosine kinase that converts ephrin-A ligand engagement into cytoskeletal remodeling, controlling cell adhesion, migration, axon guidance, and tissue morphogenesis [#2, #17]. It binds GPI-anchored ephrin-A ligands with high affinity through an N-terminal ligand-binding region encoded by exon III, while a separate C-terminal domain drives ligand-independent receptor oligomerization, defining a two-step activation mechanism [#0, #1]; assembly of signaling-competent oligomers depends on three distinct surface interfaces of the receptor [#3], and unliganded receptor dimers are additionally stabilized by the cytoplasmic SAM domain [#13]. Kinase activity is held in check by the unphosphorylated juxtamembrane segment, which engages the active site until autophosphorylation of two juxtamembrane tyrosines relieves autoinhibition through a coupled network of residues linking the juxtamembrane region to the activation loop [#6]. Activated EphA3 recruits the adaptors CrkII and Nck1 — the latter via its SH2 domain binding phosphotyrosine Y602 — and signals through Y602 together with Y779 to activate RhoA and suppress adhesion to fibronectin, cell migration, neurite outgrowth, and growth cone collapse [#2, #7, #8, #17]. A cis-interaction with ephrinA5 on the same cell surface, independent of the canonical ligand-binding domain, silences receptor phosphorylation and converts uniform EphA3 expression into a functional sensitivity gradient in retinal axons [#4], and presenilin-1/γ-secretase cleavage of EphA3 generates an intracellular domain that binds non-muscle myosin IIA and increases its Ser1943 phosphorylation to promote axon elongation while inhibiting RhoA [#14]. EphA3 is required for atrioventricular endocardial cushion development and epithelial-to-mesenchymal transformation in the heart [#5]. In cancer, EphA3 has kinase- and ephrin-dependent tumor-suppressing activity that is disrupted by somatic mutations impairing kinase activity, ligand binding, or surface localization — some acting as dominant negatives — and wild-type re-expression promotes apoptosis by suppressing AKT [#9, #10, #22]. Its expression is controlled by DNA methylation, HIF-1α, and an androgen receptor/SP1 complex binding the core promoter [#19, #21, #24], and at the cell surface neurocan blocks NCAM/EphA3 complex formation and clustering to inhibit ephrin-A5-induced growth cone collapse [#18].\",\n  \"teleology\": [\n    {\n      \"year\": 1995,\n      \"claim\": \"Establishing the ligands of an orphan-like receptor was prerequisite to any mechanistic model; this work isolated high-affinity GPI-anchored ephrin-A ligands for EphA3.\",\n      \"evidence\": \"Expression cloning using soluble EphA3-Fc as an affinity probe in a human T-lymphoma line\",\n      \"pmids\": [\"7838529\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve how ligand binding triggers receptor activation\", \"Ligand specificity overlapped with the related ELK receptor\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"How a single ligand-binding event produces an active receptor was unclear; mapping distinct ligand-binding and oligomerization subdomains established a two-step activation mechanism.\",\n      \"evidence\": \"BIAcore kinetics, zebrafish mRNA injection, and transphosphorylation of recombinant subdomains\",\n      \"pmids\": [\"9685371\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Atomic-level interfaces not yet defined\", \"Did not address downstream effectors\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"The downstream consequence of activation was undefined; ephrin-A5 clustering was shown to activate RhoA and recruit CrkII to drive cytoskeletal collapse and cell detachment.\",\n      \"evidence\": \"RhoA activation assays, CrkII Co-IP, dominant-negative CrkII rescue, and morphological assays in 293T and melanoma cells\",\n      \"pmids\": [\"11870224\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not map the receptor docking site for CrkII\", \"Link between RhoA and specific cytoskeletal machinery unresolved\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"The structural determinants of ligand-driven oligomerization were unknown; a mutagenesis screen identified three surface areas required for signaling-competent complex assembly, one outside known interaction domains.\",\n      \"evidence\": \"Random mutagenesis in yeast with selection for impaired ephrin-A5 binding and functional validation\",\n      \"pmids\": [\"14660665\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Function of the third, structurally uncharacterized interface not mechanistically explained\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"EphA3 expression predicted a role in motor axon guidance; a clean knockout showed motor axons reach correct targets without EphA3, refining the receptor's in vivo guidance role.\",\n      \"evidence\": \"EphA3 null mice with retrograde axonal labeling of motor projections\",\n      \"pmids\": [\"14585969\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not exclude redundancy with other Eph receptors\", \"Other guidance contexts left untested\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"How uniform receptor expression yields graded responsiveness was unexplained; a cis-interaction with ephrinA5 was shown to silence receptor phosphorylation and convert expression into a functional gradient.\",\n      \"evidence\": \"FRET imaging, ephrinA5 point mutants, phosphorylation assays, and retinal axon sensitivity assays\",\n      \"pmids\": [\"16491080\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of the cis-interface not defined\", \"Extent of cis-silencing in non-retinal tissues unknown\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"A developmental requirement for EphA3 was undefined; knockout mice revealed it is required for endocardial cushion development and EMT in the heart.\",\n      \"evidence\": \"EphA3 null mice with cardiac morphology, cushion explant migration assays, and ligand co-expression analysis\",\n      \"pmids\": [\"17046737\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream signaling driving cushion EMT not dissected\", \"Cell-autonomy of the requirement not established\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"How kinase activity is restrained was unknown; crystallography revealed juxtamembrane autoinhibition relieved by autophosphorylation through a coupled residue network.\",\n      \"evidence\": \"X-ray crystallography of multiple kinase-domain states plus juxtamembrane tyrosine mutagenesis\",\n      \"pmids\": [\"18547520\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not connect autoinhibition release to specific downstream substrates\", \"Kinetics of the activation transition in cells unresolved\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"The receptor effector linking activation to migration suppression was unidentified; Nck1 was shown to bind phosphoY602 via its SH2 domain to mediate inhibition of migration and process retraction.\",\n      \"evidence\": \"Yeast two-hybrid, GST pulldown, Co-IP, Y602 mutagenesis, and dominant-negative Nck1 rescue with migration assays\",\n      \"pmids\": [\"19505147\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Only partial rescue indicates additional effectors\", \"Connection between Nck1 and RhoA not directly traced\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Whether a single docking site sufficed for cytoskeletal output was untested; Y602 and Y779 were shown to act collaboratively, with full effect requiring both.\",\n      \"evidence\": \"Tyrosine-to-phenylalanine mutagenesis with migration, neurite outgrowth, and growth cone collapse assays across cell types\",\n      \"pmids\": [\"20697431\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Effectors binding Y779 not identified\", \"Mechanism of cooperativity between the two sites unresolved\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"The disease relevance of EphA3 mutations was unclear; cancer-derived mutations were shown to impair kinase activity, ligand binding, or surface localization, with some acting as dominant negatives over wild-type tumor-suppressive signaling.\",\n      \"evidence\": \"Biochemical kinase, binding, and surface-localization assays on mutant panels plus AKT signaling, apoptosis, and xenograft assays\",\n      \"pmids\": [\"22242939\", \"22829656\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Tumor-suppressor mechanism beyond AKT suppression incompletely defined\", \"Context dependence of suppressor vs oncogenic roles unresolved\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"How EphA3 contributes to tumors via the microenvironment was unknown; agonist antibody activation of EphA3+ stromal cells was shown to drive contraction, segregation, and apoptosis, disrupting tumor stroma and vasculature.\",\n      \"evidence\": \"EphA3 agonist antibody on isolated stromal cells with contraction/apoptosis assays and in vivo xenograft treatment\",\n      \"pmids\": [\"25125683\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Molecular signaling underlying stromal apoptosis not detailed\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Whether receptor dimers exist before ligand binding was untested; live-cell FRET showed unliganded dimers stabilized by the SAM domain.\",\n      \"evidence\": \"Quantitative FRET dimerization assay with SAM-deletion mutants on live cells\",\n      \"pmids\": [\"26232493\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of pre-formed dimers for signaling output not established\", \"SAM-mediated interface not structurally resolved\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"A surface mechanism restraining EphA3 clustering was unknown; neurocan was shown to block NCAM/EphA3 complex formation by binding the EphA3-binding site on NCAM Ig2, inhibiting kinase activation and growth cone collapse.\",\n      \"evidence\": \"Co-IP, NCAM Ig-domain binding-site mapping, clustering imaging, kinase activation, and growth cone collapse assays\",\n      \"pmids\": [\"29670169\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether neurocan regulation operates outside cortical interneurons untested\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Transcriptional control of EphA3 was incompletely mapped; an androgen-receptor/SP1 complex was shown to bind the core promoter and drive androgen-stimulated expression.\",\n      \"evidence\": \"Promoter luciferase deletion, AR/SP1 perturbation, Co-IP, and ChIP at the EPHA3 promoter\",\n      \"pmids\": [\"29917167\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream consequence of AR-driven EphA3 in prostate cancer not functionally tied to signaling\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"A ligand-independent signaling output of EphA3 was undefined; presenilin/γ-secretase cleavage was shown to release an ICD that binds NMIIA, raises its Ser1943 phosphorylation, and promotes axon elongation while inhibiting RhoA.\",\n      \"evidence\": \"Genetic rescue in PS1/EphA3-deficient neurons, ICD interactome proteomics, NMIIA phosphorylation assays, and pharmacological NMII inhibition\",\n      \"pmids\": [\"31577226\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Trigger for cleavage in vivo not defined\", \"Balance between full-length signaling and ICD function unresolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the receptor's context-dependent switch between tumor-suppressive full-length signaling, microenvironmental effects, and ligand-independent ICD functions is coordinated at the molecular level remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified model integrating full-length kinase signaling, cis-silencing, and ICD output\", \"Effectors downstream of Y779 not identified\", \"Mechanistic basis of EZH2/DNMT1-mediated PTEN regulation remains correlative\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [6, 9]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 6]},\n      {\"term_id\": \"GO:0001618\", \"supporting_discovery_ids\": [1, 3]},\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [2, 7, 8]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [7]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [4, 9, 13]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [2, 7, 8]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [4, 5, 14]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [9, 10, 22]}\n    ],\n    \"complexes\": [\"NCAM/EphA3 complex\"],\n    \"partners\": [\"EFNA5\", \"CRK\", \"NCK1\", \"MYH9\", \"NCAM1\", \"PSEN1\", \"EFNA1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"tie","faith_supported":8,"faith_total":8,"faith_pct":100.0}}