{"gene":"NETO2","run_date":"2026-06-10T05:19:52","timeline":{"discoveries":[{"year":2013,"finding":"Neto2 physically associates with KCC2 (K+-Cl- cotransporter 2), specifically binding the active oligomeric form of the transporter. This interaction is required to maintain normal KCC2 abundance and KCC2-mediated Cl- extrusion; loss of the Neto2:KCC2 interaction reduces Cl- extrusion and decreases synaptic inhibition in hippocampal neurons.","method":"Co-immunoprecipitation, gramicidin perforated patch clamp, Neto2-null mouse model","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP identifying specific oligomeric form, functional patch-clamp electrophysiology in neurons, replicated in subsequent studies (PMID:26441539)","pmids":["23401525"],"is_preprint":false},{"year":2015,"finding":"In Neto2-null hippocampal pyramidal neurons, EGABA is significantly depolarized, surface levels of KCC2 are reduced, and phosphorylation of KCC2 at serine 940 is reduced compared to wild-type. Neto2-null mice show reduced sIPSC amplitude and frequency, and increased susceptibility to PTZ-induced seizures.","method":"Gramicidin perforated patch clamp recordings, surface biotinylation, western blot, PTZ seizure induction in Neto2-/- mice","journal":"Frontiers in cellular neuroscience","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (electrophysiology, biochemistry, in vivo seizure model) in Neto2-null mice, extends findings of PMID:23401525","pmids":["26441539"],"is_preprint":false},{"year":2011,"finding":"Neto2 acts as an auxiliary subunit of kainate receptors containing GluK1, GluK1/5, or GluK2/5, slowing desensitization to varying degrees and markedly increasing the rate of recovery from desensitization. Effects on postsynaptic currents in neurons expressing recombinant kainate receptors were also slowed by Neto2, particularly for receptors containing GluK5.","method":"Rapid application outside-out patch clamp recordings in heterologous cells and neurons","journal":"The Journal of neuroscience","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct in vitro electrophysiology with rapid application; multiple receptor subunit compositions tested; replicated across multiple subsequent studies","pmids":["21632929"],"is_preprint":false},{"year":2012,"finding":"Neto2 co-expression with recombinant GluK2(Q) kainate receptors greatly reduces inward rectification (polyamine block) without altering calcium permeability. The extracellular LDLa domain of Neto2 is required for effects on desensitization but only partially for rectification, whereas the intracellular C-terminal domain (including positively charged residues) is required for reduction of rectification but not for effects on channel kinetics.","method":"Patch clamp electrophysiology in heterologous cells; domain deletion and point-mutation analysis of Neto2","journal":"The Journal of neuroscience","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct electrophysiology with mutagenesis identifying distinct functional domains; multiple orthogonal mutations tested","pmids":["22973017"],"is_preprint":false},{"year":2012,"finding":"Neto2 regulates synaptic localization of GluK2-containing kainate receptors in the cerebellum; Neto2-null mice show ~40% decrease in GluK2-KARs at the postsynaptic density without change in total GluK2 levels. Neto2 directly interacts with the scaffolding protein GRIP via a PDZ-dependent interaction, and Neto2 co-expression increases the amount of GRIP associated with GluK2.","method":"PSD fractionation from Neto2-null mouse cerebellum, Co-immunoprecipitation (Neto2-GRIP), western blot","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP plus fractionation in knockout mouse; two orthogonal methods linking Neto2 to synaptic KAR localization","pmids":["23236500"],"is_preprint":false},{"year":2021,"finding":"Cryo-EM structures of homotetrameric GluK2 in complex with NETO2 at inhibited and desensitized states reveal variable stoichiometry (1 or 2 NETO2 subunits per tetramer). NETO2 accesses two broad faces of the receptor, intermolecularly crosslinking the lower lobe of ATD A/C, upper lobe of LBD B/D, and lower lobe of LBD A/C, thereby stabilizing gating kinetics. The NETO2 transmembrane helix is positioned proximal to the selectivity filter and competes with the amphiphilic H1 helix after M4 for interaction with an intracellular cap domain formed by M1-M2 linkers, explaining how NETO2 regulates rectification.","method":"Cryo-electron microscopy structure determination","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 / Strong — high-resolution cryo-EM structures at multiple functional states providing atomic-level mechanistic detail, published in Nature","pmids":["34552241"],"is_preprint":false},{"year":2025,"finding":"Cryo-EM structures of rat GluK2 KAR in apo closed and agonist/PAM-activated open states, with and without Neto2, show that Neto2 binding does not alter individual or dimeric LBD behavior or ion channel conductance but prevents tightening of the interface between two LBD dimers during activation, thereby slowing deactivation kinetics.","method":"Time-resolved cryo-electron microscopy structure determination","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple cryo-EM structures at distinct functional states with and without Neto2, providing direct mechanistic insight into activation modulation","pmids":["40846810"],"is_preprint":false},{"year":2015,"finding":"The M3-S2 gating linkers of KAR subunits are critical determinants of Neto2 modulation: individual residues in these linkers bidirectionally influence Neto2 modulation of desensitization in an agonist-specific manner. A single mutation in this domain abolishes Neto2 modulation of heteromeric receptor desensitization. Neto2 also alters cation sensitivity of KAR gating (implicating the D1 dimer interface), and this modulation is eliminated by M3-S2 linker mutations, functionally correlating these two discrete structural sites.","method":"Site-directed mutagenesis of KAR M3-S2 linkers combined with patch clamp electrophysiology in heterologous cells","journal":"The Journal of physiology","confidence":"High","confidence_rationale":"Tier 1 / Strong — systematic mutagenesis with electrophysiology identifying specific structural determinants; multiple mutations and agonists tested","pmids":["26282342"],"is_preprint":false},{"year":2015,"finding":"The extracellular N-terminal region including the two CUB domains of Neto2 (vs. Neto1) is largely responsible for distinct regulatory effects on desensitization properties of GluK1 homomeric receptors, as determined using chimeric Neto1/Neto2 subunits.","method":"Chimeric Neto1/Neto2 subunit construction combined with patch clamp electrophysiology in HEK-293T cells","journal":"Neuropharmacology","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — chimeric protein approach with electrophysiology in single study; establishes domain importance but limited replication","pmids":["26277340"],"is_preprint":false},{"year":2017,"finding":"Neto2 serine 409 is phosphorylated by CaMKII and PKA both in vitro and in heterologous cells, and endogenous Neto2 Ser-409 phosphorylation is detected in brain. CaMKII-mediated Neto2 Ser-409 phosphorylation is reduced in the presence of GluK1 or GluK2. The phosphomimetic Neto2 S409D mutant (but not WT or S409A) impedes GluK1 trafficking to synapses, demonstrating that Neto2 Ser-409 phosphorylation restricts synaptic targeting of GluK1.","method":"Mass spectrometry identification of phosphorylation sites, in vitro kinase assays, phosphomimetic/phosphodeficient mutant transfection in neurons, synaptic fractionation","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro kinase assay, endogenous phosphorylation validation, mutagenesis with functional synaptic localization readout; multiple orthogonal methods in single study","pmids":["28717010"],"is_preprint":false},{"year":2017,"finding":"Neto2 protein is highly expressed in neonatal DRG neurons and assembles with endogenous KARs to modify their gating. Neto2-/- adult DRG neurons display stunted neurite outgrowth. Neto2 expression in adult DRG is upregulated via MEK/ERK signaling and after sciatic nerve crush injury.","method":"Electrophysiology in DRG neurons from Neto2-/- mice, neurite outgrowth assays, MEK inhibitor pharmacology, sciatic nerve crush in vivo","journal":"The Journal of neuroscience","confidence":"High","confidence_rationale":"Tier 2 / Strong — loss-of-function electrophysiology in native neurons with knockout, mechanistic pathway (MEK/ERK) identified for regulation, in vivo injury model","pmids":["28235897"],"is_preprint":false},{"year":2019,"finding":"In Neto2-/- mice, synaptosomal KAR GluK2/3 subunit abundance is reduced 20.8% in ventral hippocampus and 36.5% in medial prefrontal cortex, and GluK5 abundance is reduced 23.8% in ventral hippocampus and 16.9% in amygdala. These reductions are associated with higher fear expression and delayed fear extinction in cued fear conditioning.","method":"Synaptosomal fractionation western blot, behavioral fear conditioning in Neto2-/- mice, in situ hybridization for Neto2 expression","journal":"Neuropsychopharmacology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — fractionation plus behavioral phenotype in knockout; single lab, no rescue experiment","pmids":["30770891"],"is_preprint":false},{"year":2020,"finding":"Neto2 is required for maturation of the amygdala PV interneuron network; Neto2-/- adult mice show reduced PV+PNN+ cells, reduced PV staining intensity, increased glutamatergic and reduced GABAergic transmission, and increased spine density in basal amygdala compared to wild-type. These structural and functional amygdala changes are associated with increased fear expression and delayed extinction.","method":"Immunohistochemistry for PV/PNN, whole-cell patch-clamp recordings in amygdala slices, spine density analysis, c-Fos immunostaining in Neto2-/- mice","journal":"eNeuro","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple electrophysiology and histology methods in knockout; single lab","pmids":["32788298"],"is_preprint":false},{"year":2023,"finding":"Soluble LRIG3 derived from glioma cells interacts with the CUB1 domain of NETO2 in tumor-associated macrophages (TAMs). This interaction suppresses M2 polarization of TAMs. NETO2 knockout blocks the inhibitory effect of sLRIG3 on M2 polarization and promotes GBM tumor growth; CUB1-deletion mutant NETO2 does not fully recover sLRIG3's suppressive effects.","method":"Mass spectrometry, Co-immunoprecipitation, NETO2 knockout and domain-deletion mutant rescue experiments in TAMs","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus MS identification of interaction, domain-deletion mutant rescue, knockout functional phenotype; single lab","pmids":["36639372"],"is_preprint":false},{"year":2023,"finding":"GluK2-NETO2 (but not GluK2-NETO1) interaction is upregulated in ipsilateral dorsal horn neurons at 6 h post-incision. Intrathecal NETO2 siRNA pretreatment reduces pain hypersensitivity, decreases synaptic abundance of GluK2 and GluR1 (AMPA receptor subunit), and reduces PKCγ activation in ipsilateral dorsal horn, indicating that NETO2-GluK2 interaction drives PKCγ activation and synaptic AMPA receptor incorporation in postoperative pain.","method":"Co-immunoprecipitation in dorsal horn tissue, intrathecal siRNA knockdown, behavioral pain assays, western blot for synaptic fractions","journal":"Neuroscience letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP in native tissue plus siRNA knockdown with multiple readouts; single lab, single study","pmids":["37544581"],"is_preprint":false},{"year":2025,"finding":"NETO2 slows the channel-opening rate of GluK2 homomeric receptors by ~7-fold and the channel-closing rate by ~3-fold (compared to ~2-fold slowing by NETO1 for both rates), demonstrating that NETO2 is a more impactful auxiliary subunit than NETO1 on GluK2 channel-opening kinetics.","method":"Laser-pulse photolysis combined with whole-cell recording in HEK-293 cells","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — direct kinetic measurement with a specialized laser photolysis technique; single lab, single study","pmids":["41197725"],"is_preprint":false},{"year":2019,"finding":"NETO2 activates the PI3K/AKT/NF-κB/Snail signaling axis in gastric cancer cells via TNFRSF12A as a mediator, promoting invasion, migration, and EMT. Silencing NETO2 reduced phosphorylation of PI3K, AKT, NF-κB p65 and Snail expression; overexpression had opposite effects. TNFRSF12A was identified as the mediator linking NETO2 to this pathway.","method":"shRNA knockdown and overexpression in gastric cancer cells, western blot for pathway components, in vitro migration/invasion assays, in vivo metastasis model","journal":"Cell death & disease","confidence":"Low","confidence_rationale":"Tier 3 / Moderate — pathway placement by western blot and KD/OE; no direct binding assay or reconstitution to confirm TNFRSF12A as mechanistic mediator","pmids":["30770791"],"is_preprint":false},{"year":2025,"finding":"α5-nAChR physically interacts with NETO2 in lung adenocarcinoma cells, as demonstrated by Co-immunoprecipitation. Acetylcholine/nicotine stimulation via α5-nAChR upregulates NETO2, p-CaMKII, p-STAT3, and vimentin expression, and the α5-nAChR/NETO2 axis promotes LUAD cell proliferation, migration, and invasion.","method":"Co-immunoprecipitation, molecular docking, western blot, siRNA knockdown, proliferation and invasion assays","journal":"Cancer cell international","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP without reciprocal confirmation; molecular docking is computational; single lab","pmids":["40001189"],"is_preprint":false}],"current_model":"NETO2 is a single-pass transmembrane auxiliary protein that functions as an accessory subunit of kainate receptors (KARs) by directly associating with GluK1-5 subunits to slow desensitization and deactivation, increase recovery from desensitization, reduce polyamine-mediated inward rectification (through its intracellular C-terminal domain), and regulate synaptic KAR abundance via interactions with scaffolding proteins such as GRIP; cryo-EM structures reveal that NETO2 binds two faces of the KAR tetramer to prevent LBD dimer interface tightening during activation and positions its transmembrane helix near the selectivity filter to compete for an intracellular cap domain, thereby modulating gating and rectification. In addition, NETO2 binds and stabilizes the active oligomeric form of the neuronal K+-Cl- cotransporter KCC2, maintaining its surface expression and Ser940 phosphorylation to preserve the chloride gradient required for GABAergic inhibition, with loss of NETO2 causing depolarized EGABA and seizure susceptibility. NETO2 is also phosphorylated at Ser409 by CaMKII/PKA, which restricts synaptic targeting of GluK1."},"narrative":{"mechanistic_narrative":"NETO2 is a single-pass transmembrane auxiliary protein that shapes neuronal excitability by acting as an accessory subunit of kainate-type glutamate receptors (KARs) and as a binding partner of the K+-Cl- cotransporter KCC2 [PMID:21632929, PMID:23401525]. As a KAR auxiliary subunit, NETO2 associates with GluK1-, GluK2-, and GluK5-containing receptors to slow desensitization and deactivation, accelerate recovery from desensitization, and slow channel opening and closing kinetics [PMID:21632929, PMID:41197725]. Its extracellular CUB and LDLa domains govern effects on desensitization, whereas its intracellular positively charged C-terminal domain reduces polyamine-mediated inward rectification of GluK2 receptors [PMID:22973017, PMID:26277340]. Cryo-EM structures show NETO2 binding across two faces of the GluK2 tetramer with variable stoichiometry, crosslinking the ATD and LBD lobes to prevent tightening of the inter-dimer LBD interface during activation, while its transmembrane helix sits near the selectivity filter and competes for an intracellular cap domain to control rectification; the gating effects map functionally to the M3-S2 linkers [PMID:34552241, PMID:40846810, PMID:26282342]. NETO2 also controls synaptic KAR abundance, both by interacting with the PDZ scaffold GRIP to localize GluK2 receptors at the postsynaptic density and through CaMKII/PKA phosphorylation at Ser409, which restricts synaptic targeting of GluK1 [PMID:23236500, PMID:28717010]. Independently of its KAR role, NETO2 binds and stabilizes the active oligomeric form of KCC2, maintaining KCC2 surface expression and Ser940 phosphorylation; loss of NETO2 depolarizes EGABA, weakens synaptic inhibition, and increases seizure susceptibility [PMID:23401525, PMID:26441539]. These activities give NETO2 broader physiological roles in fear behavior, amygdala interneuron network maturation, DRG neurite outgrowth, and nociception [PMID:30770891, PMID:32788298, PMID:28235897, PMID:37544581]. NETO2 has additionally been implicated in cancer-related signaling outside the nervous system [PMID:36639372, PMID:30770791].","teleology":[{"year":2011,"claim":"Established NETO2 as a bona fide auxiliary subunit of kainate receptors, answering whether it directly modifies KAR gating rather than merely co-expressing with them.","evidence":"Rapid-application outside-out patch clamp of recombinant KARs in heterologous cells and neurons across multiple GluK subunit compositions","pmids":["21632929"],"confidence":"High","gaps":["Did not resolve which NETO2 domains mediate each gating effect","Native stoichiometry and structural basis unknown"]},{"year":2012,"claim":"Resolved the domain logic of NETO2 modulation, separating effects on gating kinetics from effects on rectification.","evidence":"Patch clamp with domain deletion and point mutagenesis showing the LDLa domain controls desensitization and the charged C-terminus controls polyamine block of GluK2(Q)","pmids":["22973017"],"confidence":"High","gaps":["Structural mechanism for C-terminal rectification effect not yet visualized","Did not address synaptic localization"]},{"year":2012,"claim":"Linked NETO2 to synaptic KAR abundance, showing it does more than tune gating by also positioning receptors at synapses.","evidence":"PSD fractionation from Neto2-null cerebellum and reciprocal Co-IP identifying a PDZ-dependent NETO2-GRIP interaction","pmids":["23236500"],"confidence":"High","gaps":["Mechanism by which GRIP retains receptors at the PSD not dissected","Regulation of the interaction unknown"]},{"year":2013,"claim":"Identified a KAR-independent role for NETO2 in chloride homeostasis by showing it binds and stabilizes the active oligomeric KCC2.","evidence":"Reciprocal Co-IP and gramicidin perforated patch clamp in hippocampal neurons of Neto2-null mice","pmids":["23401525"],"confidence":"High","gaps":["Structural basis of NETO2-KCC2 binding undefined","How NETO2 selectively recognizes the oligomeric form unknown"]},{"year":2015,"claim":"Connected the NETO2-KCC2 interaction to inhibitory tone and seizure threshold in vivo, establishing physiological consequence.","evidence":"Perforated patch clamp, surface biotinylation, Ser940 phospho-blot, and PTZ seizure assays in Neto2-/- mice","pmids":["26441539"],"confidence":"High","gaps":["Mechanism linking NETO2 loss to reduced Ser940 phosphorylation not established","Kinase/phosphatase pathway not identified"]},{"year":2015,"claim":"Mapped the structural determinants on the KAR side, identifying the M3-S2 gating linkers and D1 dimer interface as the substrate of NETO2 modulation.","evidence":"Systematic M3-S2 linker mutagenesis with patch clamp and cation-sensitivity analysis in heterologous cells","pmids":["26282342"],"confidence":"High","gaps":["Atomic-level contact map not yet available","Agonist-specificity mechanism unresolved"]},{"year":2015,"claim":"Attributed NETO2-versus-NETO1 functional divergence to the CUB-domain-containing N-terminal region.","evidence":"Chimeric Neto1/Neto2 constructs with patch clamp in HEK-293T cells on GluK1 homomers","pmids":["26277340"],"confidence":"Medium","gaps":["Single-study chimera approach; specific CUB residues not pinpointed","Limited replication"]},{"year":2017,"claim":"Showed NETO2 is itself a regulated protein, with Ser409 phosphorylation by CaMKII/PKA gating GluK1 synaptic targeting.","evidence":"Mass-spec site identification, in vitro kinase assays, and phosphomimetic/phosphodeficient mutant synaptic fractionation in neurons","pmids":["28717010"],"confidence":"High","gaps":["Phosphatase that reverses Ser409 not identified","Whether KCC2 association is similarly regulated unknown"]},{"year":2017,"claim":"Extended NETO2 function to peripheral sensory neurons, linking it to KAR gating and injury-induced neurite outgrowth.","evidence":"Electrophysiology in Neto2-/- DRG neurons, neurite outgrowth assays, MEK inhibition, and sciatic nerve crush in vivo","pmids":["28235897"],"confidence":"High","gaps":["Mechanism linking NETO2 to neurite outgrowth unclear","Whether outgrowth defect is KAR-dependent untested"]},{"year":2019,"claim":"Tied region-specific synaptic KAR loss in Neto2-null mice to fear behavior abnormalities.","evidence":"Synaptosomal fractionation western blot and cued fear conditioning in Neto2-/- mice","pmids":["30770891"],"confidence":"Medium","gaps":["No rescue experiment to establish causality","Single lab"]},{"year":2020,"claim":"Implicated NETO2 in amygdala parvalbumin interneuron network maturation underlying fear circuit function.","evidence":"PV/PNN immunohistochemistry, amygdala slice patch clamp, spine density and c-Fos analysis in Neto2-/- mice","pmids":["32788298"],"confidence":"Medium","gaps":["Whether interneuron phenotype is cell-autonomous unknown","Mechanistic link to KAR or KCC2 roles not dissected"]},{"year":2023,"claim":"Showed a NETO2-GluK2 axis drives postoperative pain hypersensitivity via PKCγ activation and AMPA receptor synaptic incorporation.","evidence":"Co-IP in dorsal horn tissue, intrathecal NETO2 siRNA, behavioral pain assays and synaptic-fraction western blots","pmids":["37544581"],"confidence":"Medium","gaps":["Mechanism connecting GluK2-NETO2 to PKCγ unresolved","Single study"]},{"year":2023,"claim":"Revealed a non-neuronal role in which the NETO2 CUB1 domain receives glioma-derived sLRIG3 to suppress macrophage M2 polarization.","evidence":"Mass spec, Co-IP, NETO2 knockout and CUB1-deletion rescue in tumor-associated macrophages","pmids":["36639372"],"confidence":"Medium","gaps":["Downstream signaling from CUB1 engagement unmapped","Single lab"]},{"year":2025,"claim":"Quantified NETO2's impact on GluK2 channel opening/closing rates, showing it is a more potent kinetic modulator than NETO1.","evidence":"Laser-pulse photolysis with whole-cell recording in HEK-293 cells","pmids":["41197725"],"confidence":"Medium","gaps":["Single study with specialized technique","Structural correlate of rate differences not defined"]},{"year":2025,"claim":"Provided time-resolved structural mechanism by which NETO2 slows deactivation: preventing tightening of the inter-LBD-dimer interface during activation.","evidence":"Time-resolved cryo-EM of GluK2 in apo-closed and agonist/PAM-activated states with and without Neto2","pmids":["40846810"],"confidence":"High","gaps":["Does not address KCC2-bound structure","Heteromeric KAR-NETO2 structures not resolved"]},{"year":null,"claim":"How NETO2's two distinct partnerships — KARs and KCC2 — are coordinated, regulated, and integrated within a single neuron remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structure of the NETO2-KCC2 complex","Phosphoregulation of KCC2 binding undefined","Whether cancer-associated signaling roles share the neuronal binding interfaces unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[2,3,5,6,15]},{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[2,7]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[4]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,1,5]}],"pathway":[{"term_id":"R-HSA-112316","term_label":"Neuronal System","supporting_discovery_ids":[2,0]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[2,3]}],"complexes":["Kainate receptor complex (GluK1-5/NETO2)","KCC2-NETO2 complex"],"partners":["GRIK1","GRIK2","GRIK5","SLC12A5","GRIP1","CAMK2A","PRKACA","LRIG3"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q8NC67","full_name":"Neuropilin and tolloid-like protein 2","aliases":["Brain-specific transmembrane protein containing 2 CUB and 1 LDL-receptor class A domains protein 2"],"length_aa":525,"mass_kda":59.4,"function":"Accessory subunit of neuronal kainate-sensitive glutamate receptors, GRIK2 and GRIK3. Increases kainate-receptor channel activity, slowing the decay kinetics of the receptors, without affecting their expression at the cell surface, and increasing the open probability of the receptor channels. Modulates the agonist sensitivity of kainate receptors. Slows the decay of kainate receptor-mediated excitatory postsynaptic currents (EPSCs), thus directly influencing synaptic transmission (By similarity)","subcellular_location":"Membrane","url":"https://www.uniprot.org/uniprotkb/Q8NC67/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/NETO2","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"CANX","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/NETO2","total_profiled":1310},"omim":[{"mim_id":"607974","title":"NEUROPILIN- AND TOLLOID-LIKE 2; NETO2","url":"https://www.omim.org/entry/607974"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Golgi apparatus","reliability":"Approved"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"brain","ntpm":18.9},{"tissue":"lymphoid tissue","ntpm":21.4}],"url":"https://www.proteinatlas.org/search/NETO2"},"hgnc":{"alias_symbol":["FLJ10430","NEOT2"],"prev_symbol":[]},"alphafold":{"accession":"Q8NC67","domains":[{"cath_id":"2.60.120.290","chopping":"48-168","consensus_level":"high","plddt":93.7636,"start":48,"end":168},{"cath_id":"2.60.120.290","chopping":"178-315","consensus_level":"high","plddt":92.5091,"start":178,"end":315}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8NC67","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q8NC67-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q8NC67-F1-predicted_aligned_error_v6.png","plddt_mean":72.06},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=NETO2","jax_strain_url":"https://www.jax.org/strain/search?query=NETO2"},"sequence":{"accession":"Q8NC67","fasta_url":"https://rest.uniprot.org/uniprotkb/Q8NC67.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q8NC67/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8NC67"}},"corpus_meta":[{"pmid":"33390848","id":"PMC_33390848","title":"NETO2 promotes esophageal cancer progression by inducing proliferation and metastasis via PI3K/AKT and ERK pathway.","date":"2021","source":"International journal of biological sciences","url":"https://pubmed.ncbi.nlm.nih.gov/33390848","citation_count":67,"is_preprint":false},{"pmid":"23401525","id":"PMC_23401525","title":"Neto2 is a KCC2 interacting protein required for neuronal Cl- regulation in hippocampal neurons.","date":"2013","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/23401525","citation_count":65,"is_preprint":false},{"pmid":"30770791","id":"PMC_30770791","title":"NETO2 promotes invasion and metastasis of gastric cancer cells via activation of PI3K/Akt/NF-κB/Snail axis and predicts outcome of the patients.","date":"2019","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/30770791","citation_count":60,"is_preprint":false},{"pmid":"21632929","id":"PMC_21632929","title":"Neto2 modulation of kainate receptors with different subunit compositions.","date":"2011","source":"The Journal of neuroscience : the official journal of the Society for Neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/21632929","citation_count":53,"is_preprint":false},{"pmid":"33006432","id":"PMC_33006432","title":"Exosomal lncRNA FAM225A accelerates esophageal squamous cell carcinoma progression and angiogenesis via sponging miR-206 to upregulate NETO2 and FOXP1 expression.","date":"2020","source":"Cancer medicine","url":"https://pubmed.ncbi.nlm.nih.gov/33006432","citation_count":48,"is_preprint":false},{"pmid":"22973017","id":"PMC_22973017","title":"The auxiliary subunits Neto1 and Neto2 reduce voltage-dependent inhibition of recombinant kainate receptors.","date":"2012","source":"The Journal of neuroscience : the official journal of the Society for Neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/22973017","citation_count":47,"is_preprint":false},{"pmid":"23236500","id":"PMC_23236500","title":"Neto2 interacts with the scaffolding protein GRIP and regulates synaptic abundance of kainate receptors.","date":"2012","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/23236500","citation_count":36,"is_preprint":false},{"pmid":"34552241","id":"PMC_34552241","title":"Kainate receptor modulation by NETO2.","date":"2021","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/34552241","citation_count":34,"is_preprint":false},{"pmid":"28235897","id":"PMC_28235897","title":"Neto2 Assembles with Kainate Receptors in DRG Neurons during Development and Modulates Neurite Outgrowth in Adult Sensory Neurons.","date":"2017","source":"The Journal of neuroscience : the official journal of the Society for Neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/28235897","citation_count":26,"is_preprint":false},{"pmid":"26441539","id":"PMC_26441539","title":"Neto2-null mice have impaired GABAergic inhibition and are susceptible to seizures.","date":"2015","source":"Frontiers in cellular neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/26441539","citation_count":21,"is_preprint":false},{"pmid":"26277340","id":"PMC_26277340","title":"The auxiliary subunits Neto1 and Neto2 have distinct, subunit-dependent effects at recombinant GluK1- and GluK2-containing kainate receptors.","date":"2015","source":"Neuropharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/26277340","citation_count":20,"is_preprint":false},{"pmid":"26282342","id":"PMC_26282342","title":"Identification of critical functional determinants of kainate receptor modulation by auxiliary protein Neto2.","date":"2015","source":"The Journal of physiology","url":"https://pubmed.ncbi.nlm.nih.gov/26282342","citation_count":19,"is_preprint":false},{"pmid":"31239769","id":"PMC_31239769","title":"NETO2 promotes pancreatic cancer cell proliferation, invasion and migration via activation of the STAT3 signaling pathway.","date":"2019","source":"Cancer management and research","url":"https://pubmed.ncbi.nlm.nih.gov/31239769","citation_count":17,"is_preprint":false},{"pmid":"30770891","id":"PMC_30770891","title":"Kainate receptor auxiliary subunit NETO2 is required for normal fear expression and extinction.","date":"2019","source":"Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/30770891","citation_count":15,"is_preprint":false},{"pmid":"29297384","id":"PMC_29297384","title":"Upregulation of NETO2 gene in colorectal cancer.","date":"2017","source":"BMC genetics","url":"https://pubmed.ncbi.nlm.nih.gov/29297384","citation_count":14,"is_preprint":false},{"pmid":"34671432","id":"PMC_34671432","title":"Long non-coding RNA SNHG17 promotes lung adenocarcinoma progression by targeting the microRNA-193a-5p/NETO2 axis.","date":"2021","source":"Oncology letters","url":"https://pubmed.ncbi.nlm.nih.gov/34671432","citation_count":14,"is_preprint":false},{"pmid":"33362854","id":"PMC_33362854","title":"NETO2 Is Deregulated in Breast, Prostate, and Colorectal Cancer and Participates in Cellular Signaling.","date":"2020","source":"Frontiers in genetics","url":"https://pubmed.ncbi.nlm.nih.gov/33362854","citation_count":13,"is_preprint":false},{"pmid":"36639372","id":"PMC_36639372","title":"Glioma-derived LRIG3 interacts with NETO2 in tumor-associated macrophages to modulate microenvironment and suppress tumor growth.","date":"2023","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/36639372","citation_count":12,"is_preprint":false},{"pmid":"28717010","id":"PMC_28717010","title":"Phosphorylation of the kainate receptor (KAR) auxiliary subunit Neto2 at serine 409 regulates synaptic targeting of the KAR subunit GluK1.","date":"2017","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/28717010","citation_count":10,"is_preprint":false},{"pmid":"32788298","id":"PMC_32788298","title":"Kainate Receptor Auxiliary Subunit NETO2-Related Cued Fear Conditioning Impairments Associate with Defects in Amygdala Development and Excitability.","date":"2020","source":"eNeuro","url":"https://pubmed.ncbi.nlm.nih.gov/32788298","citation_count":9,"is_preprint":false},{"pmid":"26991362","id":"PMC_26991362","title":"Pharmacological Modulation of GluK1 and GluK2 by NETO1, NETO2, and PSD95.","date":"2016","source":"Assay and drug development technologies","url":"https://pubmed.ncbi.nlm.nih.gov/26991362","citation_count":8,"is_preprint":false},{"pmid":"36509232","id":"PMC_36509232","title":"TYMSOS-miR-101-3p-NETO2 axis promotes osteosarcoma progression.","date":"2022","source":"Molecular and cellular probes","url":"https://pubmed.ncbi.nlm.nih.gov/36509232","citation_count":7,"is_preprint":false},{"pmid":"30975469","id":"PMC_30975469","title":"Reducing NETO2 expression prevents human nasopharyngeal carcinoma (NPC) progression by suppressing metastasis and inducing apoptosis.","date":"2019","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/30975469","citation_count":7,"is_preprint":false},{"pmid":"35229031","id":"PMC_35229031","title":"TTN-AS1 accelerates the growth and migration of nasopharyngeal carcinoma cells via targeting miR-876-5p/NETO2.","date":"2021","source":"Molecular therapy 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lung adenocarcinoma progression.","date":"2025","source":"Cancer cell international","url":"https://pubmed.ncbi.nlm.nih.gov/40001189","citation_count":3,"is_preprint":false},{"pmid":"40158169","id":"PMC_40158169","title":"Targeting NETO2 suppresses cell proliferation, invasion, and migration and inactivates the STAT3/C-MYC pathway in hepatocellular carcinoma.","date":"2025","source":"World journal of surgical oncology","url":"https://pubmed.ncbi.nlm.nih.gov/40158169","citation_count":2,"is_preprint":false},{"pmid":"37544581","id":"PMC_37544581","title":"NETO2-GluK2 interaction contributes to postoperative pain hypersensitivity through inducing PKCγ activation and synaptic incorporation of AMPA receptor GluR1 subunits in rat dorsal horn.","date":"2023","source":"Neuroscience letters","url":"https://pubmed.ncbi.nlm.nih.gov/37544581","citation_count":1,"is_preprint":false},{"pmid":"41197725","id":"PMC_41197725","title":"A comparative study of NETO1 and NETO2 on channel-opening kinetics of GluK2 kainate receptors.","date":"2025","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/41197725","citation_count":0,"is_preprint":false},{"pmid":"41714211","id":"PMC_41714211","title":"The role of NETO2 in neurological disorders and cancer: From molecular function to clinical relevance.","date":"2026","source":"Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie","url":"https://pubmed.ncbi.nlm.nih.gov/41714211","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":18804,"output_tokens":4976,"usd":0.065526,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":13035,"output_tokens":4324,"usd":0.086638,"stage2_stop_reason":"end_turn"},"total_usd":0.152164,"stage1_batch_id":"msgbatch_013a8chtv3AWyTqv4z4sEF4C","stage2_batch_id":"msgbatch_01AHsuW63vinsvuTgzmQ3n62","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2013,\n      \"finding\": \"Neto2 physically associates with KCC2 (K+-Cl- cotransporter 2), specifically binding the active oligomeric form of the transporter. This interaction is required to maintain normal KCC2 abundance and KCC2-mediated Cl- extrusion; loss of the Neto2:KCC2 interaction reduces Cl- extrusion and decreases synaptic inhibition in hippocampal neurons.\",\n      \"method\": \"Co-immunoprecipitation, gramicidin perforated patch clamp, Neto2-null mouse model\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP identifying specific oligomeric form, functional patch-clamp electrophysiology in neurons, replicated in subsequent studies (PMID:26441539)\",\n      \"pmids\": [\"23401525\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"In Neto2-null hippocampal pyramidal neurons, EGABA is significantly depolarized, surface levels of KCC2 are reduced, and phosphorylation of KCC2 at serine 940 is reduced compared to wild-type. Neto2-null mice show reduced sIPSC amplitude and frequency, and increased susceptibility to PTZ-induced seizures.\",\n      \"method\": \"Gramicidin perforated patch clamp recordings, surface biotinylation, western blot, PTZ seizure induction in Neto2-/- mice\",\n      \"journal\": \"Frontiers in cellular neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (electrophysiology, biochemistry, in vivo seizure model) in Neto2-null mice, extends findings of PMID:23401525\",\n      \"pmids\": [\"26441539\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Neto2 acts as an auxiliary subunit of kainate receptors containing GluK1, GluK1/5, or GluK2/5, slowing desensitization to varying degrees and markedly increasing the rate of recovery from desensitization. Effects on postsynaptic currents in neurons expressing recombinant kainate receptors were also slowed by Neto2, particularly for receptors containing GluK5.\",\n      \"method\": \"Rapid application outside-out patch clamp recordings in heterologous cells and neurons\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct in vitro electrophysiology with rapid application; multiple receptor subunit compositions tested; replicated across multiple subsequent studies\",\n      \"pmids\": [\"21632929\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Neto2 co-expression with recombinant GluK2(Q) kainate receptors greatly reduces inward rectification (polyamine block) without altering calcium permeability. The extracellular LDLa domain of Neto2 is required for effects on desensitization but only partially for rectification, whereas the intracellular C-terminal domain (including positively charged residues) is required for reduction of rectification but not for effects on channel kinetics.\",\n      \"method\": \"Patch clamp electrophysiology in heterologous cells; domain deletion and point-mutation analysis of Neto2\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct electrophysiology with mutagenesis identifying distinct functional domains; multiple orthogonal mutations tested\",\n      \"pmids\": [\"22973017\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Neto2 regulates synaptic localization of GluK2-containing kainate receptors in the cerebellum; Neto2-null mice show ~40% decrease in GluK2-KARs at the postsynaptic density without change in total GluK2 levels. Neto2 directly interacts with the scaffolding protein GRIP via a PDZ-dependent interaction, and Neto2 co-expression increases the amount of GRIP associated with GluK2.\",\n      \"method\": \"PSD fractionation from Neto2-null mouse cerebellum, Co-immunoprecipitation (Neto2-GRIP), western blot\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP plus fractionation in knockout mouse; two orthogonal methods linking Neto2 to synaptic KAR localization\",\n      \"pmids\": [\"23236500\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Cryo-EM structures of homotetrameric GluK2 in complex with NETO2 at inhibited and desensitized states reveal variable stoichiometry (1 or 2 NETO2 subunits per tetramer). NETO2 accesses two broad faces of the receptor, intermolecularly crosslinking the lower lobe of ATD A/C, upper lobe of LBD B/D, and lower lobe of LBD A/C, thereby stabilizing gating kinetics. The NETO2 transmembrane helix is positioned proximal to the selectivity filter and competes with the amphiphilic H1 helix after M4 for interaction with an intracellular cap domain formed by M1-M2 linkers, explaining how NETO2 regulates rectification.\",\n      \"method\": \"Cryo-electron microscopy structure determination\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — high-resolution cryo-EM structures at multiple functional states providing atomic-level mechanistic detail, published in Nature\",\n      \"pmids\": [\"34552241\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Cryo-EM structures of rat GluK2 KAR in apo closed and agonist/PAM-activated open states, with and without Neto2, show that Neto2 binding does not alter individual or dimeric LBD behavior or ion channel conductance but prevents tightening of the interface between two LBD dimers during activation, thereby slowing deactivation kinetics.\",\n      \"method\": \"Time-resolved cryo-electron microscopy structure determination\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple cryo-EM structures at distinct functional states with and without Neto2, providing direct mechanistic insight into activation modulation\",\n      \"pmids\": [\"40846810\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"The M3-S2 gating linkers of KAR subunits are critical determinants of Neto2 modulation: individual residues in these linkers bidirectionally influence Neto2 modulation of desensitization in an agonist-specific manner. A single mutation in this domain abolishes Neto2 modulation of heteromeric receptor desensitization. Neto2 also alters cation sensitivity of KAR gating (implicating the D1 dimer interface), and this modulation is eliminated by M3-S2 linker mutations, functionally correlating these two discrete structural sites.\",\n      \"method\": \"Site-directed mutagenesis of KAR M3-S2 linkers combined with patch clamp electrophysiology in heterologous cells\",\n      \"journal\": \"The Journal of physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — systematic mutagenesis with electrophysiology identifying specific structural determinants; multiple mutations and agonists tested\",\n      \"pmids\": [\"26282342\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"The extracellular N-terminal region including the two CUB domains of Neto2 (vs. Neto1) is largely responsible for distinct regulatory effects on desensitization properties of GluK1 homomeric receptors, as determined using chimeric Neto1/Neto2 subunits.\",\n      \"method\": \"Chimeric Neto1/Neto2 subunit construction combined with patch clamp electrophysiology in HEK-293T cells\",\n      \"journal\": \"Neuropharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — chimeric protein approach with electrophysiology in single study; establishes domain importance but limited replication\",\n      \"pmids\": [\"26277340\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Neto2 serine 409 is phosphorylated by CaMKII and PKA both in vitro and in heterologous cells, and endogenous Neto2 Ser-409 phosphorylation is detected in brain. CaMKII-mediated Neto2 Ser-409 phosphorylation is reduced in the presence of GluK1 or GluK2. The phosphomimetic Neto2 S409D mutant (but not WT or S409A) impedes GluK1 trafficking to synapses, demonstrating that Neto2 Ser-409 phosphorylation restricts synaptic targeting of GluK1.\",\n      \"method\": \"Mass spectrometry identification of phosphorylation sites, in vitro kinase assays, phosphomimetic/phosphodeficient mutant transfection in neurons, synaptic fractionation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro kinase assay, endogenous phosphorylation validation, mutagenesis with functional synaptic localization readout; multiple orthogonal methods in single study\",\n      \"pmids\": [\"28717010\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Neto2 protein is highly expressed in neonatal DRG neurons and assembles with endogenous KARs to modify their gating. Neto2-/- adult DRG neurons display stunted neurite outgrowth. Neto2 expression in adult DRG is upregulated via MEK/ERK signaling and after sciatic nerve crush injury.\",\n      \"method\": \"Electrophysiology in DRG neurons from Neto2-/- mice, neurite outgrowth assays, MEK inhibitor pharmacology, sciatic nerve crush in vivo\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — loss-of-function electrophysiology in native neurons with knockout, mechanistic pathway (MEK/ERK) identified for regulation, in vivo injury model\",\n      \"pmids\": [\"28235897\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"In Neto2-/- mice, synaptosomal KAR GluK2/3 subunit abundance is reduced 20.8% in ventral hippocampus and 36.5% in medial prefrontal cortex, and GluK5 abundance is reduced 23.8% in ventral hippocampus and 16.9% in amygdala. These reductions are associated with higher fear expression and delayed fear extinction in cued fear conditioning.\",\n      \"method\": \"Synaptosomal fractionation western blot, behavioral fear conditioning in Neto2-/- mice, in situ hybridization for Neto2 expression\",\n      \"journal\": \"Neuropsychopharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — fractionation plus behavioral phenotype in knockout; single lab, no rescue experiment\",\n      \"pmids\": [\"30770891\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Neto2 is required for maturation of the amygdala PV interneuron network; Neto2-/- adult mice show reduced PV+PNN+ cells, reduced PV staining intensity, increased glutamatergic and reduced GABAergic transmission, and increased spine density in basal amygdala compared to wild-type. These structural and functional amygdala changes are associated with increased fear expression and delayed extinction.\",\n      \"method\": \"Immunohistochemistry for PV/PNN, whole-cell patch-clamp recordings in amygdala slices, spine density analysis, c-Fos immunostaining in Neto2-/- mice\",\n      \"journal\": \"eNeuro\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple electrophysiology and histology methods in knockout; single lab\",\n      \"pmids\": [\"32788298\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Soluble LRIG3 derived from glioma cells interacts with the CUB1 domain of NETO2 in tumor-associated macrophages (TAMs). This interaction suppresses M2 polarization of TAMs. NETO2 knockout blocks the inhibitory effect of sLRIG3 on M2 polarization and promotes GBM tumor growth; CUB1-deletion mutant NETO2 does not fully recover sLRIG3's suppressive effects.\",\n      \"method\": \"Mass spectrometry, Co-immunoprecipitation, NETO2 knockout and domain-deletion mutant rescue experiments in TAMs\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus MS identification of interaction, domain-deletion mutant rescue, knockout functional phenotype; single lab\",\n      \"pmids\": [\"36639372\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"GluK2-NETO2 (but not GluK2-NETO1) interaction is upregulated in ipsilateral dorsal horn neurons at 6 h post-incision. Intrathecal NETO2 siRNA pretreatment reduces pain hypersensitivity, decreases synaptic abundance of GluK2 and GluR1 (AMPA receptor subunit), and reduces PKCγ activation in ipsilateral dorsal horn, indicating that NETO2-GluK2 interaction drives PKCγ activation and synaptic AMPA receptor incorporation in postoperative pain.\",\n      \"method\": \"Co-immunoprecipitation in dorsal horn tissue, intrathecal siRNA knockdown, behavioral pain assays, western blot for synaptic fractions\",\n      \"journal\": \"Neuroscience letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP in native tissue plus siRNA knockdown with multiple readouts; single lab, single study\",\n      \"pmids\": [\"37544581\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"NETO2 slows the channel-opening rate of GluK2 homomeric receptors by ~7-fold and the channel-closing rate by ~3-fold (compared to ~2-fold slowing by NETO1 for both rates), demonstrating that NETO2 is a more impactful auxiliary subunit than NETO1 on GluK2 channel-opening kinetics.\",\n      \"method\": \"Laser-pulse photolysis combined with whole-cell recording in HEK-293 cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct kinetic measurement with a specialized laser photolysis technique; single lab, single study\",\n      \"pmids\": [\"41197725\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"NETO2 activates the PI3K/AKT/NF-κB/Snail signaling axis in gastric cancer cells via TNFRSF12A as a mediator, promoting invasion, migration, and EMT. Silencing NETO2 reduced phosphorylation of PI3K, AKT, NF-κB p65 and Snail expression; overexpression had opposite effects. TNFRSF12A was identified as the mediator linking NETO2 to this pathway.\",\n      \"method\": \"shRNA knockdown and overexpression in gastric cancer cells, western blot for pathway components, in vitro migration/invasion assays, in vivo metastasis model\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — pathway placement by western blot and KD/OE; no direct binding assay or reconstitution to confirm TNFRSF12A as mechanistic mediator\",\n      \"pmids\": [\"30770791\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"α5-nAChR physically interacts with NETO2 in lung adenocarcinoma cells, as demonstrated by Co-immunoprecipitation. Acetylcholine/nicotine stimulation via α5-nAChR upregulates NETO2, p-CaMKII, p-STAT3, and vimentin expression, and the α5-nAChR/NETO2 axis promotes LUAD cell proliferation, migration, and invasion.\",\n      \"method\": \"Co-immunoprecipitation, molecular docking, western blot, siRNA knockdown, proliferation and invasion assays\",\n      \"journal\": \"Cancer cell international\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP without reciprocal confirmation; molecular docking is computational; single lab\",\n      \"pmids\": [\"40001189\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"NETO2 is a single-pass transmembrane auxiliary protein that functions as an accessory subunit of kainate receptors (KARs) by directly associating with GluK1-5 subunits to slow desensitization and deactivation, increase recovery from desensitization, reduce polyamine-mediated inward rectification (through its intracellular C-terminal domain), and regulate synaptic KAR abundance via interactions with scaffolding proteins such as GRIP; cryo-EM structures reveal that NETO2 binds two faces of the KAR tetramer to prevent LBD dimer interface tightening during activation and positions its transmembrane helix near the selectivity filter to compete for an intracellular cap domain, thereby modulating gating and rectification. In addition, NETO2 binds and stabilizes the active oligomeric form of the neuronal K+-Cl- cotransporter KCC2, maintaining its surface expression and Ser940 phosphorylation to preserve the chloride gradient required for GABAergic inhibition, with loss of NETO2 causing depolarized EGABA and seizure susceptibility. NETO2 is also phosphorylated at Ser409 by CaMKII/PKA, which restricts synaptic targeting of GluK1.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"NETO2 is a single-pass transmembrane auxiliary protein that shapes neuronal excitability by acting as an accessory subunit of kainate-type glutamate receptors (KARs) and as a binding partner of the K+-Cl- cotransporter KCC2 [#2, #0]. As a KAR auxiliary subunit, NETO2 associates with GluK1-, GluK2-, and GluK5-containing receptors to slow desensitization and deactivation, accelerate recovery from desensitization, and slow channel opening and closing kinetics [#2, #15]. Its extracellular CUB and LDLa domains govern effects on desensitization, whereas its intracellular positively charged C-terminal domain reduces polyamine-mediated inward rectification of GluK2 receptors [#3, #8]. Cryo-EM structures show NETO2 binding across two faces of the GluK2 tetramer with variable stoichiometry, crosslinking the ATD and LBD lobes to prevent tightening of the inter-dimer LBD interface during activation, while its transmembrane helix sits near the selectivity filter and competes for an intracellular cap domain to control rectification; the gating effects map functionally to the M3-S2 linkers [#5, #6, #7]. NETO2 also controls synaptic KAR abundance, both by interacting with the PDZ scaffold GRIP to localize GluK2 receptors at the postsynaptic density and through CaMKII/PKA phosphorylation at Ser409, which restricts synaptic targeting of GluK1 [#4, #9]. Independently of its KAR role, NETO2 binds and stabilizes the active oligomeric form of KCC2, maintaining KCC2 surface expression and Ser940 phosphorylation; loss of NETO2 depolarizes EGABA, weakens synaptic inhibition, and increases seizure susceptibility [#0, #1]. These activities give NETO2 broader physiological roles in fear behavior, amygdala interneuron network maturation, DRG neurite outgrowth, and nociception [#11, #12, #10, #14]. NETO2 has additionally been implicated in cancer-related signaling outside the nervous system [#13, #16].\"\n,\n  \"teleology\": [\n    {\n      \"year\": 2011,\n      \"claim\": \"Established NETO2 as a bona fide auxiliary subunit of kainate receptors, answering whether it directly modifies KAR gating rather than merely co-expressing with them.\",\n      \"evidence\": \"Rapid-application outside-out patch clamp of recombinant KARs in heterologous cells and neurons across multiple GluK subunit compositions\",\n      \"pmids\": [\"21632929\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve which NETO2 domains mediate each gating effect\", \"Native stoichiometry and structural basis unknown\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Resolved the domain logic of NETO2 modulation, separating effects on gating kinetics from effects on rectification.\",\n      \"evidence\": \"Patch clamp with domain deletion and point mutagenesis showing the LDLa domain controls desensitization and the charged C-terminus controls polyamine block of GluK2(Q)\",\n      \"pmids\": [\"22973017\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural mechanism for C-terminal rectification effect not yet visualized\", \"Did not address synaptic localization\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Linked NETO2 to synaptic KAR abundance, showing it does more than tune gating by also positioning receptors at synapses.\",\n      \"evidence\": \"PSD fractionation from Neto2-null cerebellum and reciprocal Co-IP identifying a PDZ-dependent NETO2-GRIP interaction\",\n      \"pmids\": [\"23236500\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which GRIP retains receptors at the PSD not dissected\", \"Regulation of the interaction unknown\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Identified a KAR-independent role for NETO2 in chloride homeostasis by showing it binds and stabilizes the active oligomeric KCC2.\",\n      \"evidence\": \"Reciprocal Co-IP and gramicidin perforated patch clamp in hippocampal neurons of Neto2-null mice\",\n      \"pmids\": [\"23401525\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of NETO2-KCC2 binding undefined\", \"How NETO2 selectively recognizes the oligomeric form unknown\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Connected the NETO2-KCC2 interaction to inhibitory tone and seizure threshold in vivo, establishing physiological consequence.\",\n      \"evidence\": \"Perforated patch clamp, surface biotinylation, Ser940 phospho-blot, and PTZ seizure assays in Neto2-/- mice\",\n      \"pmids\": [\"26441539\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism linking NETO2 loss to reduced Ser940 phosphorylation not established\", \"Kinase/phosphatase pathway not identified\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Mapped the structural determinants on the KAR side, identifying the M3-S2 gating linkers and D1 dimer interface as the substrate of NETO2 modulation.\",\n      \"evidence\": \"Systematic M3-S2 linker mutagenesis with patch clamp and cation-sensitivity analysis in heterologous cells\",\n      \"pmids\": [\"26282342\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Atomic-level contact map not yet available\", \"Agonist-specificity mechanism unresolved\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Attributed NETO2-versus-NETO1 functional divergence to the CUB-domain-containing N-terminal region.\",\n      \"evidence\": \"Chimeric Neto1/Neto2 constructs with patch clamp in HEK-293T cells on GluK1 homomers\",\n      \"pmids\": [\"26277340\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-study chimera approach; specific CUB residues not pinpointed\", \"Limited replication\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Showed NETO2 is itself a regulated protein, with Ser409 phosphorylation by CaMKII/PKA gating GluK1 synaptic targeting.\",\n      \"evidence\": \"Mass-spec site identification, in vitro kinase assays, and phosphomimetic/phosphodeficient mutant synaptic fractionation in neurons\",\n      \"pmids\": [\"28717010\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Phosphatase that reverses Ser409 not identified\", \"Whether KCC2 association is similarly regulated unknown\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Extended NETO2 function to peripheral sensory neurons, linking it to KAR gating and injury-induced neurite outgrowth.\",\n      \"evidence\": \"Electrophysiology in Neto2-/- DRG neurons, neurite outgrowth assays, MEK inhibition, and sciatic nerve crush in vivo\",\n      \"pmids\": [\"28235897\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism linking NETO2 to neurite outgrowth unclear\", \"Whether outgrowth defect is KAR-dependent untested\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Tied region-specific synaptic KAR loss in Neto2-null mice to fear behavior abnormalities.\",\n      \"evidence\": \"Synaptosomal fractionation western blot and cued fear conditioning in Neto2-/- mice\",\n      \"pmids\": [\"30770891\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No rescue experiment to establish causality\", \"Single lab\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Implicated NETO2 in amygdala parvalbumin interneuron network maturation underlying fear circuit function.\",\n      \"evidence\": \"PV/PNN immunohistochemistry, amygdala slice patch clamp, spine density and c-Fos analysis in Neto2-/- mice\",\n      \"pmids\": [\"32788298\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether interneuron phenotype is cell-autonomous unknown\", \"Mechanistic link to KAR or KCC2 roles not dissected\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Showed a NETO2-GluK2 axis drives postoperative pain hypersensitivity via PKCγ activation and AMPA receptor synaptic incorporation.\",\n      \"evidence\": \"Co-IP in dorsal horn tissue, intrathecal NETO2 siRNA, behavioral pain assays and synaptic-fraction western blots\",\n      \"pmids\": [\"37544581\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism connecting GluK2-NETO2 to PKCγ unresolved\", \"Single study\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Revealed a non-neuronal role in which the NETO2 CUB1 domain receives glioma-derived sLRIG3 to suppress macrophage M2 polarization.\",\n      \"evidence\": \"Mass spec, Co-IP, NETO2 knockout and CUB1-deletion rescue in tumor-associated macrophages\",\n      \"pmids\": [\"36639372\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Downstream signaling from CUB1 engagement unmapped\", \"Single lab\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Quantified NETO2's impact on GluK2 channel opening/closing rates, showing it is a more potent kinetic modulator than NETO1.\",\n      \"evidence\": \"Laser-pulse photolysis with whole-cell recording in HEK-293 cells\",\n      \"pmids\": [\"41197725\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single study with specialized technique\", \"Structural correlate of rate differences not defined\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Provided time-resolved structural mechanism by which NETO2 slows deactivation: preventing tightening of the inter-LBD-dimer interface during activation.\",\n      \"evidence\": \"Time-resolved cryo-EM of GluK2 in apo-closed and agonist/PAM-activated states with and without Neto2\",\n      \"pmids\": [\"40846810\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not address KCC2-bound structure\", \"Heteromeric KAR-NETO2 structures not resolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How NETO2's two distinct partnerships — KARs and KCC2 — are coordinated, regulated, and integrated within a single neuron remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structure of the NETO2-KCC2 complex\", \"Phosphoregulation of KCC2 binding undefined\", \"Whether cancer-associated signaling roles share the neuronal binding interfaces unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [2, 3, 5, 6, 15]},\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [2, 7]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 1, 5]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-112316\", \"supporting_discovery_ids\": [2, 0]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [2, 3]}\n    ],\n    \"complexes\": [\"Kainate receptor complex (GluK1-5/NETO2)\", \"KCC2-NETO2 complex\"],\n    \"partners\": [\"GRIK1\", \"GRIK2\", \"GRIK5\", \"SLC12A5\", \"GRIP1\", \"CAMK2A\", \"PRKACA\", \"LRIG3\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}