Affinage

NETO2

Neuropilin and tolloid-like protein 2 · UniProt Q8NC67

Length
525 aa
Mass
59.4 kDa
Annotated
2026-06-10
32 papers in source corpus 18 papers cited in narrative 18 extracted findings
Cross-family judge vs UniProt: Affinage preferred faithfulness: 8/8 claims corpus-supported (100%)

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

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).

Mechanistic history

Synthesis pass · year-by-year structured walk · 15 steps
  1. 2011 High

    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

    PMID:21632929

    Open questions at the time
    • Did not resolve which NETO2 domains mediate each gating effect
    • Native stoichiometry and structural basis unknown
  2. 2012 High

    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)

    PMID:22973017

    Open questions at the time
    • Structural mechanism for C-terminal rectification effect not yet visualized
    • Did not address synaptic localization
  3. 2012 High

    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

    PMID:23236500

    Open questions at the time
    • Mechanism by which GRIP retains receptors at the PSD not dissected
    • Regulation of the interaction unknown
  4. 2013 High

    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

    PMID:23401525

    Open questions at the time
    • Structural basis of NETO2-KCC2 binding undefined
    • How NETO2 selectively recognizes the oligomeric form unknown
  5. 2015 High

    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

    PMID:26441539

    Open questions at the time
    • Mechanism linking NETO2 loss to reduced Ser940 phosphorylation not established
    • Kinase/phosphatase pathway not identified
  6. 2015 High

    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

    PMID:26282342

    Open questions at the time
    • Atomic-level contact map not yet available
    • Agonist-specificity mechanism unresolved
  7. 2015 Medium

    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

    PMID:26277340

    Open questions at the time
    • Single-study chimera approach; specific CUB residues not pinpointed
    • Limited replication
  8. 2017 High

    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

    PMID:28717010

    Open questions at the time
    • Phosphatase that reverses Ser409 not identified
    • Whether KCC2 association is similarly regulated unknown
  9. 2017 High

    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

    PMID:28235897

    Open questions at the time
    • Mechanism linking NETO2 to neurite outgrowth unclear
    • Whether outgrowth defect is KAR-dependent untested
  10. 2019 Medium

    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

    PMID:30770891

    Open questions at the time
    • No rescue experiment to establish causality
    • Single lab
  11. 2020 Medium

    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

    PMID:32788298

    Open questions at the time
    • Whether interneuron phenotype is cell-autonomous unknown
    • Mechanistic link to KAR or KCC2 roles not dissected
  12. 2023 Medium

    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

    PMID:37544581

    Open questions at the time
    • Mechanism connecting GluK2-NETO2 to PKCγ unresolved
    • Single study
  13. 2023 Medium

    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

    PMID:36639372

    Open questions at the time
    • Downstream signaling from CUB1 engagement unmapped
    • Single lab
  14. 2025 Medium

    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

    PMID:41197725

    Open questions at the time
    • Single study with specialized technique
    • Structural correlate of rate differences not defined
  15. 2025 High

    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

    PMID:40846810

    Open questions at the time
    • Does not address KCC2-bound structure
    • Heteromeric KAR-NETO2 structures not resolved

Open questions

Synthesis pass · forward-looking unresolved questions
  • How NETO2's two distinct partnerships — KARs and KCC2 — are coordinated, regulated, and integrated within a single neuron remains unresolved.
  • 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

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0098772 molecular function regulator activity 5 GO:0060089 molecular transducer activity 2 GO:0060090 molecular adaptor activity 1
Localization
GO:0005886 plasma membrane 3
Pathway
R-HSA-112316 Neuronal System 2 R-HSA-162582 Signal Transduction 2
Partners
CAMK2AGRIK1GRIK2GRIK5GRIP1LRIG3PRKACASLC12A5
Complex memberships
KCC2-NETO2 complexKainate receptor complex (GluK1-5/NETO2)

Evidence

Reading pass · 18 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2013 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. Co-immunoprecipitation, gramicidin perforated patch clamp, Neto2-null mouse model Proceedings of the National Academy of Sciences of the United States of America High 23401525
2015 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. Gramicidin perforated patch clamp recordings, surface biotinylation, western blot, PTZ seizure induction in Neto2-/- mice Frontiers in cellular neuroscience High 26441539
2011 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. Rapid application outside-out patch clamp recordings in heterologous cells and neurons The Journal of neuroscience High 21632929
2012 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. Patch clamp electrophysiology in heterologous cells; domain deletion and point-mutation analysis of Neto2 The Journal of neuroscience High 22973017
2012 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. PSD fractionation from Neto2-null mouse cerebellum, Co-immunoprecipitation (Neto2-GRIP), western blot PloS one High 23236500
2021 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. Cryo-electron microscopy structure determination Nature High 34552241
2025 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. Time-resolved cryo-electron microscopy structure determination Nature structural & molecular biology High 40846810
2015 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. Site-directed mutagenesis of KAR M3-S2 linkers combined with patch clamp electrophysiology in heterologous cells The Journal of physiology High 26282342
2015 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. Chimeric Neto1/Neto2 subunit construction combined with patch clamp electrophysiology in HEK-293T cells Neuropharmacology Medium 26277340
2017 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. Mass spectrometry identification of phosphorylation sites, in vitro kinase assays, phosphomimetic/phosphodeficient mutant transfection in neurons, synaptic fractionation The Journal of biological chemistry High 28717010
2017 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. Electrophysiology in DRG neurons from Neto2-/- mice, neurite outgrowth assays, MEK inhibitor pharmacology, sciatic nerve crush in vivo The Journal of neuroscience High 28235897
2019 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. Synaptosomal fractionation western blot, behavioral fear conditioning in Neto2-/- mice, in situ hybridization for Neto2 expression Neuropsychopharmacology Medium 30770891
2020 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. Immunohistochemistry for PV/PNN, whole-cell patch-clamp recordings in amygdala slices, spine density analysis, c-Fos immunostaining in Neto2-/- mice eNeuro Medium 32788298
2023 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. Mass spectrometry, Co-immunoprecipitation, NETO2 knockout and domain-deletion mutant rescue experiments in TAMs Cell death & disease Medium 36639372
2023 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. Co-immunoprecipitation in dorsal horn tissue, intrathecal siRNA knockdown, behavioral pain assays, western blot for synaptic fractions Neuroscience letters Medium 37544581
2025 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. Laser-pulse photolysis combined with whole-cell recording in HEK-293 cells The Journal of biological chemistry Medium 41197725
2019 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. shRNA knockdown and overexpression in gastric cancer cells, western blot for pathway components, in vitro migration/invasion assays, in vivo metastasis model Cell death & disease Low 30770791
2025 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. Co-immunoprecipitation, molecular docking, western blot, siRNA knockdown, proliferation and invasion assays Cancer cell international Low 40001189

Source papers

Stage 0 corpus · 32 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2021 NETO2 promotes esophageal cancer progression by inducing proliferation and metastasis via PI3K/AKT and ERK pathway. International journal of biological sciences 67 33390848
2013 Neto2 is a KCC2 interacting protein required for neuronal Cl- regulation in hippocampal neurons. Proceedings of the National Academy of Sciences of the United States of America 65 23401525
2019 NETO2 promotes invasion and metastasis of gastric cancer cells via activation of PI3K/Akt/NF-κB/Snail axis and predicts outcome of the patients. Cell death & disease 60 30770791
2011 Neto2 modulation of kainate receptors with different subunit compositions. The Journal of neuroscience : the official journal of the Society for Neuroscience 53 21632929
2020 Exosomal lncRNA FAM225A accelerates esophageal squamous cell carcinoma progression and angiogenesis via sponging miR-206 to upregulate NETO2 and FOXP1 expression. Cancer medicine 48 33006432
2012 The auxiliary subunits Neto1 and Neto2 reduce voltage-dependent inhibition of recombinant kainate receptors. The Journal of neuroscience : the official journal of the Society for Neuroscience 47 22973017
2012 Neto2 interacts with the scaffolding protein GRIP and regulates synaptic abundance of kainate receptors. PloS one 36 23236500
2021 Kainate receptor modulation by NETO2. Nature 34 34552241
2017 Neto2 Assembles with Kainate Receptors in DRG Neurons during Development and Modulates Neurite Outgrowth in Adult Sensory Neurons. The Journal of neuroscience : the official journal of the Society for Neuroscience 26 28235897
2015 Neto2-null mice have impaired GABAergic inhibition and are susceptible to seizures. Frontiers in cellular neuroscience 21 26441539
2015 The auxiliary subunits Neto1 and Neto2 have distinct, subunit-dependent effects at recombinant GluK1- and GluK2-containing kainate receptors. Neuropharmacology 20 26277340
2015 Identification of critical functional determinants of kainate receptor modulation by auxiliary protein Neto2. The Journal of physiology 19 26282342
2019 NETO2 promotes pancreatic cancer cell proliferation, invasion and migration via activation of the STAT3 signaling pathway. Cancer management and research 17 31239769
2019 Kainate receptor auxiliary subunit NETO2 is required for normal fear expression and extinction. Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology 15 30770891
2021 Long non-coding RNA SNHG17 promotes lung adenocarcinoma progression by targeting the microRNA-193a-5p/NETO2 axis. Oncology letters 14 34671432
2017 Upregulation of NETO2 gene in colorectal cancer. BMC genetics 14 29297384
2020 NETO2 Is Deregulated in Breast, Prostate, and Colorectal Cancer and Participates in Cellular Signaling. Frontiers in genetics 13 33362854
2023 Glioma-derived LRIG3 interacts with NETO2 in tumor-associated macrophages to modulate microenvironment and suppress tumor growth. Cell death & disease 12 36639372
2017 Phosphorylation of the kainate receptor (KAR) auxiliary subunit Neto2 at serine 409 regulates synaptic targeting of the KAR subunit GluK1. The Journal of biological chemistry 10 28717010
2020 Kainate Receptor Auxiliary Subunit NETO2-Related Cued Fear Conditioning Impairments Associate with Defects in Amygdala Development and Excitability. eNeuro 9 32788298
2016 Pharmacological Modulation of GluK1 and GluK2 by NETO1, NETO2, and PSD95. Assay and drug development technologies 8 26991362
2022 TYMSOS-miR-101-3p-NETO2 axis promotes osteosarcoma progression. Molecular and cellular probes 7 36509232
2021 TTN-AS1 accelerates the growth and migration of nasopharyngeal carcinoma cells via targeting miR-876-5p/NETO2. Molecular therapy oncolytics 7 35229031
2019 Reducing NETO2 expression prevents human nasopharyngeal carcinoma (NPC) progression by suppressing metastasis and inducing apoptosis. Biochemical and biophysical research communications 7 30975469
2025 Activation of kainate receptor GluK2-Neto2 complex. Nature structural & molecular biology 6 40846810
2016 Neto2 Influences on Kainate Receptor Pharmacology and Function. Basic & clinical pharmacology & toxicology 6 26928870
2023 NETO2 promotes melanoma progression via activation of the Ca2+/CaMKII signaling pathway. Frontiers of medicine 4 36738427
2025 α5-nAChR/NETO2 contributed to chronic stress-promoted lung adenocarcinoma progression. Cancer cell international 3 40001189
2025 Targeting NETO2 suppresses cell proliferation, invasion, and migration and inactivates the STAT3/C-MYC pathway in hepatocellular carcinoma. World journal of surgical oncology 2 40158169
2023 NETO2-GluK2 interaction contributes to postoperative pain hypersensitivity through inducing PKCγ activation and synaptic incorporation of AMPA receptor GluR1 subunits in rat dorsal horn. Neuroscience letters 1 37544581
2026 The role of NETO2 in neurological disorders and cancer: From molecular function to clinical relevance. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie 0 41714211
2025 A comparative study of NETO1 and NETO2 on channel-opening kinetics of GluK2 kainate receptors. The Journal of biological chemistry 0 41197725

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