{"gene":"LNX2","run_date":"2026-06-10T02:59:50","timeline":{"discoveries":[{"year":2015,"finding":"LNX2 RING domain, flanked by two zinc-binding motifs (Zn-RING-Zn), is required for E3 ubiquitin ligase activity; the N-terminal zinc finger adopts a novel open-circle Cys2His2 conformation and is indispensable for LNX2 activity and stability. The Zn-RING-Zn domain dimerizes, undergoes autoubiquitination with all seven ubiquitin chain linkages, and directly ubiquitinates Numb.","method":"Crystal structure of Zn-RING-Zn domain; active-site mutagenesis; in vitro ubiquitination assay with full-length LNX2","journal":"Oncotarget","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure combined with mutagenesis and in vitro reconstitution of ubiquitination activity in a single rigorous study","pmids":["26451611"],"is_preprint":false},{"year":2005,"finding":"The intracellular tail of the cell-surface protein CAR (coxsackievirus and adenovirus receptor) directly interacts with LNX2 both in vivo and in vitro; interaction domains were mapped by truncation analysis and affinity chromatography, and LNX2 organizes a protein complex at specific subcellular sites.","method":"Yeast two-hybrid screen; co-immunoprecipitation; in vitro binding/affinity chromatography; truncation analysis","journal":"Experimental cell research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP plus in vitro binding and domain mapping in a single study","pmids":["15979067"],"is_preprint":false},{"year":2011,"finding":"LNX2 PDZ domains interact with the cytosolic C-terminal valine motif of CD8α; LNX2 promotes ubiquitylation of CD8α, redistribution of LNX2 from cytosol to plasma membrane, and subsequent CD8α downregulation from the plasma membrane, transport to lysosomes, and degradation.","method":"Co-immunoprecipitation in human T cells; in vitro ubiquitylation assay; heterologous expression with domain-truncation mapping; live-cell imaging/fractionation","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — in vitro ubiquitylation reconstitution, domain mapping, and cellular degradation assay combined in one study","pmids":["22045731"],"is_preprint":false},{"year":2013,"finding":"RNAi silencing of LNX2 in colorectal cancer cell lines reduces NOTCH levels, downregulates TCF7L2, and markedly reduces WNT signaling, placing LNX2 upstream of both NOTCH and WNT/β-catenin pathways.","method":"RNAi knockdown followed by global expression profiling; cell viability assay","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — RNAi plus transcriptome profiling in multiple CRC lines; no in vitro reconstitution","pmids":["23319804"],"is_preprint":false},{"year":2015,"finding":"LNX2 is required for osteoclastogenesis; its knockdown in bone marrow macrophages accumulates Numb, promotes Notch2 degradation, reduces Hes1 expression, attenuates M-CSF-induced ERK/AKT activation and RANKL-stimulated NF-κB/JNK signaling, and accelerates osteoclast apoptosis.","method":"Lentiviral shRNA knockdown in bone marrow macrophages; Western blot for pathway components; osteoclast differentiation assay","journal":"Calcified tissue international","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function with multiple defined signaling readouts in primary cells, single lab","pmids":["25712254"],"is_preprint":false},{"year":2014,"finding":"LNX2 interacts with Caspr4 (CNTNAP4) in a PDZ domain-dependent manner in neural progenitor cells; LNX2 promotes neuronal differentiation and is epistatic to Caspr4 — overexpression of LNX2 rescues the differentiation defect caused by Caspr4 knockdown, whereas C4ICD cannot rescue LNX2 knockdown.","method":"Co-immunoprecipitation; PDZ domain interaction mapping; lentiviral knockdown and overexpression in mouse NPCs; rescue epistasis experiments","journal":"Stem cells and development","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP plus genetic epistasis rescue experiments, single lab","pmids":["25279559"],"is_preprint":false},{"year":2018,"finding":"LNX1 and LNX2 localize at connexin36 (Cx36)-containing neuronal gap junctions in rodent brain; both proteins co-immunoprecipitate with Cx36 and bind Cx36 via their second PDZ domain. Expression of E3 ligase-competent LNX1/2 isoforms in cultured cells causes loss of Cx36-containing gap junctions, whereas ligase-inactive isoforms do not, indicating LNX proteins ubiquitinate Cx36 to promote its internalization.","method":"Immunofluorescence colocalization in wild-type vs. LNX-null mouse brain; co-immunoprecipitation; GST pull-down with individual PDZ domains; cotransfection with wild-type vs. ligase-dead LNX isoforms","journal":"The European journal of neuroscience","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, PDZ domain pull-down, in-cell functional assay with ligase-dead controls, validated in null mice","pmids":["30295974"],"is_preprint":false},{"year":2019,"finding":"LNX1 and LNX2 RING finger domains ubiquitinate the glycine transporter GlyT2 at a cytoplasmic C-terminal lysine cluster (K751, K773, K787, K791), regulating GlyT2 expression levels and transport activity. Genetic deletion of endogenous LNX2 in spinal cord neurons increases GlyT2 expression, and LNX2 is required for PKC-mediated control of GlyT2 transport.","method":"Unbiased substrate screen; in vitro ubiquitination assay with RING domain; site-directed mutagenesis of GlyT2 lysines; LNX2 knockout primary spinal cord neurons; PKC regulation assay","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with mutagenesis plus KO primary neurons, single lab, multiple orthogonal methods","pmids":["31628376"],"is_preprint":false},{"year":2015,"finding":"In zebrafish, Lnx2a/b double loss-of-function inhibits exocrine pancreatic cell differentiation; this phenotype is rescued by inhibition of Numb expression, placing Lnx2 upstream of Numb in a pathway that fine-tunes Notch signaling during pancreatic cell-type specification. Loss of Lnx2a/b also reduces the number of Notch-active cells in the pancreas.","method":"Splice-blocking morpholino knockdown; frameshift null mutation; Numb inhibition rescue epistasis; Notch reporter assay in zebrafish embryos","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis with morpholino and null mutation plus Numb rescue in zebrafish, single lab","pmids":["26392552"],"is_preprint":false},{"year":2023,"finding":"LNX2 knockdown in mouse preimplantation embryos blocks blastocyst formation without affecting morula development; knockdown increases expression of Oct4 and genes involved in Notch and Hippo signaling, suggesting LNX2 normally suppresses these pathways to permit inner cell mass lineage specification.","method":"siRNA knockdown in mouse preimplantation embryos; transcript analysis of lineage and signaling genes","journal":"International journal of molecular sciences","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single knockdown experiment with transcript readouts, no biochemical pathway reconstitution, single lab","pmids":["36674899"],"is_preprint":false},{"year":2023,"finding":"LNX2 is required for ghrelin-induced neuronal differentiation of adipose-derived mesenchymal stem cells; LNX2 silencing suppresses nuclear translocation of β-catenin and reduces WNT/β-catenin transcriptional activity as measured by luciferase reporter assay.","method":"siRNA knockdown; β-catenin nuclear translocation imaging; luciferase reporter assay for WNT/β-catenin transcriptional activity","journal":"Journal of bioenergetics and biomembranes","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, single knockdown approach with reporter assay, no biochemical reconstitution","pmids":["37237241"],"is_preprint":false},{"year":2026,"finding":"In mouse ameloblasts at the maturation stage, LNX2 co-localizes with Numb in aggregated intracellular vesicles and with the lysosomal marker LAMP-1, consistent with LNX2-mediated ubiquitination and lysosomal degradation of Numb and the tight-junction protein claudin-7 at these cells.","method":"Double immunofluorescence staining in paraformaldehyde-fixed, decalcified mouse incisors with antibodies against LNX2, Numb, LAMP-1, and claudin-7","journal":"Journal of oral biosciences","confidence":"Low","confidence_rationale":"Tier 3 / Weak — co-localization by immunofluorescence only, no biochemical interaction or ubiquitination assay confirmed, single lab","pmids":["41714050"],"is_preprint":false}],"current_model":"LNX2 is a RING/PDZ-domain E3 ubiquitin ligase whose catalytic Zn-RING-Zn domain (structurally resolved and mutagenically validated) ubiquitinates multiple substrates including Numb, CD8α, connexin36, and GlyT2, thereby controlling Notch/WNT signaling, immune-receptor surface levels, neuronal gap-junction plasticity, and presynaptic glycine transport, while its PDZ domains scaffold interactions with partners such as CAR and Caspr4 at specific subcellular sites."},"narrative":{"mechanistic_narrative":"LNX2 is a RING/PDZ-domain E3 ubiquitin ligase that controls cell-surface receptor levels and the strength of Notch and WNT signaling across diverse developmental and physiological contexts [PMID:26451611, PMID:23319804]. Its catalytic activity resides in a Zn-RING-Zn module in which the N-terminal zinc finger adopts a novel open-circle Cys2His2 conformation indispensable for activity and stability; this domain dimerizes, autoubiquitinates with all seven ubiquitin chain linkages, and directly ubiquitinates the Notch regulator Numb [PMID:26451611]. By targeting Numb, LNX2 acts upstream of Notch and WNT/β-catenin signaling, a role demonstrated in colorectal cancer cells where its loss reduces NOTCH and TCF7L2 levels and WNT activity [PMID:23319804], and in osteoclast precursors where its knockdown accumulates Numb and promotes Notch2 degradation [PMID:25712254]. The PDZ domains serve as substrate-recruitment and scaffolding modules: they bind the C-terminal motifs of CD8α to drive its ubiquitylation, lysosomal transport, and surface downregulation [PMID:22045731], and bind connexin36 via the second PDZ domain to promote ubiquitination and internalization of neuronal gap junctions [PMID:30295974]. LNX2 additionally ubiquitinates the glycine transporter GlyT2 at a defined C-terminal lysine cluster to regulate presynaptic glycine transport, and is required for PKC-mediated control of GlyT2 [PMID:31628376]. Through PDZ-dependent interactions with partners including CAR and Caspr4, LNX2 organizes protein complexes at specific subcellular sites and promotes neuronal differentiation [PMID:15979067, PMID:25279559].","teleology":[{"year":2005,"claim":"Established LNX2 as a PDZ-scaffold that engages a cell-surface receptor tail, raising the question of what it does to bound partners.","evidence":"Yeast two-hybrid, reciprocal Co-IP, and in vitro binding with domain mapping of the CAR intracellular tail","pmids":["15979067"],"confidence":"Medium","gaps":["No catalytic consequence of the CAR interaction shown","Functional role of the LNX2-organized complex undefined"]},{"year":2011,"claim":"Showed LNX2 PDZ domains recruit a substrate (CD8α) for ubiquitylation and lysosomal degradation, linking scaffolding to receptor turnover.","evidence":"Co-IP in T cells, in vitro ubiquitylation, domain-truncation mapping, and fractionation/imaging of CD8α downregulation","pmids":["22045731"],"confidence":"High","gaps":["Physiological context of CD8α regulation in T cells not established","E2 partner not defined"]},{"year":2013,"claim":"Placed LNX2 upstream of both NOTCH and WNT/β-catenin signaling in a disease context, broadening its role beyond single-substrate turnover.","evidence":"RNAi knockdown with global expression profiling in colorectal cancer lines","pmids":["23319804"],"confidence":"Medium","gaps":["No biochemical reconstitution linking LNX2 catalysis to pathway output","Direct substrate mediating WNT effect not identified"]},{"year":2014,"claim":"Demonstrated PDZ-dependent binding to Caspr4 and genetic epistasis driving neuronal differentiation, establishing a developmental signaling role.","evidence":"Reciprocal Co-IP, PDZ mapping, and knockdown/overexpression rescue epistasis in mouse NPCs","pmids":["25279559"],"confidence":"Medium","gaps":["Whether Caspr4 is a ubiquitination substrate unresolved","Downstream effector pathway not defined"]},{"year":2015,"claim":"Resolved the catalytic architecture, showing a novel open-circle Zn-RING-Zn fold that dimerizes, autoubiquitinates with all linkage types, and directly ubiquitinates Numb.","evidence":"Crystal structure of Zn-RING-Zn domain, active-site mutagenesis, and in vitro ubiquitination with full-length LNX2","pmids":["26451611"],"confidence":"High","gaps":["Linkage-type selectivity in cells not defined","Regulation of dimerization in vivo unknown"]},{"year":2015,"claim":"Connected LNX2 to physiological Notch tuning via Numb in two systems: osteoclastogenesis in mammals and pancreatic cell specification in zebrafish.","evidence":"shRNA knockdown with pathway Western blots in bone marrow macrophages; morpholino/null mutation with Numb-inhibition rescue and Notch reporter in zebrafish","pmids":["25712254","26392552"],"confidence":"Medium","gaps":["Direct in vivo Numb ubiquitination not shown in these tissues","Contribution of other substrates not assessed"]},{"year":2018,"claim":"Established connexin36 as a PDZ-bound substrate whose ubiquitination by LNX2 controls gap-junction internalization in neurons.","evidence":"Colocalization in LNX-null vs wild-type mouse brain, reciprocal Co-IP, PDZ-domain GST pull-down, and ligase-dead controls in cotransfection","pmids":["30295974"],"confidence":"High","gaps":["Functional consequence for electrical synapse plasticity in vivo not measured","Redundancy with LNX1 not fully separated"]},{"year":2019,"claim":"Identified GlyT2 as a RING-domain substrate ubiquitinated at a defined lysine cluster, placing LNX2 in presynaptic glycine transport control.","evidence":"Unbiased substrate screen, in vitro ubiquitination, GlyT2 lysine mutagenesis, and LNX2-knockout primary spinal cord neurons with PKC assay","pmids":["31628376"],"confidence":"High","gaps":["Mechanism linking PKC signaling to LNX2 activity undefined","In vivo behavioral relevance not tested"]},{"year":2023,"claim":"Extended LNX2's pathway-suppressive role to early embryonic lineage decisions and stem-cell neuronal differentiation via Notch/Hippo and WNT/β-catenin readouts.","evidence":"siRNA knockdown in mouse preimplantation embryos with lineage transcript analysis; siRNA with β-catenin translocation imaging and WNT luciferase reporter in adipose-derived MSCs","pmids":["36674899","37237241"],"confidence":"Low","gaps":["Single knockdown approach with no biochemical reconstitution","Direct substrate mediating these effects not identified"]},{"year":2026,"claim":"Provided localization evidence consistent with LNX2-mediated lysosomal degradation of Numb and claudin-7 in maturation-stage ameloblasts.","evidence":"Double immunofluorescence for LNX2, Numb, LAMP-1, and claudin-7 in mouse incisors","pmids":["41714050"],"confidence":"Low","gaps":["Colocalization only; no biochemical interaction or ubiquitination assay","Claudin-7 as a direct substrate not confirmed"]},{"year":null,"claim":"How LNX2 substrate selection, ubiquitin-linkage choice, and PDZ-domain recruitment are integrated and regulated across its distinct physiological roles remains unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No unified model of substrate prioritization in vivo","Upstream regulation of LNX2 catalytic activity largely unknown","Functional separation from paralog LNX1 incomplete"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[0,2,7]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,2,6,7]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[1,2,5,6]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[2,6]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[2]},{"term_id":"GO:0005764","term_label":"lysosome","supporting_discovery_ids":[2]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[3,4]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,2,7]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[5,8]}],"complexes":[],"partners":["NUMB","CD8A","GJD2","SLC6A5","CXADR","CNTNAP4"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q8N448","full_name":"Ligand of Numb protein X 2","aliases":["Numb-binding protein 2","PDZ domain-containing RING finger protein 1"],"length_aa":690,"mass_kda":76.0,"function":"","subcellular_location":"","url":"https://www.uniprot.org/uniprotkb/Q8N448/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/LNX2","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/LNX2","total_profiled":1310},"omim":[{"mim_id":"613717","title":"TREACHER COLLINS SYNDROME 2; TCS2","url":"https://www.omim.org/entry/613717"},{"mim_id":"613715","title":"POLYMERASE I, RNA, SUBUNIT D; POLR1D","url":"https://www.omim.org/entry/613715"},{"mim_id":"609733","title":"LIGAND OF NUMB PROTEIN X2; LNX2","url":"https://www.omim.org/entry/609733"},{"mim_id":"609730","title":"PDZ DOMAIN-CONTAINING RING FINGER PROTEIN 4; PDZRN4","url":"https://www.omim.org/entry/609730"},{"mim_id":"609729","title":"PDZ DOMAIN-CONTAINING RING FINGER PROTEIN 3; PDZRN3","url":"https://www.omim.org/entry/609729"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Vesicles","reliability":"Approved"},{"location":"Plasma membrane","reliability":"Additional"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/LNX2"},"hgnc":{"alias_symbol":["MGC46315"],"prev_symbol":["PDZRN1"]},"alphafold":{"accession":"Q8N448","domains":[{"cath_id":"3.30.40.10","chopping":"40-140","consensus_level":"medium","plddt":90.5267,"start":40,"end":140},{"cath_id":"2.30.42.10","chopping":"231-317","consensus_level":"high","plddt":90.146,"start":231,"end":317},{"cath_id":"2.30.42.10","chopping":"338-422","consensus_level":"high","plddt":86.3808,"start":338,"end":422},{"cath_id":"2.30.42.10","chopping":"459-560_578-584","consensus_level":"high","plddt":87.5206,"start":459,"end":584},{"cath_id":"2.30.42.10","chopping":"598-690","consensus_level":"high","plddt":88.0368,"start":598,"end":690}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8N448","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q8N448-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q8N448-F1-predicted_aligned_error_v6.png","plddt_mean":74.62},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=LNX2","jax_strain_url":"https://www.jax.org/strain/search?query=LNX2"},"sequence":{"accession":"Q8N448","fasta_url":"https://rest.uniprot.org/uniprotkb/Q8N448.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q8N448/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8N448"}},"corpus_meta":[{"pmid":"23319804","id":"PMC_23319804","title":"Genetic amplification of the NOTCH modulator LNX2 upregulates the WNT/β-catenin pathway in colorectal cancer.","date":"2013","source":"Cancer research","url":"https://pubmed.ncbi.nlm.nih.gov/23319804","citation_count":70,"is_preprint":false},{"pmid":"15979067","id":"PMC_15979067","title":"The cell surface protein coxsackie- and adenovirus receptor (CAR) directly associates with the Ligand-of-Numb Protein-X2 (LNX2).","date":"2005","source":"Experimental cell research","url":"https://pubmed.ncbi.nlm.nih.gov/15979067","citation_count":39,"is_preprint":false},{"pmid":"25712254","id":"PMC_25712254","title":"Ubiquitin E3 Ligase LNX2 is Critical for Osteoclastogenesis In Vitro by Regulating M-CSF/RANKL Signaling and Notch2.","date":"2015","source":"Calcified tissue international","url":"https://pubmed.ncbi.nlm.nih.gov/25712254","citation_count":29,"is_preprint":false},{"pmid":"25279559","id":"PMC_25279559","title":"Caspr4 interaction with LNX2 modulates the proliferation and neuronal differentiation of mouse neural progenitor cells.","date":"2014","source":"Stem cells and development","url":"https://pubmed.ncbi.nlm.nih.gov/25279559","citation_count":29,"is_preprint":false},{"pmid":"22045731","id":"PMC_22045731","title":"Ligand of Numb proteins LNX1p80 and LNX2 interact with the human glycoprotein CD8α and promote its ubiquitylation and endocytosis.","date":"2011","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/22045731","citation_count":22,"is_preprint":false},{"pmid":"30295974","id":"PMC_30295974","title":"E3 ubiquitin ligases LNX1 and LNX2 localize at neuronal gap junctions formed by connexin36 in rodent brain and molecularly interact with connexin36.","date":"2018","source":"The European journal of neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/30295974","citation_count":17,"is_preprint":false},{"pmid":"32714586","id":"PMC_32714586","title":"LNX1/LNX2 proteins: functions in neuronal signalling and beyond.","date":"2018","source":"Neuronal signaling","url":"https://pubmed.ncbi.nlm.nih.gov/32714586","citation_count":16,"is_preprint":false},{"pmid":"31628376","id":"PMC_31628376","title":"E3 ubiquitin ligases LNX1 and LNX2 are major regulators of the presynaptic glycine transporter GlyT2.","date":"2019","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/31628376","citation_count":15,"is_preprint":false},{"pmid":"26392552","id":"PMC_26392552","title":"Lnx2 ubiquitin ligase is essential for exocrine cell differentiation in the early zebrafish pancreas.","date":"2015","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/26392552","citation_count":14,"is_preprint":false},{"pmid":"26451611","id":"PMC_26451611","title":"Structural basis for the indispensable role of a unique zinc finger motif in LNX2 ubiquitination.","date":"2015","source":"Oncotarget","url":"https://pubmed.ncbi.nlm.nih.gov/26451611","citation_count":11,"is_preprint":false},{"pmid":"36674899","id":"PMC_36674899","title":"Impaired Blastocyst Formation in Lnx2-Knockdown Mouse Embryos.","date":"2023","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/36674899","citation_count":2,"is_preprint":false},{"pmid":"37237241","id":"PMC_37237241","title":"LNX2 involves in the role of ghrelin to promote the neuronal differentiation of adipose tissue-derived mesenchymal stem cells.","date":"2023","source":"Journal of bioenergetics and biomembranes","url":"https://pubmed.ncbi.nlm.nih.gov/37237241","citation_count":0,"is_preprint":false},{"pmid":"40269869","id":"PMC_40269869","title":"Differential neuronal functions of LNX1 and LNX2 revealed by behavioural analysis in single and double knockout mice.","date":"2025","source":"Behavioral and brain functions : BBF","url":"https://pubmed.ncbi.nlm.nih.gov/40269869","citation_count":0,"is_preprint":false},{"pmid":"41714050","id":"PMC_41714050","title":"Protein behavior of the ligand of Numb-protein X 2 (LNX2) in mouse ameloblasts at the maturation stage.","date":"2026","source":"Journal of oral biosciences","url":"https://pubmed.ncbi.nlm.nih.gov/41714050","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":9101,"output_tokens":3065,"usd":0.036639,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10457,"output_tokens":3459,"usd":0.06938,"stage2_stop_reason":"end_turn"},"total_usd":0.106019,"stage1_batch_id":"msgbatch_01BbTCMyrRPyhtEq6b1MdwCN","stage2_batch_id":"msgbatch_011dEed5bbxYvTAaQWKJevxu","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2015,\n      \"finding\": \"LNX2 RING domain, flanked by two zinc-binding motifs (Zn-RING-Zn), is required for E3 ubiquitin ligase activity; the N-terminal zinc finger adopts a novel open-circle Cys2His2 conformation and is indispensable for LNX2 activity and stability. The Zn-RING-Zn domain dimerizes, undergoes autoubiquitination with all seven ubiquitin chain linkages, and directly ubiquitinates Numb.\",\n      \"method\": \"Crystal structure of Zn-RING-Zn domain; active-site mutagenesis; in vitro ubiquitination assay with full-length LNX2\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure combined with mutagenesis and in vitro reconstitution of ubiquitination activity in a single rigorous study\",\n      \"pmids\": [\"26451611\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"The intracellular tail of the cell-surface protein CAR (coxsackievirus and adenovirus receptor) directly interacts with LNX2 both in vivo and in vitro; interaction domains were mapped by truncation analysis and affinity chromatography, and LNX2 organizes a protein complex at specific subcellular sites.\",\n      \"method\": \"Yeast two-hybrid screen; co-immunoprecipitation; in vitro binding/affinity chromatography; truncation analysis\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP plus in vitro binding and domain mapping in a single study\",\n      \"pmids\": [\"15979067\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"LNX2 PDZ domains interact with the cytosolic C-terminal valine motif of CD8α; LNX2 promotes ubiquitylation of CD8α, redistribution of LNX2 from cytosol to plasma membrane, and subsequent CD8α downregulation from the plasma membrane, transport to lysosomes, and degradation.\",\n      \"method\": \"Co-immunoprecipitation in human T cells; in vitro ubiquitylation assay; heterologous expression with domain-truncation mapping; live-cell imaging/fractionation\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vitro ubiquitylation reconstitution, domain mapping, and cellular degradation assay combined in one study\",\n      \"pmids\": [\"22045731\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"RNAi silencing of LNX2 in colorectal cancer cell lines reduces NOTCH levels, downregulates TCF7L2, and markedly reduces WNT signaling, placing LNX2 upstream of both NOTCH and WNT/β-catenin pathways.\",\n      \"method\": \"RNAi knockdown followed by global expression profiling; cell viability assay\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — RNAi plus transcriptome profiling in multiple CRC lines; no in vitro reconstitution\",\n      \"pmids\": [\"23319804\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"LNX2 is required for osteoclastogenesis; its knockdown in bone marrow macrophages accumulates Numb, promotes Notch2 degradation, reduces Hes1 expression, attenuates M-CSF-induced ERK/AKT activation and RANKL-stimulated NF-κB/JNK signaling, and accelerates osteoclast apoptosis.\",\n      \"method\": \"Lentiviral shRNA knockdown in bone marrow macrophages; Western blot for pathway components; osteoclast differentiation assay\",\n      \"journal\": \"Calcified tissue international\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function with multiple defined signaling readouts in primary cells, single lab\",\n      \"pmids\": [\"25712254\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"LNX2 interacts with Caspr4 (CNTNAP4) in a PDZ domain-dependent manner in neural progenitor cells; LNX2 promotes neuronal differentiation and is epistatic to Caspr4 — overexpression of LNX2 rescues the differentiation defect caused by Caspr4 knockdown, whereas C4ICD cannot rescue LNX2 knockdown.\",\n      \"method\": \"Co-immunoprecipitation; PDZ domain interaction mapping; lentiviral knockdown and overexpression in mouse NPCs; rescue epistasis experiments\",\n      \"journal\": \"Stem cells and development\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP plus genetic epistasis rescue experiments, single lab\",\n      \"pmids\": [\"25279559\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"LNX1 and LNX2 localize at connexin36 (Cx36)-containing neuronal gap junctions in rodent brain; both proteins co-immunoprecipitate with Cx36 and bind Cx36 via their second PDZ domain. Expression of E3 ligase-competent LNX1/2 isoforms in cultured cells causes loss of Cx36-containing gap junctions, whereas ligase-inactive isoforms do not, indicating LNX proteins ubiquitinate Cx36 to promote its internalization.\",\n      \"method\": \"Immunofluorescence colocalization in wild-type vs. LNX-null mouse brain; co-immunoprecipitation; GST pull-down with individual PDZ domains; cotransfection with wild-type vs. ligase-dead LNX isoforms\",\n      \"journal\": \"The European journal of neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, PDZ domain pull-down, in-cell functional assay with ligase-dead controls, validated in null mice\",\n      \"pmids\": [\"30295974\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"LNX1 and LNX2 RING finger domains ubiquitinate the glycine transporter GlyT2 at a cytoplasmic C-terminal lysine cluster (K751, K773, K787, K791), regulating GlyT2 expression levels and transport activity. Genetic deletion of endogenous LNX2 in spinal cord neurons increases GlyT2 expression, and LNX2 is required for PKC-mediated control of GlyT2 transport.\",\n      \"method\": \"Unbiased substrate screen; in vitro ubiquitination assay with RING domain; site-directed mutagenesis of GlyT2 lysines; LNX2 knockout primary spinal cord neurons; PKC regulation assay\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with mutagenesis plus KO primary neurons, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"31628376\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"In zebrafish, Lnx2a/b double loss-of-function inhibits exocrine pancreatic cell differentiation; this phenotype is rescued by inhibition of Numb expression, placing Lnx2 upstream of Numb in a pathway that fine-tunes Notch signaling during pancreatic cell-type specification. Loss of Lnx2a/b also reduces the number of Notch-active cells in the pancreas.\",\n      \"method\": \"Splice-blocking morpholino knockdown; frameshift null mutation; Numb inhibition rescue epistasis; Notch reporter assay in zebrafish embryos\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis with morpholino and null mutation plus Numb rescue in zebrafish, single lab\",\n      \"pmids\": [\"26392552\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"LNX2 knockdown in mouse preimplantation embryos blocks blastocyst formation without affecting morula development; knockdown increases expression of Oct4 and genes involved in Notch and Hippo signaling, suggesting LNX2 normally suppresses these pathways to permit inner cell mass lineage specification.\",\n      \"method\": \"siRNA knockdown in mouse preimplantation embryos; transcript analysis of lineage and signaling genes\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single knockdown experiment with transcript readouts, no biochemical pathway reconstitution, single lab\",\n      \"pmids\": [\"36674899\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"LNX2 is required for ghrelin-induced neuronal differentiation of adipose-derived mesenchymal stem cells; LNX2 silencing suppresses nuclear translocation of β-catenin and reduces WNT/β-catenin transcriptional activity as measured by luciferase reporter assay.\",\n      \"method\": \"siRNA knockdown; β-catenin nuclear translocation imaging; luciferase reporter assay for WNT/β-catenin transcriptional activity\",\n      \"journal\": \"Journal of bioenergetics and biomembranes\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, single knockdown approach with reporter assay, no biochemical reconstitution\",\n      \"pmids\": [\"37237241\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"In mouse ameloblasts at the maturation stage, LNX2 co-localizes with Numb in aggregated intracellular vesicles and with the lysosomal marker LAMP-1, consistent with LNX2-mediated ubiquitination and lysosomal degradation of Numb and the tight-junction protein claudin-7 at these cells.\",\n      \"method\": \"Double immunofluorescence staining in paraformaldehyde-fixed, decalcified mouse incisors with antibodies against LNX2, Numb, LAMP-1, and claudin-7\",\n      \"journal\": \"Journal of oral biosciences\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — co-localization by immunofluorescence only, no biochemical interaction or ubiquitination assay confirmed, single lab\",\n      \"pmids\": [\"41714050\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"LNX2 is a RING/PDZ-domain E3 ubiquitin ligase whose catalytic Zn-RING-Zn domain (structurally resolved and mutagenically validated) ubiquitinates multiple substrates including Numb, CD8α, connexin36, and GlyT2, thereby controlling Notch/WNT signaling, immune-receptor surface levels, neuronal gap-junction plasticity, and presynaptic glycine transport, while its PDZ domains scaffold interactions with partners such as CAR and Caspr4 at specific subcellular sites.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"LNX2 is a RING/PDZ-domain E3 ubiquitin ligase that controls cell-surface receptor levels and the strength of Notch and WNT signaling across diverse developmental and physiological contexts [#0, #3]. Its catalytic activity resides in a Zn-RING-Zn module in which the N-terminal zinc finger adopts a novel open-circle Cys2His2 conformation indispensable for activity and stability; this domain dimerizes, autoubiquitinates with all seven ubiquitin chain linkages, and directly ubiquitinates the Notch regulator Numb [#0]. By targeting Numb, LNX2 acts upstream of Notch and WNT/\\u03b2-catenin signaling, a role demonstrated in colorectal cancer cells where its loss reduces NOTCH and TCF7L2 levels and WNT activity [#3], and in osteoclast precursors where its knockdown accumulates Numb and promotes Notch2 degradation [#4]. The PDZ domains serve as substrate-recruitment and scaffolding modules: they bind the C-terminal motifs of CD8\\u03b1 to drive its ubiquitylation, lysosomal transport, and surface downregulation [#2], and bind connexin36 via the second PDZ domain to promote ubiquitination and internalization of neuronal gap junctions [#6]. LNX2 additionally ubiquitinates the glycine transporter GlyT2 at a defined C-terminal lysine cluster to regulate presynaptic glycine transport, and is required for PKC-mediated control of GlyT2 [#7]. Through PDZ-dependent interactions with partners including CAR and Caspr4, LNX2 organizes protein complexes at specific subcellular sites and promotes neuronal differentiation [#1, #5].\",\n  \"teleology\": [\n    {\n      \"year\": 2005,\n      \"claim\": \"Established LNX2 as a PDZ-scaffold that engages a cell-surface receptor tail, raising the question of what it does to bound partners.\",\n      \"evidence\": \"Yeast two-hybrid, reciprocal Co-IP, and in vitro binding with domain mapping of the CAR intracellular tail\",\n      \"pmids\": [\"15979067\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No catalytic consequence of the CAR interaction shown\", \"Functional role of the LNX2-organized complex undefined\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Showed LNX2 PDZ domains recruit a substrate (CD8\\u03b1) for ubiquitylation and lysosomal degradation, linking scaffolding to receptor turnover.\",\n      \"evidence\": \"Co-IP in T cells, in vitro ubiquitylation, domain-truncation mapping, and fractionation/imaging of CD8\\u03b1 downregulation\",\n      \"pmids\": [\"22045731\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological context of CD8\\u03b1 regulation in T cells not established\", \"E2 partner not defined\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Placed LNX2 upstream of both NOTCH and WNT/\\u03b2-catenin signaling in a disease context, broadening its role beyond single-substrate turnover.\",\n      \"evidence\": \"RNAi knockdown with global expression profiling in colorectal cancer lines\",\n      \"pmids\": [\"23319804\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No biochemical reconstitution linking LNX2 catalysis to pathway output\", \"Direct substrate mediating WNT effect not identified\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Demonstrated PDZ-dependent binding to Caspr4 and genetic epistasis driving neuronal differentiation, establishing a developmental signaling role.\",\n      \"evidence\": \"Reciprocal Co-IP, PDZ mapping, and knockdown/overexpression rescue epistasis in mouse NPCs\",\n      \"pmids\": [\"25279559\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether Caspr4 is a ubiquitination substrate unresolved\", \"Downstream effector pathway not defined\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Resolved the catalytic architecture, showing a novel open-circle Zn-RING-Zn fold that dimerizes, autoubiquitinates with all linkage types, and directly ubiquitinates Numb.\",\n      \"evidence\": \"Crystal structure of Zn-RING-Zn domain, active-site mutagenesis, and in vitro ubiquitination with full-length LNX2\",\n      \"pmids\": [\"26451611\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Linkage-type selectivity in cells not defined\", \"Regulation of dimerization in vivo unknown\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Connected LNX2 to physiological Notch tuning via Numb in two systems: osteoclastogenesis in mammals and pancreatic cell specification in zebrafish.\",\n      \"evidence\": \"shRNA knockdown with pathway Western blots in bone marrow macrophages; morpholino/null mutation with Numb-inhibition rescue and Notch reporter in zebrafish\",\n      \"pmids\": [\"25712254\", \"26392552\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct in vivo Numb ubiquitination not shown in these tissues\", \"Contribution of other substrates not assessed\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Established connexin36 as a PDZ-bound substrate whose ubiquitination by LNX2 controls gap-junction internalization in neurons.\",\n      \"evidence\": \"Colocalization in LNX-null vs wild-type mouse brain, reciprocal Co-IP, PDZ-domain GST pull-down, and ligase-dead controls in cotransfection\",\n      \"pmids\": [\"30295974\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence for electrical synapse plasticity in vivo not measured\", \"Redundancy with LNX1 not fully separated\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Identified GlyT2 as a RING-domain substrate ubiquitinated at a defined lysine cluster, placing LNX2 in presynaptic glycine transport control.\",\n      \"evidence\": \"Unbiased substrate screen, in vitro ubiquitination, GlyT2 lysine mutagenesis, and LNX2-knockout primary spinal cord neurons with PKC assay\",\n      \"pmids\": [\"31628376\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism linking PKC signaling to LNX2 activity undefined\", \"In vivo behavioral relevance not tested\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Extended LNX2's pathway-suppressive role to early embryonic lineage decisions and stem-cell neuronal differentiation via Notch/Hippo and WNT/\\u03b2-catenin readouts.\",\n      \"evidence\": \"siRNA knockdown in mouse preimplantation embryos with lineage transcript analysis; siRNA with \\u03b2-catenin translocation imaging and WNT luciferase reporter in adipose-derived MSCs\",\n      \"pmids\": [\"36674899\", \"37237241\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single knockdown approach with no biochemical reconstitution\", \"Direct substrate mediating these effects not identified\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Provided localization evidence consistent with LNX2-mediated lysosomal degradation of Numb and claudin-7 in maturation-stage ameloblasts.\",\n      \"evidence\": \"Double immunofluorescence for LNX2, Numb, LAMP-1, and claudin-7 in mouse incisors\",\n      \"pmids\": [\"41714050\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Colocalization only; no biochemical interaction or ubiquitination assay\", \"Claudin-7 as a direct substrate not confirmed\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How LNX2 substrate selection, ubiquitin-linkage choice, and PDZ-domain recruitment are integrated and regulated across its distinct physiological roles remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No unified model of substrate prioritization in vivo\", \"Upstream regulation of LNX2 catalytic activity largely unknown\", \"Functional separation from paralog LNX1 incomplete\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [0, 2, 7]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 2, 6, 7]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [1, 2, 5, 6]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [2, 6]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [2]},\n      {\"term_id\": \"GO:0005764\", \"supporting_discovery_ids\": [2]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [3, 4]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 2, 7]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [5, 8]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"NUMB\", \"CD8A\", \"GJD2\", \"SLC6A5\", \"CXADR\", \"CNTNAP4\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"faith_supported":5,"faith_total":6,"faith_pct":83.33333333333333}}