{"gene":"ITSN1","run_date":"2026-06-10T01:55:23","timeline":{"discoveries":[{"year":1998,"finding":"Human ITSN1 encodes two isoforms by brain-specific alternative splicing: a ubiquitous short form (1220 aa) containing two EH domains, a coiled-coil region, and five SH3 domains, and a brain-specific long form (1721 aa) that additionally contains GEF, PH, and C2 domains.","method":"cDNA cloning, alternative splicing analysis, domain architecture characterization","journal":"Genomics","confidence":"High","confidence_rationale":"Tier 1 / Strong — full-length cDNA sequencing with domain identification, replicated in independent study (PMID:10482960)","pmids":["9799604","10482960"],"is_preprint":false},{"year":2008,"finding":"In Itsn1 null mice, neurons show slowed endocytosis and increased endosome size, chromaffin cells show reduced exocytosis events, and NGF levels are reduced in the septal brain region, establishing ITSN1 as a regulator of endocytosis and vesicle trafficking in vivo.","method":"Knockout mouse generation, live-cell imaging of endocytosis, chromaffin cell amperometry, endosome size measurement by electron microscopy, NGF ELISA","journal":"Human molecular genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean KO with multiple orthogonal cellular phenotype readouts (endocytosis kinetics, exocytosis, endosome morphology, NGF levels)","pmids":["18676989"],"is_preprint":false},{"year":2008,"finding":"In C. elegans, ITSN-1 forms a stable complex with EHS-1 (Eps15) and co-localizes with it at endocytic hotspots ~300 nm from the presynaptic density; itsn-1 null mutants accumulate large irregular vesicles and membrane-associated vesicles at these sites with reduced synaptic vesicle number and reduced NMJ event frequency. Double mutants with dab-1 (Disabled) show synthetic lethality, placing itsn-1 and ehs-1 in the same genetic pathway that functions in parallel with the clathrin-associated sorting protein Disabled.","method":"C. elegans genetic null mutants, electron microscopy, electrophysiology (NMJ recordings), co-immunoprecipitation, genetic epistasis (double mutants)","journal":"Traffic","confidence":"High","confidence_rationale":"Tier 2 / Strong — null mutants with EM, electrophysiology, co-IP, and genetic epistasis in single study","pmids":["18298590"],"is_preprint":false},{"year":2010,"finding":"A neuron-specific microexon (exon 20) in ITSN1 encodes five amino acids inserted at the beginning of the n-Src loop of the SH3A domain, introducing negatively charged residues toward the interaction interface; mutational analysis confirmed that translocation of these charged residues is required for interaction with dynamin 1.","method":"Comparative sequence analysis, structural modeling, site-directed mutagenesis, pulldown/binding assays, zebrafish expression analysis","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — mutagenesis with binding assay in single lab; structural modeling supports interpretation","pmids":["20659428"],"is_preprint":false},{"year":2011,"finding":"A novel ITSN1 isoform (ITSN1-22a) with an alternative C-terminus (encoded by exon 22a) binds amphiphysin 1 SH3 domain and ITSN1 SH3A via its CTD; an intramolecular interaction within ITSN1-22a negatively regulates its binding to Cbl but not to dynamin 1, indicating isoform-specific binding selectivity controlled by the CTD.","method":"RT-PCR/cDNA cloning, co-immunoprecipitation, in vitro pulldown, Western blot","journal":"Gene","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP and in vitro pulldown with multiple partners in single lab, two orthogonal methods","pmids":["21712076"],"is_preprint":false},{"year":2012,"finding":"ITSN1 interacts with Epstein-Barr virus LMP2A via the SH3 domains of ITSN1 binding to proline-rich motifs in both N- and C-tails of LMP2A. Syk kinase promotes phosphorylation of ITSN1 in LMP2A-expressing cells. The Shb adaptor bridges ITSN1 to LMP2A by simultaneously binding phosphorylated LMP2A tyrosines and ITSN1 SH3 domains.","method":"Co-immunoprecipitation, kinase inhibitor treatment, phosphorylation assays","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — reciprocal Co-IP with pharmacological validation, single lab","pmids":["22975684"],"is_preprint":false},{"year":2012,"finding":"Yeast two-hybrid screening identified >100 new binding partners for ITSN1 and ITSN2, implicating ITSNs in regulation of Rab and Arf GTPase pathways and the DISC1 interactome; ITSN proteins also form homomeric (ITSN1-ITSN1) and heteromeric (ITSN1-ITSN2) complexes.","method":"High-throughput yeast two-hybrid screen, co-immunoprecipitation validation","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — large Y2H screen with Co-IP validation for selected partners, single lab","pmids":["22558309"],"is_preprint":false},{"year":2015,"finding":"ITSN-1s knockdown in endothelial cells impairs clathrin-mediated and caveolae-mediated endocytosis, causing TGFβ-RI (Alk5) to be internalized via compensatory enlarged endocytic structures leading to its ubiquitylation and degradation; this shifts Alk5 signaling from Smad2/3 toward Erk1/2 activation via preferential formation of the Alk5-mSos-Grb2 complex over the Alk5-Smad-SARA complex, promoting endothelial cell proliferation.","method":"shRNA knockdown in cultured endothelial cells and in vivo mouse model, co-immunoprecipitation, Western blot for signaling intermediates, microparticle transfer assays","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KD with defined signaling pathway readouts and in vivo validation, single lab","pmids":["25720380"],"is_preprint":false},{"year":2017,"finding":"CR16 (a verprolin family member) forms complexes with ITSN1 and ITSN2 in human cell lines; overexpressed CR16 promotes co-localization of ITSN1 with F-actin in MCF-7 cells, indicating CR16 modulates ITSN1 association with the actin cytoskeleton.","method":"Co-immunoprecipitation, confocal immunofluorescence co-localization","journal":"Biochemical and biophysical research communications","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP and imaging, single lab, no functional rescue","pmids":["28161632"],"is_preprint":false},{"year":2018,"finding":"WIP (WASP-interacting protein) interacts with ITSN1; the WIP/ITSN1 complex co-localizes with RAB4-positive fast recycling endosomes, participates in transferrin receptor recycling, and promotes formation of filopodia-like protrusions in MCF-7 cells. WIP enhances N-WASP interaction with ITSN1 and promotes ITSN1/β-actin association.","method":"Co-immunoprecipitation, confocal co-localization, transferrin recycling assay, overexpression/knockdown morphology analysis","journal":"Gene","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP, functional recycling assay, and morphology readout, multiple orthogonal methods in single lab","pmids":["29958948"],"is_preprint":false},{"year":2018,"finding":"The E3 ubiquitin ligase AIP4/ITCH monoubiquitinates the major isoform ITSN1-s (stabilizing it) and mono- plus oligo-ubiquitinates the minor isoform ITSN1-22a (causing its proteasomal degradation), explaining the low cellular abundance of ITSN1-22a.","method":"Ubiquitination assays, proteasome inhibitor treatment, Western blot, co-immunoprecipitation","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct ubiquitination assay with proteasome inhibitor confirmation and isoform-specific comparison, single lab","pmids":["29851086"],"is_preprint":false},{"year":2020,"finding":"A membrane-anchored ITSN1 DH-PH construct directly activates RhoJ GTPase and promotes endothelial cell sprouting; this is sensitive to phosphorylation by focal adhesion kinase (FAK) and independent of Cdc42 activation, defining an ITSN1/RhoJ signaling axis distinct from the known ITSN1/Cdc42 pathway.","method":"Gain-of-function overexpression, dominant-negative RhoJ trapping, FAK inhibitor treatment, co-immunoprecipitation, cell sprouting assay","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct interaction plus functional gain-of-function with pharmacological and dominant-negative controls, single lab","pmids":["31980169"],"is_preprint":false},{"year":2020,"finding":"ITSN1 SH3 domains interact with proline-rich motifs (PRMs) of SAM68; this interaction enhances SAM68 solubility in vitro and induces dissociation of SAM68 Nuclear Bodies (SNBs) in HeLa cells via SH3A binding to the P0 PRM. An additional SH3 domain (SH3D) of ITSN1 can bind mRNAs, suggesting ITSN1 acts as a chaperone for SAM68 within nuclear ribonucleoprotein complexes.","method":"In vitro binding/solubility assays, immunofluorescence/nuclear body imaging in HeLa cells, domain-mapping pulldowns","journal":"Cellular and molecular life sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro reconstitution of solubility plus cellular imaging with domain-specific mutants, single lab","pmids":["32780150"],"is_preprint":false},{"year":2021,"finding":"ITSN1 determines synaptojanin1 levels within clathrin-coated vesicles (CCVs) at synapses; Sgip1/AP2 excess hinders synaptojanin1 binding to ITSN1, lowering its CCV levels. ITSN1 levels in turn are determined by Eps15 (not Eps15L1), placing ITSN1 in a regulatory hierarchy: Eps15→ITSN1→synaptojanin1 for CCV life cycle control.","method":"CCV purification from mouse brain, quantitative proteomics of CCV interactome, AP1/σ1B knockout mouse comparison","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — quantitative CCV proteomics with genetic model comparison; single lab but rigorous biochemical fractionation","pmids":["33850201"],"is_preprint":false},{"year":2024,"finding":"In a Drosophila model expressing human α-synuclein, haploinsufficiency of Dap160 (ITSN1 ortholog) exacerbates α-synuclein-induced compound eye degeneration and motor deficits; in vitro assays further indicate a physical interaction between ITSN1 and α-synuclein.","method":"Drosophila genetic haploinsufficiency model, eye degeneration scoring, motor behavior assay, in vitro binding assay","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo Drosophila epistasis plus in vitro interaction assay, replicated across two independent reports (PMID:40056900, bio_10.1101_2024.07.25.24310988)","pmids":["40056900"],"is_preprint":false},{"year":2026,"finding":"Dlgap2 interacts with Itsn1 at the postsynaptic density; Dlgap2 knockdown causes specific reduction of Itsn1 via ubiquitin-mediated proteasomal degradation, resulting in synaptic deficits and autism-like behaviors in mice, defining a Dlgap2-Itsn1 regulatory axis at the synapse.","method":"Proteomics of postsynaptic density fraction, co-immunoprecipitation, ubiquitination assay, Dlgap2 knockdown mouse behavior","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — PSD proteomics with Co-IP and ubiquitination assay, in vivo behavioral phenotype, single lab","pmids":["41673270"],"is_preprint":false},{"year":2024,"finding":"Deletion of the itsn1 microexon in zebrafish produces defects in neuritogenesis, demonstrating that this alternatively spliced insert contributes a specific functional role in neuronal morphogenesis in vivo.","method":"CRISPR/Cas9 microexon deletion in zebrafish, neuritogenesis phenotype analysis","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 3 / Weak — preprint, single zebrafish line, phenotype without full mechanistic pathway placement","pmids":[],"is_preprint":true}],"current_model":"ITSN1 is a multi-domain scaffold protein (two EH domains, coiled-coil, up to five SH3 domains, and in the brain-specific long isoform, GEF/DH-PH, PH, and C2 domains) that coordinates clathrin-mediated endocytosis and synaptic vesicle recycling by scaffolding dynamin, Eps15, and synaptojanin1 into clathrin-coated vesicle complexes; its SH3 domains engage diverse proline-rich partners (dynamin 1, Cbl, SAM68, LMP2A, WIP, CR16) with binding selectivity modulated by neuron-specific microexon insertion and by isoform-specific intramolecular interactions, while its DH-PH module acts as a GEF that directly activates Cdc42 and RhoJ to drive actin-dependent membrane processes; isoform stability is differentially controlled by AIP4/ITCH-mediated ubiquitination, and loss of ITSN1 function in vivo (knockout mice, C. elegans nulls, Drosophila haploinsufficiency) produces defective endocytosis, enlarged early endosomes, reduced synaptic vesicle numbers, altered TGFβ/Erk signaling, and exacerbation of α-synuclein toxicity."},"narrative":{"mechanistic_narrative":"ITSN1 is a multi-domain scaffold that organizes clathrin-mediated endocytosis and synaptic vesicle recycling, expressed as a ubiquitous short isoform (two EH domains, coiled-coil, five SH3 domains) and a brain-specific long isoform that adds GEF/DH-PH, PH, and C2 domains [PMID:9799604, PMID:10482960]. Loss of ITSN1 in vivo slows endocytosis, enlarges endosomes, reduces exocytosis and synaptic vesicle number, and lowers septal NGF, establishing its role in vesicle trafficking [PMID:18676989, PMID:18298590]. At the synapse it operates within a regulatory hierarchy controlling the clathrin-coated vesicle life cycle, with Eps15/EHS-1 determining ITSN1 levels and ITSN1 in turn setting synaptojanin1 content in coated vesicles [PMID:18298590, PMID:33850201]. Its SH3 domains engage proline-rich partners with selectivity tuned by neuron-specific microexon insertion into the SH3A n-Src loop, which is required for dynamin 1 binding and for neuronal morphogenesis [PMID:20659428], and by isoform-specific intramolecular regulation through alternative C-termini [PMID:21712076]. SH3-mediated contacts extend ITSN1 to actin-cytoskeleton regulators including WIP/N-WASP, supporting transferrin-receptor recycling and filopodia formation [PMID:29958948], and to SAM68, whose solubility and nuclear-body dynamics ITSN1 modulates [PMID:32780150]. The DH-PH module functions as a GEF, activating RhoJ in a FAK-sensitive, Cdc42-independent axis to drive endothelial sprouting [PMID:31980169]. ITSN1 abundance is set by ubiquitination: AIP4/ITCH differentially stabilizes or degrades distinct isoforms [PMID:29851086], and Dlgap2 protects synaptic Itsn1 from proteasomal degradation [PMID:41673270]. ITSN1 also intersects disease processes, modulating TGFβ-RI (Alk5) trafficking and the Smad-versus-Erk signaling balance in endothelial cells [PMID:25720380] and exacerbating α-synuclein toxicity when reduced [PMID:40056900].","teleology":[{"year":1998,"claim":"Established the domain architecture and isoform diversity of ITSN1, defining it as a candidate multi-domain scaffold with a brain-specific signaling-capable long form.","evidence":"cDNA cloning and alternative splicing analysis identifying short and long isoforms","pmids":["9799604","10482960"],"confidence":"High","gaps":["Domain architecture alone did not establish in vivo function","Partner repertoire of individual domains unresolved at this stage"]},{"year":2008,"claim":"Demonstrated that ITSN1 is required for endocytosis and vesicle trafficking in vivo, moving it from candidate scaffold to validated trafficking regulator.","evidence":"Itsn1 knockout mice with endocytosis imaging, chromaffin cell amperometry, EM endosome sizing, and NGF ELISA; parallel C. elegans null mutants with EM, electrophysiology, co-IP, and genetic epistasis","pmids":["18676989","18298590"],"confidence":"High","gaps":["Molecular step within the coated-vesicle cycle that ITSN1 controls not pinpointed","Relationship between endocytic and exocytic phenotypes unclear"]},{"year":2010,"claim":"Showed that a neuron-specific microexon in the SH3A domain tunes partner binding, explaining how a single scaffold gene generates neuron-specialized interaction selectivity.","evidence":"Sequence/structural modeling and site-directed mutagenesis with binding assays for dynamin 1","pmids":["20659428"],"confidence":"Medium","gaps":["Single-lab binding assay","Functional consequence of microexon insertion in vivo not addressed here"]},{"year":2011,"claim":"Revealed isoform-specific binding selectivity, where an alternative C-terminus mediates an intramolecular interaction that selectively gates SH3 partner engagement.","evidence":"cDNA cloning, co-IP, and in vitro pulldown of ITSN1-22a with amphiphysin1, Cbl, and dynamin1","pmids":["21712076"],"confidence":"Medium","gaps":["Physiological role of ITSN1-22a not established","Structural basis of the intramolecular contact not resolved"]},{"year":2012,"claim":"Expanded the ITSN1 interactome and showed it engages viral and signaling adaptors plus GTPase pathways, and self-associates into homo/heteromeric complexes.","evidence":"Reciprocal co-IP with kinase inhibitors for LMP2A/Syk/Shb; high-throughput Y2H screen with co-IP validation implicating Rab/Arf and DISC1 networks","pmids":["22975684","22558309"],"confidence":"Medium","gaps":["Most Y2H partners lack functional validation","Biological significance of ITSN1 oligomerization unknown"]},{"year":2015,"claim":"Connected ITSN1 endocytic function to growth-factor signaling output, showing its loss reroutes Alk5 toward degradation and shifts TGFβ signaling from Smad to Erk.","evidence":"shRNA knockdown in endothelial cells and mouse model with co-IP and signaling Western blots","pmids":["25720380"],"confidence":"Medium","gaps":["Direct ITSN1-Alk5 contact versus indirect trafficking effect not separated","Single-lab readout"]},{"year":2017,"claim":"Linked ITSN1 to the actin cytoskeleton via CR16, indicating scaffolding beyond endocytosis into cytoskeletal organization.","evidence":"Co-IP and confocal co-localization of CR16 with ITSN1/F-actin in cell lines","pmids":["28161632"],"confidence":"Low","gaps":["Single Co-IP and imaging without functional rescue","No in vivo relevance demonstrated"]},{"year":2018,"claim":"Defined how ITSN1 couples to actin-polymerization machinery and recycling endosomes through WIP/N-WASP, and how its protein levels are controlled by isoform-selective ubiquitination.","evidence":"Co-IP, transferrin recycling assay, and morphology analysis for WIP; ubiquitination assays with proteasome inhibitor for AIP4/ITCH","pmids":["29958948","29851086"],"confidence":"Medium","gaps":["Whether WIP and endocytic scaffolding functions compete is unclear","In vivo consequence of AIP4-mediated isoform control not tested"]},{"year":2020,"claim":"Distinguished a Cdc42-independent ITSN1/RhoJ GEF axis and characterized ITSN1 as a chaperone for SAM68 in nuclear RNP complexes, broadening its mechanistic repertoire beyond endocytosis.","evidence":"Membrane-anchored DH-PH gain-of-function with dominant-negative and FAK inhibitor for RhoJ; in vitro solubility assays and nuclear-body imaging with domain mutants for SAM68","pmids":["31980169","32780150"],"confidence":"Medium","gaps":["RhoJ activation shown with membrane-anchored construct, not full-length endogenous ITSN1","Physiological role of ITSN1-SAM68 chaperoning in vivo undefined"]},{"year":2021,"claim":"Placed ITSN1 in an ordered regulatory hierarchy controlling the synaptic coated-vesicle life cycle: Eps15 sets ITSN1 levels, which set synaptojanin1 levels.","evidence":"Quantitative CCV proteomics from mouse brain with AP knockout comparisons","pmids":["33850201"],"confidence":"Medium","gaps":["Mechanism by which ITSN1 recruits synaptojanin1 to CCVs not structurally defined","Single-lab biochemical fractionation"]},{"year":2024,"claim":"Tied ITSN1 to neurodegeneration and confirmed the microexon's organismal role, showing reduced ITSN1 worsens α-synuclein toxicity and microexon loss disrupts neuritogenesis.","evidence":"Drosophila α-synuclein haploinsufficiency model with in vitro binding; CRISPR microexon deletion in zebrafish (preprint)","pmids":["40056900"],"confidence":"Medium","gaps":["Mechanism connecting endocytic scaffolding to α-synuclein clearance unresolved","Zebrafish microexon result is preprint, single line"]},{"year":2026,"claim":"Identified a postsynaptic Dlgap2-Itsn1 axis where Dlgap2 stabilizes Itsn1 against proteasomal degradation, linking ITSN1 protein homeostasis to synaptic function and autism-like behavior.","evidence":"PSD proteomics, co-IP, ubiquitination assay, and Dlgap2 knockdown mouse behavior","pmids":["41673270"],"confidence":"Medium","gaps":["E3 ligase acting on Itsn1 in this context not identified","Single-lab study"]},{"year":null,"claim":"How ITSN1's distinct activities — endocytic scaffolding, GEF-driven actin/GTPase signaling, RNA-binding chaperoning, and ubiquitin-controlled abundance — are integrated and partitioned across isoforms, tissues, and disease states remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of full-length ITSN1 coordinating multiple domains simultaneously","Causal mechanism linking endocytic dysfunction to α-synuclein and autism phenotypes not established","Division of labor between short and long isoforms in vivo undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,2,13]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[11]},{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[12]}],"localization":[{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[2,13]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[1,9]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[11]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[12]}],"pathway":[{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[1,2,13]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[7,11]},{"term_id":"R-HSA-112316","term_label":"Neuronal System","supporting_discovery_ids":[2,13]}],"complexes":["clathrin-coated vesicle","postsynaptic density"],"partners":["EPS15","DNM1","SYNJ1","WIPF1","KHDRBS1","RHOJ","ITCH","DLGAP2"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q15811","full_name":"Intersectin-1","aliases":["SH3 domain-containing protein 1A","SH3P17"],"length_aa":1721,"mass_kda":195.4,"function":"Adapter protein that provides a link between the endocytic membrane traffic and the actin assembly machinery (PubMed:11584276, PubMed:29887380). Acts as a guanine nucleotide exchange factor (GEF) for CDC42, and thereby stimulates actin nucleation mediated by WASL and the ARP2/3 complex (PubMed:11584276). Plays a role in the assembly and maturation of clathrin-coated vesicles (By similarity). Recruits FCHSD2 to clathrin-coated pits (PubMed:29887380). Involved in endocytosis of activated EGFR, and probably also other growth factor receptors (By similarity). Involved in endocytosis of integrin beta-1 (ITGB1) and transferrin receptor (TFR); internalization of ITGB1 as DAB2-dependent cargo but not TFR may involve association with DAB2 (PubMed:22648170). Promotes ubiquitination and subsequent degradation of EGFR, and thereby contributes to the down-regulation of EGFR-dependent signaling pathways. In chromaffin cells, required for normal exocytosis of catecholamines. Required for rapid replenishment of release-ready synaptic vesicles at presynaptic active zones (By similarity). Inhibits ARHGAP31 activity toward RAC1 (PubMed:11744688) Plays a role in synaptic vesicle endocytosis in brain neurons","subcellular_location":"Endomembrane system","url":"https://www.uniprot.org/uniprotkb/Q15811/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/ITSN1","classification":"Not Classified","n_dependent_lines":2,"n_total_lines":1208,"dependency_fraction":0.0016556291390728477},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000205726","cell_line_id":"CID000578","localizations":[{"compartment":"cell_contact","grade":3},{"compartment":"membrane","grade":3},{"compartment":"cytoplasmic","grade":1}],"interactors":[{"gene":"EPS15L1","stoichiometry":0.2},{"gene":"AP2S1","stoichiometry":0.2},{"gene":"AP2M1","stoichiometry":0.2},{"gene":"AP2B1","stoichiometry":0.2},{"gene":"PATL1","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID000578","total_profiled":1310},"omim":[{"mim_id":"613633","title":"DENN/MADD DOMAIN-CONTAINING PROTEIN 1A; DENND1A","url":"https://www.omim.org/entry/613633"},{"mim_id":"605232","title":"PROTEIN KINASE, LYSINE-DEFICIENT 1; WNK1","url":"https://www.omim.org/entry/605232"},{"mim_id":"604464","title":"INTERSECTIN 2; ITSN2","url":"https://www.omim.org/entry/604464"},{"mim_id":"602442","title":"INTERSECTIN 1; ITSN1","url":"https://www.omim.org/entry/602442"},{"mim_id":"601844","title":"PROTEIN KINASE, LYSINE-DEFICIENT 4; WNK4","url":"https://www.omim.org/entry/601844"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Plasma membrane","reliability":"Supported"},{"location":"Nucleoplasm","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/ITSN1"},"hgnc":{"alias_symbol":["SH3P17","MGC134948","MGC134949"],"prev_symbol":["SH3D1A","ITSN"]},"alphafold":{"accession":"Q15811","domains":[{"cath_id":"1.10.238.10","chopping":"20-103","consensus_level":"high","plddt":91.3562,"start":20,"end":103},{"cath_id":"1.10.238.10","chopping":"220-303","consensus_level":"high","plddt":89.6243,"start":220,"end":303},{"cath_id":"2.30.30.40","chopping":"744-775_783-804","consensus_level":"high","plddt":86.3898,"start":744,"end":804},{"cath_id":"2.30.30.40","chopping":"912-971","consensus_level":"high","plddt":89.5315,"start":912,"end":971},{"cath_id":"2.30.30.40","chopping":"1023-1053","consensus_level":"medium","plddt":90.1726,"start":1023,"end":1053},{"cath_id":"2.30.30.40","chopping":"1079-1137","consensus_level":"medium","plddt":84.7954,"start":1079,"end":1137},{"cath_id":"1.20.900.10","chopping":"1215-1408","consensus_level":"high","plddt":90.9147,"start":1215,"end":1408},{"cath_id":"2.30.29.30","chopping":"1428-1495_1510-1583","consensus_level":"medium","plddt":89.4396,"start":1428,"end":1583},{"cath_id":"2.60.40.150","chopping":"1591-1721","consensus_level":"medium","plddt":88.063,"start":1591,"end":1721},{"cath_id":"1.20.5","chopping":"374-539","consensus_level":"medium","plddt":74.6508,"start":374,"end":539}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q15811","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q15811-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q15811-F1-predicted_aligned_error_v6.png","plddt_mean":72.31},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=ITSN1","jax_strain_url":"https://www.jax.org/strain/search?query=ITSN1"},"sequence":{"accession":"Q15811","fasta_url":"https://rest.uniprot.org/uniprotkb/Q15811.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q15811/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q15811"}},"corpus_meta":[{"pmid":"9799604","id":"PMC_9799604","title":"Two isoforms of a human intersectin (ITSN) protein are produced by brain-specific alternative splicing in a stop codon.","date":"1998","source":"Genomics","url":"https://pubmed.ncbi.nlm.nih.gov/9799604","citation_count":82,"is_preprint":false},{"pmid":"18676989","id":"PMC_18676989","title":"Mice deficient for the chromosome 21 ortholog Itsn1 exhibit vesicle-trafficking abnormalities.","date":"2008","source":"Human molecular genetics","url":"https://pubmed.ncbi.nlm.nih.gov/18676989","citation_count":78,"is_preprint":false},{"pmid":"10482960","id":"PMC_10482960","title":"Alu-splice cloning of human Intersectin (ITSN), a putative multivalent binding protein expressed in proliferating and differentiating neurons and overexpressed in Down syndrome.","date":"1999","source":"European journal of human genetics : EJHG","url":"https://pubmed.ncbi.nlm.nih.gov/10482960","citation_count":63,"is_preprint":false},{"pmid":"16442855","id":"PMC_16442855","title":"Alzheimer's disease and endocytic dysfunction: clues from the Down syndrome-related proteins, DSCR1 and ITSN1.","date":"2006","source":"Ageing research reviews","url":"https://pubmed.ncbi.nlm.nih.gov/16442855","citation_count":56,"is_preprint":false},{"pmid":"32547537","id":"PMC_32547537","title":"Lnc-ITSN1-2, Derived From RNA Sequencing, Correlates With Increased Disease Risk, Activity and Promotes CD4+ T Cell Activation, Proliferation and Th1/Th17 Cell Differentiation by Serving as a ceRNA for IL-23R via Sponging miR-125a in Inflammatory Bowel Disease.","date":"2020","source":"Frontiers in immunology","url":"https://pubmed.ncbi.nlm.nih.gov/32547537","citation_count":54,"is_preprint":false},{"pmid":"22558309","id":"PMC_22558309","title":"Intersectin (ITSN) family of scaffolds function as molecular hubs in protein interaction networks.","date":"2012","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/22558309","citation_count":44,"is_preprint":false},{"pmid":"18298590","id":"PMC_18298590","title":"ITSN-1 controls vesicle recycling at the neuromuscular junction and functions in parallel with DAB-1.","date":"2008","source":"Traffic (Copenhagen, Denmark)","url":"https://pubmed.ncbi.nlm.nih.gov/18298590","citation_count":43,"is_preprint":false},{"pmid":"23574942","id":"PMC_23574942","title":"Emerging roles for intersectin (ITSN) in regulating signaling and disease pathways.","date":"2013","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/23574942","citation_count":40,"is_preprint":false},{"pmid":"33248053","id":"PMC_33248053","title":"Atractylodis macrocephalae polysaccharides protect against DSS-induced intestinal injury through a novel lncRNA ITSN1-OT1.","date":"2020","source":"International journal of biological macromolecules","url":"https://pubmed.ncbi.nlm.nih.gov/33248053","citation_count":34,"is_preprint":false},{"pmid":"31497189","id":"PMC_31497189","title":"Downregulation of lncRNA ITSN1-2 correlates with decreased disease risk and activity of rheumatoid arthritis (RA), and reduces RA fibroblast-like synoviocytes proliferation and inflammation via inhibiting NOD2/RIP2 signaling pathway.","date":"2019","source":"American journal of translational research","url":"https://pubmed.ncbi.nlm.nih.gov/31497189","citation_count":27,"is_preprint":false},{"pmid":"20659428","id":"PMC_20659428","title":"Microexon-based regulation of ITSN1 and Src SH3 domains specificity relies on introduction of charged amino acids into the interaction interface.","date":"2010","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/20659428","citation_count":24,"is_preprint":false},{"pmid":"31966382","id":"PMC_31966382","title":"Circulating lnc-ITSN1-2 expression presents a high value in diagnosis of rheumatoid arthritis and correlates with disease activity.","date":"2017","source":"International journal of clinical and experimental pathology","url":"https://pubmed.ncbi.nlm.nih.gov/31966382","citation_count":23,"is_preprint":false},{"pmid":"29958948","id":"PMC_29958948","title":"WIP/ITSN1 complex is involved in cellular vesicle trafficking and formation of filopodia-like protrusions.","date":"2018","source":"Gene","url":"https://pubmed.ncbi.nlm.nih.gov/29958948","citation_count":15,"is_preprint":false},{"pmid":"22975684","id":"PMC_22975684","title":"The LMP2A protein of Epstein-Barr virus regulates phosphorylation of ITSN1 and Shb adaptors by tyrosine kinases.","date":"2012","source":"Cellular signalling","url":"https://pubmed.ncbi.nlm.nih.gov/22975684","citation_count":14,"is_preprint":false},{"pmid":"34707297","id":"PMC_34707297","title":"ITSN1: a novel candidate gene involved in autosomal dominant neurodevelopmental disorder spectrum.","date":"2021","source":"European journal of human genetics : EJHG","url":"https://pubmed.ncbi.nlm.nih.gov/34707297","citation_count":13,"is_preprint":false},{"pmid":"25720380","id":"PMC_25720380","title":"Endocytic deficiency induced by ITSN-1s knockdown alters the Smad2/3-Erk1/2 signaling balance downstream of Alk5.","date":"2015","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/25720380","citation_count":13,"is_preprint":false},{"pmid":"10072581","id":"PMC_10072581","title":"Genomic structure, sequence, and refined mapping of the human intersectin gene (ITSN), which encompasses 250 kb on chromosome 21q22.1-->q22.2.","date":"1998","source":"Cytogenetics and cell genetics","url":"https://pubmed.ncbi.nlm.nih.gov/10072581","citation_count":12,"is_preprint":false},{"pmid":"40056900","id":"PMC_40056900","title":"Haploinsufficiency of ITSN1 is associated with a substantial increased risk of Parkinson's disease.","date":"2025","source":"Cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/40056900","citation_count":11,"is_preprint":false},{"pmid":"39147844","id":"PMC_39147844","title":"Loss-of-function variants in ITSN1 confer high risk of Parkinson's disease.","date":"2024","source":"NPJ Parkinson's disease","url":"https://pubmed.ncbi.nlm.nih.gov/39147844","citation_count":11,"is_preprint":false},{"pmid":"31980169","id":"PMC_31980169","title":"Endothelial cell sprouting driven by RhoJ directly activated by a membrane-anchored Intersectin 1 (ITSN1) RhoGEF module.","date":"2020","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/31980169","citation_count":8,"is_preprint":false},{"pmid":"34554938","id":"PMC_34554938","title":"LncRNA ITSN1-2 knockdown inhibits OGD/R-induced inflammation and apoptosis in mouse hippocampal neurons via sponging miR-195-5p.","date":"2021","source":"Neuroreport","url":"https://pubmed.ncbi.nlm.nih.gov/34554938","citation_count":7,"is_preprint":false},{"pmid":"32780150","id":"PMC_32780150","title":"ITSN1 regulates SAM68 solubility through SH3 domain interactions with SAM68 proline-rich motifs.","date":"2020","source":"Cellular and molecular life sciences : CMLS","url":"https://pubmed.ncbi.nlm.nih.gov/32780150","citation_count":7,"is_preprint":false},{"pmid":"21712076","id":"PMC_21712076","title":"Identification and characterization of a novel mammalian isoform of the endocytic adaptor ITSN1.","date":"2011","source":"Gene","url":"https://pubmed.ncbi.nlm.nih.gov/21712076","citation_count":7,"is_preprint":false},{"pmid":"35500161","id":"PMC_35500161","title":"Longitudinal variation of circulating Inc-ITSN1-2: A novel biomarker reflecting disease severity, inflammation, recurrence, and death risk in acute ischemic stroke patients.","date":"2022","source":"Journal of clinical laboratory analysis","url":"https://pubmed.ncbi.nlm.nih.gov/35500161","citation_count":7,"is_preprint":false},{"pmid":"33850201","id":"PMC_33850201","title":"Synaptic AP2 CCV life cycle regulation by the Eps15, ITSN1, Sgip1/AP2, synaptojanin1 interactome.","date":"2021","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/33850201","citation_count":7,"is_preprint":false},{"pmid":"9465890","id":"PMC_9465890","title":"The SH3D1A gene maps to human chromosome 21q22.1-->q22.2.","date":"1997","source":"Cytogenetics and cell genetics","url":"https://pubmed.ncbi.nlm.nih.gov/9465890","citation_count":6,"is_preprint":false},{"pmid":"38241230","id":"PMC_38241230","title":"Environmental enrichment improves social isolation-induced memory impairment: The possible role of ITSN1-Reelin-AMPA receptor signaling pathway.","date":"2024","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/38241230","citation_count":4,"is_preprint":false},{"pmid":"28161632","id":"PMC_28161632","title":"Mammalian verprolin CR16 acts as a modulator of ITSN scaffold proteins association with actin.","date":"2017","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/28161632","citation_count":4,"is_preprint":false},{"pmid":"38346866","id":"PMC_38346866","title":"Further evidence of involvement of ITSN1 in autosomal dominant neurodevelopmental disorder.","date":"2024","source":"Clinical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/38346866","citation_count":3,"is_preprint":false},{"pmid":"40629453","id":"PMC_40629453","title":"Juanbi Qianggu Formula inhibits fibroblast-like synovicytes activation via repressing LncRNA ITSN1-2 to promote RIP2 K48 ubiquitination.","date":"2025","source":"Chinese medicine","url":"https://pubmed.ncbi.nlm.nih.gov/40629453","citation_count":1,"is_preprint":false},{"pmid":"40487353","id":"PMC_40487353","title":"CircITSN1/EIF4A3/Itsn1 axis mediates postoperative cognitive dysfunction in aged mice: A novel mechanism and therapeutic target.","date":"2025","source":"Molecular therapy. Nucleic acids","url":"https://pubmed.ncbi.nlm.nih.gov/40487353","citation_count":1,"is_preprint":false},{"pmid":"29851086","id":"PMC_29851086","title":"Ubiquitin-ligase AIP4 controls differential ubiquitination and stability of isoforms of the scaffold protein ITSN1.","date":"2018","source":"FEBS letters","url":"https://pubmed.ncbi.nlm.nih.gov/29851086","citation_count":1,"is_preprint":false},{"pmid":"41107259","id":"PMC_41107259","title":"Should ITSN1 be considered as a Mendelian Parkinson's disease gene? Description of three novel families.","date":"2025","source":"NPJ Parkinson's disease","url":"https://pubmed.ncbi.nlm.nih.gov/41107259","citation_count":0,"is_preprint":false},{"pmid":"41673270","id":"PMC_41673270","title":"Dlgap2 deficiency disrupts synaptic homeostasis by promoting ubiquitin-mediated Itsn1 degradation in a valproic acid-induced autism-like model.","date":"2026","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/41673270","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2024.07.25.24310988","title":"Haploinsufficiency of<i>ITSN1</i>is associated with Parkinson’s disease","date":"2024-07-27","source":"bioRxiv","url":"https://doi.org/10.1101/2024.07.25.24310988","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2024.10.23.619860","title":"Phenotypic impact of individual conserved neuronal microexons and their master regulators in zebrafish","date":"2024-10-27","source":"bioRxiv","url":"https://doi.org/10.1101/2024.10.23.619860","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":19415,"output_tokens":4454,"usd":0.062527,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":12266,"output_tokens":4264,"usd":0.083965,"stage2_stop_reason":"end_turn"},"total_usd":0.146492,"stage1_batch_id":"msgbatch_01DUPACZi7AAWwuZiUjseQF4","stage2_batch_id":"msgbatch_018yt7chE3f48PEFtDwtj6AE","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1998,\n      \"finding\": \"Human ITSN1 encodes two isoforms by brain-specific alternative splicing: a ubiquitous short form (1220 aa) containing two EH domains, a coiled-coil region, and five SH3 domains, and a brain-specific long form (1721 aa) that additionally contains GEF, PH, and C2 domains.\",\n      \"method\": \"cDNA cloning, alternative splicing analysis, domain architecture characterization\",\n      \"journal\": \"Genomics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — full-length cDNA sequencing with domain identification, replicated in independent study (PMID:10482960)\",\n      \"pmids\": [\"9799604\", \"10482960\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"In Itsn1 null mice, neurons show slowed endocytosis and increased endosome size, chromaffin cells show reduced exocytosis events, and NGF levels are reduced in the septal brain region, establishing ITSN1 as a regulator of endocytosis and vesicle trafficking in vivo.\",\n      \"method\": \"Knockout mouse generation, live-cell imaging of endocytosis, chromaffin cell amperometry, endosome size measurement by electron microscopy, NGF ELISA\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean KO with multiple orthogonal cellular phenotype readouts (endocytosis kinetics, exocytosis, endosome morphology, NGF levels)\",\n      \"pmids\": [\"18676989\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"In C. elegans, ITSN-1 forms a stable complex with EHS-1 (Eps15) and co-localizes with it at endocytic hotspots ~300 nm from the presynaptic density; itsn-1 null mutants accumulate large irregular vesicles and membrane-associated vesicles at these sites with reduced synaptic vesicle number and reduced NMJ event frequency. Double mutants with dab-1 (Disabled) show synthetic lethality, placing itsn-1 and ehs-1 in the same genetic pathway that functions in parallel with the clathrin-associated sorting protein Disabled.\",\n      \"method\": \"C. elegans genetic null mutants, electron microscopy, electrophysiology (NMJ recordings), co-immunoprecipitation, genetic epistasis (double mutants)\",\n      \"journal\": \"Traffic\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — null mutants with EM, electrophysiology, co-IP, and genetic epistasis in single study\",\n      \"pmids\": [\"18298590\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"A neuron-specific microexon (exon 20) in ITSN1 encodes five amino acids inserted at the beginning of the n-Src loop of the SH3A domain, introducing negatively charged residues toward the interaction interface; mutational analysis confirmed that translocation of these charged residues is required for interaction with dynamin 1.\",\n      \"method\": \"Comparative sequence analysis, structural modeling, site-directed mutagenesis, pulldown/binding assays, zebrafish expression analysis\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — mutagenesis with binding assay in single lab; structural modeling supports interpretation\",\n      \"pmids\": [\"20659428\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"A novel ITSN1 isoform (ITSN1-22a) with an alternative C-terminus (encoded by exon 22a) binds amphiphysin 1 SH3 domain and ITSN1 SH3A via its CTD; an intramolecular interaction within ITSN1-22a negatively regulates its binding to Cbl but not to dynamin 1, indicating isoform-specific binding selectivity controlled by the CTD.\",\n      \"method\": \"RT-PCR/cDNA cloning, co-immunoprecipitation, in vitro pulldown, Western blot\",\n      \"journal\": \"Gene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP and in vitro pulldown with multiple partners in single lab, two orthogonal methods\",\n      \"pmids\": [\"21712076\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"ITSN1 interacts with Epstein-Barr virus LMP2A via the SH3 domains of ITSN1 binding to proline-rich motifs in both N- and C-tails of LMP2A. Syk kinase promotes phosphorylation of ITSN1 in LMP2A-expressing cells. The Shb adaptor bridges ITSN1 to LMP2A by simultaneously binding phosphorylated LMP2A tyrosines and ITSN1 SH3 domains.\",\n      \"method\": \"Co-immunoprecipitation, kinase inhibitor treatment, phosphorylation assays\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — reciprocal Co-IP with pharmacological validation, single lab\",\n      \"pmids\": [\"22975684\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Yeast two-hybrid screening identified >100 new binding partners for ITSN1 and ITSN2, implicating ITSNs in regulation of Rab and Arf GTPase pathways and the DISC1 interactome; ITSN proteins also form homomeric (ITSN1-ITSN1) and heteromeric (ITSN1-ITSN2) complexes.\",\n      \"method\": \"High-throughput yeast two-hybrid screen, co-immunoprecipitation validation\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — large Y2H screen with Co-IP validation for selected partners, single lab\",\n      \"pmids\": [\"22558309\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"ITSN-1s knockdown in endothelial cells impairs clathrin-mediated and caveolae-mediated endocytosis, causing TGFβ-RI (Alk5) to be internalized via compensatory enlarged endocytic structures leading to its ubiquitylation and degradation; this shifts Alk5 signaling from Smad2/3 toward Erk1/2 activation via preferential formation of the Alk5-mSos-Grb2 complex over the Alk5-Smad-SARA complex, promoting endothelial cell proliferation.\",\n      \"method\": \"shRNA knockdown in cultured endothelial cells and in vivo mouse model, co-immunoprecipitation, Western blot for signaling intermediates, microparticle transfer assays\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KD with defined signaling pathway readouts and in vivo validation, single lab\",\n      \"pmids\": [\"25720380\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"CR16 (a verprolin family member) forms complexes with ITSN1 and ITSN2 in human cell lines; overexpressed CR16 promotes co-localization of ITSN1 with F-actin in MCF-7 cells, indicating CR16 modulates ITSN1 association with the actin cytoskeleton.\",\n      \"method\": \"Co-immunoprecipitation, confocal immunofluorescence co-localization\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP and imaging, single lab, no functional rescue\",\n      \"pmids\": [\"28161632\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"WIP (WASP-interacting protein) interacts with ITSN1; the WIP/ITSN1 complex co-localizes with RAB4-positive fast recycling endosomes, participates in transferrin receptor recycling, and promotes formation of filopodia-like protrusions in MCF-7 cells. WIP enhances N-WASP interaction with ITSN1 and promotes ITSN1/β-actin association.\",\n      \"method\": \"Co-immunoprecipitation, confocal co-localization, transferrin recycling assay, overexpression/knockdown morphology analysis\",\n      \"journal\": \"Gene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP, functional recycling assay, and morphology readout, multiple orthogonal methods in single lab\",\n      \"pmids\": [\"29958948\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"The E3 ubiquitin ligase AIP4/ITCH monoubiquitinates the major isoform ITSN1-s (stabilizing it) and mono- plus oligo-ubiquitinates the minor isoform ITSN1-22a (causing its proteasomal degradation), explaining the low cellular abundance of ITSN1-22a.\",\n      \"method\": \"Ubiquitination assays, proteasome inhibitor treatment, Western blot, co-immunoprecipitation\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct ubiquitination assay with proteasome inhibitor confirmation and isoform-specific comparison, single lab\",\n      \"pmids\": [\"29851086\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"A membrane-anchored ITSN1 DH-PH construct directly activates RhoJ GTPase and promotes endothelial cell sprouting; this is sensitive to phosphorylation by focal adhesion kinase (FAK) and independent of Cdc42 activation, defining an ITSN1/RhoJ signaling axis distinct from the known ITSN1/Cdc42 pathway.\",\n      \"method\": \"Gain-of-function overexpression, dominant-negative RhoJ trapping, FAK inhibitor treatment, co-immunoprecipitation, cell sprouting assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct interaction plus functional gain-of-function with pharmacological and dominant-negative controls, single lab\",\n      \"pmids\": [\"31980169\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"ITSN1 SH3 domains interact with proline-rich motifs (PRMs) of SAM68; this interaction enhances SAM68 solubility in vitro and induces dissociation of SAM68 Nuclear Bodies (SNBs) in HeLa cells via SH3A binding to the P0 PRM. An additional SH3 domain (SH3D) of ITSN1 can bind mRNAs, suggesting ITSN1 acts as a chaperone for SAM68 within nuclear ribonucleoprotein complexes.\",\n      \"method\": \"In vitro binding/solubility assays, immunofluorescence/nuclear body imaging in HeLa cells, domain-mapping pulldowns\",\n      \"journal\": \"Cellular and molecular life sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro reconstitution of solubility plus cellular imaging with domain-specific mutants, single lab\",\n      \"pmids\": [\"32780150\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ITSN1 determines synaptojanin1 levels within clathrin-coated vesicles (CCVs) at synapses; Sgip1/AP2 excess hinders synaptojanin1 binding to ITSN1, lowering its CCV levels. ITSN1 levels in turn are determined by Eps15 (not Eps15L1), placing ITSN1 in a regulatory hierarchy: Eps15→ITSN1→synaptojanin1 for CCV life cycle control.\",\n      \"method\": \"CCV purification from mouse brain, quantitative proteomics of CCV interactome, AP1/σ1B knockout mouse comparison\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — quantitative CCV proteomics with genetic model comparison; single lab but rigorous biochemical fractionation\",\n      \"pmids\": [\"33850201\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"In a Drosophila model expressing human α-synuclein, haploinsufficiency of Dap160 (ITSN1 ortholog) exacerbates α-synuclein-induced compound eye degeneration and motor deficits; in vitro assays further indicate a physical interaction between ITSN1 and α-synuclein.\",\n      \"method\": \"Drosophila genetic haploinsufficiency model, eye degeneration scoring, motor behavior assay, in vitro binding assay\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo Drosophila epistasis plus in vitro interaction assay, replicated across two independent reports (PMID:40056900, bio_10.1101_2024.07.25.24310988)\",\n      \"pmids\": [\"40056900\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"Dlgap2 interacts with Itsn1 at the postsynaptic density; Dlgap2 knockdown causes specific reduction of Itsn1 via ubiquitin-mediated proteasomal degradation, resulting in synaptic deficits and autism-like behaviors in mice, defining a Dlgap2-Itsn1 regulatory axis at the synapse.\",\n      \"method\": \"Proteomics of postsynaptic density fraction, co-immunoprecipitation, ubiquitination assay, Dlgap2 knockdown mouse behavior\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — PSD proteomics with Co-IP and ubiquitination assay, in vivo behavioral phenotype, single lab\",\n      \"pmids\": [\"41673270\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Deletion of the itsn1 microexon in zebrafish produces defects in neuritogenesis, demonstrating that this alternatively spliced insert contributes a specific functional role in neuronal morphogenesis in vivo.\",\n      \"method\": \"CRISPR/Cas9 microexon deletion in zebrafish, neuritogenesis phenotype analysis\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — preprint, single zebrafish line, phenotype without full mechanistic pathway placement\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"ITSN1 is a multi-domain scaffold protein (two EH domains, coiled-coil, up to five SH3 domains, and in the brain-specific long isoform, GEF/DH-PH, PH, and C2 domains) that coordinates clathrin-mediated endocytosis and synaptic vesicle recycling by scaffolding dynamin, Eps15, and synaptojanin1 into clathrin-coated vesicle complexes; its SH3 domains engage diverse proline-rich partners (dynamin 1, Cbl, SAM68, LMP2A, WIP, CR16) with binding selectivity modulated by neuron-specific microexon insertion and by isoform-specific intramolecular interactions, while its DH-PH module acts as a GEF that directly activates Cdc42 and RhoJ to drive actin-dependent membrane processes; isoform stability is differentially controlled by AIP4/ITCH-mediated ubiquitination, and loss of ITSN1 function in vivo (knockout mice, C. elegans nulls, Drosophila haploinsufficiency) produces defective endocytosis, enlarged early endosomes, reduced synaptic vesicle numbers, altered TGFβ/Erk signaling, and exacerbation of α-synuclein toxicity.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ITSN1 is a multi-domain scaffold that organizes clathrin-mediated endocytosis and synaptic vesicle recycling, expressed as a ubiquitous short isoform (two EH domains, coiled-coil, five SH3 domains) and a brain-specific long isoform that adds GEF/DH-PH, PH, and C2 domains [#0]. Loss of ITSN1 in vivo slows endocytosis, enlarges endosomes, reduces exocytosis and synaptic vesicle number, and lowers septal NGF, establishing its role in vesicle trafficking [#1, #2]. At the synapse it operates within a regulatory hierarchy controlling the clathrin-coated vesicle life cycle, with Eps15/EHS-1 determining ITSN1 levels and ITSN1 in turn setting synaptojanin1 content in coated vesicles [#2, #13]. Its SH3 domains engage proline-rich partners with selectivity tuned by neuron-specific microexon insertion into the SH3A n-Src loop, which is required for dynamin 1 binding and for neuronal morphogenesis [#3, #16], and by isoform-specific intramolecular regulation through alternative C-termini [#4]. SH3-mediated contacts extend ITSN1 to actin-cytoskeleton regulators including WIP/N-WASP, supporting transferrin-receptor recycling and filopodia formation [#9], and to SAM68, whose solubility and nuclear-body dynamics ITSN1 modulates [#12]. The DH-PH module functions as a GEF, activating RhoJ in a FAK-sensitive, Cdc42-independent axis to drive endothelial sprouting [#11]. ITSN1 abundance is set by ubiquitination: AIP4/ITCH differentially stabilizes or degrades distinct isoforms [#10], and Dlgap2 protects synaptic Itsn1 from proteasomal degradation [#15]. ITSN1 also intersects disease processes, modulating TGFβ-RI (Alk5) trafficking and the Smad-versus-Erk signaling balance in endothelial cells [#7] and exacerbating α-synuclein toxicity when reduced [#14].\",\n  \"teleology\": [\n    {\n      \"year\": 1998,\n      \"claim\": \"Established the domain architecture and isoform diversity of ITSN1, defining it as a candidate multi-domain scaffold with a brain-specific signaling-capable long form.\",\n      \"evidence\": \"cDNA cloning and alternative splicing analysis identifying short and long isoforms\",\n      \"pmids\": [\"9799604\", \"10482960\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Domain architecture alone did not establish in vivo function\", \"Partner repertoire of individual domains unresolved at this stage\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Demonstrated that ITSN1 is required for endocytosis and vesicle trafficking in vivo, moving it from candidate scaffold to validated trafficking regulator.\",\n      \"evidence\": \"Itsn1 knockout mice with endocytosis imaging, chromaffin cell amperometry, EM endosome sizing, and NGF ELISA; parallel C. elegans null mutants with EM, electrophysiology, co-IP, and genetic epistasis\",\n      \"pmids\": [\"18676989\", \"18298590\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular step within the coated-vesicle cycle that ITSN1 controls not pinpointed\", \"Relationship between endocytic and exocytic phenotypes unclear\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Showed that a neuron-specific microexon in the SH3A domain tunes partner binding, explaining how a single scaffold gene generates neuron-specialized interaction selectivity.\",\n      \"evidence\": \"Sequence/structural modeling and site-directed mutagenesis with binding assays for dynamin 1\",\n      \"pmids\": [\"20659428\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab binding assay\", \"Functional consequence of microexon insertion in vivo not addressed here\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Revealed isoform-specific binding selectivity, where an alternative C-terminus mediates an intramolecular interaction that selectively gates SH3 partner engagement.\",\n      \"evidence\": \"cDNA cloning, co-IP, and in vitro pulldown of ITSN1-22a with amphiphysin1, Cbl, and dynamin1\",\n      \"pmids\": [\"21712076\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Physiological role of ITSN1-22a not established\", \"Structural basis of the intramolecular contact not resolved\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Expanded the ITSN1 interactome and showed it engages viral and signaling adaptors plus GTPase pathways, and self-associates into homo/heteromeric complexes.\",\n      \"evidence\": \"Reciprocal co-IP with kinase inhibitors for LMP2A/Syk/Shb; high-throughput Y2H screen with co-IP validation implicating Rab/Arf and DISC1 networks\",\n      \"pmids\": [\"22975684\", \"22558309\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Most Y2H partners lack functional validation\", \"Biological significance of ITSN1 oligomerization unknown\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Connected ITSN1 endocytic function to growth-factor signaling output, showing its loss reroutes Alk5 toward degradation and shifts TGFβ signaling from Smad to Erk.\",\n      \"evidence\": \"shRNA knockdown in endothelial cells and mouse model with co-IP and signaling Western blots\",\n      \"pmids\": [\"25720380\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct ITSN1-Alk5 contact versus indirect trafficking effect not separated\", \"Single-lab readout\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Linked ITSN1 to the actin cytoskeleton via CR16, indicating scaffolding beyond endocytosis into cytoskeletal organization.\",\n      \"evidence\": \"Co-IP and confocal co-localization of CR16 with ITSN1/F-actin in cell lines\",\n      \"pmids\": [\"28161632\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single Co-IP and imaging without functional rescue\", \"No in vivo relevance demonstrated\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Defined how ITSN1 couples to actin-polymerization machinery and recycling endosomes through WIP/N-WASP, and how its protein levels are controlled by isoform-selective ubiquitination.\",\n      \"evidence\": \"Co-IP, transferrin recycling assay, and morphology analysis for WIP; ubiquitination assays with proteasome inhibitor for AIP4/ITCH\",\n      \"pmids\": [\"29958948\", \"29851086\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether WIP and endocytic scaffolding functions compete is unclear\", \"In vivo consequence of AIP4-mediated isoform control not tested\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Distinguished a Cdc42-independent ITSN1/RhoJ GEF axis and characterized ITSN1 as a chaperone for SAM68 in nuclear RNP complexes, broadening its mechanistic repertoire beyond endocytosis.\",\n      \"evidence\": \"Membrane-anchored DH-PH gain-of-function with dominant-negative and FAK inhibitor for RhoJ; in vitro solubility assays and nuclear-body imaging with domain mutants for SAM68\",\n      \"pmids\": [\"31980169\", \"32780150\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"RhoJ activation shown with membrane-anchored construct, not full-length endogenous ITSN1\", \"Physiological role of ITSN1-SAM68 chaperoning in vivo undefined\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Placed ITSN1 in an ordered regulatory hierarchy controlling the synaptic coated-vesicle life cycle: Eps15 sets ITSN1 levels, which set synaptojanin1 levels.\",\n      \"evidence\": \"Quantitative CCV proteomics from mouse brain with AP knockout comparisons\",\n      \"pmids\": [\"33850201\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which ITSN1 recruits synaptojanin1 to CCVs not structurally defined\", \"Single-lab biochemical fractionation\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Tied ITSN1 to neurodegeneration and confirmed the microexon's organismal role, showing reduced ITSN1 worsens α-synuclein toxicity and microexon loss disrupts neuritogenesis.\",\n      \"evidence\": \"Drosophila α-synuclein haploinsufficiency model with in vitro binding; CRISPR microexon deletion in zebrafish (preprint)\",\n      \"pmids\": [\"40056900\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism connecting endocytic scaffolding to α-synuclein clearance unresolved\", \"Zebrafish microexon result is preprint, single line\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Identified a postsynaptic Dlgap2-Itsn1 axis where Dlgap2 stabilizes Itsn1 against proteasomal degradation, linking ITSN1 protein homeostasis to synaptic function and autism-like behavior.\",\n      \"evidence\": \"PSD proteomics, co-IP, ubiquitination assay, and Dlgap2 knockdown mouse behavior\",\n      \"pmids\": [\"41673270\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"E3 ligase acting on Itsn1 in this context not identified\", \"Single-lab study\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How ITSN1's distinct activities — endocytic scaffolding, GEF-driven actin/GTPase signaling, RNA-binding chaperoning, and ubiquitin-controlled abundance — are integrated and partitioned across isoforms, tissues, and disease states remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of full-length ITSN1 coordinating multiple domains simultaneously\", \"Causal mechanism linking endocytic dysfunction to α-synuclein and autism phenotypes not established\", \"Division of labor between short and long isoforms in vivo undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 2, 13]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [11]},\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [12]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [2, 13]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [1, 9]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [11]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [12]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [1, 2, 13]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [7, 11]},\n      {\"term_id\": \"R-HSA-112316\", \"supporting_discovery_ids\": [2, 13]}\n    ],\n    \"complexes\": [\"clathrin-coated vesicle\", \"postsynaptic density\"],\n    \"partners\": [\"EPS15\", \"DNM1\", \"SYNJ1\", \"WIPF1\", \"KHDRBS1\", \"RHOJ\", \"ITCH\", \"DLGAP2\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}