{"gene":"SH3GL2","run_date":"2026-06-10T07:46:31","timeline":{"discoveries":[{"year":1997,"finding":"SH3GL2 (SH3p4) binds to both synaptojanin and dynamin I via its SH3 domain (closely related to the Grb2 SH3 domain); pools of synaptojanin and dynamin I were co-precipitated from brain extracts with anti-SH3p4/8/13 antibodies, and SH3p4 transcript was detected exclusively in brain with the protein concentrated in nerve terminals.","method":"Yeast two-hybrid screening with synaptojanin proline-rich tail as bait, followed by co-immunoprecipitation from brain extracts and immunofluorescence localization","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP from brain extracts plus two-hybrid, replicated across the SH3p4/8/13 family in same study and confirmed by later work","pmids":["9238017"],"is_preprint":false},{"year":1999,"finding":"Antibody-mediated disruption of endophilin/SH3p4 function in a tonically stimulated lamprey synapse blocked invagination of clathrin-coated pits adjacent to the active zone, arresting synaptic vesicle recycling at an early-to-late endocytosis transition; in a cell-free system, endophilin was not associated with clathrin coats but functioned as a partner of dynamin.","method":"Antibody microinjection into living synapse followed by electron microscopy; cell-free biochemical fractionation assay","journal":"Neuron","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct loss-of-function in intact synapse with defined ultrastructural phenotype, combined with cell-free biochemical assay, two orthogonal methods in one study","pmids":["10677033"],"is_preprint":false},{"year":1999,"finding":"SH3GL2 (SH3p4) specifically binds the proline-rich third intracellular loop of the beta1-adrenergic receptor (but not beta2-AR) via its C-terminal SH3 domain; overexpression of SH3p4 in HEK293 cells promotes agonist-induced internalization of beta1-AR and modestly decreases Gs coupling efficacy of beta1-AR.","method":"GST pull-down assay with beta1-AR third intracellular loop, yeast two-hybrid, Co-IP in HEK293 cells, overexpression functional assay (internalization and cAMP assays)","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (pull-down, two-hybrid, Co-IP, functional overexpression) in one study with domain-level specificity demonstrated","pmids":["10535961"],"is_preprint":false},{"year":2010,"finding":"Knockdown of SH3GL2 in Hep2 laryngeal carcinoma cells upregulates EGFR expression and increases phosphorylated ERK1/2; treatment with MEK1/2 inhibitor U0126 in SH3GL2-knockdown cells reversed the increase in proliferation and decrease in apoptosis, placing SH3GL2 upstream of EGFR in the MEK-ERK signaling pathway.","method":"RNA interference knockdown, Western blot for EGFR and p-ERK1/2, pharmacological MEK inhibition (U0126), MTT proliferation assay, flow cytometry apoptosis assay","journal":"Medical science monitor","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic knockdown with pharmacological epistasis, two orthogonal readouts, single lab","pmids":["20512084"],"is_preprint":false},{"year":2012,"finding":"Forced overexpression of wild-type SH3GL2 in NSCLC cell lines increased EGFR internalization and degradation, reduced active EGFR expression, and decreased activated AKT (Ser473), STAT3 (Tyr705), and PI3K levels; it also downregulated SH3GL2 interactor USP9X and activated β-catenin, reducing in vitro and in vivo cellular growth and invasion.","method":"Stable overexpression of wild-type SH3GL2 in three NSCLC cell lines, Western blot, EGFR internalization assay, proliferation/invasion/colony formation assays, mouse xenograft in vivo","journal":"Journal of molecular medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple cell lines, in vivo validation, multiple downstream readouts, single lab","pmids":["22968441"],"is_preprint":false},{"year":2013,"finding":"Stable silencing of Sh3gl2 in RT4 urothelial carcinoma cells inhibited EGF-induced EGFR internalization, increased EGFR activation, stimulated phosphorylation of Src family kinases and STAT3, enhanced proliferation and colony formation, and promoted xenograft growth; forced re-expression of Sh3gl2 in T24 cells attenuated these oncogenic behaviors.","method":"Stable RNA interference knockdown and forced re-expression, EGFR internalization assay, Western blot for p-EGFR/p-Src/p-STAT3, proliferation/colony assays, subrenal capsule xenograft","journal":"Neoplasia","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal loss-of-function and gain-of-function experiments in multiple cell lines with mechanistic molecular readouts and in vivo validation","pmids":["23814487"],"is_preprint":false},{"year":2014,"finding":"In glioblastoma stem cells, knockdown of SH3GL2 (mimicking miR-330 overexpression) activated ERK and PI3K/AKT signaling pathways and decreased apoptotic protein expression while increasing anti-apoptotic protein expression; co-transfection with shRNA against SH3GL2 plus miR-330 mimic confirmed that miR-330 promotes malignant behavior via SH3GL2 downregulation.","method":"shRNA knockdown, miRNA mimic transfection, Western blot for ERK/AKT pathway components and apoptosis markers, orthotopic mouse xenograft","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic loss-of-function with pathway readouts and in vivo validation, single lab","pmids":["24736727"],"is_preprint":false},{"year":2017,"finding":"Knockdown of SH3GL2 in glioma cells activated STAT3 signaling and promoted expression and secretion of MMP2, enhancing cell migration and invasion; conversely, overexpression of SH3GL2 suppressed STAT3 activation and reduced MMP2 levels, inhibiting migration and invasion.","method":"siRNA knockdown and plasmid overexpression, Western blot for p-STAT3 and MMP2, ELISA/zymography for MMP2 secretion, scratch and Transwell invasion assays","journal":"Journal of cellular and molecular medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal gain/loss-of-function with defined molecular pathway readout, single lab","pmids":["28470949"],"is_preprint":false},{"year":2023,"finding":"Extracellular calcium influx in the pre-synaptic terminal triggers EndoA (endophilin A/SH3GL2 ortholog) redistribution from the plasma membrane to the cytosol, where it interacts with autophagic membranes to promote autophagosome formation; a specific residue in the flexible region of EndoA mediates this calcium-dependent mobility. A Parkinson's disease-risk mutation in SH3GL2 disrupts calcium sensing, rendering the protein immobile and unable to respond to calcium influx, thereby blocking synaptic autophagy induction.","method":"Live imaging of EndoA mobility (FRAP/fluorescence), genetic mutagenesis of calcium-sensing residue, Drosophila neuronal model, autophagosome formation assay, characterization of PD-risk variant","journal":"Autophagy","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct localization experiments with functional consequence (autophagosome formation), mutagenesis of specific residue, single lab with model organism ortholog validation","pmids":["37067454"],"is_preprint":false}],"current_model":"SH3GL2 (endophilin A1/SH3p4) is a nerve-terminal-enriched SH3-domain protein that acts as an adaptor in clathrin-mediated endocytosis by binding dynamin and synaptojanin to drive clathrin-coated pit invagination and synaptic vesicle recycling; it also promotes EGFR internalization and degradation (suppressing downstream EGFR–MEK/ERK, PI3K/AKT, and STAT3/MMP2 signaling) and, in response to calcium influx at the synapse, translocates from the plasma membrane to autophagic membranes to initiate autophagosome formation—a process disrupted by a Parkinson's disease-risk mutation that abolishes its calcium sensing."},"narrative":{"mechanistic_narrative":"SH3GL2 (endophilin A1/SH3p4) is a brain-enriched, nerve-terminal-concentrated SH3-domain adaptor that drives clathrin-mediated endocytosis and synaptic vesicle recycling [PMID:9238017, PMID:10677033]. Through its SH3 domain it binds the proline-rich tails of synaptojanin and dynamin I [PMID:9238017], and antibody-mediated disruption at the synapse arrests clathrin-coated pit invagination adjacent to the active zone at an early-to-late endocytosis transition, where it functions as a dynamin partner rather than a clathrin coat component [PMID:10677033]. Its adaptor activity extends to receptor internalization: it binds the proline-rich third intracellular loop of the beta1-adrenergic receptor and promotes its agonist-induced internalization [PMID:10535961], and it drives EGFR internalization and degradation, thereby restraining downstream EGFR-dependent MEK/ERK, PI3K/AKT, Src, and STAT3/MMP2 signaling [PMID:20512084, PMID:22968441, PMID:23814487, PMID:28470949]. Consistent with this role, loss of SH3GL2 in multiple carcinoma and glioma models elevates active EGFR and pathway output to promote proliferation, invasion, and tumor growth, while re-expression suppresses these behaviors, establishing SH3GL2 as a tumor suppressor acting upstream of EGFR [PMID:23814487, PMID:24736727, PMID:28470949]. Beyond endocytosis, calcium influx at the pre-synaptic terminal triggers SH3GL2 redistribution from the plasma membrane to autophagic membranes to initiate autophagosome formation, via a calcium-sensing residue in its flexible region; a Parkinson's disease-risk mutation abolishes this calcium sensing and blocks synaptic autophagy induction [PMID:37067454].","teleology":[{"year":1997,"claim":"Established SH3GL2 as a nerve-terminal SH3 adaptor by identifying its direct binding partners, answering what molecular machinery it engages.","evidence":"Yeast two-hybrid with synaptojanin proline-rich bait, reciprocal Co-IP from brain extracts, and immunofluorescence localization","pmids":["9238017"],"confidence":"High","gaps":["Functional consequence of dynamin/synaptojanin binding not yet shown","Domain-level binding determinants on partners not mapped"]},{"year":1999,"claim":"Demonstrated a direct functional role in clathrin-coated pit invagination, placing SH3GL2 mechanistically at an endocytic step rather than within the clathrin coat.","evidence":"Antibody microinjection into a tonically stimulated lamprey synapse with EM, plus cell-free biochemical fractionation","pmids":["10677033"],"confidence":"High","gaps":["Precise biophysical contribution to membrane curvature not resolved","Relationship to dynamin GTPase cycle not detailed"]},{"year":1999,"claim":"Extended the adaptor role beyond synaptic vesicles to GPCR internalization with receptor-subtype specificity.","evidence":"GST pull-down with beta1-AR third intracellular loop, two-hybrid, Co-IP and overexpression internalization/cAMP assays in HEK293","pmids":["10535961"],"confidence":"High","gaps":["Endogenous relevance in neurons not tested","Basis of beta1- vs beta2-AR selectivity not structurally defined"]},{"year":2010,"claim":"Linked SH3GL2 to EGFR/MEK-ERK control in cancer, positioning it upstream of EGFR signaling.","evidence":"RNAi knockdown in Hep2 cells with MEK inhibitor (U0126) epistasis, proliferation and apoptosis assays","pmids":["20512084"],"confidence":"Medium","gaps":["Direct effect on EGFR trafficking not shown in this study","Single cell line and single lab"]},{"year":2012,"claim":"Showed mechanistically that SH3GL2 drives EGFR internalization and degradation, dampening AKT/STAT3/PI3K output and tumor growth.","evidence":"Stable WT overexpression in three NSCLC lines, internalization assay, Western blot, and mouse xenograft","pmids":["22968441"],"confidence":"Medium","gaps":["Mechanism connecting EGFR degradation to USP9X and beta-catenin not fully resolved","Single lab"]},{"year":2013,"claim":"Provided reciprocal loss- and gain-of-function evidence that SH3GL2 controls EGFR internalization and Src/STAT3 activation as a tumor suppressor.","evidence":"Stable knockdown and re-expression in urothelial carcinoma lines, internalization and phospho-protein readouts, subrenal capsule xenograft","pmids":["23814487"],"confidence":"High","gaps":["Whether endocytic adaptor activity per se is required for suppression not separated from other roles"]},{"year":2017,"claim":"Tied SH3GL2 loss to STAT3-driven MMP2 secretion and invasion, defining a migration/invasion axis.","evidence":"siRNA knockdown and overexpression in glioma cells, p-STAT3/MMP2 Western blot, zymography, scratch and Transwell assays","pmids":["28470949"],"confidence":"Medium","gaps":["Direct link between EGFR trafficking and STAT3/MMP2 in this context not established","Single lab"]},{"year":2023,"claim":"Revealed a distinct, calcium-triggered role in synaptic autophagy and a disease-relevant defect, answering how SH3GL2 transitions from endocytosis to autophagosome biogenesis.","evidence":"Live FRAP imaging, mutagenesis of a calcium-sensing residue, Drosophila EndoA ortholog model, autophagosome formation assay, and PD-risk variant characterization","pmids":["37067454"],"confidence":"Medium","gaps":["Mechanism by which the flexible-region residue senses calcium not structurally defined","Validation in mammalian neurons of the human variant not shown","Single lab"]},{"year":null,"claim":"How the endocytic adaptor function, EGFR-trafficking tumor-suppressor role, and calcium-gated autophagy function are coordinated within a single neuron or cell remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model unifying SH3-domain binding, curvature, and calcium sensing","Whether the same pool of protein performs all three roles is unknown","Endogenous regulation switching between endocytosis and autophagy not defined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,1,2]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[0]},{"term_id":"GO:0140299","term_label":"molecular sensor activity","supporting_discovery_ids":[8]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[1,8]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[8]}],"pathway":[],"complexes":[],"partners":["DNM1","SYNJ1","ADRB1","EGFR","USP9X"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q99962","full_name":"Endophilin-A1","aliases":["EEN-B1","Endophilin-1","SH3 domain protein 2A","SH3 domain-containing GRB2-like protein 2"],"length_aa":352,"mass_kda":40.0,"function":"Implicated in synaptic vesicle endocytosis. May recruit other proteins to membranes with high curvature. Required for BDNF-dependent dendrite outgrowth. Cooperates with SH3GL2 to mediate BDNF-NTRK2 early endocytic trafficking and signaling from early endosomes","subcellular_location":"Cytoplasm; Membrane; Early endosome; Presynapse","url":"https://www.uniprot.org/uniprotkb/Q99962/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/SH3GL2","classification":"Not Classified","n_dependent_lines":1,"n_total_lines":1208,"dependency_fraction":0.0008278145695364238},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"SH3GL1","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/SH3GL2","total_profiled":1310},"omim":[{"mim_id":"617716","title":"RHO GTPase-ACTIVATING PROTEIN 44; ARHGAP44","url":"https://www.omim.org/entry/617716"},{"mim_id":"617361","title":"TRANSMEMBRANE PROTEIN 108; TMEM108","url":"https://www.omim.org/entry/617361"},{"mim_id":"613633","title":"DENN/MADD DOMAIN-CONTAINING PROTEIN 1A; DENND1A","url":"https://www.omim.org/entry/613633"},{"mim_id":"613004","title":"HUNTINGTIN; HTT","url":"https://www.omim.org/entry/613004"},{"mim_id":"611540","title":"SH3-DOMAIN GRB2-LIKE (ENDOPHILIN)-INTERACTING PROTEIN 1; SGIP1","url":"https://www.omim.org/entry/611540"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Microtubules","reliability":"Approved"},{"location":"Cytokinetic bridge","reliability":"Additional"},{"location":"Mitotic spindle","reliability":"Additional"}],"tissue_specificity":"Group enriched","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"brain","ntpm":227.8},{"tissue":"retina","ntpm":69.5}],"url":"https://www.proteinatlas.org/search/SH3GL2"},"hgnc":{"alias_symbol":["SH3P4","SH3D2A","CNSA2","EEN-B1"],"prev_symbol":[]},"alphafold":{"accession":"Q99962","domains":[{"cath_id":"1.20.1270.60","chopping":"27-248","consensus_level":"high","plddt":91.9323,"start":27,"end":248},{"cath_id":"2.30.30.40","chopping":"293-347","consensus_level":"high","plddt":91.3835,"start":293,"end":347}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q99962","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q99962-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q99962-F1-predicted_aligned_error_v6.png","plddt_mean":85.88},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=SH3GL2","jax_strain_url":"https://www.jax.org/strain/search?query=SH3GL2"},"sequence":{"accession":"Q99962","fasta_url":"https://rest.uniprot.org/uniprotkb/Q99962.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q99962/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q99962"}},"corpus_meta":[{"pmid":"9238017","id":"PMC_9238017","title":"The SH3p4/Sh3p8/SH3p13 protein family: binding partners for synaptojanin and dynamin via a Grb2-like Src homology 3 domain.","date":"1997","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/9238017","citation_count":335,"is_preprint":false},{"pmid":"10677033","id":"PMC_10677033","title":"Endophilin/SH3p4 is required for the transition from early to late stages in clathrin-mediated synaptic vesicle endocytosis.","date":"1999","source":"Neuron","url":"https://pubmed.ncbi.nlm.nih.gov/10677033","citation_count":276,"is_preprint":false},{"pmid":"10535961","id":"PMC_10535961","title":"Identification of the endophilins (SH3p4/p8/p13) as novel binding partners for the beta1-adrenergic receptor.","date":"1999","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/10535961","citation_count":125,"is_preprint":false},{"pmid":"23675485","id":"PMC_23675485","title":"Overexpression of EGFR in head and neck squamous cell carcinoma is associated with inactivation of SH3GL2 and CDC25A genes.","date":"2013","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/23675485","citation_count":62,"is_preprint":false},{"pmid":"23029364","id":"PMC_23029364","title":"MicroRNA-330 is an oncogenic factor in glioblastoma cells by regulating SH3GL2 gene.","date":"2012","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/23029364","citation_count":53,"is_preprint":false},{"pmid":"18239974","id":"PMC_18239974","title":"Frequent deletion and methylation in SH3GL2 and CDKN2A loci are associated with early- and late-onset breast carcinoma.","date":"2008","source":"Annals of surgical oncology","url":"https://pubmed.ncbi.nlm.nih.gov/18239974","citation_count":50,"is_preprint":false},{"pmid":"24736727","id":"PMC_24736727","title":"MiR-330-mediated regulation of SH3GL2 expression enhances malignant behaviors of glioblastoma stem cells by activating ERK and PI3K/AKT signaling pathways.","date":"2014","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/24736727","citation_count":46,"is_preprint":false},{"pmid":"19023882","id":"PMC_19023882","title":"SH3GL2 and CDKN2A/2B loci are independently altered in early dysplastic lesions of head and neck: correlation with HPV infection and tobacco habit.","date":"2009","source":"The Journal of pathology","url":"https://pubmed.ncbi.nlm.nih.gov/19023882","citation_count":45,"is_preprint":false},{"pmid":"22968441","id":"PMC_22968441","title":"SH3GL2 is frequently deleted in non-small cell lung cancer and downregulates tumor growth by modulating EGFR signaling.","date":"2012","source":"Journal of molecular medicine (Berlin, Germany)","url":"https://pubmed.ncbi.nlm.nih.gov/22968441","citation_count":30,"is_preprint":false},{"pmid":"28470949","id":"PMC_28470949","title":"Loss of SH3GL2 promotes the migration and invasion behaviours of glioblastoma cells through activating the STAT3/MMP2 signalling.","date":"2017","source":"Journal of cellular and molecular medicine","url":"https://pubmed.ncbi.nlm.nih.gov/28470949","citation_count":29,"is_preprint":false},{"pmid":"23814487","id":"PMC_23814487","title":"Loss of Sh3gl2/endophilin A1 is a common event in urothelial carcinoma that promotes malignant behavior.","date":"2013","source":"Neoplasia (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/23814487","citation_count":26,"is_preprint":false},{"pmid":"20512084","id":"PMC_20512084","title":"SH3GL2 gene participates in MEK-ERK signal pathway partly by regulating EGFR in the laryngeal carcinoma cell line Hep2.","date":"2010","source":"Medical science monitor : international medical journal of experimental and clinical research","url":"https://pubmed.ncbi.nlm.nih.gov/20512084","citation_count":16,"is_preprint":false},{"pmid":"37067454","id":"PMC_37067454","title":"Endophilin-A/SH3GL2 calcium switch for synaptic autophagy induction is impaired by a Parkinson's risk variant.","date":"2023","source":"Autophagy","url":"https://pubmed.ncbi.nlm.nih.gov/37067454","citation_count":11,"is_preprint":false},{"pmid":"26823912","id":"PMC_26823912","title":"Aberrant promoter methylation of SH3GL2 gene in vulvar squamous cell carcinoma correlates with clinicopathological characteristics and HPV infection status.","date":"2015","source":"International journal of clinical and experimental pathology","url":"https://pubmed.ncbi.nlm.nih.gov/26823912","citation_count":7,"is_preprint":false},{"pmid":"38571554","id":"PMC_38571554","title":"SH3GL2 and MMP17 as lung adenocarcinoma biomarkers: a machine-learning based approach.","date":"2024","source":"Biochemistry and biophysics reports","url":"https://pubmed.ncbi.nlm.nih.gov/38571554","citation_count":4,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.03.19.25324254","title":"DNA methylation patterns associated with prior tuberculosis infection in people with HIV: a pilot cross-sectional study","date":"2025-03-20","source":"bioRxiv","url":"https://doi.org/10.1101/2025.03.19.25324254","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":10862,"output_tokens":2601,"usd":0.0358,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9749,"output_tokens":2828,"usd":0.059722,"stage2_stop_reason":"end_turn"},"total_usd":0.095522,"stage1_batch_id":"msgbatch_013LWZZT6MWaqB9qyTSeQBmU","stage2_batch_id":"msgbatch_0194ZzQaTGNpruoNJBbEMbt3","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1997,\n      \"finding\": \"SH3GL2 (SH3p4) binds to both synaptojanin and dynamin I via its SH3 domain (closely related to the Grb2 SH3 domain); pools of synaptojanin and dynamin I were co-precipitated from brain extracts with anti-SH3p4/8/13 antibodies, and SH3p4 transcript was detected exclusively in brain with the protein concentrated in nerve terminals.\",\n      \"method\": \"Yeast two-hybrid screening with synaptojanin proline-rich tail as bait, followed by co-immunoprecipitation from brain extracts and immunofluorescence localization\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP from brain extracts plus two-hybrid, replicated across the SH3p4/8/13 family in same study and confirmed by later work\",\n      \"pmids\": [\"9238017\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Antibody-mediated disruption of endophilin/SH3p4 function in a tonically stimulated lamprey synapse blocked invagination of clathrin-coated pits adjacent to the active zone, arresting synaptic vesicle recycling at an early-to-late endocytosis transition; in a cell-free system, endophilin was not associated with clathrin coats but functioned as a partner of dynamin.\",\n      \"method\": \"Antibody microinjection into living synapse followed by electron microscopy; cell-free biochemical fractionation assay\",\n      \"journal\": \"Neuron\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct loss-of-function in intact synapse with defined ultrastructural phenotype, combined with cell-free biochemical assay, two orthogonal methods in one study\",\n      \"pmids\": [\"10677033\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"SH3GL2 (SH3p4) specifically binds the proline-rich third intracellular loop of the beta1-adrenergic receptor (but not beta2-AR) via its C-terminal SH3 domain; overexpression of SH3p4 in HEK293 cells promotes agonist-induced internalization of beta1-AR and modestly decreases Gs coupling efficacy of beta1-AR.\",\n      \"method\": \"GST pull-down assay with beta1-AR third intracellular loop, yeast two-hybrid, Co-IP in HEK293 cells, overexpression functional assay (internalization and cAMP assays)\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (pull-down, two-hybrid, Co-IP, functional overexpression) in one study with domain-level specificity demonstrated\",\n      \"pmids\": [\"10535961\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Knockdown of SH3GL2 in Hep2 laryngeal carcinoma cells upregulates EGFR expression and increases phosphorylated ERK1/2; treatment with MEK1/2 inhibitor U0126 in SH3GL2-knockdown cells reversed the increase in proliferation and decrease in apoptosis, placing SH3GL2 upstream of EGFR in the MEK-ERK signaling pathway.\",\n      \"method\": \"RNA interference knockdown, Western blot for EGFR and p-ERK1/2, pharmacological MEK inhibition (U0126), MTT proliferation assay, flow cytometry apoptosis assay\",\n      \"journal\": \"Medical science monitor\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic knockdown with pharmacological epistasis, two orthogonal readouts, single lab\",\n      \"pmids\": [\"20512084\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Forced overexpression of wild-type SH3GL2 in NSCLC cell lines increased EGFR internalization and degradation, reduced active EGFR expression, and decreased activated AKT (Ser473), STAT3 (Tyr705), and PI3K levels; it also downregulated SH3GL2 interactor USP9X and activated β-catenin, reducing in vitro and in vivo cellular growth and invasion.\",\n      \"method\": \"Stable overexpression of wild-type SH3GL2 in three NSCLC cell lines, Western blot, EGFR internalization assay, proliferation/invasion/colony formation assays, mouse xenograft in vivo\",\n      \"journal\": \"Journal of molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple cell lines, in vivo validation, multiple downstream readouts, single lab\",\n      \"pmids\": [\"22968441\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Stable silencing of Sh3gl2 in RT4 urothelial carcinoma cells inhibited EGF-induced EGFR internalization, increased EGFR activation, stimulated phosphorylation of Src family kinases and STAT3, enhanced proliferation and colony formation, and promoted xenograft growth; forced re-expression of Sh3gl2 in T24 cells attenuated these oncogenic behaviors.\",\n      \"method\": \"Stable RNA interference knockdown and forced re-expression, EGFR internalization assay, Western blot for p-EGFR/p-Src/p-STAT3, proliferation/colony assays, subrenal capsule xenograft\",\n      \"journal\": \"Neoplasia\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal loss-of-function and gain-of-function experiments in multiple cell lines with mechanistic molecular readouts and in vivo validation\",\n      \"pmids\": [\"23814487\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"In glioblastoma stem cells, knockdown of SH3GL2 (mimicking miR-330 overexpression) activated ERK and PI3K/AKT signaling pathways and decreased apoptotic protein expression while increasing anti-apoptotic protein expression; co-transfection with shRNA against SH3GL2 plus miR-330 mimic confirmed that miR-330 promotes malignant behavior via SH3GL2 downregulation.\",\n      \"method\": \"shRNA knockdown, miRNA mimic transfection, Western blot for ERK/AKT pathway components and apoptosis markers, orthotopic mouse xenograft\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic loss-of-function with pathway readouts and in vivo validation, single lab\",\n      \"pmids\": [\"24736727\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Knockdown of SH3GL2 in glioma cells activated STAT3 signaling and promoted expression and secretion of MMP2, enhancing cell migration and invasion; conversely, overexpression of SH3GL2 suppressed STAT3 activation and reduced MMP2 levels, inhibiting migration and invasion.\",\n      \"method\": \"siRNA knockdown and plasmid overexpression, Western blot for p-STAT3 and MMP2, ELISA/zymography for MMP2 secretion, scratch and Transwell invasion assays\",\n      \"journal\": \"Journal of cellular and molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal gain/loss-of-function with defined molecular pathway readout, single lab\",\n      \"pmids\": [\"28470949\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Extracellular calcium influx in the pre-synaptic terminal triggers EndoA (endophilin A/SH3GL2 ortholog) redistribution from the plasma membrane to the cytosol, where it interacts with autophagic membranes to promote autophagosome formation; a specific residue in the flexible region of EndoA mediates this calcium-dependent mobility. A Parkinson's disease-risk mutation in SH3GL2 disrupts calcium sensing, rendering the protein immobile and unable to respond to calcium influx, thereby blocking synaptic autophagy induction.\",\n      \"method\": \"Live imaging of EndoA mobility (FRAP/fluorescence), genetic mutagenesis of calcium-sensing residue, Drosophila neuronal model, autophagosome formation assay, characterization of PD-risk variant\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization experiments with functional consequence (autophagosome formation), mutagenesis of specific residue, single lab with model organism ortholog validation\",\n      \"pmids\": [\"37067454\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"SH3GL2 (endophilin A1/SH3p4) is a nerve-terminal-enriched SH3-domain protein that acts as an adaptor in clathrin-mediated endocytosis by binding dynamin and synaptojanin to drive clathrin-coated pit invagination and synaptic vesicle recycling; it also promotes EGFR internalization and degradation (suppressing downstream EGFR–MEK/ERK, PI3K/AKT, and STAT3/MMP2 signaling) and, in response to calcium influx at the synapse, translocates from the plasma membrane to autophagic membranes to initiate autophagosome formation—a process disrupted by a Parkinson's disease-risk mutation that abolishes its calcium sensing.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"SH3GL2 (endophilin A1/SH3p4) is a brain-enriched, nerve-terminal-concentrated SH3-domain adaptor that drives clathrin-mediated endocytosis and synaptic vesicle recycling [#0, #1]. Through its SH3 domain it binds the proline-rich tails of synaptojanin and dynamin I [#0], and antibody-mediated disruption at the synapse arrests clathrin-coated pit invagination adjacent to the active zone at an early-to-late endocytosis transition, where it functions as a dynamin partner rather than a clathrin coat component [#1]. Its adaptor activity extends to receptor internalization: it binds the proline-rich third intracellular loop of the beta1-adrenergic receptor and promotes its agonist-induced internalization [#2], and it drives EGFR internalization and degradation, thereby restraining downstream EGFR-dependent MEK/ERK, PI3K/AKT, Src, and STAT3/MMP2 signaling [#3, #4, #5, #7]. Consistent with this role, loss of SH3GL2 in multiple carcinoma and glioma models elevates active EGFR and pathway output to promote proliferation, invasion, and tumor growth, while re-expression suppresses these behaviors, establishing SH3GL2 as a tumor suppressor acting upstream of EGFR [#5, #6, #7]. Beyond endocytosis, calcium influx at the pre-synaptic terminal triggers SH3GL2 redistribution from the plasma membrane to autophagic membranes to initiate autophagosome formation, via a calcium-sensing residue in its flexible region; a Parkinson's disease-risk mutation abolishes this calcium sensing and blocks synaptic autophagy induction [#8].\",\n  \"teleology\": [\n    {\n      \"year\": 1997,\n      \"claim\": \"Established SH3GL2 as a nerve-terminal SH3 adaptor by identifying its direct binding partners, answering what molecular machinery it engages.\",\n      \"evidence\": \"Yeast two-hybrid with synaptojanin proline-rich bait, reciprocal Co-IP from brain extracts, and immunofluorescence localization\",\n      \"pmids\": [\"9238017\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of dynamin/synaptojanin binding not yet shown\", \"Domain-level binding determinants on partners not mapped\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Demonstrated a direct functional role in clathrin-coated pit invagination, placing SH3GL2 mechanistically at an endocytic step rather than within the clathrin coat.\",\n      \"evidence\": \"Antibody microinjection into a tonically stimulated lamprey synapse with EM, plus cell-free biochemical fractionation\",\n      \"pmids\": [\"10677033\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Precise biophysical contribution to membrane curvature not resolved\", \"Relationship to dynamin GTPase cycle not detailed\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Extended the adaptor role beyond synaptic vesicles to GPCR internalization with receptor-subtype specificity.\",\n      \"evidence\": \"GST pull-down with beta1-AR third intracellular loop, two-hybrid, Co-IP and overexpression internalization/cAMP assays in HEK293\",\n      \"pmids\": [\"10535961\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Endogenous relevance in neurons not tested\", \"Basis of beta1- vs beta2-AR selectivity not structurally defined\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Linked SH3GL2 to EGFR/MEK-ERK control in cancer, positioning it upstream of EGFR signaling.\",\n      \"evidence\": \"RNAi knockdown in Hep2 cells with MEK inhibitor (U0126) epistasis, proliferation and apoptosis assays\",\n      \"pmids\": [\"20512084\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct effect on EGFR trafficking not shown in this study\", \"Single cell line and single lab\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Showed mechanistically that SH3GL2 drives EGFR internalization and degradation, dampening AKT/STAT3/PI3K output and tumor growth.\",\n      \"evidence\": \"Stable WT overexpression in three NSCLC lines, internalization assay, Western blot, and mouse xenograft\",\n      \"pmids\": [\"22968441\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism connecting EGFR degradation to USP9X and beta-catenin not fully resolved\", \"Single lab\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Provided reciprocal loss- and gain-of-function evidence that SH3GL2 controls EGFR internalization and Src/STAT3 activation as a tumor suppressor.\",\n      \"evidence\": \"Stable knockdown and re-expression in urothelial carcinoma lines, internalization and phospho-protein readouts, subrenal capsule xenograft\",\n      \"pmids\": [\"23814487\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether endocytic adaptor activity per se is required for suppression not separated from other roles\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Tied SH3GL2 loss to STAT3-driven MMP2 secretion and invasion, defining a migration/invasion axis.\",\n      \"evidence\": \"siRNA knockdown and overexpression in glioma cells, p-STAT3/MMP2 Western blot, zymography, scratch and Transwell assays\",\n      \"pmids\": [\"28470949\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct link between EGFR trafficking and STAT3/MMP2 in this context not established\", \"Single lab\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Revealed a distinct, calcium-triggered role in synaptic autophagy and a disease-relevant defect, answering how SH3GL2 transitions from endocytosis to autophagosome biogenesis.\",\n      \"evidence\": \"Live FRAP imaging, mutagenesis of a calcium-sensing residue, Drosophila EndoA ortholog model, autophagosome formation assay, and PD-risk variant characterization\",\n      \"pmids\": [\"37067454\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which the flexible-region residue senses calcium not structurally defined\", \"Validation in mammalian neurons of the human variant not shown\", \"Single lab\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the endocytic adaptor function, EGFR-trafficking tumor-suppressor role, and calcium-gated autophagy function are coordinated within a single neuron or cell remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model unifying SH3-domain binding, curvature, and calcium sensing\", \"Whether the same pool of protein performs all three roles is unknown\", \"Endogenous regulation switching between endocytosis and autophagy not defined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 1, 2]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0140299\", \"supporting_discovery_ids\": [8]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [1, 8]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [8]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0005653\", \"supporting_discovery_ids\": []}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"DNM1\", \"SYNJ1\", \"ADRB1\", \"EGFR\", \"USP9X\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":5,"faith_pct":100.0}}