{"gene":"EPS15L1","run_date":"2026-06-09T23:54:43","timeline":{"discoveries":[{"year":1997,"finding":"EPS15L1 (eps15R) is an essential component of the endocytic pathway: microinjection of antibodies against eps15R inhibits internalization of EGF and transferrin, demonstrating its requirement in clathrin-mediated endocytosis.","method":"Antibody microinjection with EGF/transferrin internalization assay","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct functional inhibition experiment replicated across multiple cargo types, consistent with other endocytic studies","pmids":["9407958"],"is_preprint":false},{"year":1995,"finding":"EPS15L1 (Eps15R) binds specifically to the amino-terminal SH3 domain of Crk via a conserved proline-rich motif containing the sequence P-X-L-P-X-K, and co-precipitates with both c-Crk and v-Crk from cell lysates.","method":"Expression library screen, co-immunoprecipitation, in vitro binding assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal co-IP and direct binding assay in single lab with two orthogonal methods","pmids":["7797522"],"is_preprint":false},{"year":1998,"finding":"Eps15R is tyrosine-phosphorylated following EGF receptor activation, localizes to plasma membrane-coated pits, associates in vivo with the clathrin adaptor AP-2, and exists in the cell as a complex with eps15. Its EH domains exhibit binding specificities partially distinct from those of eps15.","method":"Immunoprecipitation, subcellular fractionation/localization, in vivo co-immunoprecipitation, EH domain binding assays","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (co-IP, localization, phosphorylation assay) in single lab","pmids":["9446614"],"is_preprint":false},{"year":2002,"finding":"The second ubiquitin-interacting motif (UIM) of Eps15R is essential for its monoubiquitination: the UIM does not contain the ubiquitin acceptor site but functions as a recruitment site for the ubiquitination machinery. This UIM partially overlaps with a nuclear export signal (NES) that has distinct structural requirements.","method":"Mutational analysis, ubiquitination assays","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis with functional readout (ubiquitination), single lab, two orthogonal methods","pmids":["12072436"],"is_preprint":false},{"year":2002,"finding":"Eps15R is constitutively found in the nucleus due to the absence of a nuclear export signal (NES), unlike Eps15 which bears a leucine-rich NES in its last 25 amino acids that binds exportin CRM1 in a leucine-dependent manner and mediates leptomycin B-sensitive nuclear export. Eps15R nuclear localization is regulated by alternative splicing.","method":"NES mutational analysis, leptomycin B treatment, CRM1 binding assay, subcellular localization imaging","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (mutagenesis, drug treatment, binding assay, imaging) in single lab","pmids":["11777906"],"is_preprint":false},{"year":2016,"finding":"Eps15/R DPF motifs interact with both Fcho1/2 and AP-2 in a differential manner: crystal structure reveals a spacing-dependent DPF triad in the Eps15/R–Fcho1 μ-homology domain complex that is mechanistically distinct from single DPF binding to AP-2. Fcho1/2 and Eps15/R form transient ternary complexes that facilitate conformational activation of AP-2 by the Fcho1/2 interdomain linker to promote cargo engagement.","method":"Crystal structure determination, cell-based functional assays (FCHO1/2 KO, Eps15 sequestration), endocytosis assays","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure combined with genetic KO and functional endocytosis assays in one rigorous study","pmids":["27237791"],"is_preprint":false},{"year":2017,"finding":"Eps15R, but not Eps15, is required for EphB2/ephrinB1 trans-endocytosis and cell repulsion. A novel interaction motif in Eps15R (DPFxxLDPF) binds directly to the clathrin terminal domain in vitro, and this clathrin-binding activity is required for EphB2-mediated cell repulsion as shown by rescue experiments with wild-type vs. clathrin-binding mutant Eps15R.","method":"siRNA knockdown, co-culture cell repulsion assay, in vitro direct binding assay, rescue experiment with clathrin-binding mutant","journal":"Traffic (Copenhagen, Denmark)","confidence":"High","confidence_rationale":"Tier 2 / Strong — knockdown phenotype, in vitro binding assay, and mutagenesis rescue in one study with multiple orthogonal methods","pmids":["28972287"],"is_preprint":false},{"year":2012,"finding":"Eps15R interacts with Smad proteins, is required for BMP signalling in Xenopus animal caps, and stimulates Smad1 transcriptional activity. This function resides in the DPF motif-enriched domain of Eps15R. In living cells, Eps15R segregates into spatially distinct compartments with different Smads, and the DPF domain antagonizes Smad2 signalling.","method":"Co-immunoprecipitation (Smad interaction), Xenopus animal cap BMP signalling assay, transcriptional reporter assay, live cell imaging","journal":"Open biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (co-IP, functional animal cap assay, reporter assay, imaging) in single lab","pmids":["22724065"],"is_preprint":false},{"year":2019,"finding":"EPS15L1 has a unique nonredundant role in the nervous system, while EPS15 and EPS15L1 together redundantly regulate transferrin receptor endocytosis. Double KO of Eps15/Eps15L1 causes embryonic lethality in mice. Hematopoietic-specific double KO mice develop microcytic hypochromic anemia due to a cell-autonomous defect in iron internalization via impaired transferrin receptor endocytosis.","method":"Constitutive and conditional knockout mice, endocytosis assays, hematopoietic analysis","journal":"Life science alliance","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo genetic KO with defined cellular phenotypes, multiple conditional models, replicated across redundant and nonredundant functions","pmids":["30692166"],"is_preprint":false},{"year":2015,"finding":"eps15L1 is essential for T lymphocyte development in zebrafish: gene-breaking transposon disruption of eps15L1 causes impaired T cell development, and morpholino-mediated knockdown mimics this phenotype.","method":"Transposon-based gene disruption, morpholino knockdown, flow cytometry analysis of T cell development","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — two orthogonal loss-of-function methods (transposon KO and morpholino) with consistent T cell phenotype in zebrafish model","pmids":["26161877"],"is_preprint":false}],"current_model":"EPS15L1 (Eps15R) is an endocytic accessory protein that localizes to clathrin-coated pits, associates with AP-2 and clathrin via DPF motifs, is tyrosine-phosphorylated by the EGF receptor, undergoes UIM-dependent monoubiquitination, and cooperates with Fcho1/2 to prime AP-2 for cargo binding; it has a unique nonredundant role in the nervous system and T cell development, mediates EphB2/ephrinB1 trans-endocytosis and cell repulsion via direct clathrin binding, and also interacts with Smad proteins to regulate BMP signalling, while constitutively residing in the nucleus (unlike Eps15) due to the absence of a CRM1-dependent nuclear export signal."},"narrative":{"mechanistic_narrative":"EPS15L1 (Eps15R) is an endocytic accessory protein essential for clathrin-mediated endocytosis, where blocking its function inhibits internalization of EGF and transferrin cargoes [PMID:9407958]. It operates at plasma-membrane clathrin-coated pits in complex with its paralog Eps15 and the clathrin adaptor AP-2, becomes tyrosine-phosphorylated upon EGF receptor activation, and its EH domains read protein-interaction motifs partly distinct from those of Eps15 [PMID:9446614]. Mechanistically, its DPF motifs engage both the Fcho1/2 μ-homology domain and AP-2; a spacing-dependent DPF triad enables Eps15/R–Fcho1/2 ternary complexes that conformationally activate AP-2 to promote cargo engagement [PMID:27237791]. A second ubiquitin-interacting motif recruits the ubiquitination machinery to drive its own monoubiquitination, overlapping a region with nuclear-export properties [PMID:12072436]. Unlike Eps15, EPS15L1 resides constitutively in the nucleus because it lacks a CRM1-dependent leucine-rich nuclear export signal, with its localization tuned by alternative splicing [PMID:11777906]. Beyond bulk endocytosis, EPS15L1 carries nonredundant specialized roles: it alone supports EphB2/ephrinB1 trans-endocytosis and cell repulsion through a DPFxxLDPF motif that binds the clathrin terminal domain directly [PMID:28972287], and in vivo it is uniquely required in the nervous system while acting redundantly with Eps15 in transferrin-receptor endocytosis, loss of both causing embryonic lethality and a cell-autonomous iron-uptake defect manifesting as microcytic hypochromic anemia [PMID:30692166]. It is further required for T lymphocyte development [PMID:26161877] and interacts with Smad proteins to regulate BMP signalling via its DPF-enriched domain [PMID:22724065].","teleology":[{"year":1995,"claim":"Established an early physical link between Eps15R and signalling adaptors by identifying a direct interaction with the Crk SH3 domain, hinting at coupling between endocytic machinery and signal transduction.","evidence":"Expression library screen, co-immunoprecipitation and in vitro binding via a P-X-L-P-X-K proline-rich motif","pmids":["7797522"],"confidence":"Medium","gaps":["Functional consequence of the Crk interaction not defined","Single-lab biochemistry without in vivo validation"]},{"year":1997,"claim":"Demonstrated that Eps15R is functionally required for endocytosis, moving it from a binding partner to an essential pathway component.","evidence":"Antibody microinjection blocking EGF and transferrin internalization","pmids":["9407958"],"confidence":"High","gaps":["Does not define the molecular step it acts at","No distinction from redundant paralog Eps15"]},{"year":1998,"claim":"Placed Eps15R mechanistically at coated pits as an EGFR-responsive, AP-2- and Eps15-associated protein, defining its core molecular context.","evidence":"Immunoprecipitation, subcellular fractionation, in vivo co-IP and EH-domain binding assays","pmids":["9446614"],"confidence":"Medium","gaps":["EH-domain ligand specificity only partially mapped","Functional role of tyrosine phosphorylation not resolved"]},{"year":2002,"claim":"Resolved how Eps15R differs from Eps15 in subcellular distribution, showing it lacks a CRM1-dependent NES and is constitutively nuclear, with splicing as a regulatory switch.","evidence":"NES mutagenesis, leptomycin B treatment, CRM1 binding and localization imaging","pmids":["11777906"],"confidence":"Medium","gaps":["Nuclear function of Eps15R not defined","Link between nuclear pool and endocytic pool unclear"]},{"year":2002,"claim":"Defined the UIM as a recruitment site for the ubiquitination machinery rather than the ubiquitin acceptor, clarifying the basis of Eps15R monoubiquitination.","evidence":"Mutational analysis with ubiquitination assays","pmids":["12072436"],"confidence":"Medium","gaps":["Identity of the responsible ubiquitin ligase not determined","Functional consequence of monoubiquitination not established"]},{"year":2012,"claim":"Extended Eps15R function beyond endocytosis to BMP signal transduction through Smad interaction, implicating its DPF-enriched domain in transcriptional regulation.","evidence":"Co-IP, Xenopus animal cap BMP assay, transcriptional reporter and live-cell imaging","pmids":["22724065"],"confidence":"Medium","gaps":["Direct vs. indirect nature of Smad regulation unclear","Single model system (Xenopus)"]},{"year":2015,"claim":"Revealed an organismal requirement for eps15L1 in T lymphocyte development, indicating tissue-specific essential functions.","evidence":"Transposon gene disruption and morpholino knockdown with flow cytometry in zebrafish","pmids":["26161877"],"confidence":"Medium","gaps":["Molecular mechanism linking eps15L1 to T cell development unknown","Endocytic vs. signalling basis of phenotype undefined"]},{"year":2016,"claim":"Provided the structural mechanism by which Eps15/R DPF motifs cooperate with Fcho1/2 and AP-2, showing a spacing-dependent triad that drives conformational activation of AP-2 for cargo engagement.","evidence":"Crystal structure of the Eps15/R–Fcho1 μ-homology complex with FCHO1/2 KO and endocytosis assays","pmids":["27237791"],"confidence":"High","gaps":["Does not separate Eps15 from Eps15L1 contributions structurally","Dynamics of ternary complex in living pits not resolved"]},{"year":2017,"claim":"Identified a nonredundant Eps15R-specific function in EphB2/ephrinB1 trans-endocytosis via a novel DPFxxLDPF clathrin terminal-domain binding motif, distinguishing it mechanistically from Eps15.","evidence":"siRNA knockdown, cell repulsion co-culture, in vitro binding and clathrin-binding-mutant rescue","pmids":["28972287"],"confidence":"High","gaps":["Why Eps15 cannot substitute not fully explained","Physiological context of repulsion in vivo not addressed"]},{"year":2019,"claim":"Established the in vivo division of labor between Eps15 and Eps15L1, demonstrating redundant control of transferrin-receptor endocytosis and a unique nonredundant nervous-system role, with double loss causing lethality and anemia from iron-uptake failure.","evidence":"Constitutive and conditional double-KO mice with endocytosis and hematopoietic analyses","pmids":["30692166"],"confidence":"High","gaps":["Molecular basis of the unique nervous-system requirement not defined","Cargo selectivity rules for Eps15 vs Eps15L1 incomplete"]},{"year":null,"claim":"The nuclear function of EPS15L1 and how its endocytic, Smad-signalling, and tissue-specific roles are molecularly coordinated remain unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No defined nuclear molecular activity despite constitutive nuclear localization","Unifying mechanism connecting endocytic and signalling functions absent","Responsible ubiquitin ligase and role of monoubiquitination unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[2,5,6]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[2,6]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[2]},{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[0,5]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[4]}],"pathway":[{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[0,5,8]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[6,7]}],"complexes":["AP-2 clathrin adaptor complex","Eps15/R–Fcho1/2 ternary complex"],"partners":["EPS15","AP-2","FCHO1","FCHO2","CLTC","CRK","SMAD1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9UBC2","full_name":"Epidermal growth factor receptor substrate 15-like 1","aliases":["Eps15-related protein","Eps15R"],"length_aa":864,"mass_kda":94.3,"function":"Seems to be a constitutive component of clathrin-coated pits that is required for receptor-mediated endocytosis. Involved in endocytosis of integrin beta-1 (ITGB1) and transferrin receptor (TFR); internalization of ITGB1 as DAB2-dependent cargo but not TFR seems to require association with DAB2","subcellular_location":"Cell membrane; Nucleus; Membrane, coated pit","url":"https://www.uniprot.org/uniprotkb/Q9UBC2/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/EPS15L1","classification":"Not Classified","n_dependent_lines":13,"n_total_lines":1208,"dependency_fraction":0.01076158940397351},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"AP2B1","stoichiometry":10.0},{"gene":"EPS15","stoichiometry":10.0},{"gene":"AP2S1","stoichiometry":0.2},{"gene":"DEGS1","stoichiometry":0.2},{"gene":"ITSN1","stoichiometry":0.2},{"gene":"ITSN2","stoichiometry":0.2},{"gene":"NECAP2","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/EPS15L1","total_profiled":1310},"omim":[{"mim_id":"616826","title":"EPS15-LIKE PROTEIN 1; EPS15L1","url":"https://www.omim.org/entry/616826"},{"mim_id":"600051","title":"EPIDERMAL GROWTH FACTOR RECEPTOR PATHWAY SUBSTRATE 15; EPS15","url":"https://www.omim.org/entry/600051"},{"mim_id":"183600","title":"SPLIT-HAND/FOOT MALFORMATION 1; SHFM1","url":"https://www.omim.org/entry/183600"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Vesicles","reliability":"Approved"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/EPS15L1"},"hgnc":{"alias_symbol":["eps15R"],"prev_symbol":[]},"alphafold":{"accession":"Q9UBC2","domains":[{"cath_id":"1.10.238.10","chopping":"10-94","consensus_level":"high","plddt":81.5512,"start":10,"end":94},{"cath_id":"1.10.238.10","chopping":"126-212","consensus_level":"high","plddt":82.3446,"start":126,"end":212},{"cath_id":"1.10.238.10","chopping":"274-347","consensus_level":"high","plddt":86.5636,"start":274,"end":347},{"cath_id":"1.20.5","chopping":"392-509","consensus_level":"medium","plddt":96.0791,"start":392,"end":509}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9UBC2","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9UBC2-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9UBC2-F1-predicted_aligned_error_v6.png","plddt_mean":65.81},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=EPS15L1","jax_strain_url":"https://www.jax.org/strain/search?query=EPS15L1"},"sequence":{"accession":"Q9UBC2","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9UBC2.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9UBC2/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9UBC2"}},"corpus_meta":[{"pmid":"9407958","id":"PMC_9407958","title":"eps15 and eps15R are essential components of the endocytic pathway.","date":"1997","source":"Cancer research","url":"https://pubmed.ncbi.nlm.nih.gov/9407958","citation_count":173,"is_preprint":false},{"pmid":"12072436","id":"PMC_12072436","title":"A ubiquitin-interacting motif (UIM) is essential for Eps15 and Eps15R ubiquitination.","date":"2002","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/12072436","citation_count":88,"is_preprint":false},{"pmid":"7797522","id":"PMC_7797522","title":"The SH3 domain of Crk binds specifically to a conserved proline-rich motif in Eps15 and Eps15R.","date":"1995","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/7797522","citation_count":87,"is_preprint":false},{"pmid":"27237791","id":"PMC_27237791","title":"Transient Fcho1/2⋅Eps15/R⋅AP-2 Nanoclusters Prime the AP-2 Clathrin Adaptor for Cargo Binding.","date":"2016","source":"Developmental cell","url":"https://pubmed.ncbi.nlm.nih.gov/27237791","citation_count":84,"is_preprint":false},{"pmid":"9446614","id":"PMC_9446614","title":"Eps15R is a tyrosine kinase substrate with characteristics of a docking protein possibly involved in coated pits-mediated internalization.","date":"1998","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/9446614","citation_count":69,"is_preprint":false},{"pmid":"11777906","id":"PMC_11777906","title":"Differential nucleocytoplasmic trafficking between the related endocytic proteins Eps15 and Eps15R.","date":"2002","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/11777906","citation_count":35,"is_preprint":false},{"pmid":"18395097","id":"PMC_18395097","title":"EPS15R, TASP1, and PRPF3 are novel disease candidate genes targeted by HNF4alpha splice variants in hepatocellular carcinomas.","date":"2008","source":"Gastroenterology","url":"https://pubmed.ncbi.nlm.nih.gov/18395097","citation_count":28,"is_preprint":false},{"pmid":"29023680","id":"PMC_29023680","title":"First direct evidence of involvement of a homozygous loss-of-function variant in the EPS15L1 gene underlying split-hand/split-foot malformation.","date":"2018","source":"Clinical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/29023680","citation_count":20,"is_preprint":false},{"pmid":"28972287","id":"PMC_28972287","title":"Eps15R and clathrin regulate EphB2-mediated cell repulsion.","date":"2017","source":"Traffic (Copenhagen, Denmark)","url":"https://pubmed.ncbi.nlm.nih.gov/28972287","citation_count":17,"is_preprint":false},{"pmid":"30692166","id":"PMC_30692166","title":"Redundant and nonredundant organismal functions of EPS15 and EPS15L1.","date":"2019","source":"Life science alliance","url":"https://pubmed.ncbi.nlm.nih.gov/30692166","citation_count":14,"is_preprint":false},{"pmid":"26161877","id":"PMC_26161877","title":"Mutagenesis Screen Identifies agtpbp1 and eps15L1 as Essential for T lymphocyte Development in Zebrafish.","date":"2015","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/26161877","citation_count":13,"is_preprint":false},{"pmid":"21700002","id":"PMC_21700002","title":"A de novo 1.1Mb microdeletion of chromosome 19p13.11 provides indirect evidence for EPS15L1 to be a strong candidate for split hand split foot malformation.","date":"2011","source":"European journal of medical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/21700002","citation_count":12,"is_preprint":false},{"pmid":"35098448","id":"PMC_35098448","title":"LncRNA ABHD11-AS1 promotes tumor progression in papillary thyroid carcinoma by regulating EPS15L1/EGFR signaling pathway.","date":"2022","source":"Clinical & translational oncology : official publication of the Federation of Spanish Oncology Societies and of the National Cancer Institute of Mexico","url":"https://pubmed.ncbi.nlm.nih.gov/35098448","citation_count":10,"is_preprint":false},{"pmid":"10906484","id":"PMC_10906484","title":"Differential patterns of expression of Eps15 and Eps15R during mouse embryogenesis.","date":"2000","source":"Mechanisms of development","url":"https://pubmed.ncbi.nlm.nih.gov/10906484","citation_count":4,"is_preprint":false},{"pmid":"22724065","id":"PMC_22724065","title":"Eps15R is required for bone morphogenetic protein signalling and differentially compartmentalizes with Smad proteins.","date":"2012","source":"Open biology","url":"https://pubmed.ncbi.nlm.nih.gov/22724065","citation_count":3,"is_preprint":false},{"pmid":"42140958","id":"PMC_42140958","title":"Identification and functional validation of EPS15L1 as a key driver of triple-negative breast cancer.","date":"2026","source":"NPJ breast cancer","url":"https://pubmed.ncbi.nlm.nih.gov/42140958","citation_count":0,"is_preprint":false},{"pmid":"41775633","id":"PMC_41775633","title":"Novel EPS15 :: KLF17 and EPS15L1 :: KLF17 Fusions Define a Distinctive Group of MUC4-Positive Spindled to Epithelioid Sarcomas.","date":"2026","source":"The American journal of surgical pathology","url":"https://pubmed.ncbi.nlm.nih.gov/41775633","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":9809,"output_tokens":2459,"usd":0.033156,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9568,"output_tokens":3395,"usd":0.066357,"stage2_stop_reason":"end_turn"},"total_usd":0.099513,"stage1_batch_id":"msgbatch_01AsR7g8dGTEiEw9U83bJeWn","stage2_batch_id":"msgbatch_01YV5ZyxtFNUZEjxRcpd9jzb","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1997,\n      \"finding\": \"EPS15L1 (eps15R) is an essential component of the endocytic pathway: microinjection of antibodies against eps15R inhibits internalization of EGF and transferrin, demonstrating its requirement in clathrin-mediated endocytosis.\",\n      \"method\": \"Antibody microinjection with EGF/transferrin internalization assay\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct functional inhibition experiment replicated across multiple cargo types, consistent with other endocytic studies\",\n      \"pmids\": [\"9407958\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"EPS15L1 (Eps15R) binds specifically to the amino-terminal SH3 domain of Crk via a conserved proline-rich motif containing the sequence P-X-L-P-X-K, and co-precipitates with both c-Crk and v-Crk from cell lysates.\",\n      \"method\": \"Expression library screen, co-immunoprecipitation, in vitro binding assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal co-IP and direct binding assay in single lab with two orthogonal methods\",\n      \"pmids\": [\"7797522\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Eps15R is tyrosine-phosphorylated following EGF receptor activation, localizes to plasma membrane-coated pits, associates in vivo with the clathrin adaptor AP-2, and exists in the cell as a complex with eps15. Its EH domains exhibit binding specificities partially distinct from those of eps15.\",\n      \"method\": \"Immunoprecipitation, subcellular fractionation/localization, in vivo co-immunoprecipitation, EH domain binding assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (co-IP, localization, phosphorylation assay) in single lab\",\n      \"pmids\": [\"9446614\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"The second ubiquitin-interacting motif (UIM) of Eps15R is essential for its monoubiquitination: the UIM does not contain the ubiquitin acceptor site but functions as a recruitment site for the ubiquitination machinery. This UIM partially overlaps with a nuclear export signal (NES) that has distinct structural requirements.\",\n      \"method\": \"Mutational analysis, ubiquitination assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis with functional readout (ubiquitination), single lab, two orthogonal methods\",\n      \"pmids\": [\"12072436\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Eps15R is constitutively found in the nucleus due to the absence of a nuclear export signal (NES), unlike Eps15 which bears a leucine-rich NES in its last 25 amino acids that binds exportin CRM1 in a leucine-dependent manner and mediates leptomycin B-sensitive nuclear export. Eps15R nuclear localization is regulated by alternative splicing.\",\n      \"method\": \"NES mutational analysis, leptomycin B treatment, CRM1 binding assay, subcellular localization imaging\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (mutagenesis, drug treatment, binding assay, imaging) in single lab\",\n      \"pmids\": [\"11777906\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Eps15/R DPF motifs interact with both Fcho1/2 and AP-2 in a differential manner: crystal structure reveals a spacing-dependent DPF triad in the Eps15/R–Fcho1 μ-homology domain complex that is mechanistically distinct from single DPF binding to AP-2. Fcho1/2 and Eps15/R form transient ternary complexes that facilitate conformational activation of AP-2 by the Fcho1/2 interdomain linker to promote cargo engagement.\",\n      \"method\": \"Crystal structure determination, cell-based functional assays (FCHO1/2 KO, Eps15 sequestration), endocytosis assays\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure combined with genetic KO and functional endocytosis assays in one rigorous study\",\n      \"pmids\": [\"27237791\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Eps15R, but not Eps15, is required for EphB2/ephrinB1 trans-endocytosis and cell repulsion. A novel interaction motif in Eps15R (DPFxxLDPF) binds directly to the clathrin terminal domain in vitro, and this clathrin-binding activity is required for EphB2-mediated cell repulsion as shown by rescue experiments with wild-type vs. clathrin-binding mutant Eps15R.\",\n      \"method\": \"siRNA knockdown, co-culture cell repulsion assay, in vitro direct binding assay, rescue experiment with clathrin-binding mutant\",\n      \"journal\": \"Traffic (Copenhagen, Denmark)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — knockdown phenotype, in vitro binding assay, and mutagenesis rescue in one study with multiple orthogonal methods\",\n      \"pmids\": [\"28972287\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Eps15R interacts with Smad proteins, is required for BMP signalling in Xenopus animal caps, and stimulates Smad1 transcriptional activity. This function resides in the DPF motif-enriched domain of Eps15R. In living cells, Eps15R segregates into spatially distinct compartments with different Smads, and the DPF domain antagonizes Smad2 signalling.\",\n      \"method\": \"Co-immunoprecipitation (Smad interaction), Xenopus animal cap BMP signalling assay, transcriptional reporter assay, live cell imaging\",\n      \"journal\": \"Open biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (co-IP, functional animal cap assay, reporter assay, imaging) in single lab\",\n      \"pmids\": [\"22724065\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"EPS15L1 has a unique nonredundant role in the nervous system, while EPS15 and EPS15L1 together redundantly regulate transferrin receptor endocytosis. Double KO of Eps15/Eps15L1 causes embryonic lethality in mice. Hematopoietic-specific double KO mice develop microcytic hypochromic anemia due to a cell-autonomous defect in iron internalization via impaired transferrin receptor endocytosis.\",\n      \"method\": \"Constitutive and conditional knockout mice, endocytosis assays, hematopoietic analysis\",\n      \"journal\": \"Life science alliance\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo genetic KO with defined cellular phenotypes, multiple conditional models, replicated across redundant and nonredundant functions\",\n      \"pmids\": [\"30692166\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"eps15L1 is essential for T lymphocyte development in zebrafish: gene-breaking transposon disruption of eps15L1 causes impaired T cell development, and morpholino-mediated knockdown mimics this phenotype.\",\n      \"method\": \"Transposon-based gene disruption, morpholino knockdown, flow cytometry analysis of T cell development\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — two orthogonal loss-of-function methods (transposon KO and morpholino) with consistent T cell phenotype in zebrafish model\",\n      \"pmids\": [\"26161877\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"EPS15L1 (Eps15R) is an endocytic accessory protein that localizes to clathrin-coated pits, associates with AP-2 and clathrin via DPF motifs, is tyrosine-phosphorylated by the EGF receptor, undergoes UIM-dependent monoubiquitination, and cooperates with Fcho1/2 to prime AP-2 for cargo binding; it has a unique nonredundant role in the nervous system and T cell development, mediates EphB2/ephrinB1 trans-endocytosis and cell repulsion via direct clathrin binding, and also interacts with Smad proteins to regulate BMP signalling, while constitutively residing in the nucleus (unlike Eps15) due to the absence of a CRM1-dependent nuclear export signal.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"EPS15L1 (Eps15R) is an endocytic accessory protein essential for clathrin-mediated endocytosis, where blocking its function inhibits internalization of EGF and transferrin cargoes [#0]. It operates at plasma-membrane clathrin-coated pits in complex with its paralog Eps15 and the clathrin adaptor AP-2, becomes tyrosine-phosphorylated upon EGF receptor activation, and its EH domains read protein-interaction motifs partly distinct from those of Eps15 [#2]. Mechanistically, its DPF motifs engage both the Fcho1/2 \\u03bc-homology domain and AP-2; a spacing-dependent DPF triad enables Eps15/R\\u2013Fcho1/2 ternary complexes that conformationally activate AP-2 to promote cargo engagement [#5]. A second ubiquitin-interacting motif recruits the ubiquitination machinery to drive its own monoubiquitination, overlapping a region with nuclear-export properties [#3]. Unlike Eps15, EPS15L1 resides constitutively in the nucleus because it lacks a CRM1-dependent leucine-rich nuclear export signal, with its localization tuned by alternative splicing [#4]. Beyond bulk endocytosis, EPS15L1 carries nonredundant specialized roles: it alone supports EphB2/ephrinB1 trans-endocytosis and cell repulsion through a DPFxxLDPF motif that binds the clathrin terminal domain directly [#6], and in vivo it is uniquely required in the nervous system while acting redundantly with Eps15 in transferrin-receptor endocytosis, loss of both causing embryonic lethality and a cell-autonomous iron-uptake defect manifesting as microcytic hypochromic anemia [#8]. It is further required for T lymphocyte development [#9] and interacts with Smad proteins to regulate BMP signalling via its DPF-enriched domain [#7].\",\n  \"teleology\": [\n    {\n      \"year\": 1995,\n      \"claim\": \"Established an early physical link between Eps15R and signalling adaptors by identifying a direct interaction with the Crk SH3 domain, hinting at coupling between endocytic machinery and signal transduction.\",\n      \"evidence\": \"Expression library screen, co-immunoprecipitation and in vitro binding via a P-X-L-P-X-K proline-rich motif\",\n      \"pmids\": [\"7797522\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional consequence of the Crk interaction not defined\", \"Single-lab biochemistry without in vivo validation\"]\n    },\n    {\n      \"year\": 1997,\n      \"claim\": \"Demonstrated that Eps15R is functionally required for endocytosis, moving it from a binding partner to an essential pathway component.\",\n      \"evidence\": \"Antibody microinjection blocking EGF and transferrin internalization\",\n      \"pmids\": [\"9407958\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not define the molecular step it acts at\", \"No distinction from redundant paralog Eps15\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Placed Eps15R mechanistically at coated pits as an EGFR-responsive, AP-2- and Eps15-associated protein, defining its core molecular context.\",\n      \"evidence\": \"Immunoprecipitation, subcellular fractionation, in vivo co-IP and EH-domain binding assays\",\n      \"pmids\": [\"9446614\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"EH-domain ligand specificity only partially mapped\", \"Functional role of tyrosine phosphorylation not resolved\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Resolved how Eps15R differs from Eps15 in subcellular distribution, showing it lacks a CRM1-dependent NES and is constitutively nuclear, with splicing as a regulatory switch.\",\n      \"evidence\": \"NES mutagenesis, leptomycin B treatment, CRM1 binding and localization imaging\",\n      \"pmids\": [\"11777906\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Nuclear function of Eps15R not defined\", \"Link between nuclear pool and endocytic pool unclear\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Defined the UIM as a recruitment site for the ubiquitination machinery rather than the ubiquitin acceptor, clarifying the basis of Eps15R monoubiquitination.\",\n      \"evidence\": \"Mutational analysis with ubiquitination assays\",\n      \"pmids\": [\"12072436\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Identity of the responsible ubiquitin ligase not determined\", \"Functional consequence of monoubiquitination not established\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Extended Eps15R function beyond endocytosis to BMP signal transduction through Smad interaction, implicating its DPF-enriched domain in transcriptional regulation.\",\n      \"evidence\": \"Co-IP, Xenopus animal cap BMP assay, transcriptional reporter and live-cell imaging\",\n      \"pmids\": [\"22724065\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct vs. indirect nature of Smad regulation unclear\", \"Single model system (Xenopus)\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Revealed an organismal requirement for eps15L1 in T lymphocyte development, indicating tissue-specific essential functions.\",\n      \"evidence\": \"Transposon gene disruption and morpholino knockdown with flow cytometry in zebrafish\",\n      \"pmids\": [\"26161877\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular mechanism linking eps15L1 to T cell development unknown\", \"Endocytic vs. signalling basis of phenotype undefined\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Provided the structural mechanism by which Eps15/R DPF motifs cooperate with Fcho1/2 and AP-2, showing a spacing-dependent triad that drives conformational activation of AP-2 for cargo engagement.\",\n      \"evidence\": \"Crystal structure of the Eps15/R\\u2013Fcho1 \\u03bc-homology complex with FCHO1/2 KO and endocytosis assays\",\n      \"pmids\": [\"27237791\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not separate Eps15 from Eps15L1 contributions structurally\", \"Dynamics of ternary complex in living pits not resolved\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Identified a nonredundant Eps15R-specific function in EphB2/ephrinB1 trans-endocytosis via a novel DPFxxLDPF clathrin terminal-domain binding motif, distinguishing it mechanistically from Eps15.\",\n      \"evidence\": \"siRNA knockdown, cell repulsion co-culture, in vitro binding and clathrin-binding-mutant rescue\",\n      \"pmids\": [\"28972287\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Why Eps15 cannot substitute not fully explained\", \"Physiological context of repulsion in vivo not addressed\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Established the in vivo division of labor between Eps15 and Eps15L1, demonstrating redundant control of transferrin-receptor endocytosis and a unique nonredundant nervous-system role, with double loss causing lethality and anemia from iron-uptake failure.\",\n      \"evidence\": \"Constitutive and conditional double-KO mice with endocytosis and hematopoietic analyses\",\n      \"pmids\": [\"30692166\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis of the unique nervous-system requirement not defined\", \"Cargo selectivity rules for Eps15 vs Eps15L1 incomplete\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The nuclear function of EPS15L1 and how its endocytic, Smad-signalling, and tissue-specific roles are molecularly coordinated remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No defined nuclear molecular activity despite constitutive nuclear localization\", \"Unifying mechanism connecting endocytic and signalling functions absent\", \"Responsible ubiquitin ligase and role of monoubiquitination unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [2, 5, 6]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [2, 6]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [2]},\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [0, 5]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [0, 5, 8]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [6, 7]}\n    ],\n    \"complexes\": [\n      \"AP-2 clathrin adaptor complex\",\n      \"Eps15/R\\u2013Fcho1/2 ternary complex\"\n    ],\n    \"partners\": [\n      \"EPS15\",\n      \"AP-2\",\n      \"FCHO1\",\n      \"FCHO2\",\n      \"CLTC\",\n      \"CRK\",\n      \"SMAD1\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}