{"gene":"EHD1","run_date":"2026-04-28T17:46:03","timeline":{"discoveries":[{"year":1999,"finding":"EHD1 was identified as an EH domain-containing protein that localizes to transferrin-containing endocytic vesicles in cultured cells, with predicted protein structure including an N-terminal nucleotide-binding P-loop, central coiled-coil domain, and C-terminal EH domain with EF/Ca2+-binding motif.","method":"GFP-fusion protein live imaging, Northern analysis, immunostaining","journal":"Genomics","confidence":"Medium","confidence_rationale":"Tier 2 — direct localization experiment with structural domain characterization, single lab","pmids":["10395801"],"is_preprint":false},{"year":2001,"finding":"EHD1 directly interacts with SNAP29 and associates with alpha-adaptin of the AP-2 complex and IGF-1R; overexpression of EHD1 represses IGF-1-mediated MAPK and Akt phosphorylation, indicating EHD1 acts as a downregulator of IGF-1R signaling.","method":"Co-immunoprecipitation, co-localization, overexpression with downstream signaling readouts (MAPK/Akt phosphorylation)","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2-3 — reciprocal Co-IP with functional signaling readout, single lab","pmids":["11423532"],"is_preprint":false},{"year":2002,"finding":"EHD1 associates with Arf6-containing membrane tubules and induces tubule formation to facilitate recycling of MHC-I molecules internalized via clathrin-independent endocytosis; the N-terminal P-loop domain and C-terminal EH domain are both required for tubule association and formation.","method":"Overexpression of tagged EHD1 and domain mutants, immunofluorescence, MHC-I recycling assays, dominant-negative Arf6 experiments","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods including domain mutagenesis and functional recycling assays, high citation count indicating replication","pmids":["12032069"],"is_preprint":false},{"year":2002,"finding":"EHD1 and EHD3 interact with each other (confirmed by yeast two-hybrid and co-immunoprecipitation), and the N-terminal domain of EHD3 is responsible for its tubular localization; coexpression of EHD1 and EHD3 results in their co-localization in microtubule-dependent tubules.","method":"Yeast two-hybrid, co-immunoprecipitation, GFP-fusion live imaging, domain-swap chimeras","journal":"Traffic (Copenhagen, Denmark)","confidence":"High","confidence_rationale":"Tier 1-2 — yeast two-hybrid confirmed by Co-IP, domain mutagenesis defining tubular localization signal","pmids":["12121420"],"is_preprint":false},{"year":2004,"finding":"EHD1 interacts with the divalent Rab4/Rab5 effector Rabenosyn-5 via its EH domain binding to the first two NPF motifs of Rabenosyn-5; Rabenosyn-5 acts sequentially before EHD1 in transport from early endosomes to the endosomal recycling compartment and back to the plasma membrane.","method":"GST-pulldown from brain cytosol, mass spectrometry, domain mapping, immunofluorescence, RNAi knockdown with transferrin and MHC-I recycling assays, epistasis by double RNAi","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 1-2 — biochemical pulldown with mass spectrometry, domain mapping, epistasis by sequential RNAi, functional recycling assays","pmids":["15020713"],"is_preprint":false},{"year":2004,"finding":"EHD1 interacts via its EH domain with EHBP1 and controls perinuclear localization of GLUT4-containing membranes; EHD1 is required for insulin-stimulated GLUT4 recycling to the plasma membrane in adipocytes.","method":"Co-immunoprecipitation, dominant-negative expression (ΔEH-EHD1), siRNA knockdown, immunofluorescence, glucose transport assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (Co-IP, dominant-negative, siRNA) with functional transport readout","pmids":["15247266"],"is_preprint":false},{"year":2004,"finding":"EHD1 interacts with GS32/SNAP-29 via its EH domain binding to the N-terminal NPF-containing region of GS32, and with syndapin II via the EH domain; these two interactions are mutually exclusive, suggesting EHD1 participates in pathways of both GS32 and syndapin II in a mutually exclusive manner.","method":"GST pulldown from cell extracts and brain extracts, co-immunoprecipitation, competition binding assays","journal":"Molecular membrane biology","confidence":"Medium","confidence_rationale":"Tier 2-3 — reciprocal Co-IP and pulldown with competition assay demonstrating mutual exclusivity","pmids":["15371016"],"is_preprint":false},{"year":2006,"finding":"EHD1 knockout mice exhibit delayed recycling of transferrin to the plasma membrane with accumulation in the endocytic recycling compartment, confirming EHD1's role in exit of membrane proteins from recycling endosomes in vivo.","method":"EHD1 knockout mouse model, transferrin uptake and recycling assays in embryonic fibroblasts","journal":"Traffic (Copenhagen, Denmark)","confidence":"High","confidence_rationale":"Tier 2 — clean genetic knockout with defined cellular phenotype, in vivo model","pmids":["16445686"],"is_preprint":false},{"year":2007,"finding":"EHD1 directly binds phosphatidylinositols, preferring those phosphorylated at position 3; the EH domain's Lys-483 residue (on the face opposite to the NPF-binding pocket) is critical for phosphatidylinositol binding, as determined by 2D NMR.","method":"In vitro phospholipid binding assays, 2D NMR analysis, site-directed mutagenesis (K483)","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — in vitro binding assay with NMR structural validation and mutagenesis confirming critical residue","pmids":["17412695"],"is_preprint":false},{"year":2007,"finding":"EHD1 interacts with retromer in vivo, colocalizes with retromer, and stabilizes SNX1-positive tubules; loss of EHD1 by RNAi destabilizes SNX1 tubules and inhibits endosome-to-Golgi retrieval of CIMPR; P-loop mutation of EHD1 causes dominant-negative effects on retromer localization.","method":"Comparative proteomics (endosomal fractions from WT vs retromer-deficient cells), co-immunoprecipitation, RNAi, P-loop dominant-negative mutant, CIMPR trafficking assays","journal":"Traffic (Copenhagen, Denmark)","confidence":"High","confidence_rationale":"Tier 1-2 — proteomics discovery validated by Co-IP, domain mutagenesis, and functional trafficking assays","pmids":["17868075"],"is_preprint":false},{"year":2007,"finding":"EHD1 regulates beta1 integrin endosomal recycling; RNAi knockdown of EHD1 impairs beta1 integrin recycling, EHD1-knockout MEFs show impaired cell spreading and migration on fibronectin and slower focal adhesion disassembly, phenotypes rescued by wild-type EHD1 re-expression.","method":"RNAi knockdown, EHD1 knockout MEFs, integrin recycling assays, focal adhesion analysis, migration and spreading assays, rescue with wild-type EHD1","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 — clean KO with specific cellular phenotype, RNAi confirmation, and rescue experiment","pmids":["17284518"],"is_preprint":false},{"year":2007,"finding":"EHD1 regulates cholesterol homeostasis; EHD1 knockout MEFs display reduced esterified and free cholesterol, smaller lipid droplets, and ineffectual cholesterol uptake via LDL receptor, reversed by wild-type but not dysfunctional EHD1.","method":"EHD1 knockout MEFs, cholesterol measurement assays, lipid droplet quantification, LDL receptor internalization, siRNA, rescue with WT vs. mutant EHD1","journal":"Biochemical and biophysical research communications","confidence":"High","confidence_rationale":"Tier 2 — genetic KO with functional readout, siRNA confirmation, and rescue by WT but not mutant EHD1","pmids":["17451652"],"is_preprint":false},{"year":2007,"finding":"EHD1 localizes to a tubular network containing EHD3 and Rab8a; Myosin Vb interacts with Rab8a (confirmed by yeast two-hybrid and FRET) and colocalizes with EHD1/EHD3-positive Rab8a tubules, defining a recycling pathway distinct from the Rab11a pathway.","method":"Yeast two-hybrid, FRET imaging, co-localization immunofluorescence, dominant-negative Myosin Vb expression","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 — yeast two-hybrid confirmed by FRET, with functional dominant-negative pathway analysis","pmids":["17507647"],"is_preprint":false},{"year":2007,"finding":"The solution structure of the EHD1 EH domain was solved by NMR; it resembles the second N-terminal EH domain of Eps15 but shows differences in surface charge and the NPF/DPF-binding pocket structure.","method":"NMR spectroscopy, backbone resonance assignment, solution structure determination","journal":"Journal of biomolecular NMR","confidence":"High","confidence_rationale":"Tier 1 — NMR structure determination of the EH domain","pmids":["17899392"],"is_preprint":false},{"year":2008,"finding":"EHD1 undergoes serine phosphorylation that is enhanced by serum stimulation; PKC is one of the kinases responsible; inhibitors of clathrin-mediated endocytosis (but not caveolin-mediated endocytosis) decrease EHD1 phosphorylation, placing phosphorylation between early endosomes and the endocytic recycling compartment.","method":"Phosphorylation assays, kinase inhibitor treatments, endocytosis pathway inhibitors","journal":"Cellular & molecular biology letters","confidence":"Medium","confidence_rationale":"Tier 3 — pharmacological inhibitor approach with phosphorylation readout, single lab","pmids":["18661112"],"is_preprint":false},{"year":2009,"finding":"C. elegans AMPH-1 (Amphiphysin/BIN1 ortholog) colocalizes with RME-1 (EHD1 ortholog) on recycling endosomes; AMPH-1 NPF motifs bind the RME-1 EH domain; purified AMPH-1-RME-1 complexes produce short coated membrane tubules distinct from those by either protein alone; BIN1 is required for EHD1-regulated endocytic recycling in human cells.","method":"In vivo C. elegans co-localization, deletion mutant analysis, in vitro membrane tubulation with purified proteins, human BIN1 knockdown with EHD1 recycling assays","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution with purified proteins showing tubulation, complemented by genetic analysis in C. elegans and knockdown in human cells","pmids":["19915558"],"is_preprint":false},{"year":2009,"finding":"MICAL-L1 interacts with EHD1 via its NPF motifs and the EHD1 EH domain, recruits both EHD1 and Rab8a to tubular recycling endosomes; MICAL-L1 depletion causes loss of EHD1-Rab8a interaction and absence of both proteins from membrane tubules, impairing endocytic recycling.","method":"Co-immunoprecipitation, in vitro binding, live cell imaging, siRNA depletion with recycling assays, colocalization studies","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP, live imaging, and functional recycling assays; identified mechanistic link between MICAL-L1 and EHD1 recruitment","pmids":["19864458"],"is_preprint":false},{"year":2010,"finding":"EHD1 interacts directly with Fer1L5 (via Fer1L5's second C2 domain); EHD1 (and EHD2) knockdown inhibits myoblast fusion; EHD2 is required for normal translocation of Fer1L5 to the plasma membrane.","method":"Direct binding assays, siRNA knockdown, myoblast fusion quantification, plasma membrane translocation assays","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2-3 — direct binding demonstrated with domain mapping, functional knockdown phenotype, single lab","pmids":["21177873"],"is_preprint":false},{"year":2010,"finding":"EHD1 knockout male mice are infertile due to defective spermatogenesis: abnormal acrosomal development, clumping of acrosomes, misaligned spermatids, absence of elongated spermatids, and abnormal phagocytosis of elongated spermatids by Sertoli cells.","method":"EHD1 knockout mouse model (Cre/loxP), histopathology, in situ hybridization, immunohistochemistry, light and electron microscopy","journal":"BMC developmental biology","confidence":"High","confidence_rationale":"Tier 2 — clean genetic KO with specific ultrastructural phenotype, multiple imaging modalities","pmids":["20359371"],"is_preprint":false},{"year":2010,"finding":"EHD1 regulates early transport of the GPI-anchored protein CD59 from pre-sorting endosomes to the endocytic recycling compartment; EHD1 depletion causes rapid Rab5-independent coalescence of CD59 at the ERC in a PKC-dependent manner.","method":"siRNA depletion, dominant-negative Arf6 expression, PKC inhibitor (Go6976) treatment, immunofluorescence","journal":"Traffic (Copenhagen, Denmark)","confidence":"Medium","confidence_rationale":"Tier 3 — pharmacological and genetic perturbation with imaging readout, single lab","pmids":["20961375"],"is_preprint":false},{"year":2010,"finding":"EHD1 is required for proper L1/NgCAM endocytosis and recycling in neurons; EHD1 colocalizes with L1/NgCAM predominantly in EEA1-positive early endosomes; EHD1 knockdown delays L1/NgCAM exit from EEA1-positive endosomes and impairs its axonal targeting.","method":"shRNA knockdown, live imaging, co-localization with compartment markers, EEA1-positive endosome exit assays","journal":"The Journal of neuroscience","confidence":"Medium","confidence_rationale":"Tier 2 — shRNA with live imaging and compartment-specific functional analysis, single lab","pmids":["21147988"],"is_preprint":false},{"year":2010,"finding":"Balanced levels of EHD1 and EHD4 are required for L1/NgCAM endocytosis in neurons; EHD1 overexpression impairs NgCAM internalization (but not transferrin); EHD1 oligomerization is required for this endocytosis defect; an endogenous EHD1-EHD4 complex was identified.","method":"shRNA knockdown, overexpression of EHD family members, endocytosis assays, oligomerization mutants","journal":"The Journal of neuroscience","confidence":"Medium","confidence_rationale":"Tier 2-3 — oligomerization mutant analysis with functional endocytosis readout, endogenous complex identified, single lab","pmids":["20463227"],"is_preprint":false},{"year":2010,"finding":"EHD1 interacts with snapin via its EH domain and negatively affects SNAP-25 binding to snapin; EHD1 overexpression reduces depolarization-induced exocytosis in PC12 cells, but this is not observed with an N-terminal EHD1 construct unable to bind snapin.","method":"Yeast two-hybrid, co-immunoprecipitation, binding competition assay, electrophysiological exocytosis measurement in PC12 cells","journal":"Molecular and cellular neurosciences","confidence":"Medium","confidence_rationale":"Tier 2-3 — yeast two-hybrid confirmed by Co-IP and functional exocytosis assay with domain mapping, single lab","pmids":["20696250"],"is_preprint":false},{"year":2012,"finding":"Retrograde trafficking of Shiga toxin B (STxB) from recycling endosome to TGN requires EHD1; EHD1 is not significantly required for CI-M6PR retrograde trafficking; retromer is required for exit from early endosomes to recycling endosomes for both cargoes.","method":"RNAi knockdown of EHD1 and retromer components, STxB and CI-M6PR trafficking assays, ablation of recycling endosome","journal":"Traffic (Copenhagen, Denmark)","confidence":"Medium","confidence_rationale":"Tier 2 — cargo-specific RNAi dissection of trafficking pathway, single lab","pmids":["22540229"],"is_preprint":false},{"year":2012,"finding":"EHD1 and cPLA2α interact in vivo (proximity <40 nm) and co-depletion experiments show both are involved in vesiculation of GPI-AP (CD59)-containing endosomes; cPLA2α depletion causes hypertubulation of CD59 endosomes and EHD1 depletion phenocopies this; lysophospholipid accumulation drives vesiculation.","method":"siRNA depletion, proximity ligation assay, co-immunoprecipitation, lysophospholipid acyltransferase inhibitor treatment, immunofluorescence","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2-3 — in vivo proximity assay with functional siRNA analysis, mechanistic lipid manipulation, single lab","pmids":["22456504"],"is_preprint":false},{"year":2012,"finding":"Rabankyrin-5 interacts with EHD1 via its EH domain and the NPFED motif of Rank-5; Rank-5 also colocalizes with retromer component Vps26 and is required for normal retromer distribution and CIMPR retrieval to Golgi; depletion of either Rank-5 or EHD1 impairs VSVG secretion.","method":"GST-pulldown, yeast two-hybrid, isothermal calorimetry, co-immunoprecipitation, siRNA depletion, CIMPR and VSVG trafficking assays","journal":"Traffic (Copenhagen, Denmark)","confidence":"High","confidence_rationale":"Tier 1-2 — interaction confirmed by four independent methods including isothermal calorimetry, with functional trafficking assays","pmids":["22284051"],"is_preprint":false},{"year":2014,"finding":"EHD1 mediates vesicle trafficking required for normal muscle growth; EHD1-null myoblasts have defective receptor recycling and mislocalization of caveolin-3 and Fer1L5; EHD1 localizes to T-tubules in wildtype skeletal muscle and its loss causes T-tubule overgrowth and excess vesicle accumulation; EHD1 ATPase domain is required for tubule formation in myoblasts.","method":"EHD1 knockout mouse model, immunofluorescence, electron microscopy, ATPase domain mutant expression, myoblast fusion quantification, BIN1-induced tubulation assays","journal":"Developmental biology","confidence":"High","confidence_rationale":"Tier 2 — genetic KO with multiple phenotypic readouts, ATPase domain mutant analysis, T-tubule ultrastructure characterization","pmids":["24440153"],"is_preprint":false},{"year":2014,"finding":"GRAF1 forms a complex with EHD1 and MICAL-L1; GRAF1 overexpression vesiculates MICAL-L1-containing tubular recycling endosomes, while GRAF1 depletion impairs TRE vesiculation and delays receptor recycling; co-addition of purified EHD1 and GRAF1 synergistically produces TRE vesiculation in semi-permeabilized cells.","method":"Co-immunoprecipitation, semi-permeabilized cell vesiculation assay with purified proteins, siRNA depletion, overexpression, immunofluorescence","journal":"Frontiers in cell and developmental biology","confidence":"Medium","confidence_rationale":"Tier 2 — in vitro vesiculation assay with purified proteins, confirmed by Co-IP and functional knockdown/overexpression","pmids":["25364729"],"is_preprint":false},{"year":2014,"finding":"MICAL-L1 and EHD1 regulate cytokinesis; depletion of either protein increases binucleated cells due to impaired recycling endosome transport during late cytokinesis; MICAL-L1 (but not EHD1) also regulates chromosome alignment during early mitosis and microtubule dynamics.","method":"siRNA depletion, binucleation quantification, live cell imaging, chromosome alignment analysis","journal":"Traffic (Copenhagen, Denmark)","confidence":"Medium","confidence_rationale":"Tier 2-3 — siRNA depletion with quantitative mitosis phenotype readout, distinguishes EHD1-dependent vs. independent functions, single lab","pmids":["25287187"],"is_preprint":false},{"year":2014,"finding":"EHD1 is required for Src trafficking and activation; MICAL-L1 recruits EHD1 to Src-containing recycling endosomes in response to growth factor stimulation, and EHD1 (along with MICAL-L1) is required for growth-factor- and integrin-induced Src activation, focal adhesion turnover, cell spreading and migration.","method":"siRNA depletion, co-localization, co-immunoprecipitation, Src activation assays, focal adhesion analysis, migration assays","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2-3 — multiple functional readouts with siRNA and Co-IP, but single lab","pmids":["24481818"],"is_preprint":false},{"year":2015,"finding":"EHD1 and EHD3 function in early ciliogenesis: they localize to preciliary membranes/ciliary pocket, mediate membrane tubulation essential for ciliary vesicle formation from distal appendage vesicles (DAVs), and are required for mother centriole to basal body transformation, transition zone protein and IFT20 recruitment; SNAP29 (EHD1-binding protein) is also required for DAV-mediated ciliary vesicle assembly.","method":"siRNA depletion, live cell imaging, SNAP29 knockdown, electron microscopy of centriole structures, localization of transition zone proteins and IFT20","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (siRNA, electron microscopy, protein localization), defines step-by-step ciliogenesis mechanism","pmids":["25686250"],"is_preprint":false},{"year":2015,"finding":"EHD1 recruitment to recycling endosomes requires PS flipping by the P4-ATPase ATP8A1; depletion of ATP8A1 disrupts asymmetric PS distribution in REs, dissociates EHD1 from REs, and generates aberrant endosomal tubules resistant to fission; EHD1 does not show membrane localization in cells defective in PS synthesis.","method":"siRNA depletion of ATP8A1, PS distribution assays, EHD1 localization analysis, endosomal tubule morphology by microscopy","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 — mechanistic link between P4-ATPase PS flipping and EHD1 recruitment, with multiple perturbation approaches","pmids":["25595798"],"is_preprint":false},{"year":2016,"finding":"EHD1 localizes to primary cilia and centrosomes; EHD1 knockout in mice causes embryonic lethality with neural tube closure defects, short cilia on neuroepithelium, deregulated ciliary SHH signaling, downregulation of GLI3 repressor formation, and increased ventral neuronal markers; EHD1 co-localizes with Smoothened and co-traffics with Smoothened into primary cilia, and is identified as a direct binding partner of Smoothened.","method":"EHD1 knockout mouse (B6 background), immunohistochemistry, co-immunoprecipitation, live cell imaging, ciliary length measurement","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 2 — genetic KO with multiple phenotypic and molecular readouts, direct binding partner identification","pmids":["26884322"],"is_preprint":false},{"year":2018,"finding":"EHD1 is an ATP-dependent membrane fission catalyst; ATP-bound EHD1 forms membrane-active scaffolds that bulge tubular model membranes; ATP hydrolysis promotes scaffold self-assembly causing membrane thinning and scission of tubes below 25 nm radius; deletion of N-terminal residues causes defects in scaffolding, scission and endocytic recycling; cross-complementation in C. elegans confirms membrane binding and ATP hydrolysis are necessary for recycling.","method":"Supported membrane tube assay, molecular dynamics simulations, C. elegans cross-complementation, N-terminal deletion mutagenesis, in vitro ATP hydrolysis assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 — reconstitution in vitro with mechanistic membrane deformation assay, MD simulations, mutagenesis, and in vivo genetic complementation","pmids":["30518883"],"is_preprint":false},{"year":2018,"finding":"Using a supported membrane tube scission screen, EHD1 was identified as a novel ATP-dependent membrane fission catalyst in brain cytosol; its GTP-dependent counterpart is dynamin.","method":"Supported membrane tube fission assay (microscopic screen), biochemical fractionation, mass spectrometric identification","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 — direct in vitro fission assay with biochemical fractionation and mass spectrometry identification","pmids":["30403133"],"is_preprint":false},{"year":2019,"finding":"MICAL-L1 coordinates EHD1 recruitment to the primary cilium; MICAL-L1 knockdown prevents CP110 removal from the mother centriole (similar to EHD1 knockdown) and prevents EHD1 localization to basal bodies; MICAL-L1 directly interacts with α/β-tubulin heterodimers and γ-tubulin, anchoring it to the centriole to recruit EHD1.","method":"siRNA depletion, mass spectrometry of MICAL-L1 interactors, direct interaction assays with tubulins, immunofluorescence","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2-3 — mass spectrometry discovery confirmed by direct binding, functional siRNA analysis","pmids":["31615969"],"is_preprint":false},{"year":2019,"finding":"EHD1 interacts with Cx43 (connexin 43) and regulates its internalization; EHD1 knockdown impairs Cx43 internalization and preserves gap junction-intercellular coupling; interaction is mediated by Eps15 and promoted by Cx43 phosphorylation and ubiquitination; EHD1 overexpression accelerates Cx43 internalization and exacerbates ischemia-induced Cx43 lateralization.","method":"Co-immunoprecipitation (Cx43-EHD1 interaction), siRNA knockdown, overexpression, gap junction coupling assay, ischemia model in isolated cardiomyocytes","journal":"Circulation research","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP with functional gain- and loss-of-function, mechanistic PTM requirement defined","pmids":["32138615"],"is_preprint":false},{"year":2020,"finding":"SNX17 directly interacts with EHD1; LRP1 internalization recruits cytoplasmic EHD1 to endosomal membranes; EHD1 depletion causes enlarged SNX17-containing endosomes, suggesting EHD1 catalyzes fission of SNX17-sorted receptor vesicles.","method":"Co-immunoprecipitation, in vitro binding assay, surface rendering quantification of EHD1/SNX17 overlap, EHD1 siRNA with endosome size measurement","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — direct interaction confirmed in vitro and by Co-IP, functional fission assay by endosome size measurement","pmids":["32041776"],"is_preprint":false},{"year":2020,"finding":"EHD4 is required for EHD1 recruitment to sorting endosomes; EHD4 preferentially dimerizes with EHD1; knockdown/knockout of EHD4 impairs EHD1 recruitment to sorting endosomes and causes enlarged SE; NPF-motif-containing partners Rabenosyn-5, Syndapin2 and MICAL-L1 are each required for EHD1 recruitment to SE.","method":"siRNA, shRNA and CRISPR/Cas9 EHD4 knockout, endosome size quantification, EHD1 localization assays, dimerization assays","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 — CRISPR KO and siRNA with functional EHD1 recruitment readout, single lab","pmids":["32966336"],"is_preprint":false},{"year":2020,"finding":"EHD1 and RUSC2 control cell surface display and recycling of EGFR from the Golgi; EHD1 knockdown causes EGFR accumulation at the Golgi compartment, reducing surface EGFR and impairing EGF-induced proliferation; RUSC2 knockdown phenocopies EHD1 depletion.","method":"Inducible siRNA knockdown, surface EGFR quantification, immunofluorescence, EGF-induced proliferation assay, rescue with exogenous EHD1","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 — inducible knockdown with rescue and functional proliferation readout, single lab","pmids":["31932478"],"is_preprint":false},{"year":2022,"finding":"A human founder missense mutation p.R398W in EHD1 causes autosomal recessive tubular proteinuria and sensorineural deafness; patients and Ehd1 knockout/knockin mice show impaired receptor-mediated endocytosis in proximal tubules; in silico analysis predicts R398W destabilizes EHD1 structure and impairs nucleotide binding, likely reducing EHD1 oligomerization and membrane remodeling.","method":"Genetic mapping, patient clinical analysis, Ehd1 KO and knockin mouse models, zebrafish model, proximal tubule endocytosis assays, in silico structural analysis","journal":"Journal of the American Society of Nephrology","confidence":"High","confidence_rationale":"Tier 1-2 — human genetics confirmed by multiple animal models with functional endocytosis readout and structural prediction","pmids":["35149593"],"is_preprint":false},{"year":2022,"finding":"Coronin2A (CORO2A) is a novel EHD1 interaction partner; CORO2A depletion causes enlarged endosomes and impaired recycling of clathrin-independent cargo, consistent with a role in EHD1-dependent endosomal fission.","method":"Co-immunoprecipitation, siRNA depletion, endosome size quantification, cargo recycling assays, actin protrusion analysis","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2-3 — Co-IP confirmed interaction with functional siRNA phenotype, single lab","pmids":["35921168"],"is_preprint":false},{"year":2023,"finding":"EHD1 regulates CP110 ubiquitination during ciliogenesis by transporting centriolar satellites containing the E3 ubiquitin ligase HERC2 to the mother centriole; HERC2 and MIB1 both interact with and ubiquitinate CP110; HERC2 localizes to centriolar satellites and is required for ciliogenesis.","method":"siRNA depletion, co-immunoprecipitation, ubiquitination assays, centriolar satellite tracking, immunofluorescence","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 2 — mechanistic dissection with Co-IP, ubiquitination assays, and centriolar satellite transport analysis, single lab with multiple orthogonal methods","pmids":["37074924"],"is_preprint":false},{"year":2023,"finding":"EHD1 is required for anterograde Golgi-to-plasma membrane traffic and endocytic recycling of IGF-1R in Ewing sarcoma; EHD1 overexpression-dependent oncogenic traits require IGF-1R expression and kinase activity; shRNA/CRISPR KO of EHD1 reduces tumorigenesis and metastasis.","method":"shRNA knockdown, CRISPR knockout with mouse Ehd1 rescue, RTK antibody arrays, surface IGF-1R quantification, tumor xenograft and metastasis assays","journal":"Communications biology","confidence":"Medium","confidence_rationale":"Tier 2 — CRISPR KO with rescue, receptor trafficking assays, and in vivo tumor models","pmids":["37474760"],"is_preprint":false},{"year":2023,"finding":"The EHD1 p.R398W missense mutation causes male infertility in knockin mice with defective acrosomal development, missing sperm tail midpieces, and disorganized spermatogenic epithelium; wild-type EHD1 co-localizes with acrosomal granules and the retromer component VPS35, but the p.R398W mutant does not co-localize with VPS35.","method":"Knockin mouse model, histopathology, immunofluorescence co-localization (WT vs. R398W with VPS35 and acrosomal markers), electron microscopy","journal":"Frontiers in cell and developmental biology","confidence":"High","confidence_rationale":"Tier 2 — knockin mouse model with ultrastructural analysis and mechanistically informative differential co-localization of WT vs. mutant protein","pmids":["37900275"],"is_preprint":false},{"year":2025,"finding":"EHD1 interacts with PD-L1 protein and promotes PD-L1 endocytic recycling; EHD1 knockdown inhibits PD-L1 recycling and promotes its lysosomal degradation, enhancing T cell cytotoxicity; EHD1 mRNA is m6A-modified and YTHDF1 binds EHD1 mRNA to stabilize it in an m6A-dependent manner.","method":"Molecular docking, co-immunoprecipitation, immunofluorescence, receptor internalization/recycling assays, MeRIP-qPCR, RNA immunoprecipitation, m6A-binding site mutation analysis, immunocompetent mouse tumor model","journal":"Cancer communications","confidence":"Medium","confidence_rationale":"Tier 2-3 — multiple orthogonal methods for both protein interaction and mRNA modification, with functional in vivo tumor immune assay","pmids":["40703029"],"is_preprint":false}],"current_model":"EHD1 is an ATP-dependent dynamin-family ATPase that oligomerizes on phosphatidylserine-enriched endosomal membranes (recruited via its EH domain's phosphatidylinositol-binding activity and by NPF-motif-containing partners such as MICAL-L1, Rabenosyn-5, and Syndapin2/EHD4) to catalyze membrane fission and vesicle release from tubular recycling endosomes, thereby driving receptor recycling to the plasma membrane across diverse cargo including transferrin receptor, MHC-I, integrins, GLUT4, EGFR, and IGF-1R; it additionally facilitates ciliogenesis by tubulating preciliary membranes and delivering the E3 ligase HERC2 to the mother centriole to ubiquitinate CP110, and interacts with Smoothened, Cx43, and other specialized cargoes to coordinate context-specific membrane trafficking events in development, immunity, and homeostasis."},"narrative":{"teleology":[{"year":1999,"claim":"Identification of EHD1 as an EH-domain protein on endocytic vesicles established the gene's entry into endosomal trafficking biology and revealed its domain architecture (P-loop, coiled-coil, EH domain).","evidence":"GFP-fusion live imaging and Northern analysis in cultured cells","pmids":["10395801"],"confidence":"Medium","gaps":["No function assigned beyond localization","ATPase activity of the P-loop not tested","No interacting partners identified"]},{"year":2002,"claim":"Demonstration that EHD1 associates with Arf6-containing tubules and is required for clathrin-independent recycling of MHC-I established EHD1 as a functional regulator of endosomal membrane tubulation and receptor recycling, with both the P-loop and EH domain being essential.","evidence":"Domain mutagenesis, MHC-I recycling assays, and dominant-negative Arf6 experiments in HeLa cells","pmids":["12032069","12121420"],"confidence":"High","gaps":["Mechanism of tubule formation (motor vs. scaffold) unknown","ATPase catalytic cycle not characterized"]},{"year":2004,"claim":"Mapping of the EH domain as a hub that binds NPF-motif partners (Rabenosyn-5, EHBP1) and epistasis experiments positioned EHD1 downstream of Rab4/Rab5-dependent sorting and implicated it in recycling of diverse cargo including GLUT4.","evidence":"GST pulldown with mass spectrometry, domain mapping, sequential RNAi epistasis, insulin-stimulated GLUT4 recycling assays","pmids":["15020713","15247266"],"confidence":"High","gaps":["Direct membrane-binding mechanism of EHD1 not resolved","Whether EHD1 performs fission or stabilizes tubules unclear"]},{"year":2006,"claim":"EHD1 knockout mice confirmed the in vivo requirement for EHD1 in exit of cargo from recycling endosomes, validating cell-based findings genetically.","evidence":"Transferrin recycling assays in EHD1 knockout mouse embryonic fibroblasts","pmids":["16445686"],"confidence":"High","gaps":["Organismal phenotypes beyond MEF recycling not yet characterized","Redundancy with other EHD family members not addressed"]},{"year":2007,"claim":"Multiple studies expanded EHD1's cargo repertoire to β1-integrins and cholesterol/LDL, linked EHD1 to retromer-dependent endosome-to-Golgi retrieval, and solved the EH domain NMR structure revealing a phosphoinositide-binding surface distinct from the NPF pocket.","evidence":"EHD1 KO MEFs with integrin recycling and cholesterol assays; comparative proteomics identifying retromer interaction; NMR structure and PI-binding mutagenesis (K483)","pmids":["17284518","17451652","17868075","17412695","17899392"],"confidence":"High","gaps":["Full-length EHD1 structure not determined","Mechanism of membrane scission vs. stabilization still unresolved","Relative contribution of PI binding vs. NPF-partner binding to membrane recruitment unknown"]},{"year":2009,"claim":"MICAL-L1 was identified as the key adaptor that recruits EHD1 (and Rab8a) to tubular recycling endosomes, and reconstitution of an amphiphysin–EHD1 (AMPH-1–RME-1) complex showed it generates morphologically distinct coated membrane tubules, providing the first in vitro evidence of cooperative membrane remodeling.","evidence":"Purified-protein membrane tubulation assay (C. elegans proteins), C. elegans genetics, human BIN1 knockdown; MICAL-L1 Co-IP and siRNA with recycling assays","pmids":["19915558","19864458"],"confidence":"High","gaps":["Reconstitution with human EHD1 not yet performed","Scission activity not demonstrated in this system"]},{"year":2010,"claim":"EHD1 knockout revealed essential roles in spermatogenesis (acrosome development) and showed that EHD1 and EHD4 form endogenous heteromers whose balance regulates neuronal L1/NgCAM endocytosis, broadening EHD1's physiological roles beyond generic recycling.","evidence":"EHD1 KO mouse histopathology and EM for spermatogenesis; shRNA and oligomerization mutants for neuronal trafficking","pmids":["20359371","20463227","21147988"],"confidence":"High","gaps":["Mechanism of EHD1 in acrosome biogenesis not defined at molecular level","Stoichiometry of EHD1–EHD4 complexes unknown"]},{"year":2015,"claim":"EHD1 was shown to function in ciliogenesis—tubulating preciliary membranes at distal appendage vesicles and enabling ciliary vesicle formation—and its membrane recruitment was found to depend on phosphatidylserine flipping by the P4-ATPase ATP8A1, establishing PS asymmetry as a prerequisite for EHD1 engagement.","evidence":"siRNA, EM of centriole structures, transition zone protein localization for ciliogenesis; ATP8A1 depletion with PS distribution and EHD1 localization assays","pmids":["25686250","25595798"],"confidence":"High","gaps":["How EHD1 senses PS enrichment at the molecular level unclear","Whether ATP hydrolysis is required for ciliogenesis-specific function not tested"]},{"year":2016,"claim":"EHD1 knockout on a C57BL/6 background caused embryonic lethality with neural tube defects, short cilia, and deregulated Hedgehog signaling, establishing EHD1 as essential for developmental Hedgehog pathway output and identifying Smoothened as a direct binding partner.","evidence":"EHD1 KO mouse, immunohistochemistry, Co-IP for Smoothened, ciliary length measurement, GLI3 repressor quantification","pmids":["26884322"],"confidence":"High","gaps":["Mechanism by which EHD1 regulates Smoothened ciliary entry not defined","Tissue-specific versus universal requirement for EHD1 in Hedgehog signaling not resolved"]},{"year":2018,"claim":"In vitro reconstitution on supported membrane tubes demonstrated that EHD1 is a bona fide ATP-dependent membrane fission enzyme: ATP-bound EHD1 scaffolds thin membranes and ATP hydrolysis drives self-assembly leading to scission of tubes below 25 nm radius, directly resolving the long-standing question of whether EHD1 stabilizes or severs tubules.","evidence":"Supported membrane tube assay, molecular dynamics simulation, N-terminal deletion mutagenesis, C. elegans cross-complementation","pmids":["30518883","30403133"],"confidence":"High","gaps":["Full atomic structure of the membrane-bound oligomer lacking","Regulation of ATPase cycle by partners (MICAL-L1, GRAF1) not reconstituted"]},{"year":2022,"claim":"A human founder mutation p.R398W in EHD1 was identified as the cause of autosomal recessive tubular proteinuria and sensorineural deafness, validated in KO and knockin mice showing impaired proximal tubule endocytosis and defective spermatogenesis with loss of VPS35/retromer co-localization.","evidence":"Patient genetics, Ehd1 KO and R398W knockin mice, zebrafish model, proximal tubule endocytosis assays, VPS35 co-localization","pmids":["35149593","37900275"],"confidence":"High","gaps":["Structural basis of R398W destabilization only modeled in silico","Whether hearing loss is due to ciliary or recycling defect not determined"]},{"year":2023,"claim":"The ciliogenesis mechanism was further refined: EHD1 transports HERC2-containing centriolar satellites to the mother centriole to ubiquitinate CP110, providing a molecular link between EHD1's membrane trafficking function and centriole remodeling during cilia initiation.","evidence":"siRNA, Co-IP, ubiquitination assays, centriolar satellite tracking","pmids":["37074924"],"confidence":"High","gaps":["Whether HERC2 transport is dependent on EHD1 ATPase activity not tested","Interplay between HERC2 and MIB1 at the centriole not fully resolved"]},{"year":2025,"claim":"EHD1 was shown to promote PD-L1 recycling to the cell surface; its depletion diverted PD-L1 to lysosomal degradation and enhanced anti-tumor T cell cytotoxicity, extending EHD1's cargo repertoire to immune checkpoint regulation.","evidence":"Co-IP, PD-L1 internalization/recycling assays, MeRIP-qPCR for m6A modification, immunocompetent mouse tumor model","pmids":["40703029"],"confidence":"Medium","gaps":["Direct structural basis of EHD1–PD-L1 interaction not defined","Generalizability across tumor types not established","m6A regulation of EHD1 mRNA awaits independent confirmation"]},{"year":null,"claim":"Key open questions include the high-resolution structure of the full-length membrane-bound EHD1 oligomer, the regulatory logic by which distinct NPF-motif partners select cargo-specific fission events, and the mechanism by which EHD1 ATPase activity is coordinated with partner proteins (GRAF1, MICAL-L1, BIN1) during scission.","evidence":"","pmids":[],"confidence":"High","gaps":["No high-resolution structure of full-length EHD1 on membranes","How cargo specificity is encoded by different NPF adaptors is undefined","ATPase regulation by partner proteins not reconstituted in vitro"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140657","term_label":"ATP-dependent activity","supporting_discovery_ids":[33,34]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[33,34]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[8,31]}],"localization":[{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[0,2,4,7,16,31,37]},{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[0,2,5,16,30]},{"term_id":"GO:0005929","term_label":"cilium","supporting_discovery_ids":[30,32,35]},{"term_id":"GO:0005815","term_label":"microtubule organizing center","supporting_discovery_ids":[32,35,42]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[26,36]}],"pathway":[{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[2,4,7,10,15,16,33,34]},{"term_id":"R-HSA-9609507","term_label":"Protein localization","supporting_discovery_ids":[5,39,43,45]},{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[30,32,35,42]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[1,32,43]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[18,32,44]}],"complexes":[],"partners":["MICAL-L1","RABENOSYN-5","SNAP29","EHD3","EHD4","EHBP1","BIN1","SNX17"],"other_free_text":[]},"mechanistic_narrative":"EHD1 is an ATP-dependent membrane fission enzyme that oligomerizes on phosphatidylserine-enriched endosomal tubules to catalyze vesicle scission, thereby controlling exit of diverse cargo—including transferrin receptor, MHC-I, integrins, GLUT4, EGFR, IGF-1R, and PD-L1—from recycling endosomes back to the plasma membrane [PMID:30518883, PMID:16445686, PMID:12032069, PMID:40703029]. Its C-terminal EH domain recruits EHD1 to membranes through dual recognition of NPF-motif partners (MICAL-L1, Rabenosyn-5, Syndapin2) and direct phosphatidylinositol binding, while the N-terminal P-loop ATPase domain drives scaffold assembly and membrane thinning that leads to fission of tubules below ~25 nm radius [PMID:17412695, PMID:19864458, PMID:30518883, PMID:15020713]. Beyond endocytic recycling, EHD1 functions in ciliogenesis by tubulating preciliary membranes at distal appendage vesicles and delivering the E3 ligase HERC2 to the mother centriole to ubiquitinate CP110, and its loss in mice causes neural tube defects with deregulated Hedgehog signaling [PMID:25686250, PMID:37074924, PMID:26884322]. A homozygous missense mutation (p.R398W) in EHD1 causes autosomal recessive tubular proteinuria, sensorineural deafness, and male infertility in humans and mouse models [PMID:35149593, PMID:37900275]."},"prefetch_data":{"uniprot":{"accession":"Q9H4M9","full_name":"EH domain-containing protein 1","aliases":["PAST homolog 1","hPAST1","Testilin"],"length_aa":534,"mass_kda":60.6,"function":"ATP- and membrane-binding protein that controls membrane reorganization/tubulation upon ATP hydrolysis. In vitro causes vesiculation of endocytic membranes (PubMed:24019528). Acts in early endocytic membrane fusion and membrane trafficking of recycling endosomes (PubMed:15020713, PubMed:17233914, PubMed:20801876). Recruited to endosomal membranes upon nerve growth factor stimulation, indirectly regulates neurite outgrowth (By similarity). Plays a role in myoblast fusion (By similarity). Involved in the unidirectional retrograde dendritic transport of endocytosed BACE1 and in efficient sorting of BACE1 to axons implicating a function in neuronal APP processing (By similarity). Plays a role in the formation of the ciliary vesicle (CV), an early step in cilium biogenesis (PubMed:31615969). Proposed to be required for the fusion of distal appendage vesicles (DAVs) to form the CV by recruiting SNARE complex component SNAP29. Is required for recruitment of transition zone proteins CEP290, RPGRIP1L, TMEM67 and B9D2, and of IFT20 following DAV reorganization before Rab8-dependent ciliary membrane extension. Required for the loss of CCP110 form the mother centriole essential for the maturation of the basal body during ciliogenesis (PubMed:25686250)","subcellular_location":"Recycling endosome membrane; Early endosome membrane; Cell membrane; Cell projection, cilium membrane","url":"https://www.uniprot.org/uniprotkb/Q9H4M9/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/EHD1","classification":"Not Classified","n_dependent_lines":11,"n_total_lines":1208,"dependency_fraction":0.009105960264900662},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/EHD1","total_profiled":1310},"omim":[{"mim_id":"620883","title":"FER1-LIKE FAMILY, MEMBER 5; FER1L5","url":"https://www.omim.org/entry/620883"},{"mim_id":"619563","title":"MICAL-LIKE PROTEIN 1; MICALL1","url":"https://www.omim.org/entry/619563"},{"mim_id":"614556","title":"AT-RICH INTERACTION DOMAIN-CONTAINING PROTEIN 1B; ARID1B","url":"https://www.omim.org/entry/614556"},{"mim_id":"609511","title":"RABENOSYN, RAB EFFECTOR; RBSN","url":"https://www.omim.org/entry/609511"},{"mim_id":"606540","title":"MYOSIN VB; MYO5B","url":"https://www.omim.org/entry/606540"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Plasma membrane","reliability":"Supported"},{"location":"Primary cilium","reliability":"Additional"},{"location":"Primary cilium transition zone","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"bone marrow","ntpm":143.0},{"tissue":"testis","ntpm":148.3}],"url":"https://www.proteinatlas.org/search/EHD1"},"hgnc":{"alias_symbol":["H-PAST","HPAST1","FLJ42622","FLJ44618"],"prev_symbol":["PAST1"]},"alphafold":{"accession":"Q9H4M9","domains":[{"cath_id":"1.10.268.20","chopping":"20-48_291-402","consensus_level":"high","plddt":94.7839,"start":20,"end":402},{"cath_id":"3.40.50.300","chopping":"57-265","consensus_level":"high","plddt":90.9673,"start":57,"end":265},{"cath_id":"1.10.238.10","chopping":"428-529","consensus_level":"high","plddt":93.593,"start":428,"end":529}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9H4M9","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9H4M9-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9H4M9-F1-predicted_aligned_error_v6.png","plddt_mean":89.69},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=EHD1","jax_strain_url":"https://www.jax.org/strain/search?query=EHD1"},"sequence":{"accession":"Q9H4M9","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9H4M9.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9H4M9/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9H4M9"}},"corpus_meta":[{"pmid":"12032069","id":"PMC_12032069","title":"A 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\"Genomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct localization experiment with structural domain characterization, single lab\",\n      \"pmids\": [\"10395801\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"EHD1 directly interacts with SNAP29 and associates with alpha-adaptin of the AP-2 complex and IGF-1R; overexpression of EHD1 represses IGF-1-mediated MAPK and Akt phosphorylation, indicating EHD1 acts as a downregulator of IGF-1R signaling.\",\n      \"method\": \"Co-immunoprecipitation, co-localization, overexpression with downstream signaling readouts (MAPK/Akt phosphorylation)\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — reciprocal Co-IP with functional signaling readout, single lab\",\n      \"pmids\": [\"11423532\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"EHD1 associates with Arf6-containing membrane tubules and induces tubule formation to facilitate recycling of MHC-I molecules internalized via clathrin-independent endocytosis; the N-terminal P-loop domain and C-terminal EH domain are both required for tubule association and formation.\",\n      \"method\": \"Overexpression of tagged EHD1 and domain mutants, immunofluorescence, MHC-I recycling assays, dominant-negative Arf6 experiments\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods including domain mutagenesis and functional recycling assays, high citation count indicating replication\",\n      \"pmids\": [\"12032069\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"EHD1 and EHD3 interact with each other (confirmed by yeast two-hybrid and co-immunoprecipitation), and the N-terminal domain of EHD3 is responsible for its tubular localization; coexpression of EHD1 and EHD3 results in their co-localization in microtubule-dependent tubules.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, GFP-fusion live imaging, domain-swap chimeras\",\n      \"journal\": \"Traffic (Copenhagen, Denmark)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — yeast two-hybrid confirmed by Co-IP, domain mutagenesis defining tubular localization signal\",\n      \"pmids\": [\"12121420\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"EHD1 interacts with the divalent Rab4/Rab5 effector Rabenosyn-5 via its EH domain binding to the first two NPF motifs of Rabenosyn-5; Rabenosyn-5 acts sequentially before EHD1 in transport from early endosomes to the endosomal recycling compartment and back to the plasma membrane.\",\n      \"method\": \"GST-pulldown from brain cytosol, mass spectrometry, domain mapping, immunofluorescence, RNAi knockdown with transferrin and MHC-I recycling assays, epistasis by double RNAi\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — biochemical pulldown with mass spectrometry, domain mapping, epistasis by sequential RNAi, functional recycling assays\",\n      \"pmids\": [\"15020713\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"EHD1 interacts via its EH domain with EHBP1 and controls perinuclear localization of GLUT4-containing membranes; EHD1 is required for insulin-stimulated GLUT4 recycling to the plasma membrane in adipocytes.\",\n      \"method\": \"Co-immunoprecipitation, dominant-negative expression (ΔEH-EHD1), siRNA knockdown, immunofluorescence, glucose transport assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (Co-IP, dominant-negative, siRNA) with functional transport readout\",\n      \"pmids\": [\"15247266\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"EHD1 interacts with GS32/SNAP-29 via its EH domain binding to the N-terminal NPF-containing region of GS32, and with syndapin II via the EH domain; these two interactions are mutually exclusive, suggesting EHD1 participates in pathways of both GS32 and syndapin II in a mutually exclusive manner.\",\n      \"method\": \"GST pulldown from cell extracts and brain extracts, co-immunoprecipitation, competition binding assays\",\n      \"journal\": \"Molecular membrane biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — reciprocal Co-IP and pulldown with competition assay demonstrating mutual exclusivity\",\n      \"pmids\": [\"15371016\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"EHD1 knockout mice exhibit delayed recycling of transferrin to the plasma membrane with accumulation in the endocytic recycling compartment, confirming EHD1's role in exit of membrane proteins from recycling endosomes in vivo.\",\n      \"method\": \"EHD1 knockout mouse model, transferrin uptake and recycling assays in embryonic fibroblasts\",\n      \"journal\": \"Traffic (Copenhagen, Denmark)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean genetic knockout with defined cellular phenotype, in vivo model\",\n      \"pmids\": [\"16445686\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"EHD1 directly binds phosphatidylinositols, preferring those phosphorylated at position 3; the EH domain's Lys-483 residue (on the face opposite to the NPF-binding pocket) is critical for phosphatidylinositol binding, as determined by 2D NMR.\",\n      \"method\": \"In vitro phospholipid binding assays, 2D NMR analysis, site-directed mutagenesis (K483)\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro binding assay with NMR structural validation and mutagenesis confirming critical residue\",\n      \"pmids\": [\"17412695\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"EHD1 interacts with retromer in vivo, colocalizes with retromer, and stabilizes SNX1-positive tubules; loss of EHD1 by RNAi destabilizes SNX1 tubules and inhibits endosome-to-Golgi retrieval of CIMPR; P-loop mutation of EHD1 causes dominant-negative effects on retromer localization.\",\n      \"method\": \"Comparative proteomics (endosomal fractions from WT vs retromer-deficient cells), co-immunoprecipitation, RNAi, P-loop dominant-negative mutant, CIMPR trafficking assays\",\n      \"journal\": \"Traffic (Copenhagen, Denmark)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — proteomics discovery validated by Co-IP, domain mutagenesis, and functional trafficking assays\",\n      \"pmids\": [\"17868075\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"EHD1 regulates beta1 integrin endosomal recycling; RNAi knockdown of EHD1 impairs beta1 integrin recycling, EHD1-knockout MEFs show impaired cell spreading and migration on fibronectin and slower focal adhesion disassembly, phenotypes rescued by wild-type EHD1 re-expression.\",\n      \"method\": \"RNAi knockdown, EHD1 knockout MEFs, integrin recycling assays, focal adhesion analysis, migration and spreading assays, rescue with wild-type EHD1\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean KO with specific cellular phenotype, RNAi confirmation, and rescue experiment\",\n      \"pmids\": [\"17284518\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"EHD1 regulates cholesterol homeostasis; EHD1 knockout MEFs display reduced esterified and free cholesterol, smaller lipid droplets, and ineffectual cholesterol uptake via LDL receptor, reversed by wild-type but not dysfunctional EHD1.\",\n      \"method\": \"EHD1 knockout MEFs, cholesterol measurement assays, lipid droplet quantification, LDL receptor internalization, siRNA, rescue with WT vs. mutant EHD1\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic KO with functional readout, siRNA confirmation, and rescue by WT but not mutant EHD1\",\n      \"pmids\": [\"17451652\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"EHD1 localizes to a tubular network containing EHD3 and Rab8a; Myosin Vb interacts with Rab8a (confirmed by yeast two-hybrid and FRET) and colocalizes with EHD1/EHD3-positive Rab8a tubules, defining a recycling pathway distinct from the Rab11a pathway.\",\n      \"method\": \"Yeast two-hybrid, FRET imaging, co-localization immunofluorescence, dominant-negative Myosin Vb expression\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — yeast two-hybrid confirmed by FRET, with functional dominant-negative pathway analysis\",\n      \"pmids\": [\"17507647\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"The solution structure of the EHD1 EH domain was solved by NMR; it resembles the second N-terminal EH domain of Eps15 but shows differences in surface charge and the NPF/DPF-binding pocket structure.\",\n      \"method\": \"NMR spectroscopy, backbone resonance assignment, solution structure determination\",\n      \"journal\": \"Journal of biomolecular NMR\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — NMR structure determination of the EH domain\",\n      \"pmids\": [\"17899392\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"EHD1 undergoes serine phosphorylation that is enhanced by serum stimulation; PKC is one of the kinases responsible; inhibitors of clathrin-mediated endocytosis (but not caveolin-mediated endocytosis) decrease EHD1 phosphorylation, placing phosphorylation between early endosomes and the endocytic recycling compartment.\",\n      \"method\": \"Phosphorylation assays, kinase inhibitor treatments, endocytosis pathway inhibitors\",\n      \"journal\": \"Cellular & molecular biology letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — pharmacological inhibitor approach with phosphorylation readout, single lab\",\n      \"pmids\": [\"18661112\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"C. elegans AMPH-1 (Amphiphysin/BIN1 ortholog) colocalizes with RME-1 (EHD1 ortholog) on recycling endosomes; AMPH-1 NPF motifs bind the RME-1 EH domain; purified AMPH-1-RME-1 complexes produce short coated membrane tubules distinct from those by either protein alone; BIN1 is required for EHD1-regulated endocytic recycling in human cells.\",\n      \"method\": \"In vivo C. elegans co-localization, deletion mutant analysis, in vitro membrane tubulation with purified proteins, human BIN1 knockdown with EHD1 recycling assays\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution with purified proteins showing tubulation, complemented by genetic analysis in C. elegans and knockdown in human cells\",\n      \"pmids\": [\"19915558\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"MICAL-L1 interacts with EHD1 via its NPF motifs and the EHD1 EH domain, recruits both EHD1 and Rab8a to tubular recycling endosomes; MICAL-L1 depletion causes loss of EHD1-Rab8a interaction and absence of both proteins from membrane tubules, impairing endocytic recycling.\",\n      \"method\": \"Co-immunoprecipitation, in vitro binding, live cell imaging, siRNA depletion with recycling assays, colocalization studies\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP, live imaging, and functional recycling assays; identified mechanistic link between MICAL-L1 and EHD1 recruitment\",\n      \"pmids\": [\"19864458\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"EHD1 interacts directly with Fer1L5 (via Fer1L5's second C2 domain); EHD1 (and EHD2) knockdown inhibits myoblast fusion; EHD2 is required for normal translocation of Fer1L5 to the plasma membrane.\",\n      \"method\": \"Direct binding assays, siRNA knockdown, myoblast fusion quantification, plasma membrane translocation assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — direct binding demonstrated with domain mapping, functional knockdown phenotype, single lab\",\n      \"pmids\": [\"21177873\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"EHD1 knockout male mice are infertile due to defective spermatogenesis: abnormal acrosomal development, clumping of acrosomes, misaligned spermatids, absence of elongated spermatids, and abnormal phagocytosis of elongated spermatids by Sertoli cells.\",\n      \"method\": \"EHD1 knockout mouse model (Cre/loxP), histopathology, in situ hybridization, immunohistochemistry, light and electron microscopy\",\n      \"journal\": \"BMC developmental biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean genetic KO with specific ultrastructural phenotype, multiple imaging modalities\",\n      \"pmids\": [\"20359371\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"EHD1 regulates early transport of the GPI-anchored protein CD59 from pre-sorting endosomes to the endocytic recycling compartment; EHD1 depletion causes rapid Rab5-independent coalescence of CD59 at the ERC in a PKC-dependent manner.\",\n      \"method\": \"siRNA depletion, dominant-negative Arf6 expression, PKC inhibitor (Go6976) treatment, immunofluorescence\",\n      \"journal\": \"Traffic (Copenhagen, Denmark)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — pharmacological and genetic perturbation with imaging readout, single lab\",\n      \"pmids\": [\"20961375\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"EHD1 is required for proper L1/NgCAM endocytosis and recycling in neurons; EHD1 colocalizes with L1/NgCAM predominantly in EEA1-positive early endosomes; EHD1 knockdown delays L1/NgCAM exit from EEA1-positive endosomes and impairs its axonal targeting.\",\n      \"method\": \"shRNA knockdown, live imaging, co-localization with compartment markers, EEA1-positive endosome exit assays\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — shRNA with live imaging and compartment-specific functional analysis, single lab\",\n      \"pmids\": [\"21147988\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Balanced levels of EHD1 and EHD4 are required for L1/NgCAM endocytosis in neurons; EHD1 overexpression impairs NgCAM internalization (but not transferrin); EHD1 oligomerization is required for this endocytosis defect; an endogenous EHD1-EHD4 complex was identified.\",\n      \"method\": \"shRNA knockdown, overexpression of EHD family members, endocytosis assays, oligomerization mutants\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — oligomerization mutant analysis with functional endocytosis readout, endogenous complex identified, single lab\",\n      \"pmids\": [\"20463227\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"EHD1 interacts with snapin via its EH domain and negatively affects SNAP-25 binding to snapin; EHD1 overexpression reduces depolarization-induced exocytosis in PC12 cells, but this is not observed with an N-terminal EHD1 construct unable to bind snapin.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, binding competition assay, electrophysiological exocytosis measurement in PC12 cells\",\n      \"journal\": \"Molecular and cellular neurosciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — yeast two-hybrid confirmed by Co-IP and functional exocytosis assay with domain mapping, single lab\",\n      \"pmids\": [\"20696250\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Retrograde trafficking of Shiga toxin B (STxB) from recycling endosome to TGN requires EHD1; EHD1 is not significantly required for CI-M6PR retrograde trafficking; retromer is required for exit from early endosomes to recycling endosomes for both cargoes.\",\n      \"method\": \"RNAi knockdown of EHD1 and retromer components, STxB and CI-M6PR trafficking assays, ablation of recycling endosome\",\n      \"journal\": \"Traffic (Copenhagen, Denmark)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — cargo-specific RNAi dissection of trafficking pathway, single lab\",\n      \"pmids\": [\"22540229\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"EHD1 and cPLA2α interact in vivo (proximity <40 nm) and co-depletion experiments show both are involved in vesiculation of GPI-AP (CD59)-containing endosomes; cPLA2α depletion causes hypertubulation of CD59 endosomes and EHD1 depletion phenocopies this; lysophospholipid accumulation drives vesiculation.\",\n      \"method\": \"siRNA depletion, proximity ligation assay, co-immunoprecipitation, lysophospholipid acyltransferase inhibitor treatment, immunofluorescence\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — in vivo proximity assay with functional siRNA analysis, mechanistic lipid manipulation, single lab\",\n      \"pmids\": [\"22456504\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Rabankyrin-5 interacts with EHD1 via its EH domain and the NPFED motif of Rank-5; Rank-5 also colocalizes with retromer component Vps26 and is required for normal retromer distribution and CIMPR retrieval to Golgi; depletion of either Rank-5 or EHD1 impairs VSVG secretion.\",\n      \"method\": \"GST-pulldown, yeast two-hybrid, isothermal calorimetry, co-immunoprecipitation, siRNA depletion, CIMPR and VSVG trafficking assays\",\n      \"journal\": \"Traffic (Copenhagen, Denmark)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — interaction confirmed by four independent methods including isothermal calorimetry, with functional trafficking assays\",\n      \"pmids\": [\"22284051\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"EHD1 mediates vesicle trafficking required for normal muscle growth; EHD1-null myoblasts have defective receptor recycling and mislocalization of caveolin-3 and Fer1L5; EHD1 localizes to T-tubules in wildtype skeletal muscle and its loss causes T-tubule overgrowth and excess vesicle accumulation; EHD1 ATPase domain is required for tubule formation in myoblasts.\",\n      \"method\": \"EHD1 knockout mouse model, immunofluorescence, electron microscopy, ATPase domain mutant expression, myoblast fusion quantification, BIN1-induced tubulation assays\",\n      \"journal\": \"Developmental biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic KO with multiple phenotypic readouts, ATPase domain mutant analysis, T-tubule ultrastructure characterization\",\n      \"pmids\": [\"24440153\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"GRAF1 forms a complex with EHD1 and MICAL-L1; GRAF1 overexpression vesiculates MICAL-L1-containing tubular recycling endosomes, while GRAF1 depletion impairs TRE vesiculation and delays receptor recycling; co-addition of purified EHD1 and GRAF1 synergistically produces TRE vesiculation in semi-permeabilized cells.\",\n      \"method\": \"Co-immunoprecipitation, semi-permeabilized cell vesiculation assay with purified proteins, siRNA depletion, overexpression, immunofluorescence\",\n      \"journal\": \"Frontiers in cell and developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vitro vesiculation assay with purified proteins, confirmed by Co-IP and functional knockdown/overexpression\",\n      \"pmids\": [\"25364729\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"MICAL-L1 and EHD1 regulate cytokinesis; depletion of either protein increases binucleated cells due to impaired recycling endosome transport during late cytokinesis; MICAL-L1 (but not EHD1) also regulates chromosome alignment during early mitosis and microtubule dynamics.\",\n      \"method\": \"siRNA depletion, binucleation quantification, live cell imaging, chromosome alignment analysis\",\n      \"journal\": \"Traffic (Copenhagen, Denmark)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — siRNA depletion with quantitative mitosis phenotype readout, distinguishes EHD1-dependent vs. independent functions, single lab\",\n      \"pmids\": [\"25287187\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"EHD1 is required for Src trafficking and activation; MICAL-L1 recruits EHD1 to Src-containing recycling endosomes in response to growth factor stimulation, and EHD1 (along with MICAL-L1) is required for growth-factor- and integrin-induced Src activation, focal adhesion turnover, cell spreading and migration.\",\n      \"method\": \"siRNA depletion, co-localization, co-immunoprecipitation, Src activation assays, focal adhesion analysis, migration assays\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — multiple functional readouts with siRNA and Co-IP, but single lab\",\n      \"pmids\": [\"24481818\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"EHD1 and EHD3 function in early ciliogenesis: they localize to preciliary membranes/ciliary pocket, mediate membrane tubulation essential for ciliary vesicle formation from distal appendage vesicles (DAVs), and are required for mother centriole to basal body transformation, transition zone protein and IFT20 recruitment; SNAP29 (EHD1-binding protein) is also required for DAV-mediated ciliary vesicle assembly.\",\n      \"method\": \"siRNA depletion, live cell imaging, SNAP29 knockdown, electron microscopy of centriole structures, localization of transition zone proteins and IFT20\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (siRNA, electron microscopy, protein localization), defines step-by-step ciliogenesis mechanism\",\n      \"pmids\": [\"25686250\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"EHD1 recruitment to recycling endosomes requires PS flipping by the P4-ATPase ATP8A1; depletion of ATP8A1 disrupts asymmetric PS distribution in REs, dissociates EHD1 from REs, and generates aberrant endosomal tubules resistant to fission; EHD1 does not show membrane localization in cells defective in PS synthesis.\",\n      \"method\": \"siRNA depletion of ATP8A1, PS distribution assays, EHD1 localization analysis, endosomal tubule morphology by microscopy\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — mechanistic link between P4-ATPase PS flipping and EHD1 recruitment, with multiple perturbation approaches\",\n      \"pmids\": [\"25595798\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"EHD1 localizes to primary cilia and centrosomes; EHD1 knockout in mice causes embryonic lethality with neural tube closure defects, short cilia on neuroepithelium, deregulated ciliary SHH signaling, downregulation of GLI3 repressor formation, and increased ventral neuronal markers; EHD1 co-localizes with Smoothened and co-traffics with Smoothened into primary cilia, and is identified as a direct binding partner of Smoothened.\",\n      \"method\": \"EHD1 knockout mouse (B6 background), immunohistochemistry, co-immunoprecipitation, live cell imaging, ciliary length measurement\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic KO with multiple phenotypic and molecular readouts, direct binding partner identification\",\n      \"pmids\": [\"26884322\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"EHD1 is an ATP-dependent membrane fission catalyst; ATP-bound EHD1 forms membrane-active scaffolds that bulge tubular model membranes; ATP hydrolysis promotes scaffold self-assembly causing membrane thinning and scission of tubes below 25 nm radius; deletion of N-terminal residues causes defects in scaffolding, scission and endocytic recycling; cross-complementation in C. elegans confirms membrane binding and ATP hydrolysis are necessary for recycling.\",\n      \"method\": \"Supported membrane tube assay, molecular dynamics simulations, C. elegans cross-complementation, N-terminal deletion mutagenesis, in vitro ATP hydrolysis assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstitution in vitro with mechanistic membrane deformation assay, MD simulations, mutagenesis, and in vivo genetic complementation\",\n      \"pmids\": [\"30518883\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Using a supported membrane tube scission screen, EHD1 was identified as a novel ATP-dependent membrane fission catalyst in brain cytosol; its GTP-dependent counterpart is dynamin.\",\n      \"method\": \"Supported membrane tube fission assay (microscopic screen), biochemical fractionation, mass spectrometric identification\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — direct in vitro fission assay with biochemical fractionation and mass spectrometry identification\",\n      \"pmids\": [\"30403133\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"MICAL-L1 coordinates EHD1 recruitment to the primary cilium; MICAL-L1 knockdown prevents CP110 removal from the mother centriole (similar to EHD1 knockdown) and prevents EHD1 localization to basal bodies; MICAL-L1 directly interacts with α/β-tubulin heterodimers and γ-tubulin, anchoring it to the centriole to recruit EHD1.\",\n      \"method\": \"siRNA depletion, mass spectrometry of MICAL-L1 interactors, direct interaction assays with tubulins, immunofluorescence\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — mass spectrometry discovery confirmed by direct binding, functional siRNA analysis\",\n      \"pmids\": [\"31615969\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"EHD1 interacts with Cx43 (connexin 43) and regulates its internalization; EHD1 knockdown impairs Cx43 internalization and preserves gap junction-intercellular coupling; interaction is mediated by Eps15 and promoted by Cx43 phosphorylation and ubiquitination; EHD1 overexpression accelerates Cx43 internalization and exacerbates ischemia-induced Cx43 lateralization.\",\n      \"method\": \"Co-immunoprecipitation (Cx43-EHD1 interaction), siRNA knockdown, overexpression, gap junction coupling assay, ischemia model in isolated cardiomyocytes\",\n      \"journal\": \"Circulation research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP with functional gain- and loss-of-function, mechanistic PTM requirement defined\",\n      \"pmids\": [\"32138615\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"SNX17 directly interacts with EHD1; LRP1 internalization recruits cytoplasmic EHD1 to endosomal membranes; EHD1 depletion causes enlarged SNX17-containing endosomes, suggesting EHD1 catalyzes fission of SNX17-sorted receptor vesicles.\",\n      \"method\": \"Co-immunoprecipitation, in vitro binding assay, surface rendering quantification of EHD1/SNX17 overlap, EHD1 siRNA with endosome size measurement\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct interaction confirmed in vitro and by Co-IP, functional fission assay by endosome size measurement\",\n      \"pmids\": [\"32041776\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"EHD4 is required for EHD1 recruitment to sorting endosomes; EHD4 preferentially dimerizes with EHD1; knockdown/knockout of EHD4 impairs EHD1 recruitment to sorting endosomes and causes enlarged SE; NPF-motif-containing partners Rabenosyn-5, Syndapin2 and MICAL-L1 are each required for EHD1 recruitment to SE.\",\n      \"method\": \"siRNA, shRNA and CRISPR/Cas9 EHD4 knockout, endosome size quantification, EHD1 localization assays, dimerization assays\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — CRISPR KO and siRNA with functional EHD1 recruitment readout, single lab\",\n      \"pmids\": [\"32966336\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"EHD1 and RUSC2 control cell surface display and recycling of EGFR from the Golgi; EHD1 knockdown causes EGFR accumulation at the Golgi compartment, reducing surface EGFR and impairing EGF-induced proliferation; RUSC2 knockdown phenocopies EHD1 depletion.\",\n      \"method\": \"Inducible siRNA knockdown, surface EGFR quantification, immunofluorescence, EGF-induced proliferation assay, rescue with exogenous EHD1\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — inducible knockdown with rescue and functional proliferation readout, single lab\",\n      \"pmids\": [\"31932478\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"A human founder missense mutation p.R398W in EHD1 causes autosomal recessive tubular proteinuria and sensorineural deafness; patients and Ehd1 knockout/knockin mice show impaired receptor-mediated endocytosis in proximal tubules; in silico analysis predicts R398W destabilizes EHD1 structure and impairs nucleotide binding, likely reducing EHD1 oligomerization and membrane remodeling.\",\n      \"method\": \"Genetic mapping, patient clinical analysis, Ehd1 KO and knockin mouse models, zebrafish model, proximal tubule endocytosis assays, in silico structural analysis\",\n      \"journal\": \"Journal of the American Society of Nephrology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — human genetics confirmed by multiple animal models with functional endocytosis readout and structural prediction\",\n      \"pmids\": [\"35149593\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Coronin2A (CORO2A) is a novel EHD1 interaction partner; CORO2A depletion causes enlarged endosomes and impaired recycling of clathrin-independent cargo, consistent with a role in EHD1-dependent endosomal fission.\",\n      \"method\": \"Co-immunoprecipitation, siRNA depletion, endosome size quantification, cargo recycling assays, actin protrusion analysis\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — Co-IP confirmed interaction with functional siRNA phenotype, single lab\",\n      \"pmids\": [\"35921168\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"EHD1 regulates CP110 ubiquitination during ciliogenesis by transporting centriolar satellites containing the E3 ubiquitin ligase HERC2 to the mother centriole; HERC2 and MIB1 both interact with and ubiquitinate CP110; HERC2 localizes to centriolar satellites and is required for ciliogenesis.\",\n      \"method\": \"siRNA depletion, co-immunoprecipitation, ubiquitination assays, centriolar satellite tracking, immunofluorescence\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — mechanistic dissection with Co-IP, ubiquitination assays, and centriolar satellite transport analysis, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"37074924\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"EHD1 is required for anterograde Golgi-to-plasma membrane traffic and endocytic recycling of IGF-1R in Ewing sarcoma; EHD1 overexpression-dependent oncogenic traits require IGF-1R expression and kinase activity; shRNA/CRISPR KO of EHD1 reduces tumorigenesis and metastasis.\",\n      \"method\": \"shRNA knockdown, CRISPR knockout with mouse Ehd1 rescue, RTK antibody arrays, surface IGF-1R quantification, tumor xenograft and metastasis assays\",\n      \"journal\": \"Communications biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — CRISPR KO with rescue, receptor trafficking assays, and in vivo tumor models\",\n      \"pmids\": [\"37474760\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"The EHD1 p.R398W missense mutation causes male infertility in knockin mice with defective acrosomal development, missing sperm tail midpieces, and disorganized spermatogenic epithelium; wild-type EHD1 co-localizes with acrosomal granules and the retromer component VPS35, but the p.R398W mutant does not co-localize with VPS35.\",\n      \"method\": \"Knockin mouse model, histopathology, immunofluorescence co-localization (WT vs. R398W with VPS35 and acrosomal markers), electron microscopy\",\n      \"journal\": \"Frontiers in cell and developmental biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — knockin mouse model with ultrastructural analysis and mechanistically informative differential co-localization of WT vs. mutant protein\",\n      \"pmids\": [\"37900275\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"EHD1 interacts with PD-L1 protein and promotes PD-L1 endocytic recycling; EHD1 knockdown inhibits PD-L1 recycling and promotes its lysosomal degradation, enhancing T cell cytotoxicity; EHD1 mRNA is m6A-modified and YTHDF1 binds EHD1 mRNA to stabilize it in an m6A-dependent manner.\",\n      \"method\": \"Molecular docking, co-immunoprecipitation, immunofluorescence, receptor internalization/recycling assays, MeRIP-qPCR, RNA immunoprecipitation, m6A-binding site mutation analysis, immunocompetent mouse tumor model\",\n      \"journal\": \"Cancer communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — multiple orthogonal methods for both protein interaction and mRNA modification, with functional in vivo tumor immune assay\",\n      \"pmids\": [\"40703029\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"EHD1 is an ATP-dependent dynamin-family ATPase that oligomerizes on phosphatidylserine-enriched endosomal membranes (recruited via its EH domain's phosphatidylinositol-binding activity and by NPF-motif-containing partners such as MICAL-L1, Rabenosyn-5, and Syndapin2/EHD4) to catalyze membrane fission and vesicle release from tubular recycling endosomes, thereby driving receptor recycling to the plasma membrane across diverse cargo including transferrin receptor, MHC-I, integrins, GLUT4, EGFR, and IGF-1R; it additionally facilitates ciliogenesis by tubulating preciliary membranes and delivering the E3 ligase HERC2 to the mother centriole to ubiquitinate CP110, and interacts with Smoothened, Cx43, and other specialized cargoes to coordinate context-specific membrane trafficking events in development, immunity, and homeostasis.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"EHD1 is an ATP-dependent membrane fission enzyme that oligomerizes on phosphatidylserine-enriched endosomal tubules to catalyze vesicle scission, thereby controlling exit of diverse cargo—including transferrin receptor, MHC-I, integrins, GLUT4, EGFR, IGF-1R, and PD-L1—from recycling endosomes back to the plasma membrane [PMID:30518883, PMID:16445686, PMID:12032069, PMID:40703029]. Its C-terminal EH domain recruits EHD1 to membranes through dual recognition of NPF-motif partners (MICAL-L1, Rabenosyn-5, Syndapin2) and direct phosphatidylinositol binding, while the N-terminal P-loop ATPase domain drives scaffold assembly and membrane thinning that leads to fission of tubules below ~25 nm radius [PMID:17412695, PMID:19864458, PMID:30518883, PMID:15020713]. Beyond endocytic recycling, EHD1 functions in ciliogenesis by tubulating preciliary membranes at distal appendage vesicles and delivering the E3 ligase HERC2 to the mother centriole to ubiquitinate CP110, and its loss in mice causes neural tube defects with deregulated Hedgehog signaling [PMID:25686250, PMID:37074924, PMID:26884322]. A homozygous missense mutation (p.R398W) in EHD1 causes autosomal recessive tubular proteinuria, sensorineural deafness, and male infertility in humans and mouse models [PMID:35149593, PMID:37900275].\",\n  \"teleology\": [\n    {\n      \"year\": 1999,\n      \"claim\": \"Identification of EHD1 as an EH-domain protein on endocytic vesicles established the gene's entry into endosomal trafficking biology and revealed its domain architecture (P-loop, coiled-coil, EH domain).\",\n      \"evidence\": \"GFP-fusion live imaging and Northern analysis in cultured cells\",\n      \"pmids\": [\"10395801\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No function assigned beyond localization\", \"ATPase activity of the P-loop not tested\", \"No interacting partners identified\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Demonstration that EHD1 associates with Arf6-containing tubules and is required for clathrin-independent recycling of MHC-I established EHD1 as a functional regulator of endosomal membrane tubulation and receptor recycling, with both the P-loop and EH domain being essential.\",\n      \"evidence\": \"Domain mutagenesis, MHC-I recycling assays, and dominant-negative Arf6 experiments in HeLa cells\",\n      \"pmids\": [\"12032069\", \"12121420\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of tubule formation (motor vs. scaffold) unknown\", \"ATPase catalytic cycle not characterized\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Mapping of the EH domain as a hub that binds NPF-motif partners (Rabenosyn-5, EHBP1) and epistasis experiments positioned EHD1 downstream of Rab4/Rab5-dependent sorting and implicated it in recycling of diverse cargo including GLUT4.\",\n      \"evidence\": \"GST pulldown with mass spectrometry, domain mapping, sequential RNAi epistasis, insulin-stimulated GLUT4 recycling assays\",\n      \"pmids\": [\"15020713\", \"15247266\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct membrane-binding mechanism of EHD1 not resolved\", \"Whether EHD1 performs fission or stabilizes tubules unclear\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"EHD1 knockout mice confirmed the in vivo requirement for EHD1 in exit of cargo from recycling endosomes, validating cell-based findings genetically.\",\n      \"evidence\": \"Transferrin recycling assays in EHD1 knockout mouse embryonic fibroblasts\",\n      \"pmids\": [\"16445686\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Organismal phenotypes beyond MEF recycling not yet characterized\", \"Redundancy with other EHD family members not addressed\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Multiple studies expanded EHD1's cargo repertoire to β1-integrins and cholesterol/LDL, linked EHD1 to retromer-dependent endosome-to-Golgi retrieval, and solved the EH domain NMR structure revealing a phosphoinositide-binding surface distinct from the NPF pocket.\",\n      \"evidence\": \"EHD1 KO MEFs with integrin recycling and cholesterol assays; comparative proteomics identifying retromer interaction; NMR structure and PI-binding mutagenesis (K483)\",\n      \"pmids\": [\"17284518\", \"17451652\", \"17868075\", \"17412695\", \"17899392\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full-length EHD1 structure not determined\", \"Mechanism of membrane scission vs. stabilization still unresolved\", \"Relative contribution of PI binding vs. NPF-partner binding to membrane recruitment unknown\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"MICAL-L1 was identified as the key adaptor that recruits EHD1 (and Rab8a) to tubular recycling endosomes, and reconstitution of an amphiphysin–EHD1 (AMPH-1–RME-1) complex showed it generates morphologically distinct coated membrane tubules, providing the first in vitro evidence of cooperative membrane remodeling.\",\n      \"evidence\": \"Purified-protein membrane tubulation assay (C. elegans proteins), C. elegans genetics, human BIN1 knockdown; MICAL-L1 Co-IP and siRNA with recycling assays\",\n      \"pmids\": [\"19915558\", \"19864458\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Reconstitution with human EHD1 not yet performed\", \"Scission activity not demonstrated in this system\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"EHD1 knockout revealed essential roles in spermatogenesis (acrosome development) and showed that EHD1 and EHD4 form endogenous heteromers whose balance regulates neuronal L1/NgCAM endocytosis, broadening EHD1's physiological roles beyond generic recycling.\",\n      \"evidence\": \"EHD1 KO mouse histopathology and EM for spermatogenesis; shRNA and oligomerization mutants for neuronal trafficking\",\n      \"pmids\": [\"20359371\", \"20463227\", \"21147988\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of EHD1 in acrosome biogenesis not defined at molecular level\", \"Stoichiometry of EHD1–EHD4 complexes unknown\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"EHD1 was shown to function in ciliogenesis—tubulating preciliary membranes at distal appendage vesicles and enabling ciliary vesicle formation—and its membrane recruitment was found to depend on phosphatidylserine flipping by the P4-ATPase ATP8A1, establishing PS asymmetry as a prerequisite for EHD1 engagement.\",\n      \"evidence\": \"siRNA, EM of centriole structures, transition zone protein localization for ciliogenesis; ATP8A1 depletion with PS distribution and EHD1 localization assays\",\n      \"pmids\": [\"25686250\", \"25595798\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How EHD1 senses PS enrichment at the molecular level unclear\", \"Whether ATP hydrolysis is required for ciliogenesis-specific function not tested\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"EHD1 knockout on a C57BL/6 background caused embryonic lethality with neural tube defects, short cilia, and deregulated Hedgehog signaling, establishing EHD1 as essential for developmental Hedgehog pathway output and identifying Smoothened as a direct binding partner.\",\n      \"evidence\": \"EHD1 KO mouse, immunohistochemistry, Co-IP for Smoothened, ciliary length measurement, GLI3 repressor quantification\",\n      \"pmids\": [\"26884322\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which EHD1 regulates Smoothened ciliary entry not defined\", \"Tissue-specific versus universal requirement for EHD1 in Hedgehog signaling not resolved\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"In vitro reconstitution on supported membrane tubes demonstrated that EHD1 is a bona fide ATP-dependent membrane fission enzyme: ATP-bound EHD1 scaffolds thin membranes and ATP hydrolysis drives self-assembly leading to scission of tubes below 25 nm radius, directly resolving the long-standing question of whether EHD1 stabilizes or severs tubules.\",\n      \"evidence\": \"Supported membrane tube assay, molecular dynamics simulation, N-terminal deletion mutagenesis, C. elegans cross-complementation\",\n      \"pmids\": [\"30518883\", \"30403133\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full atomic structure of the membrane-bound oligomer lacking\", \"Regulation of ATPase cycle by partners (MICAL-L1, GRAF1) not reconstituted\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"A human founder mutation p.R398W in EHD1 was identified as the cause of autosomal recessive tubular proteinuria and sensorineural deafness, validated in KO and knockin mice showing impaired proximal tubule endocytosis and defective spermatogenesis with loss of VPS35/retromer co-localization.\",\n      \"evidence\": \"Patient genetics, Ehd1 KO and R398W knockin mice, zebrafish model, proximal tubule endocytosis assays, VPS35 co-localization\",\n      \"pmids\": [\"35149593\", \"37900275\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of R398W destabilization only modeled in silico\", \"Whether hearing loss is due to ciliary or recycling defect not determined\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"The ciliogenesis mechanism was further refined: EHD1 transports HERC2-containing centriolar satellites to the mother centriole to ubiquitinate CP110, providing a molecular link between EHD1's membrane trafficking function and centriole remodeling during cilia initiation.\",\n      \"evidence\": \"siRNA, Co-IP, ubiquitination assays, centriolar satellite tracking\",\n      \"pmids\": [\"37074924\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether HERC2 transport is dependent on EHD1 ATPase activity not tested\", \"Interplay between HERC2 and MIB1 at the centriole not fully resolved\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"EHD1 was shown to promote PD-L1 recycling to the cell surface; its depletion diverted PD-L1 to lysosomal degradation and enhanced anti-tumor T cell cytotoxicity, extending EHD1's cargo repertoire to immune checkpoint regulation.\",\n      \"evidence\": \"Co-IP, PD-L1 internalization/recycling assays, MeRIP-qPCR for m6A modification, immunocompetent mouse tumor model\",\n      \"pmids\": [\"40703029\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct structural basis of EHD1–PD-L1 interaction not defined\", \"Generalizability across tumor types not established\", \"m6A regulation of EHD1 mRNA awaits independent confirmation\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key open questions include the high-resolution structure of the full-length membrane-bound EHD1 oligomer, the regulatory logic by which distinct NPF-motif partners select cargo-specific fission events, and the mechanism by which EHD1 ATPase activity is coordinated with partner proteins (GRAF1, MICAL-L1, BIN1) during scission.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No high-resolution structure of full-length EHD1 on membranes\", \"How cargo specificity is encoded by different NPF adaptors is undefined\", \"ATPase regulation by partner proteins not reconstituted in vitro\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140657\", \"supporting_discovery_ids\": [33, 34]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [33, 34]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [8, 31]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [0, 2, 4, 7, 16, 31, 37]},\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [0, 2, 5, 16, 30]},\n      {\"term_id\": \"GO:0005929\", \"supporting_discovery_ids\": [30, 32, 35]},\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [32, 35, 42]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [26, 36]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [2, 4, 7, 10, 15, 16, 33, 34]},\n      {\"term_id\": \"R-HSA-9609507\", \"supporting_discovery_ids\": [5, 39, 43, 45]},\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [30, 32, 35, 42]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [1, 32, 43]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [18, 32, 44]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"MICAL-L1\",\n      \"Rabenosyn-5\",\n      \"SNAP29\",\n      \"EHD3\",\n      \"EHD4\",\n      \"EHBP1\",\n      \"BIN1\",\n      \"SNX17\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}