{"gene":"EHD4","run_date":"2026-04-28T17:46:03","timeline":{"discoveries":[{"year":2000,"finding":"EHD4 was identified as a novel member of the EHD protein family, encoding a protein with a nucleotide-binding consensus site at the N-terminus, a bipartite nuclear localization signal, and a C-terminal EH domain with an EF-hand motif, suggesting roles in endocytosis and signaling.","method":"cDNA library screening, sequence alignment, radiation hybrid mapping","journal":"Genomics","confidence":"Medium","confidence_rationale":"Tier 3 — structural/domain characterization from sequence, no functional assay; foundational identification paper","pmids":["10673336"],"is_preprint":false},{"year":2001,"finding":"EHD4 was identified as an extracellular matrix protein that interacts with type VI collagen assembly components; it is secreted by fibroblasts into extracellular filamentous networks and forms oligomers (~220 and 158 kDa under non-denaturing conditions, ~56 kDa reduced), making it the first EH domain-containing extracellular matrix protein described.","method":"Yeast two-hybrid screen, immunofluorescence staining of developing limbs and fibroblast cultures, non-denaturing extraction and SDS-PAGE","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 3 — localization and biochemical characterization; single lab, multiple methods","pmids":["11533061"],"is_preprint":false},{"year":2008,"finding":"Endogenous EHD4 localizes to Rab5- and EEA1-containing early endosomes and Arf6-positive endosomes; knockdown of EHD4 causes enlargement of early endosomes with accumulation of internalized transferrin, MHC class I molecules, and LDL, and leads to elevated GTP-bound (active) Rab5, establishing EHD4 as a regulator of cargo exit from early endosomes toward both the recycling compartment and the late endocytic pathway.","method":"Peptide antibody localization, siRNA/shRNA knockdown, immunofluorescence, endocytic cargo uptake assays, Rab5-GTP pulldown","journal":"Traffic (Copenhagen, Denmark)","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (KD, cargo assays, active Rab5 measurement) in single lab; moderate evidence","pmids":["18331452"],"is_preprint":false},{"year":2008,"finding":"Endogenous EHD4 and EHD1 interact (co-immunoprecipitate) in cells, suggesting they cooperate in regulating transport along the early endosome to endocytic recycling compartment axis.","method":"Co-immunoprecipitation of endogenous proteins","journal":"Traffic (Copenhagen, Denmark)","confidence":"Medium","confidence_rationale":"Tier 3 — single Co-IP with endogenous proteins, single lab","pmids":["18331452"],"is_preprint":false},{"year":2009,"finding":"EHD4 interacts with cadherin 23 (CDH23) in cochlear hair cells; EHD4 co-localizes and co-immunoprecipitates with CDH23 in mammalian cells, and this interaction is calcium-sensitive, suggesting EHD4 regulates CDH23 trafficking/localization.","method":"Yeast two-hybrid screen, in situ hybridization, co-immunoprecipitation, immunofluorescence colocalization, EHD4 knockout mouse phenotyping","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2–3 — reciprocal co-IP with calcium sensitivity validated, single lab","pmids":["19487694"],"is_preprint":false},{"year":2010,"finding":"EHD4 overexpression in neurons (but not fibroblasts) impairs L1/NgCAM endocytosis; balanced levels of EHD1 and EHD4 are required for NgCAM endocytosis, and EHD1–EHD4 hetero-oligomerization is required for this effect, as simultaneous expression of EHD1 and EHD4 rescues NgCAM endocytosis.","method":"shRNA knockdown, overexpression, live-cell endocytosis assays in neurons and fibroblasts, oligomerization mutant analysis","journal":"The Journal of neuroscience","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal approaches (KD, OE, mutant rescue, cell-type comparison) in single lab with defined phenotypic readout","pmids":["20463227"],"is_preprint":false},{"year":2010,"finding":"EHD4 knockout male mice show reduced prepubertal testis size with increased germ cell apoptosis and proliferation defects; EHD4 is highly expressed in primary spermatocytes, and its deletion alters expression levels of other EHD proteins in an age-dependent manner, with EHD1 upregulation compensating in adult testis.","method":"Conditional knockout mouse generation, Western blotting, histology, sperm count, compound action potential measurement","journal":"Genesis (New York, N.Y. : 2000)","confidence":"Medium","confidence_rationale":"Tier 2 — clean KO with defined cellular phenotype; single lab","pmids":["20213691"],"is_preprint":false},{"year":2011,"finding":"Combined deletion of EHD3 and EHD4 in mice causes renal thrombotic microangiopathy with altered glomerular endothelial VEGFR2 expression and localization and increased apoptosis, establishing that EHD-mediated endocytic trafficking of VEGFR2 is essential for glomerular endothelial function.","method":"Double knockout mouse generation (Ehd3-/-; Ehd4-/-), histopathology, immunostaining, electron microscopy, proteinuria measurement","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 — double KO with defined phenotype and mechanistic link to VEGFR2 trafficking; single lab","pmids":["21408024"],"is_preprint":false},{"year":2013,"finding":"EHD4 is enriched in HIV-1 nef-deleted virions compared to wild-type virions, and simultaneous depletion of EHD4 and Ezrin in virus-producing cells reduces Nef-mediated increase in virus infectivity by ~70%, identifying EHD4 as a cofactor required by Nef to enhance viral infectivity.","method":"DiGE and iTRAQ proteomics of HIV-1 particles, siRNA knockdown, infectivity assays","journal":"Journal of virology","confidence":"Medium","confidence_rationale":"Tier 2–3 — proteomic identification plus functional KD validation; single lab","pmids":["23325686"],"is_preprint":false},{"year":2017,"finding":"EHD4 knockout mice on a C57Bl/6 background exhibit increased urine volume and reduced urine osmolality; apical membrane localization of aquaporin 2 (AQP2) and phospho-AQP2 in inner medullary collecting duct principal cells is reduced (~20%) in EHD4-KO mice, establishing EHD4 as a regulator of AQP2 trafficking and urinary water homeostasis.","method":"EHD4 knockout mice, urine volume/osmolality measurement, immunostaining of AQP2 subcellular localization, Western blotting","journal":"FASEB journal","confidence":"Medium","confidence_rationale":"Tier 2 — clean KO with functional phenotype and localization evidence; single lab","pmids":["28778975"],"is_preprint":false},{"year":2020,"finding":"EHD4 preferentially dimerizes with EHD1 and is required for recruitment of EHD1 to sorting endosomes (SE); knockdown or CRISPR/Cas9 deletion of EHD4 impairs EHD1 SE-recruitment and causes enlarged SE, establishing EHD4 as an essential component of the EHD1-mediated endosomal fission machinery.","method":"siRNA, shRNA, CRISPR/Cas9 knockout, co-immunoprecipitation, immunofluorescence of EHD1 endosomal recruitment, endosome size quantification","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 2 — three independent KD/KO approaches plus dimerization and localization assays; moderate-to-strong evidence in single lab","pmids":["32966336"],"is_preprint":false},{"year":2020,"finding":"Phostensin (PTS) associates with EHD4 (and EHD1) as demonstrated by co-immunoprecipitation and GST pull-down; the complex co-localizes at endocytic vesicles, and PTS overexpression attenuates transferrin endocytic trafficking.","method":"Co-immunoprecipitation, GST pull-down, immunofluorescence colocalization, transferrin trafficking assay","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 3 — co-IP and pulldown with functional trafficking assay; single lab","pmids":["32800345"],"is_preprint":false},{"year":2021,"finding":"EHD4 is recruited by PACSIN2 to the rear end of asymmetric adherens junctions between leader and follower endothelial cells, where it forms a PACSIN2/EHD4/MICAL-L1 complex that generates recycling endosome-like tubular structures, controls local VE-cadherin trafficking, and coordinates polarized endothelial migration and angiogenic sprouting.","method":"Co-immunoprecipitation, live imaging, siRNA knockdown, endothelial migration and sprouting assays, immunofluorescence of junction-localized complex","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 — reciprocal co-IP, live imaging, KD with defined phenotype (collective migration, angiogenesis) and cargo trafficking readout; moderate evidence","pmids":["33972531"],"is_preprint":false},{"year":2022,"finding":"EHD4, but not EHD2, is required for primary ciliogenesis; conserved residues P446 and E470 in the EH domain of EHD1 (also found in EHD3 and EHD4, but different in EHD2) are necessary for EHD1's ability to rescue ciliogenesis, and EHD1 ATP-binding is required for ciliogenesis.","method":"siRNA knockdown of EHD4, ciliogenesis rescue assays with EHD1 EH-domain point mutants, CRISPR/Cas9, immunofluorescence of cilia","journal":"Traffic (Copenhagen, Denmark)","confidence":"Medium","confidence_rationale":"Tier 2 — KD with defined phenotype and mutagenesis of key residues; single lab","pmids":["35510564"],"is_preprint":false},{"year":2024,"finding":"EHD4 possesses liposome-stimulated ATPase activity (dynamin-related); a high-throughput Malachite green assay identified small molecule inhibitors of EHD4's liposome-stimulated ATPase, confirming its enzymatic activity is druggable.","method":"Malachite green-based ATPase assay with liposomes, high-throughput screening, structure-activity relationship (SAR) analysis","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 1 — in vitro enzymatic assay with defined substrate; single lab, single study","pmids":["39074100"],"is_preprint":false},{"year":2025,"finding":"EHD4 is a negative regulator of claudin-5 (CLDN-5) expression in neural endothelial cells; CRISPR/Cas9 suppression of EHD4 leads to significant upregulation of CLDN-5 protein on the cell surface, and EHD4 appears to regulate transcriptional activity of CLDN5.","method":"Genome-wide CRISPR/Cas9 cell-sorting-based phenotypic screen, CLDN-5 surface expression measurement, transcriptional reporter assays","journal":"The FEBS journal","confidence":"Medium","confidence_rationale":"Tier 2 — genome-wide CRISPR screen with defined phenotype and mechanistic follow-up; single lab","pmids":["41361961"],"is_preprint":false},{"year":2025,"finding":"Phostensin (PTS) binds EHD4 (and EHD1) through a novel non-NPF motif, 64ILV(X)4(L/V)RL74S (residues 51–80 of PTS-α); mutation of this motif reduces binding to EHD4 and disrupts PTS-β-mediated attenuation of transferrin endocytic recycling, indicating PTS regulates endocytic recycling via association with EHD4/EHD1.","method":"GST pull-down, far western blotting, point mutagenesis, transferrin trafficking assay","journal":"Journal of biochemistry","confidence":"Medium","confidence_rationale":"Tier 1–2 — in vitro binding assay with mutagenesis and functional trafficking readout; single lab","pmids":["39776131"],"is_preprint":false}],"current_model":"EHD4 is a dynamin-related ATPase that localizes to Rab5/EEA1-positive early endosomes and, by preferentially heterodimerizing with EHD1, recruits EHD1 to sorting endosomes to catalyze vesicular fission and cargo exit toward recycling compartments and the late endocytic pathway; it additionally forms a junctional PACSIN2/EHD4/MICAL-L1 complex that controls VE-cadherin trafficking during collective endothelial migration, regulates AQP2 apical localization in renal collecting duct cells, interacts with CDH23 in a calcium-sensitive manner in cochlear hair cells, and negatively regulates claudin-5 transcription in neural endothelial cells."},"narrative":{"teleology":[{"year":2000,"claim":"Identification of EHD4 as a new member of the EHD protein family with a nucleotide-binding domain and C-terminal EH domain established it as a candidate endocytic regulator, but left its function undefined.","evidence":"cDNA library screening, sequence alignment, and radiation hybrid mapping","pmids":["10673336"],"confidence":"Medium","gaps":["No functional assay performed","Subcellular localization not determined experimentally","Enzymatic activity not tested"]},{"year":2008,"claim":"Demonstration that EHD4 resides on Rab5/EEA1-positive early endosomes and that its depletion causes endosomal enlargement with cargo accumulation and elevated Rab5-GTP established EHD4 as a regulator of cargo exit from early endosomes, answering where and how it acts in the endocytic pathway.","evidence":"siRNA/shRNA knockdown, immunofluorescence colocalization, transferrin/MHC-I/LDL uptake assays, and Rab5-GTP pulldown in cultured cells","pmids":["18331452"],"confidence":"High","gaps":["Mechanism by which EHD4 regulates Rab5-GTP levels unknown","Whether EHD4 acts through membrane fission or a scaffolding role was unresolved","Endogenous EHD4–EHD1 interaction shown by single co-IP without reciprocal validation"]},{"year":2009,"claim":"Discovery that EHD4 interacts with cadherin 23 in cochlear hair cells in a calcium-sensitive manner extended EHD4 function to specialized sensory cell trafficking, though the mechanistic consequence for hearing remained unclear.","evidence":"Yeast two-hybrid, co-immunoprecipitation, immunofluorescence in cochlear tissue, EHD4 knockout mouse analysis","pmids":["19487694"],"confidence":"Medium","gaps":["No auditory phenotype quantified in EHD4 KO mice","Whether EHD4 controls CDH23 recycling or degradation not determined","Calcium-sensitivity mechanism not structurally resolved"]},{"year":2010,"claim":"Showing that balanced EHD1–EHD4 hetero-oligomerization is required for neuronal NgCAM endocytosis revealed that EHD4 does not act alone but functions through stoichiometric partnership with EHD1, and that this requirement is cell-type-specific.","evidence":"shRNA knockdown, overexpression, oligomerization mutant rescue, live-cell endocytosis assays in neurons vs. fibroblasts","pmids":["20463227"],"confidence":"High","gaps":["Structural basis of EHD1–EHD4 hetero-oligomerization not determined","Whether other EHD family members can substitute in neurons unknown"]},{"year":2010,"claim":"EHD4 knockout mice revealed a developmental role in spermatogenesis, with increased germ cell apoptosis and compensatory EHD1 upregulation, establishing in vivo redundancy among EHD family members.","evidence":"EHD4 knockout mice, histology, Western blotting, sperm counts","pmids":["20213691"],"confidence":"Medium","gaps":["Specific trafficking cargo in spermatocytes not identified","Mechanism of EHD1 compensatory upregulation unknown"]},{"year":2011,"claim":"Combined EHD3/EHD4 deletion caused renal thrombotic microangiopathy with mislocalized VEGFR2, linking EHD4-dependent endocytic trafficking to receptor tyrosine kinase homeostasis in glomerular endothelia.","evidence":"Double knockout mice (Ehd3−/−; Ehd4−/−), histopathology, immunostaining, electron microscopy","pmids":["21408024"],"confidence":"Medium","gaps":["Individual contributions of EHD3 vs. EHD4 to VEGFR2 trafficking not separated","Whether the phenotype reflects impaired VEGFR2 recycling or degradation unclear"]},{"year":2017,"claim":"EHD4 knockout mice exhibited a urinary concentrating defect with reduced apical AQP2 in collecting duct cells, establishing an in vivo role for EHD4 in water channel trafficking and renal water homeostasis.","evidence":"EHD4 KO mice on C57Bl/6 background, urine volume/osmolality measurement, AQP2 immunostaining","pmids":["28778975"],"confidence":"Medium","gaps":["Whether EHD4 directly controls AQP2 vesicle fusion or upstream sorting is unknown","Vasopressin-stimulated recycling pathway involvement not tested"]},{"year":2020,"claim":"Demonstration that EHD4 preferentially heterodimerizes with EHD1 and is required for EHD1 recruitment to sorting endosomes resolved the mechanistic hierarchy: EHD4 acts upstream as a recruiter of the EHD1 fission machinery.","evidence":"siRNA, shRNA, CRISPR/Cas9 knockout, co-immunoprecipitation, EHD1 endosomal recruitment quantification, endosome size analysis","pmids":["32966336"],"confidence":"High","gaps":["Structural basis of preferential EHD4–EHD1 dimerization not resolved","Whether EHD4 itself possesses membrane fission activity independent of EHD1 unknown"]},{"year":2020,"claim":"Identification of Phostensin (PTS) as an EHD4 interactor that modulates transferrin recycling when overexpressed expanded the network of EHD4 regulatory partners beyond EHD family members.","evidence":"Co-immunoprecipitation, GST pull-down, transferrin trafficking assay","pmids":["32800345"],"confidence":"Medium","gaps":["Endogenous stoichiometry of PTS–EHD4 complex not assessed","Functional consequence of PTS loss on EHD4-dependent trafficking not tested"]},{"year":2021,"claim":"Discovery of a PACSIN2/EHD4/MICAL-L1 junctional complex that generates recycling tubules and controls VE-cadherin trafficking during collective endothelial migration established EHD4 as a spatially targeted endocytic regulator at cell–cell junctions.","evidence":"Co-immunoprecipitation, live imaging of tubular structures, siRNA knockdown, endothelial migration and angiogenic sprouting assays","pmids":["33972531"],"confidence":"High","gaps":["Whether EHD4 ATPase activity is required at junctions not tested","Role in angiogenesis in vivo not confirmed"]},{"year":2022,"claim":"Showing that EHD4 knockdown impairs primary ciliogenesis and that conserved EH-domain residues shared by EHD1/3/4 are required for cilium formation extended EHD4 function to organelle biogenesis beyond endosomal sorting.","evidence":"siRNA knockdown of EHD4, rescue with EHD1 EH-domain point mutants, immunofluorescence of cilia","pmids":["35510564"],"confidence":"Medium","gaps":["Direct ciliogenesis cargo trafficked by EHD4 not identified","Whether EHD4 acts at the ciliary base or at upstream endosomes is unresolved"]},{"year":2024,"claim":"Biochemical demonstration that EHD4 possesses liposome-stimulated ATPase activity and identification of small-molecule inhibitors confirmed EHD4 as an active dynamin-related enzyme and validated it as a druggable target.","evidence":"Malachite green ATPase assay with liposomes, high-throughput screening, SAR analysis","pmids":["39074100"],"confidence":"Medium","gaps":["No structural data for EHD4 ATPase domain","In-cell validation of identified inhibitors not reported"]},{"year":2025,"claim":"Mapping of PTS binding to a non-canonical motif on EHD4 and discovery that EHD4 negatively regulates claudin-5 transcription in neural endothelial cells revealed a previously unrecognized transcriptional regulatory axis for EHD4.","evidence":"GST pull-down with point mutagenesis (PTS motif mapping); genome-wide CRISPR screen with CLDN-5 surface expression and transcriptional reporter assays","pmids":["39776131","41361961"],"confidence":"Medium","gaps":["Mechanism by which an endosomal ATPase controls transcription of CLDN5 is unknown","Whether EHD4 acts on a CLDN5 transcription factor through trafficking or a direct nuclear role is unresolved"]},{"year":null,"claim":"Key unresolved questions include the structural basis of EHD4–EHD1 preferential heterodimerization, whether EHD4 possesses intrinsic membrane fission activity independent of EHD1, and the mechanism by which EHD4 regulates claudin-5 transcription.","evidence":"","pmids":[],"confidence":"Low","gaps":["No high-resolution structure of EHD4 or EHD4–EHD1 heterodimer","Reconstituted membrane fission assays with EHD4 alone not performed","Transcriptional regulatory mechanism linking endosomal EHD4 to CLDN5 promoter activity undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140657","term_label":"ATP-dependent activity","supporting_discovery_ids":[14]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[14]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[14]}],"localization":[{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[2,10,11]},{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[2,12]},{"term_id":"GO:0005929","term_label":"cilium","supporting_discovery_ids":[13]}],"pathway":[{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[2,5,10,12]},{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[13]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[7]}],"complexes":["PACSIN2/EHD4/MICAL-L1 junctional complex"],"partners":["EHD1","PACSIN2","MICAL-L1","CDH23","PPP1R18","EHD3"],"other_free_text":[]},"mechanistic_narrative":"EHD4 is a dynamin-related ATPase that functions as a central regulator of endosomal membrane trafficking, controlling cargo exit from early/sorting endosomes toward recycling and late endocytic pathways. EHD4 localizes to Rab5/EEA1-positive early endosomes, where it preferentially heterodimerizes with EHD1 and recruits EHD1 to sorting endosomes to catalyze vesicular fission; loss of EHD4 causes endosomal enlargement, elevated Rab5-GTP, and accumulation of transferrin, MHC-I, and LDL [PMID:18331452, PMID:32966336]. Beyond canonical endosomal sorting, EHD4 participates in a junctional PACSIN2/EHD4/MICAL-L1 complex that generates tubular recycling structures to control VE-cadherin trafficking during polarized endothelial migration and angiogenic sprouting [PMID:33972531], regulates aquaporin-2 apical targeting in renal collecting duct cells [PMID:28778975], is required for primary ciliogenesis [PMID:35510564], and negatively regulates claudin-5 transcription in neural endothelial cells [PMID:41361961]. EHD4 possesses liposome-stimulated ATPase activity that is pharmacologically targetable [PMID:39074100]."},"prefetch_data":{"uniprot":{"accession":"Q9H223","full_name":"EH domain-containing protein 4","aliases":["Hepatocellular carcinoma-associated protein 10/11","PAST homolog 4"],"length_aa":541,"mass_kda":61.2,"function":"ATP- and membrane-binding protein that probably controls membrane reorganization/tubulation upon ATP hydrolysis. Plays a role in early endosomal transport (PubMed:17233914, PubMed:18331452). During sprouting angiogenesis, in complex with PACSIN2 and MICALL1, forms recycling endosome-like tubular structure at asymmetric adherens junctions to control CDH5 trafficking (By similarity)","subcellular_location":"Early endosome membrane; Recycling endosome membrane; Cell membrane; Cell junction, adherens junction","url":"https://www.uniprot.org/uniprotkb/Q9H223/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/EHD4","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/EHD4","total_profiled":1310},"omim":[{"mim_id":"605892","title":"EH DOMAIN-CONTAINING 4; EHD4","url":"https://www.omim.org/entry/605892"},{"mim_id":"605891","title":"EH DOMAIN-CONTAINING 3; EHD3","url":"https://www.omim.org/entry/605891"},{"mim_id":"605890","title":"EH DOMAIN-CONTAINING 2; EHD2","url":"https://www.omim.org/entry/605890"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Uncertain","locations":[{"location":"Plasma membrane","reliability":"Uncertain"},{"location":"Primary cilium","reliability":"Additional"},{"location":"Primary cilium transition zone","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/EHD4"},"hgnc":{"alias_symbol":[],"prev_symbol":["PAST4"]},"alphafold":{"accession":"Q9H223","domains":[{"cath_id":"1.10.268.20","chopping":"23-50_294-405","consensus_level":"high","plddt":93.333,"start":23,"end":405},{"cath_id":"3.40.50.300","chopping":"62-288","consensus_level":"medium","plddt":90.773,"start":62,"end":288},{"cath_id":"1.10.238.10","chopping":"431-527","consensus_level":"high","plddt":92.3828,"start":431,"end":527}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9H223","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9H223-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9H223-F1-predicted_aligned_error_v6.png","plddt_mean":87.75},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=EHD4","jax_strain_url":"https://www.jax.org/strain/search?query=EHD4"},"sequence":{"accession":"Q9H223","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9H223.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9H223/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9H223"}},"corpus_meta":[{"pmid":"23437005","id":"PMC_23437005","title":"Ehd4 encodes a novel and Oryza-genus-specific regulator of photoperiodic flowering in rice.","date":"2013","source":"PLoS genetics","url":"https://pubmed.ncbi.nlm.nih.gov/23437005","citation_count":146,"is_preprint":false},{"pmid":"18331452","id":"PMC_18331452","title":"A role for EHD4 in the regulation of early endosomal transport.","date":"2008","source":"Traffic (Copenhagen, Denmark)","url":"https://pubmed.ncbi.nlm.nih.gov/18331452","citation_count":93,"is_preprint":false},{"pmid":"10673336","id":"PMC_10673336","title":"EHD2, EHD3, and EHD4 encode novel members of a highly conserved family of EH domain-containing proteins.","date":"2000","source":"Genomics","url":"https://pubmed.ncbi.nlm.nih.gov/10673336","citation_count":80,"is_preprint":false},{"pmid":"21408024","id":"PMC_21408024","title":"Renal thrombotic microangiopathy in mice with combined deletion of endocytic recycling regulators EHD3 and EHD4.","date":"2011","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/21408024","citation_count":41,"is_preprint":false},{"pmid":"23325686","id":"PMC_23325686","title":"Comparative proteomic analysis of HIV-1 particles reveals a role for Ezrin and EHD4 in the Nef-dependent increase of virus infectivity.","date":"2013","source":"Journal of virology","url":"https://pubmed.ncbi.nlm.nih.gov/23325686","citation_count":36,"is_preprint":false},{"pmid":"20463227","id":"PMC_20463227","title":"Alterations of EHD1/EHD4 protein levels interfere with L1/NgCAM endocytosis in neurons and disrupt axonal targeting.","date":"2010","source":"The Journal of neuroscience : the official journal of the Society for Neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/20463227","citation_count":33,"is_preprint":false},{"pmid":"33972531","id":"PMC_33972531","title":"A junctional PACSIN2/EHD4/MICAL-L1 complex coordinates VE-cadherin trafficking for endothelial migration and angiogenesis.","date":"2021","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/33972531","citation_count":31,"is_preprint":false},{"pmid":"11533061","id":"PMC_11533061","title":"Characterization of EHD4, an EH domain-containing protein expressed in the extracellular matrix.","date":"2001","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/11533061","citation_count":22,"is_preprint":false},{"pmid":"20213691","id":"PMC_20213691","title":"Ehd4 is required to attain normal prepubertal testis size but dispensable for fertility in male mice.","date":"2010","source":"Genesis (New York, N.Y. : 2000)","url":"https://pubmed.ncbi.nlm.nih.gov/20213691","citation_count":21,"is_preprint":false},{"pmid":"19487694","id":"PMC_19487694","title":"EHD4 and CDH23 are interacting partners in cochlear hair cells.","date":"2009","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/19487694","citation_count":17,"is_preprint":false},{"pmid":"32966336","id":"PMC_32966336","title":"Eps15 Homology Domain Protein 4 (EHD4) is required for Eps15 Homology Domain Protein 1 (EHD1)-mediated endosomal recruitment and fission.","date":"2020","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/32966336","citation_count":15,"is_preprint":false},{"pmid":"29534991","id":"PMC_29534991","title":"Microtubular remodeling and decreased expression of Nav1.5 with enhanced EHD4 in cells from the infarcted heart.","date":"2018","source":"Life sciences","url":"https://pubmed.ncbi.nlm.nih.gov/29534991","citation_count":8,"is_preprint":false},{"pmid":"35510564","id":"PMC_35510564","title":"Differential requirements for the Eps15 homology domain proteins EHD4 and EHD2 in the regulation of mammalian ciliogenesis.","date":"2022","source":"Traffic (Copenhagen, Denmark)","url":"https://pubmed.ncbi.nlm.nih.gov/35510564","citation_count":6,"is_preprint":false},{"pmid":"28778975","id":"PMC_28778975","title":"EHD4 is a novel regulator of urinary water homeostasis.","date":"2017","source":"FASEB journal : official publication of the Federation of American Societies for Experimental Biology","url":"https://pubmed.ncbi.nlm.nih.gov/28778975","citation_count":3,"is_preprint":false},{"pmid":"32800345","id":"PMC_32800345","title":"Identification of phostensin in association with Eps 15 homology domain-containing protein 1 (EHD1) and EHD4.","date":"2020","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/32800345","citation_count":2,"is_preprint":false},{"pmid":"39074100","id":"PMC_39074100","title":"Identification of drug-like molecules targeting the ATPase activity of dynamin-like EHD4.","date":"2024","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/39074100","citation_count":2,"is_preprint":false},{"pmid":"39776131","id":"PMC_39776131","title":"Identification of a novel Eps 15 homology domain-containing protein 1 (EHD1) and EHD4-binding motif in phostensin.","date":"2025","source":"Journal of biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/39776131","citation_count":0,"is_preprint":false},{"pmid":"41361961","id":"PMC_41361961","title":"EHD4 and ASAP2 are critical negative regulators of the claudin-5-based endothelial barrier.","date":"2025","source":"The FEBS journal","url":"https://pubmed.ncbi.nlm.nih.gov/41361961","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":10623,"output_tokens":4043,"usd":0.046257},"stage2":{"model":"claude-opus-4-6","input_tokens":7495,"output_tokens":3505,"usd":0.18765},"total_usd":0.233907,"stage1_batch_id":"msgbatch_0118RScg3WGaGkaZRtWZbtce","stage2_batch_id":"msgbatch_01Cpfzvtbj42aqp5oYZ5Te2s","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2000,\n      \"finding\": \"EHD4 was identified as a novel member of the EHD protein family, encoding a protein with a nucleotide-binding consensus site at the N-terminus, a bipartite nuclear localization signal, and a C-terminal EH domain with an EF-hand motif, suggesting roles in endocytosis and signaling.\",\n      \"method\": \"cDNA library screening, sequence alignment, radiation hybrid mapping\",\n      \"journal\": \"Genomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — structural/domain characterization from sequence, no functional assay; foundational identification paper\",\n      \"pmids\": [\"10673336\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"EHD4 was identified as an extracellular matrix protein that interacts with type VI collagen assembly components; it is secreted by fibroblasts into extracellular filamentous networks and forms oligomers (~220 and 158 kDa under non-denaturing conditions, ~56 kDa reduced), making it the first EH domain-containing extracellular matrix protein described.\",\n      \"method\": \"Yeast two-hybrid screen, immunofluorescence staining of developing limbs and fibroblast cultures, non-denaturing extraction and SDS-PAGE\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — localization and biochemical characterization; single lab, multiple methods\",\n      \"pmids\": [\"11533061\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Endogenous EHD4 localizes to Rab5- and EEA1-containing early endosomes and Arf6-positive endosomes; knockdown of EHD4 causes enlargement of early endosomes with accumulation of internalized transferrin, MHC class I molecules, and LDL, and leads to elevated GTP-bound (active) Rab5, establishing EHD4 as a regulator of cargo exit from early endosomes toward both the recycling compartment and the late endocytic pathway.\",\n      \"method\": \"Peptide antibody localization, siRNA/shRNA knockdown, immunofluorescence, endocytic cargo uptake assays, Rab5-GTP pulldown\",\n      \"journal\": \"Traffic (Copenhagen, Denmark)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (KD, cargo assays, active Rab5 measurement) in single lab; moderate evidence\",\n      \"pmids\": [\"18331452\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Endogenous EHD4 and EHD1 interact (co-immunoprecipitate) in cells, suggesting they cooperate in regulating transport along the early endosome to endocytic recycling compartment axis.\",\n      \"method\": \"Co-immunoprecipitation of endogenous proteins\",\n      \"journal\": \"Traffic (Copenhagen, Denmark)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — single Co-IP with endogenous proteins, single lab\",\n      \"pmids\": [\"18331452\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"EHD4 interacts with cadherin 23 (CDH23) in cochlear hair cells; EHD4 co-localizes and co-immunoprecipitates with CDH23 in mammalian cells, and this interaction is calcium-sensitive, suggesting EHD4 regulates CDH23 trafficking/localization.\",\n      \"method\": \"Yeast two-hybrid screen, in situ hybridization, co-immunoprecipitation, immunofluorescence colocalization, EHD4 knockout mouse phenotyping\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — reciprocal co-IP with calcium sensitivity validated, single lab\",\n      \"pmids\": [\"19487694\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"EHD4 overexpression in neurons (but not fibroblasts) impairs L1/NgCAM endocytosis; balanced levels of EHD1 and EHD4 are required for NgCAM endocytosis, and EHD1–EHD4 hetero-oligomerization is required for this effect, as simultaneous expression of EHD1 and EHD4 rescues NgCAM endocytosis.\",\n      \"method\": \"shRNA knockdown, overexpression, live-cell endocytosis assays in neurons and fibroblasts, oligomerization mutant analysis\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal approaches (KD, OE, mutant rescue, cell-type comparison) in single lab with defined phenotypic readout\",\n      \"pmids\": [\"20463227\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"EHD4 knockout male mice show reduced prepubertal testis size with increased germ cell apoptosis and proliferation defects; EHD4 is highly expressed in primary spermatocytes, and its deletion alters expression levels of other EHD proteins in an age-dependent manner, with EHD1 upregulation compensating in adult testis.\",\n      \"method\": \"Conditional knockout mouse generation, Western blotting, histology, sperm count, compound action potential measurement\",\n      \"journal\": \"Genesis (New York, N.Y. : 2000)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — clean KO with defined cellular phenotype; single lab\",\n      \"pmids\": [\"20213691\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Combined deletion of EHD3 and EHD4 in mice causes renal thrombotic microangiopathy with altered glomerular endothelial VEGFR2 expression and localization and increased apoptosis, establishing that EHD-mediated endocytic trafficking of VEGFR2 is essential for glomerular endothelial function.\",\n      \"method\": \"Double knockout mouse generation (Ehd3-/-; Ehd4-/-), histopathology, immunostaining, electron microscopy, proteinuria measurement\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — double KO with defined phenotype and mechanistic link to VEGFR2 trafficking; single lab\",\n      \"pmids\": [\"21408024\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"EHD4 is enriched in HIV-1 nef-deleted virions compared to wild-type virions, and simultaneous depletion of EHD4 and Ezrin in virus-producing cells reduces Nef-mediated increase in virus infectivity by ~70%, identifying EHD4 as a cofactor required by Nef to enhance viral infectivity.\",\n      \"method\": \"DiGE and iTRAQ proteomics of HIV-1 particles, siRNA knockdown, infectivity assays\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — proteomic identification plus functional KD validation; single lab\",\n      \"pmids\": [\"23325686\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"EHD4 knockout mice on a C57Bl/6 background exhibit increased urine volume and reduced urine osmolality; apical membrane localization of aquaporin 2 (AQP2) and phospho-AQP2 in inner medullary collecting duct principal cells is reduced (~20%) in EHD4-KO mice, establishing EHD4 as a regulator of AQP2 trafficking and urinary water homeostasis.\",\n      \"method\": \"EHD4 knockout mice, urine volume/osmolality measurement, immunostaining of AQP2 subcellular localization, Western blotting\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — clean KO with functional phenotype and localization evidence; single lab\",\n      \"pmids\": [\"28778975\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"EHD4 preferentially dimerizes with EHD1 and is required for recruitment of EHD1 to sorting endosomes (SE); knockdown or CRISPR/Cas9 deletion of EHD4 impairs EHD1 SE-recruitment and causes enlarged SE, establishing EHD4 as an essential component of the EHD1-mediated endosomal fission machinery.\",\n      \"method\": \"siRNA, shRNA, CRISPR/Cas9 knockout, co-immunoprecipitation, immunofluorescence of EHD1 endosomal recruitment, endosome size quantification\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — three independent KD/KO approaches plus dimerization and localization assays; moderate-to-strong evidence in single lab\",\n      \"pmids\": [\"32966336\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Phostensin (PTS) associates with EHD4 (and EHD1) as demonstrated by co-immunoprecipitation and GST pull-down; the complex co-localizes at endocytic vesicles, and PTS overexpression attenuates transferrin endocytic trafficking.\",\n      \"method\": \"Co-immunoprecipitation, GST pull-down, immunofluorescence colocalization, transferrin trafficking assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — co-IP and pulldown with functional trafficking assay; single lab\",\n      \"pmids\": [\"32800345\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"EHD4 is recruited by PACSIN2 to the rear end of asymmetric adherens junctions between leader and follower endothelial cells, where it forms a PACSIN2/EHD4/MICAL-L1 complex that generates recycling endosome-like tubular structures, controls local VE-cadherin trafficking, and coordinates polarized endothelial migration and angiogenic sprouting.\",\n      \"method\": \"Co-immunoprecipitation, live imaging, siRNA knockdown, endothelial migration and sprouting assays, immunofluorescence of junction-localized complex\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal co-IP, live imaging, KD with defined phenotype (collective migration, angiogenesis) and cargo trafficking readout; moderate evidence\",\n      \"pmids\": [\"33972531\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"EHD4, but not EHD2, is required for primary ciliogenesis; conserved residues P446 and E470 in the EH domain of EHD1 (also found in EHD3 and EHD4, but different in EHD2) are necessary for EHD1's ability to rescue ciliogenesis, and EHD1 ATP-binding is required for ciliogenesis.\",\n      \"method\": \"siRNA knockdown of EHD4, ciliogenesis rescue assays with EHD1 EH-domain point mutants, CRISPR/Cas9, immunofluorescence of cilia\",\n      \"journal\": \"Traffic (Copenhagen, Denmark)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — KD with defined phenotype and mutagenesis of key residues; single lab\",\n      \"pmids\": [\"35510564\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"EHD4 possesses liposome-stimulated ATPase activity (dynamin-related); a high-throughput Malachite green assay identified small molecule inhibitors of EHD4's liposome-stimulated ATPase, confirming its enzymatic activity is druggable.\",\n      \"method\": \"Malachite green-based ATPase assay with liposomes, high-throughput screening, structure-activity relationship (SAR) analysis\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 — in vitro enzymatic assay with defined substrate; single lab, single study\",\n      \"pmids\": [\"39074100\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"EHD4 is a negative regulator of claudin-5 (CLDN-5) expression in neural endothelial cells; CRISPR/Cas9 suppression of EHD4 leads to significant upregulation of CLDN-5 protein on the cell surface, and EHD4 appears to regulate transcriptional activity of CLDN5.\",\n      \"method\": \"Genome-wide CRISPR/Cas9 cell-sorting-based phenotypic screen, CLDN-5 surface expression measurement, transcriptional reporter assays\",\n      \"journal\": \"The FEBS journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genome-wide CRISPR screen with defined phenotype and mechanistic follow-up; single lab\",\n      \"pmids\": [\"41361961\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Phostensin (PTS) binds EHD4 (and EHD1) through a novel non-NPF motif, 64ILV(X)4(L/V)RL74S (residues 51–80 of PTS-α); mutation of this motif reduces binding to EHD4 and disrupts PTS-β-mediated attenuation of transferrin endocytic recycling, indicating PTS regulates endocytic recycling via association with EHD4/EHD1.\",\n      \"method\": \"GST pull-down, far western blotting, point mutagenesis, transferrin trafficking assay\",\n      \"journal\": \"Journal of biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 — in vitro binding assay with mutagenesis and functional trafficking readout; single lab\",\n      \"pmids\": [\"39776131\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"EHD4 is a dynamin-related ATPase that localizes to Rab5/EEA1-positive early endosomes and, by preferentially heterodimerizing with EHD1, recruits EHD1 to sorting endosomes to catalyze vesicular fission and cargo exit toward recycling compartments and the late endocytic pathway; it additionally forms a junctional PACSIN2/EHD4/MICAL-L1 complex that controls VE-cadherin trafficking during collective endothelial migration, regulates AQP2 apical localization in renal collecting duct cells, interacts with CDH23 in a calcium-sensitive manner in cochlear hair cells, and negatively regulates claudin-5 transcription in neural endothelial cells.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"EHD4 is a dynamin-related ATPase that functions as a central regulator of endosomal membrane trafficking, controlling cargo exit from early/sorting endosomes toward recycling and late endocytic pathways. EHD4 localizes to Rab5/EEA1-positive early endosomes, where it preferentially heterodimerizes with EHD1 and recruits EHD1 to sorting endosomes to catalyze vesicular fission; loss of EHD4 causes endosomal enlargement, elevated Rab5-GTP, and accumulation of transferrin, MHC-I, and LDL [PMID:18331452, PMID:32966336]. Beyond canonical endosomal sorting, EHD4 participates in a junctional PACSIN2/EHD4/MICAL-L1 complex that generates tubular recycling structures to control VE-cadherin trafficking during polarized endothelial migration and angiogenic sprouting [PMID:33972531], regulates aquaporin-2 apical targeting in renal collecting duct cells [PMID:28778975], is required for primary ciliogenesis [PMID:35510564], and negatively regulates claudin-5 transcription in neural endothelial cells [PMID:41361961]. EHD4 possesses liposome-stimulated ATPase activity that is pharmacologically targetable [PMID:39074100].\",\n  \"teleology\": [\n    {\n      \"year\": 2000,\n      \"claim\": \"Identification of EHD4 as a new member of the EHD protein family with a nucleotide-binding domain and C-terminal EH domain established it as a candidate endocytic regulator, but left its function undefined.\",\n      \"evidence\": \"cDNA library screening, sequence alignment, and radiation hybrid mapping\",\n      \"pmids\": [\"10673336\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"No functional assay performed\",\n        \"Subcellular localization not determined experimentally\",\n        \"Enzymatic activity not tested\"\n      ]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Demonstration that EHD4 resides on Rab5/EEA1-positive early endosomes and that its depletion causes endosomal enlargement with cargo accumulation and elevated Rab5-GTP established EHD4 as a regulator of cargo exit from early endosomes, answering where and how it acts in the endocytic pathway.\",\n      \"evidence\": \"siRNA/shRNA knockdown, immunofluorescence colocalization, transferrin/MHC-I/LDL uptake assays, and Rab5-GTP pulldown in cultured cells\",\n      \"pmids\": [\"18331452\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Mechanism by which EHD4 regulates Rab5-GTP levels unknown\",\n        \"Whether EHD4 acts through membrane fission or a scaffolding role was unresolved\",\n        \"Endogenous EHD4–EHD1 interaction shown by single co-IP without reciprocal validation\"\n      ]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Discovery that EHD4 interacts with cadherin 23 in cochlear hair cells in a calcium-sensitive manner extended EHD4 function to specialized sensory cell trafficking, though the mechanistic consequence for hearing remained unclear.\",\n      \"evidence\": \"Yeast two-hybrid, co-immunoprecipitation, immunofluorescence in cochlear tissue, EHD4 knockout mouse analysis\",\n      \"pmids\": [\"19487694\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"No auditory phenotype quantified in EHD4 KO mice\",\n        \"Whether EHD4 controls CDH23 recycling or degradation not determined\",\n        \"Calcium-sensitivity mechanism not structurally resolved\"\n      ]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Showing that balanced EHD1–EHD4 hetero-oligomerization is required for neuronal NgCAM endocytosis revealed that EHD4 does not act alone but functions through stoichiometric partnership with EHD1, and that this requirement is cell-type-specific.\",\n      \"evidence\": \"shRNA knockdown, overexpression, oligomerization mutant rescue, live-cell endocytosis assays in neurons vs. fibroblasts\",\n      \"pmids\": [\"20463227\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Structural basis of EHD1–EHD4 hetero-oligomerization not determined\",\n        \"Whether other EHD family members can substitute in neurons unknown\"\n      ]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"EHD4 knockout mice revealed a developmental role in spermatogenesis, with increased germ cell apoptosis and compensatory EHD1 upregulation, establishing in vivo redundancy among EHD family members.\",\n      \"evidence\": \"EHD4 knockout mice, histology, Western blotting, sperm counts\",\n      \"pmids\": [\"20213691\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Specific trafficking cargo in spermatocytes not identified\",\n        \"Mechanism of EHD1 compensatory upregulation unknown\"\n      ]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Combined EHD3/EHD4 deletion caused renal thrombotic microangiopathy with mislocalized VEGFR2, linking EHD4-dependent endocytic trafficking to receptor tyrosine kinase homeostasis in glomerular endothelia.\",\n      \"evidence\": \"Double knockout mice (Ehd3−/−; Ehd4−/−), histopathology, immunostaining, electron microscopy\",\n      \"pmids\": [\"21408024\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Individual contributions of EHD3 vs. EHD4 to VEGFR2 trafficking not separated\",\n        \"Whether the phenotype reflects impaired VEGFR2 recycling or degradation unclear\"\n      ]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"EHD4 knockout mice exhibited a urinary concentrating defect with reduced apical AQP2 in collecting duct cells, establishing an in vivo role for EHD4 in water channel trafficking and renal water homeostasis.\",\n      \"evidence\": \"EHD4 KO mice on C57Bl/6 background, urine volume/osmolality measurement, AQP2 immunostaining\",\n      \"pmids\": [\"28778975\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Whether EHD4 directly controls AQP2 vesicle fusion or upstream sorting is unknown\",\n        \"Vasopressin-stimulated recycling pathway involvement not tested\"\n      ]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Demonstration that EHD4 preferentially heterodimerizes with EHD1 and is required for EHD1 recruitment to sorting endosomes resolved the mechanistic hierarchy: EHD4 acts upstream as a recruiter of the EHD1 fission machinery.\",\n      \"evidence\": \"siRNA, shRNA, CRISPR/Cas9 knockout, co-immunoprecipitation, EHD1 endosomal recruitment quantification, endosome size analysis\",\n      \"pmids\": [\"32966336\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Structural basis of preferential EHD4–EHD1 dimerization not resolved\",\n        \"Whether EHD4 itself possesses membrane fission activity independent of EHD1 unknown\"\n      ]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Identification of Phostensin (PTS) as an EHD4 interactor that modulates transferrin recycling when overexpressed expanded the network of EHD4 regulatory partners beyond EHD family members.\",\n      \"evidence\": \"Co-immunoprecipitation, GST pull-down, transferrin trafficking assay\",\n      \"pmids\": [\"32800345\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Endogenous stoichiometry of PTS–EHD4 complex not assessed\",\n        \"Functional consequence of PTS loss on EHD4-dependent trafficking not tested\"\n      ]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Discovery of a PACSIN2/EHD4/MICAL-L1 junctional complex that generates recycling tubules and controls VE-cadherin trafficking during collective endothelial migration established EHD4 as a spatially targeted endocytic regulator at cell–cell junctions.\",\n      \"evidence\": \"Co-immunoprecipitation, live imaging of tubular structures, siRNA knockdown, endothelial migration and angiogenic sprouting assays\",\n      \"pmids\": [\"33972531\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether EHD4 ATPase activity is required at junctions not tested\",\n        \"Role in angiogenesis in vivo not confirmed\"\n      ]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Showing that EHD4 knockdown impairs primary ciliogenesis and that conserved EH-domain residues shared by EHD1/3/4 are required for cilium formation extended EHD4 function to organelle biogenesis beyond endosomal sorting.\",\n      \"evidence\": \"siRNA knockdown of EHD4, rescue with EHD1 EH-domain point mutants, immunofluorescence of cilia\",\n      \"pmids\": [\"35510564\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Direct ciliogenesis cargo trafficked by EHD4 not identified\",\n        \"Whether EHD4 acts at the ciliary base or at upstream endosomes is unresolved\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Biochemical demonstration that EHD4 possesses liposome-stimulated ATPase activity and identification of small-molecule inhibitors confirmed EHD4 as an active dynamin-related enzyme and validated it as a druggable target.\",\n      \"evidence\": \"Malachite green ATPase assay with liposomes, high-throughput screening, SAR analysis\",\n      \"pmids\": [\"39074100\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"No structural data for EHD4 ATPase domain\",\n        \"In-cell validation of identified inhibitors not reported\"\n      ]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Mapping of PTS binding to a non-canonical motif on EHD4 and discovery that EHD4 negatively regulates claudin-5 transcription in neural endothelial cells revealed a previously unrecognized transcriptional regulatory axis for EHD4.\",\n      \"evidence\": \"GST pull-down with point mutagenesis (PTS motif mapping); genome-wide CRISPR screen with CLDN-5 surface expression and transcriptional reporter assays\",\n      \"pmids\": [\"39776131\", \"41361961\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Mechanism by which an endosomal ATPase controls transcription of CLDN5 is unknown\",\n        \"Whether EHD4 acts on a CLDN5 transcription factor through trafficking or a direct nuclear role is unresolved\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the structural basis of EHD4–EHD1 preferential heterodimerization, whether EHD4 possesses intrinsic membrane fission activity independent of EHD1, and the mechanism by which EHD4 regulates claudin-5 transcription.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"No high-resolution structure of EHD4 or EHD4–EHD1 heterodimer\",\n        \"Reconstituted membrane fission assays with EHD4 alone not performed\",\n        \"Transcriptional regulatory mechanism linking endosomal EHD4 to CLDN5 promoter activity undefined\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140657\", \"supporting_discovery_ids\": [14]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [14]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [14]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [2, 10, 11]},\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [2, 12]},\n      {\"term_id\": \"GO:0005929\", \"supporting_discovery_ids\": [13]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [2, 5, 10, 12]},\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [13]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [7]}\n    ],\n    \"complexes\": [\n      \"PACSIN2/EHD4/MICAL-L1 junctional complex\"\n    ],\n    \"partners\": [\n      \"EHD1\",\n      \"PACSIN2\",\n      \"MICAL-L1\",\n      \"CDH23\",\n      \"PPP1R18\",\n      \"EHD3\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}