Affinage

EHD4

EH domain-containing protein 4 · UniProt Q9H223

Length
541 aa
Mass
61.2 kDa
Annotated
2026-04-28
18 papers in source corpus 16 papers cited in narrative 17 extracted findings

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

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).

Mechanistic history

Synthesis pass · year-by-year structured walk · 13 steps
  1. 2000 Medium

    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

    PMID:10673336

    Open questions at the time
    • No functional assay performed
    • Subcellular localization not determined experimentally
    • Enzymatic activity not tested
  2. 2008 High

    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

    PMID:18331452

    Open questions at the time
    • 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
  3. 2009 Medium

    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

    PMID:19487694

    Open questions at the time
    • No auditory phenotype quantified in EHD4 KO mice
    • Whether EHD4 controls CDH23 recycling or degradation not determined
    • Calcium-sensitivity mechanism not structurally resolved
  4. 2010 High

    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

    PMID:20463227

    Open questions at the time
    • Structural basis of EHD1–EHD4 hetero-oligomerization not determined
    • Whether other EHD family members can substitute in neurons unknown
  5. 2010 Medium

    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

    PMID:20213691

    Open questions at the time
    • Specific trafficking cargo in spermatocytes not identified
    • Mechanism of EHD1 compensatory upregulation unknown
  6. 2011 Medium

    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

    PMID:21408024

    Open questions at the time
    • Individual contributions of EHD3 vs. EHD4 to VEGFR2 trafficking not separated
    • Whether the phenotype reflects impaired VEGFR2 recycling or degradation unclear
  7. 2017 Medium

    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

    PMID:28778975

    Open questions at the time
    • Whether EHD4 directly controls AQP2 vesicle fusion or upstream sorting is unknown
    • Vasopressin-stimulated recycling pathway involvement not tested
  8. 2020 High

    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

    PMID:32966336

    Open questions at the time
    • Structural basis of preferential EHD4–EHD1 dimerization not resolved
    • Whether EHD4 itself possesses membrane fission activity independent of EHD1 unknown
  9. 2020 Medium

    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

    PMID:32800345

    Open questions at the time
    • Endogenous stoichiometry of PTS–EHD4 complex not assessed
    • Functional consequence of PTS loss on EHD4-dependent trafficking not tested
  10. 2021 High

    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

    PMID:33972531

    Open questions at the time
    • Whether EHD4 ATPase activity is required at junctions not tested
    • Role in angiogenesis in vivo not confirmed
  11. 2022 Medium

    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

    PMID:35510564

    Open questions at the time
    • Direct ciliogenesis cargo trafficked by EHD4 not identified
    • Whether EHD4 acts at the ciliary base or at upstream endosomes is unresolved
  12. 2024 Medium

    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

    PMID:39074100

    Open questions at the time
    • No structural data for EHD4 ATPase domain
    • In-cell validation of identified inhibitors not reported
  13. 2025 Medium

    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

    PMID:39776131 PMID:41361961

    Open questions at the time
    • 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

Open questions

Synthesis pass · forward-looking unresolved questions
  • 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.
  • 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

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0008289 lipid binding 1 GO:0016787 hydrolase activity 1 GO:0140657 ATP-dependent activity 1
Localization
GO:0005768 endosome 3 GO:0031410 cytoplasmic vesicle 2 GO:0005929 cilium 1
Pathway
R-HSA-5653656 Vesicle-mediated transport 4 R-HSA-162582 Signal Transduction 1 R-HSA-1852241 Organelle biogenesis and maintenance 1
Partners
CDH23EHD1EHD3MICAL-L1PACSIN2PPP1R18
Complex memberships
PACSIN2/EHD4/MICAL-L1 junctional complex

Evidence

Reading pass · 17 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2000 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. cDNA library screening, sequence alignment, radiation hybrid mapping Genomics Medium 10673336
2001 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. Yeast two-hybrid screen, immunofluorescence staining of developing limbs and fibroblast cultures, non-denaturing extraction and SDS-PAGE The Journal of biological chemistry Medium 11533061
2008 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. Peptide antibody localization, siRNA/shRNA knockdown, immunofluorescence, endocytic cargo uptake assays, Rab5-GTP pulldown Traffic (Copenhagen, Denmark) High 18331452
2008 Endogenous EHD4 and EHD1 interact (co-immunoprecipitate) in cells, suggesting they cooperate in regulating transport along the early endosome to endocytic recycling compartment axis. Co-immunoprecipitation of endogenous proteins Traffic (Copenhagen, Denmark) Medium 18331452
2009 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. Yeast two-hybrid screen, in situ hybridization, co-immunoprecipitation, immunofluorescence colocalization, EHD4 knockout mouse phenotyping The Journal of biological chemistry Medium 19487694
2010 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. shRNA knockdown, overexpression, live-cell endocytosis assays in neurons and fibroblasts, oligomerization mutant analysis The Journal of neuroscience High 20463227
2010 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. Conditional knockout mouse generation, Western blotting, histology, sperm count, compound action potential measurement Genesis (New York, N.Y. : 2000) Medium 20213691
2011 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. Double knockout mouse generation (Ehd3-/-; Ehd4-/-), histopathology, immunostaining, electron microscopy, proteinuria measurement PloS one Medium 21408024
2013 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. DiGE and iTRAQ proteomics of HIV-1 particles, siRNA knockdown, infectivity assays Journal of virology Medium 23325686
2017 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. EHD4 knockout mice, urine volume/osmolality measurement, immunostaining of AQP2 subcellular localization, Western blotting FASEB journal Medium 28778975
2020 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. siRNA, shRNA, CRISPR/Cas9 knockout, co-immunoprecipitation, immunofluorescence of EHD1 endosomal recruitment, endosome size quantification PloS one High 32966336
2020 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. Co-immunoprecipitation, GST pull-down, immunofluorescence colocalization, transferrin trafficking assay Biochemical and biophysical research communications Medium 32800345
2021 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. Co-immunoprecipitation, live imaging, siRNA knockdown, endothelial migration and sprouting assays, immunofluorescence of junction-localized complex Nature communications High 33972531
2022 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. siRNA knockdown of EHD4, ciliogenesis rescue assays with EHD1 EH-domain point mutants, CRISPR/Cas9, immunofluorescence of cilia Traffic (Copenhagen, Denmark) Medium 35510564
2024 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. Malachite green-based ATPase assay with liposomes, high-throughput screening, structure-activity relationship (SAR) analysis PloS one Medium 39074100
2025 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. Genome-wide CRISPR/Cas9 cell-sorting-based phenotypic screen, CLDN-5 surface expression measurement, transcriptional reporter assays The FEBS journal Medium 41361961
2025 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. GST pull-down, far western blotting, point mutagenesis, transferrin trafficking assay Journal of biochemistry Medium 39776131

Source papers

Stage 0 corpus · 18 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2013 Ehd4 encodes a novel and Oryza-genus-specific regulator of photoperiodic flowering in rice. PLoS genetics 146 23437005
2008 A role for EHD4 in the regulation of early endosomal transport. Traffic (Copenhagen, Denmark) 93 18331452
2000 EHD2, EHD3, and EHD4 encode novel members of a highly conserved family of EH domain-containing proteins. Genomics 80 10673336
2011 Renal thrombotic microangiopathy in mice with combined deletion of endocytic recycling regulators EHD3 and EHD4. PloS one 41 21408024
2013 Comparative proteomic analysis of HIV-1 particles reveals a role for Ezrin and EHD4 in the Nef-dependent increase of virus infectivity. Journal of virology 36 23325686
2010 Alterations of EHD1/EHD4 protein levels interfere with L1/NgCAM endocytosis in neurons and disrupt axonal targeting. The Journal of neuroscience : the official journal of the Society for Neuroscience 33 20463227
2021 A junctional PACSIN2/EHD4/MICAL-L1 complex coordinates VE-cadherin trafficking for endothelial migration and angiogenesis. Nature communications 31 33972531
2001 Characterization of EHD4, an EH domain-containing protein expressed in the extracellular matrix. The Journal of biological chemistry 22 11533061
2010 Ehd4 is required to attain normal prepubertal testis size but dispensable for fertility in male mice. Genesis (New York, N.Y. : 2000) 21 20213691
2009 EHD4 and CDH23 are interacting partners in cochlear hair cells. The Journal of biological chemistry 17 19487694
2020 Eps15 Homology Domain Protein 4 (EHD4) is required for Eps15 Homology Domain Protein 1 (EHD1)-mediated endosomal recruitment and fission. PloS one 15 32966336
2018 Microtubular remodeling and decreased expression of Nav1.5 with enhanced EHD4 in cells from the infarcted heart. Life sciences 8 29534991
2022 Differential requirements for the Eps15 homology domain proteins EHD4 and EHD2 in the regulation of mammalian ciliogenesis. Traffic (Copenhagen, Denmark) 6 35510564
2017 EHD4 is a novel regulator of urinary water homeostasis. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 3 28778975
2024 Identification of drug-like molecules targeting the ATPase activity of dynamin-like EHD4. PloS one 2 39074100
2020 Identification of phostensin in association with Eps 15 homology domain-containing protein 1 (EHD1) and EHD4. Biochemical and biophysical research communications 2 32800345
2025 Identification of a novel Eps 15 homology domain-containing protein 1 (EHD1) and EHD4-binding motif in phostensin. Journal of biochemistry 0 39776131
2025 EHD4 and ASAP2 are critical negative regulators of the claudin-5-based endothelial barrier. The FEBS journal 0 41361961