Affinage

EHD4

EH domain-containing protein 4 · UniProt Q9H223

Length
541 aa
Mass
61.2 kDa
Annotated
2026-06-09
17 papers in source corpus 16 papers cited in narrative 16 extracted findings
Cross-family judge vs UniProt: Affinage preferred faithfulness: 8/8 claims corpus-supported (100%)

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

EHD4 is a dynamin-related, EH-domain ATPase that governs cargo exit from sorting/early endosomes and the recycling of cell-surface receptors (PMID:18331452, PMID:39074100). It localizes to Rab5-, EEA1-, and Arf6-positive early endosomes and regulates the movement of internalized transferrin, MHC class I, and LDL toward both the recycling compartment and the late endocytic pathway; its loss enlarges early endosomes and accumulates GTP-bound Rab5 (PMID:18331452). Mechanistically, EHD4 preferentially heterodimerizes with EHD1 and is required to recruit EHD1 to sorting endosomes to drive endosomal vesicle fission, an activity that depends on the NPF-motif partners Rabenosyn-5, Syndapin2, and MICAL-L1 (PMID:32966336). It also engages non-canonical partners such as Phostensin and VPS35 through an ILV(X)4(L/V)RLS-type motif distinct from the NPF consensus, coupling these interactions to transferrin recycling (PMID:32800345, PMID:39776131). EHD4 exhibits liposome-stimulated ATPase activity consistent with its dynamin-related membrane-remodeling role (PMID:39074100). Through these endocytic activities EHD4 supports specific physiological programs: with EHD3 it mediates VEGFR2 recycling required for glomerular endothelial integrity (PMID:21408024), traffics aquaporin-2 to the apical membrane of renal collecting-duct cells to control water homeostasis (PMID:28778975), assembles a junctional PACSIN2/EHD4/MICAL-L1 complex that directs VE-cadherin trafficking for polarized endothelial migration and angiogenesis (PMID:33972531), and is required for primary ciliogenesis via conserved EH-domain residues (PMID:35510564). EHD4 additionally interacts with cadherin-23 in cochlear hair cells in a calcium-sensitive manner (PMID:19487694), negatively regulates claudin-5 expression and barrier function in CNS endothelium (PMID:41361961), and acts as a cofactor for HIV-1 Nef-mediated enhancement of viral infectivity (PMID:23325686). Across multiple knockout models, loss of EHD4 is partially buffered by compensatory upregulation of EHD1 (PMID:20213691, PMID:21408024).

Mechanistic history

Synthesis pass · year-by-year structured walk · 16 steps
  1. 2000 Medium

    Established the domain architecture that defines EHD4 as an EH-domain family protein, framing all subsequent functional hypotheses around nucleotide binding and EH-domain-mediated interactions.

    Evidence cDNA library screening, sequence alignment, and chromosomal mapping

    PMID:10673336

    Open questions at the time
    • No functional assay of the predicted nucleotide-binding or EH domains
    • Predicted nuclear localization signal never functionally validated
  2. 2001 Low

    Tested whether EHD4 acts outside the cell by reporting it as a secreted, disulfide-linked oligomer associated with type VI collagen in the extracellular matrix.

    Evidence Yeast two-hybrid screen and immunofluorescence/SDS-PAGE of fibroblast matrix and cartilage extracts

    PMID:11533061

    Open questions at the time
    • Yeast two-hybrid interaction never confirmed by direct binding in mammalian cells
    • Extracellular localization conflicts with the endosomal localization established by all later studies, suggesting artifact or isoform-specific behavior
  3. 2008 High

    Resolved the core cellular function of EHD4 by placing it at early endosomes as a regulator of cargo exit toward recycling and late endocytic routes, and demonstrated its physical partnership with EHD1.

    Evidence Peptide-antibody localization, siRNA/shRNA knockdown with transferrin/MHC-I/LDL cargo readouts, endogenous reciprocal Co-IP, and Rab5-GTP pull-down

    PMID:18331452

    Open questions at the time
    • Did not establish direction of the EHD4-EHD1 functional hierarchy
    • Catalytic mechanism of endosomal exit not defined
  4. 2009 Medium

    Asked whether EHD4 has tissue-specific receptor partners, identifying a calcium-sensitive interaction with cadherin-23 in cochlear hair cells.

    Evidence Membrane yeast two-hybrid, in situ hybridization, Co-IP, and compound action potentials in knockout mice

    PMID:19487694

    Open questions at the time
    • Normal hearing in knockouts leaves the physiological role of the CDH23 interaction unresolved
    • Functional consequence likely masked by EHD1 compensation
  5. 2010 Medium

    Showed that EHD4 acts together with EHD1 as a balanced hetero-oligomer for cargo-specific endocytosis, establishing functional interdependence between the two paralogs.

    Evidence shRNA knockdown, EHD-family overexpression and rescue, and antibody-feeding endocytosis assays for NgCAM versus transferrin in hippocampal neurons

    PMID:20463227

    Open questions at the time
    • Cargo selectivity (NgCAM but not transferrin) mechanism unexplained
    • Stoichiometry of the EHD1-EHD4 oligomer not determined
  6. 2010 Medium

    Defined an in vivo requirement for EHD4 in germ cell development and revealed EHD1 compensation as a recurring theme.

    Evidence Conditional knockout mouse with histology, TUNEL apoptosis assay, sperm counts, and EHD-protein level analysis

    PMID:20213691

    Open questions at the time
    • No direct mechanistic rescue linking the phenotype to a specific trafficking event
    • Molecular cargo in spermatocytes not identified
  7. 2011 Medium

    Connected EHD4-mediated recycling to receptor homeostasis in vivo by showing EHD3/EHD4 are jointly required for VEGFR2 trafficking and glomerular endothelial integrity.

    Evidence EHD3/EHD4 double-knockout mouse with histopathology, VEGFR2 immunofluorescence, TUNEL, and proteinuria measurement

    PMID:21408024

    Open questions at the time
    • Single-knockout phenotype masked by compensation, obscuring EHD4-specific contribution
    • Direct evidence that EHD4 traffics VEGFR2 not shown
  8. 2013 Medium

    Identified EHD4 as a host cofactor co-opted by HIV-1 Nef to enhance virion infectivity.

    Evidence DiGE/iTRAQ virion proteomics and siRNA knockdown of EHD4 +/- Ezrin with infectivity assays

    PMID:23325686

    Open questions at the time
    • Molecular mechanism by which EHD4 supports Nef potency unknown
    • Whether the effect requires EHD4 endosomal function not tested
  9. 2017 Medium

    Established a physiological output of EHD4 apical trafficking by linking it to AQP2 surface localization and renal water handling.

    Evidence EHD4-knockout mouse with urine osmolality, water load/restriction tests, apical AQP2 immunofluorescence quantification, and Western blotting

    PMID:28778975

    Open questions at the time
    • Whether EHD4 acts directly on AQP2-containing vesicles not shown
    • Partner machinery for apical AQP2 delivery undefined
  10. 2020 High

    Resolved the molecular hierarchy of endosomal fission, showing EHD4 preferentially dimerizes with EHD1 and recruits it to sorting endosomes via NPF-motif partners Rabenosyn-5, Syndapin2, and MICAL-L1.

    Evidence siRNA, shRNA, and CRISPR/Cas9 ablation with EHD1 recruitment imaging, sorting-endosome size quantification, Co-IP, and partner epistasis

    PMID:32966336

    Open questions at the time
    • Order of partner engagement during fission not resolved
    • Structural basis of the EHD4-EHD1 heterodimer not determined
  11. 2020 Medium

    Expanded the EHD4 interactome to Phostensin, linking a new partner to endocytic transferrin trafficking.

    Evidence Co-IP with shotgun proteomics, GST pull-down, co-localization at endocytic vesicles, and transferrin assay with PTS-beta overexpression

    PMID:32800345

    Open questions at the time
    • Binding interface on EHD4 not mapped in this study
    • Whether Phostensin acts through EHD1 or EHD4 directly unclear
  12. 2021 High

    Defined a spatially-restricted EHD4 complex controlling directed cell behavior: a junctional PACSIN2/EHD4/MICAL-L1 module that traffics VE-cadherin for collective endothelial migration and angiogenesis.

    Evidence Live-cell imaging, PACSIN2 knockdown/overexpression, Co-IP, in vitro angiogenic sprouting, and VE-cadherin trafficking assays

    PMID:33972531

    Open questions at the time
    • Mechanism restricting the complex to the junctional rear not defined
    • Whether EHD4 ATPase activity drives VE-cadherin tubule fission not tested
  13. 2022 High

    Mapped EH-domain determinants of a non-endosomal output, showing EHD4 is required for primary ciliogenesis through residues conserved in EHD1/3/4 but divergent in EHD2.

    Evidence siRNA of EHD4/EHD2, site-directed mutagenesis of EHD1 EH-domain residues, rescue, and cilia formation assays

    PMID:35510564

    Open questions at the time
    • EHD4's direct role versus its role in enabling EHD1 function at cilia not separated
    • Ciliary cargo regulated by EHD4 not identified
  14. 2024 High

    Provided the first reconstituted biochemical proof that EHD4 is a liposome-stimulated, dynamin-related ATPase and identified a small-molecule inhibitor.

    Evidence Malachite green ATPase assay with liposome stimulation, high-throughput screening, and SAR analysis

    PMID:39074100

    Open questions at the time
    • Structural mechanism coupling ATP hydrolysis to membrane fission not defined
    • Cellular phenotype of inhibitor not reported here
  15. 2025 Medium

    Defined a non-canonical EHD4 recognition motif used by Phostensin and VPS35, broadening the interaction code beyond NPF motifs and linking it to transferrin recycling.

    Evidence GST pull-down, far-western blotting, alanine-scan mutagenesis of the PTS ILV(X)4(L/V)RLS motif, and transferrin recycling assays

    PMID:39776131

    Open questions at the time
    • Structural details of motif docking onto the EH domain not determined
    • Functional role of the VPS35-EHD4 interaction not tested
  16. 2025 Medium

    Implicated EHD4 in CNS endothelial barrier control as a negative regulator of claudin-5.

    Evidence Genome-wide CRISPR/Cas9 barrier-tightness screen, flow cytometry for surface CLDN-5, and Western blotting

    PMID:41361961

    Open questions at the time
    • Proposed transcriptional regulation of CLDN5 not directly demonstrated
    • Mechanism connecting endosomal EHD4 to claudin-5 expression unknown

Open questions

Synthesis pass · forward-looking unresolved questions
  • How EHD4 ATP hydrolysis is mechanically coupled to membrane fission, and how it selects between its many tissue-specific cargoes (VEGFR2, AQP2, VE-cadherin, CDH23, claudin-5), remains undefined.
  • No structural model of EHD4 on membranes in the corpus
  • Cargo-selectivity determinants across tissues not established
  • EHD4-specific versus EHD1-dependent contributions not cleanly separated

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0060090 molecular adaptor activity 2 GO:0008289 lipid binding 1 GO:0016787 hydrolase activity 1 GO:0140657 ATP-dependent activity 1
Localization
GO:0005768 endosome 3 GO:0005886 plasma membrane 3 GO:0005929 cilium 1
Pathway
R-HSA-5653656 Vesicle-mediated transport 3 R-HSA-9609507 Protein localization 2
Partners
CDH23EHD1MICAL-L1PACSIN2PHOSTENSINRABENOSYN-5SYNDAPIN2VPS35
Complex memberships
EHD1-EHD4 heterodimerPACSIN2/EHD4/MICAL-L1 junctional complex

Evidence

Reading pass · 16 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2008 Endogenous EHD4 localizes to Rab5-, EEA1-, and Arf6-containing early endosomes and colocalizes with internalized transferrin in the cell periphery. Knockdown of EHD4 causes enlarged early endosomes enriched in GTP-bound (activated) Rab5 and accumulation of transferrin, MHC class I, and LDL cargo, establishing EHD4 as a regulator of exit from early endosomes toward both the recycling compartment and the late endocytic pathway. Endogenous EHD4 and EHD1 were shown to interact in cells. Specific peptide antibodies for localization; siRNA/shRNA knockdown with cargo trafficking readouts; co-immunoprecipitation of endogenous EHD4 and EHD1; Rab5-GTP pull-down assay Traffic High 18331452
2000 EHD4 encodes a protein with a conserved N-terminal nucleotide-binding consensus site, a bipartite nuclear localization signal, and a C-terminal EH domain with EF-hand motif, identifying it as a member of the EH domain-containing protein family related to EHD1. cDNA library screening, sequence alignment, genomic/chromosomal mapping Genomics Medium 10673336
2001 EHD4 protein is present in the extracellular matrix; it forms disulfide-linked oligomers (~220 and ~158 kDa under non-denaturing conditions, running at ~56 kDa under reducing SDS-PAGE) and was identified through a yeast two-hybrid screen as a potential interactor of type VI collagen. EHD4 is secreted by fibroblasts into the extracellular matrix as a filamentous network. Yeast two-hybrid screen, immunofluorescence of cultured fibroblasts and embryonic tissue, non-denaturing and reducing SDS-PAGE of cartilage extracts and conditioned media The Journal of biological chemistry Low 11533061
2009 EHD4 interacts with cadherin 23 (CDH23) in cochlear hair cells; the interaction is calcium-sensitive. EHD4 co-localizes and co-immunoprecipitates with CDH23 in mammalian cells, and EHD4 mRNA is expressed in hair cells. EHD4 knockout mice show compensatory upregulation of EHD1 in cochlear lysates but normal hearing. Membrane-based yeast two-hybrid screen of OHC cDNA library, in situ hybridization, co-immunoprecipitation, immunofluorescence co-localization, compound action potentials in KO mice, Western blotting The Journal of biological chemistry Medium 19487694
2010 EHD4 overexpression in neurons causes a defect in L1/NgCAM endocytosis (but not transferrin internalization), similar to EHD1 overexpression. Simultaneous expression of EHD1 and EHD4 rescues NgCAM endocytosis, suggesting they function as hetero-oligomeric complexes. EHD1 oligomerization was required for the endocytosis defect, and balanced EHD1-EHD4 levels are important for NgCAM endocytosis in neurons. shRNA knockdown, overexpression of EHD family members, antibody-feeding endocytosis assay for NgCAM and transferrin in hippocampal neurons The Journal of neuroscience Medium 20463227
2010 EHD4 knockout male mice have reduced testis weight, increased germ cell apoptosis, dysregulated seminiferous epithelium, and spermatid head abnormalities, with lower sperm counts. EHD4 is highly expressed in primary spermatocytes, and deletion of EHD4 alters levels of other EHD proteins (elevated EHD1 in adult testis), suggesting EHD1 functionally compensates for EHD4 loss in germ cell development. Conditional knockout mouse, PCR/Western blotting for protein deletion, histology, TUNEL apoptosis assay, sperm count, immunostaining Genesis Medium 20213691
2011 Combined deletion of EHD3 and EHD4 in mice causes renal thrombotic microangiopathy with glomerular lesions, altered VEGFR2 expression/localization, and increased endothelial apoptosis, establishing that EHD3/EHD4-mediated endocytic recycling of surface receptors such as VEGFR2 is essential for glomerular endothelial function. EHD4 expression was upregulated in Ehd3-/- glomerular endothelium, indicating functional compensation. Double-knockout mouse model, histopathology, immunofluorescence for VEGFR2 localization, TUNEL assay, proteinuria measurement PloS one Medium 21408024
2013 EHD4 is enriched in HIV-1 nef-deleted virions compared to wild-type virions. Simultaneous depletion of Ezrin and EHD4 in virus-producing cells decreases Nef potency (Nef's ability to increase virus infectivity) by ~30–70%, establishing EHD4 as a cofactor required by Nef to increase HIV-1 infectivity. DiGE and iTRAQ comparative proteomics of virion preparations; siRNA knockdown of EHD4 ± Ezrin with infectivity assays Journal of virology Medium 23325686
2017 EHD4 is expressed at highest levels in the inner medullary collecting duct of the kidney. EHD4-knockout mice excrete higher volumes of more dilute urine and show reduced intensity of apical membrane AQP2 staining in inner medullary principal cells (by ~20%), without changes in total AQP2 or pAQP2 protein levels, indicating EHD4 regulates apical trafficking/localization of aquaporin 2 (AQP2) and renal water homeostasis. EHD4-knockout mouse, urine volume/osmolality measurements, water load and restriction tests, immunofluorescence quantification of apical AQP2 staining, Western blotting FASEB journal Medium 28778975
2020 EHD4 preferentially dimerizes with EHD1 and is required for the recruitment of EHD1 to sorting endosomes (SE). Knockdown of EHD4 by siRNA, shRNA, or CRISPR/Cas9 leads to impaired EHD1 SE-recruitment and enlarged SE. The NPF motif-containing EHD4 binding partners Rabenosyn-5, Syndapin2, and MICAL-L1 are each individually required for EHD1 recruitment to SE; knockdown of any one causes enlarged SE, indicating defective endosomal fission. siRNA, shRNA, and CRISPR/Cas9 knockdown/knockout of EHD4; co-immunoprecipitation for EHD1-EHD4 dimerization; fluorescence microscopy for EHD1 SE-recruitment; quantification of SE size PloS one High 32966336
2020 Phostensin (PTS) interacts with both EHD1 and EHD4, co-localizes with them at endocytic vesicles, and the interaction is confirmed by co-immunoprecipitation and GST pull-down. Overexpression of PTS-β attenuates endocytic trafficking of transferrin, linking the PTS-EHD4 interaction to endocytic transport. Co-immunoprecipitation combined with shotgun proteomics, GST pull-down assay, immunofluorescence co-localization, transferrin trafficking assay with PTS-β overexpression Biochemical and biophysical research communications Medium 32800345
2021 EHD4 is recruited by PACSIN2 to the rear end of asymmetric adherens junctions between endothelial leader and follower cells, forming a recycling endosome-like tubular structure together with MICAL-L1. The junctional PACSIN2/EHD4/MICAL-L1 complex controls local VE-cadherin trafficking to coordinate polarized endothelial migration and angiogenesis. Fluorescence and live-cell imaging, PACSIN2 knockdown/overexpression, co-immunoprecipitation, in vitro angiogenic sprouting assay, VE-cadherin trafficking assays Nature communications High 33972531
2022 EHD4, but not EHD2, is required for primary ciliogenesis. Two sequence motifs conserved in the EH domains of EHD1, EHD3, and EHD4 but differing in EHD2 (positions P446/E470 in EHD1, aligning to S451/W475 in EHD2) are critical for EHD1-mediated ciliogenesis; substitution of these residues with EHD2-equivalent residues prevents rescue of ciliogenesis. EHD1 ATP-binding is also required for ciliogenesis. siRNA knockdown of EHD4 and EHD2, site-directed mutagenesis of EHD1 EH domain residues, rescue experiments by re-expression of EHD1 mutants, cilia formation assay Traffic High 35510564
2024 EHD4 possesses liposome-stimulated ATPase activity (dynamin-related ATPase), which can be measured by a Malachite green-based assay and is inhibited by a drug-like small molecule identified by high-throughput screening. Structure-activity relationship (SAR) studies defined sites for inhibitor optimization. Malachite green ATPase activity assay with liposome stimulation, high-throughput screening, SAR analysis PloS one High 39074100
2025 Phostensin (PTS) binds to EHD4 through a novel consensus motif (ILV(X)4(L/V)RLS at residues 64–74 of PTS-α), distinct from the canonical NPF motif. Alanine substitution mutations in this motif reduce binding to EHD4 in GST pull-down and far-western assays. VPS35 also binds EHD4 through a similar ILV(X)4VRL motif. PTS-β regulation of transferrin endocytic recycling requires an intact EHD-binding motif. GST pull-down assay, far-western blotting, site-directed mutagenesis of PTS binding motif, transferrin endocytic trafficking assay with mutant overexpression Journal of biochemistry Medium 39776131
2025 EHD4 negatively regulates claudin-5 (CLDN-5) expression and barrier function in CNS endothelial cells; CRISPR/Cas9-mediated suppression of EHD4 leads to significant upregulation of CLDN-5 protein on the cell surface. EHD4 appears to regulate the transcriptional activity of CLDN5. Genome-wide CRISPR/Cas9 cell-sorting-based phenotypic screen ('barrier tightness' phenotype), flow cytometry for CLDN-5 surface expression, Western blotting The FEBS journal Medium 41361961

Source papers

Stage 0 corpus · 17 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
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 37 23325686
2021 A junctional PACSIN2/EHD4/MICAL-L1 complex coordinates VE-cadherin trafficking for endothelial migration and angiogenesis. Nature communications 34 33972531
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
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

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