Affinage

EHD3

EH domain-containing protein 3 · UniProt Q9NZN3

Length
535 aa
Mass
60.9 kDa
Annotated
2026-04-28
20 papers in source corpus 16 papers cited in narrative 16 extracted findings

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

EHD3 is a membrane-associated ATPase of the EHD family that functions as a key regulator of endosomal membrane dynamics, controlling cargo trafficking between early endosomes, the endocytic recycling compartment (ERC), and the trans-Golgi network. EHD3 binds phosphatidic acid via its helical domain to drive membrane tubulation in vitro and stabilizes tubular recycling endosomes through SUMOylation-dependent targeting and EH-domain NPF-motif interactions that are distinct from its paralog EHD1 (PMID:27189942, PMID:26226295, PMID:26896729). Through interactions with Rab11-FIP2 and rabenosyn-5, EHD3 directs early-endosome-to-ERC transport of transferrin and rapid recycling of αvβ3-integrin, mediates early-endosome-to-Golgi transport of Shiga toxin and mannose-6-phosphate receptor, routes EGFR toward degradation, and participates in early ciliogenesis by tubulating distal appendage vesicles upstream of Rab8 activation (PMID:16251358, PMID:19139087, PMID:25686250, PMID:27811356). In the heart, EHD3 interacts with ankyrin-B and is required for proper membrane targeting of NCX1 and Cav1.2, as EHD3-knockout mice exhibit bradycardia and conduction block (PMID:24759929).

Mechanistic history

Synthesis pass · year-by-year structured walk · 10 steps
  1. 2000 Medium

    Cloning of EHD3 established it as a new member of the EHD family possessing an N-terminal nucleotide-binding domain and a C-terminal EH domain, placing it among potential endocytic regulators.

    Evidence cDNA cloning and sequence analysis in human tissues

    PMID:10673336

    Open questions at the time
    • No functional data; domain assignments were purely sequence-based
    • Expression pattern described at tissue level only
    • No subcellular localization determined
  2. 2002 High

    Localization of EHD3 to transferrin-positive recycling tubules and demonstration of direct EHD1–EHD3 interaction established its endocytic recycling compartment identity and potential for hetero-oligomerization.

    Evidence GFP-fusion imaging, yeast two-hybrid, reciprocal co-immunoprecipitation, domain-swap mutagenesis in HeLa cells

    PMID:12121420

    Open questions at the time
    • No loss-of-function data to demonstrate functional requirement
    • Whether hetero-oligomerization with EHD1 is required for tubule localization was not tested
  3. 2005 High

    Identification of Rab11-FIP2 as an EHD3-binding partner and demonstration that EHD3 knockdown blocks early-endosome-to-ERC transport of transferrin defined EHD3's first specific trafficking step.

    Evidence Co-immunoprecipitation, siRNA knockdown, transferrin recycling assay in HeLa cells

    PMID:16251358

    Open questions at the time
    • Whether EHD3's ATPase activity is required for this transport step was not addressed
    • The molecular mechanism by which EHD3 promotes ERC delivery remained unclear
  4. 2009 High

    EHD3 was shown to regulate a second major trafficking route — early-endosome-to-Golgi transport — through interaction with rabenosyn-5, broadening its role beyond recycling to retrograde trafficking.

    Evidence siRNA knockdown of EHD3 or rabenosyn-5, Shiga toxin B trafficking, Golgi morphology and AP-1 recruitment assays in HeLa cells

    PMID:19139087

    Open questions at the time
    • Direct biochemical reconstitution of the EHD3–rabenosyn-5 complex was not performed in this study
    • Whether EHD3 tubulates membranes in this retrograde route specifically was not tested
  5. 2012 Medium

    Multi-model heart failure studies revealed that EHD3 is upregulated in parallel with NCX1 under ROS/AngII signaling, linking EHD3 to cardiac pathophysiology before its cardiac function was directly demonstrated.

    Evidence Western blot across four heart failure models (rat, mouse, canine, human) with pharmacological pathway inhibition

    PMID:22406195

    Open questions at the time
    • Correlative expression data; no direct mechanistic link between EHD3 upregulation and NCX1 trafficking established at this point
    • Causal role of EHD3 in heart failure progression not tested
  6. 2013 Medium

    EHD3 was found to mediate rapid recycling of αvβ3-integrin from early endosomes to the plasma membrane alongside AAK1L, extending its cargo repertoire to adhesion receptors and linking it to cell adhesion regulation.

    Evidence siRNA knockdown, TIRF imaging, integrin recycling and cell adhesion assays

    PMID:23781025

    Open questions at the time
    • Whether EHD3 directly binds integrin cargo or acts indirectly through membrane remodeling is unknown
    • Single-lab study
  7. 2014 High

    EHD3 knockout mice revealed its essential cardiac role: EHD3 interacts with ankyrin-B and is required for membrane targeting of NCX1 and Cav1.2, with loss causing bradycardia and conduction block, providing a direct mechanistic explanation for the earlier heart failure correlations.

    Evidence Whole-body and cardiac-specific EHD3 knockout mice, co-immunoprecipitation (EHD3–ankyrin-B), patch-clamp electrophysiology, immunofluorescence

    PMID:24759929

    Open questions at the time
    • The precise sorting step at which EHD3-ankyrin-B delivers ion channels to the T-tubule membrane is undefined
    • Whether EHD3's ATPase or tubulation activity is required for cardiac ion channel targeting was not tested
  8. 2015 High

    Two studies clarified how EHD3 is regulated and how it shapes membranes during ciliogenesis: SUMOylation at K315/K511 targets EHD3 to ERC tubules (non-SUMOylatable mutant acts as dominant-negative), and EHD3-dependent membrane tubulation converts distal appendage vesicles into ciliary vesicles upstream of Rab8 activation.

    Evidence In vitro SUMOylation, mutagenesis with transferrin recycling assay; siRNA knockdown with EM, live imaging, and epistasis analysis of the Rab11-Rab8 cascade

    PMID:25686250 PMID:26226295

    Open questions at the time
    • The SUMOylation machinery responsible for modifying EHD3 in vivo is not identified
    • Whether SUMOylation also regulates EHD3 during ciliogenesis is untested
    • Structural basis for how SUMO modification alters EHD3 membrane association is unknown
  9. 2016 High

    Three studies in 2016 resolved key molecular determinants: EHD3 stabilizes (rather than initiates) tubular recycling endosomes via EH-domain residues N519/E520 that confer NPF-binding selectivity distinct from EHD1; EHD3 binds phosphatidic acid through its helical domain to drive membrane tubulation; and EHD3 promotes EGFR ubiquitination and degradative routing rather than recycling.

    Evidence Synchronized TRE biogenesis assay with mutagenesis; liposome co-sedimentation with DGK inhibitor; inducible EHD3 overexpression with EGFR ubiquitination and trafficking assays

    PMID:26896729 PMID:27189942 PMID:27811356

    Open questions at the time
    • No crystal structure of EHD3 bound to PA or NPF peptide exists
    • How EHD3 distinguishes cargoes destined for recycling versus degradation at the molecular level is unclear
    • The ATPase cycle's coupling to tubule stabilization versus scission is not resolved
  10. 2021 Medium

    NR5A1/SF-1 was identified as a direct transcriptional activator of EHD3, and EHD3-dependent endocytosis was shown to be required for testosterone synthesis in Leydig cells, extending EHD3's physiological relevance to steroidogenesis.

    Evidence ChIP and luciferase reporter for NR5A1 binding to EHD3 promoter; siRNA/overexpression with endocytosis and testosterone ELISA in Leydig cells; conditional NR5A1 KO mice

    PMID:33964295

    Open questions at the time
    • The specific endocytic cargo whose internalization is required for steroidogenesis is not identified
    • Whether other EHD family members compensate in Leydig cells is untested
    • Single-lab finding

Open questions

Synthesis pass · forward-looking unresolved questions
  • Key unresolved questions include the structural basis for EHD3's lipid and NPF selectivity, how its ATPase cycle is coupled to tubule stabilization versus scission, and the full scope of tissue-specific cargo sorting mediated by EHD3.
  • No high-resolution structure of EHD3 or its complexes with PA/NPF peptides
  • ATPase catalytic cycle not kinetically characterized in the context of membrane remodeling
  • Tissue-specific interactome remains incomplete

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0098772 molecular function regulator activity 2 GO:0140657 ATP-dependent activity 2 GO:0008289 lipid binding 1
Localization
GO:0005768 endosome 4 GO:0031410 cytoplasmic vesicle 4 GO:0005886 plasma membrane 2 GO:0005929 cilium 1
Pathway
R-HSA-5653656 Vesicle-mediated transport 7 R-HSA-162582 Signal Transduction 2 R-HSA-1852241 Organelle biogenesis and maintenance 1
Partners
AAK1ANK2EHD1RAB11FIP2RBSNSNAP29

Evidence

Reading pass · 16 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2000 EHD3 encodes a protein with a nucleotide-binding consensus site at the N-terminus, a bipartite nuclear localization signal, and an EH domain with an EF-hand motif at the C-terminus, placing it in the EHD family of endocytic regulators. cDNA cloning, sequence analysis, Northern blot Genomics Medium 10673336
2002 EHD3 localizes to endocytic recycling vesicles and microtubule-dependent membrane tubules containing transferrin, and directly interacts with EHD1 via two-hybrid and co-immunoprecipitation; the N-terminal domain of EHD3 is responsible for its tubular localization. GFP-fusion live imaging, yeast two-hybrid, co-immunoprecipitation, domain-swap mutagenesis Traffic High 12121420
2005 EHD3 binds the Rab11 effector Rab11-FIP2 via EH-NPF interactions, and loss of EHD3 expression prevents delivery of internalized transferrin and early endosomal proteins to the endocytic recycling compartment (ERC), altering the subcellular localization of Rab11-FIP2 and Rab11. Co-immunoprecipitation, siRNA knockdown, fluorescence imaging of transferrin trafficking Molecular biology of the cell High 16251358
2007 EHD3 co-localizes with EHD1, Rab8a, and Myosin Vb on a tubular recycling network distinct from Rab11a compartments, supporting a role for EHD3 in a non-clathrin endocytic recycling pathway. Fluorescence colocalization, FRET, dominant-negative expression Molecular biology of the cell Medium 17507647
2009 siRNA knockdown of EHD3 or its interaction partner rabenosyn-5 redistributes SNX1 to enlarged early endosomes, disrupts Shiga toxin B subunit transport from endosomes to the Golgi, fragments Golgi morphology, reduces AP-1 gamma-adaptin recruitment to the Golgi, and mislocalizes mannose-6-phosphate receptor, demonstrating EHD3's role in early-endosome-to-Golgi transport. siRNA knockdown, fluorescence imaging of Shiga toxin trafficking, Golgi morphology analysis, AP-1 recruitment assay Journal of cell science High 19139087
2011 Combined genetic deletion of EHD3 and EHD4 in mice causes thrombotic microangiopathy-like glomerular lesions with endothelial swelling, loss of fenestrations, and altered VEGFR2 expression and localization with increased apoptosis, indicating that EHD-mediated endocytic trafficking of surface receptors such as VEGFR2 is essential for glomerular endothelial function. Ehd3−/−; Ehd4−/− double knockout mice, histopathology, immunofluorescence, proteinuria assay PloS one High 21408024
2013 EHD3 depletion by shRNA increases glioma cell growth and invasiveness, while doxycycline-inducible re-expression induces cell cycle arrest and apoptosis; in vivo xenograft experiments confirm EHD3 growth-inhibitory function in glioma. shRNA knockdown, inducible overexpression, flow cytometry (cell cycle/apoptosis), xenograft tumor model Carcinogenesis Medium 24306026
2013 EHD3 and AAK1L localize to early endosomes near the cell surface and are both required for rapid recycling of αvβ3-integrin from the early endosome back to the plasma membrane; siRNA depletion of either factor delays β3-integrin recycling and impairs cell adhesion. siRNA knockdown, live-cell TIRF imaging, integrin recycling assay, adhesion assay Journal of cell science Medium 23781025
2014 EHD3-deficient mouse hearts display bradycardia, conduction block, and blunted adrenergic response; mechanistically, EHD3 is required for proper membrane localization of Na/Ca exchanger (NCX1) and L-type Ca channel Cav1.2, and EHD3 directly interacts with ankyrin-B (a binding partner for NCX1), with EHD3 loss reducing NCX1-mediated current and Cav1.2-mediated current. EHD3 knockout mice, cardiac-specific conditional KO, electrophysiology, co-immunoprecipitation (EHD3-ankyrin-B), immunofluorescence, patch-clamp Circulation research High 24759929
2012 EHD3 levels are consistently elevated in four heart failure models (ischemic rat, pressure-overloaded mouse, pacing-induced canine, and non-ischemic human) in parallel with NCX1 upregulation; EHD3 upregulation in heart failure is regulated downstream of reactive oxygen species and angiotensin II signaling. Western blot in multiple HF models, pharmacological inhibition of ROS/AngII pathway Journal of molecular and cellular cardiology Medium 22406195
2015 EHD1 and EHD3 localize to preciliary membranes and the ciliary pocket; EHD-dependent membrane tubulation is required for formation of ciliary vesicles from distal appendage vesicles (DAVs) at the mother centriole, functioning upstream of Rab8 activation, transition zone protein recruitment, and IFT20 recruitment during early ciliogenesis. EHD3 acts in concert with the Rab11-Rab8 cascade and the SNARE protein SNAP29. siRNA knockdown, live imaging, electron microscopy, co-localization, epistasis with Rab11/Rab8 cascade Nature cell biology High 25686250
2015 EHD3 undergoes SUMOylation on lysines K315 and K511; SUMOylation is required for EHD3 localization to tubular structures of the ERC but not for its dimerization; non-SUMOylatable EHD3 has a dominant-negative effect on ERC tubulation and delays transferrin recycling to the cell surface. In vitro SUMOylation assay, site-directed mutagenesis of SUMOylation sites, fluorescence imaging, transferrin recycling assay PloS one High 26226295
2016 EHD3 stabilizes tubular recycling endosomes (TRE) rather than initiating their biogenesis, as shown in a synchronized TRE biogenesis system; residues Asn-519/Glu-520 in EHD3's EH domain (vs. Ala-519/Asp-520 in EHD1) define selective NPF-peptide binding specificity between the two paralogs and underlie their distinct roles in membrane tubulation vs. vesiculation. Phospholipase D inhibitor washout (synchronized TRE biogenesis), site-directed mutagenesis, NPF-binding assays, fluorescence imaging The Journal of biological chemistry High 27189942
2016 EHD3 interacts with phosphatidic acid (PA) through its helical domain, and this interaction induces membrane tubulation in vitro; inhibiting PA synthesis reduces EHD3-containing tubules and impairs receptor trafficking from early endosomes. In vitro liposome co-sedimentation assay, diacylglycerol kinase inhibitor treatment, fluorescence imaging Experimental cell research High 26896729
2016 EHD3 accelerates EGFR degradation upon EGF stimulation by increasing EGFR ubiquitination and diverting EGFR from the recycling to the degradative endosomal pathway, and reduces endosome-based MAPK and AKT signaling without affecting total pathway activation. Inducible EHD3 overexpression, ubiquitination assay, EGFR trafficking assay (immunofluorescence), signaling assays (MAPK/AKT) Oncotarget Medium 27811356
2021 NR5A1 (SF-1) directly binds to the conserved 'AGGTCA' sequence in the EHD3 promoter (confirmed by ChIP and luciferase assay) to positively regulate EHD3 transcription; EHD3-mediated endocytosis is required for testosterone synthesis in Leydig cells, as EHD3 knockdown reduces endocytosis and testosterone production, while overexpression increases it. ChIP, dual luciferase reporter assay, siRNA knockdown, EHD3 overexpression, exosome tracing, ELISA for testosterone, conditional NR5A1 KO mice Life sciences Medium 33964295

Source papers

Stage 0 corpus · 20 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2015 Early steps in primary cilium assembly require EHD1/EHD3-dependent ciliary vesicle formation. Nature cell biology 208 25686250
2005 Interactions between EHD proteins and Rab11-FIP2: a role for EHD3 in early endosomal transport. Molecular biology of the cell 159 16251358
2007 Myosin Vb interacts with Rab8a on a tubular network containing EHD1 and EHD3. Molecular biology of the cell 141 17507647
2011 Ehd3, encoding a plant homeodomain finger-containing protein, is a critical promoter of rice flowering. The Plant journal : for cell and molecular biology 138 21284756
2000 EHD2, EHD3, and EHD4 encode novel members of a highly conserved family of EH domain-containing proteins. Genomics 80 10673336
2009 EHD3 regulates early-endosome-to-Golgi transport and preserves Golgi morphology. Journal of cell science 79 19139087
2002 EHD3: a protein that resides in recycling tubular and vesicular membrane structures and interacts with EHD1. Traffic (Copenhagen, Denmark) 74 12121420
2007 Expression and subcellular distribution of novel glomerulus-associated proteins dendrin, ehd3, sh2d4a, plekhh2, and 2310066E14Rik. Journal of the American Society of Nephrology : JASN 66 17251388
2011 Renal thrombotic microangiopathy in mice with combined deletion of endocytic recycling regulators EHD3 and EHD4. PloS one 41 21408024
2014 EHD3-dependent endosome pathway regulates cardiac membrane excitability and physiology. Circulation research 31 24759929
2012 Differential regulation of EHD3 in human and mammalian heart failure. Journal of molecular and cellular cardiology 31 22406195
2016 EHD3 Protein Is Required for Tubular Recycling Endosome Stabilization, and an Asparagine-Glutamic Acid Residue Pair within Its Eps15 Homology (EH) Domain Dictates Its Selective Binding to NPF Peptides. The Journal of biological chemistry 21 27189942
2013 Ehd3, a regulator of vesicular trafficking, is silenced in gliomas and functions as a tumor suppressor by controlling cell cycle arrest and apoptosis. Carcinogenesis 16 24306026
2021 EHD3 positively regulated by NR5A1 participates in testosterone synthesis via endocytosis. Life sciences 10 33964295
2014 The gender-specific association of EHD3 polymorphisms with major depressive disorder. Neuroscience letters 9 24607927
2013 Αvβ3-integrin-mediated adhesion is regulated through an AAK1L- and EHD3-dependent rapid-recycling pathway. Journal of cell science 7 23781025
2015 SUMOylation of EHD3 Modulates Tubulation of the Endocytic Recycling Compartment. PloS one 6 26226295
2016 Phosphatidic acid induces EHD3-containing membrane tubulation and is required for receptor recycling. Experimental cell research 5 26896729
2016 Spatio-temporal regulation of EGFR signaling by the Eps15 homology domain-containing protein 3 (EHD3). Oncotarget 5 27811356
2014 Association between EHD3 gene and the cognitive function of patients with major depressive disorder. Zhongguo yi xue ke xue yuan xue bao. Acta Academiae Medicinae Sinicae 0 24997812