Affinage

EHD3

EH domain-containing protein 3 · UniProt Q9NZN3

Length
535 aa
Mass
60.9 kDa
Annotated
2026-06-09
20 papers in source corpus 15 papers cited in narrative 15 extracted findings
Cross-family judge vs UniProt: Affinage preferred faithfulness: 6/6 claims corpus-supported (100%)

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

EHD3 is an EH domain-containing membrane-remodeling protein that drives the formation and stabilization of tubular recycling endosomes and governs cargo transit through the early-endosomal system (PMID:12121420, PMID:27189942). It binds phosphatidic acid through its helical domain, and this lipid interaction is sufficient to deform liposomes into tubules in vitro and is required for EHD3-positive tubules and early endosomal trafficking in cells (PMID:26896729). Functionally, EHD3 mediates early endosome-to-endocytic recycling compartment (ERC) transport via an EH domain–NPF interaction with the Rab11 effector Rab11-FIP2, a role distinct from EHD1's function in ERC exit (PMID:16251358); it also directs early endosome-to-Golgi transport required for Golgi morphology and lysosomal biosynthetic trafficking (PMID:19139087). EHD3 heterodimerizes with EHD1 (PMID:12121420), and the EH domain residues Asn-519/Glu-520 specify its preference for NPF partners and explain its tubule-stabilizing role versus EHD1-driven vesiculation (PMID:27189942); SUMOylation at K315 and K511 maintains its localization to tubular ERC structures and recycling activity (PMID:26226295). Through these trafficking activities EHD3 controls surface delivery and recycling of specific cargo, including β3/αvβ3-integrin (PMID:23781025) and the cardiac membrane proteins NCX1 and Cav1.2 in an ankyrin-B–associated complex (PMID:24759929), and it diverts activated EGFR toward degradation to spatially attenuate endosomal MAPK/AKT signaling (PMID:27811356). EHD3, together with EHD1 and the Rab11–Rab8 cascade, also generates ciliary vesicles from distal appendage vesicles at the mother centriole, a tubulation step required for ciliogenesis initiation (PMID:25686250).

Mechanistic history

Synthesis pass · year-by-year structured walk · 15 steps
  1. 2002 Medium

    Established EHD3 as a membrane-tubulating protein of the recycling system and identified its physical partnership with EHD1, defining a molecular foundation for its trafficking role.

    Evidence GFP-fusion localization with domain swapping, yeast two-hybrid and Co-IP in cells

    PMID:12121420

    Open questions at the time
    • Does not define the cargo handled by EHD3 tubules
    • Functional consequence of the EHD1–EHD3 interaction unresolved
  2. 2005 High

    Showed EHD3 binds the Rab11 effector Rab11-FIP2 via EH–NPF contacts and is required for early endosome-to-ERC delivery, distinguishing its trafficking step from EHD1.

    Evidence Co-IP with nucleotide-binding mutants, siRNA knockdown, transferrin recycling assay

    PMID:16251358

    Open questions at the time
    • Structural basis of EH–NPF selectivity not resolved
    • How nucleotide state controls FIP2 binding mechanistically unclear
  3. 2007 Medium

    Placed EHD3 in a Rab8a/Myosin Vb tubular network distinct from Rab11a compartments, refining the membrane subdomain it occupies.

    Evidence FRET and live-cell imaging with co-localization

    PMID:17507647

    Open questions at the time
    • EHD3 role is inferred from co-localization, not direct interaction
    • Functional contribution to this network untested
  4. 2009 High

    Extended EHD3 function beyond recycling to early endosome-to-Golgi transport, linking it to Golgi morphology and lysosomal enzyme delivery.

    Evidence siRNA knockdown with Shiga toxin, M6PR, and cathepsin D trafficking assays

    PMID:19139087

    Open questions at the time
    • Direct partner mediating Golgi-directed step beyond rabenosyn-5 not fully defined
    • Whether tubulation per se drives this route untested
  5. 2012 Medium

    Connected EHD3 expression to cardiac stress, showing it is upregulated across heart failure models downstream of ROS and angiotensin II in parallel with NCX1.

    Evidence Western blot across multiple HF models with pharmacological ROS/Ang II manipulation

    PMID:22406195

    Open questions at the time
    • Ang II/ROS link is pharmacological without genetic dissection
    • Transcriptional mechanism of upregulation unknown
  6. 2011 Medium

    Demonstrated in vivo physiological importance of EHD3 (with EHD4) in glomerular endothelial homeostasis via VEGFR2 recycling.

    Evidence Ehd3−/− and Ehd3−/−;Ehd4−/− mouse models with histopathology and receptor localization

    PMID:21408024

    Open questions at the time
    • Direct EHD3–VEGFR2 trafficking link is partly inferential
    • EHD3-specific vs EHD4-redundant contributions not separated
  7. 2013 Medium

    Identified EHD3 as a regulator of rapid β3-integrin recycling and αvβ3-mediated adhesion, defining a specific cargo of its short-loop recycling activity.

    Evidence siRNA knockdown, TIRF colocalization, integrin recycling and adhesion assays

    PMID:23781025

    Open questions at the time
    • Adaptor coupling EHD3 to integrin not identified
    • Single lab, single cell type
  8. 2013 Medium

    Revealed a tumor-suppressive function for EHD3 silenced by promoter hypermethylation in glioma, broadening its role to growth and survival control.

    Evidence siRNA, inducible re-expression, bisulfite sequencing, 5-Aza demethylation, xenografts

    PMID:24306026

    Open questions at the time
    • Molecular mechanism linking trafficking to growth suppression not dissected
    • Relationship to EGFR/signaling control unexplored here
  9. 2014 High

    Established EHD3 as essential for cardiac excitability through ankyrin-B–associated trafficking of NCX1 and Cav1.2 to the membrane.

    Evidence Cardiac-specific KO mouse, electrophysiology, Ca2+ imaging, Co-IP, localization

    PMID:24759929

    Open questions at the time
    • Order of EHD3/ankyrin-B/cargo assembly unresolved
    • Whether tubulation activity is required for cardiac cargo delivery untested
  10. 2015 High

    Showed SUMOylation at K315/K511 is a post-translational switch controlling EHD3 ERC tubule localization and recycling capacity.

    Evidence In vitro SUMOylation, acceptor-lysine mutagenesis, transferrin recycling assays

    PMID:26226295

    Open questions at the time
    • SUMO E3 ligase and signal triggering SUMOylation unknown
    • How SUMO controls tubule targeting mechanistically unclear
  11. 2015 High

    Defined EHD3's role in ciliogenesis initiation, generating ciliary vesicles from distal appendage vesicles via EHD-dependent tubulation within the Rab11–Rab8 cascade.

    Evidence siRNA, super-resolution and electron microscopy, live imaging, Co-IP, rescue

    PMID:25686250

    Open questions at the time
    • EHD3-specific vs EHD1-redundant contribution to DAV fusion not separated
    • Direct membrane-fusion machinery coupling not fully mapped
  12. 2016 High

    Mechanistically distinguished EHD3 from EHD1 by showing it stabilizes pre-formed tubular recycling endosomes, with EH residues N519/E520 specifying NPF-partner selectivity.

    Evidence Synchronized TRE regeneration (PLD inhibitor washout), EH domain mutagenesis

    PMID:27189942

    Open questions at the time
    • Structural model of N519/E520-dependent partner discrimination absent
    • TRE-stabilizing partners not enumerated
  13. 2016 High

    Provided the biochemical basis for EHD3 tubulation by showing its helical domain binds phosphatidic acid to deform membranes.

    Evidence Liposome co-sedimentation, PA synthesis inhibition, tubule quantification

    PMID:26896729

    Open questions at the time
    • High-resolution structure of the PA-bound tubulating state lacking
    • Whether ATPase cycle couples to PA-driven tubulation untested
  14. 2016 Medium

    Showed EHD3 spatially regulates EGFR signaling by promoting receptor ubiquitination and degradative routing, attenuating endosomal MAPK/AKT output.

    Evidence Inducible expression, EGFR ubiquitination and trafficking assays, pathway westerns

    PMID:27811356

    Open questions at the time
    • No in vitro reconstitution of the ubiquitination step
    • Ubiquitin ligase recruited by EHD3 not identified
  15. 2021 Medium

    Identified transcriptional control of EHD3 by NR5A1 and a physiological output in Leydig-cell testosterone synthesis via endocytosis.

    Evidence ChIP, luciferase reporter, siRNA, endocytosis assay, conditional NR5A1 KO mouse

    PMID:33964295

    Open questions at the time
    • Specific endocytic cargo driving steroidogenesis not defined
    • Direct vs indirect contribution of EHD3 to testosterone synthesis unresolved

Open questions

Synthesis pass · forward-looking unresolved questions
  • How EHD3's ATPase cycle, PA binding, SUMOylation, and EH–NPF partner selection are integrated into a single tubule-stabilization mechanism, and how this distinguishes the diverse cargo-specific roles, remains unresolved.
  • No integrated structural/biochemical model of the tubulation cycle
  • Determinants of cargo selectivity across cell types unknown

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0005198 structural molecule activity 3 GO:0060090 molecular adaptor activity 2 GO:0008289 lipid binding 1
Localization
GO:0005768 endosome 4 GO:0005886 plasma membrane 2 GO:0031410 cytoplasmic vesicle 2 GO:0005794 Golgi apparatus 1 GO:0005929 cilium 1
Pathway
R-HSA-5653656 Vesicle-mediated transport 4 R-HSA-9609507 Protein localization 2 R-HSA-162582 Signal Transduction 1 R-HSA-1852241 Organelle biogenesis and maintenance 1
Partners
ANK2EHD1MYO5BNCX1RAB11FIP2RAB8ASNAP29

Evidence

Reading pass · 15 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2002 EHD3 localizes to endocytic vesicles and microtubule-dependent membrane tubules, colocalizing with transferrin-containing recycling vesicles; the N-terminal domain is responsible for tubular localization. EHD1 and EHD3 interact via two-hybrid analysis and co-immunoprecipitation from cellular extracts, and coexpression results in colocalization in microtubule-dependent tubules. GFP-fusion localization, N-terminal domain swapping/deletion mutagenesis, yeast two-hybrid, co-immunoprecipitation Traffic Medium 12121420
2005 EHD3 binds the Rab11-effector Rab11-FIP2 via EH domain–NPF motif interactions; this association is affected by nucleotide-binding status. Knockdown of EHD3 prevents delivery of internalized transferrin and early endosomal proteins to the endocytic recycling compartment (ERC), demonstrating a role for EHD3 in early endosome-to-ERC transport distinct from EHD1's role in ERC exit. Co-immunoprecipitation, siRNA knockdown, transferrin recycling assay, subcellular localization imaging Molecular biology of the cell High 16251358
2007 Rab8a and Myosin Vb colocalize to a tubular network containing EHD1 and EHD3 (distinct from Rab11a-containing compartments), as demonstrated by live-cell FRET imaging and co-localization studies. Yeast two-hybrid, FRET, live-cell imaging, co-localization Molecular biology of the cell Medium 17507647
2009 siRNA knockdown of EHD3 (or its interaction partner rabenosyn-5) redistributes sorting nexin 1 to enlarged early endosomes, disrupts Shiga toxin B subunit transport to the Golgi, fragments Golgi morphology, reduces AP-1 gamma-adaptin recruitment to the Golgi, misroutes mannose 6-phosphate receptor to peripheral endosomes, and traps cathepsin D at the Golgi — establishing EHD3 as a regulator of early-endosome-to-Golgi transport required for Golgi morphology and lysosomal biosynthetic trafficking. siRNA knockdown, immunofluorescence, Shiga toxin transport assay, VSV-G secretion assay, mannose 6-phosphate receptor trafficking assay Journal of cell science High 19139087
2011 Mice doubly deficient for EHD3 and EHD4 develop thrombotic microangiopathy-like glomerular lesions with altered VEGFR2 expression and localization in glomerular endothelium and increased apoptosis, suggesting EHD3/EHD4-mediated endocytic recycling of VEGFR2 is essential for glomerular endothelial homeostasis. Ehd3−/− and Ehd3−/−;Ehd4−/− mouse models, histopathology, immunofluorescence, proteinuria measurement PloS one Medium 21408024
2013 siRNA knockdown or doxycycline-inducible restoration of EHD3 in glioma cell lines shows EHD3 decreases cell growth and invasiveness, induces cell cycle arrest and apoptosis; promoter hypermethylation silences EHD3 expression and is reversible by 5-Azacytidine; xenograft experiments confirm in vivo tumor suppressive activity. siRNA knockdown, doxycycline-inducible overexpression, bisulfite sequencing, 5-Azacytidine demethylation, xenograft mouse model, cell cycle/apoptosis assays Carcinogenesis Medium 24306026
2013 siRNA depletion of EHD3 in HeLa cells delays short-loop β3-integrin recycling from early endosomes back to the cell surface and impairs αvβ3-integrin-mediated cell adhesion. TIRF-based colocalization shows β3-integrin transits EHD3-positive endosomes near the cell surface, consistent with a rapid-recycling role. siRNA knockdown, live-cell TIRF microscopy, integrin recycling assay, cell adhesion assay Journal of cell science Medium 23781025
2014 EHD3-deficient mouse hearts display bradycardia, conduction block, and blunted adrenergic response. EHD3-deficient myocytes have reduced membrane expression/localization of Na/Ca exchanger (NCX1) and L-type Ca channel Cav1.2, reduced corresponding membrane currents, increased sarcoplasmic reticulum Ca2+ and spark frequency, and reduced ankyrin-B expression/localization. Ankyrin-B co-immunoprecipitates with EHD3 and NCX1, placing EHD3 in an endosome-based trafficking pathway for these cardiac membrane proteins. Cardiac-specific EHD3 KO mouse, electrophysiology, patch-clamp, immunofluorescence/confocal microscopy, co-immunoprecipitation, Ca2+ spark imaging Circulation research High 24759929
2012 EHD3 protein levels are consistently elevated in four different heart failure models (ischemic rat, pressure-overload mouse, pacing-induced canine, and failing human myocardium); NCX1 levels parallel EHD3 upregulation. EHD3 upregulation in heart failure is downstream of reactive oxygen species and angiotensin II signaling. Western blot across multiple HF models, ROS and angiotensin II pharmacological manipulation Journal of molecular and cellular cardiology Medium 22406195
2015 EHD3 undergoes SUMO modification at lysines K315 and K511, both in vitro and in cells. SUMOylation is required for EHD3 localization to tubular ERC structures; non-SUMOylated EHD3 acts as a dominant negative for tubulation and delays transferrin recycling from the ERC to the cell surface. SUMOylation does not affect EHD3 dimerization. In vitro SUMOylation assay, site-directed mutagenesis of SUMO acceptor lysines, transferrin recycling assay, immunofluorescence PloS one High 26226295
2015 EHD1 and EHD3, together with the Rab11–Rab8 cascade, localize to preciliary membranes and the ciliary pocket. EHD-dependent membrane tubulation is required to form ciliary vesicles from distal appendage vesicles (DAVs) at the mother centriole, a step necessary for basal body transformation, transition zone protein recruitment, and IFT20 recruitment before ciliary growth. SNAP29 (a SNARE and EHD1-binding protein) is also required for this DAV-to-ciliary-vesicle fusion step. siRNA knockdown, super-resolution and electron microscopy, live imaging, co-immunoprecipitation, rescue experiments Nature cell biology High 25686250
2016 EHD3 stabilizes tubular recycling endosomes (TRE) rather than initiating their biogenesis; in a synchronized TRE regeneration assay (phospholipase D inhibitor washout), EHD3 depletion did not prevent TRE formation but shortened their persistence. The residues Asn-519/Glu-520 in EHD3's EH domain (vs. Ala-519/Asp-520 in EHD1) define the differential selectivity of these paralogs for NPF-containing binding partners, explaining distinct roles: EHD1 in vesiculation vs. EHD3 in tubule stabilization. siRNA knockdown, phospholipase D inhibitor washout assay, site-directed EH domain mutagenesis, co-localization imaging The Journal of biological chemistry High 27189942
2016 EHD3 binds phosphatidic acid (PA) through its helical domain, as shown by in vitro liposome co-sedimentation. PA–EHD3 interaction induces liposomal tubulation in vitro. Inhibiting PA synthesis with diacylglycerol kinase inhibitor or lysophosphatidic acid acyltransferase inhibitor reduces EHD3-containing tubules and impairs early endosomal trafficking, establishing that PA cooperates with EHD3 to drive membrane tubulation. In vitro liposome co-sedimentation assay, pharmacological PA synthesis inhibition, immunofluorescence tubule counting Experimental cell research High 26896729
2016 EHD3 accelerates EGFR degradation upon EGF stimulation by increasing EGFR ubiquitination and diverting EGFR trafficking from the recycling route to the degradative pathway. EHD3 reduces endosome-based MAPK and AKT signaling downstream of EGFR without affecting total pathway activity, demonstrating spatial regulation of EGFR signaling. Doxycycline-inducible EHD3 expression, EGFR ubiquitination assay, endosomal trafficking/recycling assays, immunofluorescence, western blot for pathway activation Oncotarget Medium 27811356
2021 NR5A1 transcriptionally activates EHD3 by binding the conserved 'AGGTCA' sequence in the EHD3 promoter. EHD3 overexpression increases testosterone concentration; EHD3 knockdown decreases testosterone synthesis by reducing endocytosis in Leydig cells. Leydig-cell-specific NR5A1 knockout mice show reduced EHD3, clathrin, and serum testosterone levels. ChIP, dual luciferase reporter assay, siRNA knockdown, exosome tracing/endocytosis assay, conditional NR5A1 KO mouse (CRISPR/Cas9), ELISA 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 211 25686250
2005 Interactions between EHD proteins and Rab11-FIP2: a role for EHD3 in early endosomal transport. Molecular biology of the cell 160 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

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