{"gene":"EHD2","run_date":"2026-04-28T17:46:03","timeline":{"discoveries":[{"year":2003,"finding":"EHD2 contains an N-terminal P-loop and a C-terminal EH domain that interacts with NPF repeats in EHBP1; disruption of EHD2 or EHBP1 by siRNA inhibits transferrin and GLUT4 endocytosis into EEA1-positive endosomes; EHD2 localizes with cortical actin filaments and high expression causes extensive actin reorganization, linking clathrin-mediated endocytosis to the actin cytoskeleton.","method":"siRNA knockdown, Co-IP/pulldown, subcellular localization by fluorescence microscopy, endocytosis assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — reciprocal interaction mapping, siRNA KD with defined endocytic phenotype, actin localization; replicated by multiple subsequent studies","pmids":["14676205"],"is_preprint":false},{"year":2004,"finding":"EHD2 is present in purified GLUT4 vesicles of rat adipocytes and co-immunoprecipitates with GLUT4; insulin treatment selectively enhances this interaction in exocytic vesicle fractions; antibody or peptide blockade of EHD2 suppresses insulin-induced plasma membrane GLUT4 recruitment by up to 75%.","method":"MALDI-TOF MS identification from purified GLUT4 vesicles, co-immunoprecipitation, GST pulldown, SLO-permeabilized adipocyte trafficking assay","journal":"Biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 — multiple orthogonal methods in a single lab, functional inhibition assay confirming role","pmids":["15182197"],"is_preprint":false},{"year":2008,"finding":"EHD2 directly binds the second C2 domain of myoferlin; introduction of dominant-negative EHD2 sequesters myoferlin and inhibits myoblast fusion; myoferlin-null myoblasts accumulate transferrin and show delayed recycling, implicating EHD2 in endocytic recycling during myoblast membrane fusion.","method":"Direct binding assay (pulldown), dominant-negative overexpression, transferrin recycling assay, immunofluorescence","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — direct binding demonstrated, dominant-negative phenotype with specific cellular readout; replicated with Fer1L5 in follow-up study","pmids":["18502764"],"is_preprint":false},{"year":2010,"finding":"EHD2 (and EHD1) bind directly to the second C2 domain of Fer1L5; reduction of EHD1 and/or EHD2 inhibits myoblast fusion; EHD2 is required for normal translocation of Fer1L5 to the plasma membrane.","method":"Direct binding assay, siRNA knockdown, myoblast fusion assay, immunofluorescence localization","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — direct binding plus KD phenotype, single lab","pmids":["21177873"],"is_preprint":false},{"year":2010,"finding":"Prohibitin (PHB) undergoes palmitoylation at Cys69, which is required for its plasma membrane translocation; membrane-translocated PHB undergoes tyrosine phosphorylation and interacts with EHD2.","method":"Palmitoylation assay, site-directed mutagenesis (Cys69), co-immunoprecipitation, membrane fractionation","journal":"Biochemistry and cell biology","confidence":"Medium","confidence_rationale":"Tier 2 — mutagenesis plus co-IP, single lab, moderate mechanistic detail","pmids":["20555396"],"is_preprint":false},{"year":2011,"finding":"EHD2 interacts with Nek3 kinase and Vav1 (a GEF for Rho GTPases) as identified by yeast two-hybrid; wild-type EHD2 (but not P-loop mutants) reduces Rac1 activity; inhibitory effect on Rac1 partially rescued by Rac1 co-expression, placing EHD2 upstream of Rac1 in trafficking from the plasma membrane.","method":"Yeast two-hybrid, Rac1 activity assay (G-LISA/pull-down), dominant-negative/P-loop mutant overexpression, cholera toxin trafficking assay","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 2 — yeast two-hybrid plus functional Rac1 assay with mutants; single lab","pmids":["21756249"],"is_preprint":false},{"year":2012,"finding":"EHD2 is specifically and stably associated with caveolae at the plasma membrane (not clathrin-mediated endocytosis or endosomal recycling); EHD2 interacts with pacsin2 and cavin1; ordered membrane assembly requires cavin1 and caveolar integrity; a loop in the nucleotide-binding domain and ATP binding are required for caveolar localization; EHD2 stabilizes caveolae at the plasma membrane—its depletion results in more dynamic, short-lived, endocytic caveolae.","method":"Fluorescence microscopy, TIRF, siRNA knockdown, Co-IP, dominant-negative/mutant expression, live-cell imaging of caveolar dynamics","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods, reciprocal Co-IPs, live-cell imaging with KD phenotype, replicated independently","pmids":["22323287"],"is_preprint":false},{"year":2012,"finding":"EHD2 (an ATPase) associates with the static population of plasma membrane caveolae; recruitment requires ATP binding, anionic lipid interaction, and oligomerization into large (60-75S) complexes via EH domain interactions with intrinsic NPF/KPF motifs; ATP hydrolysis is essential for binding to caveolae; EHD2 undergoes dynamic exchange at caveolae dependent on the ATPase cycle; depletion increases mobile caveolar vesicles; overexpression confines cholera toxin B in caveolae; confining role relies on linking caveolae to actin filaments.","method":"Sedimentation (sucrose gradient), siRNA knockdown, dominant-negative mutant expression, FRAP, TIRF live imaging, actin co-localization","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1-2 — reconstitution-level biochemistry combined with live-cell imaging and mutagenesis; independently replicated","pmids":["22505029"],"is_preprint":false},{"year":2012,"finding":"EHD2 shuttles to the nucleus via a nuclear localization signal (NLS); nuclear export depends partially on a nuclear export signal (NES); elimination of a SUMOylation site causes major nuclear accumulation and SUMOylation is confirmed by co-immunoprecipitation and yeast two-hybrid; nuclear EHD2 represses transcription including p21WAF1/Cip1 (CDKN1A) as shown by GAL4 transactivation and KLF7-dependent transcription assays, confirmed by qRT-PCR in KD and OE cells.","method":"Nuclear export inhibitor treatment, NLS/NES mutagenesis, co-immunoprecipitation, yeast two-hybrid, GAL4 transactivation assay, qRT-PCR","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (localization, mutagenesis, transcription assay, KD/OE qRT-PCR) in single lab","pmids":["22448906"],"is_preprint":false},{"year":2012,"finding":"EHD2 accumulates at the site of laser-induced injury in human myotubes and at repair domes during sarcolemmal remodeling; EHD1 and a dominant-negative EHD2 mutant do not accumulate at injury sites, indicating a specific and ATPase-dependent role for EHD2 in sarcolemmal membrane repair.","method":"Live-cell laser injury assay, GFP-tagged protein localization, dominant-negative mutant expression, time-lapse fluorescence microscopy","journal":"Traffic","confidence":"Medium","confidence_rationale":"Tier 2 — direct localization with functional consequence and mutagenesis; single lab","pmids":["22679923"],"is_preprint":false},{"year":2013,"finding":"Phosphatidylinositol 4,5-bisphosphate (PIP2) is required for EHD2 plasma membrane localization; pharmacologic perturbation of PIP2 metabolism redistributes EHD2 away from the plasma membrane; EHD2 colocalizes with PIP2-enriched Arf6-generated vacuoles; actin disruption by cytochalasin D co-redistributes both PIP2 and EHD2 to actin aggregates, indicating PIP2 rather than actin per se controls EHD2 localization.","method":"Confocal microscopy, pharmacologic PIP2 perturbation, Arf6 dominant-active expression, cytochalasin D treatment","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 — multiple pharmacologic/genetic perturbations with localization readout; single lab","pmids":["24040268"],"is_preprint":false},{"year":2014,"finding":"EHD2 residues at the tip of the helical domain insert into the membrane (wedging mechanism creating curvature); the N terminus is folded into a hydrophobic pocket of the GTPase domain in solution and is released into the membrane upon membrane binding; cryo-EM shows N terminus is not essential for oligomerization but regulates targeting and stable association of EHD2 to caveolae.","method":"Electron paramagnetic resonance (EPR), X-ray crystallography, cryo-electron microscopy, site-directed mutagenesis","journal":"Structure","confidence":"High","confidence_rationale":"Tier 1 — crystal structure, cryo-EM, EPR with mutagenesis; multiple structural methods","pmids":["24508342"],"is_preprint":false},{"year":2015,"finding":"The NPF motif of the EHD2 unstructured loop is required for homo-dimerization and Syndapin2 binding, whereas the KPF motif phenylalanine is essential for plasma membrane localization; NPF-to-NAF mutation abolishes dimerization and Syndapin2 binding but maintains plasma membrane association; NPF-to-APA mutation preserves dimerization and Syndapin2 binding but increases nuclear localization.","method":"Mutagenesis (NPF→NAF, NPF→APA), co-immunoprecipitation, confocal microscopy, nuclear localization quantification","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 — systematic mutagenesis with binding and localization readouts; single lab","pmids":["25875965"],"is_preprint":false},{"year":2016,"finding":"A novel Rab10–EHBP1–EHD2 complex is essential for autophagic engulfment of lipid droplets (lipophagy) in hepatocytes; Rab10 activity increases during autophagy and recruits EHBP1 and EHD2 to nascent autophagic membranes at the lipid droplet surface; siRNA knockdown or GTPase-defective Rab10 leads to LD accumulation and impairs LC3 recruitment to the autophagosome.","method":"siRNA knockdown, GTPase-defective Rab10 expression, co-immunoprecipitation, immunofluorescence co-localization, autophagy assay","journal":"Science advances","confidence":"High","confidence_rationale":"Tier 2 — complex identified by Co-IP, functional KD phenotype with specific lipophagy readout, replicated with dominant-negative approach","pmids":["28028537"],"is_preprint":false},{"year":2017,"finding":"EHD2 is autoinhibited in solution via N-terminal residues and EH domain interactions; upon membrane binding EHD2 adopts an open conformation by tilting helical domains (shown by infrared reflection-absorption spectroscopy); ATP binding enables partial insertion into the membrane and G-domain-mediated oligomerization; ATP hydrolysis is coupled to detachment from the membrane; oligomerization in the membrane-bound state is required to restrict caveolae dynamics in cells.","method":"Infrared reflection-absorption spectroscopy (IRRAS), ATPase assay, mutagenesis, live-cell caveolae dynamics imaging","journal":"PNAS","confidence":"High","confidence_rationale":"Tier 1 — novel biophysical method combined with mutagenesis and cell-based functional validation; mechanistic cycle defined","pmids":["28223496"],"is_preprint":false},{"year":2018,"finding":"Under mechanical stress, EHD2 is rapidly released from caveolae, SUMOylated, and translocated to the nucleus where it regulates transcription of genes including caveolae constituents; EHD2 is required to maintain the caveolae reservoir during membrane tension variations; breast cancer cells lacking EHD2 show complete absence of caveolae and loss of mechanostress gene regulation, both rescued by EHD2 re-expression.","method":"Live-cell imaging of EHD2 under stretch, SUMOylation assay, nuclear fractionation, transcriptomics, metal-replica EM, EHD2 knockout/rescue in breast cancer cells","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (live imaging, biochemistry, EM, KO/rescue); mechanistic pathway from membrane to nucleus defined","pmids":["30348749"],"is_preprint":false},{"year":2018,"finding":"EHD2 positively regulates surface expression of cardiac KATP channels by stabilizing caveolar structures and reducing endocytosis rate; this effect is specific to EHD2 (EHD1, EHD3, EHD4 have no effect); dominant-negative EHD2 sensitizes cardiomyocytes to ischemic damage without changing unitary conductance or ATP sensitivity of KATP channels.","method":"Immunofluorescence, surface biotinylation, patch clamping, dominant-negative expression, cardiomyocyte ischemia assay","journal":"FASEB journal","confidence":"Medium","confidence_rationale":"Tier 2 — multiple assays (surface biotinylation, electrophysiology, ischemia readout) in single lab; EHD paralog specificity tested","pmids":["29133341"],"is_preprint":false},{"year":2019,"finding":"EHD2 controls caveolae-dependent fatty acid uptake; EHD2 global knockout in mice increases lipid droplet size in fat tissue and fatty acid uptake via a caveolae- and CD36-dependent pathway involving dynamin; elevated numbers of detached caveolae are found in adipose tissue lacking EHD2, and caveolar mobility is increased in EHD2-null MEFs; EHD2 expression is down-regulated in visceral fat of obese mouse models and obese patients.","method":"EHD2 knockout mouse, fatty acid uptake assay, electron microscopy, TIRF live imaging of caveolae mobility, tissue fractionation","journal":"PNAS","confidence":"High","confidence_rationale":"Tier 2 — in vivo KO model with multiple orthogonal cellular and biochemical readouts; replicated in MEFs and adipose tissue","pmids":["32170013"],"is_preprint":false},{"year":2019,"finding":"EHD2 controls caveolar dynamics to orchestrate eNOS activity; EHD2 deletion leads to increased detached caveolae in arteries, redistribution of eNOS from plasma membrane to internalized caveolae, decreased NO production, impaired mesenteric artery relaxation, and reduced cytosolic Ca2+ peaks in HUVECs after ATP stimulus.","method":"EHD2 knockout mouse, super-resolution imaging, NO measurement, pressure myography, Ca2+ imaging (HUVECs), eNOS immunolocalization","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 2 — in vivo KO with multiple functional readouts (vascular physiology, NO, Ca2+, eNOS localization)","pmids":["31600286"],"is_preprint":false},{"year":2019,"finding":"EHD2 protein expression is up-regulated at onset of adipocyte differentiation; siRNA-mediated EHD2 silencing impairs insulin sensitivity, lipid storage, and lipolysis; EHD2 localizes to caveolae near cell surface-associated lipid droplets; EHD2 overexpression increases lipolytic signaling and suppresses PPARγ transcription factor activity.","method":"siRNA knockdown, fluorescence imaging, insulin sensitivity assay, lipolysis assay, PPARγ activity assay, adipocyte differentiation model","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 — multiple functional assays in adipocyte model; single lab","pmids":["30811273"],"is_preprint":false},{"year":2021,"finding":"EHD2 localizes to plasma membrane-bound Dll4 and caveolae in endothelial cells; disruption of caveolae prevents EHD2 organization around Dll4 and reduces Dll4 internalization; EHD2 knockout in zebrafish increases dysmorphic vascular sprouts and reduces downstream Notch signaling, identifying EHD2 as a modulator of Dll4 endocytosis and Notch activation during blood vessel development.","method":"In vitro co-localization, caveolae disruption, EHD2 morpholino/knockout in zebrafish, Notch reporter assay, in vivo vascular imaging","journal":"Microcirculation","confidence":"Medium","confidence_rationale":"Tier 2 — in vivo zebrafish KO with signaling readout plus in vitro mechanistic experiments; single lab","pmids":["34820962"],"is_preprint":false},{"year":2022,"finding":"Super-resolution single-molecule localization shows PACSIN2 and EHD2 co-localize with caveolin-1 at typically sized caveolae; PACSIN2 F-BAR domain is positioned closer to the plasma membrane than EHD2 and caveolin-1, suggesting PACSIN2 connects caveolae to the plasma membrane while EHD2 is positioned more internally.","method":"3D single-molecule localization super-resolution microscopy (STORM/PALM), geometric clustering analysis","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 1-2 — super-resolution structural localization; single lab, no direct functional validation of spatial arrangement","pmids":["35834519"],"is_preprint":false},{"year":2023,"finding":"EHD2-dependent stabilization of plasma membrane caveolae maintains high cell surface expression of the SOCE-linked calcium channel Orai1; EHD2 shRNA knockdown and CRISPR-Cas9 knockout reduce store-operated calcium entry (SOCE) and impair tumorigenesis and metastasis in TNBC cells, rescued by mouse Ehd2 re-expression.","method":"shRNA knockdown, CRISPR-Cas9 KO, mouse Ehd2 rescue, Ca2+ imaging (SOCE assay), Orai1 surface expression, tumorigenesis and metastasis assays","journal":"eLife","confidence":"Medium","confidence_rationale":"Tier 2 — KO with rescue and multiple functional readouts; mechanistic link to Orai1 surface expression established; single lab","pmids":["36625722"],"is_preprint":false},{"year":2023,"finding":"EHD2 deficiency in adipocytes (EHD2 KO mice on high-fat diet and 3T3-L1 cells) is associated with deterioration of insulin signal transduction and impaired insulin-stimulated GLUT4 translocation; lack of EHD2 alters plasma membrane lipid and protein composition, reduces insulin receptor expression, and diminishes insulin-dependent SNARE protein complex formation.","method":"EHD2 KO mouse (high-fat diet), 3T3-L1 adipocyte siRNA KD, GLUT4 translocation assay, plasma membrane lipidomics/proteomics, insulin receptor expression, SNARE complex co-IP","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 — in vivo KO plus cell-based assays; multiple molecular readouts; single lab","pmids":["37703099"],"is_preprint":false},{"year":2025,"finding":"Cryo-electron tomography of EHD2 filaments on tubulated liposomes shows EHD2 forms a highly curved membrane scaffold that stabilizes a tubular geometry with periodic undulations; the N-terminal sequence inserts into the membrane and acts as a spacer between adjacent filaments; in endothelial cells lacking EHD2, caveolar necks become narrower and elongated.","method":"Cryo-electron tomography, tubulated liposome reconstitution, N-terminal deletion mutagenesis, EM of EHD2-null endothelial caveolae","journal":"bioRxiv (preprint)","confidence":"High","confidence_rationale":"Tier 1 — cryo-ET structural determination with reconstitution and mutagenesis plus cellular ultrastructural validation","pmids":["bio_10.1101_2025.06.05.658037"],"is_preprint":true}],"current_model":"EHD2 is a dynamin-related ATPase that forms ATP-dependent oligomeric filaments at caveolar necks to stabilize caveolae at the plasma membrane, restraining their dynamics and endocytosis; ATP binding promotes membrane insertion and open-conformation oligomerization while hydrolysis drives detachment; EHD2 interacts with pacsin2/PACSIN2, cavin1, EHBP1, and PIP2 for membrane targeting; under mechanical stress EHD2 is released from caveolae, SUMOylated, and translocated to the nucleus where it represses transcription; EHD2 also regulates endocytic recycling through interactions with myoferlin/Fer1L5, controls caveolae-dependent fatty acid uptake and Orai1/SOCE-mediated calcium entry, and modulates Dll4 endocytosis for Notch signaling in vascular development."},"narrative":{"teleology":[{"year":2003,"claim":"The first functional characterization of EHD2 established that its EH domain binds NPF motifs in EHBP1, that it localizes with cortical actin, and that loss of EHD2 or EHBP1 impairs clathrin-mediated endocytosis, placing EHD2 at the interface of endocytosis and actin remodeling.","evidence":"siRNA knockdown, Co-IP/pulldown, transferrin/GLUT4 endocytosis assays in cultured cells","pmids":["14676205"],"confidence":"High","gaps":["Whether EHD2 acts directly on clathrin-coated pits versus caveolae was not distinguished","Mechanism of actin reorganization by EHD2 undefined"]},{"year":2004,"claim":"Identification of EHD2 on purified GLUT4 vesicles and demonstration that blocking EHD2 suppresses insulin-stimulated GLUT4 surface delivery established a role in regulated exocytic trafficking in adipocytes.","evidence":"MALDI-TOF MS, co-IP, antibody/peptide blockade in SLO-permeabilized rat adipocytes","pmids":["15182197"],"confidence":"Medium","gaps":["Whether EHD2 acts on GLUT4 vesicle fusion or on caveolae-mediated surface retention was unresolved","Binding interface between EHD2 and GLUT4 vesicle components not mapped"]},{"year":2008,"claim":"Discovery of direct EHD2–myoferlin binding and the demonstration that dominant-negative EHD2 blocks myoblast fusion linked EHD2 to endocytic recycling and membrane fusion events during muscle differentiation.","evidence":"Direct binding assay, dominant-negative overexpression, transferrin recycling assay in myoblasts","pmids":["18502764"],"confidence":"High","gaps":["Whether EHD2 delivers specific membrane cargo for fusion was not determined","Relative contribution of EHD2 versus EHD1 to recycling unclear"]},{"year":2012,"claim":"Three independent studies converged to redefine EHD2 as a caveolae-specific stabilizer: EHD2 associates with static plasma membrane caveolae via ATP-dependent oligomerization and interactions with PACSIN2/cavin1; its depletion increases caveolar mobility and endocytosis; and a parallel study revealed that EHD2 also shuttles to the nucleus upon SUMOylation to repress transcription.","evidence":"TIRF/live-cell imaging, siRNA KD, FRAP, sedimentation, Co-IP, NLS/NES/SUMO mutagenesis, GAL4 transactivation and qRT-PCR","pmids":["22323287","22505029","22448906"],"confidence":"High","gaps":["Whether nuclear translocation is mechanically triggered was unknown","Structural basis of the autoinhibited-to-oligomeric transition not yet resolved","Direct transcriptional targets beyond CDKN1A not comprehensively identified"]},{"year":2014,"claim":"Crystal structure, cryo-EM, and EPR spectroscopy revealed that EHD2 inserts into membranes via helical-domain tip residues and that the N-terminal sequence is autoinhibitory in solution, released upon membrane binding, providing the structural basis for regulated membrane association.","evidence":"X-ray crystallography, cryo-EM, EPR, site-directed mutagenesis","pmids":["24508342"],"confidence":"High","gaps":["Full oligomeric filament structure on membranes not resolved","Structural coupling between ATP hydrolysis and membrane detachment unknown"]},{"year":2017,"claim":"Biophysical reconstitution defined the complete ATPase-coupled membrane cycle: autoinhibition in solution → ATP-driven open conformation and membrane insertion → G-domain oligomerization on the membrane → ATP hydrolysis triggering detachment, explaining how EHD2 dynamically maintains caveolar stability.","evidence":"IRRAS, ATPase assay, mutagenesis, live-cell caveolae dynamics imaging","pmids":["28223496"],"confidence":"High","gaps":["Whether hydrolysis occurs cooperatively within the oligomer was unresolved","Filament geometry on native caveolar necks not visualized"]},{"year":2018,"claim":"Mechanical stress was shown to trigger rapid EHD2 release from caveolae, SUMOylation, and nuclear translocation to regulate mechanoresponsive gene expression including caveolae components, establishing EHD2 as a mechanotransducer linking caveolar flattening to transcriptional programs.","evidence":"Live-cell imaging under stretch, SUMOylation assay, transcriptomics, metal-replica EM, KO/rescue in breast cancer cells","pmids":["30348749"],"confidence":"High","gaps":["Identity of the SUMO E3 ligase acting on EHD2 unknown","Whether nuclear EHD2 binds DNA directly or via cofactors not defined"]},{"year":2019,"claim":"EHD2 global knockout mice revealed that caveolae stabilization by EHD2 controls whole-organism lipid metabolism (fatty acid uptake via CD36), endothelial eNOS/NO production, and vascular relaxation, establishing physiological consequences of caveolar destabilization in vivo.","evidence":"EHD2 KO mouse, fatty acid uptake assay, TIRF, electron microscopy, pressure myography, Ca2+ imaging, NO measurement","pmids":["32170013","31600286"],"confidence":"High","gaps":["Whether phenotypes arise solely from increased caveolar endocytosis or also from altered signaling platform composition was unresolved","Compensatory mechanisms among EHD paralogs in vivo not characterized"]},{"year":2021,"claim":"EHD2 was shown to organize around Dll4 at caveolae in endothelial cells, and its knockout in zebrafish impaired Dll4 internalization and Notch signaling, revealing a role for EHD2-stabilized caveolae in vascular developmental signaling.","evidence":"Co-localization, caveolae disruption, EHD2 morpholino/KO in zebrafish, Notch reporter assay","pmids":["34820962"],"confidence":"Medium","gaps":["Direct physical interaction between EHD2 and Dll4 not demonstrated","Whether EHD2 promotes or restricts Dll4 endocytosis is context-dependent and mechanistically unresolved"]},{"year":2023,"claim":"EHD2 was found to maintain Orai1 surface expression and store-operated calcium entry via caveolar stabilization, and separately to be required for insulin-stimulated GLUT4 translocation and SNARE complex formation in adipocytes, broadening the set of physiological processes controlled by EHD2-dependent caveolar homeostasis.","evidence":"CRISPR-Cas9 KO with rescue, Ca2+ imaging, Orai1 surface expression; EHD2 KO mice on high-fat diet, GLUT4 translocation assay, plasma membrane proteomics/lipidomics","pmids":["36625722","37703099"],"confidence":"Medium","gaps":["Whether EHD2 directly interacts with Orai1 or acts indirectly via caveolae not resolved","How caveolar destabilization changes plasma membrane lipid composition mechanistically is unclear"]},{"year":null,"claim":"Key unresolved questions include: (1) the high-resolution structure of the EHD2 filament on native caveolar necks; (2) the identity of the SUMO ligase and chromatin targets mediating nuclear EHD2 transcriptional repression; and (3) how EHD2 coordinates with dynamin and other caveolar regulators to control the balance between caveolar stability and endocytic release.","evidence":"","pmids":[],"confidence":"Low","gaps":["In vivo filament architecture on caveolae at near-atomic resolution pending","Nuclear EHD2 transcriptional mechanism remains largely undefined","Functional interplay between EHD2 and dynamin at caveolae not dissected"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140657","term_label":"ATP-dependent activity","supporting_discovery_ids":[7,14,17]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[8,15]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[10,11]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[7,14,24]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,6,7,10,11,17]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[8,15]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[0,7]}],"pathway":[{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[6,7,17,22]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[15,20]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[13]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[17,19,23]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[8,15]}],"complexes":["Caveolae coat complex (with caveolin-1, cavin1, PACSIN2)"],"partners":["EHBP1","PACSIN2","CAVIN1","MYOF","FER1L5","CAV1","RAB10"],"other_free_text":[]},"mechanistic_narrative":"EHD2 is a dynamin-related ATPase that stabilizes caveolae at the plasma membrane by forming ATP-dependent oligomeric filament scaffolds around caveolar necks, thereby restraining caveolar dynamics, endocytosis, and membrane trafficking. ATP binding triggers release of an autoinhibitory N-terminal segment, enabling membrane insertion and G-domain-mediated oligomerization into large ring-like assemblies that tether caveolae to the cortical actin network via PIP2-dependent targeting and interactions with PACSIN2 and cavin1; ATP hydrolysis drives membrane detachment and dynamic exchange at caveolae [PMID:22505029, PMID:28223496, PMID:24508342]. By controlling caveolar stability, EHD2 regulates fatty acid uptake via CD36, Orai1-mediated store-operated calcium entry, eNOS-dependent NO production, Dll4/Notch signaling, KATP channel surface expression, and insulin-stimulated GLUT4 translocation [PMID:32170013, PMID:36625722, PMID:31600286, PMID:34820962, PMID:29133341, PMID:37703099]. Under mechanical stress EHD2 is released from caveolae, SUMOylated, and translocated to the nucleus where it functions as a transcriptional repressor regulating genes including caveolae components and CDKN1A [PMID:30348749, PMID:22448906]."},"prefetch_data":{"uniprot":{"accession":"Q9NZN4","full_name":"EH domain-containing protein 2","aliases":["PAST homolog 2"],"length_aa":543,"mass_kda":61.2,"function":"ATP- and membrane-binding protein that controls membrane reorganization/tubulation upon ATP hydrolysis (By similarity). Plays a role in membrane trafficking between the plasma membrane and endosomes (PubMed:17233914). Important for the internalization of GLUT4. Required for fusion of myoblasts to skeletal muscle myotubes. Required for normal translocation of FER1L5 to the plasma membrane (By similarity). Regulates the equilibrium between cell surface-associated and cell surface-dissociated caveolae by constraining caveolae at the cell membrane (PubMed:25588833)","subcellular_location":"Cell membrane; Membrane, caveola; Endosome membrane; Cytoplasm, cytosol","url":"https://www.uniprot.org/uniprotkb/Q9NZN4/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/EHD2","classification":"Not Classified","n_dependent_lines":3,"n_total_lines":1208,"dependency_fraction":0.0024834437086092716},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/EHD2","total_profiled":1310},"omim":[{"mim_id":"620883","title":"FER1-LIKE FAMILY, MEMBER 5; FER1L5","url":"https://www.omim.org/entry/620883"},{"mim_id":"618303","title":"CAVEOLAE-ASSOCIATED PROTEIN 3; CAVIN3","url":"https://www.omim.org/entry/618303"},{"mim_id":"614556","title":"AT-RICH INTERACTION DOMAIN-CONTAINING PROTEIN 1B; ARID1B","url":"https://www.omim.org/entry/614556"},{"mim_id":"609922","title":"EH DOMAIN-BINDING PROTEIN 1; EHBP1","url":"https://www.omim.org/entry/609922"},{"mim_id":"605892","title":"EH DOMAIN-CONTAINING 4; EHD4","url":"https://www.omim.org/entry/605892"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Plasma membrane","reliability":"Supported"},{"location":"Primary cilium","reliability":"Additional"},{"location":"Primary cilium transition zone","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in many","driving_tissues":[],"url":"https://www.proteinatlas.org/search/EHD2"},"hgnc":{"alias_symbol":[],"prev_symbol":["PAST2"]},"alphafold":{"accession":"Q9NZN4","domains":[{"cath_id":"1.10.268.20","chopping":"20-46_291-401","consensus_level":"high","plddt":94.8123,"start":20,"end":401},{"cath_id":"3.40.50.300","chopping":"60-284","consensus_level":"high","plddt":90.4301,"start":60,"end":284},{"cath_id":"1.10.238.10","chopping":"446-534","consensus_level":"high","plddt":90.9956,"start":446,"end":534}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9NZN4","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9NZN4-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9NZN4-F1-predicted_aligned_error_v6.png","plddt_mean":86.75},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=EHD2","jax_strain_url":"https://www.jax.org/strain/search?query=EHD2"},"sequence":{"accession":"Q9NZN4","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9NZN4.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9NZN4/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9NZN4"}},"corpus_meta":[{"pmid":"18790997","id":"PMC_18790997","title":"Ehd2, a rice ortholog of the maize INDETERMINATE1 gene, promotes flowering by up-regulating Ehd1.","date":"2008","source":"Plant physiology","url":"https://pubmed.ncbi.nlm.nih.gov/18790997","citation_count":198,"is_preprint":false},{"pmid":"22323287","id":"PMC_22323287","title":"EHD2 regulates caveolar dynamics via ATP-driven targeting and oligomerization.","date":"2012","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/22323287","citation_count":156,"is_preprint":false},{"pmid":"28028537","id":"PMC_28028537","title":"A novel Rab10-EHBP1-EHD2 complex essential for the autophagic engulfment of lipid droplets.","date":"2016","source":"Science advances","url":"https://pubmed.ncbi.nlm.nih.gov/28028537","citation_count":152,"is_preprint":false},{"pmid":"22505029","id":"PMC_22505029","title":"Oligomers of the ATPase EHD2 confine caveolae to the plasma membrane through association with actin.","date":"2012","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/22505029","citation_count":135,"is_preprint":false},{"pmid":"14676205","id":"PMC_14676205","title":"EHD2 and the novel EH domain binding protein EHBP1 couple endocytosis to the actin cytoskeleton.","date":"2003","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/14676205","citation_count":133,"is_preprint":false},{"pmid":"18502764","id":"PMC_18502764","title":"The endocytic recycling protein EHD2 interacts with myoferlin to regulate myoblast fusion.","date":"2008","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/18502764","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":"19392695","id":"PMC_19392695","title":"EHD2 inhibits ligand-induced endocytosis and signaling of the leucine-rich repeat receptor-like protein LeEix2.","date":"2009","source":"The Plant journal : for cell and molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/19392695","citation_count":73,"is_preprint":false},{"pmid":"30348749","id":"PMC_30348749","title":"EHD2 is a mechanotransducer connecting caveolae dynamics with gene transcription.","date":"2018","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/30348749","citation_count":61,"is_preprint":false},{"pmid":"21177873","id":"PMC_21177873","title":"Endocytic recycling proteins EHD1 and EHD2 interact with fer-1-like-5 (Fer1L5) and mediate myoblast fusion.","date":"2010","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/21177873","citation_count":51,"is_preprint":false},{"pmid":"32170013","id":"PMC_32170013","title":"EHD2-mediated restriction of caveolar dynamics regulates cellular fatty acid uptake.","date":"2020","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/32170013","citation_count":49,"is_preprint":false},{"pmid":"24508342","id":"PMC_24508342","title":"Structural insights into membrane interaction and caveolar targeting of dynamin-like EHD2.","date":"2014","source":"Structure (London, England : 1993)","url":"https://pubmed.ncbi.nlm.nih.gov/24508342","citation_count":43,"is_preprint":false},{"pmid":"22679923","id":"PMC_22679923","title":"Sarcolemmal repair is a slow process and includes EHD2.","date":"2012","source":"Traffic (Copenhagen, Denmark)","url":"https://pubmed.ncbi.nlm.nih.gov/22679923","citation_count":43,"is_preprint":false},{"pmid":"28223496","id":"PMC_28223496","title":"EHD2 restrains dynamics of caveolae by an ATP-dependent, membrane-bound, open conformation.","date":"2017","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/28223496","citation_count":43,"is_preprint":false},{"pmid":"15182197","id":"PMC_15182197","title":"EHD2 interacts with the insulin-responsive glucose transporter (GLUT4) in rat adipocytes and may participate in insulin-induced GLUT4 recruitment.","date":"2004","source":"Biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/15182197","citation_count":31,"is_preprint":false},{"pmid":"19936242","id":"PMC_19936242","title":"The coiled-coil domain of EHD2 mediates inhibition of LeEix2 endocytosis and signaling.","date":"2009","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/19936242","citation_count":31,"is_preprint":false},{"pmid":"23354948","id":"PMC_23354948","title":"Effects of EHD2 interference on migration of esophageal squamous cell carcinoma.","date":"2013","source":"Medical oncology (Northwood, London, England)","url":"https://pubmed.ncbi.nlm.nih.gov/23354948","citation_count":27,"is_preprint":false},{"pmid":"27572108","id":"PMC_27572108","title":"Phosphatidylinositol glycan anchor biosynthesis, class X containing complex promotes cancer cell proliferation through suppression of EHD2 and ZIC1, putative tumor suppressors.","date":"2016","source":"International journal of oncology","url":"https://pubmed.ncbi.nlm.nih.gov/27572108","citation_count":27,"is_preprint":false},{"pmid":"21756249","id":"PMC_21756249","title":"EHD2 mediates trafficking from the plasma membrane by modulating Rac1 activity.","date":"2011","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/21756249","citation_count":27,"is_preprint":false},{"pmid":"24040268","id":"PMC_24040268","title":"Role of phosphatidylinositol 4,5-bisphosphate in regulating EHD2 plasma membrane localization.","date":"2013","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/24040268","citation_count":26,"is_preprint":false},{"pmid":"20555396","id":"PMC_20555396","title":"Palmitoylation of prohibitin at cysteine 69 facilitates its membrane translocation and interaction with Eps 15 homology domain protein 2 (EHD2).","date":"2010","source":"Biochemistry and cell biology = Biochimie et biologie cellulaire","url":"https://pubmed.ncbi.nlm.nih.gov/20555396","citation_count":26,"is_preprint":false},{"pmid":"22448906","id":"PMC_22448906","title":"EHD2 shuttles to the nucleus and represses transcription.","date":"2012","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/22448906","citation_count":25,"is_preprint":false},{"pmid":"25557791","id":"PMC_25557791","title":"Role of EHD2 in migration and invasion of human breast cancer cells.","date":"2015","source":"Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine","url":"https://pubmed.ncbi.nlm.nih.gov/25557791","citation_count":23,"is_preprint":false},{"pmid":"30811273","id":"PMC_30811273","title":"EHD2 regulates adipocyte function and is enriched at cell surface-associated lipid droplets in primary human adipocytes.","date":"2019","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/30811273","citation_count":21,"is_preprint":false},{"pmid":"29133341","id":"PMC_29133341","title":"The trafficking protein, EHD2, positively regulates cardiac sarcolemmal KATP channel surface expression: role in cardioprotection.","date":"2018","source":"FASEB journal : official publication of the Federation of American Societies for Experimental Biology","url":"https://pubmed.ncbi.nlm.nih.gov/29133341","citation_count":21,"is_preprint":false},{"pmid":"37399924","id":"PMC_37399924","title":"Exogenous abscisic acid represses rice flowering via SAPK8-ABF1-Ehd1/Ehd2 pathway.","date":"2023","source":"Journal of advanced research","url":"https://pubmed.ncbi.nlm.nih.gov/37399924","citation_count":19,"is_preprint":false},{"pmid":"31600286","id":"PMC_31600286","title":"eNOS-NO-induced small blood vessel relaxation requires EHD2-dependent caveolae stabilization.","date":"2019","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/31600286","citation_count":16,"is_preprint":false},{"pmid":"24664435","id":"PMC_24664435","title":"Upregulation of EHD2 after intracerebral hemorrhage in adult rats.","date":"2014","source":"Journal of molecular neuroscience : MN","url":"https://pubmed.ncbi.nlm.nih.gov/24664435","citation_count":15,"is_preprint":false},{"pmid":"24347515","id":"PMC_24347515","title":"Scratching the surface: actin' and other roles for the C-terminal Eps15 homology domain protein, EHD2.","date":"2013","source":"Histology and histopathology","url":"https://pubmed.ncbi.nlm.nih.gov/24347515","citation_count":14,"is_preprint":false},{"pmid":"24280027","id":"PMC_24280027","title":"A point mutation in the zinc finger motif of RID1/EHD2/OsID1 protein leads to outstanding yield-related traits in japonica rice variety Wuyunjing 7.","date":"2013","source":"Rice (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/24280027","citation_count":14,"is_preprint":false},{"pmid":"32945345","id":"PMC_32945345","title":"TUSC8 inhibits the development of osteosarcoma by sponging miR‑197‑3p and targeting EHD2.","date":"2020","source":"International journal of molecular medicine","url":"https://pubmed.ncbi.nlm.nih.gov/32945345","citation_count":11,"is_preprint":false},{"pmid":"36625722","id":"PMC_36625722","title":"EHD2 overexpression promotes tumorigenesis and metastasis in triple-negative breast cancer by regulating store-operated calcium entry.","date":"2023","source":"eLife","url":"https://pubmed.ncbi.nlm.nih.gov/36625722","citation_count":10,"is_preprint":false},{"pmid":"19820301","id":"PMC_19820301","title":"EHD2 inhibits signaling of leucine rich repeat receptor-like proteins.","date":"2009","source":"Plant signaling & behavior","url":"https://pubmed.ncbi.nlm.nih.gov/19820301","citation_count":10,"is_preprint":false},{"pmid":"33356637","id":"PMC_33356637","title":"EHD2 Overexpression Suppresses the Proliferation, Migration, and Invasion in Human Colon Cancer.","date":"2021","source":"Cancer investigation","url":"https://pubmed.ncbi.nlm.nih.gov/33356637","citation_count":7,"is_preprint":false},{"pmid":"25875965","id":"PMC_25875965","title":"Role of the EHD2 unstructured loop in dimerization, protein binding and subcellular localization.","date":"2015","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/25875965","citation_count":7,"is_preprint":false},{"pmid":"33520362","id":"PMC_33520362","title":"Ese-3 contributes to colon cancer progression by downregulating EHD2 and transactivating INPP4B.","date":"2021","source":"American journal of cancer research","url":"https://pubmed.ncbi.nlm.nih.gov/33520362","citation_count":7,"is_preprint":false},{"pmid":"24154852","id":"PMC_24154852","title":"The function of EHD2 in endocytosis and defense signaling is affected by SUMO.","date":"2013","source":"Plant molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/24154852","citation_count":7,"is_preprint":false},{"pmid":"35834519","id":"PMC_35834519","title":"Super-resolution analysis of PACSIN2 and EHD2 at caveolae.","date":"2022","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/35834519","citation_count":6,"is_preprint":false},{"pmid":"34820962","id":"PMC_34820962","title":"EHD2 modulates Dll4 endocytosis during blood vessel development.","date":"2021","source":"Microcirculation (New York, N.Y. : 1994)","url":"https://pubmed.ncbi.nlm.nih.gov/34820962","citation_count":6,"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":"32409676","id":"PMC_32409676","title":"EHD2 is a Predictive Biomarker of Chemotherapy Efficacy in Triple Negative Breast Carcinoma.","date":"2020","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/32409676","citation_count":4,"is_preprint":false},{"pmid":"37703099","id":"PMC_37703099","title":"EHD2 regulates plasma membrane integrity and downstream insulin receptor signaling events.","date":"2023","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/37703099","citation_count":3,"is_preprint":false},{"pmid":"36041562","id":"PMC_36041562","title":"qHD5 encodes an AP2 factor that suppresses rice heading by down-regulating Ehd2 expression.","date":"2022","source":"Plant science : an international journal of experimental plant biology","url":"https://pubmed.ncbi.nlm.nih.gov/36041562","citation_count":3,"is_preprint":false},{"pmid":"38884792","id":"PMC_38884792","title":"OsCOL5 suppresses heading through modulation of Ghd7 and Ehd2, enhancing rice yield.","date":"2024","source":"TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik","url":"https://pubmed.ncbi.nlm.nih.gov/38884792","citation_count":3,"is_preprint":false},{"pmid":"36581344","id":"PMC_36581344","title":"Novel MYCBP::EHD2 and RUNX1::ZNF780A Fusion Genes in T-cell Acute Lymphoblastic Leukemia.","date":"2023","source":"Cancer genomics & proteomics","url":"https://pubmed.ncbi.nlm.nih.gov/36581344","citation_count":2,"is_preprint":false},{"pmid":"38613224","id":"PMC_38613224","title":"EH domain-containing protein 2 (EHD2): Overview, biological function, and therapeutic potential.","date":"2024","source":"Cell biochemistry and function","url":"https://pubmed.ncbi.nlm.nih.gov/38613224","citation_count":1,"is_preprint":false},{"pmid":"38059172","id":"PMC_38059172","title":"EHD2, a novel HIF target gene, is a promising biomarker in clear cell renal cell carcinoma.","date":"2023","source":"International journal of clinical and experimental pathology","url":"https://pubmed.ncbi.nlm.nih.gov/38059172","citation_count":0,"is_preprint":false},{"pmid":"32727647","id":"PMC_32727647","title":"[Unsaturated fatty acid of Actinidia chinesis planch seed oil (kiwi fruit essence) inhibits growth and metastasis of transplanted tumor in lung adenocarcinoma mice by up-regulating EHD2 expression].","date":"2020","source":"Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology","url":"https://pubmed.ncbi.nlm.nih.gov/32727647","citation_count":0,"is_preprint":false},{"pmid":"39861650","id":"PMC_39861650","title":"A Series of Novel Alleles of Ehd2 Modulating Heading and Salt Tolerance in Rice.","date":"2025","source":"Plants (Basel, Switzerland)","url":"https://pubmed.ncbi.nlm.nih.gov/39861650","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.06.05.658037","title":"Structures of EHD2 filaments on curved membranes provide a model for caveolar neck stabilization","date":"2025-06-07","source":"bioRxiv","url":"https://doi.org/10.1101/2025.06.05.658037","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":24575,"output_tokens":6437,"usd":0.08514},"stage2":{"model":"claude-opus-4-6","input_tokens":10039,"output_tokens":3162,"usd":0.193867},"total_usd":0.279007,"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\": 2003,\n      \"finding\": \"EHD2 contains an N-terminal P-loop and a C-terminal EH domain that interacts with NPF repeats in EHBP1; disruption of EHD2 or EHBP1 by siRNA inhibits transferrin and GLUT4 endocytosis into EEA1-positive endosomes; EHD2 localizes with cortical actin filaments and high expression causes extensive actin reorganization, linking clathrin-mediated endocytosis to the actin cytoskeleton.\",\n      \"method\": \"siRNA knockdown, Co-IP/pulldown, subcellular localization by fluorescence microscopy, endocytosis assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal interaction mapping, siRNA KD with defined endocytic phenotype, actin localization; replicated by multiple subsequent studies\",\n      \"pmids\": [\"14676205\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"EHD2 is present in purified GLUT4 vesicles of rat adipocytes and co-immunoprecipitates with GLUT4; insulin treatment selectively enhances this interaction in exocytic vesicle fractions; antibody or peptide blockade of EHD2 suppresses insulin-induced plasma membrane GLUT4 recruitment by up to 75%.\",\n      \"method\": \"MALDI-TOF MS identification from purified GLUT4 vesicles, co-immunoprecipitation, GST pulldown, SLO-permeabilized adipocyte trafficking assay\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods in a single lab, functional inhibition assay confirming role\",\n      \"pmids\": [\"15182197\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"EHD2 directly binds the second C2 domain of myoferlin; introduction of dominant-negative EHD2 sequesters myoferlin and inhibits myoblast fusion; myoferlin-null myoblasts accumulate transferrin and show delayed recycling, implicating EHD2 in endocytic recycling during myoblast membrane fusion.\",\n      \"method\": \"Direct binding assay (pulldown), dominant-negative overexpression, transferrin recycling assay, immunofluorescence\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct binding demonstrated, dominant-negative phenotype with specific cellular readout; replicated with Fer1L5 in follow-up study\",\n      \"pmids\": [\"18502764\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"EHD2 (and EHD1) bind directly to the second C2 domain of Fer1L5; reduction of EHD1 and/or EHD2 inhibits myoblast fusion; EHD2 is required for normal translocation of Fer1L5 to the plasma membrane.\",\n      \"method\": \"Direct binding assay, siRNA knockdown, myoblast fusion assay, immunofluorescence localization\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct binding plus KD phenotype, single lab\",\n      \"pmids\": [\"21177873\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Prohibitin (PHB) undergoes palmitoylation at Cys69, which is required for its plasma membrane translocation; membrane-translocated PHB undergoes tyrosine phosphorylation and interacts with EHD2.\",\n      \"method\": \"Palmitoylation assay, site-directed mutagenesis (Cys69), co-immunoprecipitation, membrane fractionation\",\n      \"journal\": \"Biochemistry and cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — mutagenesis plus co-IP, single lab, moderate mechanistic detail\",\n      \"pmids\": [\"20555396\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"EHD2 interacts with Nek3 kinase and Vav1 (a GEF for Rho GTPases) as identified by yeast two-hybrid; wild-type EHD2 (but not P-loop mutants) reduces Rac1 activity; inhibitory effect on Rac1 partially rescued by Rac1 co-expression, placing EHD2 upstream of Rac1 in trafficking from the plasma membrane.\",\n      \"method\": \"Yeast two-hybrid, Rac1 activity assay (G-LISA/pull-down), dominant-negative/P-loop mutant overexpression, cholera toxin trafficking assay\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — yeast two-hybrid plus functional Rac1 assay with mutants; single lab\",\n      \"pmids\": [\"21756249\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"EHD2 is specifically and stably associated with caveolae at the plasma membrane (not clathrin-mediated endocytosis or endosomal recycling); EHD2 interacts with pacsin2 and cavin1; ordered membrane assembly requires cavin1 and caveolar integrity; a loop in the nucleotide-binding domain and ATP binding are required for caveolar localization; EHD2 stabilizes caveolae at the plasma membrane—its depletion results in more dynamic, short-lived, endocytic caveolae.\",\n      \"method\": \"Fluorescence microscopy, TIRF, siRNA knockdown, Co-IP, dominant-negative/mutant expression, live-cell imaging of caveolar dynamics\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods, reciprocal Co-IPs, live-cell imaging with KD phenotype, replicated independently\",\n      \"pmids\": [\"22323287\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"EHD2 (an ATPase) associates with the static population of plasma membrane caveolae; recruitment requires ATP binding, anionic lipid interaction, and oligomerization into large (60-75S) complexes via EH domain interactions with intrinsic NPF/KPF motifs; ATP hydrolysis is essential for binding to caveolae; EHD2 undergoes dynamic exchange at caveolae dependent on the ATPase cycle; depletion increases mobile caveolar vesicles; overexpression confines cholera toxin B in caveolae; confining role relies on linking caveolae to actin filaments.\",\n      \"method\": \"Sedimentation (sucrose gradient), siRNA knockdown, dominant-negative mutant expression, FRAP, TIRF live imaging, actin co-localization\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — reconstitution-level biochemistry combined with live-cell imaging and mutagenesis; independently replicated\",\n      \"pmids\": [\"22505029\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"EHD2 shuttles to the nucleus via a nuclear localization signal (NLS); nuclear export depends partially on a nuclear export signal (NES); elimination of a SUMOylation site causes major nuclear accumulation and SUMOylation is confirmed by co-immunoprecipitation and yeast two-hybrid; nuclear EHD2 represses transcription including p21WAF1/Cip1 (CDKN1A) as shown by GAL4 transactivation and KLF7-dependent transcription assays, confirmed by qRT-PCR in KD and OE cells.\",\n      \"method\": \"Nuclear export inhibitor treatment, NLS/NES mutagenesis, co-immunoprecipitation, yeast two-hybrid, GAL4 transactivation assay, qRT-PCR\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (localization, mutagenesis, transcription assay, KD/OE qRT-PCR) in single lab\",\n      \"pmids\": [\"22448906\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"EHD2 accumulates at the site of laser-induced injury in human myotubes and at repair domes during sarcolemmal remodeling; EHD1 and a dominant-negative EHD2 mutant do not accumulate at injury sites, indicating a specific and ATPase-dependent role for EHD2 in sarcolemmal membrane repair.\",\n      \"method\": \"Live-cell laser injury assay, GFP-tagged protein localization, dominant-negative mutant expression, time-lapse fluorescence microscopy\",\n      \"journal\": \"Traffic\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct localization with functional consequence and mutagenesis; single lab\",\n      \"pmids\": [\"22679923\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Phosphatidylinositol 4,5-bisphosphate (PIP2) is required for EHD2 plasma membrane localization; pharmacologic perturbation of PIP2 metabolism redistributes EHD2 away from the plasma membrane; EHD2 colocalizes with PIP2-enriched Arf6-generated vacuoles; actin disruption by cytochalasin D co-redistributes both PIP2 and EHD2 to actin aggregates, indicating PIP2 rather than actin per se controls EHD2 localization.\",\n      \"method\": \"Confocal microscopy, pharmacologic PIP2 perturbation, Arf6 dominant-active expression, cytochalasin D treatment\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple pharmacologic/genetic perturbations with localization readout; single lab\",\n      \"pmids\": [\"24040268\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"EHD2 residues at the tip of the helical domain insert into the membrane (wedging mechanism creating curvature); the N terminus is folded into a hydrophobic pocket of the GTPase domain in solution and is released into the membrane upon membrane binding; cryo-EM shows N terminus is not essential for oligomerization but regulates targeting and stable association of EHD2 to caveolae.\",\n      \"method\": \"Electron paramagnetic resonance (EPR), X-ray crystallography, cryo-electron microscopy, site-directed mutagenesis\",\n      \"journal\": \"Structure\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure, cryo-EM, EPR with mutagenesis; multiple structural methods\",\n      \"pmids\": [\"24508342\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"The NPF motif of the EHD2 unstructured loop is required for homo-dimerization and Syndapin2 binding, whereas the KPF motif phenylalanine is essential for plasma membrane localization; NPF-to-NAF mutation abolishes dimerization and Syndapin2 binding but maintains plasma membrane association; NPF-to-APA mutation preserves dimerization and Syndapin2 binding but increases nuclear localization.\",\n      \"method\": \"Mutagenesis (NPF→NAF, NPF→APA), co-immunoprecipitation, confocal microscopy, nuclear localization quantification\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — systematic mutagenesis with binding and localization readouts; single lab\",\n      \"pmids\": [\"25875965\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"A novel Rab10–EHBP1–EHD2 complex is essential for autophagic engulfment of lipid droplets (lipophagy) in hepatocytes; Rab10 activity increases during autophagy and recruits EHBP1 and EHD2 to nascent autophagic membranes at the lipid droplet surface; siRNA knockdown or GTPase-defective Rab10 leads to LD accumulation and impairs LC3 recruitment to the autophagosome.\",\n      \"method\": \"siRNA knockdown, GTPase-defective Rab10 expression, co-immunoprecipitation, immunofluorescence co-localization, autophagy assay\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — complex identified by Co-IP, functional KD phenotype with specific lipophagy readout, replicated with dominant-negative approach\",\n      \"pmids\": [\"28028537\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"EHD2 is autoinhibited in solution via N-terminal residues and EH domain interactions; upon membrane binding EHD2 adopts an open conformation by tilting helical domains (shown by infrared reflection-absorption spectroscopy); ATP binding enables partial insertion into the membrane and G-domain-mediated oligomerization; ATP hydrolysis is coupled to detachment from the membrane; oligomerization in the membrane-bound state is required to restrict caveolae dynamics in cells.\",\n      \"method\": \"Infrared reflection-absorption spectroscopy (IRRAS), ATPase assay, mutagenesis, live-cell caveolae dynamics imaging\",\n      \"journal\": \"PNAS\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — novel biophysical method combined with mutagenesis and cell-based functional validation; mechanistic cycle defined\",\n      \"pmids\": [\"28223496\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Under mechanical stress, EHD2 is rapidly released from caveolae, SUMOylated, and translocated to the nucleus where it regulates transcription of genes including caveolae constituents; EHD2 is required to maintain the caveolae reservoir during membrane tension variations; breast cancer cells lacking EHD2 show complete absence of caveolae and loss of mechanostress gene regulation, both rescued by EHD2 re-expression.\",\n      \"method\": \"Live-cell imaging of EHD2 under stretch, SUMOylation assay, nuclear fractionation, transcriptomics, metal-replica EM, EHD2 knockout/rescue in breast cancer cells\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (live imaging, biochemistry, EM, KO/rescue); mechanistic pathway from membrane to nucleus defined\",\n      \"pmids\": [\"30348749\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"EHD2 positively regulates surface expression of cardiac KATP channels by stabilizing caveolar structures and reducing endocytosis rate; this effect is specific to EHD2 (EHD1, EHD3, EHD4 have no effect); dominant-negative EHD2 sensitizes cardiomyocytes to ischemic damage without changing unitary conductance or ATP sensitivity of KATP channels.\",\n      \"method\": \"Immunofluorescence, surface biotinylation, patch clamping, dominant-negative expression, cardiomyocyte ischemia assay\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple assays (surface biotinylation, electrophysiology, ischemia readout) in single lab; EHD paralog specificity tested\",\n      \"pmids\": [\"29133341\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"EHD2 controls caveolae-dependent fatty acid uptake; EHD2 global knockout in mice increases lipid droplet size in fat tissue and fatty acid uptake via a caveolae- and CD36-dependent pathway involving dynamin; elevated numbers of detached caveolae are found in adipose tissue lacking EHD2, and caveolar mobility is increased in EHD2-null MEFs; EHD2 expression is down-regulated in visceral fat of obese mouse models and obese patients.\",\n      \"method\": \"EHD2 knockout mouse, fatty acid uptake assay, electron microscopy, TIRF live imaging of caveolae mobility, tissue fractionation\",\n      \"journal\": \"PNAS\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — in vivo KO model with multiple orthogonal cellular and biochemical readouts; replicated in MEFs and adipose tissue\",\n      \"pmids\": [\"32170013\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"EHD2 controls caveolar dynamics to orchestrate eNOS activity; EHD2 deletion leads to increased detached caveolae in arteries, redistribution of eNOS from plasma membrane to internalized caveolae, decreased NO production, impaired mesenteric artery relaxation, and reduced cytosolic Ca2+ peaks in HUVECs after ATP stimulus.\",\n      \"method\": \"EHD2 knockout mouse, super-resolution imaging, NO measurement, pressure myography, Ca2+ imaging (HUVECs), eNOS immunolocalization\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — in vivo KO with multiple functional readouts (vascular physiology, NO, Ca2+, eNOS localization)\",\n      \"pmids\": [\"31600286\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"EHD2 protein expression is up-regulated at onset of adipocyte differentiation; siRNA-mediated EHD2 silencing impairs insulin sensitivity, lipid storage, and lipolysis; EHD2 localizes to caveolae near cell surface-associated lipid droplets; EHD2 overexpression increases lipolytic signaling and suppresses PPARγ transcription factor activity.\",\n      \"method\": \"siRNA knockdown, fluorescence imaging, insulin sensitivity assay, lipolysis assay, PPARγ activity assay, adipocyte differentiation model\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple functional assays in adipocyte model; single lab\",\n      \"pmids\": [\"30811273\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"EHD2 localizes to plasma membrane-bound Dll4 and caveolae in endothelial cells; disruption of caveolae prevents EHD2 organization around Dll4 and reduces Dll4 internalization; EHD2 knockout in zebrafish increases dysmorphic vascular sprouts and reduces downstream Notch signaling, identifying EHD2 as a modulator of Dll4 endocytosis and Notch activation during blood vessel development.\",\n      \"method\": \"In vitro co-localization, caveolae disruption, EHD2 morpholino/knockout in zebrafish, Notch reporter assay, in vivo vascular imaging\",\n      \"journal\": \"Microcirculation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vivo zebrafish KO with signaling readout plus in vitro mechanistic experiments; single lab\",\n      \"pmids\": [\"34820962\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Super-resolution single-molecule localization shows PACSIN2 and EHD2 co-localize with caveolin-1 at typically sized caveolae; PACSIN2 F-BAR domain is positioned closer to the plasma membrane than EHD2 and caveolin-1, suggesting PACSIN2 connects caveolae to the plasma membrane while EHD2 is positioned more internally.\",\n      \"method\": \"3D single-molecule localization super-resolution microscopy (STORM/PALM), geometric clustering analysis\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 — super-resolution structural localization; single lab, no direct functional validation of spatial arrangement\",\n      \"pmids\": [\"35834519\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"EHD2-dependent stabilization of plasma membrane caveolae maintains high cell surface expression of the SOCE-linked calcium channel Orai1; EHD2 shRNA knockdown and CRISPR-Cas9 knockout reduce store-operated calcium entry (SOCE) and impair tumorigenesis and metastasis in TNBC cells, rescued by mouse Ehd2 re-expression.\",\n      \"method\": \"shRNA knockdown, CRISPR-Cas9 KO, mouse Ehd2 rescue, Ca2+ imaging (SOCE assay), Orai1 surface expression, tumorigenesis and metastasis assays\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — KO with rescue and multiple functional readouts; mechanistic link to Orai1 surface expression established; single lab\",\n      \"pmids\": [\"36625722\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"EHD2 deficiency in adipocytes (EHD2 KO mice on high-fat diet and 3T3-L1 cells) is associated with deterioration of insulin signal transduction and impaired insulin-stimulated GLUT4 translocation; lack of EHD2 alters plasma membrane lipid and protein composition, reduces insulin receptor expression, and diminishes insulin-dependent SNARE protein complex formation.\",\n      \"method\": \"EHD2 KO mouse (high-fat diet), 3T3-L1 adipocyte siRNA KD, GLUT4 translocation assay, plasma membrane lipidomics/proteomics, insulin receptor expression, SNARE complex co-IP\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vivo KO plus cell-based assays; multiple molecular readouts; single lab\",\n      \"pmids\": [\"37703099\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Cryo-electron tomography of EHD2 filaments on tubulated liposomes shows EHD2 forms a highly curved membrane scaffold that stabilizes a tubular geometry with periodic undulations; the N-terminal sequence inserts into the membrane and acts as a spacer between adjacent filaments; in endothelial cells lacking EHD2, caveolar necks become narrower and elongated.\",\n      \"method\": \"Cryo-electron tomography, tubulated liposome reconstitution, N-terminal deletion mutagenesis, EM of EHD2-null endothelial caveolae\",\n      \"journal\": \"bioRxiv (preprint)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — cryo-ET structural determination with reconstitution and mutagenesis plus cellular ultrastructural validation\",\n      \"pmids\": [\"bio_10.1101_2025.06.05.658037\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"EHD2 is a dynamin-related ATPase that forms ATP-dependent oligomeric filaments at caveolar necks to stabilize caveolae at the plasma membrane, restraining their dynamics and endocytosis; ATP binding promotes membrane insertion and open-conformation oligomerization while hydrolysis drives detachment; EHD2 interacts with pacsin2/PACSIN2, cavin1, EHBP1, and PIP2 for membrane targeting; under mechanical stress EHD2 is released from caveolae, SUMOylated, and translocated to the nucleus where it represses transcription; EHD2 also regulates endocytic recycling through interactions with myoferlin/Fer1L5, controls caveolae-dependent fatty acid uptake and Orai1/SOCE-mediated calcium entry, and modulates Dll4 endocytosis for Notch signaling in vascular development.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"EHD2 is a dynamin-related ATPase that stabilizes caveolae at the plasma membrane by forming ATP-dependent oligomeric filament scaffolds around caveolar necks, thereby restraining caveolar dynamics, endocytosis, and membrane trafficking. ATP binding triggers release of an autoinhibitory N-terminal segment, enabling membrane insertion and G-domain-mediated oligomerization into large ring-like assemblies that tether caveolae to the cortical actin network via PIP2-dependent targeting and interactions with PACSIN2 and cavin1; ATP hydrolysis drives membrane detachment and dynamic exchange at caveolae [PMID:22505029, PMID:28223496, PMID:24508342]. By controlling caveolar stability, EHD2 regulates fatty acid uptake via CD36, Orai1-mediated store-operated calcium entry, eNOS-dependent NO production, Dll4/Notch signaling, KATP channel surface expression, and insulin-stimulated GLUT4 translocation [PMID:32170013, PMID:36625722, PMID:31600286, PMID:34820962, PMID:29133341, PMID:37703099]. Under mechanical stress EHD2 is released from caveolae, SUMOylated, and translocated to the nucleus where it functions as a transcriptional repressor regulating genes including caveolae components and CDKN1A [PMID:30348749, PMID:22448906].\",\n  \"teleology\": [\n    {\n      \"year\": 2003,\n      \"claim\": \"The first functional characterization of EHD2 established that its EH domain binds NPF motifs in EHBP1, that it localizes with cortical actin, and that loss of EHD2 or EHBP1 impairs clathrin-mediated endocytosis, placing EHD2 at the interface of endocytosis and actin remodeling.\",\n      \"evidence\": \"siRNA knockdown, Co-IP/pulldown, transferrin/GLUT4 endocytosis assays in cultured cells\",\n      \"pmids\": [\"14676205\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether EHD2 acts directly on clathrin-coated pits versus caveolae was not distinguished\", \"Mechanism of actin reorganization by EHD2 undefined\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Identification of EHD2 on purified GLUT4 vesicles and demonstration that blocking EHD2 suppresses insulin-stimulated GLUT4 surface delivery established a role in regulated exocytic trafficking in adipocytes.\",\n      \"evidence\": \"MALDI-TOF MS, co-IP, antibody/peptide blockade in SLO-permeabilized rat adipocytes\",\n      \"pmids\": [\"15182197\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether EHD2 acts on GLUT4 vesicle fusion or on caveolae-mediated surface retention was unresolved\", \"Binding interface between EHD2 and GLUT4 vesicle components not mapped\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Discovery of direct EHD2–myoferlin binding and the demonstration that dominant-negative EHD2 blocks myoblast fusion linked EHD2 to endocytic recycling and membrane fusion events during muscle differentiation.\",\n      \"evidence\": \"Direct binding assay, dominant-negative overexpression, transferrin recycling assay in myoblasts\",\n      \"pmids\": [\"18502764\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether EHD2 delivers specific membrane cargo for fusion was not determined\", \"Relative contribution of EHD2 versus EHD1 to recycling unclear\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Three independent studies converged to redefine EHD2 as a caveolae-specific stabilizer: EHD2 associates with static plasma membrane caveolae via ATP-dependent oligomerization and interactions with PACSIN2/cavin1; its depletion increases caveolar mobility and endocytosis; and a parallel study revealed that EHD2 also shuttles to the nucleus upon SUMOylation to repress transcription.\",\n      \"evidence\": \"TIRF/live-cell imaging, siRNA KD, FRAP, sedimentation, Co-IP, NLS/NES/SUMO mutagenesis, GAL4 transactivation and qRT-PCR\",\n      \"pmids\": [\"22323287\", \"22505029\", \"22448906\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether nuclear translocation is mechanically triggered was unknown\", \"Structural basis of the autoinhibited-to-oligomeric transition not yet resolved\", \"Direct transcriptional targets beyond CDKN1A not comprehensively identified\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Crystal structure, cryo-EM, and EPR spectroscopy revealed that EHD2 inserts into membranes via helical-domain tip residues and that the N-terminal sequence is autoinhibitory in solution, released upon membrane binding, providing the structural basis for regulated membrane association.\",\n      \"evidence\": \"X-ray crystallography, cryo-EM, EPR, site-directed mutagenesis\",\n      \"pmids\": [\"24508342\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full oligomeric filament structure on membranes not resolved\", \"Structural coupling between ATP hydrolysis and membrane detachment unknown\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Biophysical reconstitution defined the complete ATPase-coupled membrane cycle: autoinhibition in solution → ATP-driven open conformation and membrane insertion → G-domain oligomerization on the membrane → ATP hydrolysis triggering detachment, explaining how EHD2 dynamically maintains caveolar stability.\",\n      \"evidence\": \"IRRAS, ATPase assay, mutagenesis, live-cell caveolae dynamics imaging\",\n      \"pmids\": [\"28223496\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether hydrolysis occurs cooperatively within the oligomer was unresolved\", \"Filament geometry on native caveolar necks not visualized\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Mechanical stress was shown to trigger rapid EHD2 release from caveolae, SUMOylation, and nuclear translocation to regulate mechanoresponsive gene expression including caveolae components, establishing EHD2 as a mechanotransducer linking caveolar flattening to transcriptional programs.\",\n      \"evidence\": \"Live-cell imaging under stretch, SUMOylation assay, transcriptomics, metal-replica EM, KO/rescue in breast cancer cells\",\n      \"pmids\": [\"30348749\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the SUMO E3 ligase acting on EHD2 unknown\", \"Whether nuclear EHD2 binds DNA directly or via cofactors not defined\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"EHD2 global knockout mice revealed that caveolae stabilization by EHD2 controls whole-organism lipid metabolism (fatty acid uptake via CD36), endothelial eNOS/NO production, and vascular relaxation, establishing physiological consequences of caveolar destabilization in vivo.\",\n      \"evidence\": \"EHD2 KO mouse, fatty acid uptake assay, TIRF, electron microscopy, pressure myography, Ca2+ imaging, NO measurement\",\n      \"pmids\": [\"32170013\", \"31600286\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether phenotypes arise solely from increased caveolar endocytosis or also from altered signaling platform composition was unresolved\", \"Compensatory mechanisms among EHD paralogs in vivo not characterized\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"EHD2 was shown to organize around Dll4 at caveolae in endothelial cells, and its knockout in zebrafish impaired Dll4 internalization and Notch signaling, revealing a role for EHD2-stabilized caveolae in vascular developmental signaling.\",\n      \"evidence\": \"Co-localization, caveolae disruption, EHD2 morpholino/KO in zebrafish, Notch reporter assay\",\n      \"pmids\": [\"34820962\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct physical interaction between EHD2 and Dll4 not demonstrated\", \"Whether EHD2 promotes or restricts Dll4 endocytosis is context-dependent and mechanistically unresolved\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"EHD2 was found to maintain Orai1 surface expression and store-operated calcium entry via caveolar stabilization, and separately to be required for insulin-stimulated GLUT4 translocation and SNARE complex formation in adipocytes, broadening the set of physiological processes controlled by EHD2-dependent caveolar homeostasis.\",\n      \"evidence\": \"CRISPR-Cas9 KO with rescue, Ca2+ imaging, Orai1 surface expression; EHD2 KO mice on high-fat diet, GLUT4 translocation assay, plasma membrane proteomics/lipidomics\",\n      \"pmids\": [\"36625722\", \"37703099\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether EHD2 directly interacts with Orai1 or acts indirectly via caveolae not resolved\", \"How caveolar destabilization changes plasma membrane lipid composition mechanistically is unclear\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include: (1) the high-resolution structure of the EHD2 filament on native caveolar necks; (2) the identity of the SUMO ligase and chromatin targets mediating nuclear EHD2 transcriptional repression; and (3) how EHD2 coordinates with dynamin and other caveolar regulators to control the balance between caveolar stability and endocytic release.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"In vivo filament architecture on caveolae at near-atomic resolution pending\", \"Nuclear EHD2 transcriptional mechanism remains largely undefined\", \"Functional interplay between EHD2 and dynamin at caveolae not dissected\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140657\", \"supporting_discovery_ids\": [7, 14, 17]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [8, 15]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [10, 11]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [7, 14, 24]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 6, 7, 10, 11, 17]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [8, 15]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [0, 7]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [6, 7, 17, 22]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [15, 20]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [13]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [17, 19, 23]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [8, 15]}\n    ],\n    \"complexes\": [\n      \"Caveolae coat complex (with caveolin-1, cavin1, PACSIN2)\"\n    ],\n    \"partners\": [\n      \"EHBP1\",\n      \"PACSIN2\",\n      \"CAVIN1\",\n      \"MYOF\",\n      \"FER1L5\",\n      \"CAV1\",\n      \"RAB10\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}