{"gene":"EHD2","run_date":"2026-06-09T23:54:42","timeline":{"discoveries":[{"year":2003,"finding":"EHD2 localizes to cortical actin filaments at the plasma membrane and couples clathrin-mediated endocytosis to the actin cytoskeleton. Its C-terminal EH domain interacts with NPF repeats in the novel binding partner EHBP1, which contains a calponin homology actin-binding domain. siRNA-mediated silencing of EHD2 or EHBP1 inhibits transferrin and GLUT4 endocytosis into EEA1-positive endosomes. High expression of EHD2 or EHBP1 causes extensive actin reorganization.","method":"siRNA knockdown, co-immunoprecipitation, subcellular localization by fluorescence microscopy, endocytosis assays (transferrin, GLUT4)","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal binding assays and functional knockdown with defined phenotypic readout, single lab, multiple orthogonal methods","pmids":["14676205"],"is_preprint":false},{"year":2004,"finding":"EHD2 is present in purified GLUT4 vesicles of rat adipocytes and physically co-immunoprecipitates with GLUT4. Insulin selectively enhances EHD2-GLUT4 interaction in an endosomal fraction containing GLUT4 exocytic vesicles. EHD2 also interacts with clathrin adaptor subunits µ1, µ2, and rCALM in GST pull-down experiments. An anti-EHD2 antibody and an EHD2-derived peptide suppressed insulin-induced plasma membrane GLUT4 recruitment in permeabilized adipocytes, indicating EHD2 plays a key role in insulin-induced GLUT4 trafficking.","method":"MALDI-TOF MS identification, co-immunoprecipitation, GST pull-down, subcellular fractionation, antibody inhibition assay in SLO-permeabilized adipocytes","journal":"Biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal biochemical methods (Co-IP, pulldown, antibody inhibition), single lab","pmids":["15182197"],"is_preprint":false},{"year":2008,"finding":"EHD2 directly binds the second C2 domain of myoferlin. Myoferlin-null myoblasts show reduced EHD2 levels, accumulate labeled transferrin, and have delayed recycling. Introduction of dominant-negative EHD2 into myoblasts leads to sequestration of myoferlin and inhibition of myoblast fusion, identifying a molecular link between endocytic recycling and myoblast membrane fusion machinery.","method":"Direct binding assay (C2 domain mapping), dominant-negative EHD2 expression, transferrin recycling assay, myoblast fusion assay, immunofluorescence","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct binding mapped to specific domain, dominant-negative functional assay, single lab, multiple orthogonal methods","pmids":["18502764"],"is_preprint":false},{"year":2010,"finding":"EHD2 (and EHD1) directly binds Fer1L5 via the second C2 domain of Fer1L5. Reduction of EHD1 and/or EHD2 by siRNA inhibits myoblast fusion, and EHD2 is specifically required for normal translocation of Fer1L5 to the plasma membrane.","method":"Direct binding assay (C2 domain mapping), siRNA knockdown, plasma membrane translocation assay, myoblast fusion assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct binding mapped to specific domain, functional knockdown with defined readout, single lab","pmids":["21177873"],"is_preprint":false},{"year":2011,"finding":"EHD2 regulates trafficking from the plasma membrane by controlling Rac1 activity. Using yeast two-hybrid, EHD2 was found to interact with Nek3 kinase and associate with Vav1 (a Nek3-regulated GEF for Rho GTPases). Overexpression of wild-type EHD2, but not P-loop (nucleotide-binding) mutants, reduced Rac1 activity. The inhibitory effect of EHD2 on cholera toxin trafficking was partially rescued by co-expression of Rac1.","method":"Yeast two-hybrid, co-immunoprecipitation, Rac1 activity assay, cholera toxin trafficking assay, P-loop mutagenesis","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple methods including activity assay and mutagenesis, single lab","pmids":["21756249"],"is_preprint":false},{"year":2012,"finding":"EHD2 is specifically and stably associated with caveolae at the plasma membrane. EHD2 dimers oligomerize into rings on highly curved membranes, stimulating intrinsic ATPase activity. EHD2 interacts with pacsin2 and cavin1; ordered membrane assembly requires cavin1 and caveolar integrity. A loop in the nucleotide-binding domain, together with ATP binding, is required for caveolar localization. High EHD2 levels distort/reduce caveolae; depletion results in endocytic, more dynamic and short-lived caveolae. EHD2 constitutes a third structural component of caveolae controlling stability and turnover.","method":"Live-cell imaging, electron microscopy, ATPase assay, siRNA depletion, overexpression, co-immunoprecipitation (pacsin2, cavin1), domain mutagenesis","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal methods (EM, ATPase assay, mutagenesis, siRNA, Co-IP), replicated across labs","pmids":["22323287"],"is_preprint":false},{"year":2012,"finding":"EHD2 associates with the static population of plasma membrane caveolae via ATP binding, interaction with anionic lipids, and oligomerization into large complexes (~60–75S) via EH domain interactions with intrinsic NPF/KPF motifs. ATP hydrolysis is essential for EHD2 binding to caveolae. EHD2 undergoes dynamic exchange at caveolae dependent on a functional ATPase cycle. Depletion of EHD2 by siRNA or dominant-negative expression dramatically increases the fraction of mobile caveolar vesicles from the PM; overexpression confines cholera toxin B to caveolae. The confining role relies on EHD2's capacity to link caveolae to actin filaments.","method":"Live-cell imaging (TIRF, FRAP), siRNA knockdown, dominant-negative expression, ATPase mutant analysis, sucrose gradient ultracentrifugation, cholera toxin tracking assay","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal methods, replication of key findings across labs (consistent with PMID:22323287)","pmids":["22505029"],"is_preprint":false},{"year":2012,"finding":"EHD2 can shuttle to the nucleus in a NLS-dependent manner; nuclear exit depends partially on a NES. SUMOylation of EHD2 was confirmed by co-immunoprecipitation and yeast two-hybrid, and elimination of the SUMOylation site causes major nuclear accumulation. Nuclear EHD2 represses transcription, including repression of the p21WAF1/Cip1 (CDKN1A) gene via a KLF7-dependent transcription assay, confirmed by qRT-PCR in EHD2 overexpression and knockdown cells.","method":"Nuclear export inhibition, NLS/NES mutagenesis, co-immunoprecipitation (SUMOylation), yeast two-hybrid, GAL4-based transactivation assay, KLF7-p21 transcription assay, qRT-PCR","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (mutagenesis, Co-IP, transcription assay, qRT-PCR), single lab","pmids":["22448906"],"is_preprint":false},{"year":2012,"finding":"EHD2 participates in sarcolemmal membrane repair. Following laser wounding of human myotubes, EHD2 (but not EHD1) accumulates at the injury site and at the repair dome structure. A mutant EHD2 does not accumulate, indicating the ATPase function is required for this role.","method":"Live fluorescence imaging of GFP-tagged and endogenous EHD2 after laser wounding, comparison of EHD2 vs EHD1 and mutant EHD2","journal":"Traffic (Copenhagen, Denmark)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct localization with functional consequence via mutagenesis, single lab","pmids":["22679923"],"is_preprint":false},{"year":2013,"finding":"Plasma membrane phospholipid PIP2 plays a crucial role in regulating EHD2 subcellular localization. Pharmacological perturbation of PIP2 metabolism causes EHD2 to redistribute away from the plasma membrane. EHD2 localizes to PIP2-enriched vacuoles generated by active Arf6. Cytochalasin D-induced actin collapse does not displace EHD2 from the PM, but both PIP2 and EHD2 co-redistribute to actin aggregates, supporting PIP2 as the primary determinant. EHD2 plasma membrane targeting is independent of syndapin2, EHBP1, or its EH domain.","method":"Confocal microscopy, pharmacological PIP2 perturbation, Arf6 overexpression (PIP2-enriched vacuoles), cytochalasin D treatment, siRNA depletion of partner proteins, domain deletion mutants","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal perturbations in single lab establishing PIP2 role in localization","pmids":["24040268"],"is_preprint":false},{"year":2014,"finding":"Using EPR spectroscopy and X-ray crystallography, the N-terminus of EHD2 is folded into a hydrophobic pocket of the GTPase domain in solution and can be released into the membrane. Residues at the tip of the helical domain can insert into the membrane and may create membrane curvature by a wedging mechanism. Cryo-EM showed the N-terminus is not essential for oligomerization but regulates targeting and stable association of EHD2 with caveolae.","method":"Electron paramagnetic resonance (EPR) spectroscopy, X-ray crystallography, cryo-electron microscopy, mutagenesis of N-terminal region","journal":"Structure (London, England : 1993)","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure, EPR, and cryo-EM with mutagenesis in single study, multiple orthogonal structural methods","pmids":["24508342"],"is_preprint":false},{"year":2015,"finding":"The EHD2 unstructured loop contains two PF motifs (NPF and KPF). The NPF phenylalanine residue is crucial for EHD2 plasma membrane localization, whereas the NPF proline residue is essential for EHD2 dimerization and binding to Syndapin2. The KPF motif NPF-to-APA mutation increases nuclear localization and reduces plasma membrane association. These results support a model in which the N-terminal region regulates availability of the unstructured loop for oligomerization.","method":"Site-directed mutagenesis, co-immunoprecipitation (dimerization, Syndapin2 binding), confocal microscopy (localization), nuclear fractionation","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis with binding and localization readouts, single lab, multiple orthogonal methods","pmids":["25875965"],"is_preprint":false},{"year":2016,"finding":"Rab10, EHBP1, and EHD2 form a novel complex essential for lipophagy in hepatocytes. During autophagy, Rab10 activity is amplified and recruits EHBP1 and EHD2 to nascent autophagic membranes at the lipid droplet surface. Disruption of Rab10 by siRNA or GTPase-defective variant causes lipid droplet accumulation. Rab10 activation is essential for LC3 recruitment to the autophagosome and stimulates association with EHBP1 and EHD2, driving engulfment of lipid droplets.","method":"siRNA knockdown, GTPase-defective mutant expression, fluorescence imaging, co-immunoprecipitation, autophagy assays (LC3 recruitment, lipid droplet accumulation)","journal":"Science advances","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple complementary functional assays plus complex identification, single lab","pmids":["28028537"],"is_preprint":false},{"year":2017,"finding":"EHD2 is kept in an autoinhibited conformation in solution by its N-terminal residues and EH domain. Upon membrane binding, EHD2 adopts an open conformation by tilting its helical domains (demonstrated by infrared reflection-absorption spectroscopy). ATP binding enables partial insertion of EHD2 into the membrane, where G-domain-mediated oligomerization occurs. ATP hydrolysis is coupled to detachment of EHD2 from the membrane. Regulation of EHD2 oligomerization in the membrane-bound state is required for restricting caveolae dynamics.","method":"Infrared reflection-absorption spectroscopy (IRRAS), ATPase assays, mutagenesis, cell-based caveolae dynamics assays","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Moderate — novel biophysical method (IRRAS) combined with ATPase assay and mutagenesis in single study with multiple orthogonal methods","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 several genes including those coding for caveolae constituents. EHD2 is required to maintain the caveolae reservoir at the plasma membrane during membrane tension variations. Breast cancer cells lacking EHD2 show a complete absence of caveolae and lack gene regulation under mechanical stress; re-expression of EHD2 restores both functions.","method":"Live-cell imaging, metal-replica electron microscopy, mechanical stress assays, SUMOylation detection, nuclear fractionation, transcriptome analysis (gene expression), EHD2 KO and rescue","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (EM, imaging, transcriptomics, KO+rescue), establishes mechanotransduction axis","pmids":["30348749"],"is_preprint":false},{"year":2018,"finding":"EHD2 positively regulates the surface expression of cardiac sarcolemmal KATP channels by stabilizing KATP channel-containing caveolar structures, reducing the rate of endocytosis. EHD2 is specific to KATP channels among EHD family members (EHD1, EHD3, EHD4 had no effect) and does not alter channel unitary conductance or ATP sensitivity. A dominant-negative EHD2 mutant sensitizes cardiomyocytes to ischemic damage.","method":"Surface biotinylation, immunofluorescence, patch clamping, dominant-negative EHD2 expression, cardiomyocyte ischemia assay, comparison across EHD paralogs","journal":"FASEB journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal functional assays (biotinylation, patch clamp, imaging) in single lab","pmids":["29133341"],"is_preprint":false},{"year":2019,"finding":"EHD2 controls a caveolae- and CD36-dependent fatty acid uptake pathway in adipocytes. Global genetic ablation of EHD2 in mice leads to increased lipid droplet size in fat tissue and elevated fatty acid uptake. EHD2-null adipose tissue shows increased numbers of detached caveolae. EHD2 expression is down-regulated in visceral fat of obese mouse models and obese patients.","method":"EHD2 knockout mice, fatty acid uptake assay, electron microscopy (caveolae counting), live-cell caveolar mobility assay, CD36/dynamin inhibitor pharmacology","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic knockout model with multiple orthogonal cellular and organismal readouts, consistent with prior caveolae mechanistic findings","pmids":["32170013"],"is_preprint":false},{"year":2019,"finding":"EHD2-controlled caveolar dynamics orchestrates eNOS/NO activity. Loss of EHD2 in small arteries increases detached caveolae numbers, impairs mesenteric artery relaxation, and decreases nitric oxide production without changing eNOS expression levels. Super-resolution imaging shows eNOS redistributes from the plasma membrane to internalized detached caveolae in EHD2-lacking tissues/cells. EHD2 deletion or knockdown also reduces cytosolic Ca2+ peaks following ATP stimulus in HUVECs.","method":"EHD2 knockout mice, vascular relaxation assay, NO measurement, super-resolution microscopy (eNOS localization), Ca2+ imaging, siRNA knockdown in HUVECs","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO plus multiple orthogonal methods (imaging, functional vessel assay, Ca2+ assay, NO measurement), replicated in vivo and in vitro","pmids":["31600286"],"is_preprint":false},{"year":2019,"finding":"EHD2 protein expression is up-regulated at the onset of triglyceride accumulation during adipocyte differentiation. EHD2 localizes to caveolae adjacent to cell surface-associated lipid droplets in primary human adipocytes. siRNA silencing of EHD2 impairs differentiation, insulin sensitivity, lipid storage, and lipolysis. EHD2 overexpression increases lipolytic signaling and suppresses PPARγ transcription factor activity.","method":"siRNA knockdown, EHD2 overexpression, fluorescence imaging, insulin sensitivity assay, lipolysis assay, PPARγ activity assay","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple functional readouts in adipocytes with KD and OE, single lab","pmids":["30811273"],"is_preprint":false},{"year":2021,"finding":"EHD2 is a novel modulator of Notch activation in endothelial cells through controlling endocytosis of Dll4. EHD2 co-localizes with plasma membrane Dll4 and caveolae. Chemical disruption of caveolae prevents EHD2 organization around Dll4 and blocks Dll4 internalization, blunting Notch activation. EHD2 knockout in zebrafish increases dysmorphic intersomitic vessel sprouts and reduces downstream Notch signaling.","method":"In vitro co-localization (immunofluorescence), caveolae disruption pharmacology, Dll4 internalization assay, Notch signaling readouts, zebrafish EHD2 knockout","journal":"Microcirculation (New York, N.Y. : 1994)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo zebrafish KO plus in vitro mechanistic assays, single lab","pmids":["34820962"],"is_preprint":false},{"year":2022,"finding":"Super-resolution single-molecule localization microscopy revealed that PACSIN2 and EHD2 both co-localize with caveolin-1 at caveolae. The mean centroid of the PACSIN2 F-BAR domain is positioned closer to the plasma membrane than EHD2 and caveolin-1 centroids, suggesting PACSIN2 connects caveolae to the plasma membrane while EHD2 is positioned at the caveolar body/neck.","method":"Super-resolution single-molecule localization microscopy (SMLM), 3D spatial analysis of PACSIN2, EHD2, and caveolin-1 relative positions","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — quantitative super-resolution structural data, single lab, no functional mutagenesis","pmids":["35834519"],"is_preprint":false},{"year":2023,"finding":"EHD2 deficiency in adipocytes is associated with deterioration of insulin signal transduction and impaired insulin-stimulated GLUT4 translocation. EHD2 loss is linked to altered plasma membrane lipid and protein composition, reduced insulin receptor expression, and diminished insulin-dependent SNARE protein complex formation, indicating EHD2 is required for plasma membrane integrity and downstream insulin receptor signaling.","method":"EHD2 knockout mice (high-fat diet), 3T3-L1 adipocytes, GLUT4 translocation assay, insulin signaling assays, membrane lipid/protein composition analysis, SNARE complex co-immunoprecipitation","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO model plus multiple biochemical readouts, single lab","pmids":["37703099"],"is_preprint":false},{"year":2023,"finding":"EHD2 stabilizes plasma membrane caveolae to ensure high cell surface expression of the SOCE-linked calcium channel Orai1. EHD2 shRNA knockdown and CRISPR-Cas9 knockout with mouse Ehd2 rescue in TNBC cell lines demonstrates that EHD2 promotes tumorigenesis and metastasis through store-operated calcium entry (SOCE), defining an EHD2-SOCE oncogenic axis.","method":"shRNA knockdown, CRISPR-Cas9 knockout, Ehd2 rescue, surface expression assays, SOCE assay, caveolae imaging, tumorigenesis and metastasis assays","journal":"eLife","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO with rescue and multiple functional assays, single lab","pmids":["36625722"],"is_preprint":false},{"year":2025,"finding":"Cryo-electron tomography of EHD2 oligomerized on tubulated liposomes revealed that EHD2 forms filaments creating a highly curved membrane scaffold that stabilizes a tubular membrane geometry with undulations along the tube axis. The amino-terminal sequence facilitates this geometry by inserting into the membrane, acting as a spacer between adjacent filaments. In endothelial cells lacking EHD2, caveolar necks become narrower and elongated, providing a cellular correlate to the structural model.","method":"Cryo-electron tomography, in vitro tubulated liposome reconstitution with EHD2 oligomers, endothelial cells from EHD2 knockout with electron microscopy of caveolar neck morphology","journal":"bioRxiv","confidence":"High","confidence_rationale":"Tier 1 / Moderate — cryo-ET structure of reconstituted filaments plus cell biology validation in EHD2 KO, multiple orthogonal methods in single preprint study","pmids":["bio_10.1101_2025.06.05.658037"],"is_preprint":true},{"year":2010,"finding":"Palmitoylation of prohibitin (PHB) at Cys69 is required for PHB membrane translocation, which in turn facilitates PHB tyrosine phosphorylation and its interaction with EHD2. Thus, the EHD2-PHB interaction at the plasma membrane is dependent on PHB palmitoylation-driven membrane recruitment.","method":"Palmitoylation site mutagenesis (Cys69), membrane fractionation, co-immunoprecipitation (PHB-EHD2), phosphorylation detection","journal":"Biochemistry and cell biology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP with mutagenesis, single lab, no deeper mechanistic follow-up on EHD2's role","pmids":["20555396"],"is_preprint":false}],"current_model":"EHD2 is a dynamin-related ATPase that forms oligomeric filament scaffolds around caveolar necks at the plasma membrane, where ATP binding promotes membrane insertion and an open conformation, oligomerization stabilizes caveolae and restricts their endocytosis, and ATP hydrolysis drives EHD2 detachment; under mechanical stress EHD2 is released from caveolae, SUMOylated, and translocated to the nucleus where it represses gene transcription, thereby functioning as a mechanotransducer that links caveolae dynamics to transcriptional regulation, fatty acid uptake, eNOS/NO signaling, KATP channel surface expression, GLUT4 trafficking, Dll4/Notch endocytosis, lipophagy, and myoblast fusion."},"narrative":{"mechanistic_narrative":"EHD2 is a dynamin-related ATPase that functions as the principal structural stabilizer of plasma membrane caveolae, controlling their dynamics and turnover and thereby gating a wide range of caveolae-dependent cellular processes [PMID:22323287, PMID:22505029]. EHD2 dimers oligomerize into ring- and filament-shaped scaffolds on highly curved membranes, where ATP binding drives membrane insertion and an open conformation, oligomerization stabilizes caveolae and confines them to the cell surface, and ATP hydrolysis triggers EHD2 detachment [PMID:22505029, PMID:28223496, PMID:bio_10.1101_2025.06.05.658037]. Its membrane targeting is governed by an autoinhibitory N-terminus that folds into the GTPase domain and is released to wedge into the bilayer, by ATP binding, and by the anionic phospholipid PIP2, while an unstructured loop bearing NPF/KPF motifs directs both oligomerization and partner binding [PMID:24040268, PMID:24508342, PMID:28223496]. At caveolae EHD2 cooperates with the structural components cavin1, caveolin-1, and the F-BAR protein pacsin2/syndapin2, occupying the caveolar body/neck while pacsin2 links the structure to the plasma membrane [PMID:22323287, PMID:35834519]. By restricting caveolar internalization, EHD2 supports caveola-dependent fatty acid/CD36 uptake and adipocyte lipid handling [PMID:32170013, PMID:30811273], eNOS surface localization and nitric oxide signaling [PMID:31600286], sarcolemmal KATP channel and Orai1/SOCE surface expression [PMID:29133341, PMID:36625722], and Dll4 endocytosis controlling Notch activation [PMID:34820962]. EHD2 additionally acts as a mechanotransducer: under mechanical stress it is released from caveolae, SUMOylated, and translocated to the nucleus, where it represses transcription of target genes including caveolae constituents and CDKN1A/p21 via a KLF7-dependent mechanism [PMID:22448906, PMID:30348749]. Independent of caveolae, EHD2 couples endocytic recycling to the actin cytoskeleton and membrane-fusion/repair machinery through its EH-domain partner EHBP1 and the ferlins myoferlin and Fer1L5, contributing to myoblast fusion and sarcolemmal repair [PMID:14676205, PMID:18502764, PMID:22679923], and forms a Rab10–EHBP1–EHD2 complex driving lipophagy of lipid droplets [PMID:28028537].","teleology":[{"year":2003,"claim":"Established EHD2's earliest known function—linking endocytosis to the actin cytoskeleton—by identifying its EH-domain partner and a defined trafficking defect.","evidence":"siRNA knockdown, co-IP with EHBP1, and transferrin/GLUT4 endocytosis assays","pmids":["14676205"],"confidence":"Medium","gaps":["Did not address caveolar localization or ATPase mechanism","EHBP1-actin coupling not structurally resolved"]},{"year":2004,"claim":"Connected EHD2 to insulin-regulated GLUT4 trafficking, implying a role in metabolic membrane transport.","evidence":"MS identification in GLUT4 vesicles, Co-IP, GST pull-down, antibody inhibition in permeabilized adipocytes","pmids":["15182197"],"confidence":"Medium","gaps":["Mechanistic link to caveolae not yet known","Adaptor interactions not validated in vivo"]},{"year":2008,"claim":"Linked EHD2-driven endocytic recycling to the membrane-fusion machinery of myoblasts via direct ferlin binding.","evidence":"C2-domain binding mapping to myoferlin, dominant-negative EHD2, fusion and recycling assays","pmids":["18502764"],"confidence":"Medium","gaps":["Whether caveolae are involved in ferlin trafficking unclear","Single lab"]},{"year":2010,"claim":"Extended the ferlin connection to Fer1L5, showing EHD2 is specifically required for ferlin delivery to the plasma membrane.","evidence":"C2-domain binding assay, siRNA knockdown, plasma membrane translocation and fusion assays","pmids":["21177873"],"confidence":"Medium","gaps":["Molecular basis of selective EHD2 vs EHD1 roles unresolved"]},{"year":2010,"claim":"Reported a palmitoylation-dependent prohibitin–EHD2 plasma membrane interaction.","evidence":"Cys69 palmitoylation mutagenesis, membrane fractionation, PHB-EHD2 Co-IP","pmids":["20555396"],"confidence":"Low","gaps":["Single Co-IP with no functional follow-up on EHD2's role","No reciprocal validation"]},{"year":2011,"claim":"Placed EHD2 upstream of Rho-GTPase signaling, showing its trafficking control depends on nucleotide binding and Rac1.","evidence":"Yeast two-hybrid (Nek3, Vav1), Rac1 activity assay, cholera toxin trafficking, P-loop mutagenesis","pmids":["21756249"],"confidence":"Medium","gaps":["Whether Rac1 regulation acts through caveolae not addressed","Direct vs indirect Vav1 link unclear"]},{"year":2012,"claim":"Redefined EHD2 as a third structural component of caveolae, establishing the central caveolar stabilization function and its ATPase/oligomerization basis.","evidence":"Live imaging, EM, ATPase assays, sucrose gradients, FRAP, siRNA/dominant-negative, Co-IP with pacsin2 and cavin1","pmids":["22323287","22505029"],"confidence":"High","gaps":["Atomic-resolution scaffold geometry not yet resolved","How ATPase cycle is regulated in cells unclear"]},{"year":2012,"claim":"Identified a non-caveolar role in sarcolemmal membrane repair requiring EHD2 ATPase activity.","evidence":"Laser wounding of myotubes, GFP-EHD2 imaging, EHD2 vs EHD1 and mutant comparison","pmids":["22679923"],"confidence":"Medium","gaps":["Repair partners at the wound site not identified","Relationship to ferlin function not tested"]},{"year":2012,"claim":"Revealed an unexpected nuclear function for EHD2, showing SUMOylation-gated shuttling and transcriptional repression of p21/CDKN1A.","evidence":"NLS/NES mutagenesis, SUMOylation Co-IP, yeast two-hybrid, GAL4 transactivation and KLF7-p21 transcription assays, qRT-PCR","pmids":["22448906"],"confidence":"Medium","gaps":["Trigger for nuclear shuttling not yet linked to caveolae","Direct DNA/chromatin engagement undefined"]},{"year":2013,"claim":"Defined PIP2 as the primary determinant of EHD2 plasma membrane targeting, independent of actin and EH-domain partners.","evidence":"Confocal imaging with PIP2 pharmacology, Arf6 vacuoles, cytochalasin D, partner siRNA, domain deletions","pmids":["24040268"],"confidence":"Medium","gaps":["Specific PIP2-binding residues not mapped here","Single lab"]},{"year":2014,"claim":"Provided the structural basis for membrane insertion, showing the N-terminus folds into the GTPase domain and can wedge into the bilayer to create curvature.","evidence":"EPR, X-ray crystallography, cryo-EM, N-terminal mutagenesis","pmids":["24508342"],"confidence":"High","gaps":["In-cell conformational triggering not directly observed","Filament geometry at caveolar necks not resolved"]},{"year":2015,"claim":"Dissected the NPF/KPF loop motifs controlling localization, dimerization, and syndapin2 binding versus nuclear partitioning.","evidence":"Site-directed mutagenesis, Co-IP, confocal localization, nuclear fractionation","pmids":["25875965"],"confidence":"Medium","gaps":["How motif occupancy is regulated dynamically unknown"]},{"year":2016,"claim":"Identified a Rab10–EHBP1–EHD2 complex essential for lipophagy, extending EHD2 to autophagic lipid droplet clearance.","evidence":"siRNA, GTPase-defective Rab10, imaging, Co-IP, LC3 and lipid droplet assays","pmids":["28028537"],"confidence":"Medium","gaps":["EHD2's specific membrane-shaping step in lipophagy undefined","Single lab"]},{"year":2017,"claim":"Resolved the conformational cycle: autoinhibited in solution, opening and inserting upon ATP binding, oligomerizing, then detaching on hydrolysis to restrict caveolae dynamics.","evidence":"IRRAS, ATPase assays, mutagenesis, caveolae dynamics assays","pmids":["28223496"],"confidence":"High","gaps":["In-cell timing of the cycle not directly measured","What regulates hydrolysis-driven release unclear"]},{"year":2018,"claim":"Established EHD2 as a mechanotransducer coupling caveolar tension sensing to nuclear gene regulation and showed its loss abolishes caveolae in cancer cells.","evidence":"Live imaging, metal-replica EM, mechanical stress, SUMOylation, transcriptomics, KO and rescue","pmids":["30348749"],"confidence":"High","gaps":["Direct transcriptional targets and DNA engagement incompletely defined","Mechanosensing threshold mechanism unknown"]},{"year":2018,"claim":"Demonstrated EHD2 stabilizes caveolae to sustain cardiac KATP channel surface expression, with protective relevance to ischemia.","evidence":"Surface biotinylation, patch clamp, dominant-negative EHD2, ischemia assay, EHD paralog comparison","pmids":["29133341"],"confidence":"Medium","gaps":["In vivo cardiac KO phenotype not tested here","Direct KATP–caveolae binding interface undefined"]},{"year":2019,"claim":"Linked EHD2-controlled caveolae stability to metabolic fatty acid uptake and obesity via a CD36-dependent pathway, using a whole-animal knockout.","evidence":"EHD2 KO mice, fatty acid uptake, EM caveolae counting, caveolar mobility, CD36/dynamin pharmacology","pmids":["32170013"],"confidence":"High","gaps":["Whether nuclear EHD2 contributes to the metabolic phenotype unclear"]},{"year":2019,"claim":"Showed EHD2-dependent caveolar stability governs eNOS surface localization and endothelial NO/Ca2+ signaling.","evidence":"EHD2 KO mice, vascular relaxation, NO measurement, super-resolution eNOS imaging, Ca2+ imaging, HUVEC siRNA","pmids":["31600286"],"confidence":"High","gaps":["Direct eNOS–caveolae retention mechanism not biochemically dissected"]},{"year":2019,"claim":"Connected EHD2 to adipocyte differentiation and insulin sensitivity, including suppression of PPARγ activity.","evidence":"siRNA, overexpression, imaging, insulin sensitivity, lipolysis, PPARγ activity assays","pmids":["30811273"],"confidence":"Medium","gaps":["Mechanism linking caveolar EHD2 to PPARγ transcription undefined"]},{"year":2021,"claim":"Identified EHD2 as a modulator of Notch signaling through caveolae-dependent Dll4 endocytosis, with an in vivo vascular phenotype.","evidence":"Co-localization, caveolae disruption pharmacology, Dll4 internalization, Notch readouts, zebrafish KO","pmids":["34820962"],"confidence":"Medium","gaps":["Direct EHD2–Dll4 interaction not demonstrated","Single lab"]},{"year":2022,"claim":"Refined the molecular architecture of caveolae by positioning EHD2 at the caveolar body/neck relative to PACSIN2 and caveolin-1.","evidence":"Super-resolution single-molecule localization microscopy and 3D spatial analysis","pmids":["35834519"],"confidence":"Medium","gaps":["No functional mutagenesis tied to the spatial map","Static snapshot, not dynamic"]},{"year":2023,"claim":"Showed EHD2 maintains plasma membrane integrity required for insulin receptor signaling and GLUT4 translocation in adipocytes.","evidence":"EHD2 KO mice on high-fat diet, 3T3-L1 adipocytes, GLUT4 translocation, insulin signaling, membrane composition, SNARE Co-IP","pmids":["37703099"],"confidence":"Medium","gaps":["Causal chain from caveolae loss to insulin receptor downregulation incompletely defined"]},{"year":2023,"claim":"Defined an EHD2–SOCE oncogenic axis whereby caveolar stabilization sustains Orai1 surface expression to drive tumorigenesis.","evidence":"shRNA, CRISPR KO with mouse Ehd2 rescue, surface expression, SOCE, caveolae imaging, tumor/metastasis assays","pmids":["36625722"],"confidence":"Medium","gaps":["Direct Orai1–caveolae interaction not mapped","Single lab"]},{"year":2025,"claim":"Provided a near-atomic structural model of EHD2 filaments shaping a highly curved membrane scaffold, with the N-terminus acting as an inter-filament spacer and a KO correlate at caveolar necks.","evidence":"Cryo-electron tomography of reconstituted EHD2-tubulated liposomes plus EM of EHD2 KO endothelial caveolar necks (preprint)","pmids":["bio_10.1101_2025.06.05.658037"],"confidence":"High","gaps":["Preprint, not peer-reviewed","In-cell filament organization around native caveolar necks not directly resolved"]},{"year":null,"claim":"How EHD2 nuclear translocation and its transcriptional targets are mechanistically coupled to specific physiological mechanical stimuli, and whether nuclear EHD2 contributes to its metabolic and oncogenic phenotypes, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["Direct DNA/chromatin engagement by nuclear EHD2 undefined","Full target gene set across tissues unknown","Relative contribution of caveolar vs nuclear EHD2 to disease phenotypes unquantified"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140657","term_label":"ATP-dependent activity","supporting_discovery_ids":[5,6,13]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[9,10,13]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[5,23]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[7,14]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[0,6]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[6,13]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,5,6,9]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[0,6]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[7,14]},{"term_id":"GO:0005811","term_label":"lipid droplet","supporting_discovery_ids":[12,18]}],"pathway":[{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[5,6,0]},{"term_id":"R-HSA-8953897","term_label":"Cellular responses to stimuli","supporting_discovery_ids":[14]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[12]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[16,18]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[7,14]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[17,19]}],"complexes":["caveolae","Rab10-EHBP1-EHD2 complex"],"partners":["EHBP1","CAVIN1","PACSIN2","CAV1","MYOF","FER1L5","RAB10","GLUT4"],"other_free_text":[]}},"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, 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Its C-terminal EH domain interacts with NPF repeats in the novel binding partner EHBP1, which contains a calponin homology actin-binding domain. siRNA-mediated silencing of EHD2 or EHBP1 inhibits transferrin and GLUT4 endocytosis into EEA1-positive endosomes. High expression of EHD2 or EHBP1 causes extensive actin reorganization.\",\n      \"method\": \"siRNA knockdown, co-immunoprecipitation, subcellular localization by fluorescence microscopy, endocytosis assays (transferrin, GLUT4)\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal binding assays and functional knockdown with defined phenotypic readout, single lab, multiple orthogonal methods\",\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 physically co-immunoprecipitates with GLUT4. Insulin selectively enhances EHD2-GLUT4 interaction in an endosomal fraction containing GLUT4 exocytic vesicles. EHD2 also interacts with clathrin adaptor subunits µ1, µ2, and rCALM in GST pull-down experiments. An anti-EHD2 antibody and an EHD2-derived peptide suppressed insulin-induced plasma membrane GLUT4 recruitment in permeabilized adipocytes, indicating EHD2 plays a key role in insulin-induced GLUT4 trafficking.\",\n      \"method\": \"MALDI-TOF MS identification, co-immunoprecipitation, GST pull-down, subcellular fractionation, antibody inhibition assay in SLO-permeabilized adipocytes\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal biochemical methods (Co-IP, pulldown, antibody inhibition), single lab\",\n      \"pmids\": [\"15182197\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"EHD2 directly binds the second C2 domain of myoferlin. Myoferlin-null myoblasts show reduced EHD2 levels, accumulate labeled transferrin, and have delayed recycling. Introduction of dominant-negative EHD2 into myoblasts leads to sequestration of myoferlin and inhibition of myoblast fusion, identifying a molecular link between endocytic recycling and myoblast membrane fusion machinery.\",\n      \"method\": \"Direct binding assay (C2 domain mapping), dominant-negative EHD2 expression, transferrin recycling assay, myoblast fusion assay, immunofluorescence\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct binding mapped to specific domain, dominant-negative functional assay, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"18502764\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"EHD2 (and EHD1) directly binds Fer1L5 via the second C2 domain of Fer1L5. Reduction of EHD1 and/or EHD2 by siRNA inhibits myoblast fusion, and EHD2 is specifically required for normal translocation of Fer1L5 to the plasma membrane.\",\n      \"method\": \"Direct binding assay (C2 domain mapping), siRNA knockdown, plasma membrane translocation assay, myoblast fusion assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct binding mapped to specific domain, functional knockdown with defined readout, single lab\",\n      \"pmids\": [\"21177873\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"EHD2 regulates trafficking from the plasma membrane by controlling Rac1 activity. Using yeast two-hybrid, EHD2 was found to interact with Nek3 kinase and associate with Vav1 (a Nek3-regulated GEF for Rho GTPases). Overexpression of wild-type EHD2, but not P-loop (nucleotide-binding) mutants, reduced Rac1 activity. The inhibitory effect of EHD2 on cholera toxin trafficking was partially rescued by co-expression of Rac1.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, Rac1 activity assay, cholera toxin trafficking assay, P-loop mutagenesis\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple methods including activity assay and mutagenesis, 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. EHD2 dimers oligomerize into rings on highly curved membranes, stimulating intrinsic ATPase activity. EHD2 interacts with pacsin2 and cavin1; ordered membrane assembly requires cavin1 and caveolar integrity. A loop in the nucleotide-binding domain, together with ATP binding, is required for caveolar localization. High EHD2 levels distort/reduce caveolae; depletion results in endocytic, more dynamic and short-lived caveolae. EHD2 constitutes a third structural component of caveolae controlling stability and turnover.\",\n      \"method\": \"Live-cell imaging, electron microscopy, ATPase assay, siRNA depletion, overexpression, co-immunoprecipitation (pacsin2, cavin1), domain mutagenesis\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal methods (EM, ATPase assay, mutagenesis, siRNA, Co-IP), replicated across labs\",\n      \"pmids\": [\"22323287\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"EHD2 associates with the static population of plasma membrane caveolae via ATP binding, interaction with anionic lipids, and oligomerization into large complexes (~60–75S) via EH domain interactions with intrinsic NPF/KPF motifs. ATP hydrolysis is essential for EHD2 binding to caveolae. EHD2 undergoes dynamic exchange at caveolae dependent on a functional ATPase cycle. Depletion of EHD2 by siRNA or dominant-negative expression dramatically increases the fraction of mobile caveolar vesicles from the PM; overexpression confines cholera toxin B to caveolae. The confining role relies on EHD2's capacity to link caveolae to actin filaments.\",\n      \"method\": \"Live-cell imaging (TIRF, FRAP), siRNA knockdown, dominant-negative expression, ATPase mutant analysis, sucrose gradient ultracentrifugation, cholera toxin tracking assay\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal methods, replication of key findings across labs (consistent with PMID:22323287)\",\n      \"pmids\": [\"22505029\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"EHD2 can shuttle to the nucleus in a NLS-dependent manner; nuclear exit depends partially on a NES. SUMOylation of EHD2 was confirmed by co-immunoprecipitation and yeast two-hybrid, and elimination of the SUMOylation site causes major nuclear accumulation. Nuclear EHD2 represses transcription, including repression of the p21WAF1/Cip1 (CDKN1A) gene via a KLF7-dependent transcription assay, confirmed by qRT-PCR in EHD2 overexpression and knockdown cells.\",\n      \"method\": \"Nuclear export inhibition, NLS/NES mutagenesis, co-immunoprecipitation (SUMOylation), yeast two-hybrid, GAL4-based transactivation assay, KLF7-p21 transcription assay, qRT-PCR\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (mutagenesis, Co-IP, transcription assay, qRT-PCR), single lab\",\n      \"pmids\": [\"22448906\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"EHD2 participates in sarcolemmal membrane repair. Following laser wounding of human myotubes, EHD2 (but not EHD1) accumulates at the injury site and at the repair dome structure. A mutant EHD2 does not accumulate, indicating the ATPase function is required for this role.\",\n      \"method\": \"Live fluorescence imaging of GFP-tagged and endogenous EHD2 after laser wounding, comparison of EHD2 vs EHD1 and mutant EHD2\",\n      \"journal\": \"Traffic (Copenhagen, Denmark)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization with functional consequence via mutagenesis, single lab\",\n      \"pmids\": [\"22679923\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Plasma membrane phospholipid PIP2 plays a crucial role in regulating EHD2 subcellular localization. Pharmacological perturbation of PIP2 metabolism causes EHD2 to redistribute away from the plasma membrane. EHD2 localizes to PIP2-enriched vacuoles generated by active Arf6. Cytochalasin D-induced actin collapse does not displace EHD2 from the PM, but both PIP2 and EHD2 co-redistribute to actin aggregates, supporting PIP2 as the primary determinant. EHD2 plasma membrane targeting is independent of syndapin2, EHBP1, or its EH domain.\",\n      \"method\": \"Confocal microscopy, pharmacological PIP2 perturbation, Arf6 overexpression (PIP2-enriched vacuoles), cytochalasin D treatment, siRNA depletion of partner proteins, domain deletion mutants\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal perturbations in single lab establishing PIP2 role in localization\",\n      \"pmids\": [\"24040268\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Using EPR spectroscopy and X-ray crystallography, the N-terminus of EHD2 is folded into a hydrophobic pocket of the GTPase domain in solution and can be released into the membrane. Residues at the tip of the helical domain can insert into the membrane and may create membrane curvature by a wedging mechanism. Cryo-EM showed the N-terminus is not essential for oligomerization but regulates targeting and stable association of EHD2 with caveolae.\",\n      \"method\": \"Electron paramagnetic resonance (EPR) spectroscopy, X-ray crystallography, cryo-electron microscopy, mutagenesis of N-terminal region\",\n      \"journal\": \"Structure (London, England : 1993)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure, EPR, and cryo-EM with mutagenesis in single study, multiple orthogonal structural methods\",\n      \"pmids\": [\"24508342\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"The EHD2 unstructured loop contains two PF motifs (NPF and KPF). The NPF phenylalanine residue is crucial for EHD2 plasma membrane localization, whereas the NPF proline residue is essential for EHD2 dimerization and binding to Syndapin2. The KPF motif NPF-to-APA mutation increases nuclear localization and reduces plasma membrane association. These results support a model in which the N-terminal region regulates availability of the unstructured loop for oligomerization.\",\n      \"method\": \"Site-directed mutagenesis, co-immunoprecipitation (dimerization, Syndapin2 binding), confocal microscopy (localization), nuclear fractionation\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis with binding and localization readouts, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"25875965\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Rab10, EHBP1, and EHD2 form a novel complex essential for lipophagy in hepatocytes. During autophagy, Rab10 activity is amplified and recruits EHBP1 and EHD2 to nascent autophagic membranes at the lipid droplet surface. Disruption of Rab10 by siRNA or GTPase-defective variant causes lipid droplet accumulation. Rab10 activation is essential for LC3 recruitment to the autophagosome and stimulates association with EHBP1 and EHD2, driving engulfment of lipid droplets.\",\n      \"method\": \"siRNA knockdown, GTPase-defective mutant expression, fluorescence imaging, co-immunoprecipitation, autophagy assays (LC3 recruitment, lipid droplet accumulation)\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple complementary functional assays plus complex identification, single lab\",\n      \"pmids\": [\"28028537\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"EHD2 is kept in an autoinhibited conformation in solution by its N-terminal residues and EH domain. Upon membrane binding, EHD2 adopts an open conformation by tilting its helical domains (demonstrated by infrared reflection-absorption spectroscopy). ATP binding enables partial insertion of EHD2 into the membrane, where G-domain-mediated oligomerization occurs. ATP hydrolysis is coupled to detachment of EHD2 from the membrane. Regulation of EHD2 oligomerization in the membrane-bound state is required for restricting caveolae dynamics.\",\n      \"method\": \"Infrared reflection-absorption spectroscopy (IRRAS), ATPase assays, mutagenesis, cell-based caveolae dynamics assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — novel biophysical method (IRRAS) combined with ATPase assay and mutagenesis in single study with multiple orthogonal methods\",\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 several genes including those coding for caveolae constituents. EHD2 is required to maintain the caveolae reservoir at the plasma membrane during membrane tension variations. Breast cancer cells lacking EHD2 show a complete absence of caveolae and lack gene regulation under mechanical stress; re-expression of EHD2 restores both functions.\",\n      \"method\": \"Live-cell imaging, metal-replica electron microscopy, mechanical stress assays, SUMOylation detection, nuclear fractionation, transcriptome analysis (gene expression), EHD2 KO and rescue\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (EM, imaging, transcriptomics, KO+rescue), establishes mechanotransduction axis\",\n      \"pmids\": [\"30348749\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"EHD2 positively regulates the surface expression of cardiac sarcolemmal KATP channels by stabilizing KATP channel-containing caveolar structures, reducing the rate of endocytosis. EHD2 is specific to KATP channels among EHD family members (EHD1, EHD3, EHD4 had no effect) and does not alter channel unitary conductance or ATP sensitivity. A dominant-negative EHD2 mutant sensitizes cardiomyocytes to ischemic damage.\",\n      \"method\": \"Surface biotinylation, immunofluorescence, patch clamping, dominant-negative EHD2 expression, cardiomyocyte ischemia assay, comparison across EHD paralogs\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal functional assays (biotinylation, patch clamp, imaging) in single lab\",\n      \"pmids\": [\"29133341\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"EHD2 controls a caveolae- and CD36-dependent fatty acid uptake pathway in adipocytes. Global genetic ablation of EHD2 in mice leads to increased lipid droplet size in fat tissue and elevated fatty acid uptake. EHD2-null adipose tissue shows increased numbers of detached caveolae. EHD2 expression is down-regulated in visceral fat of obese mouse models and obese patients.\",\n      \"method\": \"EHD2 knockout mice, fatty acid uptake assay, electron microscopy (caveolae counting), live-cell caveolar mobility assay, CD36/dynamin inhibitor pharmacology\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic knockout model with multiple orthogonal cellular and organismal readouts, consistent with prior caveolae mechanistic findings\",\n      \"pmids\": [\"32170013\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"EHD2-controlled caveolar dynamics orchestrates eNOS/NO activity. Loss of EHD2 in small arteries increases detached caveolae numbers, impairs mesenteric artery relaxation, and decreases nitric oxide production without changing eNOS expression levels. Super-resolution imaging shows eNOS redistributes from the plasma membrane to internalized detached caveolae in EHD2-lacking tissues/cells. EHD2 deletion or knockdown also reduces cytosolic Ca2+ peaks following ATP stimulus in HUVECs.\",\n      \"method\": \"EHD2 knockout mice, vascular relaxation assay, NO measurement, super-resolution microscopy (eNOS localization), Ca2+ imaging, siRNA knockdown in HUVECs\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO plus multiple orthogonal methods (imaging, functional vessel assay, Ca2+ assay, NO measurement), replicated in vivo and in vitro\",\n      \"pmids\": [\"31600286\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"EHD2 protein expression is up-regulated at the onset of triglyceride accumulation during adipocyte differentiation. EHD2 localizes to caveolae adjacent to cell surface-associated lipid droplets in primary human adipocytes. siRNA silencing of EHD2 impairs differentiation, insulin sensitivity, lipid storage, and lipolysis. EHD2 overexpression increases lipolytic signaling and suppresses PPARγ transcription factor activity.\",\n      \"method\": \"siRNA knockdown, EHD2 overexpression, fluorescence imaging, insulin sensitivity assay, lipolysis assay, PPARγ activity assay\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple functional readouts in adipocytes with KD and OE, single lab\",\n      \"pmids\": [\"30811273\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"EHD2 is a novel modulator of Notch activation in endothelial cells through controlling endocytosis of Dll4. EHD2 co-localizes with plasma membrane Dll4 and caveolae. Chemical disruption of caveolae prevents EHD2 organization around Dll4 and blocks Dll4 internalization, blunting Notch activation. EHD2 knockout in zebrafish increases dysmorphic intersomitic vessel sprouts and reduces downstream Notch signaling.\",\n      \"method\": \"In vitro co-localization (immunofluorescence), caveolae disruption pharmacology, Dll4 internalization assay, Notch signaling readouts, zebrafish EHD2 knockout\",\n      \"journal\": \"Microcirculation (New York, N.Y. : 1994)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo zebrafish KO plus in vitro mechanistic assays, single lab\",\n      \"pmids\": [\"34820962\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Super-resolution single-molecule localization microscopy revealed that PACSIN2 and EHD2 both co-localize with caveolin-1 at caveolae. The mean centroid of the PACSIN2 F-BAR domain is positioned closer to the plasma membrane than EHD2 and caveolin-1 centroids, suggesting PACSIN2 connects caveolae to the plasma membrane while EHD2 is positioned at the caveolar body/neck.\",\n      \"method\": \"Super-resolution single-molecule localization microscopy (SMLM), 3D spatial analysis of PACSIN2, EHD2, and caveolin-1 relative positions\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — quantitative super-resolution structural data, single lab, no functional mutagenesis\",\n      \"pmids\": [\"35834519\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"EHD2 deficiency in adipocytes is associated with deterioration of insulin signal transduction and impaired insulin-stimulated GLUT4 translocation. EHD2 loss is linked to altered plasma membrane lipid and protein composition, reduced insulin receptor expression, and diminished insulin-dependent SNARE protein complex formation, indicating EHD2 is required for plasma membrane integrity and downstream insulin receptor signaling.\",\n      \"method\": \"EHD2 knockout mice (high-fat diet), 3T3-L1 adipocytes, GLUT4 translocation assay, insulin signaling assays, membrane lipid/protein composition analysis, SNARE complex co-immunoprecipitation\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO model plus multiple biochemical readouts, single lab\",\n      \"pmids\": [\"37703099\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"EHD2 stabilizes plasma membrane caveolae to ensure high cell surface expression of the SOCE-linked calcium channel Orai1. EHD2 shRNA knockdown and CRISPR-Cas9 knockout with mouse Ehd2 rescue in TNBC cell lines demonstrates that EHD2 promotes tumorigenesis and metastasis through store-operated calcium entry (SOCE), defining an EHD2-SOCE oncogenic axis.\",\n      \"method\": \"shRNA knockdown, CRISPR-Cas9 knockout, Ehd2 rescue, surface expression assays, SOCE assay, caveolae imaging, tumorigenesis and metastasis assays\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO with rescue and multiple functional assays, single lab\",\n      \"pmids\": [\"36625722\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Cryo-electron tomography of EHD2 oligomerized on tubulated liposomes revealed that EHD2 forms filaments creating a highly curved membrane scaffold that stabilizes a tubular membrane geometry with undulations along the tube axis. The amino-terminal sequence facilitates this geometry by inserting into the membrane, acting as a spacer between adjacent filaments. In endothelial cells lacking EHD2, caveolar necks become narrower and elongated, providing a cellular correlate to the structural model.\",\n      \"method\": \"Cryo-electron tomography, in vitro tubulated liposome reconstitution with EHD2 oligomers, endothelial cells from EHD2 knockout with electron microscopy of caveolar neck morphology\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — cryo-ET structure of reconstituted filaments plus cell biology validation in EHD2 KO, multiple orthogonal methods in single preprint study\",\n      \"pmids\": [\"bio_10.1101_2025.06.05.658037\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Palmitoylation of prohibitin (PHB) at Cys69 is required for PHB membrane translocation, which in turn facilitates PHB tyrosine phosphorylation and its interaction with EHD2. Thus, the EHD2-PHB interaction at the plasma membrane is dependent on PHB palmitoylation-driven membrane recruitment.\",\n      \"method\": \"Palmitoylation site mutagenesis (Cys69), membrane fractionation, co-immunoprecipitation (PHB-EHD2), phosphorylation detection\",\n      \"journal\": \"Biochemistry and cell biology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP with mutagenesis, single lab, no deeper mechanistic follow-up on EHD2's role\",\n      \"pmids\": [\"20555396\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"EHD2 is a dynamin-related ATPase that forms oligomeric filament scaffolds around caveolar necks at the plasma membrane, where ATP binding promotes membrane insertion and an open conformation, oligomerization stabilizes caveolae and restricts their endocytosis, and ATP hydrolysis drives EHD2 detachment; under mechanical stress EHD2 is released from caveolae, SUMOylated, and translocated to the nucleus where it represses gene transcription, thereby functioning as a mechanotransducer that links caveolae dynamics to transcriptional regulation, fatty acid uptake, eNOS/NO signaling, KATP channel surface expression, GLUT4 trafficking, Dll4/Notch endocytosis, lipophagy, and myoblast fusion.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"EHD2 is a dynamin-related ATPase that functions as the principal structural stabilizer of plasma membrane caveolae, controlling their dynamics and turnover and thereby gating a wide range of caveolae-dependent cellular processes [#5, #6]. EHD2 dimers oligomerize into ring- and filament-shaped scaffolds on highly curved membranes, where ATP binding drives membrane insertion and an open conformation, oligomerization stabilizes caveolae and confines them to the cell surface, and ATP hydrolysis triggers EHD2 detachment [#6, #13, #23]. Its membrane targeting is governed by an autoinhibitory N-terminus that folds into the GTPase domain and is released to wedge into the bilayer, by ATP binding, and by the anionic phospholipid PIP2, while an unstructured loop bearing NPF/KPF motifs directs both oligomerization and partner binding [#9, #10, #13]. At caveolae EHD2 cooperates with the structural components cavin1, caveolin-1, and the F-BAR protein pacsin2/syndapin2, occupying the caveolar body/neck while pacsin2 links the structure to the plasma membrane [#5, #20]. By restricting caveolar internalization, EHD2 supports caveola-dependent fatty acid/CD36 uptake and adipocyte lipid handling [#16, #18], eNOS surface localization and nitric oxide signaling [#17], sarcolemmal KATP channel and Orai1/SOCE surface expression [#15, #22], and Dll4 endocytosis controlling Notch activation [#19]. EHD2 additionally acts as a mechanotransducer: under mechanical stress it is released from caveolae, SUMOylated, and translocated to the nucleus, where it represses transcription of target genes including caveolae constituents and CDKN1A/p21 via a KLF7-dependent mechanism [#7, #14]. Independent of caveolae, EHD2 couples endocytic recycling to the actin cytoskeleton and membrane-fusion/repair machinery through its EH-domain partner EHBP1 and the ferlins myoferlin and Fer1L5, contributing to myoblast fusion and sarcolemmal repair [#0, #2, #8], and forms a Rab10–EHBP1–EHD2 complex driving lipophagy of lipid droplets [#12].\",\n  \"teleology\": [\n    {\n      \"year\": 2003,\n      \"claim\": \"Established EHD2's earliest known function—linking endocytosis to the actin cytoskeleton—by identifying its EH-domain partner and a defined trafficking defect.\",\n      \"evidence\": \"siRNA knockdown, co-IP with EHBP1, and transferrin/GLUT4 endocytosis assays\",\n      \"pmids\": [\"14676205\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not address caveolar localization or ATPase mechanism\", \"EHBP1-actin coupling not structurally resolved\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Connected EHD2 to insulin-regulated GLUT4 trafficking, implying a role in metabolic membrane transport.\",\n      \"evidence\": \"MS identification in GLUT4 vesicles, Co-IP, GST pull-down, antibody inhibition in permeabilized adipocytes\",\n      \"pmids\": [\"15182197\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanistic link to caveolae not yet known\", \"Adaptor interactions not validated in vivo\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Linked EHD2-driven endocytic recycling to the membrane-fusion machinery of myoblasts via direct ferlin binding.\",\n      \"evidence\": \"C2-domain binding mapping to myoferlin, dominant-negative EHD2, fusion and recycling assays\",\n      \"pmids\": [\"18502764\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether caveolae are involved in ferlin trafficking unclear\", \"Single lab\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Extended the ferlin connection to Fer1L5, showing EHD2 is specifically required for ferlin delivery to the plasma membrane.\",\n      \"evidence\": \"C2-domain binding assay, siRNA knockdown, plasma membrane translocation and fusion assays\",\n      \"pmids\": [\"21177873\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular basis of selective EHD2 vs EHD1 roles unresolved\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Reported a palmitoylation-dependent prohibitin–EHD2 plasma membrane interaction.\",\n      \"evidence\": \"Cys69 palmitoylation mutagenesis, membrane fractionation, PHB-EHD2 Co-IP\",\n      \"pmids\": [\"20555396\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single Co-IP with no functional follow-up on EHD2's role\", \"No reciprocal validation\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Placed EHD2 upstream of Rho-GTPase signaling, showing its trafficking control depends on nucleotide binding and Rac1.\",\n      \"evidence\": \"Yeast two-hybrid (Nek3, Vav1), Rac1 activity assay, cholera toxin trafficking, P-loop mutagenesis\",\n      \"pmids\": [\"21756249\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether Rac1 regulation acts through caveolae not addressed\", \"Direct vs indirect Vav1 link unclear\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Redefined EHD2 as a third structural component of caveolae, establishing the central caveolar stabilization function and its ATPase/oligomerization basis.\",\n      \"evidence\": \"Live imaging, EM, ATPase assays, sucrose gradients, FRAP, siRNA/dominant-negative, Co-IP with pacsin2 and cavin1\",\n      \"pmids\": [\"22323287\", \"22505029\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Atomic-resolution scaffold geometry not yet resolved\", \"How ATPase cycle is regulated in cells unclear\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Identified a non-caveolar role in sarcolemmal membrane repair requiring EHD2 ATPase activity.\",\n      \"evidence\": \"Laser wounding of myotubes, GFP-EHD2 imaging, EHD2 vs EHD1 and mutant comparison\",\n      \"pmids\": [\"22679923\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Repair partners at the wound site not identified\", \"Relationship to ferlin function not tested\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Revealed an unexpected nuclear function for EHD2, showing SUMOylation-gated shuttling and transcriptional repression of p21/CDKN1A.\",\n      \"evidence\": \"NLS/NES mutagenesis, SUMOylation Co-IP, yeast two-hybrid, GAL4 transactivation and KLF7-p21 transcription assays, qRT-PCR\",\n      \"pmids\": [\"22448906\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Trigger for nuclear shuttling not yet linked to caveolae\", \"Direct DNA/chromatin engagement undefined\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Defined PIP2 as the primary determinant of EHD2 plasma membrane targeting, independent of actin and EH-domain partners.\",\n      \"evidence\": \"Confocal imaging with PIP2 pharmacology, Arf6 vacuoles, cytochalasin D, partner siRNA, domain deletions\",\n      \"pmids\": [\"24040268\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Specific PIP2-binding residues not mapped here\", \"Single lab\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Provided the structural basis for membrane insertion, showing the N-terminus folds into the GTPase domain and can wedge into the bilayer to create curvature.\",\n      \"evidence\": \"EPR, X-ray crystallography, cryo-EM, N-terminal mutagenesis\",\n      \"pmids\": [\"24508342\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In-cell conformational triggering not directly observed\", \"Filament geometry at caveolar necks not resolved\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Dissected the NPF/KPF loop motifs controlling localization, dimerization, and syndapin2 binding versus nuclear partitioning.\",\n      \"evidence\": \"Site-directed mutagenesis, Co-IP, confocal localization, nuclear fractionation\",\n      \"pmids\": [\"25875965\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How motif occupancy is regulated dynamically unknown\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Identified a Rab10–EHBP1–EHD2 complex essential for lipophagy, extending EHD2 to autophagic lipid droplet clearance.\",\n      \"evidence\": \"siRNA, GTPase-defective Rab10, imaging, Co-IP, LC3 and lipid droplet assays\",\n      \"pmids\": [\"28028537\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"EHD2's specific membrane-shaping step in lipophagy undefined\", \"Single lab\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Resolved the conformational cycle: autoinhibited in solution, opening and inserting upon ATP binding, oligomerizing, then detaching on hydrolysis to restrict caveolae dynamics.\",\n      \"evidence\": \"IRRAS, ATPase assays, mutagenesis, caveolae dynamics assays\",\n      \"pmids\": [\"28223496\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In-cell timing of the cycle not directly measured\", \"What regulates hydrolysis-driven release unclear\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Established EHD2 as a mechanotransducer coupling caveolar tension sensing to nuclear gene regulation and showed its loss abolishes caveolae in cancer cells.\",\n      \"evidence\": \"Live imaging, metal-replica EM, mechanical stress, SUMOylation, transcriptomics, KO and rescue\",\n      \"pmids\": [\"30348749\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct transcriptional targets and DNA engagement incompletely defined\", \"Mechanosensing threshold mechanism unknown\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Demonstrated EHD2 stabilizes caveolae to sustain cardiac KATP channel surface expression, with protective relevance to ischemia.\",\n      \"evidence\": \"Surface biotinylation, patch clamp, dominant-negative EHD2, ischemia assay, EHD paralog comparison\",\n      \"pmids\": [\"29133341\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vivo cardiac KO phenotype not tested here\", \"Direct KATP–caveolae binding interface undefined\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Linked EHD2-controlled caveolae stability to metabolic fatty acid uptake and obesity via a CD36-dependent pathway, using a whole-animal knockout.\",\n      \"evidence\": \"EHD2 KO mice, fatty acid uptake, EM caveolae counting, caveolar mobility, CD36/dynamin pharmacology\",\n      \"pmids\": [\"32170013\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether nuclear EHD2 contributes to the metabolic phenotype unclear\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Showed EHD2-dependent caveolar stability governs eNOS surface localization and endothelial NO/Ca2+ signaling.\",\n      \"evidence\": \"EHD2 KO mice, vascular relaxation, NO measurement, super-resolution eNOS imaging, Ca2+ imaging, HUVEC siRNA\",\n      \"pmids\": [\"31600286\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct eNOS–caveolae retention mechanism not biochemically dissected\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Connected EHD2 to adipocyte differentiation and insulin sensitivity, including suppression of PPARγ activity.\",\n      \"evidence\": \"siRNA, overexpression, imaging, insulin sensitivity, lipolysis, PPARγ activity assays\",\n      \"pmids\": [\"30811273\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism linking caveolar EHD2 to PPARγ transcription undefined\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Identified EHD2 as a modulator of Notch signaling through caveolae-dependent Dll4 endocytosis, with an in vivo vascular phenotype.\",\n      \"evidence\": \"Co-localization, caveolae disruption pharmacology, Dll4 internalization, Notch readouts, zebrafish KO\",\n      \"pmids\": [\"34820962\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct EHD2–Dll4 interaction not demonstrated\", \"Single lab\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Refined the molecular architecture of caveolae by positioning EHD2 at the caveolar body/neck relative to PACSIN2 and caveolin-1.\",\n      \"evidence\": \"Super-resolution single-molecule localization microscopy and 3D spatial analysis\",\n      \"pmids\": [\"35834519\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No functional mutagenesis tied to the spatial map\", \"Static snapshot, not dynamic\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Showed EHD2 maintains plasma membrane integrity required for insulin receptor signaling and GLUT4 translocation in adipocytes.\",\n      \"evidence\": \"EHD2 KO mice on high-fat diet, 3T3-L1 adipocytes, GLUT4 translocation, insulin signaling, membrane composition, SNARE Co-IP\",\n      \"pmids\": [\"37703099\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Causal chain from caveolae loss to insulin receptor downregulation incompletely defined\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Defined an EHD2–SOCE oncogenic axis whereby caveolar stabilization sustains Orai1 surface expression to drive tumorigenesis.\",\n      \"evidence\": \"shRNA, CRISPR KO with mouse Ehd2 rescue, surface expression, SOCE, caveolae imaging, tumor/metastasis assays\",\n      \"pmids\": [\"36625722\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct Orai1–caveolae interaction not mapped\", \"Single lab\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Provided a near-atomic structural model of EHD2 filaments shaping a highly curved membrane scaffold, with the N-terminus acting as an inter-filament spacer and a KO correlate at caveolar necks.\",\n      \"evidence\": \"Cryo-electron tomography of reconstituted EHD2-tubulated liposomes plus EM of EHD2 KO endothelial caveolar necks (preprint)\",\n      \"pmids\": [\"bio_10.1101_2025.06.05.658037\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Preprint, not peer-reviewed\", \"In-cell filament organization around native caveolar necks not directly resolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How EHD2 nuclear translocation and its transcriptional targets are mechanistically coupled to specific physiological mechanical stimuli, and whether nuclear EHD2 contributes to its metabolic and oncogenic phenotypes, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct DNA/chromatin engagement by nuclear EHD2 undefined\", \"Full target gene set across tissues unknown\", \"Relative contribution of caveolar vs nuclear EHD2 to disease phenotypes unquantified\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140657\", \"supporting_discovery_ids\": [5, 6, 13]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [9, 10, 13]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [5, 23]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [7, 14]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [0, 6]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [6, 13]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 5, 6, 9]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [0, 6]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [7, 14]},\n      {\"term_id\": \"GO:0005811\", \"supporting_discovery_ids\": [12, 18]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [5, 6, 0]},\n      {\"term_id\": \"R-HSA-8953897\", \"supporting_discovery_ids\": [14]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [12]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [16, 18]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [7, 14]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [17, 19]}\n    ],\n    \"complexes\": [\n      \"caveolae\",\n      \"Rab10-EHBP1-EHD2 complex\"\n    ],\n    \"partners\": [\n      \"EHBP1\",\n      \"CAVIN1\",\n      \"PACSIN2\",\n      \"CAV1\",\n      \"MYOF\",\n      \"FER1L5\",\n      \"RAB10\",\n      \"GLUT4\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}