{"gene":"GAPVD1","run_date":"2026-06-10T01:55:21","timeline":{"discoveries":[{"year":2018,"finding":"GAPVD1 physically interacts with active RAB5 (endosomal regulator) and with ANKFY1, as demonstrated by coimmunoprecipitation; patient-derived missense mutations in GAPVD1 alter binding affinity for active RAB5 and reduce nephrin-GAPVD1 binding. GAPVD1 also physically interacts with nephrin (the slit diaphragm protein) and partially colocalizes with it in rat glomeruli. Silencing GAPVD1 diminishes podocyte migration rate, and in Drosophila nephrocytes silencing Gapvd1 impairs endocytosis and causes mistrafficking of the nephrin ortholog.","method":"Coimmunoprecipitation, colocalization by immunofluorescence, siRNA knockdown with migration assay, Drosophila nephrocyte endocytosis assay, ectopic expression of patient-derived mutants","journal":"Journal of the American Society of Nephrology : JASN","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, functional rescue with patient mutations, orthogonal Drosophila endocytosis assay, multiple methods in one study","pmids":["29959197"],"is_preprint":false},{"year":2020,"finding":"GAPVD1 is a direct substrate of CK1δ and CK1ε: it was identified as one of the most abundant interacting partners of endogenously tagged CK1δ/ε by mass spectrometry. In vitro kinase assay demonstrated up to 38 phosphorylated residues on GAPVD1 by CK1δ/ε. Phosphorylation of GAPVD1 is required for its function in endocytosis: a phosphomimetic mutant of GAPVD1, but not a phospho-ablating mutant, rescued defects in transferrin and EGF internalization caused by loss of endogenous GAPVD1.","method":"Mass spectrometry co-purification, in vitro kinase assay, phosphomimetic/phospho-ablating mutant rescue of transferrin and EGF endocytosis in GAPVD1-deficient cells","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — in vitro kinase assay plus mutagenesis-based functional rescue, single lab but multiple orthogonal methods","pmids":["32321936"],"is_preprint":false},{"year":2017,"finding":"GAPVD1 is a component of cytoplasmic PER complexes in mouse liver, where it functions as a cytoplasmic trafficking factor that regulates the assembly pathway of PER/CRY/CK1δ complexes (~0.9–1.1 MDa) prior to their incorporation into the nuclear repressor complex.","method":"Biochemical fractionation, single-particle electron microscopy of purified endogenous PER complexes from mouse liver","journal":"Molecular cell","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — single lab, fractionation and EM of endogenous complexes but functional role of GAPVD1 inferred from complex composition rather than direct loss-of-function in this study","pmids":["28886335"],"is_preprint":false},{"year":2019,"finding":"GAPVD1 (Gapex-5) is phosphorylated by AMPK on Ser902 in primary mouse hepatocytes upon AMPK activation, identifying it as a novel AMPK substrate involved in vesicle trafficking.","method":"Chemical genetic screen with specific AMPK activator (991) in primary hepatocytes, mass spectrometry, immunoblotting with phosphorylation site-specific antibody","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — chemical genetics plus MS identification of phosphosite, confirmed by phospho-specific antibody, single lab","pmids":["30772465"],"is_preprint":false},{"year":2021,"finding":"GAPVD1 is a bona fide component of human PER complexes (not only mouse). CSNK1D (CK1δ) regulates the phosphorylation of GAPVD1 in situ, and phosphorylation state determines the kinetics of GAPVD1 degradation. PER2 and the C-terminal autoinhibitory domain of CSNK1D control GAPVD1 phosphorylation, indicating that regulation of GAPVD1 phosphorylation is a function of cytoplasmic PER complexes.","method":"Biochemical screen for PER2-interacting proteins, immunoprecipitation, immunoblotting with phospho-specific antibodies, pulse-chase/degradation assays","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — Co-IP plus phosphorylation/degradation assays, single lab, no in vitro reconstitution","pmids":["33917494"],"is_preprint":false},{"year":2018,"finding":"Evolutionary and functional analysis places GAPVD1 in the Rabex5+GAPVD1 subfamily of Vps9-domain GTPase regulators, conserved across eukaryotes. The VPS9 domain enables guanine nucleotide exchange on endocytic RAB GTPases (Rab5, Rab21, Rab22), linking GAPVD1 to regulation of early endosomes.","method":"Molecular evolutionary analysis combined with functional characterization of the ortholog in T. brucei (simultaneous knockdown prevents membrane recruitment of Rab5 and Rab21)","journal":"Traffic (Copenhagen, Denmark)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ortholog knockdown with Rab5/Rab21 membrane recruitment readout in T. brucei; functional conservation supports mechanistic inference for mammalian GAPVD1","pmids":["29603841"],"is_preprint":false},{"year":2024,"finding":"GAPVD1 mediates downstream signaling from the NRP-1 receptor activated by VEGFA in TNBC cells, acting through the Wnt/β-catenin pathway to promote cancer stem cell phenotype. NRP-1 receptor engagement by VEGFA leads to GAPVD1-dependent activation of Wnt/β-catenin signaling.","method":"GAPVD1 knockdown/overexpression in TNBC cells, Western blotting for Wnt/β-catenin pathway components, co-culture macrophage-TNBC assays, cancer stemness functional assays","journal":"International journal of biological sciences","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, pathway placement inferred from KD/OE and Western blot without direct biochemical interaction between NRP-1 and GAPVD1","pmids":["38169627"],"is_preprint":false},{"year":2025,"finding":"GAPVD1 knockdown in TNBC cells reduces cell proliferation and alters cell cycle progression, associated with decreased levels of PCNA, Cyclin A, and reduced ERK/MAPK pathway activity; GAPVD1 overexpression has the opposite effect. In vivo, GAPVD1 inhibition impedes tumor growth.","method":"CCK-8, colony formation, flow cytometry (cell cycle), Western blotting for PCNA/Cyclin A/ERK pathway, xenograft mouse model","journal":"Current cancer drug targets","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, phenotypic KD/OE with Western blot, no direct biochemical mechanism linking GAPVD1 to ERK pathway established","pmids":["39021189"],"is_preprint":false}],"current_model":"GAPVD1 is a cytoplasmic GTPase-activating and VPS9-domain protein that functions as a RAB5 guanine nucleotide exchange factor/regulator to promote early endocytosis; it is directly phosphorylated by CK1δ/ε (with up to 38 sites), and this phosphorylation is required for its role in receptor internalization (transferrin, EGF); it is also an AMPK substrate (Ser902); it associates with cytoplasmic PER complexes where CK1δ regulates its phosphorylation and consequent degradation; and loss-of-function mutations that impair its interaction with active RAB5 or nephrin cause nephrotic syndrome in humans."},"narrative":{"mechanistic_narrative":"GAPVD1 is a cytoplasmic VPS9-domain guanine nucleotide exchange regulator for endocytic RAB GTPases that promotes early endosome formation and receptor internalization [PMID:29603841, PMID:32321936]. Through its VPS9 domain it acts on RAB5, RAB21, and RAB22, and its ortholog is required for membrane recruitment of Rab5 and Rab21, placing GAPVD1 in the conserved Rabex5+GAPVD1 subfamily of endocytic Rab regulators [PMID:29603841]. GAPVD1 physically associates with active RAB5 and with ANKFY1, and its activity supports internalization of the transferrin and EGF receptors [PMID:29959197, PMID:32321936]. This endocytic function is gated by phosphorylation: CK1δ/ε directly phosphorylate GAPVD1 at numerous residues, and a phosphomimetic but not a phospho-ablating form rescues transferrin and EGF uptake in GAPVD1-deficient cells, establishing phosphorylation as a requirement for its trafficking role [PMID:32321936]. GAPVD1 is also a substrate of AMPK at Ser902 [PMID:30772465], and it is a component of cytoplasmic PER complexes in which CK1δ/CSNK1D and PER2 control its phosphorylation state and consequent degradation kinetics, coupling its trafficking activity to circadian-clock machinery [PMID:28886335, PMID:33917494]. In the kidney, GAPVD1 binds nephrin and partially colocalizes with it at the glomerular slit diaphragm, and loss-of-function missense mutations that weaken its binding to active RAB5 or to nephrin cause human nephrotic syndrome, with parallel endocytic and nephrin-trafficking defects in Drosophila nephrocytes [PMID:29959197].","teleology":[{"year":2017,"claim":"Established that GAPVD1 is not solely an endocytic factor but a stable cytoplasmic component of PER complexes, linking it to circadian-clock assembly machinery.","evidence":"Biochemical fractionation and single-particle EM of endogenous PER complexes from mouse liver","pmids":["28886335"],"confidence":"Medium","gaps":["Functional role inferred from complex composition rather than direct loss-of-function","No mechanism for how a trafficking factor contributes to PER complex assembly","Human relevance not tested in this study"]},{"year":2018,"claim":"Defined GAPVD1's disease relevance and molecular partners by showing it binds active RAB5 and nephrin and that patient mutations weaken these interactions to cause nephrotic syndrome.","evidence":"Reciprocal Co-IP, immunofluorescence colocalization in rat glomeruli, siRNA podocyte migration assay, Drosophila nephrocyte endocytosis assay, patient-mutant expression","pmids":["29959197"],"confidence":"High","gaps":["Whether GAPVD1 acts catalytically as a GEF on RAB5 in this context not directly assayed","Mechanistic link between nephrin endocytosis and slit-diaphragm maintenance not fully resolved"]},{"year":2018,"claim":"Placed GAPVD1 in the conserved Rabex5+GAPVD1 VPS9-domain subfamily and assigned it nucleotide exchange activity toward endocytic Rab GTPases.","evidence":"Molecular evolutionary analysis with T. brucei ortholog knockdown reading out Rab5/Rab21 membrane recruitment","pmids":["29603841"],"confidence":"Medium","gaps":["GEF activity for mammalian GAPVD1 inferred by orthology rather than measured biochemically","Substrate Rab specificity in human cells not directly ranked"]},{"year":2019,"claim":"Identified GAPVD1 as an AMPK substrate, connecting its trafficking function to energy-sensing signaling.","evidence":"Chemical genetic AMPK activation in primary hepatocytes, mass spectrometry, phospho-Ser902-specific immunoblotting","pmids":["30772465"],"confidence":"Medium","gaps":["Functional consequence of Ser902 phosphorylation on endocytosis not established","Single lab, single cell system"]},{"year":2020,"claim":"Demonstrated that CK1δ/ε directly phosphorylate GAPVD1 and that this phosphorylation is required for its receptor-internalization function.","evidence":"MS co-purification with endogenously tagged CK1δ/ε, in vitro kinase assay, phosphomimetic/phospho-ablating mutant rescue of transferrin and EGF endocytosis","pmids":["32321936"],"confidence":"High","gaps":["Which of the up-to-38 sites are functionally critical not resolved","How phosphorylation alters GEF activity or partner binding not defined"]},{"year":2021,"claim":"Extended the PER-complex finding to human cells and showed CSNK1D and PER2 control GAPVD1 phosphorylation, which in turn sets its degradation kinetics.","evidence":"Biochemical screen for PER2-interacting proteins, Co-IP, phospho-specific immunoblotting, degradation/pulse-chase assays","pmids":["33917494"],"confidence":"Medium","gaps":["No in vitro reconstitution of the regulatory circuit","Whether circadian regulation of GAPVD1 feeds back on endocytosis untested"]},{"year":2024,"claim":"Proposed a signaling role for GAPVD1 downstream of NRP-1/VEGFA via Wnt/β-catenin in triple-negative breast cancer stemness.","evidence":"GAPVD1 knockdown/overexpression in TNBC cells, Western blotting, macrophage co-culture and stemness assays","pmids":["38169627"],"confidence":"Low","gaps":["Pathway placement inferred from KD/OE and Western blot without direct NRP-1–GAPVD1 interaction","Mechanism linking endocytic regulator to Wnt signaling unestablished"]},{"year":2025,"claim":"Associated GAPVD1 levels with TNBC proliferation, cell-cycle progression, and ERK/MAPK activity in vitro and tumor growth in vivo.","evidence":"CCK-8, colony formation, flow cytometry, Western blotting for PCNA/Cyclin A/ERK, xenograft model","pmids":["39021189"],"confidence":"Low","gaps":["No direct biochemical mechanism linking GAPVD1 to ERK pathway","Phenotypic correlation only; causal molecular step undefined"]},{"year":null,"claim":"It remains unresolved how GAPVD1 phosphorylation by CK1δ/ε and AMPK mechanistically modulates its GEF activity toward specific Rab GTPases, and whether its circadian and cancer-signaling roles share a common biochemical basis.","evidence":"","pmids":[],"confidence":"Low","gaps":["No direct measurement of GAPVD1 GEF kinetics as a function of phosphorylation","No structural model of the VPS9 domain engaging Rab5/Rab21/Rab22","Connection between PER-complex regulation and endocytic output untested"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,5]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[2]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[0,5]}],"pathway":[{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[0,1,5]},{"term_id":"R-HSA-9909396","term_label":"Circadian clock","supporting_discovery_ids":[2,4]}],"complexes":["PER/CRY/CK1δ cytoplasmic complex"],"partners":["RAB5","ANKFY1","NPHS1","CSNK1D","CSNK1E","PER2"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q14C86","full_name":"GTPase-activating protein and VPS9 domain-containing protein 1","aliases":["GAPex-5","Rab5-activating protein 6"],"length_aa":1478,"mass_kda":165.0,"function":"Acts both as a GTPase-activating protein (GAP) and a guanine nucleotide exchange factor (GEF), and participates in various processes such as endocytosis, insulin receptor internalization or LC2A4/GLUT4 trafficking. Acts as a GEF for the Ras-related protein RAB31 by exchanging bound GDP for free GTP, leading to regulate LC2A4/GLUT4 trafficking. In the absence of insulin, it maintains RAB31 in an active state and promotes a futile cycle between LC2A4/GLUT4 storage vesicles and early endosomes, retaining LC2A4/GLUT4 inside the cells. Upon insulin stimulation, it is translocated to the plasma membrane, releasing LC2A4/GLUT4 from intracellular storage vesicles. Also involved in EGFR trafficking and degradation, possibly by promoting EGFR ubiquitination and subsequent degradation by the proteasome. Has GEF activity for Rab5 and GAP activity for Ras","subcellular_location":"Membrane; Endosome","url":"https://www.uniprot.org/uniprotkb/Q14C86/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/GAPVD1","classification":"Not Classified","n_dependent_lines":36,"n_total_lines":1208,"dependency_fraction":0.029801324503311258},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"CSNK1A1","stoichiometry":10.0},{"gene":"CSNK1D","stoichiometry":10.0},{"gene":"CSNK1E","stoichiometry":4.0}],"url":"https://opencell.sf.czbiohub.org/search/GAPVD1","total_profiled":1310},"omim":[{"mim_id":"611714","title":"GTPase-ACTIVATING PROTEIN AND VPS9 DOMAINS 1; GAPVD1","url":"https://www.omim.org/entry/611714"},{"mim_id":"607927","title":"ANKYRIN REPEATS- AND FYVE DOMAIN-CONTAINING PROTEIN 1; ANKFY1","url":"https://www.omim.org/entry/607927"},{"mim_id":"256300","title":"NEPHROTIC SYNDROME, TYPE 1; NPHS1","url":"https://www.omim.org/entry/256300"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Plasma membrane","reliability":"Supported"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/GAPVD1"},"hgnc":{"alias_symbol":["DKFZP434C212","KIAA1521"],"prev_symbol":[]},"alphafold":{"accession":"Q14C86","domains":[{"cath_id":"-","chopping":"2-63_1115-1135_1155-1324","consensus_level":"medium","plddt":88.4872,"start":2,"end":1324},{"cath_id":"1.10.506.10","chopping":"190-349_360-369","consensus_level":"medium","plddt":88.0315,"start":190,"end":369},{"cath_id":"1.20.1050.80","chopping":"1332-1477","consensus_level":"high","plddt":91.5306,"start":1332,"end":1477}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q14C86","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q14C86-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q14C86-F1-predicted_aligned_error_v6.png","plddt_mean":62.19},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=GAPVD1","jax_strain_url":"https://www.jax.org/strain/search?query=GAPVD1"},"sequence":{"accession":"Q14C86","fasta_url":"https://rest.uniprot.org/uniprotkb/Q14C86.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q14C86/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q14C86"}},"corpus_meta":[{"pmid":"28886335","id":"PMC_28886335","title":"Macromolecular Assemblies of the Mammalian Circadian Clock.","date":"2017","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/28886335","citation_count":208,"is_preprint":false},{"pmid":"33243845","id":"PMC_33243845","title":"Genetic analysis of obstructive sleep apnoea discovers a strong association with cardiometabolic health.","date":"2021","source":"The European respiratory journal","url":"https://pubmed.ncbi.nlm.nih.gov/33243845","citation_count":164,"is_preprint":false},{"pmid":"22864814","id":"PMC_22864814","title":"Selection of novel reference genes for use in the human central nervous system: a BrainNet Europe Study.","date":"2012","source":"Acta neuropathologica","url":"https://pubmed.ncbi.nlm.nih.gov/22864814","citation_count":98,"is_preprint":false},{"pmid":"29959197","id":"PMC_29959197","title":"GAPVD1 and ANKFY1 Mutations Implicate RAB5 Regulation in Nephrotic Syndrome.","date":"2018","source":"Journal of the American Society of Nephrology : JASN","url":"https://pubmed.ncbi.nlm.nih.gov/29959197","citation_count":50,"is_preprint":false},{"pmid":"38169627","id":"PMC_38169627","title":"VEGFA/NRP-1/GAPVD1 axis promotes progression and cancer stemness of triple-negative breast cancer by enhancing tumor cell-macrophage crosstalk.","date":"2024","source":"International journal of biological sciences","url":"https://pubmed.ncbi.nlm.nih.gov/38169627","citation_count":49,"is_preprint":false},{"pmid":"27001119","id":"PMC_27001119","title":"Response to interferon-beta treatment in multiple sclerosis patients: a genome-wide association study.","date":"2016","source":"The pharmacogenomics journal","url":"https://pubmed.ncbi.nlm.nih.gov/27001119","citation_count":25,"is_preprint":false},{"pmid":"30772465","id":"PMC_30772465","title":"Chemical genetic screen identifies Gapex-5/GAPVD1 and STBD1 as novel AMPK substrates.","date":"2019","source":"Cellular signalling","url":"https://pubmed.ncbi.nlm.nih.gov/30772465","citation_count":23,"is_preprint":false},{"pmid":"36094435","id":"PMC_36094435","title":"Role of syndecan-4 in breast cancer pathophysiology.","date":"2022","source":"American journal of physiology. Cell physiology","url":"https://pubmed.ncbi.nlm.nih.gov/36094435","citation_count":15,"is_preprint":false},{"pmid":"32321936","id":"PMC_32321936","title":"CRISPR-mediated gene targeting of CK1δ/ε leads to enhanced understanding of their role in endocytosis via phosphoregulation of GAPVD1.","date":"2020","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/32321936","citation_count":13,"is_preprint":false},{"pmid":"29603841","id":"PMC_29603841","title":"Regulation of early endosomes across eukaryotes: Evolution and functional homology of Vps9 proteins.","date":"2018","source":"Traffic (Copenhagen, Denmark)","url":"https://pubmed.ncbi.nlm.nih.gov/29603841","citation_count":11,"is_preprint":false},{"pmid":"32356940","id":"PMC_32356940","title":"Interaction of Plasmodium falciparum casein kinase 1 with components of host cell protein trafficking machinery.","date":"2020","source":"IUBMB life","url":"https://pubmed.ncbi.nlm.nih.gov/32356940","citation_count":7,"is_preprint":false},{"pmid":"33917494","id":"PMC_33917494","title":"Phosphorylation of GAPVD1 Is Regulated by the PER Complex and Linked to GAPVD1 Degradation.","date":"2021","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/33917494","citation_count":6,"is_preprint":false},{"pmid":"39021189","id":"PMC_39021189","title":"GAPVD1 Promotes the Proliferation of Triple-negative Breast Cancer Cells by Regulating the ERK/MAPK Signaling Pathway.","date":"2025","source":"Current cancer drug targets","url":"https://pubmed.ncbi.nlm.nih.gov/39021189","citation_count":2,"is_preprint":false},{"pmid":"40545108","id":"PMC_40545108","title":"circGAPVD1 inhibits the progression of gastric cancer through miR-4424/STK4 axis and encoding GAPVD1-137aa protein.","date":"2025","source":"International journal of biological macromolecules","url":"https://pubmed.ncbi.nlm.nih.gov/40545108","citation_count":2,"is_preprint":false},{"pmid":"40311696","id":"PMC_40311696","title":"Comprehensive genetic analysis based on multi - omics reveals novel therapeutic targets for mitral valve prolapse and drug molecular dynamics simulation.","date":"2025","source":"International journal of cardiology","url":"https://pubmed.ncbi.nlm.nih.gov/40311696","citation_count":1,"is_preprint":false},{"pmid":"38011643","id":"PMC_38011643","title":"Screening and Identifying Reference Genes for Erythrocyte Production from Cord Blood CD34+ Cells Exposed to Hypoxia.","date":"2023","source":"DNA and cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/38011643","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":10376,"output_tokens":2388,"usd":0.033474,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9450,"output_tokens":3195,"usd":0.063562,"stage2_stop_reason":"end_turn"},"total_usd":0.097036,"stage1_batch_id":"msgbatch_01DUPACZi7AAWwuZiUjseQF4","stage2_batch_id":"msgbatch_018yt7chE3f48PEFtDwtj6AE","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2018,\n      \"finding\": \"GAPVD1 physically interacts with active RAB5 (endosomal regulator) and with ANKFY1, as demonstrated by coimmunoprecipitation; patient-derived missense mutations in GAPVD1 alter binding affinity for active RAB5 and reduce nephrin-GAPVD1 binding. GAPVD1 also physically interacts with nephrin (the slit diaphragm protein) and partially colocalizes with it in rat glomeruli. Silencing GAPVD1 diminishes podocyte migration rate, and in Drosophila nephrocytes silencing Gapvd1 impairs endocytosis and causes mistrafficking of the nephrin ortholog.\",\n      \"method\": \"Coimmunoprecipitation, colocalization by immunofluorescence, siRNA knockdown with migration assay, Drosophila nephrocyte endocytosis assay, ectopic expression of patient-derived mutants\",\n      \"journal\": \"Journal of the American Society of Nephrology : JASN\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, functional rescue with patient mutations, orthogonal Drosophila endocytosis assay, multiple methods in one study\",\n      \"pmids\": [\"29959197\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"GAPVD1 is a direct substrate of CK1δ and CK1ε: it was identified as one of the most abundant interacting partners of endogenously tagged CK1δ/ε by mass spectrometry. In vitro kinase assay demonstrated up to 38 phosphorylated residues on GAPVD1 by CK1δ/ε. Phosphorylation of GAPVD1 is required for its function in endocytosis: a phosphomimetic mutant of GAPVD1, but not a phospho-ablating mutant, rescued defects in transferrin and EGF internalization caused by loss of endogenous GAPVD1.\",\n      \"method\": \"Mass spectrometry co-purification, in vitro kinase assay, phosphomimetic/phospho-ablating mutant rescue of transferrin and EGF endocytosis in GAPVD1-deficient cells\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro kinase assay plus mutagenesis-based functional rescue, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"32321936\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"GAPVD1 is a component of cytoplasmic PER complexes in mouse liver, where it functions as a cytoplasmic trafficking factor that regulates the assembly pathway of PER/CRY/CK1δ complexes (~0.9–1.1 MDa) prior to their incorporation into the nuclear repressor complex.\",\n      \"method\": \"Biochemical fractionation, single-particle electron microscopy of purified endogenous PER complexes from mouse liver\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — single lab, fractionation and EM of endogenous complexes but functional role of GAPVD1 inferred from complex composition rather than direct loss-of-function in this study\",\n      \"pmids\": [\"28886335\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"GAPVD1 (Gapex-5) is phosphorylated by AMPK on Ser902 in primary mouse hepatocytes upon AMPK activation, identifying it as a novel AMPK substrate involved in vesicle trafficking.\",\n      \"method\": \"Chemical genetic screen with specific AMPK activator (991) in primary hepatocytes, mass spectrometry, immunoblotting with phosphorylation site-specific antibody\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — chemical genetics plus MS identification of phosphosite, confirmed by phospho-specific antibody, single lab\",\n      \"pmids\": [\"30772465\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"GAPVD1 is a bona fide component of human PER complexes (not only mouse). CSNK1D (CK1δ) regulates the phosphorylation of GAPVD1 in situ, and phosphorylation state determines the kinetics of GAPVD1 degradation. PER2 and the C-terminal autoinhibitory domain of CSNK1D control GAPVD1 phosphorylation, indicating that regulation of GAPVD1 phosphorylation is a function of cytoplasmic PER complexes.\",\n      \"method\": \"Biochemical screen for PER2-interacting proteins, immunoprecipitation, immunoblotting with phospho-specific antibodies, pulse-chase/degradation assays\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — Co-IP plus phosphorylation/degradation assays, single lab, no in vitro reconstitution\",\n      \"pmids\": [\"33917494\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Evolutionary and functional analysis places GAPVD1 in the Rabex5+GAPVD1 subfamily of Vps9-domain GTPase regulators, conserved across eukaryotes. The VPS9 domain enables guanine nucleotide exchange on endocytic RAB GTPases (Rab5, Rab21, Rab22), linking GAPVD1 to regulation of early endosomes.\",\n      \"method\": \"Molecular evolutionary analysis combined with functional characterization of the ortholog in T. brucei (simultaneous knockdown prevents membrane recruitment of Rab5 and Rab21)\",\n      \"journal\": \"Traffic (Copenhagen, Denmark)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ortholog knockdown with Rab5/Rab21 membrane recruitment readout in T. brucei; functional conservation supports mechanistic inference for mammalian GAPVD1\",\n      \"pmids\": [\"29603841\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"GAPVD1 mediates downstream signaling from the NRP-1 receptor activated by VEGFA in TNBC cells, acting through the Wnt/β-catenin pathway to promote cancer stem cell phenotype. NRP-1 receptor engagement by VEGFA leads to GAPVD1-dependent activation of Wnt/β-catenin signaling.\",\n      \"method\": \"GAPVD1 knockdown/overexpression in TNBC cells, Western blotting for Wnt/β-catenin pathway components, co-culture macrophage-TNBC assays, cancer stemness functional assays\",\n      \"journal\": \"International journal of biological sciences\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, pathway placement inferred from KD/OE and Western blot without direct biochemical interaction between NRP-1 and GAPVD1\",\n      \"pmids\": [\"38169627\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"GAPVD1 knockdown in TNBC cells reduces cell proliferation and alters cell cycle progression, associated with decreased levels of PCNA, Cyclin A, and reduced ERK/MAPK pathway activity; GAPVD1 overexpression has the opposite effect. In vivo, GAPVD1 inhibition impedes tumor growth.\",\n      \"method\": \"CCK-8, colony formation, flow cytometry (cell cycle), Western blotting for PCNA/Cyclin A/ERK pathway, xenograft mouse model\",\n      \"journal\": \"Current cancer drug targets\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, phenotypic KD/OE with Western blot, no direct biochemical mechanism linking GAPVD1 to ERK pathway established\",\n      \"pmids\": [\"39021189\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"GAPVD1 is a cytoplasmic GTPase-activating and VPS9-domain protein that functions as a RAB5 guanine nucleotide exchange factor/regulator to promote early endocytosis; it is directly phosphorylated by CK1δ/ε (with up to 38 sites), and this phosphorylation is required for its role in receptor internalization (transferrin, EGF); it is also an AMPK substrate (Ser902); it associates with cytoplasmic PER complexes where CK1δ regulates its phosphorylation and consequent degradation; and loss-of-function mutations that impair its interaction with active RAB5 or nephrin cause nephrotic syndrome in humans.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"GAPVD1 is a cytoplasmic VPS9-domain guanine nucleotide exchange regulator for endocytic RAB GTPases that promotes early endosome formation and receptor internalization [#5, #1]. Through its VPS9 domain it acts on RAB5, RAB21, and RAB22, and its ortholog is required for membrane recruitment of Rab5 and Rab21, placing GAPVD1 in the conserved Rabex5+GAPVD1 subfamily of endocytic Rab regulators [#5]. GAPVD1 physically associates with active RAB5 and with ANKFY1, and its activity supports internalization of the transferrin and EGF receptors [#0, #1]. This endocytic function is gated by phosphorylation: CK1\\u03b4/\\u03b5 directly phosphorylate GAPVD1 at numerous residues, and a phosphomimetic but not a phospho-ablating form rescues transferrin and EGF uptake in GAPVD1-deficient cells, establishing phosphorylation as a requirement for its trafficking role [#1]. GAPVD1 is also a substrate of AMPK at Ser902 [#3], and it is a component of cytoplasmic PER complexes in which CK1\\u03b4/CSNK1D and PER2 control its phosphorylation state and consequent degradation kinetics, coupling its trafficking activity to circadian-clock machinery [#2, #4]. In the kidney, GAPVD1 binds nephrin and partially colocalizes with it at the glomerular slit diaphragm, and loss-of-function missense mutations that weaken its binding to active RAB5 or to nephrin cause human nephrotic syndrome, with parallel endocytic and nephrin-trafficking defects in Drosophila nephrocytes [#0].\",\n  \"teleology\": [\n    {\n      \"year\": 2017,\n      \"claim\": \"Established that GAPVD1 is not solely an endocytic factor but a stable cytoplasmic component of PER complexes, linking it to circadian-clock assembly machinery.\",\n      \"evidence\": \"Biochemical fractionation and single-particle EM of endogenous PER complexes from mouse liver\",\n      \"pmids\": [\"28886335\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Functional role inferred from complex composition rather than direct loss-of-function\",\n        \"No mechanism for how a trafficking factor contributes to PER complex assembly\",\n        \"Human relevance not tested in this study\"\n      ]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Defined GAPVD1's disease relevance and molecular partners by showing it binds active RAB5 and nephrin and that patient mutations weaken these interactions to cause nephrotic syndrome.\",\n      \"evidence\": \"Reciprocal Co-IP, immunofluorescence colocalization in rat glomeruli, siRNA podocyte migration assay, Drosophila nephrocyte endocytosis assay, patient-mutant expression\",\n      \"pmids\": [\"29959197\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether GAPVD1 acts catalytically as a GEF on RAB5 in this context not directly assayed\",\n        \"Mechanistic link between nephrin endocytosis and slit-diaphragm maintenance not fully resolved\"\n      ]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Placed GAPVD1 in the conserved Rabex5+GAPVD1 VPS9-domain subfamily and assigned it nucleotide exchange activity toward endocytic Rab GTPases.\",\n      \"evidence\": \"Molecular evolutionary analysis with T. brucei ortholog knockdown reading out Rab5/Rab21 membrane recruitment\",\n      \"pmids\": [\"29603841\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"GEF activity for mammalian GAPVD1 inferred by orthology rather than measured biochemically\",\n        \"Substrate Rab specificity in human cells not directly ranked\"\n      ]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Identified GAPVD1 as an AMPK substrate, connecting its trafficking function to energy-sensing signaling.\",\n      \"evidence\": \"Chemical genetic AMPK activation in primary hepatocytes, mass spectrometry, phospho-Ser902-specific immunoblotting\",\n      \"pmids\": [\"30772465\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Functional consequence of Ser902 phosphorylation on endocytosis not established\",\n        \"Single lab, single cell system\"\n      ]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Demonstrated that CK1\\u03b4/\\u03b5 directly phosphorylate GAPVD1 and that this phosphorylation is required for its receptor-internalization function.\",\n      \"evidence\": \"MS co-purification with endogenously tagged CK1\\u03b4/\\u03b5, in vitro kinase assay, phosphomimetic/phospho-ablating mutant rescue of transferrin and EGF endocytosis\",\n      \"pmids\": [\"32321936\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Which of the up-to-38 sites are functionally critical not resolved\",\n        \"How phosphorylation alters GEF activity or partner binding not defined\"\n      ]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Extended the PER-complex finding to human cells and showed CSNK1D and PER2 control GAPVD1 phosphorylation, which in turn sets its degradation kinetics.\",\n      \"evidence\": \"Biochemical screen for PER2-interacting proteins, Co-IP, phospho-specific immunoblotting, degradation/pulse-chase assays\",\n      \"pmids\": [\"33917494\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"No in vitro reconstitution of the regulatory circuit\",\n        \"Whether circadian regulation of GAPVD1 feeds back on endocytosis untested\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Proposed a signaling role for GAPVD1 downstream of NRP-1/VEGFA via Wnt/\\u03b2-catenin in triple-negative breast cancer stemness.\",\n      \"evidence\": \"GAPVD1 knockdown/overexpression in TNBC cells, Western blotting, macrophage co-culture and stemness assays\",\n      \"pmids\": [\"38169627\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"Pathway placement inferred from KD/OE and Western blot without direct NRP-1\\u2013GAPVD1 interaction\",\n        \"Mechanism linking endocytic regulator to Wnt signaling unestablished\"\n      ]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Associated GAPVD1 levels with TNBC proliferation, cell-cycle progression, and ERK/MAPK activity in vitro and tumor growth in vivo.\",\n      \"evidence\": \"CCK-8, colony formation, flow cytometry, Western blotting for PCNA/Cyclin A/ERK, xenograft model\",\n      \"pmids\": [\"39021189\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"No direct biochemical mechanism linking GAPVD1 to ERK pathway\",\n        \"Phenotypic correlation only; causal molecular step undefined\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unresolved how GAPVD1 phosphorylation by CK1\\u03b4/\\u03b5 and AMPK mechanistically modulates its GEF activity toward specific Rab GTPases, and whether its circadian and cancer-signaling roles share a common biochemical basis.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"No direct measurement of GAPVD1 GEF kinetics as a function of phosphorylation\",\n        \"No structural model of the VPS9 domain engaging Rab5/Rab21/Rab22\",\n        \"Connection between PER-complex regulation and endocytic output untested\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 5]},\n      {\"term_id\": \"GO:0005085\", \"supporting_discovery_ids\": [5]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [2]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [0, 5]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [0, 1, 5]},\n      {\"term_id\": \"R-HSA-9909396\", \"supporting_discovery_ids\": [2, 4]}\n    ],\n    \"complexes\": [\n      \"PER/CRY/CK1\\u03b4 cytoplasmic complex\"\n    ],\n    \"partners\": [\n      \"RAB5\",\n      \"ANKFY1\",\n      \"NPHS1\",\n      \"CSNK1D\",\n      \"CSNK1E\",\n      \"PER2\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}