{"gene":"ARF5","run_date":"2026-06-09T22:02:44","timeline":{"discoveries":[{"year":2012,"finding":"BRAG2/GEP100/IQSec1 activates endogenous ARF5 (as well as Arf4 and Arf6), and it is specifically ARF5 (not Arf6 or Arf4) that mediates clathrin-mediated endocytosis of β1 integrins. BRAG2 and ARF5 co-localize at clathrin-coated pits; depletion of ARF5 slows β1 integrin internalization without affecting transferrin receptor uptake, and a rapid-cycling ARF5 mutant (T161A) but not the corresponding ARF6 mutant rescues spreading in BRAG2-depleted cells.","method":"siRNA knockdown of ARF5/ARF4/ARF6, rapid-cycling mutant rescue assay, co-localization with clathrin/AP-2, integrin internalization assay, cell spreading on fibronectin","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (KD phenotype, mutant rescue, co-localization, cargo-specific internalization assay) in a single focused study with rigorous controls","pmids":["22815487"],"is_preprint":false},{"year":2012,"finding":"ARAP1, an Arf GAP, uses ARF1 and ARF5 as substrates to control the ring size of circular dorsal ruffles (CDRs). Expression of dominant-negative ARF1 and ARF5 expands CDR size, placing ARF1 and ARF5 downstream of ARAP1 in a pathway regulating actin ring structure.","method":"ARAP1 overexpression/knockdown, dominant-negative ARF1/ARF5 mutant expression, CDR size measurement, Arf GAP activity-dependent mutant analysis","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis (dominant-negative mutants) plus KD/OE with defined phenotypic readout, single lab","pmids":["22573888"],"is_preprint":false},{"year":2015,"finding":"ARF5 (in its active, GTP-bound form) interacts with the GORAB golgin via the IGRAB domain, and this interaction is required for Golgi targeting of GORAB. A patient-derived GORAB missense mutation (p.Ser175Phe) selectively abolishes ARF5 binding while retaining RAB6 binding, displacing GORAB from the Golgi to vesicular structures.","method":"Yeast two-hybrid screening, co-localization with trans-Golgi markers, Brefeldin A treatment, disease-associated mutant analysis, subcellular localization imaging","journal":"The Journal of investigative dermatology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — yeast two-hybrid plus functional validation with disease mutants and localization studies, single lab","pmids":["26000619"],"is_preprint":false},{"year":2023,"finding":"ARF5 is a novel regulator of mTORC1 at plasma membrane ruffles. ARF5 co-localizes with endogenous mTOR, Rheb, and PI3,4P2 at membrane ruffles; ARF5 was identified as an interacting partner of the mTORC1 subunit Raptor by immunoprecipitation/MS. Knockdown of ARF5 reduces mTOR recruitment to ruffles, decreases phosphorylation of a plasma membrane-targeted mTORC1 biosensor substrate, and impairs rapid mTORC1-mediated S6 phosphorylation upon nutrient refeeding.","method":"Immunoprecipitation/mass spectrometry (Raptor interactome), ARF5-GFP co-localization with mTOR/Rheb, ARF5 siRNA knockdown, plasma membrane-targeted mTORC1 biosensor phosphorylation assay, S6 phosphorylation upon nutrient starvation/refeeding","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal IP/MS identification plus multiple orthogonal functional assays (localization, biosensor, nutrient signaling) in a single focused study","pmids":["36735494"],"is_preprint":false},{"year":2024,"finding":"ARF5 (together with ARF1 and ARF4) is required for ER-to-Golgi export of receptor tyrosine kinases (KIT, PDGFRA, EGFR, MET). Simultaneous siRNA knockdown of ARF1, ARF4, and ARF5 (but not single knockdown of any one) blocks RTK ER export, mimicking the effect of BFA/M-COPA. In vitro pulldown assays confirmed that BFA/M-COPA directly blocks ARF1, ARF4, and ARF5 function.","method":"siRNA triple knockdown (ARF1/ARF4/ARF5), RTK ER-export assay, in vitro pulldown with BFA/M-COPA, cell apoptosis assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — in vitro pulldown plus genetic knockdown with defined cargo-trafficking phenotype and pharmacological validation, single lab but multiple orthogonal methods","pmids":["38679330"],"is_preprint":false},{"year":2019,"finding":"ARF5 directly binds foot-and-mouth disease virus IRES RNA (domain 3) and diminishes IRES-driven translation activity. ARF5 co-localizes with IRES-driven RNA near ER-Golgi membranes, suggesting ARF5 participates in IRES-RNA localization to this ribosome-rich compartment.","method":"Proteomic identification of IRES-binding proteins, direct RNA-binding assay, RNA-FISH co-localization with ARF5, IRES activity reporter assay","journal":"Life science alliance","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct binding assay plus functional IRES activity measurement and RNA-FISH co-localization, single lab","pmids":["30655362"],"is_preprint":false},{"year":1999,"finding":"Transcription of the human ARF5 gene is dependent on Sp1 or an Sp1-like factor binding to two GC boxes within 169 bp of the transcription initiation site. Mutation of either GC box reduced reporter expression, and EMSA showed specific protein–DNA complexes competed by canonical Sp1-binding oligonucleotides.","method":"Deletion/mutant luciferase reporter transfection, primer extension analysis, EMSA/electrophoretic mobility-shift assay, site-directed mutagenesis","journal":"Biochimica et biophysica acta","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis of cis elements combined with EMSA and reporter assay, single lab","pmids":["10366714"],"is_preprint":false},{"year":2023,"finding":"Rab11-FIP4 physically interacts with ARF5 and this complex influences CDK1/cyclin B levels to promote cancer stem cell-like characteristics in hepatocellular carcinoma cells.","method":"Co-immunoprecipitation (Rab11-FIP4 and ARF5), sphere formation assay, stemness marker expression, CDK1/cyclin B western blot, Rab11-FIP4 knockdown","journal":"Journal of physiology and biochemistry","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP with limited mechanistic follow-up on ARF5's direct role; phenotype attributed to the complex but ARF5-specific mechanism not dissected","pmids":["37458957"],"is_preprint":false},{"year":2024,"finding":"ARF5 knockout mice (complete ARF5 KO) show no significant change in Zika virus load in serum or tissues and no significant difference in pathological changes compared to wild-type, indicating ARF5 is not essential for ZIKV infection in vivo (negative finding). ARF4, but not ARF5, was found to be essential for ZIKV infection.","method":"CRISPR-Cas9 knockout mice, ZIKV infection, RT-qPCR viral load measurement, H&E histopathology","journal":"Sheng wu gong cheng xue bao = Chinese journal of biotechnology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean genetic KO with direct in vivo viral load readout; negative result for ARF5 is the primary finding","pmids":["39722520"],"is_preprint":false},{"year":2025,"finding":"Rab11-FIP4 interacts with ARF5 in mouse oocytes (confirmed by co-immunoprecipitation), and this interaction is implicated in the endocytic/recycling endosome pathway for nanoparticle internalization.","method":"Co-immunoprecipitation (Rab11fip4 and Arf5), Rab11fip4 knockdown, endocytic inhibitor treatment, transcriptome/WGCNA analysis","journal":"ACS applied materials & interfaces","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP in an oocyte context; ARF5-specific mechanistic role not directly tested beyond the interaction","pmids":["40720586"],"is_preprint":false}],"current_model":"Human/mammalian ARF5 is a class II ADP-ribosylation factor GTPase that functions in vesicular trafficking: it is activated by the ArfGEF BRAG2 at clathrin-coated pits to mediate clathrin-dependent endocytosis of β1 integrins (but not bulk transferrin), acts redundantly with ARF1 and ARF4 to support ER-to-Golgi export of receptor tyrosine kinases (inhibited by BFA/M-COPA), interacts with the GORAB golgin via an IGRAB domain to regulate Golgi targeting, serves as a substrate of the Arf GAP ARAP1 to control actin-ring size at circular dorsal ruffles, and regulates mTORC1 signaling by recruiting the mTORC1 complex to PI3,4P2-enriched plasma membrane ruffles; additionally, ARF5 transcription is driven by Sp1/Sp1-like factors binding GC boxes in a TATA-less promoter."},"narrative":{"mechanistic_narrative":"ARF5 is a small ADP-ribosylation factor GTPase that operates across multiple membrane-trafficking and signaling steps [PMID:22815487, PMID:38679330]. At clathrin-coated pits it is activated by the ArfGEF BRAG2/GEP100/IQSec1 to drive clathrin-mediated endocytosis of β1 integrins, a cargo-selective role distinct from bulk transferrin uptake and not substituted by ARF6 [PMID:22815487]. It also acts redundantly with ARF1 and ARF4 in ER-to-Golgi export of receptor tyrosine kinases (KIT, PDGFRA, EGFR, MET), a function blocked by the ARF inhibitors BFA/M-COPA [PMID:38679330]. In its GTP-bound state ARF5 binds the GORAB golgin through the IGRAB domain to direct GORAB Golgi targeting [PMID:26000619], and it serves as a substrate of the Arf GAP ARAP1 to control actin-ring size at circular dorsal ruffles [PMID:22573888]. Beyond trafficking, ARF5 co-localizes with mTOR, Rheb, and PI3,4P2 at plasma membrane ruffles and interacts with the mTORC1 subunit Raptor to recruit mTORC1 and support rapid nutrient-stimulated S6 phosphorylation [PMID:36735494]. ARF5 transcription is driven by Sp1/Sp1-like factors binding GC boxes in a TATA-less promoter [PMID:10366714].","teleology":[{"year":1999,"claim":"Established how ARF5 expression is controlled, identifying the cis-elements and trans-factors that drive its transcription.","evidence":"deletion/mutant luciferase reporters, primer extension, and EMSA on the human ARF5 promoter","pmids":["10366714"],"confidence":"Medium","gaps":["Does not address conditional or signal-responsive regulation of ARF5","No link between transcriptional output and ARF5 protein function"]},{"year":2012,"claim":"Resolved whether ARF5 has a cargo-specific trafficking role, showing it mediates BRAG2-driven clathrin-dependent endocytosis of β1 integrins distinct from other Arfs.","evidence":"siRNA knockdown, rapid-cycling mutant rescue, clathrin/AP-2 co-localization, and cargo-specific internalization assays","pmids":["22815487"],"confidence":"High","gaps":["Mechanism of cargo selectivity over transferrin not defined","Downstream effectors at the coated pit not identified"]},{"year":2012,"claim":"Placed ARF5 in an actin-regulatory pathway, identifying it as an ARAP1 GAP substrate controlling dorsal ruffle ring size.","evidence":"ARAP1 overexpression/knockdown with dominant-negative ARF1/ARF5 mutants and CDR size measurement","pmids":["22573888"],"confidence":"Medium","gaps":["ARF5-specific vs ARF1 contribution not separated","Effectors linking ARF5 to actin ring geometry unknown"]},{"year":2015,"claim":"Defined a GTP-dependent ARF5 effector interaction, showing ARF5 binding through the IGRAB domain is required for GORAB Golgi targeting.","evidence":"yeast two-hybrid, trans-Golgi co-localization, BFA treatment, and disease-mutant (GORAB p.Ser175Phe) analysis","pmids":["26000619"],"confidence":"Medium","gaps":["Functional consequence of GORAB mislocalization for ARF5 trafficking roles not tested","Structural basis of IGRAB recognition undetermined"]},{"year":2019,"claim":"Revealed an unexpected RNA-binding capacity, showing ARF5 directly binds viral IRES RNA and modulates IRES-driven translation.","evidence":"proteomic identification, direct RNA-binding assay, RNA-FISH co-localization, and IRES reporter activity","pmids":["30655362"],"confidence":"Medium","gaps":["Whether RNA binding depends on GTP state untested","Physiological relevance beyond viral IRES unknown"]},{"year":2023,"claim":"Connected ARF5 to nutrient signaling, identifying it as a Raptor partner that recruits mTORC1 to plasma membrane ruffles.","evidence":"Raptor IP/MS, ARF5-GFP co-localization with mTOR/Rheb/PI3,4P2, knockdown, biosensor phosphorylation, and S6 refeeding assays","pmids":["36735494"],"confidence":"High","gaps":["Whether ARF5 GTPase activity is required for mTORC1 recruitment not established","Relationship between ruffle trafficking and mTORC1 activation undefined"]},{"year":2023,"claim":"Implicated an ARF5–Rab11-FIP4 complex in cancer stem cell properties, linking it to CDK1/cyclin B levels in hepatocellular carcinoma.","evidence":"Co-IP, sphere formation, stemness markers, and CDK1/cyclin B western blot with Rab11-FIP4 knockdown","pmids":["37458957"],"confidence":"Low","gaps":["Single Co-IP with no reciprocal validation of ARF5's direct role","Phenotype attributed to the complex, not dissected for ARF5 specifically"]},{"year":2024,"claim":"Defined ARF5's redundant role in early secretion, showing it cooperates with ARF1/ARF4 for ER-to-Golgi export of receptor tyrosine kinases.","evidence":"triple siRNA knockdown, RTK ER-export assay, in vitro BFA/M-COPA pulldown, and apoptosis assays","pmids":["38679330"],"confidence":"High","gaps":["Non-redundant ARF5-specific cargo at this step not identified","Order of ARF action in the export pathway unresolved"]},{"year":2024,"claim":"Tested ARF5 requirement in vivo for flavivirus infection, showing complete ARF5 knockout does not alter Zika virus load, unlike ARF4.","evidence":"CRISPR-Cas9 ARF5 knockout mice with RT-qPCR viral load and histopathology","pmids":["39722520"],"confidence":"Medium","gaps":["Negative result does not exclude redundancy with other Arfs in vivo","No assessment of ARF5 loss on its established trafficking cargoes in this model"]},{"year":2025,"claim":"Extended the ARF5–Rab11-FIP4 interaction to oocyte endocytic recycling for nanoparticle internalization.","evidence":"Co-IP in oocytes, Rab11fip4 knockdown, endocytic inhibitor treatment, and WGCNA analysis","pmids":["40720586"],"confidence":"Low","gaps":["Single Co-IP without reciprocal validation","ARF5-specific mechanistic contribution not directly tested"]},{"year":null,"claim":"How ARF5's distinct effector interactions and cargo selectivities are coordinated across endocytosis, secretion, and mTORC1 signaling remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model distinguishing ARF5 from related Arfs in effector choice","GEF/GAP regulation integrating its multiple roles not mapped","Determinants of redundancy vs non-redundancy with ARF1/ARF4 unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0003924","term_label":"GTPase activity","supporting_discovery_ids":[0,1,4]},{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[5]},{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[3]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,3]},{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[2]},{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[4]}],"pathway":[{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[0,4]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[3]},{"term_id":"R-HSA-9609507","term_label":"Protein localization","supporting_discovery_ids":[4]}],"complexes":[],"partners":["BRAG2","GORAB","ARAP1","RPTOR","RAB11FIP4","ARF1","ARF4"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P84085","full_name":"ADP-ribosylation factor 5","aliases":[],"length_aa":180,"mass_kda":20.5,"function":"GTP-binding protein involved in protein trafficking; may modulate vesicle budding and uncoating within the Golgi apparatus (Microbial infection) Functions as an allosteric activator of the cholera toxin catalytic subunit, an ADP-ribosyltransferase","subcellular_location":"Golgi apparatus; Cytoplasm, perinuclear region; Membrane; Golgi apparatus, trans-Golgi network membrane","url":"https://www.uniprot.org/uniprotkb/P84085/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/ARF5","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000004059","cell_line_id":"CID000656","localizations":[{"compartment":"er","grade":3},{"compartment":"nucleoplasm","grade":1}],"interactors":[{"gene":"ARF4","stoichiometry":0.2},{"gene":"RRP36","stoichiometry":0.2},{"gene":"COPA","stoichiometry":0.2},{"gene":"COPB2","stoichiometry":0.2},{"gene":"COPE","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID000656","total_profiled":1310},"omim":[{"mim_id":"616594","title":"ARF GTPase-ACTIVATING PROTEIN WITH SH3 DOMAIN, ANKYRIN REPEAT, AND PH DOMAIN 3; ASAP3","url":"https://www.omim.org/entry/616594"},{"mim_id":"611999","title":"RAB11 FAMILY-INTERACTING PROTEIN 4; RAB11FIP4","url":"https://www.omim.org/entry/611999"},{"mim_id":"610166","title":"IQ MOTIF- AND SEC7 DOMAIN-CONTAINING PROTEIN 1; IQSEC1","url":"https://www.omim.org/entry/610166"},{"mim_id":"608738","title":"RAB11 FAMILY-INTERACTING PROTEIN 3; RAB11FIP3","url":"https://www.omim.org/entry/608738"},{"mim_id":"608651","title":"ARF GTPase-ACTIVATING PROTEIN WITH GTPase DOMAIN, ANKYRIN REPEAT, AND PLECKSTRIN HOMOLOGY DOMAIN 1; AGAP1","url":"https://www.omim.org/entry/608651"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/ARF5"},"hgnc":{"alias_symbol":[],"prev_symbol":[]},"alphafold":{"accession":"P84085","domains":[{"cath_id":"3.40.50.300","chopping":"24-178","consensus_level":"high","plddt":88.5635,"start":24,"end":178}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P84085","model_url":"https://alphafold.ebi.ac.uk/files/AF-P84085-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P84085-F1-predicted_aligned_error_v6.png","plddt_mean":86.12},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=ARF5","jax_strain_url":"https://www.jax.org/strain/search?query=ARF5"},"sequence":{"accession":"P84085","fasta_url":"https://rest.uniprot.org/uniprotkb/P84085.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P84085/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P84085"}},"corpus_meta":[{"pmid":"25274430","id":"PMC_25274430","title":"Irrepressible MONOPTEROS/ARF5 promotes de novo shoot formation.","date":"2014","source":"The New phytologist","url":"https://pubmed.ncbi.nlm.nih.gov/25274430","citation_count":61,"is_preprint":false},{"pmid":"22815487","id":"PMC_22815487","title":"BRAG2/GEP100/IQSec1 interacts with clathrin and regulates α5β1 integrin endocytosis through activation of ADP ribosylation factor 5 (Arf5).","date":"2012","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/22815487","citation_count":47,"is_preprint":false},{"pmid":"25145395","id":"PMC_25145395","title":"Distinct subclades of Aux/IAA genes are direct targets of ARF5/MP transcriptional regulation.","date":"2014","source":"The New phytologist","url":"https://pubmed.ncbi.nlm.nih.gov/25145395","citation_count":44,"is_preprint":false},{"pmid":"22573888","id":"PMC_22573888","title":"ARAP1 regulates the ring size of circular dorsal ruffles through Arf1 and Arf5.","date":"2012","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/22573888","citation_count":32,"is_preprint":false},{"pmid":"26000619","id":"PMC_26000619","title":"GORAB Missense Mutations Disrupt RAB6 and ARF5 Binding and Golgi Targeting.","date":"2015","source":"The Journal of investigative dermatology","url":"https://pubmed.ncbi.nlm.nih.gov/26000619","citation_count":30,"is_preprint":false},{"pmid":"37658649","id":"PMC_37658649","title":"E3 ubiquitin ligases SINA4 and SINA11 regulate anthocyanin biosynthesis by targeting the IAA29-ARF5-1-ERF3 module in apple.","date":"2023","source":"Plant, cell & environment","url":"https://pubmed.ncbi.nlm.nih.gov/37658649","citation_count":29,"is_preprint":false},{"pmid":"31508562","id":"PMC_31508562","title":"Auxin promotion of seedling growth via ARF5 is dependent on the brassinosteroid-regulated transcription factors BES1 and BEH4.","date":"2019","source":"Plant direct","url":"https://pubmed.ncbi.nlm.nih.gov/31508562","citation_count":20,"is_preprint":false},{"pmid":"22751359","id":"PMC_22751359","title":"A dominant mutation reveals asymmetry in MP/ARF5 function along the adaxial-abaxial axis of shoot lateral organs.","date":"2012","source":"Plant signaling & behavior","url":"https://pubmed.ncbi.nlm.nih.gov/22751359","citation_count":20,"is_preprint":false},{"pmid":"33180161","id":"PMC_33180161","title":"ARF4 regulates shoot regeneration through coordination with ARF5 and IAA12.","date":"2020","source":"Plant cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/33180161","citation_count":19,"is_preprint":false},{"pmid":"32305460","id":"PMC_32305460","title":"Determinants of PB1 Domain Interactions in Auxin Response Factor ARF5 and Repressor IAA17.","date":"2020","source":"Journal of molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/32305460","citation_count":17,"is_preprint":false},{"pmid":"30655362","id":"PMC_30655362","title":"Rab1b and ARF5 are novel RNA-binding proteins involved in FMDV IRES-driven RNA localization.","date":"2019","source":"Life science alliance","url":"https://pubmed.ncbi.nlm.nih.gov/30655362","citation_count":16,"is_preprint":false},{"pmid":"10366714","id":"PMC_10366714","title":"Transcriptional regulation of the human ADP-ribosylation factor 5 (ARF5) gene.","date":"1999","source":"Biochimica et biophysica acta","url":"https://pubmed.ncbi.nlm.nih.gov/10366714","citation_count":8,"is_preprint":false},{"pmid":"9169151","id":"PMC_9169151","title":"Localization and characterization of the human ADP-ribosylation factor 5 (ARF5) gene.","date":"1997","source":"Genomics","url":"https://pubmed.ncbi.nlm.nih.gov/9169151","citation_count":7,"is_preprint":false},{"pmid":"38679330","id":"PMC_38679330","title":"Brefeldin A and M-COPA block the export of RTKs from the endoplasmic reticulum via simultaneous inactivation of ARF1, ARF4, and ARF5.","date":"2024","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/38679330","citation_count":6,"is_preprint":false},{"pmid":"37458957","id":"PMC_37458957","title":"Rab11-FIP4 interacts with ARF5 to promote cancer stemness in hepatocellular carcinoma.","date":"2023","source":"Journal of physiology and biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/37458957","citation_count":6,"is_preprint":false},{"pmid":"36735494","id":"PMC_36735494","title":"Arf5-mediated regulation of mTORC1 at the plasma membrane.","date":"2023","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/36735494","citation_count":5,"is_preprint":false},{"pmid":"37329689","id":"PMC_37329689","title":"Mutant IAA21 genes from Dendrocalamus sinicus Chia et J. L. Sun inhibit stem and root growth in transgenic tobacco by interacting with ARF5.","date":"2023","source":"Plant physiology and biochemistry : PPB","url":"https://pubmed.ncbi.nlm.nih.gov/37329689","citation_count":5,"is_preprint":false},{"pmid":"39150252","id":"PMC_39150252","title":"Molecular Mechanism of ARF5-AHL15-Mediated Auxin-Induced Embryogenic Cell Formation in Apples.","date":"2024","source":"Journal of agricultural and food chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/39150252","citation_count":4,"is_preprint":false},{"pmid":"38617932","id":"PMC_38617932","title":"Pan-cancer analysis of ARFs family and ARF5 promoted the progression of hepatocellular carcinoma.","date":"2024","source":"Heliyon","url":"https://pubmed.ncbi.nlm.nih.gov/38617932","citation_count":3,"is_preprint":false},{"pmid":"40720586","id":"PMC_40720586","title":"Resveratrol-Loaded PLGA Enhanced Vitrified Oocyte Viability through Rab11fip4/Arf5-Mediated Internalization Route.","date":"2025","source":"ACS applied materials & interfaces","url":"https://pubmed.ncbi.nlm.nih.gov/40720586","citation_count":2,"is_preprint":false},{"pmid":"39722520","id":"PMC_39722520","title":"[Characterization of host factors ARF4 and ARF5 upon Zika virus infection in vivo by construction of gene knockout mice].","date":"2024","source":"Sheng wu gong cheng xue bao = Chinese journal of biotechnology","url":"https://pubmed.ncbi.nlm.nih.gov/39722520","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":12312,"output_tokens":2938,"usd":0.040503,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":20412,"output_tokens":4949,"usd":0.179678,"resynth_parse_fail":true,"stage2_stop_reason":"refusal"},"total_usd":0.220181,"stage1_batch_id":"msgbatch_0179Z2pxRYBsXi2xADneafRw","stage2_batch_id":"msgbatch_01LaY9ZaVdgjWArcZo9eqQE5","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2012,\n      \"finding\": \"BRAG2/GEP100/IQSec1 activates endogenous ARF5 (as well as Arf4 and Arf6), and it is specifically ARF5 (not Arf6 or Arf4) that mediates clathrin-mediated endocytosis of β1 integrins. BRAG2 and ARF5 co-localize at clathrin-coated pits; depletion of ARF5 slows β1 integrin internalization without affecting transferrin receptor uptake, and a rapid-cycling ARF5 mutant (T161A) but not the corresponding ARF6 mutant rescues spreading in BRAG2-depleted cells.\",\n      \"method\": \"siRNA knockdown of ARF5/ARF4/ARF6, rapid-cycling mutant rescue assay, co-localization with clathrin/AP-2, integrin internalization assay, cell spreading on fibronectin\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (KD phenotype, mutant rescue, co-localization, cargo-specific internalization assay) in a single focused study with rigorous controls\",\n      \"pmids\": [\"22815487\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"ARAP1, an Arf GAP, uses ARF1 and ARF5 as substrates to control the ring size of circular dorsal ruffles (CDRs). Expression of dominant-negative ARF1 and ARF5 expands CDR size, placing ARF1 and ARF5 downstream of ARAP1 in a pathway regulating actin ring structure.\",\n      \"method\": \"ARAP1 overexpression/knockdown, dominant-negative ARF1/ARF5 mutant expression, CDR size measurement, Arf GAP activity-dependent mutant analysis\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis (dominant-negative mutants) plus KD/OE with defined phenotypic readout, single lab\",\n      \"pmids\": [\"22573888\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"ARF5 (in its active, GTP-bound form) interacts with the GORAB golgin via the IGRAB domain, and this interaction is required for Golgi targeting of GORAB. A patient-derived GORAB missense mutation (p.Ser175Phe) selectively abolishes ARF5 binding while retaining RAB6 binding, displacing GORAB from the Golgi to vesicular structures.\",\n      \"method\": \"Yeast two-hybrid screening, co-localization with trans-Golgi markers, Brefeldin A treatment, disease-associated mutant analysis, subcellular localization imaging\",\n      \"journal\": \"The Journal of investigative dermatology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast two-hybrid plus functional validation with disease mutants and localization studies, single lab\",\n      \"pmids\": [\"26000619\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"ARF5 is a novel regulator of mTORC1 at plasma membrane ruffles. ARF5 co-localizes with endogenous mTOR, Rheb, and PI3,4P2 at membrane ruffles; ARF5 was identified as an interacting partner of the mTORC1 subunit Raptor by immunoprecipitation/MS. Knockdown of ARF5 reduces mTOR recruitment to ruffles, decreases phosphorylation of a plasma membrane-targeted mTORC1 biosensor substrate, and impairs rapid mTORC1-mediated S6 phosphorylation upon nutrient refeeding.\",\n      \"method\": \"Immunoprecipitation/mass spectrometry (Raptor interactome), ARF5-GFP co-localization with mTOR/Rheb, ARF5 siRNA knockdown, plasma membrane-targeted mTORC1 biosensor phosphorylation assay, S6 phosphorylation upon nutrient starvation/refeeding\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal IP/MS identification plus multiple orthogonal functional assays (localization, biosensor, nutrient signaling) in a single focused study\",\n      \"pmids\": [\"36735494\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ARF5 (together with ARF1 and ARF4) is required for ER-to-Golgi export of receptor tyrosine kinases (KIT, PDGFRA, EGFR, MET). Simultaneous siRNA knockdown of ARF1, ARF4, and ARF5 (but not single knockdown of any one) blocks RTK ER export, mimicking the effect of BFA/M-COPA. In vitro pulldown assays confirmed that BFA/M-COPA directly blocks ARF1, ARF4, and ARF5 function.\",\n      \"method\": \"siRNA triple knockdown (ARF1/ARF4/ARF5), RTK ER-export assay, in vitro pulldown with BFA/M-COPA, cell apoptosis assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vitro pulldown plus genetic knockdown with defined cargo-trafficking phenotype and pharmacological validation, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"38679330\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"ARF5 directly binds foot-and-mouth disease virus IRES RNA (domain 3) and diminishes IRES-driven translation activity. ARF5 co-localizes with IRES-driven RNA near ER-Golgi membranes, suggesting ARF5 participates in IRES-RNA localization to this ribosome-rich compartment.\",\n      \"method\": \"Proteomic identification of IRES-binding proteins, direct RNA-binding assay, RNA-FISH co-localization with ARF5, IRES activity reporter assay\",\n      \"journal\": \"Life science alliance\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct binding assay plus functional IRES activity measurement and RNA-FISH co-localization, single lab\",\n      \"pmids\": [\"30655362\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Transcription of the human ARF5 gene is dependent on Sp1 or an Sp1-like factor binding to two GC boxes within 169 bp of the transcription initiation site. Mutation of either GC box reduced reporter expression, and EMSA showed specific protein–DNA complexes competed by canonical Sp1-binding oligonucleotides.\",\n      \"method\": \"Deletion/mutant luciferase reporter transfection, primer extension analysis, EMSA/electrophoretic mobility-shift assay, site-directed mutagenesis\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis of cis elements combined with EMSA and reporter assay, single lab\",\n      \"pmids\": [\"10366714\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Rab11-FIP4 physically interacts with ARF5 and this complex influences CDK1/cyclin B levels to promote cancer stem cell-like characteristics in hepatocellular carcinoma cells.\",\n      \"method\": \"Co-immunoprecipitation (Rab11-FIP4 and ARF5), sphere formation assay, stemness marker expression, CDK1/cyclin B western blot, Rab11-FIP4 knockdown\",\n      \"journal\": \"Journal of physiology and biochemistry\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP with limited mechanistic follow-up on ARF5's direct role; phenotype attributed to the complex but ARF5-specific mechanism not dissected\",\n      \"pmids\": [\"37458957\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ARF5 knockout mice (complete ARF5 KO) show no significant change in Zika virus load in serum or tissues and no significant difference in pathological changes compared to wild-type, indicating ARF5 is not essential for ZIKV infection in vivo (negative finding). ARF4, but not ARF5, was found to be essential for ZIKV infection.\",\n      \"method\": \"CRISPR-Cas9 knockout mice, ZIKV infection, RT-qPCR viral load measurement, H&E histopathology\",\n      \"journal\": \"Sheng wu gong cheng xue bao = Chinese journal of biotechnology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean genetic KO with direct in vivo viral load readout; negative result for ARF5 is the primary finding\",\n      \"pmids\": [\"39722520\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Rab11-FIP4 interacts with ARF5 in mouse oocytes (confirmed by co-immunoprecipitation), and this interaction is implicated in the endocytic/recycling endosome pathway for nanoparticle internalization.\",\n      \"method\": \"Co-immunoprecipitation (Rab11fip4 and Arf5), Rab11fip4 knockdown, endocytic inhibitor treatment, transcriptome/WGCNA analysis\",\n      \"journal\": \"ACS applied materials & interfaces\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP in an oocyte context; ARF5-specific mechanistic role not directly tested beyond the interaction\",\n      \"pmids\": [\"40720586\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"Human/mammalian ARF5 is a class II ADP-ribosylation factor GTPase that functions in vesicular trafficking: it is activated by the ArfGEF BRAG2 at clathrin-coated pits to mediate clathrin-dependent endocytosis of β1 integrins (but not bulk transferrin), acts redundantly with ARF1 and ARF4 to support ER-to-Golgi export of receptor tyrosine kinases (inhibited by BFA/M-COPA), interacts with the GORAB golgin via an IGRAB domain to regulate Golgi targeting, serves as a substrate of the Arf GAP ARAP1 to control actin-ring size at circular dorsal ruffles, and regulates mTORC1 signaling by recruiting the mTORC1 complex to PI3,4P2-enriched plasma membrane ruffles; additionally, ARF5 transcription is driven by Sp1/Sp1-like factors binding GC boxes in a TATA-less promoter.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ARF5 is a small ADP-ribosylation factor GTPase that operates across multiple membrane-trafficking and signaling steps [#0, #4]. At clathrin-coated pits it is activated by the ArfGEF BRAG2/GEP100/IQSec1 to drive clathrin-mediated endocytosis of β1 integrins, a cargo-selective role distinct from bulk transferrin uptake and not substituted by ARF6 [#0]. It also acts redundantly with ARF1 and ARF4 in ER-to-Golgi export of receptor tyrosine kinases (KIT, PDGFRA, EGFR, MET), a function blocked by the ARF inhibitors BFA/M-COPA [#4]. In its GTP-bound state ARF5 binds the GORAB golgin through the IGRAB domain to direct GORAB Golgi targeting [#2], and it serves as a substrate of the Arf GAP ARAP1 to control actin-ring size at circular dorsal ruffles [#1]. Beyond trafficking, ARF5 co-localizes with mTOR, Rheb, and PI3,4P2 at plasma membrane ruffles and interacts with the mTORC1 subunit Raptor to recruit mTORC1 and support rapid nutrient-stimulated S6 phosphorylation [#3]. ARF5 transcription is driven by Sp1/Sp1-like factors binding GC boxes in a TATA-less promoter [#6].\",\n  \"teleology\": [\n    {\n      \"year\": 1999,\n      \"claim\": \"Established how ARF5 expression is controlled, identifying the cis-elements and trans-factors that drive its transcription.\",\n      \"evidence\": \"deletion/mutant luciferase reporters, primer extension, and EMSA on the human ARF5 promoter\",\n      \"pmids\": [\"10366714\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Does not address conditional or signal-responsive regulation of ARF5\", \"No link between transcriptional output and ARF5 protein function\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Resolved whether ARF5 has a cargo-specific trafficking role, showing it mediates BRAG2-driven clathrin-dependent endocytosis of β1 integrins distinct from other Arfs.\",\n      \"evidence\": \"siRNA knockdown, rapid-cycling mutant rescue, clathrin/AP-2 co-localization, and cargo-specific internalization assays\",\n      \"pmids\": [\"22815487\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of cargo selectivity over transferrin not defined\", \"Downstream effectors at the coated pit not identified\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Placed ARF5 in an actin-regulatory pathway, identifying it as an ARAP1 GAP substrate controlling dorsal ruffle ring size.\",\n      \"evidence\": \"ARAP1 overexpression/knockdown with dominant-negative ARF1/ARF5 mutants and CDR size measurement\",\n      \"pmids\": [\"22573888\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"ARF5-specific vs ARF1 contribution not separated\", \"Effectors linking ARF5 to actin ring geometry unknown\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Defined a GTP-dependent ARF5 effector interaction, showing ARF5 binding through the IGRAB domain is required for GORAB Golgi targeting.\",\n      \"evidence\": \"yeast two-hybrid, trans-Golgi co-localization, BFA treatment, and disease-mutant (GORAB p.Ser175Phe) analysis\",\n      \"pmids\": [\"26000619\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional consequence of GORAB mislocalization for ARF5 trafficking roles not tested\", \"Structural basis of IGRAB recognition undetermined\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Revealed an unexpected RNA-binding capacity, showing ARF5 directly binds viral IRES RNA and modulates IRES-driven translation.\",\n      \"evidence\": \"proteomic identification, direct RNA-binding assay, RNA-FISH co-localization, and IRES reporter activity\",\n      \"pmids\": [\"30655362\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether RNA binding depends on GTP state untested\", \"Physiological relevance beyond viral IRES unknown\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Connected ARF5 to nutrient signaling, identifying it as a Raptor partner that recruits mTORC1 to plasma membrane ruffles.\",\n      \"evidence\": \"Raptor IP/MS, ARF5-GFP co-localization with mTOR/Rheb/PI3,4P2, knockdown, biosensor phosphorylation, and S6 refeeding assays\",\n      \"pmids\": [\"36735494\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether ARF5 GTPase activity is required for mTORC1 recruitment not established\", \"Relationship between ruffle trafficking and mTORC1 activation undefined\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Implicated an ARF5–Rab11-FIP4 complex in cancer stem cell properties, linking it to CDK1/cyclin B levels in hepatocellular carcinoma.\",\n      \"evidence\": \"Co-IP, sphere formation, stemness markers, and CDK1/cyclin B western blot with Rab11-FIP4 knockdown\",\n      \"pmids\": [\"37458957\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single Co-IP with no reciprocal validation of ARF5's direct role\", \"Phenotype attributed to the complex, not dissected for ARF5 specifically\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Defined ARF5's redundant role in early secretion, showing it cooperates with ARF1/ARF4 for ER-to-Golgi export of receptor tyrosine kinases.\",\n      \"evidence\": \"triple siRNA knockdown, RTK ER-export assay, in vitro BFA/M-COPA pulldown, and apoptosis assays\",\n      \"pmids\": [\"38679330\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Non-redundant ARF5-specific cargo at this step not identified\", \"Order of ARF action in the export pathway unresolved\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Tested ARF5 requirement in vivo for flavivirus infection, showing complete ARF5 knockout does not alter Zika virus load, unlike ARF4.\",\n      \"evidence\": \"CRISPR-Cas9 ARF5 knockout mice with RT-qPCR viral load and histopathology\",\n      \"pmids\": [\"39722520\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Negative result does not exclude redundancy with other Arfs in vivo\", \"No assessment of ARF5 loss on its established trafficking cargoes in this model\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Extended the ARF5–Rab11-FIP4 interaction to oocyte endocytic recycling for nanoparticle internalization.\",\n      \"evidence\": \"Co-IP in oocytes, Rab11fip4 knockdown, endocytic inhibitor treatment, and WGCNA analysis\",\n      \"pmids\": [\"40720586\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single Co-IP without reciprocal validation\", \"ARF5-specific mechanistic contribution not directly tested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How ARF5's distinct effector interactions and cargo selectivities are coordinated across endocytosis, secretion, and mTORC1 signaling remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model distinguishing ARF5 from related Arfs in effector choice\", \"GEF/GAP regulation integrating its multiple roles not mapped\", \"Determinants of redundancy vs non-redundancy with ARF1/ARF4 unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0003924\", \"supporting_discovery_ids\": [0, 1, 4]},\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [5]},\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [3]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 3]},\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [2]},\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [0, 4]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [3]},\n      {\"term_id\": \"R-HSA-9609507\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"BRAG2\", \"GORAB\", \"ARAP1\", \"RPTOR\", \"RAB11FIP4\", \"ARF1\", \"ARF4\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}