Affinage

ARFGEF3

Brefeldin A-inhibited guanine nucleotide-exchange protein 3 · UniProt Q5TH69

Length
2177 aa
Mass
240.7 kDa
Annotated
2026-06-09
17 papers in source corpus 14 papers cited in narrative 14 extracted findings
Cross-family judge vs UniProt: Affinage preferred faithfulness: 7/7 claims corpus-supported (100%)

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

ARFGEF3 (BIG3) is a multifunctional scaffold protein that controls the subcellular localization and activity of its principal partner PHB2/REA across several tissue contexts (PMID:19496786, PMID:28555617). Despite carrying a Sec7 domain, BIG3 lacks the conserved catalytic glutamate required for guanine nucleotide exchange and instead functions through an armadillo-type alpha-helical repeat that mediates its PHB2 interaction (PMID:24997568). In ERα-positive breast cancer cells, cytoplasmic BIG3 sequesters PHB2, blocking its estrogen-dependent binding to karyopherin-alpha import factors (KPNA1, KPNA5, KPNA6) and thereby preventing PHB2 nuclear translocation and its suppression of ERα (PMID:19496786, PMID:26052702). Mechanistically, BIG3 acts as an A-kinase anchoring protein that simultaneously binds PKA and the phosphatase catalytic subunit PP1Cα; E2-induced PKA phosphorylation of BIG3 stimulates PP1Cα-mediated dephosphorylation of PHB2-S39, inactivating PHB2 and sustaining estrogen-dependent growth (PMID:28555617). Disrupting the BIG3-PHB2 complex with the cell-permeable ERAP peptide releases PHB2 to inhibit genomic and non-genomic ERα signalling and ERα/growth-factor-receptor crosstalk, overcoming tamoxifen resistance (PMID:24051437, PMID:25736224). Independently of this cancer role, BIG3 localizes to the trans-Golgi network in pancreatic beta- and alpha-cells where it negatively regulates secretory-granule biogenesis, limiting insulin and glucagon content and secretion without altering individual granule exocytotic kinetics (PMID:24711543, PMID:25139048, PMID:25737957). A mitochondrial pool of the BIG3-PHB2 complex regulates inner mitochondrial membrane function and suppresses PARP-1/AIF-dependent apoptosis in osteosarcoma cells, and Arfgef3 loss is protective against ischemia-reperfusion kidney injury via improved mitochondrial function (PMID:34363714, PMID:41087303).

Mechanistic history

Synthesis pass · year-by-year structured walk · 13 steps
  1. 2003 Medium

    Established the first functional role for BIG3, linking it to skeletal cell differentiation downstream of BMP signalling.

    Evidence Stable overexpression in ATDC5 chondrogenic cells with proteoglycan, ALP, and mineralization readouts

    PMID:14657013

    Open questions at the time
    • Mechanism connecting BIG3 to BMP/chondrocyte program not defined
    • Gain-of-function only, no loss-of-function in this system
    • No molecular partner identified
  2. 2004 Medium

    Extended BIG3's pro-differentiation role to osteoblasts and placed it upstream of Smad1 phosphorylation independent of endogenous BMPs.

    Evidence Overexpression in MC3T3-E1 cells with noggin epistasis and phosphoSmad1 nuclear localization assays

    PMID:15707593

    Open questions at the time
    • Direct molecular link between BIG3 and Smad1 activation unresolved
    • Overexpression-based, no knockout validation
    • Relationship to later PHB2-based functions unclear
  3. 2009 High

    Defined the core BIG3 mechanism in breast cancer: cytoplasmic sequestration of PHB2 to keep ERα active.

    Evidence siRNA knockdown, reciprocal Co-IP, and nuclear translocation assays in breast cancer cells

    PMID:19496786

    Open questions at the time
    • Import machinery blocked by BIG3 not yet identified
    • Post-translational regulation of the complex unknown
  4. 2013 High

    Demonstrated the BIG3-PHB2 interaction is therapeutically druggable, converting a scaffold mechanism into a strategy to overcome tamoxifen resistance.

    Evidence Cell-permeable ERAP peptide disruption with Co-IP and in vitro/in vivo growth assays

    PMID:24051437

    Open questions at the time
    • Structural basis of peptide-mediated disruption not solved
    • Off-target scaffold effects of ERAP not excluded
  5. 2014 High

    Revealed a distinct BIG3 function at the trans-Golgi network as a negative regulator of insulin granule biogenesis, acting at the biogenesis rather than docking/fusion stage.

    Evidence BIG3-knockout mice and cell lines with immunofluorescence, secretion assays, electron microscopy, and patch-clamp electrophysiology

    PMID:24711543 PMID:25139048

    Open questions at the time
    • Molecular machinery by which BIG3 restricts granule formation unknown
    • Whether PHB2 participates in the TGN role not addressed
  6. 2014 Medium

    Reconciled BIG3's Sec7 domain with its apparent lack of GEF activity by showing absence of the catalytic glutamate and an ARM-repeat scaffold fold.

    Evidence Homology modeling and fold recognition with experimental verification of the predicted PHB2 interaction site

    PMID:24997568

    Open questions at the time
    • GEF inactivity inferred computationally, not by direct nucleotide-exchange assay
    • Full-length structure not determined
  7. 2015 Medium

    Identified the karyopherin-alpha members through which BIG3 controls PHB2 nuclear import, completing the import-blockade mechanism.

    Evidence Individual KPNA siRNA knockdown, Co-IP, and translocation assays in breast cancer cells

    PMID:26052702

    Open questions at the time
    • Direct competition between BIG3 and KPNAs for PHB2 not biochemically resolved
    • Single lab
  8. 2015 Medium

    Extended the secretory-granule role to alpha-cells, establishing BIG3 as a conserved negative regulator of hormone granule biogenesis.

    Evidence Immunofluorescence and glucagon measurements in BIG3-knockout mice, islets, and cells

    PMID:25737957

    Open questions at the time
    • Shared molecular mechanism between alpha- and beta-cell roles not defined
    • Single lab
  9. 2015 Medium

    Showed PHB2 released from BIG3 also disrupts membrane ERα/growth-factor-receptor crosstalk, broadening the signalling consequences of complex disruption.

    Evidence ERAP peptide disruption with Co-IP and phosphorylation assays in ERα-positive cells

    PMID:25736224

    Open questions at the time
    • Direct versus indirect effects on each receptor not separated
    • Single lab
  10. 2017 High

    Defined BIG3 as an AKAP coordinating PKA and PP1Cα to phospho-regulate PHB2-S39, providing the enzymatic basis for BIG3-dependent PHB2 inactivation.

    Evidence Reciprocal Co-IP of the tri-complex, phosphorylation-site mutagenesis, kinase/phosphatase assays, and cohort correlation

    PMID:28555617

    Open questions at the time
    • Structural organization of the BIG3-PKA-PP1Cα complex unknown
    • Whether the AKAP role operates in non-breast tissues untested
  11. 2019 Medium

    Mapped a BIG3-unique loop absent in BIG1/BIG2 that governs protein stability and the kinetics of PHB2 binding.

    Evidence Biophysical secondary-structure, stability, and binding-kinetics measurements with comparative analysis of BIG-family N-terminal domains

    PMID:31421830

    Open questions at the time
    • In vitro only; cellular relevance of the loop untested
    • No high-resolution structure
  12. 2021 Medium

    Localized a mitochondrial BIG3-PHB2 pool that maintains inner-membrane function and suppresses PARP-1/AIF apoptosis, revealing a context-specific localization distinct from breast cancer.

    Evidence siRNA and peptide disruption with cell-cycle, PARP/AIF, proteomic, and subcellular fractionation analyses in osteosarcoma cells

    PMID:34363714

    Open questions at the time
    • How BIG3 targets to mitochondria not defined
    • Single lab
  13. 2025 Medium

    Linked BIG3 to mitochondrial function in vivo, showing its loss protects against ischemia-reperfusion kidney injury.

    Evidence Arfgef3 knockout mice with renal ischemia-reperfusion and measurements of mitochondrial biogenesis, ATP, inflammation, and apoptosis

    PMID:41087303

    Open questions at the time
    • Molecular mechanism connecting BIG3 to renal mitochondrial function unknown
    • Role of PHB2 in this context untested
    • Limited mechanistic detail

Open questions

Synthesis pass · forward-looking unresolved questions
  • How a single scaffold partitions between cytoplasmic, trans-Golgi, and mitochondrial pools to execute its distinct breast-cancer, secretory, and apoptotic functions remains unresolved.
  • Determinants of BIG3 localization not identified
  • Whether AKAP/PHB2 mechanism applies outside breast cancer untested
  • No experimental structure of full-length BIG3 or its complexes

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0060090 molecular adaptor activity 3 GO:0140313 molecular sequestering activity 2 GO:0098772 molecular function regulator activity 1
Localization
GO:0005739 mitochondrion 2 GO:0005794 Golgi apparatus 1 GO:0005829 cytosol 1
Pathway
R-HSA-162582 Signal Transduction 3 R-HSA-5653656 Vesicle-mediated transport 3 R-HSA-5357801 Programmed Cell Death 1
Partners
KPNA1KPNA5KPNA6PHB2PPP1CAPRKACA
Complex memberships
BIG3-PHB2 complexBIG3-PKA-PP1Cα (AKAP) complex

Evidence

Reading pass · 14 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2009 BIG3 (ARFGEF3) binds PHB2/REA in the cytoplasm and inhibits its estrogen-dependent nuclear translocation. When BIG3 is absent, estradiol stimulation causes PHB2 to translocate to the nucleus, interact with ERα, and suppress ERα transcriptional activity. When BIG3 is present, it traps PHB2 in the cytoplasm, thereby enhancing ERα transcriptional activity. siRNA knockdown of BIG3, co-immunoprecipitation, immunoblotting, nuclear translocation assays in breast cancer cells Cancer science High 19496786
2013 A cell-permeable peptide inhibitor (ERAP) derived from BIG3 disrupts the BIG3-PHB2 interaction, releasing PHB2 to bind directly to nuclear- and membrane-associated ERα, thereby suppressing multiple ERα-signalling pathways (genomic, non-genomic ERα activation and ERα phosphorylation) and overcoming tamoxifen resistance in ERα-positive breast cancer cells. Cell-permeable peptide inhibitor (ERAP), co-immunoprecipitation, in vitro and in vivo breast cancer cell growth assays Nature communications High 24051437
2014 BIG3 (ARFGEF3) is predominantly localized to insulin- and clathrin-positive trans-Golgi network (TGN) compartments in insulin-secreting cells. BIG3 deficiency increases insulin content, granule number, insulin secretion upon stimulation, and accelerates proinsulin-to-insulin processing, demonstrating that BIG3 negatively modulates insulin granule biogenesis and maturation. BIG3-knockout mice and BIG3-deficient insulin-secreting cell lines; immunofluorescence localization; insulin content/secretion assays; electron microscopy EMBO reports High 24711543
2014 BIG3 negatively regulates insulin granule exocytosis by restricting insulin granule biogenesis without affecting the release kinetics of individual granules at final exocytotic steps. BKO β-cells have >60% more insulin granules but an unaltered number of morphologically docked granules, indicating BIG3 acts at the biogenesis stage rather than the docking/fusion stage. Patch-clamp electrophysiology (ATP-elicited currents), electron microscopy of BIG3-knockout mouse β-cells American journal of physiology. Endocrinology and metabolism High 25139048
2014 Bioinformatic structural analysis reveals that BIG3's Sec7 domain lacks the conserved critical glutamate residue required for GEF activity, suggesting BIG3 has no guanine nucleotide-exchange factor activity. BIG3 is predicted to adopt an armadillo (ARM)-type α-helical repeat structure that mediates interaction with PHB2, and this interaction site was subsequently verified experimentally. Homology modeling, fold recognition, bioinformatic interaction prediction with experimental verification of predicted BIG3-PHB2 interaction site BMC research notes Medium 24997568
2015 BIG3 blocks the estrogen-dependent nuclear import of PHB2 by interfering with PHB2 binding to karyopherin-alpha family members (KPNA1, KPNA5, KPNA6). PHB2 interacts with KPNA1, KPNA5, and KPNA6 to enable E2-dependent nuclear translocation, and siRNA knockdown of each KPNA inhibits PHB2 translocation even in BIG3-depleted cells. siRNA knockdown of individual KPNAs, co-immunoprecipitation, nuclear translocation assays in breast cancer cells PloS one Medium 26052702
2015 PHB2 released from BIG3 by ERAP disrupts interactions between membrane-associated ERα and growth factor receptors (IGF-1Rβ, EGFR, PI3K, HER2), inhibiting their activation and reducing Akt, MAPK, and ERα phosphorylation, thereby suppressing crosstalk between estrogen and growth factor signaling associated with tamoxifen resistance. ERAP peptide inhibitor, co-immunoprecipitation, phosphorylation assays, in vitro and in vivo proliferation assays in ERα-positive breast cancer cells Cancer science Medium 25736224
2015 BIG3 is highly expressed in pancreatic alpha-cells in addition to beta-cells. Depletion of BIG3 in alpha-cells leads to elevated glucagon production and secretion, and BIG3-knockout mice display increased glucagon release under hypoglycemic conditions, indicating a conserved role for BIG3 in negatively regulating hormone secretory granule biogenesis in both alpha and beta cells. Immunofluorescence/confocal microscopy, glucagon measurement in BIG3-KO mice, islets, and cells Molecular metabolism Medium 25737957
2017 BIG3 functions as an A-kinase anchoring protein (AKAP) that simultaneously binds PKA and the alpha isoform of the catalytic subunit of PP1 (PP1Cα). E2-induced PKA-mediated phosphorylation of BIG3-S305 and BIG3-S1208 enhances PP1Cα activity, which dephosphorylates PHB2-S39, inactivating PHB2 and thereby activating E2/ERα signaling in breast cancer cells. Co-immunoprecipitation of BIG3-PKA-PP1Cα complex, phosphorylation-site mutagenesis (S305, S1208 of BIG3; S39 of PHB2), kinase/phosphatase activity assays, breast cancer cohort correlation analysis Nature communications High 28555617
2019 Biophysical characterization shows that a non-conserved loop region unique to BIG3 (not present in BIG1/BIG2 paralogs) significantly affects the colloidal and thermodynamic stability of BIG3 protein and the thermodynamic and kinetic profile of its interaction with PHB2. Biophysical characterization (secondary structure calculation, stability and binding kinetics measurements), comparative analysis of BIG family N-terminal domains Biochemical and biophysical research communications Medium 31421830
2021 In osteosarcoma (OS) cells, the BIG3-PHB2 complex localizes predominantly to mitochondria (distinct from its cytoplasmic localization in breast cancer cells). Disruption of the BIG3-PHB2 complex causes G2/M-phase arrest and induces apoptosis via PARP-1/AIF pathway activation, and downregulates inner mitochondrial membrane protein complex activity. siRNA depletion of BIG3, peptide inhibitor disruption of BIG3-PHB2, cell growth/migration/invasion assays, flow cytometry cell cycle analysis, PARP cleavage/AIF assays, proteomic and bioinformatic pathway analyses, subcellular fractionation/localization Cancer science Medium 34363714
2003 BIG3/BIG-3 (ARFGEF3) protein levels increase during chondrocyte differentiation and in response to BMP-2 treatment. Stable expression of BIG-3 in ATDC5 cells accelerates matrix proteoglycan synthesis, increases alkaline phosphatase and osteopontin mRNA, and promotes mineralized matrix formation, demonstrating a functional role in chondrocyte differentiation. Stable transfection of full-length BIG-3 in ATDC5 chondrogenic cells, proteoglycan synthesis assay, alkaline phosphatase activity, mRNA expression, mineralization assay Endocrinology Medium 14657013
2004 BIG-3 (ARFGEF3) accelerates osteoblast differentiation in MC3T3-E1 cells by inducing phosphorylation and nuclear translocation of Smad1 independently of endogenously produced BMPs. Noggin treatment (BMP inhibition) inhibited differentiation markers in control cells but not in BIG-3-overexpressing cells, and BIG-3 expression prevented noggin-induced reduction in phosphoSmad1 nuclear localization. Stable transfection of BIG-3 in MC3T3-E1 osteoblastic cells, noggin treatment, Smad1 phosphorylation assay, nuclear localization of phosphoSmad1, alkaline phosphatase activity, Runx2/collagen mRNA, mineralization assay Experimental cell research Medium 15707593
2025 Arfgef3 knockout in mice ameliorates ischemia-reperfusion-induced acute kidney injury by improving mitochondrial function, reducing mitochondrial biogenesis marker dysregulation, restoring ATP production capacity, and attenuating renal inflammation, oxidative stress, and apoptosis. Arfgef3 knockout mice subjected to renal ischemia-reperfusion injury; measurement of mitochondrial biogenesis markers, ATP production, inflammatory cytokines (IL-6, MCP-1, TNF-α), oxidative stress markers, and apoptosis Renal failure Medium 41087303

Source papers

Stage 0 corpus · 17 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2013 Targeting BIG3-PHB2 interaction to overcome tamoxifen resistance in breast cancer cells. Nature communications 66 24051437
2014 Xanthohumol suppresses oestrogen-signalling in breast cancer through the inhibition of BIG3-PHB2 interactions. Scientific reports 60 25483453
2009 Activation of an estrogen/estrogen receptor signaling by BIG3 through its inhibitory effect on nuclear transport of PHB2/REA in breast cancer. Cancer science 54 19496786
2014 BIG3 inhibits insulin granule biogenesis and insulin secretion. EMBO reports 25 24711543
2015 BIG3 Inhibits the Estrogen-Dependent Nuclear Translocation of PHB2 via Multiple Karyopherin-Alpha Proteins in Breast Cancer Cells. PloS one 23 26052702
2003 BIG-3, a novel WD-40 repeat protein, is expressed in the developing growth plate and accelerates chondrocyte differentiation in vitro. Endocrinology 23 14657013
2017 A-kinase anchoring protein BIG3 coordinates oestrogen signalling in breast cancer cells. Nature communications 20 28555617
2019 Involvement of BIG5 and BIG3 in BRI1 Trafficking Reveals Diverse Functions of BIG-subfamily ARF-GEFs in Plant Growth and Gravitropism. International journal of molecular sciences 16 31083521
2015 Therapeutic advances in BIG3-PHB2 inhibition targeting the crosstalk between estrogen and growth factors in breast cancer. Cancer science 15 25736224
2021 The survival and proliferation of osteosarcoma cells are dependent on the mitochondrial BIG3-PHB2 complex formation. Cancer science 14 34363714
2017 Stapled BIG3 helical peptide ERAP potentiates anti-tumour activity for breast cancer therapeutics. Scientific reports 12 28500289
2014 Detection of insulin granule exocytosis by an electrophysiology method with high temporal resolution reveals enlarged insulin granule pool in BIG3-knockout mice. American journal of physiology. Endocrinology and metabolism 12 25139048
2015 Increased biogenesis of glucagon-containing secretory granules and glucagon secretion in BIG3-knockout mice. Molecular metabolism 8 25737957
2014 Brefeldin A-inhibited guanine nucleotide-exchange protein 3 (BIG3) is predicted to interact with its partner through an ARM-type α-helical structure. BMC research notes 8 24997568
2019 Biophysical characterization of the breast cancer-related BIG3-PHB2 interaction: Effect of non-conserved loop region of BIG3 on the structure and the interaction. Biochemical and biophysical research communications 3 31421830
2004 The effects of BIG-3 on osteoblast differentiation are not dependent upon endogenously produced BMPs. Experimental cell research 3 15707593
2025 Arfgef3 knockout ameliorates ischemia reperfusion-induced acute kidney injury via mitochondrial dysfunction alleviation in mice. Renal failure 0 41087303

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