Affinage

ARL4D

ADP-ribosylation factor-like protein 4D · UniProt P49703

Length
201 aa
Mass
22.2 kDa
Annotated
2026-06-09
12 papers in source corpus 10 papers cited in narrative 10 extracted findings
Cross-family judge vs UniProt: Affinage preferred faithfulness: 6/6 claims corpus-supported (100%)

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

ARL4D is an N-terminally myristoylated small GTPase whose subcellular distribution and effector engagement are governed by its nucleotide state, allowing it to coordinate actin remodeling, cell migration, microtubule organization, and mitochondrial morphology (PMID:17804820, PMID:22927989, PMID:32755434). In its GTP-bound state ARL4D localizes to the plasma membrane and recruits cytohesin-2/ARNO through that protein's PH and polybasic domains, driving ARF6 activation, actin stress fiber disassembly, and migration; this same axis acts upstream of cytohesin-2 and ARF6 to promote neurite outgrowth (PMID:17804820, PMID:19327349). At the plasma membrane ARL4D additionally functions as a scaffold that assembles Erk1/2 and Pak1 into a complex, enabling Erk1/2 to phosphorylate Pak1 in response to PDGF and thereby driving migration (PMID:40309925); this signaling output is amplified by PI(4,5)P2- and Pak1-dependent self-association of ARL4D, which increases its membrane residency and stability and is required for Pak1 activation (PMID:41779780). GTP-bound ARL4D also binds the microtubule plus-end protein EB1 via an SxLP motif, promoting centrosomal EB1 recruitment, EB1–dynactin association, and microtubule nucleation (PMID:32755434). Conversely, the GDP-bound/GTP-binding-defective form localizes to endosomes to regulate transferrin receptor recycling and translocates to mitochondria—a transition driven by the GAP TBC1D15—where it lowers mitochondrial membrane potential and causes fragmentation (PMID:15049518, PMID:22927989, PMID:41709823). ARL4D protein levels are controlled post-transcriptionally by Akt/mTOR signaling downstream of PTEN loss, and ARL4D restrains IL-2 production and Akt phosphorylation in CD8 T cells (PMID:18240926, PMID:30382149).

Mechanistic history

Synthesis pass · year-by-year structured walk · 10 steps
  1. 2004 Medium

    Established that ARL4D's nucleotide state dictates its localization and that it regulates membrane trafficking, the founding observation linking GTP/GDP cycling to function.

    Evidence Immunocytochemistry with Q80L/T35N mutants and transferrin receptor localization assays

    PMID:15049518

    Open questions at the time
    • No effector identified for the recycling function
    • Single study without in vitro reconstitution
  2. 2007 High

    Identified cytohesin-2/ARNO as a GTP-dependent effector, explaining how active ARL4D drives ARF6 activation, actin remodeling, and migration.

    Evidence Co-IP with domain mapping, GTP-dependent binding, dominant-negative and siRNA rescue, migration and actin assays

    PMID:17804820

    Open questions at the time
    • Did not address how nucleotide state is regulated in vivo
    • ARF6 downstream targets not enumerated
  3. 2008 Medium

    Showed ARL4D abundance is set post-transcriptionally by Akt/mTOR after PTEN loss, connecting it to growth-factor and tumor-suppressor signaling.

    Evidence Western/Northern blot, qPCR, polysomal fractionation, rapamycin treatment in PTEN-defined glioma lines

    PMID:18240926

    Open questions at the time
    • Mechanism of translational control not defined
    • Correlative transcript/polysome data only
  4. 2009 Medium

    Extended the ARL4D–cytohesin-2–ARF6 axis to a physiological readout, neuronal morphogenesis, via neurite outgrowth.

    Evidence siRNA KD, constitutively active mutant, cell-permeable peptide inhibition, SecinH3 in N1E-115 cells

    PMID:19327349

    Open questions at the time
    • In vivo relevance to neuronal development untested
    • Single cell-line context
  5. 2012 Medium

    Revealed a distinct GDP-bound function: mitochondrial localization with effects on membrane potential and morphology, indicating dual organelle targeting.

    Evidence T35N overexpression, subcellular fractionation, immunofluorescence, membrane potential assays, NLS deletion

    PMID:22927989

    Open questions at the time
    • Mitochondrial binding partners unknown at this stage
    • Mechanism linking ARL4D to membrane potential unresolved
  6. 2018 Medium

    Demonstrated an immunoregulatory role in which ARL4D limits CD8 T cell IL-2 production and Akt signaling, broadening its physiological scope.

    Evidence Arl4d-deficient mice, in vitro T cell stimulation, viral infection in vivo, IL-2 ELISA, flow cytometry

    PMID:30382149

    Open questions at the time
    • Molecular pathway connecting ARL4D to IL-2/Akt not defined
    • Whether GTPase activity is required is untested
  7. 2020 High

    Identified EB1 as a GTP-dependent effector, defining a microtubule/centrosomal function distinct from the actin pathway.

    Evidence Reciprocal Co-IP, SxLP motif mapping, siRNA depletion, centrosomal MT nucleation assays, colocalization

    PMID:32755434

    Open questions at the time
    • How ARL4D is targeted to centrosomes not resolved
    • Relationship to its plasma-membrane pool unclear
  8. 2025 Medium

    Defined ARL4D as a plasma-membrane scaffold that assembles Erk1/2 and Pak1, explaining how it targets Erk1/2 substrate phosphorylation during PDGF-driven migration.

    Evidence Co-IP, plasma membrane recruitment, phosphorylation assays, loss-of-function and rescue, migration assays

    PMID:40309925

    Open questions at the time
    • Whether scaffolding requires the cytohesin-2/ARF6 arm is unclear
    • Single-lab biochemistry
  9. 2026 Medium

    Identified TBC1D15 as the ARL4D GAP, providing the regulatory switch that drives GDP-bound ARL4D to mitochondria under serum starvation.

    Evidence Co-IP, in vitro GAP activity assays, TBC1D15 siRNA KD, subcellular fractionation

    PMID:41709823

    Open questions at the time
    • GEF for ARL4D still unidentified
    • Single-lab in vitro GAP assay
  10. 2026 High

    Showed that PI(4,5)P2- and Pak1-phosphorylation-dependent self-association is a feedback mechanism that stabilizes ARL4D at the membrane and is required for downstream Pak1 activation and migration.

    Evidence AlphaFold-guided mutagenesis, lipid binding, Ser144 phosphorylation mapping, self-association Co-IP, migration assays

    PMID:41779780

    Open questions at the time
    • Stoichiometry/structure of the self-associated oligomer not experimentally determined
    • Whether self-association applies to non-migratory functions untested

Open questions

Synthesis pass · forward-looking unresolved questions
  • The activating GEF for ARL4D and how its competing plasma-membrane, centrosomal, endosomal, and mitochondrial pools are spatially partitioned remain unresolved.
  • No GEF identified
  • No structural model of full-length ARL4D bound to effectors
  • Integration of actin, microtubule, and mitochondrial functions unexplained

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0003924 GTPase activity 2 GO:0098772 molecular function regulator activity 2 GO:0008289 lipid binding 1 GO:0060090 molecular adaptor activity 1
Localization
GO:0005886 plasma membrane 4 GO:0005739 mitochondrion 2 GO:0005768 endosome 1 GO:0005815 microtubule organizing center 1
Pathway
R-HSA-162582 Signal Transduction 3 R-HSA-168256 Immune System 1 R-HSA-9609507 Protein localization 1
Partners
ARF6CYTH2EB1MAPK3PAK1TBC1D15

Evidence

Reading pass · 10 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2007 ARL4D interacts with cytohesin-2/ARNO in a GTP-dependent manner, binding to its C-terminal pleckstrin homology (PH) and polybasic c domains. GTP-bound ARL4D localizes to the plasma membrane (requiring N-terminal myristoylation) and recruits cytohesin-2/ARNO there, leading to ARF6 activation, actin stress fiber disassembly, and increased cell migration. Knockdown of cytohesin-2/ARNO or expression of its inactive form (E156K) blocked ARL4D-mediated actin remodeling. ARL4D-induced translocation of cytohesin-2/ARNO did not require PI3K activation. Co-immunoprecipitation, GTP-dependent binding assays, constitutively active/dominant-negative mutants (Q80L, E156K), siRNA knockdown, cell migration assays, actin staining Molecular biology of the cell High 17804820
2004 GTP-bound ARL4D (Q80L) localizes to the plasma membrane and promotes transport of cargo (e.g., transferrin receptor) to the plasma membrane, while GDP-bound ARL4D (T35N) localizes to endosomes and causes accumulation of transferrin receptors in the endosomal compartment, establishing ARL4D as a regulator of recycling between endosomes and the plasma membrane. Immunocytochemistry with GTP/GDP-binding mutants (Q80L, T35N), transferrin receptor localization assays Cellular and molecular neurobiology Medium 15049518
2009 Arl4D acts upstream of cytohesin-2 and ARF6 to regulate neurite outgrowth in N1E-115 neuroblastoma cells. Constitutively active Arl4D induced neurite outgrowth, Arl4D knockdown inhibited VPA-induced outgrowth, and a cell-permeable peptide corresponding to the cytohesin-2-binding region of Arl4D blocked VPA effects, establishing the Arl4D–cytohesin-2–ARF6 axis in neuronal morphogenesis. siRNA knockdown, constitutively active mutant overexpression, cell-permeable peptide inhibition, pharmacological inhibition (SecinH3) Experimental cell research Medium 19327349
2012 GTP-binding-defective ARL4D (T35N) localizes to mitochondria (requiring N-terminal myristoylation), where it reduces mitochondrial membrane potential (ΔΨm) and causes mitochondrial fragmentation. The C-terminal NLS region of ARL4D(T35N) is required for these mitochondrial effects. A portion of endogenous ARL4D also resides in mitochondria. Mutant overexpression (T35N), subcellular fractionation, immunofluorescence, mitochondrial membrane potential assays, NLS deletion mutants PloS one Medium 22927989
2008 ARL4D (ARF4L) protein expression is controlled post-transcriptionally by the Akt/mTOR pathway downstream of PTEN loss; rapamycin treatment decreased ARF4L protein levels, and the ARF4L transcript preferentially associated with the polysomal compartment following PTEN loss or Akt activation, with no change at the transcript level. Western blot, Northern blot, qPCR, polysomal fractionation, rapamycin treatment in glioma cell lines with defined PTEN status Journal of neurosurgery Medium 18240926
2018 Arl4D expression is induced by PD-L1-dependent signals in LSEC-primed CD8 T cells and limits IL-2 production and Akt phosphorylation. Arl4D-deficient T cells overproduced IL-2 upon stimulation, showed enhanced expansion, effector function, and preferential development into KLRG1+CD127- short-lived effector cells during viral infection in vivo. Genetic knockout (Arl4d-deficient mice), in vitro T cell stimulation, viral infection in vivo, IL-2 ELISA, flow cytometry, Akt phosphorylation assays Scientific reports Medium 30382149
2020 Arl4D interacts with the microtubule plus-end tracking protein EB1 in a GTP-dependent manner, via EB1's C-terminal EB homology domain and an SxLP motif in Arl4D. Arl4D colocalizes with γ-tubulin at centrosomes; Arl4D depletion causes centrosomal MT nucleation defects. Arl4D–EB1 interaction promotes centrosomal EB1 recruitment and increases the association between EB1 and the p150 subunit of dynactin, important for MT stabilization. Co-immunoprecipitation, GTP-dependence assays, domain-mapping mutants (SxLP motif), siRNA depletion, centrosomal MT nucleation assays, immunofluorescence colocalization Molecular biology of the cell High 32755434
2025 Arl4D acts as a scaffolding protein at the plasma membrane that recruits both Erk1/2 and Pak1, assembling them into a functional complex. This complex enables Erk1/2 to phosphorylate Pak1 at the plasma membrane in response to PDGF signaling, driving cell migration. Arl4D thereby functions as a novel regulator of Erk1/2 substrate targeting. Co-immunoprecipitation, plasma membrane recruitment assays, phosphorylation assays, loss-of-function and rescue experiments, cell migration assays Journal of cell science Medium 40309925
2026 TBC1D15, a known Rab7 GAP, also functions as a GTPase-activating protein (GAP) for Arl4D: it interacts with Arl4D through its TBC domain and promotes GTP hydrolysis of Arl4D. Under serum starvation, GDP-bound Arl4D translocates to mitochondria; knockdown of TBC1D15 increases Arl4D GTP-bound activity and decreases its mitochondrial translocation, placing TBC1D15 as the Arl4D GAP regulating mitochondrial targeting. Co-immunoprecipitation, GTPase activity assays (GAP activity), TBC1D15 siRNA knockdown, subcellular fractionation, mitochondrial localization assays Journal of cell science Medium 41709823
2026 PI(4,5)P2 promotes GTP-dependent Arl4D self-association at the plasma membrane via a conserved C-terminal polybasic motif. Fibronectin stimulation enhances Arl4D self-association through two cooperative mechanisms: direct PI(4,5)P2 binding and phosphorylation of Arl4D at Ser144 by its effector kinase Pak1 (a positive feedback). Arl4D self-association increases membrane residency and protein stability, and is required for downstream Pak1 activation and cell migration. An AlphaFold-guided mutant defective in self-association but retaining GTP binding and membrane targeting failed to activate Pak1 or support migration; forced self-association of this mutant restored these functions. AlphaFold structural prediction with mutagenesis, lipid-binding assays, phosphorylation mapping (Ser144), Co-immunoprecipitation for self-association, cell migration assays, GTP-binding assays Proceedings of the National Academy of Sciences of the United States of America High 41779780

Source papers

Stage 0 corpus · 12 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2007 ARL4D recruits cytohesin-2/ARNO to modulate actin remodeling. Molecular biology of the cell 82 17804820
2009 Valproic acid-inducible Arl4D and cytohesin-2/ARNO, acting through the downstream Arf6, regulate neurite outgrowth in N1E-115 cells. Experimental cell research 29 19327349
2012 GTP-binding-defective ARL4D alters mitochondrial morphology and membrane potential. PloS one 19 22927989
2018 The PDL1-inducible GTPase Arl4d controls T effector function by limiting IL-2 production. Scientific reports 16 30382149
2008 Increased expression of the glioma-associated antigen ARF4L after loss of the tumor suppressor PTEN. Laboratory investigation. Journal of neurosurgery 14 18240926
1998 Expression of an ADP-ribosylation factor like gene, ARF4L, is induced after transient forebrain ischemia in the gerbil. Brain research. Molecular brain research 11 9602063
2011 Overexpression of the small GTPase Arl4D suppresses adipogenesis. International journal of molecular medicine 9 21769420
2004 Role of ARF4L in recycling between endosomes and the plasma membrane. Cellular and molecular neurobiology 9 15049518
2020 Arl4D-EB1 interaction promotes centrosomal recruitment of EB1 and microtubule growth. Molecular biology of the cell 8 32755434
2025 The phosphorylation of Pak1 by Erk1/2 to drive cell migration requires Arl4D acting as a scaffolding protein. Journal of cell science 2 40309925
2026 TBC1D15 functions as an Arl4D GAP and promotes the mitochondrial translocation of Arl4D for organelle homeostasis. Journal of cell science 0 41709823
2026 Phosphatidylinositol 4,5-bisphosphate mediates Arl4D self-interaction to promote Pak1 signaling. Proceedings of the National Academy of Sciences of the United States of America 0 41779780

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