Affinage

CYTH3

Cytohesin-3 · UniProt O43739

Length
400 aa
Mass
46.3 kDa
Annotated
2026-04-28
36 papers in source corpus 21 papers cited in narrative 21 extracted findings

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

CYTH3 (GRP1/ARNO3) is a PtdIns(3,4,5)P3-regulated ARF guanine nucleotide exchange factor that couples PI3K signaling to membrane trafficking and insulin-dependent metabolic homeostasis. Its Sec7 domain catalyzes GDP/GTP exchange on ARF1, ARF5, and ARF6, while its PH domain selectively recognizes PtdIns(3,4,5)P3 via a diglycine motif and a sentry glutamate (E345), driving PI3K-dependent translocation to the plasma membrane where nonspecific electrostatic interactions with background anionic lipids facilitate a two-step membrane search mechanism (PMID:9442017, PMID:10913124, PMID:21932773, PMID:15610010). Akt-mediated phosphorylation switches PH domain specificity from PtdIns(3,4,5)P3 to PI4P and releases autoinhibition, redirecting GRP1 from the plasma membrane to the recycling endosome where it drives ARF6-dependent GLUT4 vesicle recycling (PMID:33026967, PMID:22609160). Cyth3-knockout mice exhibit impaired insulin receptor signaling in liver and adipose tissue and resistance to diet-induced obesity, establishing an in vivo requirement for CYTH3 in metabolic regulation (PMID:30837656).

Mechanistic history

Synthesis pass · year-by-year structured walk · 9 steps
  1. 1998 High

    Establishing that CYTH3 is a PtdIns(3,4,5)P3-activated ARF GEF resolved the question of how PI3K lipid products are decoded to regulate ARF-dependent membrane traffic.

    Evidence In vitro GEF assays on ARF1/ARF5, competitive lipid binding, GFP-PH live-cell translocation with PI3K inhibitors, Golgi disruption upon overexpression

    PMID:9442017 PMID:9707577 PMID:9742223

    Open questions at the time
    • Relative preference among ARF substrates in vivo not determined
    • Mechanism of PIP3-induced GEF activation (allosteric vs. membrane recruitment) not resolved
  2. 1999 High

    Identification of ARF6 as a GRP1 substrate and definition of the diglycine motif as the PIP3-specificity determinant established that GRP1 links PI3K signaling to plasma membrane ARF6 activation with uniquely high PIP3 selectivity.

    Evidence Cell-free GEF assay on ARF6, co-localization at membrane ruffles, mutagenesis of beta1/beta2 loop glycine motif with quantitative binding assays, live-cell PH domain specificity discrimination

    PMID:10480924 PMID:10585883 PMID:10913124

    Open questions at the time
    • Whether ARF6 is the preferred in vivo substrate over ARF1 not quantitatively established
    • Downstream effectors of GRP1-activated ARF6 not identified
  3. 2001 High

    Discovery that GRSP1 forms a stable coiled-coil complex with GRP1 and co-translocates to the plasma membrane upon insulin stimulation revealed a dedicated binding partner that may scaffold GRP1 function.

    Evidence Probe library screening, reciprocal co-immunoprecipitation, domain mapping, immunodepletion from lung tissue, confocal co-localization in insulin-stimulated CHO cells

    PMID:11445584

    Open questions at the time
    • Functional consequence of GRSP1-GRP1 complex on GEF activity or ARF signaling not determined
    • Whether GRSP1 contributes to substrate selection or membrane targeting unclear
  4. 2004 High

    Crystal structures of PH domain–lipid complexes and biophysical membrane binding studies revealed the structural basis for PIP3 selectivity and a two-step electrostatic search mechanism for membrane recruitment.

    Evidence X-ray crystallography of GRP1 PH domain with PIP3/PIP2 head groups, FRET equilibrium and stopped-flow kinetics on defined lipid bilayers

    PMID:15359279 PMID:15610010

    Open questions at the time
    • In vivo contribution of nonspecific electrostatic interactions to membrane residence time not measured
    • Role of membrane curvature not addressed
  5. 2010 High

    Biophysical characterization of GRP1 homodimerization and antiparallel GRP1–GRSP1 heterodimerization clarified the oligomeric state but showed that dimerization does not alter PIP3 binding, leaving the functional role of dimerization open.

    Evidence Analytical ultracentrifugation, FRET-based orientation assays, lipid headgroup binding and liposome co-sedimentation

    PMID:20527794

    Open questions at the time
    • Functional consequence of homodimerization vs. heterodimerization in cells not established
    • Whether dimerization affects GEF activity not tested
  6. 2011 High

    Identification of a sentry glutamate (E345) that excludes PIP2 binding and characterization of hydrophobic loop contacts with the bilayer refined the dual-recognition model of PH domain membrane engagement.

    Evidence E345K charge-reversal mutagenesis with 8-fold PIP2 affinity gain and constitutive membrane targeting; NMR, MD simulations, and monolayer penetration assays for loop residues

    PMID:21893292 PMID:21932773

    Open questions at the time
    • Whether E345 mutations occur in disease contexts not explored
    • Quantitative contribution of each loop residue to in vivo membrane dwell time unknown
  7. 2012 High

    Connecting GRP1 to insulin-stimulated GLUT4 recycling via Akt phosphorylation and ARF6 GEF activity established the first physiological trafficking pathway regulated by GRP1, while EPR studies defined the shallow membrane docking geometry.

    Evidence GLUT4 trafficking assays with phosphomimetic mutants and loss-of-function, Akt phosphorylation assays, EPR spin labeling at 18 positions on PIP3-containing bilayers

    PMID:22479423 PMID:22609160

    Open questions at the time
    • Identity of phosphorylation sites on GRP1 not fully mapped
    • How GRP1 specifically recognizes GLUT4-containing vesicles not addressed
  8. 2019 High

    Cyth3 knockout mice demonstrated that CYTH3 is required for full insulin receptor signaling and normal metabolic homeostasis in vivo, extending cell-based findings to an organismal context.

    Evidence Genetic knockout mice with insulin signaling readouts in liver/adipose, metabolic phenotyping including body weight, fat mass, and lipid excretion under normal and HFD conditions

    PMID:30837656

    Open questions at the time
    • Whether the metabolic phenotype is attributable to ARF6 GEF activity specifically or additional ARF substrates not resolved
    • Tissue-specific contributions (liver vs. adipose vs. muscle) not dissected
  9. 2020 High

    Demonstrating that phosphorylation switches GRP1 PH domain specificity from PIP3 to PI4P and releases coiled-coil autoinhibition resolved how a single GEF is redirected from the plasma membrane to the recycling endosome.

    Evidence Phosphomimetic and phosphoablative mutants, lipid binding assays, subcellular fractionation, co-immunoprecipitation with recycling endosome proteins, confocal localization

    PMID:33026967

    Open questions at the time
    • Identity of the kinase(s) responsible for phosphorylation in endogenous context not confirmed beyond Akt
    • Whether PI4P binding is direct or scaffolded not fully resolved
    • Structural basis of autoinhibition release not determined

Open questions

Synthesis pass · forward-looking unresolved questions
  • Key unresolved questions include: the structural basis of Akt-triggered autoinhibition release, the relative in vivo substrate preference among ARF family members in different tissues, and the functional role of GRP1 homodimerization and GRSP1 heterodimerization in regulating GEF activity or subcellular targeting.
  • No crystal structure of full-length GRP1 in autoinhibited vs. active state
  • In vivo ARF substrate preference not determined by loss-of-function approaches
  • Functional consequence of GRSP1 binding on GLUT4 or other trafficking pathways untested

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0008289 lipid binding 6 GO:0098772 molecular function regulator activity 4
Localization
GO:0005886 plasma membrane 6 GO:0005768 endosome 2 GO:0005829 cytosol 2
Pathway
R-HSA-5653656 Vesicle-mediated transport 4 R-HSA-162582 Signal Transduction 3 R-HSA-1430728 Metabolism 2
Partners
AKT1ARF1ARF5ARF6GRSP1
Complex memberships
GRP1-GRSP1 heterodimer

Evidence

Reading pass · 21 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
1998 GRP1 (CYTH3) Sec7 domain catalyzes guanine nucleotide exchange on ARF1 and ARF5 in vitro; PtdIns(3,4,5)P3 binding to the PH domain markedly enhances this ARF exchange activity, while PtdIns(4,5)P2 does not; inositol 1,3,4,5-tetrakisphosphate competitively blocks PtdIns(3,4,5)P3-mediated activation by binding the PH domain. In vitro GEF assay with recombinant proteins, lipid binding competition assay The Journal of biological chemistry High 9442017
1998 GRP1 PH domain binds the inositol head group of PtdIns(3,4,5)P3 with high affinity (Kd ~32 nM for Ins(1,3,4,5)P4), and growth factor (NGF/EGF) stimulation causes PI3K-dependent translocation of GFP-GRP1 from cytosol to plasma membrane in PC12 cells via its PH domain. Inositol phosphate binding assay, confocal live-cell microscopy of GFP fusion protein, PI3K inhibitors (wortmannin, LY294002), dominant-negative p85 co-expression The Biochemical journal High 9742223
1998 ARNO3 (CYTH3/GRP1 family member, also called ARNO3) acts as an ARF1 GEF via its Sec7 domain; overexpression in mammalian cells causes Golgi fragmentation, redistribution of beta-COP, and inhibition of secretory transport, establishing the Sec7 domain alone as sufficient for ARF1 substrate specificity. In vitro GEF assay, overexpression in mammalian cells with Golgi morphology readout, SEAP secretion assay Proceedings of the National Academy of Sciences of the United States of America High 9707577
1999 GRP1 co-localizes with ARF6 at plasma membrane ruffles in response to insulin/EGF; GRP1 catalyzes GTP/GDP exchange on ARF6 in a cell-free system and elevates GTP-loaded ARF6 in intact cells, establishing ARF6 as a physiological substrate of GRP1. Co-localization by confocal microscopy, in vitro GEF assay with recombinant proteins, GTP-loading assay in intact cells using epitope-tagged ARF6 The Journal of biological chemistry High 10480924
1999 The GRP1 PH domain binds PtdIns(3,4,5)P3 with ~650-fold higher selectivity over PtdIns(4,5)P2; this selectivity is determined by a diglycine motif in the beta1/beta2 loop, whereas the related proteins ARNO and cytohesin-1 contain a triglycine motif conferring dual specificity. Lipid binding affinity measurements (Kd determination), site-directed mutagenesis (glycine insertion/deletion), cell-based PH domain translocation assays The Journal of biological chemistry High 10913124
1999 The GRP1 PH domain is a selective sensor for PtdIns(3,4,5)P3 in vivo; GFP-PH(GRP1) translocates to the plasma membrane in response to insulin or PDGF but not to H2O2 (which generates only PtdIns(3,4)P2), establishing that the GRP1 PH domain discriminates PtdIns(3,4,5)P3 from PtdIns(3,4)P2 in intact cells. GFP fusion protein confocal microscopy in live HEK293 and Swiss 3T3 cells, radioligand displacement assay for lipid levels The Biochemical journal High 10585883
2001 GRP1 forms a stable complex with GRSP1 (a FERM domain-containing protein) via coiled-coil domain interactions; endogenous GRSP1 exists almost entirely in complex with GRP1 in lung tissue, and both proteins co-translocate to plasma membrane ruffles upon insulin stimulation. 32P-labeled GRP1 probe library screening, co-immunoprecipitation, domain mapping, immunodepletion, confocal co-localization in insulin-stimulated CHO cells The Journal of biological chemistry High 11445584
2004 Crystal structures of dual-specificity splice variants of GRP1 and ARNO PH domains in complex with PtdIns(4,5)P2 and PtdIns(3,4,5)P3 head groups reveal that glycine insertion in the beta1/beta2 loop alleviates unfavorable contacts for PtdIns(4,5)P2 binding while reducing PtdIns(3,4,5)P3 affinity by losing loop contacts, explaining the specificity switch. X-ray crystallography, systematic mutagenesis, lipid binding affinity measurements The EMBO journal High 15359279
2004 GRP1 PH domain binds membrane-embedded PIP3 with Kd ~50 nM; background anionic lipids (phosphatidylserine, phosphatidylinositol) facilitate membrane targeting by increasing the PIP3 on-rate through nonspecific electrostatic interactions, constituting a two-step search mechanism. Protein-to-membrane FRET equilibrium and stopped-flow kinetic assays with defined lipid bilayers Biochemistry High 15610010
2008 GRP1 PH domain membrane association involves multivalent docking: specific PtdIns(3,4,5)P3 recognition triggers membrane insertion; His355 protonation at acidic pH is required for robust PtdIns(3,4,5)P3 binding and membrane penetration; nonspecific electrostatic interactions with PS/PI amplify affinity ~6-fold. NMR spectroscopy, monolayer surface tension, surface plasmon resonance, site-directed mutagenesis (H355 mutant), live-cell confocal microscopy Journal of lipid research High 18469301
2011 The GRP1 PH domain contains a sentry glutamate (E345) that excludes PtdIns(4,5)P2 binding; the E345K charge-reversal mutation enhances PI(4,5)P2 affinity 8-fold and causes constitutive plasma membrane targeting in cells, analogous to the AKT1 E17K oncogenic mutation. Site-directed mutagenesis, lipid binding assays, live-cell confocal microscopy, PI(4,5)P2 hydrolysis experiments Biochemistry High 21932773
2011 Loops flanking the PIP3-binding site of GRP1 PH domain make additional contacts with the lipid bilayer; mutating hydrophobic residues in these loops reduces membrane penetration, supporting a dual-recognition model where specific PIP3 binding is supplemented by lipid bilayer interactions. Molecular dynamics simulations, NMR chemical shift perturbation, monolayer penetration assays, mutagenesis Structure High 21893292
2012 EPR site-directed spin labeling determined the membrane docking geometry of GRP1 PH domain bound to bilayer-embedded PIP3; the domain adopts a shallow membrane position, engulfing the PIP3 headgroup with minimal bilayer penetration, and undergoes rapid lateral diffusion on the membrane surface. EPR site-directed spin labeling and relaxation measurements on 18 spin-labeled positions, with high-resolution PH domain structure as constraint PloS one High 22479423
2012 Grp1 acts as a GEF for ARF6 to promote GLUT4 vesicle formation and subsequent recycling steps; Akt phosphorylates Grp1 in response to insulin signaling, and phosphomimetic mutations in Grp1 bypass upstream insulin signaling to induce GLUT4 recycling. Co-immunoprecipitation, GLUT4 trafficking assays, Akt phosphorylation assays, phosphomimetic mutant expression, loss-of-function experiments Developmental cell High 22609160
2012 GRASP co-localizes with constitutively inactive ARF6 in the endocytic recycling compartment (ERC); co-expression of GRASP and Grp1 promotes membrane ruffling; GRASP accumulation in ERC blocks MHC-I recycling (an ARF6-dependent pathway) but not transferrin receptor recycling (clathrin-dependent pathway). Co-localization confocal microscopy, overexpression and dominant-negative ARF6 assays, MHC-I and transferrin recycling assays Cell biology international Medium 22931251
2013 MD simulations and FRET kinetic studies reveal that GRP1 PH domain undergoes a two-step membrane search: initial nonspecific electrostatic 'hopping' on PS-containing membranes followed by specific PIP3 docking; background PS lipids are critical for initial membrane recruitment. All-atom and coarse-grained MD simulations, EPR membrane docking structure, FRET kinetic experiments Journal of molecular biology High 23747485
2014 C. elegans GRP-1 (sole cytohesin ortholog) controls asymmetric neuroblast divisions producing apoptotic cells; GRP-1 GEF activity (Sec7 domain) is required; GRP-1 functions at the plasma membrane and possibly at the cytokinetic furrow; genetic interactions with ARF GAP CNT-2 and ARF GEFs EFA-6 and BRIS-1 place GRP-1 in an ARF GTPase regulatory network controlling division asymmetry. Genetic loss-of-function, domain rescue experiments, GFP-tagged protein localization, epistasis analysis with cnt-2, efa-6, bris-1 mutants Genetics Medium 25053664
2019 Cytohesin-3 (CYTH3) is required for full insulin receptor signaling in mouse liver and adipose tissue; cyth3-knockout mice show significantly reduced insulin receptor-dependent signaling events after insulin injection, and display reduced age- and HFD-induced weight gain with increased lipid excretion. Genetic knockout mice, insulin stimulation with signaling readouts (Western blot for downstream effectors), metabolic phenotyping (body weight, fat mass, energy expenditure, lipid excretion), HFD challenge Scientific reports High 30837656
2020 Phosphorylation of Grp1 switches its PH domain specificity from PtdIns(3,4,5)P3 (plasma membrane) to phosphatidylinositol 4-phosphate (PI4P; recycling endosome) and releases an autoinhibitory mechanism allowing the coiled-coil domain to engage two peripheral membrane proteins of the recycling endosome, redirecting Grp1 from plasma membrane to recycling endosome. Phosphomimetic mutants, lipid binding assays, subcellular fractionation, co-immunoprecipitation with recycling endosome peripheral membrane proteins, confocal localization Molecular biology of the cell High 33026967
2010 Grp1 N-terminal heptad repeats mediate homodimerization; Grsp1 forms heterodimers with Grp1 and cytohesin-1 (but not as efficiently with ARNO) in an antiparallel orientation; formation of Grsp1-Grp1 heterodimers does not affect PtdIns(3,4,5)P3 or PtdIns(4,5)P2 headgroup binding or liposome partitioning. Analytical ultracentrifugation, FRET-based orientation assays, lipid headgroup binding assays, liposome co-sedimentation Biochemistry High 20527794
1998 The human ARNO3/CYTH3 gene was mapped to chromosome 7p21 by radiation hybrid mapping. Radiation hybrid panel PCR mapping, somatic cell hybrid analysis Annals of human genetics Medium 10363132

Source papers

Stage 0 corpus · 36 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2000 The hsp110 and Grp1 70 stress proteins: newly recognized relatives of the Hsp70s. Cell stress & chaperones 239 11048651
1999 The pleckstrin homology domains of protein kinase B and GRP1 (general receptor for phosphoinositides-1) are sensitive and selective probes for the cellular detection of phosphatidylinositol 3,4-bisphosphate and/or phosphatidylinositol 3,4,5-trisphosphate in vivo. The Biochemical journal 180 10585883
1998 Regulation of GRP1-catalyzed ADP ribosylation factor guanine nucleotide exchange by phosphatidylinositol 3,4,5-trisphosphate. The Journal of biological chemistry 140 9442017
2000 Distinct polyphosphoinositide binding selectivities for pleckstrin homology domains of GRP1-like proteins based on diglycine versus triglycine motifs. The Journal of biological chemistry 119 10913124
1998 Nerve growth factor- and epidermal growth factor-stimulated translocation of the ADP-ribosylation factor-exchange factor GRP1 to the plasma membrane of PC12 cells requires activation of phosphatidylinositol 3-kinase and the GRP1 pleckstrin homology domain. The Biochemical journal 114 9742223
1999 ADP-ribosylation factor 6 as a target of guanine nucleotide exchange factor GRP1. The Journal of biological chemistry 102 10480924
1998 ARNO3, a Sec7-domain guanine nucleotide exchange factor for ADP ribosylation factor 1, is involved in the control of Golgi structure and function. Proceedings of the National Academy of Sciences of the United States of America 89 9707577
2004 Structural determinants of phosphoinositide selectivity in splice variants of Grp1 family PH domains. The EMBO journal 85 15359279
2004 GRP1 pleckstrin homology domain: activation parameters and novel search mechanism for rare target lipid. Biochemistry 71 15610010
2003 Vav mediates Ras stimulation by direct activation of the GDP/GTP exchange factor Ras GRP1. The EMBO journal 60 12839994
2013 Molecular mechanism of membrane binding of the GRP1 PH domain. Journal of molecular biology 55 23747485
2008 Molecular mechanism of membrane targeting by the GRP1 PH domain. Journal of lipid research 49 18469301
2011 Biophysical and computational studies of membrane penetration by the GRP1 pleckstrin homology domain. Structure (London, England : 1993) 48 21893292
2012 Grp1 plays a key role in linking insulin signaling to glut4 recycling. Developmental cell 44 22609160
2016 Association of Peripheral Membrane Proteins with Membranes: Free Energy of Binding of GRP1 PH Domain with Phosphatidylinositol Phosphate-Containing Model Bilayers. The journal of physical chemistry letters 41 26977543
2001 Signaling complexes of the FERM domain-containing protein GRSP1 bound to ARF exchange factor GRP1. The Journal of biological chemistry 40 11445584
2021 LncRNA Neat1 expedites the progression of liver fibrosis in mice through targeting miR-148a-3p and miR-22-3p to upregulate Cyth3. Cell cycle (Georgetown, Tex.) 30 33550894
2006 Use of the GRP1 PH domain as a tool to measure the relative levels of PtdIns(3,4,5)P3 through a protein-lipid overlay approach. Journal of lipid research 27 17130283
2019 Microscopic Characterization of GRP1 PH Domain Interaction with Anionic Membranes. Journal of computational chemistry 26 31762060
2011 The GRP1 PH domain, like the AKT1 PH domain, possesses a sentry glutamate residue essential for specific targeting to plasma membrane PI(3,4,5)P(3). Biochemistry 22 21932773
2012 Membrane docking geometry of GRP1 PH domain bound to a target lipid bilayer: an EPR site-directed spin-labeling and relaxation study. PloS one 20 22479423
2014 Asymmetric neuroblast divisions producing apoptotic cells require the cytohesin GRP-1 in Caenorhabditis elegans. Genetics 19 25053664
2001 Hydrophobic interactions of the structural protein GRP1.8 in the cell wall of protoxylem elements. Plant physiology 19 11161025
1994 Vascular expression of the grp1.8 promoter is controlled by three specific regulatory elements and one unspecific activating sequence. Plant molecular biology 17 7948928
2011 Parathyroid hormone (PTH) regulates the sodium chloride cotransporter via Ras guanyl releasing protein 1 (Ras-GRP1) and extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein kinase (MAPK) pathway. Translational research : the journal of laboratory and clinical medicine 15 22005268
2014 Cytohesin-3 is upregulated in hepatocellular carcinoma and contributes to tumor growth and vascular invasion. International journal of clinical and experimental pathology 14 24966920
2009 A high-resolution map of the Grp1 locus on chromosome V of potato harbouring broad-spectrum resistance to the cyst nematode species Globodera pallida and Globodera rostochiensis. TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik 14 19363662
2010 5-Stabilized phosphatidylinositol 3,4,5-trisphosphate analogues bind Grp1 PH, inhibit phosphoinositide phosphatases, and block neutrophil migration. Chembiochem : a European journal of chemical biology 10 20052709
2010 Specificity and membrane partitioning of Grsp1 signaling complexes with Grp1 family Arf exchange factors. Biochemistry 9 20527794
2019 Cytohesin-3 is required for full insulin receptor signaling and controls body weight via lipid excretion. Scientific reports 6 30837656
2012 Grp1-associated scaffold protein (GRASP) is a regulator of the ADP ribosylation factor 6 (Arf6)-dependent membrane trafficking pathway. Cell biology international 6 22931251
2011 Design and synthesis of biotinylated inositol 1,3,4,5-tetrakisphosphate targeting Grp1 pleckstrin homology domain. Bioorganic & medicinal chemistry 5 21996606
2020 Coordination of Grp1 recruitment mechanisms by its phosphorylation. Molecular biology of the cell 3 33026967
2014 Grp1-associated scaffold protein regulates skin homeostasis after ultraviolet irradiation. Photochemical & photobiological sciences : Official journal of the European Photochemistry Association and the European Society for Photobiology 3 24407555
1998 Assignment of the human ARNO3 gene (PSCD3) to chromosome 7p21 by radiation hybrid mapping. Annals of human genetics 1 10363132
2020 The cytosolic protein GRP1 facilitates abscisic acid- and darkness-induced stomatal closure in Salvia miltiorrhiza. Journal of plant physiology 0 31926459