title: Insulin-dependent translocation of ARNO to the plasma membrane of adipocytes requires phosphatidylinositol 3-kinase.
abstract: ADP-ribosylation factors (ARFs) are small GTP-binding proteins that are regulators of vesicle trafficking in eukaryotic cells [1]. ARNO is a member of the family of guanine nucleotide exchange factors for ARFs which includes cytohesin-1 and GRP-1 [2] [3-5]. Members of this family contain a carboxy-terminal pleckstrin homology (PH) domain which, in the case of GRP-1, has been shown to bind the second messenger phosphatidylinositol 3,4,5-trisphosphate (PIP3) in preference to phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2) in vitro [3,4]. Here, we show that recombinant ARNO has the binding characteristics of a PIP3 receptor and that this activity is restricted to the PH domain. When expressed in murine 3T3 L1 adipocytes, ARNO tagged using green fluorescent protein (GFP) is localised exclusively in the cytoplasm. Stimulation with insulin, however, causes a rapid (< 50 second) PH-domain-dependent translocation of GFP-ARNO to the plasma membrane. This translocation is blocked by the PI(4,5)P2 3-kinase (PI 3-kinase) inhibitors wortmannin and LY294002, and by co-expression with a dominant-negative p85 mutant, suggesting that the translocation is a consequence of insulin stimulation of PI 3-kinase. Our data strongly suggest that ARNO binds PIP3 in vivo and that this interaction causes a translocation of ARNO to the plasma membrane where it might activate ARF6 and regulate subsequent plasma membrane cycling events.
authors.affiliation: Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol, BS8 1TD, UK.
authors.forename: K
authors.initials: K
authors.lastname: Venkateswarlu
authors.forename: P B
authors.initials: PB
authors.lastname: Oatey
authors.forename: J M
authors.initials: JM
authors.lastname: Tavaré
authors.forename: P J
authors.initials: PJ
authors.lastname: Cullen
mesh_terms: D016475:3T3 Cells; D017667:Adipocytes / Q000201:enzymology / Q000378:metabolism*; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport; D001798:Blood Proteins / Q000235:genetics; D002462:Cell Membrane / Q000378:metabolism; D002867:Chromones / Q000494:pharmacology; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000737:chemistry; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D020690:GTPase-Activating Proteins; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D007328:Insulin / Q000494:pharmacology*; D051379:Mice; D009025:Morpholines / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology*; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010750:Phosphoproteins; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D011993:Recombinant Fusion Proteins; D017386:Sequence Homology, Amino Acid; D000077191:Wortmannin
chemical_list: D000730:Androstadienes; D001798:Blood Proteins; D002867:Chromones; D004791:Enzyme Inhibitors; D019204:GTP-Binding Proteins; D020690:GTPase-Activating Proteins; D007295:Inositol Phosphates; D007328:Insulin; D009025:Morpholines; D019869:Phosphatidylinositol 3-Kinases; D018129:Phosphatidylinositol Phosphates; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010750:Phosphoproteins; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D000077191:Wortmannin; C102474:cytohesin-2; C060974:phosphatidylinositol 3,4,5-triphosphate; C040985:phosphatidylinositol receptors; C039077:platelet protein P47; C081655:inositol-1,3,4,5-tetrakisphosphate; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one