title: EGF-and NGF-stimulated translocation of cytohesin-1 to the plasma membrane of PC12 cells requires PI 3-kinase activation and a functional cytohesin-1 PH domain.
abstract: ADP-ribosylation factors (ARFs) are small GTP-binding proteins that function as regulators of eukaryotic vesicle trafficking. Cytohesin-1 is a member of a family of ARF guanine nucleotide-exchange factors that contain a C-terminal pleckstrin homology (PH) domain which has been proposed to bind the lipid second messenger phosphatidylinositol 3,4,5-trisphosphate (PIP3). Here we demonstrate that in vitro, recombinant cytohesin-1 binds, via its PH domain, the inositol head group of PIP3, inositol 1,3,4, 5-tetrakisphosphate (IP4), with an affinity greater than 200-fold higher than the inositol head group of either phosphatidylinositol 4, 5-bisphosphate or phosphatidylinositol 3,4-bisphosphate. Moreover, addition of glycerol or diacetylglycerol to the 1-phosphate of IP4 does not alter the ability to interact with cytohesin-1, data which is entirely consistent with cytohesin-1 functioning as a putative PIP3 receptor. To address whether cytohesin-1 binds PIP3 in vivo, we have expressed a chimera of green fluorescent protein (GFP) fused to the N terminus of cytohesin-1 in PC12 cells. Using laser scanning confocal microscopy we demonstrate that either EGF- or NGF-stimulation of transiently transfected PC12 cells results in a rapid translocation of GFP-cytohesin-1 from the cytosol to the plasma membrane. This translocation is dependent on the cytohesin-1 PH domain and occurs with a time course that parallels the rate of plasma membrane PIP3 production. Furthermore, the translocation requires the ability of either agonist to activate PI 3-kinase, since it is inhibited by wortmannin (100 nM), LY294002 (50 microM) and by coexpression with a dominant negative p85. This data therefore suggests that in vivo cytohesin-1 can interact with PIP3 via its PH domain.
authors.affiliation: Department of Biochemistry, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, UK.
authors.forename: K
authors.initials: K
authors.lastname: Venkateswarlu
authors.forename: F
authors.initials: F
authors.lastname: Gunn-Moore
authors.forename: J M
authors.initials: JM
authors.lastname: Tavaré
authors.forename: P J
authors.initials: PJ
authors.lastname: Cullen
mesh_terms: D000818:Animals; D001692:Biological Transport; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D049452:Green Fluorescent Proteins; D020662:Guanine Nucleotide Exchange Factors; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D018613:Microscopy, Confocal; D009414:Nerve Growth Factors / Q000494:pharmacology*; D016716:PC12 Cells; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D015815:Cell Adhesion Molecules; D020662:Guanine Nucleotide Exchange Factors; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D009414:Nerve Growth Factors; D010761:Phosphorus Radioisotopes; D011994:Recombinant Proteins; C100723:cytohesin-1; D049452:Green Fluorescent Proteins; D004815:Epidermal Growth Factor; D019869:Phosphatidylinositol 3-Kinases