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

ADP-ribosylation factors (ARFs) are small GTP-binding proteins that functio n as regulators of eukaryotic vesicle trafficking. Cytohesin-1 is a member of a family of ARF guanine nucleotide-exchange factors that contain a C-ter minal pleckstrin homology (PH) domain which has been proposed to bind the l ipid second messenger phosphatidylinositol 3,4,5-trisphosphate (PIP3). Here we demonstrate that in vitro, recombinant cytohesin-1 binds, via its PH do main, 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 phosphatidylinosit ol 3,4-bisphosphate. Moreover, addition of glycerol or diacetylglycerol to the 1-phosphate of IP4 does not alter the ability to interact with cytohesi n-1, data which is entirely consistent with cytohesin-1 functioning as a pu tative 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 micro scopy we demonstrate that either EGF- or NGF-stimulation of transiently tra nsfected PC12 cells results in a rapid translocation of GFP-cytohesin-1 fro m the cytosol to the plasma membrane, This translocation is dependent on th e cytohesin-1 PH domain and occurs with a time course that parallels the ra te of plasma membrane PIE production. Furthermore, the translocation requir es the ability of either agonist to activate PI 3-kinase, since it is inhib ited by wortmannin (100 nM), LY294002 (50 mu M) 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.