INSULIN-DEPENDENT TRANSLOCATION OF ARNO TO THE PLASMA-MEMBRANE OF ADIPOCYTES REQUIRES PHOSPHATIDYLINOSITOL 3-KINASE

ADP-ribosylation factors (ARFs) are small GTP-binding proteins that ar e 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 [2-5]. Members of this family con tain a carboxy-terminal pleckstrin homology (PH) domain which, in the case of GRP-1, has been shown to bind the second messenger phosphatidy linositol 3,4,5-trisphosphate (PIP3) in preference to phosphatidylinos itol 4,5-bisphosphate (PI(4,5)P-2) and phosphatidylinositol 3,4-bispho sphate (PI(3,4)P-2) in vitro [3,4]. Here, we show that recombinant ARN O has the binding characteristics of a PIP3 receptor and that this act ivity is restricted to the PH domain. When expressed in murine 3T3 L1 adipocytes, ARNO tagged using green fluorescent protein (GFP) is local ised 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)P-2 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-kina se. Our data strongly suggest that ARNO binds PIP3 in vivo and that th is interaction causes a translocation of ARNO to the plasma membrane w here it might activate ARF6 and regulate subsequent plasma membrane cy cling events. (C) Current Biology Ltd ISSN 0960-9822.