Venkateswarlu K, Oatey P B, Tavaré J M, Cullen P J
Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol, BS8 1TD, UK.
Curr Biol. 1998 Apr 9;8(8):463-6. doi: 10.1016/s0960-9822(98)70181-2.
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.
ADP-核糖基化因子（ARFs）是一类小的GTP结合蛋白，是真核细胞中囊泡运输的调节因子[1]。ARNO是ARF鸟嘌呤核苷酸交换因子家族的成员，该家族包括细胞粘附素-1和GRP-1[2][3 - 5]。这个家族的成员含有一个羧基末端的普列克底物蛋白同源（PH）结构域，就GRP-1而言，在体外实验中已表明它优先结合第二信使磷脂酰肌醇3,4,5-三磷酸（PIP3），而不是磷脂酰肌醇4,5-二磷酸（PI(4,5)P2）和磷脂酰肌醇3,4-二磷酸（PI(3,4)P2）[3,4]。在此，我们表明重组ARNO具有PIP3受体的结合特性，且这种活性仅限于PH结构域。当在小鼠3T3 L1脂肪细胞中表达时，用绿色荧光蛋白（GFP）标记的ARNO仅定位于细胞质中。然而，胰岛素刺激会导致GFP-ARNO快速（<50秒）依赖于PH结构域的向质膜的转位。这种转位被PI(4,5)P2 3-激酶（PI 3-激酶）抑制剂渥曼青霉素和LY294002以及与显性负性p85突变体共表达所阻断，这表明这种转位是胰岛素刺激PI 3-激酶的结果。我们的数据有力地表明，ARNO在体内结合PIP3，并且这种相互作用导致ARNO向质膜转位，在质膜上它可能激活ARF6并调节随后的质膜循环事件。
