Insulin-dependent translocation of ARNO to the plasma membrane of adipocytes requires phosphatidylinositol 3-kinase
Insulin-dependent translocation of ARNO to the plasma membrane of adipocytes requires phosphatidylinositol 3-kinase
复制标题
胰岛素依赖性 ARNO 易位至脂肪细胞质膜需要磷脂酰肌醇 3-激酶
DOI：
10.1016/s0960-9822(98)70181-2
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发表时间：
1998
期刊：
影响因子：
9.2
通讯作者：
P. Cullen
中科院分区：
文献类型：
--
作者：
K. Venkateswarlu;P. Oatey;J. Tavaré;P. Cullen
ADP-核糖基化因子（ARF）是小的GTP结合蛋白，其是真核细胞中囊泡运输的调节剂[1]。ARNO是ARF的鸟嘌呤核苷酸交换因子家族的成员，包括细胞粘连素-1和GRP-1 [2-5]。该家族的成员含有羧基末端普列克底物蛋白同源（PH）结构域，在GRP-1的情况下，已显示其在体外优先于磷脂酰肌醇4，5-二磷酸（PI（4，5）P2）和磷脂酰肌醇3，4-二磷酸（PI（3，4）P2）结合第二信使磷脂酰肌醇3，4，5-三磷酸（PIP 3）[3，4]。在这里，我们表明，重组ARNO具有PIP 3受体的结合特性，这种活性仅限于PH结构域。当在鼠3 T3 L1脂肪细胞中表达时，使用绿色荧光蛋白（GFP）标记的ARNO仅定位于细胞质中。然而，用胰岛素刺激导致GFP-ARNO快速（< 50秒）PH结构域依赖性转运至质膜。该易位被PI（4，5）P23-激酶（PI 3-激酶）抑制剂渥曼青霉素和LY 294002阻断，并通过与显性负性p85突变体共表达而阻断，表明该易位是胰岛素刺激PI 3-激酶的结果。我们的数据强烈表明，ARNO在体内结合PIP 3，这种相互作用导致ARNO易位到质膜，在那里它可能激活ARF 6并调节随后的质膜循环事件。
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 [2–5]. 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 PIP3receptor 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)P23-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 PIP3in vivoand that this interaction causes a translocation of ARNO to the plasma membrane where it might activate ARF6 and regulate subsequent plasma membrane cycling events.