Nerve growth factor- and epidermal growth factor-stimulated translocation of the ADP ribosylation factor exchange factor GRP1 to the plasma membrane of PC12 cells requires activation of phosphatidylinositol 3-kinase and the GRP1 pleckstrin homology domain
Nerve growth factor- and epidermal growth factor-stimulated translocation of the ADP ribosylation factor exchange factor GRP1 to the plasma membrane of PC12 cells requires activation of phosphatidylinositol 3-kinase and the GRP1 pleckstrin homology domain
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DOI：
10.1042/bj3350139
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发表时间：
1998-10-01
影响因子：
4.1
通讯作者：
Cullen, PJ
中科院分区：
文献类型：
--
作者：
Venkateswarlu, K;Gunn-Moore, F;Cullen, PJ
ADP核糖基化因子（ARF）是一种小的GTP结合蛋白，是真核细胞中囊泡运输的调节因子。GRP 1是ARF鸟苷酸交换因子家族的成员，在体外结合脂质第二信使3，4，5-三磷酸磷脂酰肌醇[PtdIns（3，4，5）P-3]。为了研究PtdIns（3，4，5）P-3对GRP 1功能的影响，我们克隆了GRP 1的人类同源物，其编码的蛋白与小鼠脑GRP 1具有98.8%的同一性。人GRP 1通过其普列克底物蛋白同源（PH）结构域以高亲和力（32.2+/-5.2 nM）和肌醇磷酸特异性（Ins的K-d值）结合PtdIns（3，4，5）P-3的肌醇头基，肌醇1，3，4，5-四磷酸[Ins（1，3，4，5）P-4]（1，3，4，5，6）P-5、InsP（6）、Ins（1，3，4）P-3和Ins（1，4，5）P-3：分别为283+/-32、>10000、> 10000和> 10000 nM）。此外，GRP 1可以容纳甘油或二乙酰甘油添加到Ins（1，3，4，5）P-4的1-磷酸，数据与其作为推定的PtdIns（3，4，5）P-3受体的拟议作用一致。为了解决GRP 1是否结合PtdIns（3，4，5）P-3在体内，我们已经表达了嵌合体的绿色荧光蛋白（GFP）融合的N-末端的GRP 1在PC 12细胞，并使用共聚焦显微镜，检查其在活细胞中的定位。用神经生长因子或表皮生长因子（均为100 ng/ml）刺激导致GFP-GRP 1从胞质溶胶到质膜的快速的PH结构域依赖性易位，其发生的时间过程与PtdIns（3，4，5）P-3的产生平行。这种易位依赖于磷脂酰肌醇3-激酶的活化，因为它被渥曼青霉素（100 nM）、LY 294002（50 μ M）和与显性负性p85的共表达抑制。总之，这些数据有力地表明，GRP 1在体内与位于质膜的PtdIns（3，4，5）P-3相互作用，因此构成真正的PtdIns（3，4，5）P-3受体。
ADP-ribosylation factors (ARFs) are small GTP-binding proteins that are regulators of vesicle trafficking in eukaryotic cells. GRP1 is a member of a family of ARF guanine-nucleotide-exchange factors that binds in vitro the lipid second messenger phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P-3]. In order to study the effects of PtdIns(3,4,5)P-3 on the function of GRP1, we have cloned the human homologue of GRP1, encoding for a protein which is 98.8 % identical to mouse brain GRP1. Human GRP1 binds, via its pleckstrin homology (PH) domain, the inositol head group of PtdIns(3,4,5)P-3, inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P-4], with high affinity 32.2+/-5.2 nM) and inositol phosphate specificity [K-d values for Ins(1,3,4,5,6)P-5, InsP(6), Ins(1,3,4)P-3 and Ins(1,4,5)P-3: 283+/-32, >10000, > 10000 and > 10000 nM, respectively). Furthermore, GRP1 can accommodate addition of glycerol or diacetylglycerol to the 1-phosphate of Ins(1,3,4,5)P-4, data that are consistent with its proposed role as a putative PtdIns(3,4,5)P-3 receptor. To address whether GRP1 binds PtdIns(3,4,5)P-3 in vivo, we have expressed a chimaera of green fluorescent protein (GFP) fused to the N-terminus of GRP1 in PC12 cells and, using confocal microscopy, examined its resultant localization in live cells. Stimulation with either nerve growth factor or epidermal growth factor (both at 100 ng/ml) results in a rapid, PH-domain dependent, translocation of GFP-GRP1 from the cytosol to the plasma membrane, which occurs with a time course that parallels the production of PtdIns(3,4,5)P-3. This translocation is dependent on the activation of phosphatidylinositol 3-kinase, since it is inhibited by wortmannin (100 nM), LY294002 (50 mu M) and by the co-expression with dominant negative p85. Taken together these data strongly suggest that GRP1 interacts in vivo with plasma membrane-located PtdIns(3,4,5)P-3 and hence constitutes a true PtdIns(3,4,5)P-3 receptor.