Langille S E, Patki V, Klarlund J K, Buxton J M, Holik J J, Chawla A, Corvera S, Czech M P
Program in Molecular Medicine, Worcester, Massachusetts 01605, USA.
J Biol Chem. 1999 Sep 17;274(38):27099-104. doi: 10.1074/jbc.274.38.27099.
The GRP1 protein contains a Sec7 homology domain that catalyzes guanine nucleotide exchange on ADP-ribosylation factors (ARF) 1 and 5 as well as a pleckstrin homology domain that binds phosphatidylinositol(3,4,5)P(3), an intermediate in cell signaling by insulin and other extracellular stimuli (Klarlund, J. K., Guilherme, A., Holik, J. J., Virbasius, J. V., Chawla, A., and Czech, M. P. (1997) Science 275, 1927-1930). Here we show that both endogenous GRP1 and ARF6 rapidly co-localize in plasma membrane ruffles in Chinese hamster ovary (CHO-T) cells expressing human insulin receptors and COS-1 cells in response to insulin and epidermal growth factor, respectively. The pleckstrin homology domain of GRP1 appears to be sufficient for regulated membrane localization. Using a novel method to estimate GTP loading of expressed HA epitope-tagged ARF proteins in intact cells, levels of biologically active, GTP-bound ARF6 as well as GTP-bound ARF1 were elevated when these ARF proteins were co-expressed with GRP1 or the related protein cytohesin-1. GTP loading of ARF6 in both control cells and in response to GRP1 or cytohesin-1 was insensitive to brefeldin A, consistent with previous data on endogenous ARF6 exchange activity. The ability of GRP1 to catalyze GTP/GDP exchange on ARF6 was confirmed using recombinant proteins in a cell-free system. Taken together, these results suggest that phosphatidylinositol(3,4,5)P(3) may be generated in cell membrane ruffles where receptor tyrosine kinases are concentrated in response to growth factors, causing recruitment of endogenous GRP1. Further, co-localization of GRP1 with ARF6, combined with its demonstrated ability to activate ARF6, suggests a physiological role for GRP1 in regulating ARF6 functions.
GRP1蛋白包含一个Sec7同源结构域，可催化ADP核糖基化因子（ARF）1和5上的鸟嘌呤核苷酸交换，以及一个普列克底物蛋白同源结构域，该结构域可结合磷脂酰肌醇（3,4,5）三磷酸酯，这是胰岛素和其他细胞外刺激引发细胞信号传导过程中的一种中间体（克拉伦德，J.K.，吉列尔梅，A.，霍利克，J.J.，维尔巴西乌斯，J.V.，乔拉，A.，以及捷克，M.P.（1997年）《科学》275卷，1927 - 1930页）。在此我们表明，在分别表达人胰岛素受体的中国仓鼠卵巢（CHO - T）细胞以及COS - 1细胞中，内源性GRP1和ARF6会分别响应胰岛素和表皮生长因子，迅速共定位于质膜皱褶处。GRP1的普列克底物蛋白同源结构域似乎足以实现对膜定位的调控。运用一种新方法来估计完整细胞中表达的带有HA表位标签的ARF蛋白的GTP负载量，当这些ARF蛋白与GRP1或相关蛋白细胞黏附分子1共表达时，具有生物活性的GTP结合型ARF6以及GTP结合型ARF1的水平会升高。在对照细胞以及响应GRP1或细胞黏附分子1时，ARF6的GTP负载量对布雷菲德菌素A不敏感，这与之前关于内源性ARF6交换活性的数据一致。利用无细胞系统中的重组蛋白证实了GRP1催化ARF6上GTP / GDP交换的能力。综上所述，这些结果表明，磷脂酰肌醇（3,4,5）三磷酸酯可能在质膜皱褶处生成，此处受体酪氨酸激酶因生长因子而聚集，从而导致内源性GRP1的募集。此外，GRP1与ARF6的共定位，连同其已证实的激活ARF6的能力，表明GRP1在调节ARF6功能中具有生理作用。
