Rümenapp U, Geiszt M, Wahn F, Schmidt M, Jakobs K H
Institut für Pharmakologie, Universität GH Essen, Germany.
Eur J Biochem. 1995 Nov 15;234(1):240-4. doi: 10.1111/j.1432-1033.1995.240_c.x.
Activation of phospholipase D (PLD) is a cellular response to a wide variety of extracellular ligands. However, the exact mechanisms that link cell surface receptors to PLD remain unclear. In this study, we report the involvement of the small-molecular-mass guanine-nucleotide-binding protein, ADP-ribosylation factor (ARF), in the activation of PLD by the muscarinic acetylcholine receptor (mAChR) in human embryonic kidney cells stably expressing the human m3 subtype. PLD stimulation in permeabilized cells by guanosine 5'-O-[gamma-thio]triphosphate (GTP[S]) was dependent on a cytosolic factor and reconstituted by purified recombinant ARF 1. Brefeldin A, a known inhibitor of the ARF guanine-nucleotide-exchange-factor activity in Golgi membranes, inhibited mAChR-stimulated PLD, whereas basal PLD activity and stimulation by GTP[S] were not affected. Upon cell permeabilization without the addition of stimulus, ARF proteins were released. However, the addition of GTP[S] during permeabilization and mAChR activation before permeabilization caused an almost complete and partial (about 60%) inhibition, respectively, of ARF release, indicating that ARF proteins are activated and thereby translocated to membranes. The results indicate that ARF proteins and their nucleotide-exchange factor are apparently involved in the signalling pathway leading from mAChR activation to PLD stimulation in human embryonic kidney cells.
磷脂酶D（PLD）的激活是细胞对多种细胞外配体的一种反应。然而，将细胞表面受体与PLD联系起来的确切机制仍不清楚。在本研究中，我们报道了小分子质量鸟嘌呤核苷酸结合蛋白——ADP核糖基化因子（ARF），在稳定表达人m3亚型的人胚肾细胞中，参与毒蕈碱型乙酰胆碱受体（mAChR）对PLD的激活过程。在通透细胞中，鸟苷5'-O-[γ-硫代]三磷酸（GTP[S]）对PLD的刺激依赖于一种胞质因子，并且可被纯化的重组ARF 1重建。布雷菲德菌素A是一种已知的高尔基体膜中ARF鸟嘌呤核苷酸交换因子活性的抑制剂，它抑制了mAChR刺激的PLD，而基础PLD活性和GTP[S]刺激不受影响。在不添加刺激物的情况下使细胞通透化后，ARF蛋白被释放出来。然而，在通透化过程中添加GTP[S]以及在通透化之前激活mAChR，分别导致ARF释放几乎完全抑制和部分抑制（约60%），这表明ARF蛋白被激活并因此转位到膜上。结果表明，ARF蛋白及其核苷酸交换因子显然参与了人胚肾细胞中从mAChR激活到PLD刺激的信号通路。
