Schmidt M, Rümenapp U, Bienek C, Keller J, von Eichel-Streiber C, Jakobs K H
Institut für Pharmacologie, Universität GH Essen, Federal Republic of Germany.
J Biol Chem. 1996 Feb 2;271(5):2422-6. doi: 10.1074/jbc.271.5.2422.
Rho proteins have been reported to activate phospholipase D (PLD) in in vitro preparations. To examine the role of Rho proteins in receptor signaling to PLD, we studied the effect of Clostridium difficile toxin B, which glucosylates Rho proteins, on the regulation of PLD activity in human embryonic kidney (HEK) cells stably expressing the m3 muscarinic acetylcholine receptor (mAChR). Toxin B treatment of HEK cells potently and efficiently blocked mAChR-stimulated PLD. In contrast, basal and phorbol ester-stimulated PLD activities were not or only slightly reduced. Cytochalasin B and Clostridium botulinum C2 toxin, mimicking the effect of toxin B on the actin cytoskeleton but without involving Rho proteins, had no effect on mAChR-stimulated PLD. Toxin B did not alter cell surface mAChR number and mAChR-stimulated binding of (guanosine 5'-O-(thio)triphosphate (GTP gamma S) to G proteins. In addition to mAChR-stimulated PLD, toxin B treatment also inhibited PLD activation by the direct G protein activators, AlF4- and GTP gamma S, studied in intact and permeabilized cells, respectively. Finally, C. botulinum C3 exoenzyme, which ADP-ribosylates Rho proteins, mimicked the inhibitory effect of toxin B on GTP gamma S-stimulated PLD activity. In conclusion, the data presented indicate that toxin B potently and selectively interferes with receptor coupling mechanisms to PLD, and furthermore suggest an essential role for Rho proteins in receptor signaling to PLD.
据报道，Rho蛋白可在体外制剂中激活磷脂酶D（PLD）。为了研究Rho蛋白在受体向PLD信号传导中的作用，我们研究了艰难梭菌毒素B（可使Rho蛋白糖基化）对稳定表达m3毒蕈碱型乙酰胆碱受体（mAChR）的人胚肾（HEK）细胞中PLD活性调节的影响。用毒素B处理HEK细胞可有效且有力地阻断mAChR刺激的PLD。相比之下，基础的和佛波酯刺激的PLD活性未降低或仅略有降低。细胞松弛素B和肉毒梭菌C2毒素模拟毒素B对肌动蛋白细胞骨架的作用，但不涉及Rho蛋白，对mAChR刺激的PLD没有影响。毒素B不会改变细胞表面mAChR的数量以及mAChR刺激的（鸟苷5'-O-（硫代）三磷酸（GTPγS）与G蛋白的结合。除了mAChR刺激的PLD外，毒素B处理还分别在完整细胞和通透细胞中抑制了直接G蛋白激活剂AlF4-和GTPγS对PLD的激活。最后，可使Rho蛋白ADP核糖基化的肉毒梭菌C3外毒素模拟了毒素B对GTPγS刺激的PLD活性的抑制作用。总之，所呈现的数据表明毒素B有力且选择性地干扰了受体与PLD的偶联机制，并且进一步表明Rho蛋白在受体向PLD的信号传导中起重要作用。
