EVIDENCE FOR ADP-RIBOSYLATION-FACTOR-MEDIATED ACTIVATION OF PHOSPHOLIPASE-D BY M3 MUSCARINIC ACETYLCHOLINE-RECEPTOR

被引:79
作者
RUMENAPP, U [1 ]
GEISZT, M [1 ]
WAHN, F [1 ]
SCHMIDT, M [1 ]
JAKOBS, KH [1 ]
机构
[1] UNIV ESSEN GESAMTHSCH KLINIKUM,INST PHARMAKOL,D-45122 ESSEN,GERMANY
来源
EUROPEAN JOURNAL OF BIOCHEMISTRY | 1995年 / 234卷 / 01期
关键词
PHOSPHOLIPASE D; ADP-RIBOSYLATION FACTOR; MUSCARINIC ACETYLCHOLINE RECEPTOR; BREFELDIN A; HUMAN EMBRYONIC KIDNEY CELLS;
D O I
10.1111/j.1432-1033.1995.240_c.x
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
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页码:240 / 244
页数:5
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