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

Activation of phospholipase D (PLD) is a cellular response to a wide v ariety of extracellular ligands. However, the exact mechanisms that li nk cell surface receptors to PLD remain unclear. In this study, we rep ort the involvement of the small-molecular-mass guanine-nucleotide-bin ding protein, ADP-ribosylation factor (ARF), in the activation of PLD by the muscarinic acetylcholine receptor (mAChR) in human embryonic ki dney 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 re combinant ARF 1. Brefeldin A, a known inhibitor of the ARF guanine-nuc leotide-exchange factor activity in Golgi membranes, inhibited mAChR-s timulated PLD, whereas basal PLD activity and stimulation by GTP[S] we re not affected. Upon cell permeabilization without the addition of st imulus, ARF proteins were released. However, the addition of GTP[S] du ring permeabilization and mAChR activation before permeabilization cau sed an almost complete and partial (about 60%) inhibition, respectivel y, of ARF release, indicating that ARF proteins are activated and ther eby translocated to membranes. The results indicate that ARF proteins and their nucleotide-exchange factor are apparently involved in the si gnalling pathway leading from mAChR activation to PLD stimulation in h uman embryonic kidney cells.