The M3 Muscarinic Acetylcholine Receptor Expressed in HEK-293 Cells Signals to Phospholipase D via G12 but Not Gq-type G Proteins REGULATORS OF G PROTEINS AS TOOLS TO DISSECT PERTUSSIS TOXIN-RESISTANT G PROTEINS IN RECEPTOR-EFFECTOR COUPLING

Abstract The M3 muscarinic acetylcholine receptor (mAChR) expressed in HEK-293 cells couples to Gqand G12 proteins and stimulates phospholipase C (PLC) and phospholipase D (PLD) in a pertussis toxin-insensitive manner. To determine the type of G protein mediating M3 mAChR-PLD coupling in comparison to M3 mAChR-PLC coupling, we expressed various Gα proteins and regulators of the G protein signaling (RGS), which act as GTPase-activating proteins for Gq- or G12-type G proteins. PLD stimulation by the M3 mAChR was enhanced by the overexpression of Gα12 and Gα13, whereas the overexpression of Gαq strongly increased PLC activity without affecting PLD activity. Expression of the RGS homology domain of Lsc, which acts specifically on Gα12 and Gα13, blunted the M3 mAChR-induced PLD stimulation without affecting PLC stimulation. On the other hand, overexpression of RGS4, which acts on Gαq- but not Gα12-type G proteins, suppressed the M3 mAChR-induced PLC stimulation without altering PLD stimulation. We conclude that the M3 mAChR in HEK-293 cells apparently signals to PLD via G12- but not Gq-type G proteins and that G protein subtype-selective RGS proteins can be used as powerful tools to dissect the pertussis toxin-resistant G proteins and their role in receptor-effector coupling.