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

The M-3 muscarinic acetylcholine receptor (mAChR) expressed in HEK-293 cells couples to G(q) and G(12) proteins and stimulates phospholipase C (PLC) and phospholipase D (PLD) in a pertussis toxin-insensitive manner. To determine the type of G protein mediating M-3 mAChR-PLD coupling in comparison to M-3 mAChR-PLC coupling, we expressed various G alpha proteins and regulators of the G protein signaling (RGS), which act as GTPase-activating proteins for G(q)- or G(12)-type G proteins. PLD stimulation by the M-3 mAChR was enhanced by the overexpression of G alpha (12), and G alpha (13), whereas the overexpression of G alpha (q) strongly increased PLC activity without affecting PLD activity. Expression of the RGS homology domain of Lsc, which acts specifically on G alpha (12) and G alpha (13), blunted the M-3 mAChR-induced PLD stimulation without affecting PLC stimulation. On the other hand, overexpression of RGS4, which acts on G alpha (q)- but not G alpha (12)-type G proteins, suppressed the M-3 mAChR-induced PLC stimulation without altering PLD stimulation. We conclude that the M-3 mAChR in HEK-293 cells apparently signals to PLD via G alpha (q)- but not G alpha (12)-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.