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 Galpha 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 Galpha(12) and Galpha(13), whereas the overexpression of Galpha(q) strongly increased PLC activity without affecting PLD activity. Expression of the RGS homology domain of Lsc, which acts specifically on Galpha(12) and Galpha(13), blunted the M(3) mAChR-induced PLD stimulation without affecting PLC stimulation. On the other hand, overexpression of RGS4, which acts on Galpha(q)- but not Galpha(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(12)- but not G(q)-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.
Rümenapp
U
U
Institut für Pharmakologie, Universitätsklinikum Essen, Hufelandstrasse 55, D-45122 Essen, Germany. ulrich.ruemenapp@uni-essen.de
Asmus
M
M
Schablowski
H
H
Woznicki
M
M
Han
L
L
Jakobs
K H
KH
Fahimi-Vahid
M
M
Michalek
C
C
Wieland
T
T
Schmidt
M
M
D002217:Carbachol / Q000494:pharmacology; D004351:Drug Resistance; D044365:GTP-Binding Protein alpha Subunits, G12-G13; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D018721:Muscarinic Agonists / Q000494:pharmacology; D037342:Pertussis Toxin; D010739:Phospholipase D / Q000378:metabolism*; D020710:RGS Proteins / Q000378:metabolism; D043587:Receptor, Muscarinic M3; D011976:Receptors, Muscarinic / Q000378:metabolism*; D015398:Signal Transduction; D010738:Type C Phospholipases / Q000378:metabolism; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D018721:Muscarinic Agonists; D020710:RGS Proteins; D043587:Receptor, Muscarinic M3; D011976:Receptors, Muscarinic; D010566:Virulence Factors, Bordetella; D002217:Carbachol; D037342:Pertussis Toxin; D010738:Type C Phospholipases; D010739:Phospholipase D; D044365:GTP-Binding Protein alpha Subunits, G12-G13; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins