Stimulation of phospholipase C-epsilon by the M3 muscarinic acetylcholine receptor mediated by cyclic AMP and the GTPase Rap2B.
Stimulation of phospholipase C (PLC) by G(q)-coupled receptors such as the M(3) muscarinic acetylcholine receptor (mAChR) is caused by direct activation of PLC-beta enzymes by Galpha(q) proteins. We have recently shown that G(s)-coupled receptors can stimulate PLC-epsilon, apparently via formation of cyclic AMP and activation of the Ras-related GTPase Rap2B. Here we report that PLC stimulation by the M(3) mAChR expressed in HEK-293 cells also involves, in part, similar mechanisms. M(3) mAChR-mediated PLC stimulation and [Ca(2+)](i) increase were reduced by 2',5'-dideoxyadenosine (dd-Ado), a direct adenylyl cyclase inhibitor. On the other hand, overexpression of Galpha(s) or Epac1, a cyclic AMP-regulated guanine nucleotide exchange factor for Rap GTPases, enhanced M(3) mAChR-mediated PLC stimulation. Inactivation of Ras-related GTPases with clostridial toxins suppressed the M(3) mAChR responses. The inhibitory toxin effects were mimicked by expression of inactive Rap2B, but not of other inactive GTPases (Rac1, Ras, RalA, Rap1A, and Rap2A). Activation of the M(3) mAChR induced GTP loading of Rap2B, an effect strongly enhanced by overexpression of Galpha(s) and inhibited by dd-Ado. Overexpression of PLC-epsilon and PLC-beta1, but not PLC-gamma1 or PLC-delta1, enhanced M(3) mAChR-mediated PLC stimulation and [Ca(2+)](i) increase. In contrast, expression of a catalytically inactive PLC-epsilon mutant reduced PLC stimulation by the M(3) mAChR and abrogated the potentiating effect of Galpha(s). In conclusion, our findings suggest that PLC stimulation by the M(3) mAChR is a composite action of PLC-beta1 stimulation by Galpha(q) and stimulation of PLC-epsilon apparently mediated by G(s)-dependent cyclic AMP formation and subsequent activation of Rap2B.
Evellin
Sandrine
S
Institut für Pharmakologie, Universitätsklinikum Essen, D-45122 Essen, Germany.
Nolte
Jan
J
Tysack
Karina
K
vom Dorp
Frank
F
Thiel
Markus
M
Weernink
Paschal A Oude
PA
Jakobs
Karl H
KH
Webb
Edwin J
EJ
Lomasney
Jon W
JW
Schmidt
Martina
M
D002118:Calcium / Q000378:metabolism; D002217:Carbachol / Q000494:pharmacology; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism*; D018076:DNA, Complementary / Q000378:metabolism; D003838:Deoxyadenine Nucleotides / Q000494:pharmacology; D054306:Dideoxynucleotides; D020558:GTP Phosphohydrolases / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D009154:Mutation; D054801:Phosphoinositide Phospholipase C; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D020033:Protein Isoforms; D043587:Receptor, Muscarinic M3; D011976:Receptors, Muscarinic / Q000378:metabolism*; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection; D010738:Type C Phospholipases / Q000737:chemistry* / Q000378:metabolism*; D020668:rap GTP-Binding Proteins / Q000378:metabolism*
D018076:DNA, Complementary; D003838:Deoxyadenine Nucleotides; D054306:Dideoxynucleotides; D020662:Guanine Nucleotide Exchange Factors; D020033:Protein Isoforms; C116122:RAPGEF3 protein, human; D043587:Receptor, Muscarinic M3; D011976:Receptors, Muscarinic; C037773:2',3'-dideoxyadenosine triphosphate; D002217:Carbachol; D000242:Cyclic AMP; D010738:Type C Phospholipases; D054801:Phosphoinositide Phospholipase C; C423320:phospholipase C epsilon; D020558:GTP Phosphohydrolases; C065562:RAP2B protein, human; D020668:rap GTP-Binding Proteins; D002118:Calcium