Stimulation of Phospholipase C-ε by the M3Muscarinic Acetylcholine Receptor Mediated by Cyclic AMP and the GTPase Rap2B

Abstract Stimulation of phospholipase C (PLC) by Gq-coupled receptors such as the M3muscarinic acetylcholine receptor (mAChR) is caused by direct activation of PLC-β enzymes by Gαq proteins. We have recently shown that Gs-coupled receptors can stimulate PLC-e, apparently via formation of cyclic AMP and activation of the Ras-related GTPase Rap2B. Here we report that PLC stimulation by the M3 mAChR expressed in HEK-293 cells also involves, in part, similar mechanisms. M3 mAChR-mediated PLC stimulation and [Ca2+]i increase were reduced by 2′,5′-dideoxyadenosine (dd-Ado), a direct adenylyl cyclase inhibitor. On the other hand, overexpression of Gαs or Epac1, a cyclic AMP-regulated guanine nucleotide exchange factor for Rap GTPases, enhanced M3 mAChR-mediated PLC stimulation. Inactivation of Ras-related GTPases with clostridial toxins suppressed the M3 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 M3 mAChR induced GTP loading of Rap2B, an effect strongly enhanced by overexpression of Gαs and inhibited by dd-Ado. Overexpression of PLC-e and PLC-β1, but not PLC-γ1 or PLC-δ1, enhanced M3 mAChR-mediated PLC stimulation and [Ca2+]i increase. In contrast, expression of a catalytically inactive PLC-e mutant reduced PLC stimulation by the M3 mAChR and abrogated the potentiating effect of Gαs. In conclusion, our findings suggest that PLC stimulation by the M3 mAChR is a composite action of PLC-β1 stimulation by Gαq and stimulation of PLC-e apparently mediated by Gs-dependent cyclic AMP formation and subsequent activation of Rap2B.