Phospholipase C, protein kinase C and Ca2+/calmodulin-dependent protein kinase II are involved in carbachol-induced phospholipase D activation in Chinese hamster ovary cells expressing muscarinic acetylcholine receptor of Caenorhabditid elegans.

Abstract

Recently, we have isolated a cDNA encoding a muscarinic acetylcholine receptor (mAChR) from Caenorhabditis elegans. To investigate the regulation of phospholipase D (PLD) signaling via a muscarinic receptor, we generated stable transfected Chinese hamster ovary (CHO) cells that overexpress the mAChR of C. elegans (CHO‐GAR‐3). Carbachol (CCh) induced inositol phosphate formation and a significantly higher Ca2+ elevation and stimulated PLD activity through the mAChR ; this was insensitive to pertussis toxin, but its activity was abolished by the phospholipase C (PLC) inhibitor U73122. Western blot analysis revealed several apparent tyrosine‐phosphorylated protein bands after CCh treatment. The CCh‐induced PLD activation and tyrosine phosphorylation were significantly reduced by the protein kinase C (PKC) inhibitor calphostin C and down‐regulation of PKC and the tyrosine kinase inhibitor genistein. Moreover, the Ca2+‐calmodulin‐dependent protein kinase II (CaM kinase II) inhibitor KN62, in addition to chelation of extracellular or intracellular Ca2+ by EGTA and BAPTA/AM, abolished CCh‐induced PLD activation and protein tyrosine phosphorylation. Taken together, these results suggest that the PLC/PKC‐PLD pathway and the CaM kinase II/tyrosine kinase‐PLD pathway are involved in the activation of PLD through mAChRs of C. elegans.