Phospholipase C, Protein Kinase C, Ca2+/Calmodulin‐Dependent Protein Kinase II, and Tyrosine Phosphorylation Are Involved in Carbachol‐Induced Phospholipase D Activation in Chinese Hamster Ovary Cells Expressing Muscarinic Acetylcholine Receptor of Caenorhabditis elegans

• Published in: Journal of neurochemistry 2000-07, Vol.75 (1), p.274-281
• Main Authors: Min, Do Sik, Cho, Nam Jeong, Yoon, Shin Hee, Lee, Young Han, Hahn, Sang‐June, Lee, Kweon‐Haeng, Kim, Myung‐Suk, Jo, Yang‐Hyeok
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; Ca2+/calmodulin‐dependent protein; Caenorhabditis elegans; Cell receptors; Cell structures and functions; Fundamental and applied biological sciences. Psychology; kinase II; Molecular and cellular biology; Monoamines receptors (catecholamine, serotonine, histamine, acetylcholine); Muscarinic acetylcholine receptor; Phospholipase D
• ISSN: 0022-3042; 1471-4159
• DOI: 10.1046/j.1471-4159.2000.0750274.x

: 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.