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.
| Original language | English |
|---|---|
| Pages (from-to) | 274-281 |
| Number of pages | 8 |
| Journal | Journal of Neurochemistry |
| Volume | 75 |
| Issue number | 1 |
| DOIs | |
| Publication status | Published - 2000 Jul 11 |
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All Science Journal Classification (ASJC) codes
- Biochemistry
- Cellular and Molecular Neuroscience
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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. / Min, Do Sik; Cho, Nam Jeong; Yoon, Shin Hee; Lee, Young Han; Hahn, Sang June; Lee, Kweon Haeng; Kim, Myung Suk; Jo, Yang Hyeok.
In: Journal of Neurochemistry, Vol. 75, No. 1, 11.07.2000, p. 274-281.Research output: Contribution to journal › Article
TY - JOUR
T1 - 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
AU - Min, Do Sik
AU - Cho, Nam Jeong
AU - Yoon, Shin Hee
AU - Lee, Young Han
AU - Hahn, Sang June
AU - Lee, Kweon Haeng
AU - Kim, Myung Suk
AU - Jo, Yang Hyeok
PY - 2000/7/11
Y1 - 2000/7/11
N2 - 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.
AB - 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.
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UR - http://www.scopus.com/inward/citedby.url?scp=0001557269&partnerID=8YFLogxK
U2 - 10.1046/j.1471-4159.2000.0750274.x
DO - 10.1046/j.1471-4159.2000.0750274.x
M3 - Article
C2 - 10854271
AN - SCOPUS:0001557269
VL - 75
SP - 274
EP - 281
JO - Journal of Neurochemistry
JF - Journal of Neurochemistry
SN - 0022-3042
IS - 1
ER -