Mechanisms of phospholipase D stimulation by m3 muscarinic acetylcholine receptors

Abstract

In human embryonic kidney cells stably expressing the human m3 muscarinic acetylcholine receptor (mAChR) subtype, agonist (carbachol) activation stimulated phospholipase C, increased cytoplasmic calcium concentration, induced tyrosine phosphorylation of various cellular proteins and activated phospholipase D. Bypassing membrane receptors, phospholipase D was activated in these cells by direct activation of protein kinase C by phorbol esters, by direct activation of GTPbinding proteins by AlFy and a stable GTP analogue (in permeabilized cells), by increasing cytoplasmic calcium concentration with the calcium ionophore A23187 and also apparently by tyrosine phosphorylation. In order to identify possible mechanisms by which the m3 mAChR couples to phospholipase D, various inhibitors of protein kinase C, tyrosine kinases and calcium-dependent events were studied. Prevention of an agonist-induced increase in cytoplasmic calcium concentration did not alter the mAChR-induced phospholipase D stimulation. The protein kinase C inhibitors, calphostin C and staurosporine, efficiently prevented phospholipase D activation by phorbol 12-myristate 13-acetate but only partially inhibited the activation induced by the mAChR agonist. Additionally, down-regulation of protein kinase C by prolonged exposure to phorbol 12-myristate 13-acetate abrogated phospholipase D activation by this effector but had only minor or no effects on the response to the mAChR agonist and direct activators of GTP-binding proteins. In contrast, the tyrosine kinase inhibitor genistein abolished the carbachol-induced and A1F;-induced phospholipase D activation but had no effect on enzyme activation by phorbol 12-myristate 13-acetate. The data indicate that phospholipase D in m3 mAChR-expressing human embryonic kidney cells can be activated by various different mechanisms, i.e. receptor agonists, GTP-binding proteins, protein kinase C-dependent and calcium-dependent events and tyrosine phosphorylation. The coupling of m3 mAChR to phospholipase D appears to be largely independent of concomitant phospholipase C activation with subsequent increase in cytoplasmic calcium concentration and protein kinase C activity. The data instead suggest the involvement of an essential protein tyrosine phosphorylation mechanism in phospholipase D activation by the m3 mAChR and heterotrimeric GTP-binding proteins. Abbreviations. mAChR, muscarinic acetylcholine receptor; BAPTA/AM, bis-(0-aminophenoxy)-ethane N, N, N' , "-tetraacetic acid tetra(acetoxymethy1ester); Fura-2/AM, Enzymes. Phospholipase C (EC 3.1.4.3); phospholipase D (EC 3.1.4.4); protein kinase C (EC 2.7.1.37).gers, inositol 1,4,5-trisphosphate, which releases sequestered calcium from a subpopulation of the endoplasmic reticulum, and diacylglycerol, which activates protein kinases C [l, 21. These two signals apparently also participate in the transduction of mitogenic signals across the plasma membrane into the nucleus leading to the stimulation of cell growth, DNA synthesis and cell division [2]. Phosphatidylcholine...

Phospholipase D Stimulation by Receptor Tyrosine Kinases Mediated by Protein Kinase C and a Ras/Ral Signaling Cascade

“…The M 3 mAChR expressed in HEK-293 cells mediates marked phospholipase C stimulation (10). Nevertheless, PLD stimulation induced by this G protein-coupled receptor was not affected by inhibition or down-regulation of PKC or by expression of dominant-negative Ras and RalA mutants.…”

“…Cell Culture and Transfection-Culture conditions of HEK-293 cells stably expressing the M 3 mAChR were as reported in detail before (10). For experiments, cells subcultured in Dulbecco's modified Eagle's medium/F-12 medium were grown to near confluence (145-mm culture dishes).…”

Activation of Type I Phosphatidylinositol 4-Phosphate 5-Kinase Isoforms by the Rho GTPases, RhoA, Rac1, and Cdc42

“…Cell Culture and Transfection-HEK-293 cells were routinely passaged in Dulbecco's modified Eagle's medium/F-12 medium with 10% fetal calf serum (46). For transient expression of proteins, subconfluent monolayers on 145-mm culture dishes were transfected with cDNAs encoding PIP5K-I␣, PIP5K-I␣ A138, PIP5K-I␤ (all in pCMV), PIP5K-I␥ (in pCDNA3), RhoA and RhoA N19 (in pRK5), Rac1, Rac1 N17, Cdc42, and Cdc42 N17 (all in pEX), Rho-kinase, Rho-kinase-CAT, RB/PH (TT), and C3 transferase (all in pEF), and RalA, RalA A26, Rap1A, and Rap1A N17 (all in pRK5) by the calcium phosphate method (47).…”

Matrix metalloproteinase-7-catalyzed release of HB-EGF mediates deoxycholyltaurine-induced proliferation of a human colon cancer cell line

“…Phosphorylation of p44/42 MAPK, EGFR and p90RSK was determined by methods described previously [32]. Briefly, cells were subcultured in 6-well plates (10 6 cells/well).…”