MECHANISMS OF PHOSPHOLIPASE-D STIMULATION BY M3 MUSCARINIC ACETYLCHOLINE-RECEPTORS - EVIDENCE FOR INVOLVEMENT OF TYROSINE PHOSPHORYLATION

In human embryonic kidney cells stably expressing the human m3 muscari nic acetylcholine receptor (mAChR) subtype, agonist (carbachol) activa tion stimulated phospholipase C, increased cytoplasmic calcium concent ration, induced tyrosine phosphorylation of various cellular proteins and activated phospholipase D. Bypassing membrane receptors, phospholi pase D was activated in these cells by direct activation of protein ki nase C by phorbol esters, by direct activation of GTP-binding proteins by AIF(4)(-) and a stable GTP analogue (in permeabilized cells), by i ncreasing cytoplasmic calcium concentration with the calcium ionophore A23187 and also apparently by tyrosine phosphorylation. In order to i dentify possible mechanisms by which the m3 mAChR couples to phospholi pase D, various inhibitors of protein kinase C, tyrosine kinases and c alcium-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 inhi bited the activation induced by the mAChR agonist. Additionally, down- regulation of protein kinase C by prolonged exposure to phorbol 12-myr istate 13-acetate abrogated phospholipase D activation by this effecto r but had only minor or no effects on the response to the mAChR agonis t and direct activators of GTP-binding proteins. In contrast, the tyro sine kinase inhibitor genistein abolished the carbachol-induced and Al F4--induced phospholipase D activation but had no effect on enzyme act ivation by phorbol 12-myristate 13-acetate. The data indicate that pho spholipase D in m3 mAChR-expressing human embryonic kidney cells can b e activated by various different mechanisms, i.e. receptor agonists, G TP-binding proteins, protein kinase C-dependent and calcium-dependent events and tyrosine phosphorylation. The coupling of m3 mAChR to phosp holipase D appears to be largely independent of concomitant phospholip ase C activation with subsequent increase in cytoplasmic calcium conce ntration and protein kinase C activity. The data instead suggest the i nvolvement of an essential protein tyrosine phosphorylation mechanism in phospholipase D activation by the m3 mAChR and heterotrimeric GTP-b inding proteins.