REGULATION OF PHOSPHOLIPASE-C AND PHOSPHOLIPASE-D ACTIVITIES BY SMALLMOLECULAR-WEIGHT G-PROTEINS AND MUSCARINIC RECEPTORS

The role of small molecular weight guanine nucleotide-binding proteins (G proteins) of the Rho family in muscarinic acetylcholine receptor ( mAChR) signaling to phospholipase C (PLC) and phospholipase D (PLD) wa s studied in human embryonic kidney (HEK) cells, stably expressing the human m3 receptor subtype. Evidence for the involvement of Rho protei ns in m3 mAChR signaling to both phospholipases is based on findings o btained with Clostridium (C.) difficile toxin B and C. botulinum C3 ex oenzyme, both of which specifically, although by different mechanisms, inactivate Rho family G proteins. Toxin B potently inhibited both the mAChR-stimulated PLC and PLD activities in intact cells as well as th e stimulation of both phospholipases by the stable GTP analog GTP gamm a S in permeabilized cells, the latter effect being mimicked by C3 exo enzyme. In contrast, PLC and PLD activities, measured in the presence of exogenous phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P-2], a substrate and cofactor for PLC and PLD, respectively, were not altere d. These data suggested that the Rho-inactivating toxins inhibit stimu lation of PLC and PLD by reducing the cellular level of PtdIns(4,5)P-2 , which was indeed found with both toxin B and C3 exoenzyme. In agreem ent with a crucial role of cellular PtdIns(4,5)P-2 supply for PLC sign aling, we observed that short-term agonist (carbachol) treatment of HE K cells caused a long-lasting increase in PtdIns(4,5)P-2 level, accomp anied by a potentiation of receptor- and G protein-stimulated inositol phosphate formation. Finally, studies with tyrosine kinase and tyrosi ne phosphatase inhibitors strongly suggest that PtdIns(4,5)P-2 synthes is and mAChR-stimulated PLD activity in HEK cells apparently also invo lve a tyrosine phosphorylation-dependent mechanism(s). Thus, m3 mAChR signaling to PLC and PLD in HEK cells requires the concerted action of various intracellular components, most notably the complex regulation of PtdIns(4,5)P-2 synthesis.