G protein-coupled receptor-induced sensitization of phospholipase C stimulation by receptor tyrosine kinases

Activation of stably expressed M-2 and M-3 muscarinic acetylcholine recepto rs (mAChRs) as well as of endogenously expressed lysophosphatidic acid and purinergic receptors in HEK-293 cells can induce a long lasting potentiatio n of phospholipase C (PLC) stimulation by these and other G protein-coupled receptors (GPCRs), Here, we report that GPCRs can induce an up-regulation of PLC stimulation by receptor tyrosine kinases (RTKs) as well and provide essential mechanistic characteristics of this sensitization process. Pretre atment of HEK-293 cells for 2 min with carbachol, a mAChR agonist, lysophos phatidic acid, or ATP, followed by agonist washout, strongly increased (by 23-fold) maximal PLC stimulation (measured greater than or equal to 40 min later) by epidermal growth factor and platelet-derived growth factor, but n ot insulin, and largely enhanced PLC sensitivity to these RTK agonists, The up-regulation of RTK-induced PLC stimulation was cycloheximide-insensitive and was observed for up to similar to 90 min after removal of the GPCR ago nist, Sensitization of receptor-induced PLC stimulation caused by prior M-2 mAChR activation was fully prevented by pertussis toxin and strongly reduc ed by expression of G beta gamma scavengers. Furthermore, inhibition of con ventional protein kinase C (PKC) isoenzymes and chelation of intracellular Ca2+ suppressed the sensitization process, while overexpression of PKC-alph a, but not PKC-beta1, further enhanced the M-2 mAChR-induced sensitization of PLC stimulation. None of these treatments affected acute PLC stimulation by either GPCR or RTK agonists, Taken together, short term activation of G PCRs can induce a strong and long lasting sensitization of PLC stimulation by RTKs, a process apparently involving G(i)-derived G beta gammas as well as increases in intracellular Ca2+ and activation of a PKC isoenzyme, most likely PKC-alpha.