G Protein-coupled Receptor-induced Sensitization of Phospholipase C Stimulation by Receptor Tyrosine Kinases

Abstract Activation of stably expressed M2 and M3 muscarinic acetylcholine receptors (mAChRs) as well as of endogenously expressed lysophosphatidic acid and purinergic receptors in HEK-293 cells can induce a long lasting potentiation 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. Pretreatment of HEK-293 cells for 2 min with carbachol, a mAChR agonist, lysophosphatidic acid, or ATP, followed by agonist washout, strongly increased (by 2–3-fold) maximal PLC stimulation (measured ≥40 min later) by epidermal growth factor and platelet-derived growth factor, but not 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 ∼90 min after removal of the GPCR agonist. Sensitization of receptor-induced PLC stimulation caused by prior M2 mAChR activation was fully prevented by pertussis toxin and strongly reduced by expression of Gβγ scavengers. Furthermore, inhibition of conventional protein kinase C (PKC) isoenzymes and chelation of intracellular Ca2+ suppressed the sensitization process, while overexpression of PKC-α, but not PKC-βI, further enhanced the M2 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 GPCRs can induce a strong and long lasting sensitization of PLC stimulation by RTKs, a process apparently involving Gi-derived Gβγs as well as increases in intracellular Ca2+ and activation of a PKC isoenzyme, most likely PKC-α.