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

Abstract

Activation of stably expressed M 2 and M 3 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 ϳ90 min after removal of the GPCR agonist. Sensitization of receptor-induced PLC stimulation caused by prior M 2 mAChR activation was fully prevented by pertussis toxin and strongly reduced by expression of G␤␥ scavengers. Furthermore, inhibition of conventional protein kinase C (PKC) isoenzymes and chelation of intracellular Ca 2؉ suppressed the sensitization process, while overexpression of PKC-␣, but not PKC-␤I, 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 GPCRs can induce a strong and long lasting sensitization of PLC stimulation by RTKs, a process apparently involving G i -derived G␤␥s as well as increases in intracellular Ca 2؉ and activation of a PKC isoenzyme, most likely PKC-␣. Stimulation of phosphoinositide-hydrolyzing phospholipase C (PLC)1 is a cellular response to activation of a large variety of membrane receptors, including numerous G protein-coupled receptors (GPCRs) as well as several receptor tyrosine kinases (RTKs). These two types of membrane receptors generally stimulate distinct PLC isoenzymes. GPCRs activate PLC-␤ isoenzymes, either via GTP-liganded ␣ subunits of the G q class of G proteins or by ␤␥ dimers liberated from G i type G proteins. In contrast, RTKs, such as those for epidermal growth factor (EGF) and platelet-derived growth factor (PDGF), activate PLC-␥ isoenzymes by recruitment of these PLC enzymes to the autophosphorylated RTKs and subsequent tyrosine phosphorylation (1, 2). The hydrolysis of phosphatidylinositol 4,5-bisphosphate by PLC enzymes results in the generation of the two second messengers, inositol 1,4,5-trisphosphate (InsP 3 ) and diacylglycerol, which induce Ca 2ϩ release from intracellular stores and activation of protein kinase C (PKC) isoforms, respectively. It is generally accepted that by these functional consequences stimulation of PLC enzymes plays a major role in many early and late cellular responses to receptor activation, such as smooth m...

Roles of PLC-γ2 and PKCα in TPA-induced apoptosis of gastric cancer cells

“…Our results showed a definite linkage between PLC-γ2 and PKCα translocation, and the synergistic effect of PLC-γ2 and PKCα in the initiation of apoptosis in MGC80-3 cells induced by TPA. In addition, the fact that pretreatment of cells with PKC inhibitor did not affect PLC-γ2 activation and translocation by TPA was consistent with the observation in which pretreatment of 293 cells for 30 min with PKC inhibitor Gö6976 did not stimulate PLC activation by EGF [46] , demonstrating that PKC is a downstream molecule of PLCγ pathway. Taken together, PLC-γ2 functions in signal transmission to initiate TPAinduced apoptosis via PKCα pathway.…”

Activation of Epidermal Growth Factor Receptor Inhibits KCNQ2/3 Current through Two Distinct Pathways: Membrane PtdIns(4,5)P2Hydrolysis and Channel Phosphorylation

“…Activation of the EGF receptor can also hydrolyze PtdIns(4,5)P 2 through PLC ␥ (Schmidt et al, 2000). Thus, we hypothesized that EGF-induced fast inhibition of KCNQ2/3 currents might be the result of PtdIns(4,5)P 2 hydrolysis.…”

Rap2B-Dependent Stimulation of Phospholipase C-ε by Epidermal Growth Factor Receptor Mediated by c-Src Phosphorylation of RasGRP3

“…An aim of the present study was to analyze the mechanisms of PLC-ε stimulation by the EGF receptor in a cellular setting (in which a possible interaction of PLC-␥1 and PLC-ε can be assessed). For this, we used HEK-293 cells endogenously expressing PLC-coupled EGF receptors as well as PLC-␥1 and PLC-ε (7,17,31). We report here that the EGF receptor induces stimulation of PLC-ε in these cells in a Rap2B-dependent manner and that this process apparently involves PLC-␥1, c-Src, and the Ras/ Rap-GEF RasGRP3.…”

“…Thus, we wondered whether the EGF receptor (known to activate PLC-␥1) (8,24,26) might stimulate PLC-ε in addition as well. For this, we first studied the effects of wild-type PLC-␥1 and PLC-ε and their lipaseinactive mutants, H335Q PLC-␥1 and H1144L PLC-ε (15), respectively, in HEK-293 cells endogenously expressing PLCcoupled EGF receptors and PLC-ε (7,17,31). As expected, overexpression of wild-type PLC-␥1 largely enhanced (by about twofold) IP 3 formation induced by EGF (100 ng/ml), leaving basal IP 3 levels unaffected whereas expression of the lipase-inactive H335Q PLC-␥1 reduced EGF-stimulated IP 3 formation by about 50% (Fig.…”

“…The aim of the present study was to analyze whether the EGF receptor stimulates two distinct PLC isoforms, i.e., PLC-␥1 and PLC-ε, as well as to identify the mechanisms of PLC-ε activation, particularly whether and how PLC-␥1, which is directly activated by the EGF receptor (8,24,26,29), contributes to activation of PLC-ε. Using HEK-293 cells endogenously expressing PLCcoupled EGF receptors and PLC-ε (7,17,31) as a cellular model, we report here that the EGF receptor stimulates both PLC-␥1 and PLC-ε and that stimulation of PLC-ε is mediated by Rap2B activated by the Ca 2ϩ /DAG-regulated GEF RasGRP3 in response to PLC-␥1 activation and tyrosine phosphorylation of RasGRP3 by c-Src.…”