G Protein-coupled Receptor-induced Sensitization of Phospholipase C Stimulation by Receptor Tyrosine Kinases
2000; Elsevier BV; Volume: 275; Issue: 42 Linguagem: Inglês
10.1074/jbc.m004784200
ISSN1083-351X
AutoresMartina Schmidt, Markus Frings, Marie-Luise Mono, Yuanjian Guo, Paschal A. Oude Weernink, Sandrine Evellin, Han Li, Karl H. Jakobs,
Tópico(s)Neuroscience and Neuropharmacology Research
ResumoActivation 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. 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. phospholipase C G protein-coupled receptor receptor tyrosine kinase epidermal growth factor platelet-derived growth factor inositol 1,4,5-trisphosphate protein kinase C muscarinic acetylcholine receptor lysophosphatidic acid pertussis toxin N-methylscopolamine carboxyl terminus of the β-adrenergic receptor kinase 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetyl methylester 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-β isoenzymes, either via GTP-liganded α subunits of the Gqclass of G proteins or by βγ dimers liberated from Gitype G proteins. In contrast, RTKs, such as those for epidermal growth factor (EGF) and platelet-derived growth factor (PDGF), activate PLC-γ isoenzymes by recruitment of these PLC enzymes to the autophosphorylated RTKs and subsequent tyrosine phosphorylation (1Exton J.H. Annu. Rev. Pharmacol. Toxicol. 1997; 36: 481-509Crossref Scopus (299) Google Scholar, 2Rhee S.G. Bae Y.S. J. Biol. Chem. 1997; 272: 15045-15048Abstract Full Text Full Text PDF PubMed Scopus (809) Google Scholar). The hydrolysis of phosphatidylinositol 4,5-bisphosphate by PLC enzymes results in the generation of the two second messengers, inositol 1,4,5-trisphosphate (InsP3) and diacylglycerol, which induce Ca2+ 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 muscle contraction, secretion, neuronal signaling, and cell growth and differentiation, to name but a few (3Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6147) Google Scholar, 4Nishizuka Y. FASEB J. 1995; 9: 484-496Crossref PubMed Scopus (2352) Google Scholar, 5Berridge M.J. Neuron. 1998; 21: 13-26Abstract Full Text Full Text PDF PubMed Scopus (1735) Google Scholar, 6Ji Q.-S. Winnier G.E. Niswender K.D. Horstman D. Wisdom R. Magnuson M.A. Carpenter G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2999-3003Crossref PubMed Scopus (217) Google Scholar). Thus, alteration in receptor signaling to PLC enzymes is expected to have a major impact on cellular responses evoked by this receptor.We reported recently that short term activation of GPCRs in HEK-293 cells stably expressing the M2 or M3 subtypes of muscarinic acetylcholine receptors (mAChRs) can induce a long lasting potentiation of PLC stimulation by these and other GPCRs, including the endogenously expressed lysophosphatidic acid (LPA) and purinergic receptors (7Schmidt M. Fasselt B. Rümenapp U. Bienek C. Wieland T. van Koppen C.J. Jakobs K.H. J. Biol. Chem. 1995; 270: 19949-19956Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 8Schmidt M. Nehls C. Rümenapp U. Jakobs K.H. Mol. Pharmacol. 1996; 50: 1038-1046PubMed Google Scholar, 9Schmidt M. Lohmann B. Hammer K. Haupenthal S. Voß M. Nehls C. Jakobs K.H. Mol. Pharmacol. 1998; 53: 1139-1148PubMed Google Scholar). Studies with pertussis toxin (PTX) and PKC inhibitors, furthermore, suggested that this potentiation of PLC stimulation by GPCRs is mediated by Gi type G proteins and involves activation of a PKC isoenzyme (8Schmidt M. Nehls C. Rümenapp U. Jakobs K.H. Mol. Pharmacol. 1996; 50: 1038-1046PubMed Google Scholar, 9Schmidt M. Lohmann B. Hammer K. Haupenthal S. Voß M. Nehls C. Jakobs K.H. Mol. Pharmacol. 1998; 53: 1139-1148PubMed Google Scholar). Since GPCRs and RTKs activate distinct PLC isoenzymes and by distinct mechanisms, we wondered whether GPCRs may also induce sensitization of PLC stimulation by RTKs endogenously expressed in HEK-293 cells (10Tsai W. Morielli A.D. Peralta E.G. EMBO J. 1997; 16: 4597-4605Crossref PubMed Scopus (188) Google Scholar, 11Voß M. Oude Weernink P.A. Haupenthal S. Möller U. Cool R.H. Bauer B. Camonis J.H. Jakobs K.H. Schmidt M. J. Biol. Chem. 1999; 274: 34691-34698Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). We report here that short term activation of GPCRs can induce a long lasting up-regulation of PLC stimulation by EGF and PDGF but not insulin. Furthermore, evidence is provided suggesting that this sensitization of PLC stimulation is mediated by Gi-derived Gβγ dimers and that increases in cytosolic Ca2+ and activation of a conventional PKC enzyme, most likely PKC-α, are required for this novel PLC regulatory mechanism.DISCUSSIONWe reported before that short term activation of GPCRs in HEK-293 cells stably expressing the M2 or M3 mAChR subtypes can induce a long lasting sensitization of PLC stimulation by these and other GPCRs. The GPCR-induced up-regulation of PLC stimulation was prevented by PTX and inhibition of PKC enzymes (7Schmidt M. Fasselt B. Rümenapp U. Bienek C. Wieland T. van Koppen C.J. Jakobs K.H. J. Biol. Chem. 1995; 270: 19949-19956Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 8Schmidt M. Nehls C. Rümenapp U. Jakobs K.H. Mol. Pharmacol. 1996; 50: 1038-1046PubMed Google Scholar, 9Schmidt M. Lohmann B. Hammer K. Haupenthal S. Voß M. Nehls C. Jakobs K.H. Mol. Pharmacol. 1998; 53: 1139-1148PubMed Google Scholar). Since GPCRs and RTKs activate distinct PLC isoenzymes, GPCRs mainly PLC-β enzymes and RTKs PLC-γ enzymes, particularly the widely expressed PLC-γ1 (1Exton J.H. Annu. Rev. Pharmacol. Toxicol. 1997; 36: 481-509Crossref Scopus (299) Google Scholar, 2Rhee S.G. Bae Y.S. J. Biol. Chem. 1997; 272: 15045-15048Abstract Full Text Full Text PDF PubMed Scopus (809) Google Scholar), a major aim of the present report was to examine whether GPCRs can induce potentiation of PLC stimulation by RTKs as well. Furthermore, the mechanisms involved in this up-regulation were explored, particularly whether up-regulation of PLC stimulation by GPCRs and RTKs involves identical or distinct mechanisms. For the study, we used wild-type HEK-293 cells as well as HEK-293 cells stably expressing the M2 or M3mAChR subtypes and endogenously expressing various other GPCRs as well as RTKs for EGF, PDGF, and insulin (8Schmidt M. Nehls C. Rümenapp U. Jakobs K.H. Mol. Pharmacol. 1996; 50: 1038-1046PubMed Google Scholar, 9Schmidt M. Lohmann B. Hammer K. Haupenthal S. Voß M. Nehls C. Jakobs K.H. Mol. Pharmacol. 1998; 53: 1139-1148PubMed Google Scholar, 10Tsai W. Morielli A.D. Peralta E.G. EMBO J. 1997; 16: 4597-4605Crossref PubMed Scopus (188) Google Scholar, 11Voß M. Oude Weernink P.A. Haupenthal S. Möller U. Cool R.H. Bauer B. Camonis J.H. Jakobs K.H. Schmidt M. J. Biol. Chem. 1999; 274: 34691-34698Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). We report here that short term activation of the overexpressed M2 and M3 mAChRs and the endogenously expressed LPA and purinergic receptors can induce a strong and long lasting up-regulation and sensitization of PLC stimulation by EGF and PDGF but not insulin. Furthermore, evidence is provided that the GPCR-induced sensitization of PLC stimulation is apparently mediated by Gβγ dimers liberated from PTX-sensitive Gi type G proteins and requires increases in cytosolic Ca2+ concentration and activation of a conventional PKC enzyme, most likely PKC-α. Finally, extensive comparison of PLC stimulation by the M2 mAChR and the EGF receptor strongly suggests that very similar or even identical mechanisms are involved in the process of sensitization of PLC stimulation by these two distinct receptor types.The enhancement of PLC stimulation by EGF and PDGF induced by prior GPCR activation was apparently not due to a block or inhibition of a desensitization process. First, accumulation of inositol phosphates induced by EGF and PDGF in control cells was rather linear with time for up to 30 min; thus, there was no major desensitization of PLC stimulation during this time period. Second, similar to the enhancement of inositol phosphate accumulation measured over a 30-min period, prior GPCR treatment of HEK-293 cells also strongly increased EGF- and PDGF-stimulated formation of InsP3, measured 15 s after challenge of the cells with the RTK agonists. Interestingly, PLC stimulation in HEK-293 cells by insulin was not increased by prior activation of the M2 mAChR. The reason for this discrepancy is presently not clear. While stimulation of PLC activity, particularly of the PLC-γ1 enzyme, is a well established and rather general response to EGF and PDGF receptor activation, it is not so for insulin (1Exton J.H. Annu. Rev. Pharmacol. Toxicol. 1997; 36: 481-509Crossref Scopus (299) Google Scholar, 2Rhee S.G. Bae Y.S. J. Biol. Chem. 1997; 272: 15045-15048Abstract Full Text Full Text PDF PubMed Scopus (809) Google Scholar, 26Saltiel A.R. Am. J. Physiol. 1996; 270: E375-E385PubMed Google Scholar, 27Whitehead J.P. Clark S.F. Ursø B. James D.E. Curr. Opin. Cell Biol. 2000; 12: 222-228Crossref PubMed Scopus (100) Google Scholar), although stimulation of PLC activity by insulin has been described in some cell types, and PLC-γ has recently been reported to participate in metabolic signaling by the insulin receptor in adipocytes (28Kellerer M. Machicao F. Seffer E. Mushack J. Ullrich A. Häring H.U. Biochem. Biophys. Res. Commun. 1991; 181: 566-572Crossref PubMed Scopus (14) Google Scholar, 29Van Epps-Fung M. Gupta K. Hardy R.W. Wells A. Endocrinology. 1997; 138: 5170-5175Crossref PubMed Scopus (37) Google Scholar, 30Kayali A.G. Eichhorn J. Haruta T. Morris A.J. Nelson J.G. Vollenweider P. Olefsky J.M. Webster N.J.G. J. Biol. Chem. 1998; 273: 13808-13818Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Regardless of the underlying mechanisms, the insensitivity of PLC stimulation by insulin to prior GPCR activation indicates that the sensitization of PLC stimulation by EGF and PDGF is not an unspecific response to any PLC stimulatory receptor.The experimental paradigm used in the present study to demonstrate GPCR-induced sensitization of PLC stimulation by EGF and PDGF (i.e. first treatment of HEK-293 cells for a short period (2 min) with a GPCR agonist and then washout of this agonist and subsequent incubation of the cells for 40 min or longer without any agonist before actual measurement of PLC activity) is based on previous data on up-regulation of PLC stimulation by GPCRs. These studies on M2 and M3 mAChR-induced sensitization of PLC stimulation demonstrated that maximal up-regulation of PLC stimulation by these mAChRs is observed at about 40 min after washout of the initial stimulus and slowly disappears thereafter (8Schmidt M. Nehls C. Rümenapp U. Jakobs K.H. Mol. Pharmacol. 1996; 50: 1038-1046PubMed Google Scholar, 9Schmidt M. Lohmann B. Hammer K. Haupenthal S. Voß M. Nehls C. Jakobs K.H. Mol. Pharmacol. 1998; 53: 1139-1148PubMed Google Scholar). 2M. Schmidt, M. Frings, M.-L. Mono, Y. Guo, P. A. Oude Weernink, S. Evellin, L. Han, and K. H. Jakobs, unpublished data. Using these experimental conditions, it is demonstrated that sensitization of PLC stimulation by EGF and PDGF caused by prior M2 mAChR activation is also a long lasting process, with a maximum at 40 min after the initial treatment with carbachol and a slow decline thereafter, reaching control values at ∼150 min.Not only the time courses but also the mechanisms involved in the GPCR-induced sensitization of PLC stimulation by GPCRs and RTKs are apparently very similar. Specifically, it is shown that the M2 mAChR-induced up-regulation of PLC stimulation by either carbachol or EGF is completely abrogated by PTX treatment of the cells and largely reduced by expression of the two Gβγ scavengers, βARK-CT and Gαt. In addition, inhibition of conventional PKC enzymes with Gö 6976 and chelation of intracellular Ca2+ with BAPTA/AM fully blocked the M2 mAChR-induced up-regulation of PLC stimulation by either carbachol or EGF. None of these treatments had an effect on PLC stimulation by carbachol or EGF in naive cells. Furthermore, as demonstrated for the M2 mAChR, the sensitization of PLC stimulation caused by prior GPCR activation was apparently not due to a corresponding up-regulation of cell surface receptor number. Thus, the sensitizing GPCRs apparently generate two distinct signals mediating the long lasting sensitization process of PLC stimulation. One signal is apparently caused by activation of PLC, which is PTX-insensitive in HEK-293 cells, thus most likely mediated by Gq type G proteins, and finally results in Ca2+ mobilization and PKC activation. The results obtained with Gö 6976 and BAPTA/AM prompted us to investigate which of the conventional Ca2+-dependent PKC isoenzymes known to be inhibited by Gö 6976, PKC-α or PKC-βI (24Martiny-Baron G. Kazanietz M.G. Mischak H. Blumberg P.M. Hug H. Marmé D. Schächtele C. J. Biol. Chem. 1993; 268: 9194-9197Abstract Full Text PDF PubMed Google Scholar), mediates the up-regulation of PLC stimulation. It is shown that overexpression of PKC-α, which had no effect on PLC stimulation in naive cells, largely enhanced the M2 mAChR-induced sensitization of PLC stimulation by carbachol and EGF, whereas overexpression of PKC-βI was without any effect. Similar negative results were obtained in cells overexpressing PKC-βII, PKC-ε, or PKC-ζ (data not shown). Thus, one major signal involved in and mediating the GPCR-induced sensitization of PLC stimulation is apparently the activation of a conventional Ca2+-dependent PKC isoenzyme, most likely PKC-α. It remains to be studied whether Ca2+ acts solely by activating the PKC enzyme or whether additional Ca2+-dependent steps are involved in the PLC sensitization process. The second signal generated by the sensitizing GPCR is apparently dependent on Gβγ dimers derived from receptor-activated Gi type G proteins (31Offermanns S. Wieland T. Homann D. Sandmann J. Bombien E. Spicher K. Schultz G. Jakobs K.H. Mol. Pharmacol. 1994; 45: 890-898PubMed Google Scholar). During the last few years, various direct and indirect effectors of Gβγ dimers have been identified (22Clapham D.E. Neer E. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 167-203Crossref PubMed Scopus (699) Google Scholar, 23Gautam N. Downes G.B. Yan K. Kisselev O. Cell. Signal. 1998; 7: 447-455Crossref Scopus (156) Google Scholar). Since the M2 mAChR-induced PLC stimulation in naive cells was affected neither by PTX nor by expression of Gβγ scavengers, it is highly unlikely that a PLC-β isoenzyme known to be controlled by Gβγs (1Exton J.H. Annu. Rev. Pharmacol. Toxicol. 1997; 36: 481-509Crossref Scopus (299) Google Scholar, 2Rhee S.G. Bae Y.S. J. Biol. Chem. 1997; 272: 15045-15048Abstract Full Text Full Text PDF PubMed Scopus (809) Google Scholar) is the relevant Gβγ effector. Thus, overall, the GPCR-induced sensitization of PLC stimulation by GPCRs and RTKs apparently requires the two PLC-derived signals (i.e. increase in intracellular Ca2+concentration and activation of a conventional PKC isoenzyme) and an as yet unidentified Gβγ effector, which then in combination induce a long lasting cellular memory for receptor-mediated PLC stimulation.During the last years, various GPCRs have been reported to cause "transactivation" of RTKs, specifically of the EGF and PDGF receptors, in different cellular systems (for a review, see Ref. 32Zwick E. Hackel P.O. Prenzel N. Ullrich A. Trends Pharmacol. Sci. 1999; 20: 408-412Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar). These studies demonstrated that tyrosine phosphorylation of the EGF or PDGF receptor is an essential intermediate step particularly for mitogenic signaling by these GPCRs. The results presented in this report demonstrating GPCR-induced sensitization of PLC stimulation by EGF and PDGF receptors may also be termed "transactivation," however with a completely different meaning. First, the experimental paradigm used to demonstrate GPCR-induced sensitization of PLC stimulation by RTKs is quite distinct from that used in the above mentioned "transactivation" studies, in which acute GPCR-induced cellular responses were shown to involve activation of a RTK. Second, in contrast to the GPCR-induced "transactivation" of EGF or PDGF receptors, which was independent of exogenous RTK agonists (32Zwick E. Hackel P.O. Prenzel N. Ullrich A. Trends Pharmacol. Sci. 1999; 20: 408-412Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar), the GPCR-induced PLC sensitization was only observed upon the addition of exogenous RTK (or GPCR) ligands, whereas agonist-independent basal PLC activity was not altered in GPCR-pretreated cells.In conclusion, the data presented in this report demonstrate that short term activation of various GPCRs expressed in HEK-293 cells can induce a strong and long lasting up-regulation and sensitization of PLC stimulation by EGF and PDGF receptors, two prototypical RTKs. This novel cellular response apparently involves the complex interplay of at least two distinct signaling pathways induced by the GPCRs. The up-regulation and sensitization of PLC stimulation by RTKs described herein most likely has a major impact on physiological and possibly also pathological cellular responses triggered by these growth factor receptors. 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-β isoenzymes, either via GTP-liganded α subunits of the Gqclass of G proteins or by βγ dimers liberated from Gitype G proteins. In contrast, RTKs, such as those for epidermal growth factor (EGF) and platelet-derived growth factor (PDGF), activate PLC-γ isoenzymes by recruitment of these PLC enzymes to the autophosphorylated RTKs and subsequent tyrosine phosphorylation (1Exton J.H. Annu. Rev. Pharmacol. Toxicol. 1997; 36: 481-509Crossref Scopus (299) Google Scholar, 2Rhee S.G. Bae Y.S. J. Biol. Chem. 1997; 272: 15045-15048Abstract Full Text Full Text PDF PubMed Scopus (809) Google Scholar). The hydrolysis of phosphatidylinositol 4,5-bisphosphate by PLC enzymes results in the generation of the two second messengers, inositol 1,4,5-trisphosphate (InsP3) and diacylglycerol, which induce Ca2+ 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 muscle contraction, secretion, neuronal signaling, and cell growth and differentiation, to name but a few (3Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6147) Google Scholar, 4Nishizuka Y. FASEB J. 1995; 9: 484-496Crossref PubMed Scopus (2352) Google Scholar, 5Berridge M.J. Neuron. 1998; 21: 13-26Abstract Full Text Full Text PDF PubMed Scopus (1735) Google Scholar, 6Ji Q.-S. Winnier G.E. Niswender K.D. Horstman D. Wisdom R. Magnuson M.A. Carpenter G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2999-3003Crossref PubMed Scopus (217) Google Scholar). Thus, alteration in receptor signaling to PLC enzymes is expected to have a major impact on cellular responses evoked by this receptor. We reported recently that short term activation of GPCRs in HEK-293 cells stably expressing the M2 or M3 subtypes of muscarinic acetylcholine receptors (mAChRs) can induce a long lasting potentiation of PLC stimulation by these and other GPCRs, including the endogenously expressed lysophosphatidic acid (LPA) and purinergic receptors (7Schmidt M. Fasselt B. Rümenapp U. Bienek C. Wieland T. van Koppen C.J. Jakobs K.H. J. Biol. Chem. 1995; 270: 19949-19956Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 8Schmidt M. Nehls C. Rümenapp U. Jakobs K.H. Mol. Pharmacol. 1996; 50: 1038-1046PubMed Google Scholar, 9Schmidt M. Lohmann B. Hammer K. Haupenthal S. Voß M. Nehls C. Jakobs K.H. Mol. Pharmacol. 1998; 53: 1139-1148PubMed Google Scholar). Studies with pertussis toxin (PTX) and PKC inhibitors, furthermore, suggested that this potentiation of PLC stimulation by GPCRs is mediated by Gi type G proteins and involves activation of a PKC isoenzyme (8Schmidt M. Nehls C. Rümenapp U. Jakobs K.H. Mol. Pharmacol. 1996; 50: 1038-1046PubMed Google Scholar, 9Schmidt M. Lohmann B. Hammer K. Haupenthal S. Voß M. Nehls C. Jakobs K.H. Mol. Pharmacol. 1998; 53: 1139-1148PubMed Google Scholar). Since GPCRs and RTKs activate distinct PLC isoenzymes and by distinct mechanisms, we wondered whether GPCRs may also induce sensitization of PLC stimulation by RTKs endogenously expressed in HEK-293 cells (10Tsai W. Morielli A.D. Peralta E.G. EMBO J. 1997; 16: 4597-4605Crossref PubMed Scopus (188) Google Scholar, 11Voß M. Oude Weernink P.A. Haupenthal S. Möller U. Cool R.H. Bauer B. Camonis J.H. Jakobs K.H. Schmidt M. J. Biol. Chem. 1999; 274: 34691-34698Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). We report here that short term activation of GPCRs can induce a long lasting up-regulation of PLC stimulation by EGF and PDGF but not insulin. Furthermore, evidence is provided suggesting that this sensitization of PLC stimulation is mediated by Gi-derived Gβγ dimers and that increases in cytosolic Ca2+ and activation of a conventional PKC enzyme, most likely PKC-α, are required for this novel PLC regulatory mechanism. DISCUSSIONWe reported before that short term activation of GPCRs in HEK-293 cells stably expressing the M2 or M3 mAChR subtypes can induce a long lasting sensitization of PLC stimulation by these and other GPCRs. The GPCR-induced up-regulation of PLC stimulation was prevented by PTX and inhibition of PKC enzymes (7Schmidt M. Fasselt B. Rümenapp U. Bienek C. Wieland T. van Koppen C.J. Jakobs K.H. J. Biol. Chem. 1995; 270: 19949-19956Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 8Schmidt M. Nehls C. Rümenapp U. Jakobs K.H. Mol. Pharmacol. 1996; 50: 1038-1046PubMed Google Scholar, 9Schmidt M. Lohmann B. Hammer K. Haupenthal S. Voß M. Nehls C. Jakobs K.H. Mol. Pharmacol. 1998; 53: 1139-1148PubMed Google Scholar). Since GPCRs and RTKs activate distinct PLC isoenzymes, GPCRs mainly PLC-β enzymes and RTKs PLC-γ enzymes, particularly the widely expressed PLC-γ1 (1Exton J.H. Annu. Rev. Pharmacol. Toxicol. 1997; 36: 481-509Crossref Scopus (299) Google Scholar, 2Rhee S.G. Bae Y.S. J. Biol. Chem. 1997; 272: 15045-15048Abstract Full Text Full Text PDF PubMed Scopus (809) Google Scholar), a major aim of the present report was to examine whether GPCRs can induce potentiation of PLC stimulation by RTKs as well. Furthermore, the mechanisms involved in this up-regulation were explored, particularly whether up-regulation of PLC stimulation by GPCRs and RTKs involves identical or distinct mechanisms. For the study, we used wild-type HEK-293 cells as well as HEK-293 cells stably expressing the M2 or M3mAChR subtypes and endogenously expressing various other GPCRs as well as RTKs for EGF, PDGF, and insulin (8Schmidt M. Nehls C. Rümenapp U. Jakobs K.H. Mol. Pharmacol. 1996; 50: 1038-1046PubMed Google Scholar, 9Schmidt M. Lohmann B. Hammer K. Haupenthal S. Voß M. Nehls C. Jakobs K.H. Mol. Pharmacol. 1998; 53: 1139-1148PubMed Google Scholar, 10Tsai W. Morielli A.D. Peralta E.G. EMBO J. 1997; 16: 4597-4605Crossref PubMed Scopus (188) Google Scholar, 11Voß M. Oude Weernink P.A. Haupenthal S. Möller U. Cool R.H. Bauer B. Camonis J.H. Jakobs K.H. Schmidt M. J. Biol. Chem. 1999; 274: 34691-34698Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). We report here that short term activation of the overexpressed M2 and M3 mAChRs and the endogenously expressed LPA and purinergic receptors can induce a strong and long lasting up-regulation and sensitization of PLC stimulation by EGF and PDGF but not insulin. Furthermore, evidence is provided that the GPCR-induced sensitization of PLC stimulation is apparently mediated by Gβγ dimers liberated from PTX-sensitive Gi type G proteins and requires increases in cytosolic Ca2+ concentration and activation of a conventional PKC enzyme, most likely PKC-α. Finally, extensive comparison of PLC stimulation by the M2 mAChR and the EGF receptor strongly suggests that very similar or even identical mechanisms are involved in the process of sensitization of PLC stimulation by these two distinct receptor types.The enhancement of PLC stimulation by EGF and PDGF induced by prior GPCR activation was apparently not due to a block or inhibition of a desensitization process. First, accumulation of inositol phosphates induced by EGF and PDGF in control cells was rather linear with time for up to 30 min; thus, there was no major desensitization of PLC stimulation during this time period. Second, similar to the enhancement of inositol phosphate accumulation measured over a 30-min period, prior GPCR treatment of HEK-293 cells also strongly increased EGF- and PDGF-stimulated formation of InsP3, measured 15 s after challenge of the cells with the RTK agonists. Interestingly, PLC stimulation in HEK-293 cells by insulin was not increased by prior activation of the M2 mAChR. The reason for this discrepancy is presently not clear. While stimulation of PLC activity, particularly of the PLC-γ1 enzyme, is a well established and rather general response to EGF and PDGF receptor activation, it is not so for insulin (1Exton J.H. Annu. Rev. Pharmacol. Toxicol. 1997; 36: 481-509Crossref Scopus (299) Google Scholar, 2Rhee S.G. Bae Y.S. J. Biol. Chem. 1997; 272: 15045-15048Abstract Full Text Full Text PDF PubMed Scopus (809) Google Scholar, 26Saltiel A.R. Am. J. Physiol. 1996; 270: E375-E385PubMed Google Scholar, 27Whitehead J.P. Clark S.F. Ursø B. James D.E. Curr. Opin. Cell Biol. 2000; 12: 222-228Crossref PubMed Scopus (100) Google Scholar), although stimulation of PLC activity by insulin has been described in some cell types, and PLC-γ has recently been reported to participate in metabolic signaling by the insulin receptor in adipocytes (28Kellerer M. Machicao F. Seffer E. Mushack J. Ullrich A. Häring H.U. Biochem. Biophys. Res. Commun. 1991; 181: 566-572Crossref PubMed Scopus (14) Google Scholar, 29Van Epps-Fung M. Gupta K. Hardy R.W. Wells A. Endocrinology. 1997; 138: 5170-5175Crossref PubMed Scopus (37) Google Scholar, 30Kayali A.G. Eichhorn J. Haruta T. Morris A.J. Nelson J.G. Vollenweider P. Olefsky J.M. Webster N.J.G. J. Biol. Chem. 1998; 273: 13808-13818Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Regardless of the underlying mechanisms, the insensitivity of PLC stimulation by insulin to prior GPCR activation indicates that the sensitization of PLC stimulation by EGF and PDGF is not an unspecific response to any PLC stimulatory receptor.The experimental paradigm used in the present study to demonstrate GPCR-induced sensitization of PLC stimulation by EGF and PDGF (i.e. first treatment of HEK-293 cells for a short period (2 min) with a GPCR agonist and then washout of this agonist and subsequent incubation of the cells for 40 min or longer without any agonist before actual measurement of PLC activity) is based on previous data on up-regulation of PLC stimulation by GPCRs. These studies on M2 and M3 mAChR-induced sensitization of PLC stimulation demonstrated that maximal up-regulation of PLC stimulation by these mAChRs is observed at about 40 min after washout of the initial stimulus and slowly disappears thereafter (8Schmidt M. Nehls C. Rümenapp U. Jakobs K.H. Mol. Pharmacol. 1996; 50: 1038-1046PubMed Google Scholar, 9Schmidt M. Lohmann B. Hammer K. Haupenthal S. Voß M. Nehls C. Jakobs K.H. Mol. Pharmacol. 1998; 53: 1139-1148PubMed Google Scholar). 2M. Schmidt, M. Frings, M.-L. Mono, Y. Guo, P. A. Oude Weernink, S. Evellin, L. Han, and K. H. Jakobs, unpublished data. Using these experimental conditions, it is demonstrated that sensitization of PLC stimulation by EGF and PDGF caused by prior M2 mAChR activation is also a long lasting process, with a maximum at 40 min after the initial treatment with carbachol and a slow decline thereafter, reaching control values at ∼150 min.Not only the time courses but also the mechanisms involved in the GPCR-induced sensitization of PLC stimulation by GPCRs and RTKs are apparently very similar. Specifically, it is shown that the M2 mAChR-induced up-regulation of PLC stimulation by either carbachol or EGF is completely abrogated by PTX treatment of the cells and largely reduced by expression of the two Gβγ scavengers, βARK-CT and Gαt. In addition, inhibition of conventional PKC enzymes with Gö 6976 and chelation of intracellular Ca2+ with BAPTA/AM fully blocked the M2 mAChR-induced up-regulation of PLC stimulation by either carbachol or EGF. None of these treatments had an effect on PLC stimulation by carbachol or EGF in naive cells. Furthermore, as demonstrated for the M2 mAChR, the sensitization of PLC stimulation caused by prior GPCR activation was apparently not due to a corresponding up-regulation of cell surface receptor number. Thus, the sensitizing GPCRs apparently generate two distinct signals mediating the long lasting sensitization process of PLC stimulation. One signal is apparently caused by activation of PLC, which is PTX-insensitive in HEK-293 cells, thus most likely mediated by Gq type G proteins, and finally results in Ca2+ mobilization and PKC activation. The results obtained with Gö 6976 and BAPTA/AM prompted us to investigate which of the conventional Ca2+-dependent PKC isoenzymes known to be inhibited by Gö 6976, PKC-α or PKC-βI (24Martiny-Baron G. Kazanietz M.G. Mischak H. Blumberg P.M. Hug H. Marmé D. Schächtele C. J. Biol. Chem. 1993; 268: 9194-9197Abstract Full Text PDF PubMed Google Scholar), mediates the up-regulation of PLC stimulation. It is shown that overexpression of PKC-α, which had no effect on PLC stimulation in naive cells, largely enhanced the M2 mAChR-induced sensitization of PLC stimulation by carbachol and EGF, whereas overexpression of PKC-βI was without any effect. Similar negative results were obtained in cells overexpressing PKC-βII, PKC-ε, or PKC-ζ (data not shown). Thus, one major signal involved in and mediating the GPCR-induced sensitization of PLC stimulation is apparently the activation of a conventional Ca2+-dependent PKC isoenzyme, most likely PKC-α. It remains to be studied whether Ca2+ acts solely by activating the PKC enzyme or whether additional Ca2+-dependent steps are involved in the PLC sensitization process. The second signal generated by the sensitizing GPCR is apparently dependent on Gβγ dimers derived from receptor-activated Gi type G proteins (31Offermanns S. Wieland T. Homann D. Sandmann J. Bombien E. Spicher K. Schultz G. Jakobs K.H. Mol. Pharmacol. 1994; 45: 890-898PubMed Google Scholar). During the last few years, various direct and indirect effectors of Gβγ dimers have been identified (22Clapham D.E. Neer E. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 167-203Crossref PubMed Scopus (699) Google Scholar, 23Gautam N. Downes G.B. Yan K. Kisselev O. Cell. Signal. 1998; 7: 447-455Crossref Scopus (156) Google Scholar). Since the M2 mAChR-induced PLC stimulation in naive cells was affected neither by PTX nor by expression of Gβγ scavengers, it is highly unlikely that a PLC-β isoenzyme known to be controlled by Gβγs (1Exton J.H. Annu. Rev. Pharmacol. Toxicol. 1997; 36: 481-509Crossref Scopus (299) Google Scholar, 2Rhee S.G. Bae Y.S. J. Biol. Chem. 1997; 272: 15045-15048Abstract Full Text Full Text PDF PubMed Scopus (809) Google Scholar) is the relevant Gβγ effector. Thus, overall, the GPCR-induced sensitization of PLC stimulation by GPCRs and RTKs apparently requires the two PLC-derived signals (i.e. increase in intracellular Ca2+concentration and activation of a conventional PKC isoenzyme) and an as yet unidentified Gβγ effector, which then in combination induce a long lasting cellular memory for receptor-mediated PLC stimulation.During the last years, various GPCRs have been reported to cause "transactivation" of RTKs, specifically of the EGF and PDGF receptors, in different cellular systems (for a review, see Ref. 32Zwick E. Hackel P.O. Prenzel N. Ullrich A. Trends Pharmacol. Sci. 1999; 20: 408-412Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar). These studies demonstrated that tyrosine phosphorylation of the EGF or PDGF receptor is an essential intermediate step particularly for mitogenic signaling by these GPCRs. The results presented in this report demonstrating GPCR-induced sensitization of PLC stimulation by EGF and PDGF receptors may also be termed "transactivation," however with a completely different meaning. First, the experimental paradigm used to demonstrate GPCR-induced sensitization of PLC stimulation by RTKs is quite distinct from that used in the above mentioned "transactivation" studies, in which acute GPCR-induced cellular responses were shown to involve activation of a RTK. Second, in contrast to the GPCR-induced "transactivation" of EGF or PDGF receptors, which was independent of exogenous RTK agonists (32Zwick E. Hackel P.O. Prenzel N. Ullrich A. Trends Pharmacol. Sci. 1999; 20: 408-412Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar), the GPCR-induced PLC sensitization was only observed upon the addition of exogenous RTK (or GPCR) ligands, whereas agonist-independent basal PLC activity was not altered in GPCR-pretreated cells.In conclusion, the data presented in this report demonstrate that short term activation of various GPCRs expressed in HEK-293 cells can induce a strong and long lasting up-regulation and sensitization of PLC stimulation by EGF and PDGF receptors, two prototypical RTKs. This novel cellular response apparently involves the complex interplay of at least two distinct signaling pathways induced by the GPCRs. The up-regulation and sensitization of PLC stimulation by RTKs described herein most likely has a major impact on physiological and possibly also pathological cellular responses triggered by these growth factor receptors. We reported before that short term activation of GPCRs in HEK-293 cells stably expressing the M2 or M3 mAChR subtypes can induce a long lasting sensitization of PLC stimulation by these and other GPCRs. The GPCR-induced up-regulation of PLC stimulation was prevented by PTX and inhibition of PKC enzymes (7Schmidt M. Fasselt B. Rümenapp U. Bienek C. Wieland T. van Koppen C.J. Jakobs K.H. J. Biol. Chem. 1995; 270: 19949-19956Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 8Schmidt M. Nehls C. Rümenapp U. Jakobs K.H. Mol. Pharmacol. 1996; 50: 1038-1046PubMed Google Scholar, 9Schmidt M. Lohmann B. Hammer K. Haupenthal S. Voß M. Nehls C. Jakobs K.H. Mol. Pharmacol. 1998; 53: 1139-1148PubMed Google Scholar). Since GPCRs and RTKs activate distinct PLC isoenzymes, GPCRs mainly PLC-β enzymes and RTKs PLC-γ enzymes, particularly the widely expressed PLC-γ1 (1Exton J.H. Annu. Rev. Pharmacol. Toxicol. 1997; 36: 481-509Crossref Scopus (299) Google Scholar, 2Rhee S.G. Bae Y.S. J. Biol. Chem. 1997; 272: 15045-15048Abstract Full Text Full Text PDF PubMed Scopus (809) Google Scholar), a major aim of the present report was to examine whether GPCRs can induce potentiation of PLC stimulation by RTKs as well. Furthermore, the mechanisms involved in this up-regulation were explored, particularly whether up-regulation of PLC stimulation by GPCRs and RTKs involves identical or distinct mechanisms. For the study, we used wild-type HEK-293 cells as well as HEK-293 cells stably expressing the M2 or M3mAChR subtypes and endogenously expressing various other GPCRs as well as RTKs for EGF, PDGF, and insulin (8Schmidt M. Nehls C. Rümenapp U. Jakobs K.H. Mol. Pharmacol. 1996; 50: 1038-1046PubMed Google Scholar, 9Schmidt M. Lohmann B. Hammer K. Haupenthal S. Voß M. Nehls C. Jakobs K.H. Mol. Pharmacol. 1998; 53: 1139-1148PubMed Google Scholar, 10Tsai W. Morielli A.D. Peralta E.G. EMBO J. 1997; 16: 4597-4605Crossref PubMed Scopus (188) Google Scholar, 11Voß M. Oude Weernink P.A. Haupenthal S. Möller U. Cool R.H. Bauer B. Camonis J.H. Jakobs K.H. Schmidt M. J. Biol. Chem. 1999; 274: 34691-34698Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). We report here that short term activation of the overexpressed M2 and M3 mAChRs and the endogenously expressed LPA and purinergic receptors can induce a strong and long lasting up-regulation and sensitization of PLC stimulation by EGF and PDGF but not insulin. Furthermore, evidence is provided that the GPCR-induced sensitization of PLC stimulation is apparently mediated by Gβγ dimers liberated from PTX-sensitive Gi type G proteins and requires increases in cytosolic Ca2+ concentration and activation of a conventional PKC enzyme, most likely PKC-α. Finally, extensive comparison of PLC stimulation by the M2 mAChR and the EGF receptor strongly suggests that very similar or even identical mechanisms are involved in the process of sensitization of PLC stimulation by these two distinct receptor types. The enhancement of PLC stimulation by EGF and PDGF induced by prior GPCR activation was apparently not due to a block or inhibition of a desensitization process. First, accumulation of inositol phosphates induced by EGF and PDGF in control cells was rather linear with time for up to 30 min; thus, there was no major desensitization of PLC stimulation during this time period. Second, similar to the enhancement of inositol phosphate accumulation measured over a 30-min period, prior GPCR treatment of HEK-293 cells also strongly increased EGF- and PDGF-stimulated formation of InsP3, measured 15 s after challenge of the cells with the RTK agonists. Interestingly, PLC stimulation in HEK-293 cells by insulin was not increased by prior activation of the M2 mAChR. The reason for this discrepancy is presently not clear. While stimulation of PLC activity, particularly of the PLC-γ1 enzyme, is a well established and rather general response to EGF and PDGF receptor activation, it is not so for insulin (1Exton J.H. Annu. Rev. Pharmacol. Toxicol. 1997; 36: 481-509Crossref Scopus (299) Google Scholar, 2Rhee S.G. Bae Y.S. J. Biol. Chem. 1997; 272: 15045-15048Abstract Full Text Full Text PDF PubMed Scopus (809) Google Scholar, 26Saltiel A.R. Am. J. Physiol. 1996; 270: E375-E385PubMed Google Scholar, 27Whitehead J.P. Clark S.F. Ursø B. James D.E. Curr. Opin. Cell Biol. 2000; 12: 222-228Crossref PubMed Scopus (100) Google Scholar), although stimulation of PLC activity by insulin has been described in some cell types, and PLC-γ has recently been reported to participate in metabolic signaling by the insulin receptor in adipocytes (28Kellerer M. Machicao F. Seffer E. Mushack J. Ullrich A. Häring H.U. Biochem. Biophys. Res. Commun. 1991; 181: 566-572Crossref PubMed Scopus (14) Google Scholar, 29Van Epps-Fung M. Gupta K. Hardy R.W. Wells A. Endocrinology. 1997; 138: 5170-5175Crossref PubMed Scopus (37) Google Scholar, 30Kayali A.G. Eichhorn J. Haruta T. Morris A.J. Nelson J.G. Vollenweider P. Olefsky J.M. Webster N.J.G. J. Biol. Chem. 1998; 273: 13808-13818Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Regardless of the underlying mechanisms, the insensitivity of PLC stimulation by insulin to prior GPCR activation indicates that the sensitization of PLC stimulation by EGF and PDGF is not an unspecific response to any PLC stimulatory receptor. The experimental paradigm used in the present study to demonstrate GPCR-induced sensitization of PLC stimulation by EGF and PDGF (i.e. first treatment of HEK-293 cells for a short period (2 min) with a GPCR agonist and then washout of this agonist and subsequent incubation of the cells for 40 min or longer without any agonist before actual measurement of PLC activity) is based on previous data on up-regulation of PLC stimulation by GPCRs. These studies on M2 and M3 mAChR-induced sensitization of PLC stimulation demonstrated that maximal up-regulation of PLC stimulation by these mAChRs is observed at about 40 min after washout of the initial stimulus and slowly disappears thereafter (8Schmidt M. Nehls C. Rümenapp U. Jakobs K.H. Mol. Pharmacol. 1996; 50: 1038-1046PubMed Google Scholar, 9Schmidt M. Lohmann B. Hammer K. Haupenthal S. Voß M. Nehls C. Jakobs K.H. Mol. Pharmacol. 1998; 53: 1139-1148PubMed Google Scholar). 2M. Schmidt, M. Frings, M.-L. Mono, Y. Guo, P. A. Oude Weernink, S. Evellin, L. Han, and K. H. Jakobs, unpublished data. Using these experimental conditions, it is demonstrated that sensitization of PLC stimulation by EGF and PDGF caused by prior M2 mAChR activation is also a long lasting process, with a maximum at 40 min after the initial treatment with carbachol and a slow decline thereafter, reaching control values at ∼150 min. Not only the time courses but also the mechanisms involved in the GPCR-induced sensitization of PLC stimulation by GPCRs and RTKs are apparently very similar. Specifically, it is shown that the M2 mAChR-induced up-regulation of PLC stimulation by either carbachol or EGF is completely abrogated by PTX treatment of the cells and largely reduced by expression of the two Gβγ scavengers, βARK-CT and Gαt. In addition, inhibition of conventional PKC enzymes with Gö 6976 and chelation of intracellular Ca2+ with BAPTA/AM fully blocked the M2 mAChR-induced up-regulation of PLC stimulation by either carbachol or EGF. None of these treatments had an effect on PLC stimulation by carbachol or EGF in naive cells. Furthermore, as demonstrated for the M2 mAChR, the sensitization of PLC stimulation caused by prior GPCR activation was apparently not due to a corresponding up-regulation of cell surface receptor number. Thus, the sensitizing GPCRs apparently generate two distinct signals mediating the long lasting sensitization process of PLC stimulation. One signal is apparently caused by activation of PLC, which is PTX-insensitive in HEK-293 cells, thus most likely mediated by Gq type G proteins, and finally results in Ca2+ mobilization and PKC activation. The results obtained with Gö 6976 and BAPTA/AM prompted us to investigate which of the conventional Ca2+-dependent PKC isoenzymes known to be inhibited by Gö 6976, PKC-α or PKC-βI (24Martiny-Baron G. Kazanietz M.G. Mischak H. Blumberg P.M. Hug H. Marmé D. Schächtele C. J. Biol. Chem. 1993; 268: 9194-9197Abstract Full Text PDF PubMed Google Scholar), mediates the up-regulation of PLC stimulation. It is shown that overexpression of PKC-α, which had no effect on PLC stimulation in naive cells, largely enhanced the M2 mAChR-induced sensitization of PLC stimulation by carbachol and EGF, whereas overexpression of PKC-βI was without any effect. Similar negative results were obtained in cells overexpressing PKC-βII, PKC-ε, or PKC-ζ (data not shown). Thus, one major signal involved in and mediating the GPCR-induced sensitization of PLC stimulation is apparently the activation of a conventional Ca2+-dependent PKC isoenzyme, most likely PKC-α. It remains to be studied whether Ca2+ acts solely by activating the PKC enzyme or whether additional Ca2+-dependent steps are involved in the PLC sensitization process. The second signal generated by the sensitizing GPCR is apparently dependent on Gβγ dimers derived from receptor-activated Gi type G proteins (31Offermanns S. Wieland T. Homann D. Sandmann J. Bombien E. Spicher K. Schultz G. Jakobs K.H. Mol. Pharmacol. 1994; 45: 890-898PubMed Google Scholar). During the last few years, various direct and indirect effectors of Gβγ dimers have been identified (22Clapham D.E. Neer E. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 167-203Crossref PubMed Scopus (699) Google Scholar, 23Gautam N. Downes G.B. Yan K. Kisselev O. Cell. Signal. 1998; 7: 447-455Crossref Scopus (156) Google Scholar). Since the M2 mAChR-induced PLC stimulation in naive cells was affected neither by PTX nor by expression of Gβγ scavengers, it is highly unlikely that a PLC-β isoenzyme known to be controlled by Gβγs (1Exton J.H. Annu. Rev. Pharmacol. Toxicol. 1997; 36: 481-509Crossref Scopus (299) Google Scholar, 2Rhee S.G. Bae Y.S. J. Biol. Chem. 1997; 272: 15045-15048Abstract Full Text Full Text PDF PubMed Scopus (809) Google Scholar) is the relevant Gβγ effector. Thus, overall, the GPCR-induced sensitization of PLC stimulation by GPCRs and RTKs apparently requires the two PLC-derived signals (i.e. increase in intracellular Ca2+concentration and activation of a conventional PKC isoenzyme) and an as yet unidentified Gβγ effector, which then in combination induce a long lasting cellular memory for receptor-mediated PLC stimulation. During the last years, various GPCRs have been reported to cause "transactivation" of RTKs, specifically of the EGF and PDGF receptors, in different cellular systems (for a review, see Ref. 32Zwick E. Hackel P.O. Prenzel N. Ullrich A. Trends Pharmacol. Sci. 1999; 20: 408-412Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar). These studies demonstrated that tyrosine phosphorylation of the EGF or PDGF receptor is an essential intermediate step particularly for mitogenic signaling by these GPCRs. The results presented in this report demonstrating GPCR-induced sensitization of PLC stimulation by EGF and PDGF receptors may also be termed "transactivation," however with a completely different meaning. First, the experimental paradigm used to demonstrate GPCR-induced sensitization of PLC stimulation by RTKs is quite distinct from that used in the above mentioned "transactivation" studies, in which acute GPCR-induced cellular responses were shown to involve activation of a RTK. Second, in contrast to the GPCR-induced "transactivation" of EGF or PDGF receptors, which was independent of exogenous RTK agonists (32Zwick E. Hackel P.O. Prenzel N. Ullrich A. Trends Pharmacol. Sci. 1999; 20: 408-412Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar), the GPCR-induced PLC sensitization was only observed upon the addition of exogenous RTK (or GPCR) ligands, whereas agonist-independent basal PLC activity was not altered in GPCR-pretreated cells. In conclusion, the data presented in this report demonstrate that short term activation of various GPCRs expressed in HEK-293 cells can induce a strong and long lasting up-regulation and sensitization of PLC stimulation by EGF and PDGF receptors, two prototypical RTKs. This novel cellular response apparently involves the complex interplay of at least two distinct signaling pathways induced by the GPCRs. The up-regulation and sensitization of PLC stimulation by RTKs described herein most likely has a major impact on physiological and possibly also pathological cellular responses triggered by these growth factor receptors. We thank K. Rehder, M. Hagedorn, and H. Geldermann for expert technical assistance and Drs. R. J. Lefkowitz, M. Kellerer, H. Mischak, and T. Wieland for providing various DNA constructs.
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