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

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.

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 4,5bisphosphate 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 muscle contraction, secretion, neuronal signaling, and cell growth and differentiation, to name but a few (3)(4)(5)(6). 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 M 2 or M 3 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 (7)(8)(9). Studies with pertussis toxin (PTX) and PKC inhibitors, furthermore, suggested that this potentiation of PLC stimulation by GPCRs is mediated by G i type G proteins and involves activation of a PKC isoenzyme (8,9). 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 (10,11). 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 G i -derived G␤␥ dimers and that increases in cytosolic Ca 2ϩ and activation of a conventional PKC enzyme, most likely PKC-␣, are required for this novel PLC regulatory mechanism. from NEN Life Science Products. Unlabeled D-myo-InsP 3 , PDGF-BB, and EGF were from Biomol; insulin (I-2767; human recombinant expressed in Escherichia coli, sodium salt, crystalline), LPA, and cycloheximide were from Sigma; and Gö 6976 and BAPTA/AM were from Calbiochem. The antibodies, MC5, which recognizes PKC-␣, -␤, and -␥ isoforms, and C-20, which recognizes all G␣ i isoforms including G␣ t , were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). All other materials were from previously described sources (7)(8)(9).
Cell Culture and Transfection-DNAs encoding PKC-␣ and PKC-␤I both subcloned into pRK5 were kindly provided by Drs. M. Kellerer and H. Mischak. DNA encoding the carboxyl terminus of the ␤-adrenergic receptor kinase (␤-ARK-CT) subcloned into pRK5 (12) was donated by Dr. R. J. Lefkowitz. DNA encoding G␣ t subcloned into pCIS was donated by Dr. T. Wieland. Wild-type HEK-293 cells and HEK-293 cells stably expressing the M 2 or M 3 mAChR (13) were cultured as reported before (9,14). Transfection of cells grown to near confluence on 145-mm culture dishes with the indicated DNAs or the corresponding empty vectors was performed with the calcium phosphate method with a transfection efficiency of 50 -80% (15). Expression of the encoded proteins was checked by immunoblotting of cell lysates with specific antibodies. Assays of PLC activity were performed 48 h after transfection.
Agonist Pretreatment and Measurement of Inositol Phosphate Formation-Cellular phospholipids were labeled by incubating cells for 36 h with myo-[ 3 H]inositol (0.5 Ci/ml) in serum-free medium. Where indicated, the cells were incubated during the last 16 h of the labeling period with 100 ng/ml PTX. Afterward, the labeling medium was removed, and the adherent cells were equilibrated for 10 min at 37°C in Hanks' balanced salt solution, containing 118 mM NaCl, 5 mM KCl, 1 mM CaCl 2 , 1 mM MgCl 2 , and 5 mM D-glucose, buffered at pH 7.4 with 15 mM HEPES. Thereafter, the cells were incubated for 2 min at 37°C in Hanks' balanced salt solution with and without the indicated receptor agonist in the absence of LiCl, followed by thorough washout of the agonist and further incubation of the cells for 30 min or the indicated periods of time without agonist as reported before (8,9). Then the adherent cells were incubated for 10 min at 37°C with 10 mM LiCl in Hanks' balanced salt solution, immediately followed by the addition of stimulatory agents in the presence of 10 mM LiCl to measure the formation of total [ 3 H]inositol phosphates (usually for 30 min at 37°C) as described before (16). To study the effects of cycloheximide, Gö 6976, and BAPTA/AM on PLC stimulation, the cells were pretreated for 60 min (cycloheximide) or 30 min (Gö 6976, BAPTA/AM) with these agents or their solvent, dimethyl sulfoxide (0.1 or 0.2%). These agents were also present during agonist pretreatment, subsequent incubation without agonist, and final PLC assays.
InsP 3 Mass Determination-Unlabeled HEK-293 cells serum-starved for 36 h were treated for 2 min with and without receptor agonist, followed by agonist washout and, 30 min later, treatment for 10 min with 10 mM LiCl as described above. Then the adherent cells were incubated for 15 s at 37°C with and without EGF or PDGF. InsP 3 mass was determined by a radioreceptor assay as described before (9,17,18).
Immunoblot Analysis-For detection of PKC-␣, PKC-␤I, and G␣ t , equal amounts of protein from cell lysates were separated by SDSpolyacrylamide gel electrophoresis on 10% acrylamide gels. After a transfer to nitrocellulose membranes and a 1 h incubation with the antibodies, MC5 (dilution 1:400) and C-20 (dilution 1:1000), the proteins were visualized by enhanced chemiluminescence.
Data Presentation-Data shown in the figures are mean Ϯ S.D. from one representative experiment performed in triplicate and repeated as indicated. Results mentioned in the text (see "Results") are mean Ϯ S.E., with n providing the number of independent experiments. Comparisons between means were either with Student's paired t test or a one-way analysis of variance test, with the significance level set at p Ͻ 0.05. Concentration-response curves were analyzed by fitting sigmoidal functions (iterative nonlinear regression analysis) to the experimental data with the GraphPadPrism program (version 2.0).

RESULTS
Basal Characteristics of RTK-induced PLC Stimulation in HEK-293 Cells-Activation of RTKs for EGF, PDGF, and insulin endogenously expressed in HEK-293 cells (10, 11) resulted in rapid and concentration-dependent accumulation of [ 3 H]inositol phosphates. As illustrated in Fig. 1 for M 2 mAChRexpressing HEK-293 cells, at maximally effective concentrations, EGF (50 ng/ml), PDGF (20 ng/ml), and insulin (10 g/ml) increased [ 3 H]inositol phosphate production determined 30 min after agonist addition by 2-3-fold above basal level. The formation of [ 3 H]inositol phosphates induced by the three RTK agonists was rather linear with time for up to 30 min of incubation. PLC stimulation by EGF and PDGF, which was also monitored as rapid InsP 3 accumulation (see Fig. 3), was specifically inhibited by the EGF receptor-specific tyrphostin AG 1478 (1 M) and the PDGF receptor-specific tyrphostin AG 1296 (10 M) (20,21), respectively, without altering PLC stimulation by other RTK agonists (data not shown). Treatment of the cells with PTX (100 ng/ml, 16 h) did not affect PLC stimulation by any of the three RTK agonists (data not shown; see Fig. 8).
GPCR-induced Sensitization of PLC Stimulation by RTKs-To study whether GPCRs can induce sensitization of PLC stimulation by RTKs, M 2 mAChR-expressing HEK-293 cells were first treated for 2 min with the mAChR agonist, carbachol (1 mM), followed by agonist washout, a further 40-min incubation without any agonist, and then measurement of basal and agonist-stimulated accumulation of [ 3 H]inositol phosphates. As reported before (9), basal [ 3 H]inositol phosphate accumulation was not altered in carbachol-pretreated compared with control cells, while [ 3 H]inositol phosphate formation induced by restimulation of the cells with 1 mM carbachol was significantly increased by about 60% (n ϭ 4, p Ͻ 0.01). As illustrated in Fig.  2, prestimulation of the cells with carbachol also markedly enhanced PLC stimulation by EGF and PDGF. At 40 min after the 2-min treatment with carbachol, [ 3 H]inositol phosphate production induced by EGF (50 ng/ml) was enhanced 2-fold, from 2.95 Ϯ 0.45 to 5.9 Ϯ 0.51 ϫ 10 3 cpm/mg of protein (n ϭ 4, p Ͻ 0.01) ( Fig. 2A). Similarly, [ 3 H]inositol phosphate formation induced by PDGF (20 ng/ml) was increased from 3.05 Ϯ 0.23 ϫ 10 3 cpm/mg of protein in untreated control cells to 5.85 Ϯ 0.25 ϫ 10 3 cpm/mg of protein in carbachol-pretreated cells (n ϭ 4, p Ͻ 0.01) (Fig. 2B). Under the same conditions, PLC stimulation by insulin was not altered in carbachol-pretreated compared with control cells (Fig. 2C). The up-regulation of agonistinduced [ 3 H]inositol phosphate formation was fully blocked by the mAChR antagonist, atropine (10 M), added during pretreatment of the cells with 1 mM carbachol (data not shown). Similar to the enhancement of EGF-and PDGF-stimulated [ 3 H]inositol phosphate formation, pretreatment of the cells with carbachol also markedly increased rapid formation of InsP 3 by the RTK agonists. As shown in Fig. 3, stimulation of Under the conditions studied, the M 2 mAChR-induced upregulation of PLC stimulation by EGF and PDGF was maximal at 40 min after carbachol removal, the earliest time point examined, and declined thereafter (Fig. 4). Even at 85 min after carbachol removal, EGF-and PDGF-induced [ 3 H]inositol phosphate formation was significantly (p Ͻ 0.05) enhanced compared with untreated control cells, while at 145 min, control responses were again obtained. Thus, short term M 2 mAChR activation of HEK-293 cells caused a long lasting upregulation of PLC stimulation by the RTK agonists, EGF and PDGF. The up-regulation of PLC stimulation induced by carbachol pretreatment was apparently not dependent on the synthesis of a protein causing this long lasting effect. As studied for EGF-stimulated PLC activity, pretreatment of HEK-293 cells for 1 h with 350 M cycloheximide decreased [ 3 H]inositol phosphate formation stimulated by EGF (50 ng/ml) in control cells from 2.58 Ϯ 0.16 to 1.83 Ϯ 0.05 ϫ 10 3 cpm/mg of protein (n ϭ 3). However, the up-regulation of EGF-stimulated PLC activity induced by a 2-min pretreatment of the cells with 1 mM carbachol was not altered by prior cycloheximide treatment. In cells pretreated with carbachol, EGF increased [ 3 H]inositol phosphate formation in control and cycloheximide-pretreated cells by 4.13 Ϯ 0.07 and 3.38 Ϯ 0.08 ϫ 10 3 cpm/mg of protein, respectively (n ϭ 3) (data not shown).
EGF-and PDGF-induced PLC stimulation in M 2 mAChRexpressing HEK-293 cells was also up-regulated by activation of the endogenously expressed LPA receptor. Similar to carbachol, pretreatment of the cells for 2 min with 10 M LPA, followed by agonist washout and measurement of [ 3 H]inositol phosphate formation 40 min later, increased PLC stimulation by EGF and PDGF, without altering basal [ 3 H]inositol phosphate accumulation. Maximal EGF-induced [ 3 H]inositol phosphate formation was increased by 55% (n ϭ 5, p Ͻ 0.01) (Fig.  5A), and that induced by PDGF was increased by 62% (n ϭ 3, p Ͻ 0.01) (Fig. 5B). The up-regulation of PLC stimulation by EGF and PDGF caused by pretreatment of the cells with LPA was even more evident at low concentrations of the RTK agonists. For example, 7.5 ng/ml PDGF only slightly increased [ 3 H]inositol phosphate formation in control cells, whereas in LPA-pretreated cells PDGF at the same concentration caused a strong increase in [ 3 H]inositol phosphate formation, reaching the same level as the maximal PDGF-induced stimulation in control cells (Fig. 5B).
Sensitization of RTK-induced PLC stimulation caused by short term activation of GPCRs was also observed in wild-type cells and HEK-293 cells overexpressing the M 3 mAChR. As shown in Fig. 6, treatment of either cell type for 2 min with 10 M LPA, followed by washout of LPA and measurement of [ 3 H]inositol phosphate formation 40 min later, strongly increased EGF-induced PLC stimulation. In wild-type cells, maximal EGF-induced [ 3 H]inositol phosphate formation was increased by 157% (n ϭ 3, p Ͻ 0.01) (Fig. 6A), and that induced by EGF in M 3 mAChR-expressing cells was increased by prior treatment with LPA by 134% (n ϭ 4, p Ͻ 0.01) (Fig. 6B). Furthermore, pretreatment of M 3 mAChR-expressing HEK-293 cells for 2 min with 1 mM carbachol increased PLC stimulation by EGF (50 ng/ml) measured 40 min later by 90% (n ϭ 3, p Ͻ 0.01) (data not shown). Finally, as demonstrated in Fig. 7, short term (2 min) activation of the endogenously expressed purinergic receptor with ATP (1 mM), followed by washout of ATP and measurement of [ 3 H]inositol phosphate formation 70 min later, not only enhanced PLC stimulation by carbachol (1 mM) in M 2 mAChR-expressing cells (by 60%, n ϭ 4, p Ͻ 0.01) as reported before (9) but also largely increased EGF-stimulated PLC activity in these as well as in wild-type HEK-293 cells. Maximal EGF-induced [ 3 H]inositol phosphate formation was increased in M 2 mAChR-expressing cells by 88% (n ϭ 4, p Ͻ 0.01) (Fig. 7A), and that induced by EGF in wild-type cells was increased by prior treatment with ATP by 182% (n ϭ 3, p Ͻ 0.01) (Fig. 7B). Thus, short term activation of various endogenously expressed or overexpressed GPCRs in HEK-293 cells strongly increased maximal PLC stimulation by EGF and PDGF as well as the sensitivity to the RTK agonists.
Since prior GPCR activation increased subsequent PLC stimulation by both RTKs (for EGF and PDGF) and GPCRs Participation of G i -derived G␤␥s in M 2 mAChR-induced Sensitization of PLC Stimulation-In the following studies on the mechanisms of GPCR-induced sensitization of PLC stimulation, we analyzed and compared PLC stimulation by carbachol (1 mM) and EGF (50 ng/ml) in M 2 mAChR-expressing HEK-293 cells pretreated or not for 2 min with 1 mM carbachol. First, we examined whether G i proteins are involved in M 2 mAChRinduced sensitization of PLC stimulation by EGF, as reported before for M 2 and M 3 mAChR-induced sensitization of PLC stimulation by GPCRs (8,9). Pretreatment of the cells for 16 h with PTX (100 ng/ml) did not alter [ 3 H]inositol phosphate formation induced by carbachol or EGF in control cells (Fig. 8). However, in cells pretreated with PTX, the up-regulation of PLC stimulation by carbachol and EGF caused by prior carbachol treatment was completely prevented. Since the M 2 mAChR inhibits adenylyl cyclase via PTX-sensitive G i proteins and decreases cAMP levels in these cells (13), we examined whether the M 2 mAChR-induced up-regulation of PLC stimulation may be caused by a fall in cAMP levels. However, treatment of the cells for 30 min with the membrane-permeable cAMP analog, dibutyryl cAMP (1 mM), neither altered PLC stimulation by carbachol and EGF in control cells nor affected the up-regulation of PLC stimulation caused by prior carbachol treatment (data not shown).
Receptor-activated G proteins transmit the signal to effectors either by the GTP-liganded ␣ subunits or by the released free G␤␥ dimers (22,23). To study whether G␤␥ dimers mediate the PTX-sensitive up-regulation of PLC responses, we examined the effects of expression of the two G␤␥ scavengers, ␤-ARK-CT and G␣ t (12), on acute PLC stimulation and its sensitization caused by pretreatment of the cells with carbachol. Expression of ␤-ARK-CT or G␣ t did not alter basal PLC activity (data not shown) and PLC stimulation by carbachol and EGF in control untreated cells (Fig. 9). In contrast, the up-regulation of PLC stimulation by carbachol and EGF induced by pretreatment of the cells with carbachol was largely reduced by expression of ␤-ARK-CT or G␣ t . In ␤-ARK-CTexpressing HEK-293 cells, carbachol increased [ 3 H]inositol phosphate formation in control and carbachol-pretreated cells by 4.32 Ϯ 0.49 and 4.55 Ϯ 0.20 ϫ 10 3 cpm/mg of protein (n ϭ 4), respectively, and that induced by EGF amounted to 2.82 Ϯ 0.23 and 3.37 Ϯ 0.47 ϫ 10 3 cpm/mg of protein (n ϭ 4), respectively (Fig. 9A). In G␣ t -expressing cells, [ 3 H]inositol phosphate formation was increased by carbachol in control and carbacholpretreated cells by 6.00 Ϯ 0.50 and 6.37 Ϯ 0.42 ϫ 10 3 cpm/mg of protein (n ϭ 3), respectively, and that induced by EGF amounted to 2.51 Ϯ 0.51 and 2.76 Ϯ 0.32 ϫ 10 3 cpm/mg of protein (n ϭ 3), respectively (Fig. 9B).

Role of PKC and Ca 2ϩ in M 2 mAChR-induced Sensitization of PLC Stimulation-Pretreatment of HEK-293 cells for 30 min
with the PKC inhibitor, Gö 6976 (100 nM), did not affect PLC stimulation by either carbachol or EGF in control cells. However, the up-regulation of PLC stimulation by these two receptor agonists caused by prior carbachol treatment was fully prevented in cells pretreated with Gö 6976 (Fig. 10). Gö 6976 has been reported to preferentially inhibit the conventional Ca 2ϩ -dependent PKC isoenzymes, PKC-␣ and PKC-␤I (24). To study which of these two PKC isoenzymes is involved in M 2 mAChR-induced sensitization of PLC stimulation, we examined the effects of overexpression of PKC-␣ and PKC-␤I on PLC stimulation. Overexpression of either PKC isoenzyme had no effect on PLC stimulation by carbachol or EGF in naive cells (Fig. 11). However, in cells overexpressing PKC-␣, the increase in carbachol-and EGF-induced PLC stimulation caused by pretreatment of the cells with carbachol was strongly enhanced (Fig. 11A). In carbachol-pretreated cells, rechallenge with carbachol increased [ 3 H]inositol phosphate formation by 12.9 Ϯ 0.78 ϫ 10 3 cpm/mg of protein in control cells and by 16.2 Ϯ 0.93 ϫ 10 3 cpm/mg of protein in cells overexpressing PKC-␣ (n ϭ 4, p Ͻ 0.01). Similarly, in carbachol-pretreated cells, EGF-induced [ 3 H]inositol phosphate formation was enhanced from 4.85 Ϯ 0.62 ϫ 10 3 cpm/mg of protein in control cells to 7.52 Ϯ 0.49 ϫ 10 3 cpm/mg of protein in cells overexpressing PKC-␣ (n ϭ 4, p Ͻ 0.01). This enhancement of M 2 mAChRinduced sensitization of PLC stimulation was fully blocked by Gö 6976 (data not shown). In contrast to PKC-␣, overexpression of PKC-␤I did not alter the M 2 mAChR-induced sensitization of PLC stimulation (Fig. 11B).
Since the GPCRs that induced sensitization of PLC stimulation also markedly increase cytosolic Ca 2ϩ concentration in HEK-293 cells (16,25), we finally examined whether this increase is involved in sensitization of PLC stimulation. For this, the cells were treated before carbachol treatment with the intracellular Ca 2ϩ chelator, BAPTA/AM (20 M, 30 min), which completely prevented the agonist-induced increase in cytosolic Ca 2ϩ concentration (data not shown). As shown in Fig. 12, in cells pretreated with BAPTA/AM, PLC stimulation caused by carbachol or EGF in control cells was not altered. However, the BAPTA/AM treatment completely abolished the carbachol-induced up-regulation of PLC stimulation caused by either carbachol or EGF. DISCUSSION We reported before that short term activation of GPCRs in HEK-293 cells stably expressing the M 2 or M 3 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 (7)(8)(9). Since GPCRs and RTKs activate distinct PLC isoenzymes, GPCRs mainly PLC-␤ enzymes and RTKs PLC-␥ enzymes, particularly the widely expressed PLC-␥1 (1, 2), 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 M 2 or M 3 mAChR subtypes and endogenously expressing various other GPCRs as well as RTKs for EGF, PDGF, and insulin (8 -11). We report here that short term activation of the overexpressed M 2 and M 3 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 G␤␥ dimers liberated from PTX-sensitive G i type G proteins and requires increases in cytosolic Ca 2ϩ concentration and activation of a conventional PKC enzyme, most likely PKC-␣. Finally, extensive comparison of PLC stimulation by the M 2 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 InsP 3 , 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 M 2 mAChR. The reason for this discrepancy is presently not clear. While stimulation of PLC activity, particularly of the PLC-␥1 enzyme, is a well established and rather general response to EGF and PDGF receptor activation, it is not so for insulin (1,2,26,27), although stimulation of PLC activity by insulin has been described in some cell types, and PLC-␥ has recently been reported to participate in metabolic signaling by the insulin receptor in adipocytes (28 -30). 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 M 2 and M 3 mAChRinduced 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 (8,9). 2 Using these experimental conditions, it is demonstrated that sensitization of PLC stimulation by EGF and PDGF caused by prior M 2 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 ϳ150 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 M 2 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 G␤␥ scavengers, ␤ARK-CT and G␣ t . In addition, inhibition of conventional PKC enzymes with Gö 6976 and chelation of intracellular Ca 2ϩ with BAPTA/AM fully blocked the M 2 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 M 2 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 G q type G proteins, and finally results in Ca 2ϩ mobilization and PKC activation. The results obtained with Gö 6976 and BAPTA/AM prompted us to investigate which of the conventional Ca 2ϩ -dependent PKC isoenzymes known to be inhibited by Gö 6976, PKC-␣ or PKC-␤I (24), mediates the up-regulation of PLC stimulation. It is shown that overexpression of PKC-␣, which had no effect on PLC stimulation in naive cells, largely enhanced the M 2 mAChR-induced sensitization of PLC stimulation by carbachol and EGF, whereas overexpression of PKC-␤I was without any effect. Similar negative results were obtained in cells overexpressing PKC-␤II, PKC-⑀, or PKC-(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 Ca 2ϩ -dependent PKC isoenzyme, most likely PKC-␣. It remains to be studied whether Ca 2ϩ acts solely by activating the PKC enzyme or whether additional Ca 2ϩ -dependent steps are involved in the PLC sensitization process. The second signal generated by the sensitizing GPCR is apparently dependent on G␤␥ dimers derived from receptor-activated G i type G proteins (31). During the last few years, various direct and indirect effectors of G␤␥ dimers have been identified (22,23). Since the M 2 mAChR-induced PLC stimulation in naive cells was affected neither by PTX nor by expression of G␤␥ scavengers, it is highly unlikely that a PLC-␤ isoenzyme known to be controlled by G␤␥s (1, 2) is the relevant G␤␥ 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 Ca 2ϩ concentration and activation of a conventional PKC isoenzyme) and an as yet unidentified G␤␥ 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. 32). 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 GPCRinduced 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 (32), 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.