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Originally published In Press as doi:10.1074/jbc.M112024200 on March 4, 2002

J. Biol. Chem., Vol. 277, Issue 19, 16805-16813, May 10, 2002
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Stimulation of Phospholipase C-epsilon by the M3 Muscarinic Acetylcholine Receptor Mediated by Cyclic AMP and the GTPase Rap2B*

Sandrine EvellinDagger , Jan NolteDagger , Karina TysackDagger , Frank vom DorpDagger , Markus ThielDagger , Paschal A. Oude WeerninkDagger , Karl H. JakobsDagger , Edwin J. Webb§, Jon W. Lomasney§, and Martina SchmidtDagger

From the Dagger  Institut für Pharmakologie, Universitätsklinikum Essen, D-45122 Essen, Germany and the § Departments of Pathology and Pharmacology, Northwestern University Medical School, Chicago, Illinois 60611-3008

Received for publication, December 17, 2001

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Stimulation of phospholipase C (PLC) by Gq-coupled receptors such as the M3 muscarinic acetylcholine receptor (mAChR) is caused by direct activation of PLC-beta enzymes by Galpha q proteins. We have recently shown that Gs-coupled receptors can stimulate PLC-epsilon , apparently via formation of cyclic AMP and activation of the Ras-related GTPase Rap2B. Here we report that PLC stimulation by the M3 mAChR expressed in HEK-293 cells also involves, in part, similar mechanisms. M3 mAChR-mediated PLC stimulation and [Ca2+]i increase were reduced by 2',5'-dideoxyadenosine (dd-Ado), a direct adenylyl cyclase inhibitor. On the other hand, overexpression of Galpha s or Epac1, a cyclic AMP-regulated guanine nucleotide exchange factor for Rap GTPases, enhanced M3 mAChR-mediated PLC stimulation. Inactivation of Ras-related GTPases with clostridial toxins suppressed the M3 mAChR responses. The inhibitory toxin effects were mimicked by expression of inactive Rap2B, but not of other inactive GTPases (Rac1, Ras, RalA, Rap1A, and Rap2A). Activation of the M3 mAChR induced GTP loading of Rap2B, an effect strongly enhanced by overexpression of Galpha s and inhibited by dd-Ado. Overexpression of PLC-epsilon and PLC-beta 1, but not PLC-gamma 1 or PLC-delta 1, enhanced M3 mAChR-mediated PLC stimulation and [Ca2+]i increase. In contrast, expression of a catalytically inactive PLC-epsilon mutant reduced PLC stimulation by the M3 mAChR and abrogated the potentiating effect of Galpha s. In conclusion, our findings suggest that PLC stimulation by the M3 mAChR is a composite action of PLC-beta 1 stimulation by Galpha q and stimulation of PLC-epsilon apparently mediated by Gs-dependent cyclic AMP formation and subsequent activation of Rap2B.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Stimulation of phosphatidylinositol 4,5-bisphosphate (PIP2)1-hydrolyzing phospholipase C (PLC) is a major signal transduction system used by a wide variety of membrane receptors and apparently regulates various cellular functions, such as smooth muscle contraction, secretion, neuronal signaling, and cell growth and differentiation (1, 2). The eleven PLC isoforms (PLC-beta 1-4, PLC-gamma 1-2, PLC-delta 1-4, and PLC-epsilon ) identified so far differ largely in their structure and regulatory mechanisms. Receptor regulation of PLC-beta and PLC-gamma isozymes is well established. While tyrosine kinase receptors, such as those for epidermal growth factor and platelet-derived growth factor, activate PLC-gamma enzymes by recruitment to the autophosphorylated receptor and subsequent tyrosine phosphorylation, G protein-coupled receptors (GPCRs) activate PLC-beta enzymes, either via GTP-liganded alpha  subunits of the Gq class of G proteins or by beta gamma dimers liberated from Gi type G proteins (3, 4). Regulation of PLC-delta has been assumed to involve the G protein Gh (transglutaminase II) and/or capacitative Ca2+ influx (5, 6). The very recently identified PLC-epsilon contains, in addition to the PLC-defining catalytic and calcium-binding domains, two Ras-binding domains and a Ras-specific guanine nucleotide exchange factor (GEF) domain. Initial data suggest that the activity of PLC-epsilon is controlled by Galpha 12 and Ras and Rap GTPases, by yet unresolved mechanisms; thus, PLC-epsilon may link signaling by heterotrimeric G proteins and Ras-related GTPases (7-10).

The M3 muscarinic acetylcholine receptor (mAChR) is a prototypical GPCR known to stimulate PLC via pertussis toxin (PTX)-insensitive Galpha q type G proteins (11-14). We very recently observed that Gs-coupled receptors, i.e. the beta 2-adrenoreceptor expressed in HEK-293 cells and the prostaglandin E1 receptor endogenously expressed in N1E-115 neuroblastoma cells, can induce PLC stimulation and PLC-dependent calcium signaling (15). These receptor responses were apparently dependent on Gs-mediated cyclic AMP formation and under control of the Ras-related GTPase Rap2B. As activation of the M3 mAChR expressed in HEK-293 can increase cyclic AMP levels (11), we examined whether M3 mAChR signaling to PLC may involve cyclic AMP and Rap GTPases as well. We report here that the M3 mAChR can stimulate PLC-epsilon and that this PLC stimulation is apparently mediated by Gs-dependent formation of cyclic AMP and subsequent activation of the GTPase Rap2B.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- myo-[3H]Inositol (10-25 Ci/mmol) and D-myo-[3H]inositol 1,4,5-trisphosphate ([3H]IP3; 21 Ci/mmol) were from PerkinElmer Life Sciences. Unlabeled IP3 was from Biomol, 2'5'-dideoxyadenosine (dd-Ado) was from Calbiochem-Novabiochem, and Fura-2/AM was from Molecular Probes. The antibodies against Rac1, Rap1, Rap2, PLC-beta 1, PLC-gamma 1, and PLC-delta 1 were from Santa Cruz. The antibodies against RalA and Ras were from Transduction Laboratories. The antibody against HA-tagged proteins (12CA5) was a kind gift of Dr. J. L. Bos. The polyclonal rabbit anti-PLC-epsilon antibody raised against the unique N-terminal 600 amino acids of PLC-epsilon did not recognize other PLC isoforms (not shown). Clostridium difficile toxin B-1470 and Clostridium sordellii lethal toxin (strains 82 and 1522, respectively) were kind gifts of Dr. C. von Eichel-Streiber.

Expression Plasmids and Transfection-- cDNAs encoding the inactive GTPase mutants of Rac1 (T17N Rac1; subcloned into pEXV), RalA (G26A RalA), Rap1A (S17N Rap1A), Rap2A (S17N Rap2A), and Rap2B (S17N Rap2B; each subcloned into pRK5) were kindly provided by Drs. A. Hall, J. H. Camonis, and J. de Gunzburg. cDNAs encoding the inactive GTPase mutant of Ras (S17N Ras; subcloned into pRSV) and wild-type HA-tagged Epac1 (subcloned into pMT2) were kindly provided by Drs. J. L. Bos and J. de Rooij. cDNAs encoding the constitutively active mutant of Galpha 12 (Q229L Galpha 12, subcloned into pCis), FLAG-tagged RGS4 (subcloned into pCMV), and wild-type HA-tagged Galpha s (subcloned into pcDNA3) were kindly provided by Drs. T. Wieland and C. Kleuss. cDNAs encoding PLC-beta 1 and PLC-gamma 1 (both subcloned into pRK5) were kindly provided by Drs. D. Illenberger and A. Ullrich. PLC-delta 1, wild-type PLC-epsilon , and catalytically inactive PLC-epsilon (H1144L PLC-epsilon ) were each subcloned into pcDNA3. HEK-293 cells stably expressing the M3 mAChR at high density (11) were cultured as reported previously (14). Transfection of cells grown to near confluence on 145-mm culture dishes with the indicated amounts of either plasmid DNA or the corresponding empty vectors was performed with the calcium phosphate method, reaching a transfection efficiency of 50-60% (14). Expression of the encoded proteins was verified by the immunoblotting of cell lysates with specific antibodies. Assays were performed 48 h after transfection.

Measurement of PLC Activity-- For measurement of inositol phosphate formation, cellular phospholipids were labeled by incubating cells for 24 h with myo-[3H]inositol (0.5 µCi/ml) in growth medium. Thereafter, the adherent cells were first treated for 10 min at 37 °C in Hanks' balanced salt solution, containing 118 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, and 5 mM D-glucose, buffered at pH 7.4 with 15 mM HEPES, plus 10 mM LiCl, followed then by further incubation for 30 min at 37 °C in the presence of stimulatory agents and determination of [3H]inositol phosphate accumulation. For measurement of IP3 formation, unlabeled cells were incubated for the indicated periods of time at 37 °C with and without carbachol. Stop of the PLC assays, extraction, and analysis of [3H]inositol phosphates or IP3 mass were performed as described previously (16). To study the effects of dd-Ado, the cells were pretreated for 30 min with the agent or its solvent, dimethyl sulfoxide (0.1%); dd-Ado was also present during the PLC assays. To study the effects of clostridial toxins, the cells were treated for 24 h without and with the toxins at the indicated concentrations, followed by PLC activity assays.

Calcium Measurements-- Intracellular free Ca2+ concentration ([Ca2+]i) was determined in cell suspensions with the fluorescent Ca2+ indicator dye Fura-2 in a Hitachi spectrofluorometer as described previously (17).

Activation of Rap2B-- Cells were stimulated without and with carbachol for 5 min at 37 °C, followed by two washes with ice-cold phosphate-buffered saline and lysis in a buffer containing 10% glycerol, 1% Nonidet P-40, 50 mM Tris/HCl, pH 7.4, 200 mM NaCl, 2 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride, 2 µM leupeptin, 1 µM aprotinin, 0.1 µM trypsin inhibitor, 10 mM NaF, and 1 mM Na3VO4. After centrifugation, the supernatants were incubated with 15 µg of purified glutathione S-transferase-tagged RalGDS-RBD (Rap-binding domain of the Ral guanine nucleotide dissociation stimulator) bound to glutathione-Sepharose beads for 1 h at 4 °C. Then, the beads were washed three times with lysis buffer and finally incubated in Laemmli buffer for 10 min at 95 °C. Bound Rap2-GTP was determined by immunoblotting with an anti-Rap2 antibody (15, 18). Densitometric analysis of the bands was performed with ImageQuant software (Molecular Dynamics).

Immunoblot Analysis-- For detection of Ras (dilution of 1:400), RalA (dilution 1:5000), Rac1, Rap1A, Rap2A, Rap2B, HA-tagged Epac1, HA-tagged Galpha s, PLC-beta 1, PLC-delta 1, PLC-gamma 1 (each at a dilution of 1:500), and PLC-epsilon (dilution 1:2500), equal amounts of protein from cell lysates were separated by SDS-polyacrylamide gel electrophoresis on 10 or 15% acrylamide gels. After a transfer to nitrocellulose membranes and a 1-h incubation with the antibodies at the above given dilution factors, the proteins were visualized by enhanced chemiluminescence.

Data Presentation-- Data shown in figures are means ± S.E. of n independent experiments, each performed in triplicate. Comparisons between means were either with the Student's paired t test or one-way analysis of variance test, and a difference was regarded significant at p < 0.05. Curves were analyzed by fitting iterative nonlinear regression analysis to the experimental data with the GraphPad Prism program (Version 2.0, 1995).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Role of Cyclic AMP in M3 mAChR-mediated PLC Stimulation-- We have recently reported that stimulation of cyclic AMP formation by the beta 2-adrenoreceptor expressed in HEK-293 cells or direct activation of adenylyl cyclase by forskolin results in PLC stimulation (15). To examine whether cyclic AMP and cyclic AMP-dependent processes are involved in PLC stimulation by the M3 mAChR as well, several approaches were used. First, we studied the effect of the P-site adenylyl cyclase inhibitor, dd-Ado (19), on PLC stimulation by the M3 mAChR stably expressed in HEK-293 cells. Treatment of the cells with dd-Ado (10 µM) had no effect on unstimulated inositol phosphate accumulation or IP3 levels. However, dd-Ado strongly reduced, by 30-40%, IP3 formation or inositol phosphate accumulation (see below) stimulated by the mAChR agonist, carbachol (Fig. 1A). In line with this inhibition, dd-Ado significantly (p < 0.0001) reduced the carbachol (1 µM)-induced increase in [Ca2+]i from 485 ± 25 nM to 246 ± 35 nM (n = 8-10). Second, we examined whether overexpression of Galpha s, the adenylyl cyclase-stimulatory G protein, alters PLC stimulation by the M3 mAChR. In cells overexpressing Galpha s, basal inositol phosphate formation was not altered. However, PLC stimulation induced by carbachol (1 µM) was increased by about 50%. This increase in PLC stimulation was almost fully suppressed by treatment of the cells with 10 µM dd-Ado (Fig. 1B). Together, these data suggested that PLC stimulation by the M3 mAChR is, at least in part, dependent on cyclic AMP formation.


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  		Fig. 1.   Effects of dd-Ado and Galpha s on M3 mAChR-mediated PLC and calcium signaling. A, M3 mAChR-expressing HEK-293 cells were treated for 30 min without (Control) and with 10 µM dd-Ado, followed by measurement of IP3 formation (1 min) in the absence and presence of 1 µM or 100 µM carbachol (left panel) or of carbachol (1 µM)-induced [Ca2+]i increase (right panel). Data in the left panel are means ± S.E. (n = 3), while in the right panel superimposed tracings of [Ca2+]i are shown. B, HEK-293 cells were transfected with empty vector (Vector, V) or Galpha s (100 µg of DNA) and labeled with myo-[3H]inositol. At 48 h after transfection, [3H]inositol phosphate formation without (Basal) and with 1 µM carbachol was determined directly (left panel) or after additional 30 min treatment without (Ctr) and with 10 µM dd-Ado (right panel). Inset, immunoblot detection of HA-tagged Galpha s. Data are means ± S.E. (n = 4-6).

As observed with beta 2-adrenoreceptor-induced PLC stimulation (15), treatment of HEK-293 cells with the protein kinase A inhibitor, H-89 (10 µM), had no significant effect on PLC stimulation induced by carbachol, neither in control cells nor in cells overexpressing Galpha s (data not shown), suggesting that the action of cyclic AMP is mediated by another effector. Therefore, we overexpressed Epac1, a cyclic AMP-activated GEF for Rap GTPases (18, 20). As illustrated in Fig. 2A, similar to Galpha s, overexpression of Epac1 had no effect on basal PLC activity, but strongly enhanced PLC stimulation induced by carbachol (1 µM). Overexpression of Epac1 also strongly enhanced the potency of carbachol to increase [Ca2+]i in HEK-293 cells. While carbachol increased [Ca2+]i in control cells with an EC50 value of 450 ± 14 nM, this value was reduced by about one order of magnitude, to 26 ± 2 nM, in cells overexpressing Epac1 (Fig. 2A). These data suggested that cyclic AMP/Epac1-controlled Rap GTPases are involved in PLC stimulation by the M3 mAChR.


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  		Fig. 2.   Effects of Epac1 and clostridial toxins on M3 mAChR-mediated PLC and calcium signaling. A, HEK-293 cells were transfected with empty vector (Control) or Epac1 (25 µg of DNA). At 48 h after transfection, [3H]inositol phosphate formation without (Basal) and with 1 µM carbachol (left panel) or [Ca2+]i increase induced by carbachol at the indicated concentrations was determined (right panel). Data are means ± S.E. (n = 6-8). B, HEK-293 cells were treated for 24 h without (Control) and with 300 pg/ml toxin B-1470 or 100 ng/ml lethal toxin, followed by measurement of IP3 formation (15 s) in the absence (Basal) and presence of 1 µM carbachol (left panel) or of carbachol (1 µM)-induced [Ca2+]i increase (right panel). Data in the left panel are means ± S.E. (n = 3), while in the right panel superimposed tracings of [Ca2+]i are shown.

Involvement of Rap2B in M3 mAChR-mediated PLC Stimulation-- To study whether and which type of Rap GTPases are involved in PLC stimulation by the M3 mAChR, we first examined the effects of C. difficile toxin B-1470 and C. sordellii lethal toxin, known to inactivate Ras-related GTPases (21, 22). Treatment of HEK-293 cells for 24 h with 300 pg/ml toxin B-1470 and 100 ng/ml lethal toxin strongly reduced inositol phosphate accumulation (data not shown) and IP3 formation induced by carbachol (Fig. 2B). PLC stimulation induced by the direct G protein activators, AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> (intact cells) and GTPgamma S (permeabilized cells), was also strongly reduced in cells treated with the toxins (data not shown). The reduction in M3 mAChR-mediated IP3 formation was paralleled by an attenuation of receptor-mediated [Ca2+]i increase. While carbachol (1 µM) increased [Ca2+]i by 485 ± 50 nM (n = 8) in control cells, this increase was significantly (p < 0.001) reduced to 305 ± 35 nM (n = 10) and 265 ± 25 nM (n = 6) in cells treated with toxin B-1470 and lethal toxin, respectively (Fig. 2B). Treatment of the cells with the toxins, however, did not alter the number of cell surface M3 mAChRs, determined by binding of the membrane-impermeant mAChR antagonist, N-[3H]methylscopolamine, in intact cells (16), and the carbachol-induced binding of [35S]guanosine 5'-O-(3-thiotriphosphate) to G proteins, measured in permeabilized cells (23). Furthermore, the toxins had no effect on the cellular PIP2 levels, measured as [3H]PIP2 or PIP2 mass in control and toxin-treated cells (24), and did not reduce Ca2+ (1 µM)-stimulated PLC activity, measured with exogenous PIP2 in cell lysates (17) (data not shown). Thus, inhibition of PLC and calcium signaling by the GTPase-inactivating toxins was apparently not due to a loss of cell curface receptors, a defective receptor-G protein coupling, a fall in PLC substrate levels, or a general reduction in PLC activities, suggesting that inhibition of M3 mAChR signaling to PLC by the toxins is caused by inactivation of small GTPases specifically involved in PLC stimulation.

To identify the specific GTPase, we expressed inactive mutants of the GTPases serving as toxin substrates. Compared with the expression of the endogenous GTPases, the various GTPase mutants were overexpressed to a comparable level (Fig. 3A). However, the GTPase mutants largely differed in their effects on PLC signaling. Expression of inactive Rac1, H-Ras, RalA, RalB (not shown), Rap1A, and Rap2A did neither change basal PLC activity nor PLC stimulation by the M3 mAChR. In contrast, in cells expressing S17N Rap2B PLC stimulation induced by carbachol was reduced by 40-50% (Fig. 3A). In line with their distinct effects on PLC stimulation, expression of S17N Rap2B, but not S17N Ras or S17N Rap1A (not shown), strongly (p < 0.0001) reduced the carbachol (1 µM)-induced [Ca2+]i increase, from 535 ± 55 nM in control cells to 310 ± 45 nM (n = 6-8) in cells expressing S17N Rap2B (Fig. 3B).


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  		Fig. 3.   Effects of various inactive GTPase mutants on M3 mAChR-induced PLC and calcium signaling. HEK-293 cells were transfected with empty vector (Control, V), T17N Rac1, S17N Ras, G26A RalA, S17N Rap1A, S17N Rap2A, or S17N Rap2B (100 µg of DNA each). At 48 h after transfection, [3H]inositol phosphate formation without (Basal) and with 1 µM carbachol (A) or [Ca2+]i increase induced by 1 µM carbachol was determined (B). Insets, immunoblot detection of the GTPases in lysates of transfected cells. Data in A are means ± S.E. (n = 5), while in B superimposed tracings of [Ca2+]i are shown.

Next, we studied whether Rap2B is activated by the M3 mAChR and whether this activation is affected by agents inhibiting or enhancing receptor-mediated PLC stimulation. Carbachol (1 µM) treatment of HEK-293 cells enhanced GTP loading of endogenous and overexpressed Rap2B, as determined by extraction of the GTPase from cell lysates with immobilized RalGDS-RBD (Fig. 4). Expression of constitutively active Galpha 12 (Q229L Galpha 12) or overexpression of wild-type Galpha i2 (not shown), both of which did not alter carbachol-induced PLC stimulation (14, not shown), had no effect on M3 mAChR-induced Rap2B activation. In contrast, overexpression of Galpha s or Epac1 (not shown), which by themselves did not alter the activity state of Rap2B, strongly enhanced the stimulatory effect of carbachol (Fig. 4). Treatment of the cells with dd-Ado (10 µM) almost completely (by 90 ± 5%; n = 4) abrogated the potentiating effect of Galpha s (data not shown). Thus, the M3 mAChR activates Rap2B and, similarly as observed for PLC stimulation, this activation is apparently controlled by cyclic AMP.


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  		Fig. 4.   Activation of Rap2B by the M3 mAChR. HEK-293 cells were transfected with empty vector (Control), Q229L Galpha 12 (25 µg of DNA) or Galpha s (100 µg of DNA), either alone (A) or with wild-type Rap2B (50 µg of DNA) (B). At 48 h after transfection, the cells were stimulated for 5 min without (-, Basal) and with 1 µM carbachol (+), followed by extraction of GTP-loaded Rap GTPases with RalGDS-RBD, SDS-PAGE, and immunoblotting with an anti-Rap2 antibody as described under "Experimental Procedures." Representative immunoblots are shown in the upper panels, while in the lower panels means ± S.E. (n = 3-6) are presented, with the amount of GTP-loaded Rap2 in unstimulated control cells set to 1. Note, the exposure time of the immunoblots in A and B was 10-15 min and 30 s, respectively.

Stimulation of PLC-epsilon by the M3 mAChR-- We next sought to determine which PLC isozyme is activated by the M3 mAChR. For this, we examined the effects of overexpression of PLC-beta 1, PLC-gamma 1, PLC-delta 1, and PLC-epsilon on basal and receptor-stimulated PLC activities. As illustrated in Fig. 5A, overexpression of PLC-gamma 1, which increased PLC stimulation by epidermal growth factor (not shown), did neither change basal PLC activity nor its stimulation by carbachol (1 µM). In cells overexpressing PLC-delta 1, basal PLC activity was increased by 2.5-fold, whereas carbachol-stimulated PLC activity was not altered. In contrast, overexpression of PLC-beta 1 and PLC-epsilon enhanced PLC stimulation by carbachol by 50 and 70%, respectively, without altering basal PLC activity. In line with these data, carbachol (1 µM)-induced [Ca2+]i increase was strongly potentiated in cells overexpressing PLC-beta 1 or PLC-epsilon , from 470 ± 55 nM in control cells to 700 ± 45 nM in cells overexpressing PLC-beta 1 (n = 6-8; p < 0.0001) and from 524 ± 35 nM to 940 ± 65 nM in cells overexpressing PLC-epsilon (n = 8-10; p < 0.0001) (Fig. 5B).


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  		Fig. 5.   Potentiation of M3 mAChR-induced PLC and calcium signaling by PLC-beta 1 and PLC-epsilon . HEK-293 cells were transfected with empty vector (Control, V), PLC-beta 1, PLC-delta 1, PLC-gamma 1, or PLC-epsilon (25 µg of DNA each). At 48 h after transfection, [3H]inositol phosphate formation without (Basal) and with 1 µM carbachol (A) or [Ca2+]i increase induced by 1 µM carbachol was determined (B). Insets, immunoblot detection of the PLC isozymes in lysates of transfected cells. Data in A are means ± S.E. (n = 5-6), while in B superimposed tracings of [Ca2+]i are shown.

In contrast to wild-type PLC-epsilon , expression of the catalytically inactive PLC-epsilon mutant, H1144L PLC-epsilon (7), reduced PLC stimulation by carbachol by 25% (Fig. 6A). Interestingly, expression of H1144L PLC-epsilon almost fully reversed the potentiating effect of co-expressed Galpha s, suggesting that potentiation of PLC stimulation by Galpha s is due to activation of the PLC-epsilon isozyme. This assumption was corroborated by studies with the adenylyl cyclase inhibitor, dd-Ado, in cells overexpressing PLC-epsilon or PLC-beta 1. As shown in Fig. 6B, dd-Ado (10 µM) strongly reduced the potentiating effect of overexpressed PLC-epsilon on carbachol-stimulated inositol phosphate formation, whereas dd-Ado was without effect in cells overexpressing PLC-beta 1, suggesting that cyclic AMP is involved in stimulation of PLC-epsilon , but not PLC-beta 1, by the M3 mAChR.


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  		Fig. 6.   Signaling of the M3 mAChR to PLC-epsilon is mediated by cyclic AMP. HEK-293 cells were transfected with empty vector (Vector, V), H1144L PLC-epsilon , Galpha s (100 µg of DNA each), or H1144L PLC-epsilon plus Galpha s (A) or with PLC-beta 1 or PLC-epsilon (25 µg of DNA each) (B) as indicated. At 48 after transfection, [3H]inositol phosphate formation without (Basal) and with 1 µM carbachol was determined directly (A) or after additional 30 min treatment without (Ctr) and with 10 µM dd-Ado (B). Inset, immunoblot detection of PLC-epsilon . Expression of H1144L PLC-epsilon did not alter the expression level of Galpha s (not shown). Data are means ± S.E. (n = 5-6).

We have recently reported that overexpression of the regulator of G protein signaling 4 (RGS4), which acts as a GTPase-activating protein for Galpha q and Galpha i proteins (25, 26), strongly reduces M3 mAChR-mediated PLC stimulation (14). As shown in Fig. 7A, in cells overexpressing RGS4 M3 mAChR-mediated PLC stimulation was reduced by about 40%. Most important, the inhibitory effect of dd-Ado (10 µM) on PLC stimulation was fully retained in cells overexpressing RGS4. In contrast, in cells expressing S17N Rap2B, in which the M3 mAChR response was reduced to a similar extent as in cells overexpressing RGS4, treatment with dd-Ado did not cause a further reduction in receptor-mediated PLC stimulation (Fig. 7B).


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  		Fig. 7.   Effect of dd-Ado on M3 mAChR-mediated PLC stimulation suppressed by RGS4 or S17N Rap2B. HEK-293 cells were transfected with empty vector (Vector), RGS4 (A), or S17N Rap2B (100 µg of DNA each) (B). At 48 h after transfection, the cells were treated for 30 min without (Ctr) and with 10 µM dd-Ado, followed by measurement of [3H]inositol phosphate formation in the absence (Basal) and presence of 1 µM carbachol. Data are means ± S.E. (n = 3-4).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

PTX-insensitive stimulation of PLC by GPCRs is generally assumed to be caused by direct activation of PLC-beta isozymes by activated alpha -subunits of Gq type G proteins (3, 4). The M3 mAChR is a prototypical example of such GPCRs. PLC stimulation by the M3 mAChR that preferentially couples to G proteins of the Gq family is PTX-insensitive (11-13). Furthermore, we have recently reported that PLC stimulation by the M3 mAChR is specifically suppressed by RGS proteins inactivating PTX-resistant Galpha q, but not Galpha 12 type G proteins (14). Interestingly, overexpression of the Galpha q-inactivating RGS4 reduced PLC stimulation by the M3 mAChR only partially, by 60-70% (14). In studies with Gs-coupled receptors, we made very recently the unexpected observation that such receptors, i.e. the beta 2-adrenoreceptor expressed in HEK-293 cells and the prostanoid receptor endogenously expressed in N1E-115 neuroblastoma cells, can also mediate PLC stimulation (15). This PLC stimulation was PTX-insensitive and apparently mediated by Gs-dependent formation of cyclic AMP and activation of the Ras-related GTPase Rap2B, finally resulting in stimulation of the PLC-epsilon isozyme (15). As the M3 mAChR can couple to Gs and increase cyclic AMP formation (11, 27), we examined in the present study whether PLC stimulation by the M3 mAChR may involve similar mechanisms. We report here that PLC stimulation by the M3 mAChR expressed in HEK-293 cells is a composite action on PLC-beta 1 and PLC-epsilon isozymes and that stimulation of PLC-epsilon by the M3 mAChR is apparently mediated by Gs-dependent cyclic AMP formation and activation of the GTPase Rap2B.

First, treatment of the cells with the P-site adenylyl cyclase inhibitor, dd-Ado, reduced PLC stimulation by the M3 mAChR. Second, overexpression of Galpha s, which by itself had no effect on PLC activity, strongly enhanced M3 mAChR signaling to PLC. Third, a similar enhancement of M3 mAChR-mediated PLC stimulation was observed in cells overexpressing the cyclic AMP-activated GEF for Rap GTPases, Epac1, while inhibition of cyclic AMP-dependent protein kinase A by H-89 was without effect. Fourth, inactivation of Ras-related GTPases with C. difficile toxin B-1470 and C. sordellii lethal toxin strongly reduced PLC stimulation. Fifth, the inhibitory toxin effects were mimicked by expression of an inactive Rap2B mutant, but not by inactive mutants of other GTPases serving as toxin substrates. Sixth, the M3 mAChR induced activation of Rap2B, and this activation was enhanced by overexpression of Galpha s or Epac1 and suppressed by dd-Ado. Seventh, PLC stimulation by the M3 mAChR was enhanced by overexpression of PLC-epsilon , similar to overexpression of PLC-beta 1, and reduced by expression of a catalytically inactive PLC-epsilon mutant. Finally, using various combinations, i.e. Galpha s with H1144L PLC-epsilon and dd-Ado with PLC-beta 1, PLC-epsilon , RGS4, and S17N Rap2B, evidence is provided that cyclic AMP-dependent PLC stimulation by the M3 mAChR involves Rap2B and the PLC-epsilon isozyme and that this stimulation is largely independent of PLC-beta 1 stimulation by Galpha q.

Activation of Gs and stimulation of adenylyl cyclase is not considered a primary function of the M3 mAChR, compared with coupling to Gq and stimulation PLC-beta isozymes (12). Therefore, we were surprised to observe that inhibition of adenylyl cyclase by dd-Ado and overexpression of cyclic AMP-activated Epac1 had such marked effects on M3 mAChR-mediated PLC stimulation. Similarly as reported before by others (11), increases in total cellular cyclic AMP levels in HEK-293 cells expressing the M3 mAChR were observed only at rather high carbachol concentrations (>1 µM) (data not shown). However, as shown herein, inhibition of M3 mAChR-mediated PLC stimulation by dd-Ado was largely independent of the carbachol concentration used (Fig. 1A), and overexpression of Epac1 markedly increased PLC stimulation and [Ca2+]i increase induced by carbachol at low concentrations. A possible explanation for this finding is that, in contrast to many other GEFs for other small GTPases, Epac1 is a membrane-associated protein even in its basal activity state, i.e. in the absence of cyclic AMP, and is activated by cyclic AMP at the plasma membrane (28). Thus, similarly as recently described for activation of the L-type Ca2+ channel Cav 1.2 by the Gs- and adenylyl cyclase-coupled beta 2-adrenoreceptor (29), Epac1 and the adenylyl cyclase may be assembled into a signaling complex at the plasma membrane, and local increases in cyclic AMP concentration not detected by measuring total cellular cyclic AMP levels may suffice to activate Epac1 and in consequence Rap2B. The existence of such a signaling complex is under investigation.

The magnitude of PLC inhibition caused by expression of S17N Rap2B was in the similar range as the inhibition induced by treatment of the cells with dd-Ado. Taking the transfection efficiency of 50-60% into consideration, these data suggest that S17N Rap2B, in addition to inhibiting cyclic AMP-dependent stimulation of PLC-epsilon by the M3 mAChR, may also interfere with receptor stimulation of PLC-beta 1 via Gq proteins. However, PLC-beta 1, in contrast to PLC-epsilon , does not contain binding domains for Ras/Rap proteins, making a direct interaction of PLC-beta 1 with Rap2B unlikely. Recently, RGS14 has been shown to interact with Rap GTPases via a RBD domain also found in RGS12 (30-32). However, no data have been reported whether Rap GTPases alter the GTPase-activating activities of these RGS proteins. Furthermore, RGS12 and RGS14 preferentially act on PTX-sensitive Gi/o proteins (33, 34), whereas PLC stimulation by the M3 mAChR is fully PTX-resistant. Thus, although an indirect action of Rap2B on Galpha q cannot be excluded, an alternative explanation for the pronounced inhibitory effect of S17N Rap2B is that Rap2B is also activated by Galpha q, independent of Gs-dependent cyclic AMP formation, finally resulting in stimulation of PLC-epsilon . Activation of Rap GTPases (Rap1 and Rap2) is not only achieved by the cyclic AMP-activated GEFs, Epac1 and Epac2 (18, 20, 28), but also by other Rap-specific GEFs, such as the CalDAG-GEFs, which are presumably activated by the second messengers, Ca2+ and diacylglycerol (35), and the PDZ-GEFs, for which the activation mechanisms are not yet known (36). Thus, it is conceivable to assume that Galpha q, e.g. by increasing Ca2+ and diacylglycerol levels via stimulation of PLC-beta isozymes, activates Rap2B and thereby PLC-epsilon . Such a reaction may also explain why overexpression of PLC-epsilon enhanced M3 mAChR-mediated PLC stimulation at least as efficiently as overexpression of PLC-beta 1 (see Fig. 5). The involvement of additional Rap-GEFs in the M3 mAChR response will be addressed in future studies.

In conclusion, we report here that PLC and calcium signaling by the M3 mAChR is mediated by the two PTX-insensitive G proteins, Gq and Gs, finally resulting in stimulation of PLC-beta 1 and PLC-epsilon , respectively. While stimulation of PLC-beta 1 is most likely caused by direct interaction with activated Galpha q proteins, stimulation of PLC-epsilon is apparently dependent on cyclic AMP formation and activation of Epac1 and in consequence Rap2B. This GTPase seems also to be involved in overall PLC stimulation by Gq, suggesting that Rap2B and its exchange factors play a major role in PLC stimulation by GPCRs.

    ACKNOWLEDGEMENTS

We thank K. Baden, M. Hagedorn, H. Geldermann, and D. Petermeyer for expert technical assistance and Drs. J. L. Bos, J. H. Camonis, C. von Eichel-Streiber, J. de Gunzburg, A. Hall, D. Illenberger, C. Kleuss, J. de Rooij, A. Ullrich, and T. Wieland for providing various toxins and cDNA constructs.

    FOOTNOTES

* This work was supported by the Deutsche Forschungsgemeinschaft and the Interne Forschungsförderung Essen.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Inst. für Pharmakologie, Universitätsklinikum Essen, Hufelandstrasse 55, D-45122 Essen, Germany, Tel.: 49-201-723-3457; Fax: 49-201-723-5968; E-mail: martina.schmidt@uni-essen.de.

Published, JBC Papers in Press, March 4, 2002, DOI 10.1074/jbc.M112024200

    ABBREVIATIONS

The abbreviations used are: PIP2, phosphatidylinositol 4,5-bisphosphate; PLC, phospholipase C; GPCR, G protein-coupled receptor; GEF, guanine nucleotide exchange factor; PTX, pertussis toxin; mAChR, muscarinic acetylcholine receptor; IP3, inositol 1,4,5-trisphosphate; dd-Ado, 2'5'-dideoxyadenosine; RalGDS-RBD, Rap-binding domain of the Ral guanine nucleotide dissociation stimulator; RGS, regulator of G protein signaling; HA, hemagglutinin; GTPgamma S, guanosine 5'-O-(thiotriphosphate).

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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