The M3 Muscarinic Acetylcholine Receptor Expressed in HEK-293 Cells Signals to Phospholipase D via G12 but Not Gq-type G Proteins

REGULATORS OF G PROTEINS AS TOOLS TO DISSECT PERTUSSIS TOXIN-RESISTANT G PROTEINS IN RECEPTOR-EFFECTOR COUPLING*

Ulrich RümenappDagger §, Melanie AsmusDagger , Helge SchablowskiDagger , Markus WoznickiDagger , Li HanDagger , Karl H. JakobsDagger , Mercedeh Fahimi-Vahid, Christina Michalek, Thomas Wieland, and Martina SchmidtDagger

From the Dagger  Institut für Pharmakologie, Universitätsklinikum Essen, Hufelandstrasse 55, D-45122 Essen, Germany and the  Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Universitäts-Krankenhaus Eppendorf, D-20246 Hamburg, Germany

Received for publication, June 7, 2000, and in revised form, October 16, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The M3 muscarinic acetylcholine receptor (mAChR) expressed in HEK-293 cells couples to Gq and G12 proteins and stimulates phospholipase C (PLC) and phospholipase D (PLD) in a pertussis toxin-insensitive manner. To determine the type of G protein mediating M3 mAChR-PLD coupling in comparison to M3 mAChR-PLC coupling, we expressed various Galpha proteins and regulators of the G protein signaling (RGS), which act as GTPase-activating proteins for Gq- or G12-type G proteins. PLD stimulation by the M3 mAChR was enhanced by the overexpression of Galpha 12 and Galpha 13, whereas the overexpression of Galpha q strongly increased PLC activity without affecting PLD activity. Expression of the RGS homology domain of Lsc, which acts specifically on Galpha 12 and Galpha 13, blunted the M3 mAChR-induced PLD stimulation without affecting PLC stimulation. On the other hand, overexpression of RGS4, which acts on Galpha q- but not Galpha 12-type G proteins, suppressed the M3 mAChR-induced PLC stimulation without altering PLD stimulation. We conclude that the M3 mAChR in HEK-293 cells apparently signals to PLD via G12- but not Gq-type G proteins and that G protein subtype-selective RGS proteins can be used as powerful tools to dissect the pertussis toxin-resistant G proteins and their role in receptor-effector coupling.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Stimulation of phosphatidylcholine-specific phospholipase D (PLD),1 leading to the formation of the putative second messenger phosphatidic acid, has been described in a wide range of cell types in response to stimulation of a large variety of different membrane receptors (1-5). Particularly, numerous receptors coupled to heterotrimeric G proteins have been shown to cause PLD stimulation. Interestingly, almost every G protein-coupled receptor (GPCR) known to stimulate phosphoinositide-specific phospholipase C (PLC) also stimulates PLD activity. This concomitant activation of the two phospholipases may be caused at several levels of signal transduction by these GPCRs.

First, stimulation of PLD activity may be secondary to PLC stimulation, and the cellular signals generated by the PLC reaction specifically increase in the cytosolic Ca2+ concentration and activation of protein kinase C isoforms. In fact, evidence has been provided for some GPCRs in different cell types that PLD stimulation is apparently a consequence of the primary PLC stimulation (1-3).

Second, the same receptor-activated G protein may stimulate both PLC and PLD. There is ample evidence that one type of G protein can regulate different effectors (6, 7). Two distinct G protein subtypes mediate GPCR-PLC coupling, the pertussis toxin (PTX)-insensitive Gq-type G proteins and the PTX-sensitive Gi-type G proteins (2, 8). Studies on stimulation of PLD activity by chemoattractants in neutrophils suggest that in these cells GPCRs couple to PLD and PLC via the same Gi-type G proteins (4, 9). On the other hand, PLC-independent coupling of GPCRs to PLD via Gq-type G proteins has not yet been reported. However, it has recently been reported that the expression of constitutively active Gq can strongly increase PLD activity (10), suggesting that this possibility should be considered.

Third, the receptor may couple to PLC and PLD by activating two distinct types of heterotrimeric G proteins. For example, GPCR-PLC coupling may be mediated by Gq proteins and GPCR-PLD coupling may be mediated by Gi proteins or vice versa. Other potential candidates for mediating specific GPCR-PLD coupling are the PTX-insensitive G12-type G proteins G12 and G13, which are not directly involved in GPCR-PLC coupling (2, 11, 12). Expression of constitutively active Galpha 12 and Galpha 13 has been shown to strongly increase the activity of a coexpressed PLD1 enzyme like the constitutively active Galpha q (10).

The M3 muscarinic acetylcholine receptor (mAChR) stably expressed in HEK-293 cells is coupled to both PLD and PLC (13), and stimulation of either phospholipase by this GPCR is resistant to the treatment of the cells with PTX (14), which indicates that Gi-type G proteins are not involved in coupling M3 mAChR to either PLC or PLD. Previous studies furthermore demonstrate that the M3 mAChR-induced PLD stimulation is not affected by protein kinase C inhibition or down-regulation or by chelation of intracellular Ca2+ (15, 16), suggesting that it is not secondary to PLC stimulation. Thus, because the M3 mAChR coupled to Gq-type G proteins in these cells (14), we had to consider that the receptor coupled to PLD and PLC via the same type of G proteins, i.e. Gq proteins, or that two distinct PTX-insensitive G proteins mediate GPCR coupling to the two phospholipases.

To resolve this question, we took advantage of the regulators of G protein signaling (RGS). These are GTPase-activating proteins (GAPs) that accelerate GTP hydrolysis by Galpha proteins and thereby attenuate signaling via heterotrimeric G proteins (17-19). More than 20 different RGS proteins have been identified, of which RGS4 has received the most extensive biochemical characterization (20-24). RGS4 exhibits GAP activity for Galpha i- and Galpha q-type G proteins but not for Galpha 12-type proteins (21). Furthermore, structural data suggest that an interaction of either Galpha 12 or Galpha 13 with RGS4 is unlikely (23). Recently, an NH2-terminal RGS homology domain has been identified in the Rho-specific guanine nucleotide exchange factor (GEF), p115 RhoGEF, its murine homolog Lsc, and some closely related RhoGEFs (25). This RGS homology domain exhibited GAP activity for Galpha 12 and Galpha 13 but not for other Galpha proteins. Thus, the use of the RGS homology domain of these RhoGEFs inactivating G12-type G proteins in comparison to RGS4 inactivating Gq-proteins should help to clarify whether Gq- and/or G12-type G proteins mediate M3 mAChR-PLD coupling in HEK-293 cells. Using this approach in combination with the overexpression of relevant Galpha proteins, strong evidence is provided that G12 but not Gq proteins mediate M3 mAChR-PLD coupling.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Expression Plasmids-- The pCis vectors carrying cDNAs for wild-type Galpha q, Galpha 12, Galpha 13, and the constitutively active Galpha q mutant, Galpha q R183C, were described previously (26, 27). FLAG-tagged RGS4 in a pCMV-based expression vector and the anti-RGS4 antibody were kind gifts of Dr. J. H. Kehrl. cDNA encoding Lsc was donated by Dr. R. Kay. For the expression of a Myc-tagged variant of Lsc containing the amino terminus with the RGS homology domain but lacking the Dbl and pleckstrin homology domains (Lsc-RGS, amino acids 1-283), the corresponding cDNA fragment was subcloned into pCMV3-Tag3 expression vector (Stratagene).

Cell Culture and Transient Transfection-- HEK-293 cells stably expressing the M3 mAChR were cultured as reported previously (15). For experiments, cells were grown to about 50% confluence on 145-mm culture dishes and transfected with the indicated amounts of either plasmid DNA or empty expression vectors using the calcium phosphate precipitation method. As studied by the expression of the green fluorescent protein, transfection efficiency ranged from 50 to 70%. All assays were performed 48 h after transfection. Expression of the proteins was verified by the immunoblotting of cell lysates with specific antibodies. The antibodies against Galpha q, Galpha 12, Galpha 13, and His6 were from Santa Cruz, the anti-Myc antibody was from Roche Molecular Biochemicals, and the anti-FLAG antibody was from Stratagene.

Construction of and Infection with Recombinant Adenoviruses-- For construction of the recombinant adenoviruses encoding human RGS4 or mouse Lsc-RGS, the Adeasy system kindly provided by Dr. B. Vogelstein was used (28). The constructs were transfected into HEK-293 cells using LipofectAMINE, and recombinant viruses were amplified in several steps of this cell line. After purification over a CsCl2 gradient, high titer viral stocks of 3-4 × 108 plaque-forming units/µl were obtained. Infection of subconfluent monolayers of HEK-293 cells stably expressing the M3 mAChR was performed at a multiplicity of infection of 30 for 24 h before labeling. Before the PLC and PLD assays, infected cells were visualized and quantified by fluorescence microscopy as the constructs additionally expressed green fluorescent protein under the control of an independent CMV promoter. For control, an adenovirus encoding bacterial beta -galactosidase LacZ, a kind gift of Dr. T. Eschenhagen, was used.

Photoaffinity Labeling of G Proteins with [gamma -32P]GTP-azidoanilide-- Synthesis and purification of [gamma -32P]GTP-azidoanilide were performed as described previously (29). G proteins in the membranes of HEK-293 cells (100-200 µg of protein) were photoaffinity-labeled as described (29) with the following modifications: the reaction mixture (50 µl) contained 60 mM HEPES, pH 7.4, 60 mM NaCl, 10 mM MgCl2, 0.2 mM EDTA, and 2 µCi of [gamma -32P]GTP-azidoanilide. Incubation was for 5 min at 30 °C in the absence and presence of 1 mM carbachol. Solubilization of irradiated membranes and immunoprecipitation of Galpha q and Galpha 12 were exactly as described previously (30) using the antibodies AS 370 (5 µl/tube) and AS 233 (10 µl/tube), respectively (kindly provided by Dr. S. Offermanns). Immunoprecipitates were eluted from protein A-Sepharose with sample buffer and resolved by SDS-polyacrylamide gel electrophoresis (9% acrylamide plus 6 M urea). Gels were stained with Coomassie Brilliant Blue and dried. Labeled proteins were visualized by phosphoimaging (PhosphorImager, Molecular Dynamics) (29).

Assays of PLD and PLC Activities-- For measurement of PLD and PLC activities, the transfected cells were replated 24 h after transfection on 145-mm culture dishes. Cellular phospholipids were labeled by incubation for 20-24 h with [3H]oleic acid (2 µCi/ml) and myo-[3H]inositol (1 µCi/ml) in inositol-free growth medium. Thereafter, the cells were detached from the dishes, washed once with Hanks' balanced salt solution (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), supplemented with 10 mM LiCl, and resuspended at a density of 1 × 107 cells/ml. Then phospholipase activities were measured for 30 min at 37 °C in a total volume of 200 µl containing 100 µl of the cell suspension (1 × 106 cells), 1.75% ethanol, and the indicated stimulatory agents. Alternatively, the transfected cells were split to poly-L-lysine-coated 35-mm dishes, and after labeling PLD and PLC activities were determined in adherent cells (15). Stop of the enzyme reactions and analysis of [3H]inositol phosphates and labeled phospholipids including the specific PLD product [3H]phosphatidylethanol were carried out as described previously (15). Protein levels were measured by the Bradford method in separate culture dishes. The formation of [3H]phosphatidylethanol was expressed as the percentage of total labeled phospholipids. The formation of [3H]inositol phosphates was given as counts/min per 106 cells or per mg of protein. Similar results were obtained whether the cells were in suspension or were adherent.

Data Presentation-- Data shown are means ± S.D. from one representative experiment performed in triplicate and repeated as indicated or means ± S.E. with n providing the number of independent experiments. Concentration response curves were analyzed using iterative nonlinear regression analysis (GraphPAD Prism, GraphPAD Software for Science, San Diego, CA).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Effects of Various Galpha Proteins on PLD and PLC Activities-- Agonist activation of the M3 mAChR stably expressed in HEK-293 cells results in rapid and strong stimulation of PLD and PLC activities that are insensitive to PTX treatment (13-15). As a first approach to determine the type of G proteins mediating M3 mAChR-PLD coupling in comparison to M3 mAChR-PLC coupling, HEK-293 cells were transiently transfected with expression vectors for wild-type Galpha 12, Galpha 13, and Galpha q as well as the constitutively active Galpha q R183C. In cells overexpressing either Galpha 12 or Galpha 13, the stimulation of PLD activity by carbachol (1 mM) was increased by 60-100% compared with vector-transfected control cells (Fig. 1A), whereas the overexpression of Galpha q did not alter PLD activity (Fig. 1B). In contrast to M3 mAChR-mediated PLD stimulation, basal PLD activity and PLD stimulation by 100 nM phorbol 12-myristate 13-acetate (PMA) were not affected by the overexpression of Galpha 12 or Galpha 13 (Fig. 2). On the other hand, basal and carbachol-stimulated PLC activities were not altered by the overexpression of Galpha 12 or Galpha 13 (Fig. 3A) but were enhanced by 3-4-fold in cells overexpressing Galpha q (Fig. 3B). Expression of the constitutively active Galpha q R183C caused an even larger increase in basal PLC activity, which was not further enhanced by carbachol (Fig. 4A). However, despite this marked PLC stimulation, neither basal nor carbachol-stimulated PLD activities were altered by the expression of Galpha q R183C (Fig. 4B).



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 1.   Effects of overexpression of various Galpha proteins on PLD stimulation by the M3 mAChR. M3 mAChR-expressing HEK-293 cells were transfected with empty expression vectors (Control) or with expression plasmids for Galpha 12, Galpha 13 (A), or Galpha q (B) (100 µg each). After 48 h, [3H]phosphatidylethanol (PtdEtOH) formation was determined in [3H]oleic acid-labeled cells in the absence (Basal) and the presence of 1 mM carbachol. C, immunoblots of Galpha 12, Galpha 13, and Galpha q with specific antibodies. Data are representative of 3-5 experiments.



View larger version (48K):
[in this window]
[in a new window]
 
Fig. 2.   Lack of effect of Galpha 12 and Galpha 13 on PLD stimulation by PMA. M3 mAChR-expressing HEK-293 cells were transfected with empty expression vector (Control) or with expression plasmids for Galpha 12 or Galpha 13 (100 µg each). After 48 h, PLD activity was determined in the absence (Basal) and presence of 100 nM PMA. Data are representative of three experiments.



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 3.   Effects of overexpression of various Galpha proteins on PLC stimulation by the M3 mAChR. M3 mAChR-expressing HEK-293 cells were transfected with empty expression vectors (Control) or with expression plasmids for Galpha 12, Galpha 13 (A), or Galpha q (B) (100 µg each). After 48 h, [3H]inositol phosphate formation was determined in [3H] inositol-labeled cells in the absence (Basal) and presence of 1 mM carbachol. Data are representative of 3-5 experiments.



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 4.   Effects of expression of Galpha q R183C on PLC and PLD activities. M3 mAChR-expressing HEK-293 cells were transfected with empty expression vector (Control) or with expression plasmid for Galpha q R183C (50 µg each). After 48 h, PLC (A) and PLD (B) activities were determined in the absence (Basal) and presence of 1 mM carbachol (n = 3).

To investigate whether the M3 mAChR is able to activate Gq- and G12-type G proteins, the incorporation of the photoreactive GTP analog [gamma -32P]GTP-azidoanilide into Galpha q and Galpha 12 proteins overexpressed in HEK-293 cells was studied. In line with previous findings (14), the addition of carbachol (1 mM) strongly increased the incorporation of [gamma -32P]GTP-azidoanilide into Galpha q proteins (Fig. 5A). Binding of the GTP analog to Galpha 12 was also, but less efficiently, enhanced by carbachol (Fig. 5B). Thus, the M3 mAChR stably expressed in HEK-293 cells can activate both Gq- and G12-type G proteins.



View larger version (51K):
[in this window]
[in a new window]
 
Fig. 5.   M3 mAChR-induced activation of Galpha q and Galpha 12. Photoaffinity labeling of G proteins with [gamma -32P]GTP-azidoanilide was performed in membranes of HEK-293 cells overexpressing either Galpha q (A) or Galpha 12 (B) in the absence (-) and presence (+) of 1 mM carbachol (Carb) using either 100 µg (A) or 200 µg (B) of membrane protein. Thereafter, Galpha q (A) and Galpha 12 (B) proteins were immunoprecipitated with the antisera AS 370- and AS 233-specific for Galpha q and Galpha 12, respectively, and the precipitates were resolved by SDS-polyacrylamide gel electrophoresis. PhosphorImager images (Molecular Dynamics) of representative experiments are shown.

Effects of RGS Proteins on M3 mAChR-induced PLD and PLC Stimulation-- To determine the endogenous G protein subtype involved in the coupling of M3 mAChR to the stimulation of PLD and PLC, we made use of the two RGS proteins, RGS4 and the RGS homology domain of Lsc (Lsc-RGS), which act as GAPs for Galpha q and Galpha 12 family members, respectively (17-25). As shown in Fig. 6A, M3 mAChR-stimulated PLD activity was strongly reduced (by about 50%) in cells transiently expressing Lsc-RGS, whereas the expression of RGS4 was without effect. On the other hand, the expression of RGS4 markedly reduced (by about 50%) 1 mM carbachol-stimulated PLC activity, whereas the expression of Lsc-RGS was without effect (Fig. 6B). In contrast to M3 mAChR-mediated PLD stimulation, expression of Lsc-RGS did not alter 100 nM PMA-induced PLD stimulation (Fig. 7).



View larger version (25K):
[in this window]
[in a new window]
 
Fig. 6.   Effects of transient expression of Lsc-RGS and RGS4 on PLD and PLC stimulation by the M3 mAChR. M3 mAChR-expressing HEK-293 cells were transfected with empty expression vectors (Control, open bars) or expression plasmids for RGS4 (black bars) or Lsc-RGS (dotted bars) (100 µg each). After 48 h, PLD (A) and PLC (B) activities were determined in the absence (Basal) and presence of 1 mM carbachol. C, expression of RGS4 and Lsc-RGS verified by immunoblotting with an anti-FLAG and anti-Myc antibody, respectively. Data are representative of four experiments.



View larger version (33K):
[in this window]
[in a new window]
 
Fig. 7.   Lack of effect of Lsc-RGS on PMA-induced PLD stimulation. M3 mAChR-expressing HEK-293 cells were transfected with empty expression vector (Control, open bar) or with expression plasmid for Lsc-RGS (dotted bar) (100 µg each). After 48 h, PLD activity was determined in the absence (Basal) and presence of 100 nM PMA. Data are representative of four experiments.

The extent of inhibition of carbachol-stimulated PLD and PLC activities observed upon the transient expression of Lsc-RGS and RGS4, respectively, roughly reflects the transfection efficiency. However, a small effect of Lsc-RGS and RGS4 on PLC and PLD activities, respectively, may have escaped detection. Therefore, the expression of RGS proteins was increased by a second approach, i.e. infection of cells with recombinant adenoviruses encoding Lsc-RGS or RGS4. As judged by the expression of green fluorescent protein, the efficiency of adenoviral gene transfer into HEK-293 cells was >95% (data not shown). The expression of RGS4 by adenoviral infection was without any effect on M3 mAChR-stimulated PLD activity (Fig. 8A). Neither the maximal extent nor the concentration dependence of carbachol-induced PLD stimulation was altered in RGS4 expression compared with control cells infected with an adenovirus encoding LacZ. In contrast, the carbachol-induced PLD stimulation was blunted (by about 80%) by the expression of Lsc-RGS. On the other hand, adenoviral expression of Lsc-RGS did not affect the stimulation of PLC activity at any carbachol concentration that had been examined, whereas PLC stimulation was strongly reduced (by about 70%) by the expression of RGS4 (Fig. 8B).



View larger version (21K):
[in this window]
[in a new window]
 
Fig. 8.   Effects of adenoviral expression of Lsc-RGS and RGS4 on PLD and PLC stimulation by the M3 mAChR. M3 mAChR-expressing HEK-293 cells were infected with recombinant adenoviruses encoding LacZ, Lsc-RGS, or RGS4 at a multiplicity of infection = 30. At 48 h later, PLD (A) and PLC (B) activities were determined at the indicated concentrations of carbachol. C, expression of RGS4 and Lsc-RGS was verified by immunoblotting with an anti-RGS4 and anti-(His)6 antibody, respectively. Data are representative of two experiments.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

A large variety of GPCRs that stimulate phosphoinositide-hydrolyzing PLC also activates PLD. This concomitant stimulation of the two phospholipases may be a sequential reaction in that the receptor initially leads to PLC stimulation followed by the increase in PLD activity that is caused by intracellular Ca2+ and/or activated protein kinase C, consequences of the PLC reaction that are in fact reported for some GPCRs (1-3). Alternatively, PLC and PLD enzymes are independently regulated by the receptor either by activating one heterotrimeric G protein that then leads to the stimulation of both PLC and PLD or two distinct G proteins, one responsible for PLC stimulation and the other responsible for mediating PLD stimulation. The M3 mAChR expressed in HEK-293 cells is a prototypical example of a GPCR that stimulates both PLC and PLD (13-15). Previous studies indicate that PLD stimulation is apparently independent of changes in intracellular Ca2+ and protein kinase C activation (15, 16), suggesting that it is not a consequence of the PLC stimulation. Thus, two other possibilities were considered for mediating M3 mAChR-PLD coupling. As the M3 mAChR couples to Gq-type G proteins in HEK-293 cells and stimulation of either phospholipase is resistant to PTX (14), we first studied whether Gq proteins may mediate M3 mAChR-PLD coupling. However, as shown here, the basal PLD activity and its stimulation by the M3 mAChR were not affected by the overexpression of Galpha q (wild type or constitutively active), which on the other hand strongly increased PLC activity. Furthermore, overexpression (transient or by infection with a recombinant adenovirus) of RGS4, which is known to act as a GAP for Gq proteins (17-24), suppressed the M3 mAChR-mediated PLC stimulation but did not affect PLD stimulation by this GPCR. These results are in line with previously published data on the regulation of PLC stimulation by Galpha q and RGS4 (2, 12, 17-19, 31-33) and strongly corroborate the idea that PLD stimulation by the M3 mAChR in HEK-293 cells is in fact not a consequence of the concomitant PLC stimulation.

As the M3 mAChR couples to PLD but apparently not by activating the PLC-stimulatory Gq proteins, we examined whether the PTX-resistant G12-type G proteins may act as specific transducers for PLD stimulation. The overexpression of Galpha 12 or Galpha 13 enhanced PLD stimulation by the M3 mAChR by up to 2-fold. On the other hand, the expression of Lsc-RGS, the murine homolog of the RGS domain of p115 RhoGEF that acts as a specific GAP for Galpha 12 and Galpha 13 (25), suppressed the M3 mAChR-mediated PLD stimulation. In contrast, the basal and M3 mAChR-stimulated PLC activities were not altered by the overexpression of Galpha 12 or Galpha 13 or by the expression of Lsc-RGS. These results are in line with our knowledge that G12-type G proteins do not directly influence the activities of PLC enzymes (2, 11, 12) and indicate that the expression of the PLD-inhibitory Lsc-RGS does not unspecifically inhibit the M3 mAChR. As the expression of Galpha 12, Galpha 13, or Lsc-RGS did not alter phorbol ester-stimulated PLD activity, it can be concluded that these proteins, such as Galpha 12 and Galpha 13, specifically enhance or interfere, such as Lsc-RGS, with M3 mAChR-PLD coupling. Thus, together with the finding that the M3 mAChR induced the incorporation of [gamma -32P]GTP-azidoanilide into G12 proteins, these results strongly suggest that G12-type G proteins specifically mediate the coupling of the M3 mAChR to the PLD signaling pathway in HEK-293 cells.

Recently, the G protein specificities for coupling of the angiotensin II type 1 receptor to PLD and PLC in vascular smooth muscle cells were reported (34, 35). Stimulation of PLD activity by this GPCR was inhibited (by 50%) by antibodies against Galpha 12 but not Galpha q/11. However, both types of antibodies suppressed in an additive manner angiotensin II stimulation of PLC-beta 1. Thus, in contrast to the specific function of G12-type G proteins in M3 mAChR-PLD coupling in HEK-293 cells, G12 proteins are apparently involved in GPCR-induced stimulation of both phospholipases, PLD and PLC, in vascular smooth muscle cells.

As direct activation of PLD enzymes by heterotrimeric G proteins including G12-type G proteins has not been observed (1-5), these G proteins most probably activate intermediate signaling pathways and proteins, finally leading to PLD stimulation. The best characterized intermediates involved in receptor-induced PLD stimulation are small GTPases of the ADP-ribosylation factor and Rho families as well as protein kinase C isoenzymes (1-5). As G12-type G proteins do not stimulate PLC (11, 12), PLD stimulation by these G proteins most probably is not caused by protein kinase C activation. Many of the diverse cellular responses observed upon expression of constitutively active Galpha 12 or Galpha 13 including PLD1 stimulation, actin stress fiber formation, neurite retraction, activation of Na+-H+-exchanger, and serum response element-dependent gene transcription were inhibited by dominant-negative Rho GTPases (10, 36-40), suggesting that Rho GTPases are major targets of the action of G12-type G proteins. However, from the finding that Rho GTPases are involved in a cellular response, it cannot be concluded that G12-type G proteins mediate the receptor action as Rho-dependent cellular actions as well as direct Rho activation can also be induced by activated Galpha q proteins (37, 40-43). Also, in HEK-293 cells, the expression of mutationally activated Galpha 12 and Galpha q family members caused strong and comparable stimulation of serum response factor-mediated gene transcription that was sensitive to Clostridium botulinum C3 transferase (data not shown), indicating the involvement and probably activation of Rho by either type of heterotrimeric G protein. Thus, although Gq proteins can cause Rho activation, the Rho-dependent PLD stimulation by the M3 mAChR in HEK-293 cells (44) is apparently independent of this reaction but mediated by G12-type G proteins. In combination with the finding that the M3 mAChR activates PLC and protein kinase C (data not shown) but stimulates PLD that is apparently independent of this reaction, these data suggest that the M3 mAChR-PLD coupling occurs in a highly organized subcellular compartment.

PLD stimulation by the M3 mAChR-expressed HEK-293 cells is dependent on Rho GTPases apparently acting via Rho kinase (44, 45). Thus, from the data discussed above and those data presented in this study, it seems reasonable to assume that the M3 mAChR activates endogenous G12-type G proteins, which then through an unidentified GEF activate Rho and subsequently PLD. In addition to and apparently independent of Rho proteins, ADP-ribosylation factor GTPases are required for M3 mAChR-induced PLD stimulation in HEK-293 cells (46, 47). As PLD stimulation was not affected by the inactivation of Gq proteins (by expression of RGS4), it has to be concluded that the activation of ADP-ribosylation factor GTPases is probably also mediated by G12-type G proteins. Experiments are in progress to study whether both G12-type G proteins, G12 and G13, are involved in M3 mAChR-induced PLD stimulation and may even exhibit selectivity for coupling the receptor to the activation of Rho and ADP-ribosylation factor GTPases.

In summary, our data indicate that distinct heterotrimeric G proteins couple the M3 mAChR to the stimulation of PLD and PLC in HEK-293 cells. Whereas Gq-type G proteins mediate M3 mAChR-PLC coupling, the stimulation of PLD activity is apparently mediated specifically by G12-type G proteins. Moreover, we demonstrate that G protein subtype-specific RGS proteins can be used as powerful tools to dissect the PTX-resistant G protein families, Gq and G12, and their role in receptor-effector coupling.


    ACKNOWLEDGEMENTS

We thank C. Moorkamp, K. Rehder, M. Hagedorn, and H. Geldermann for expert technical assistance.


    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: Institut für Pharmakologie, Universitätsklinikum Essen, Hufelandstrasse 55, D-45122 Essen, Germany. Tel.: 49-201-723-3463; Fax: 49-201-723-5968; E-mail: ulrich.ruemenapp@uni-essen.de.

Published, JBC Papers in Press, October 17, 2000, DOI 10.1074/jbc.M004957200


    ABBREVIATIONS

The abbreviations used are: PLD, phospholipase D; GPCR, G protein-coupled receptor; PLC, phospholipase C; PTX, pertussis toxin; mAChR, muscarinic acetylcholine receptor; RGS, regulator of G protein signaling; GAP, GTPase-activating protein; GEF, guanine nucleotide exchange factor; HEK, human embryonic kidney; PMA, phorbol 12-myristate 13-acetate.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


1. Exton, J. H. (1997) Physiol. Rev. 77, 303-320[Abstract/Free Full Text]
2. Singer, W. D., Brown, H. A., and Sternweis, P. C. (1997) Annu. Rev. Biochem. 66, 475-509[CrossRef][Medline] [Order article via Infotrieve]
3. Exton, J. H. (1999) Biochim. Biophys. Acta 1439, 121-133[Medline] [Order article via Infotrieve]
4. Jones, D., Morgan, C., and Cockcroft, S. (1999) Biochim. Biophys. Acta 1439, 229-244[Medline] [Order article via Infotrieve]
5. Liscovitch, M., Czarny, M., Fiucci, G., and Tang, X. (2000) Biochem. J. 345, 401-415[CrossRef][Medline] [Order article via Infotrieve]
6. Neer, E. J. (1995) Cell 80, 249-257[Medline] [Order article via Infotrieve]
7. Gudermann, T., Kalkbrenner, F., and Schultz, G. (1996) Annu. Rev. Pharmacol. Toxicol. 36, 429-459[CrossRef][Medline] [Order article via Infotrieve]
8. Rhee, S. G., and Bae, Y. S. (1997) J. Biol. Chem. 272, 15045-15048[Free Full Text]
9. Fensome, A., Whatmore, J., Morgan, C. P., Jones, D., and Cockcroft, S. (1998) J. Biol. Chem. 273, 13157-13164[Abstract/Free Full Text]
10. Plonk, S. G., Park, S.-K., and Exton, J. H. (1998) J. Biol. Chem. 273, 4823-4826[Abstract/Free Full Text]
11. Dhanasekaran, N., and Dermott, J. M. (1996) Cell. Signal. 8, 235-245[CrossRef][Medline] [Order article via Infotrieve]
12. Fields, T. A., and Casey, P. J. (1997) Biochem. J. 321, 561-571[Medline] [Order article via Infotrieve]
13. Sandmann, J., Peralta, E. G., and Wurtman, R. J. (1991) J. Biol. Chem. 266, 6031-6034[Abstract/Free Full Text]
14. Offermanns, S., Wieland, T., Homann, D., Sandmann, J., Bombien, E., Spicher, K., Schultz, G., and Jakobs, K. H. (1994) Mol. Pharmacol. 45, 890-898[Abstract]
15. Schmidt, M., Hüwe, S. M., Fasselt, B., Homann, D., Rümenapp, U., Sandmann, J., and Jakobs, K. H. (1994) Eur. J. Biochem. 225, 667-675[Abstract]
16. Rümenapp, U., Schmidt, M., Wahn, F., Tapp, E., Granass, A., and Jakobs, K. H. (1997) Eur. J. Biochem. 248, 407-414[Abstract]
17. Berman, D. M., and Gilman, A. G. (1998) J. Biol. Chem. 273, 1269-1272[Free Full Text]
18. Wieland, T., and Chen, C.-K. (1999) Naunyn-Schmiedeberg's Arch. Pharmacol. 360, 14-26[CrossRef][Medline] [Order article via Infotrieve]
19. De Vries, L., and Farquhar, M. G. (1999) Trends Cell Biol. 9, 138-144[CrossRef][Medline] [Order article via Infotrieve]
20. Berman, D. M., Wilkie, T. M., and Gilman, A. G. (1996) Cell 86, 445-452[Medline] [Order article via Infotrieve]
21. Berman, D. M., Kozasa, T., and Gilman, A. G. (1996) J. Biol. Chem. 271, 27209-27212[Abstract/Free Full Text]
22. Popov, S., Yu, K., Kozasa, T., and Wilkie, T. M. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 7216-7220[Abstract/Free Full Text]
23. Tesmer, J. J. G., Berman, D. M., Gilman, A. G., and Sprang, S. R. (1997) Cell 89, 251-261[Medline] [Order article via Infotrieve]
24. Srinivasa, S. P., Watson, N., Overton, M. C., and Blumer, K. J. (1998) J. Biol. Chem. 273, 1529-1533[Abstract/Free Full Text]
25. Kozasa, T., Jiang, X. J., Hart, M. J., Sternweis, P. M., Singer, W. D., Gilman, A. G., Bollag, G., and Sternweis, P. C. (1998) Science 280, 2109-2111[Abstract/Free Full Text]
26. Strathmann, M. P., and Simon, M. I. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 9113-9117[Abstract]
27. Strathmann, M. P., and Simon, M. I. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 5582-5586[Abstract]
28. He, T.-C., Zhou, S., Da Costa, L. T., Yu, J., Kinzler, K. W., and Vogelstein, B. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 2509-2514[Abstract/Free Full Text]
29. Gsell, S., Eschenhagen, T., Kaspareit, G., Nose, M., Scholz, H., Behrends, O., and Wieland, T. (2000) FASEB J. 14, 17-26[Abstract/Free Full Text]
30. Wieland, T., Nürnberg, B., Ulibarri, I., Kaldenberg-Stasch, S., Schultz, G., and Jakobs, K. H. (1993) J. Biol. Chem. 268, 18111-18118[Abstract/Free Full Text]
31. Yan, Y., Chi, P. P., and Bourne, H. R. (1997) J. Biol. Chem. 272, 11924-11921[Abstract/Free Full Text]
32. Huang, C., Hepler, J. R., Gilman, A. G., and Mumby, S. M. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 6159-6163[Abstract/Free Full Text]
33. Xu, X., Zeng, W., Popov, S., Berman, D. M., Davignon, I., Yu, K., Yowe, D., Offermanns, S., Muallem, S., and Wilkie, T. M. (1999) J. Biol. Chem. 274, 3549-3556[Abstract/Free Full Text]
34. Ushio-Fukai, M., Alexander, R. W., Akers, M., Lyons, P. R., Lassègue, B., and Griendling, K. K. (1999) Mol. Pharmacol. 55, 142-149[Abstract/Free Full Text]
35. Ushio-Fukai, M., Griendling, K. K., Akers, M., Lyons, P. R., and Alexander, R. W. (1998) J. Biol. Chem. 273, 19772-19777[Abstract/Free Full Text]
36. Buhl, A. M., Johnson, M. L., Dhanasekaran, N., and Johnson, G. L. (1995) J. Biol. Chem. 270, 24631-24634[Abstract/Free Full Text]
37. Katoh, H., Aoki, J., Yamaguchi, Y., Kitano, Y., Ichikawa, A., and Negishi, M. (1998) J. Biol. Chem. 273, 28700-28707[Abstract/Free Full Text]
38. Hooley, R., Yu, C.-Y., Symons, M., and Barber, D. L. (1996) J. Biol. Chem. 271, 6152-6158[Abstract/Free Full Text]
39. Fromm, C., Coso, O. A., Montaner, S., Xu, N., and Gutkind, J. S. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 10098-10103[Abstract/Free Full Text]
40. Mao, J., Yuan, H., Xie, W., and Wu, D. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 12973-12976[Abstract/Free Full Text]
41. Sah, V. P., Hoshijima, M., Chien, K. R., and Brown, J. H. (1996) J. Biol. Chem. 271, 31185-31190[Abstract/Free Full Text]
42. Kjøller, L., and Hall, A. (1999) Exp. Cell Res. 253, 166-179[CrossRef][Medline] [Order article via Infotrieve]
43. Mao, J., Yuan, H., Xie, W., Simon, M. I., and Wu, D. (1998) J. Biol. Chem. 273, 27118-27123[Abstract/Free Full Text]
44. Schmidt, M., Rümenapp, U., Bienek, C., Keller, J., von Eichel-Streiber, C., and Jakobs, K. H. (1996) J. Biol. Chem. 271, 2422-2426[Abstract/Free Full Text]
45. Schmidt, M., Vobeta , M., Oude Weernink, P. A., Wetzel, J., Amano, M., Kaibuchi, K., and Jakobs, K. H. (1999) J. Biol. Chem. 274, 14648-14654[Abstract/Free Full Text]
46. Rümenapp, U., Geiszt, M., Wahn, F., Schmidt, M., and Jakobs, K. H. (1995) Eur. J. Biochem. 234, 240-244[Abstract]
47. Schürmann, A., Schmidt, M., Asmus, M., Bayer, S., Fliegert, F., Koling, S., Mabeta mann, S., Schilf, C., Subauste, M. C., Vobeta , M., Jakobs, K. H., and Joost, H.-G. (1999) J. Biol. Chem. 274, 9744-9751[Abstract/Free Full Text]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.