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INTRODUCTION |
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 G
12 and G
13 has been shown to
strongly increase the activity of a coexpressed PLD1 enzyme like the
constitutively active G
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 G
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 G
i- and G
q-type G
proteins but not for G
12-type proteins (21).
Furthermore, structural data suggest that an interaction of either
G
12 or G
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
G
12 and G
13 but not for other G
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 G
proteins, strong evidence is provided that G12 but not Gq
proteins mediate M3 mAChR-PLD coupling.
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EXPERIMENTAL PROCEDURES |
Expression Plasmids--
The pCis vectors carrying cDNAs for
wild-type G
q, G
12, G
13,
and the constitutively active G
q mutant,
G
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
G
q, G
12, G
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
-galactosidase LacZ, a
kind gift of Dr. T. Eschenhagen, was used.
Photoaffinity Labeling of G Proteins with
[
-32P]GTP-azidoanilide--
Synthesis and
purification of [
-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 [
-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 G
q and G
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).
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RESULTS |
Effects of Various G
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 G
12,
G
13, and G
q as well as the constitutively
active G
q R183C. In cells overexpressing either
G
12 or G
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 G
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 G
12 or G
13 (Fig.
2). On the other hand, basal and
carbachol-stimulated PLC activities were not altered by the
overexpression of G
12 or G
13 (Fig.
3A) but were enhanced by
3-4-fold in cells overexpressing G
q (Fig.
3B). Expression of the constitutively active
G
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 G
q R183C
(Fig. 4B).

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Fig. 1.
Effects of overexpression of various
G 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
G 12, G 13 (A), or
G 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 G 12, G 13,
and G q with specific antibodies. Data are representative
of 3-5 experiments.
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Fig. 2.
Lack of effect of
G 12 and
G 13 on PLD stimulation by
PMA. M3 mAChR-expressing HEK-293 cells were
transfected with empty expression vector (Control) or with
expression plasmids for G 12 or G 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.
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Fig. 3.
Effects of overexpression of various
G 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
G 12, G 13 (A), or
G 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.
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Fig. 4.
Effects of expression of
G 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 G 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).
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To investigate whether the M3 mAChR is able to activate
Gq- and G12-type G proteins, the incorporation
of the photoreactive GTP analog [
-32P]GTP-azidoanilide
into G
q and G
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 [
-32P]GTP-azidoanilide into
G
q proteins (Fig.
5A). Binding of the GTP analog
to G
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.

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Fig. 5.
M3 mAChR-induced activation of
G q and
G 12. Photoaffinity labeling
of G proteins with [ -32P]GTP-azidoanilide was
performed in membranes of HEK-293 cells overexpressing either
G q (A) or G 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, G q
(A) and G 12 (B) proteins were
immunoprecipitated with the antisera AS 370- and AS 233-specific for
G q and G 12, respectively, and the
precipitates were resolved by SDS-polyacrylamide gel electrophoresis.
PhosphorImager images (Molecular Dynamics) of representative
experiments are shown.
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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
G
q and G
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).

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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.
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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.
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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).

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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.
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DISCUSSION |
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 G
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 G
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
G
12 or G
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 G
12 and
G
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 G
12 or G
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 G
12,
G
13, or Lsc-RGS did not alter phorbol ester-stimulated PLD activity, it can be concluded that these proteins, such as G
12 and G
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
[
-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 G
12 but not
G
q/11. However, both types of antibodies suppressed in an additive manner angiotensin II stimulation of PLC-
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 G
12 or
G
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 G
q proteins (37, 40-43). Also, in HEK-293 cells, the expression of mutationally activated G
12 and
G
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.