INTRODUCTION

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The family of G-protein-coupled receptors (GPCRs) is the largest and most complex group of integral membrane proteins involved in signal transduction. These receptors can be activated by a diverse array of external stimuli, including growth factors, vasoactive peptides, chemoattractants, neurotransmitters, hormones, phospholipids, photons, odorants, and taste ligands. Following ligand binding they promote the GDP-GTP exchange of heterotrimeric G proteins. In turn, GTP-bound  subunits and released complexes initiate a broad range of intracellular signaling events, including the activation of classical effectors such as adenylyl cyclases, phosphodiesterases, and phospholipases and the regulation of the activity of ion channels, ion transporters, and several kinases (22, 23, 41, 59). Recently, it has become increasingly apparent that, like receptor tyrosine kinases, GPCRs and G proteins are also involved in the regulation of cell growth and differentiation. A number of human proliferative diseases have been linked to mutations of GPCRs or G proteins (5, 15, 16). Furthermore, overexpression of constitutively active GPCRs or G proteins, as well as prolonged agonist stimulation of GPCRs, can induce cellular transformation in cultured fibroblasts (2, 15, 25).

The question of how GPCRs control signals that regulate gene expression in the nucleus, even though intensively studied during the last years, is not yet fully answered. It has been shown that GPCRs can activate mitogen-activated kinase (MAPK) pathways, which is sufficient and necessary for the control of proliferation in different cellular systems (26, 39). Mechanisms by which GPCRs activate MAPK cascades appear to be different. G_i-coupled receptors preferentially utilize a G-dependent route via phosphatidylinositol (PI) 3-kinase , Src, and Ras (12, 37). In contrast, G_q/11-coupled receptors employ protein kinase C (PKC) to directly target Raf-1 (33, 50) or calcium to activate the MAPK module via Pyk2, Src, and Ras (17, 34). Furthermore, in certain cells transactivation of epidermal growth factor (EGF) or platelet-derived growth factor receptors has been shown to be essential for extracellular-regulated-kinase (ERK) activation by G_i- as well as by G_q/11-coupled receptors (13, 30). The vast majority of the currently described pathways leading to MAPK stimulation have been considered as linear, initiated either by G_q/11 or G protein subunits (26, 57). However, most GPCRs can couple to several G proteins within a single cell (22, 23). For example m₁ and m₃ muscarinic receptors, ₂-adrenergic receptors, and receptors for thrombin and lysophosphatidic acid have been shown to stimulate G_i and G_q/11 proteins even though efficacies could differ among cell types (22, 23, 59). It is unknown at present whether one pathway initiated by a distinct G protein subfamily dominates over the other(s) or whether these receptors signal via parallel routes that might converge at a certain point. Coupling to multiple G proteins has rarely been considered with respect to MAPK activation, even though it should have an important impact on specific cellular responses elicited by GPCRs.

In order to address the question whether multiple G proteins are involved in ERK activation, we have analyzed signaling pathways linking the bradykinin B₂ receptor (B₂R) to the ERK/MAPK cascade. Even though often described as a prototypical G_q/11-coupled receptor, the B₂R can also catalyze the GDP-GTP exchange of G_i/o, G_s, and G_12/13 proteins (20, 27, 28, 35, 36). Activation of the ERK/MAPK cascade by bradykinin has been reported to occur via PKC and/or calcium-dependent pathways, involving the protein tyrosine kinases Pyk2 and Src or the EGF receptor (1, 17, 58, 62). In this study we demonstrate that a cooperative action of G_q/11 and G_i is required for efficient signal transmission to the ERK/MAPK cascade by the B₂R and other dually coupled GPCRs.

	MATERIALS AND METHODS

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Antibodies and reagents. An antiserum against the C terminus of ARK1/2 was provided by R. J. Lefkowitz (Durham, N.C.), and the antiserum against Csk was a gift from S. Courtneidge (Sugen, Inc.). A polyclonal antiserum against ERK2 and the hemagglutinin (HA) tag were provided by L. Rönnstrand (Uppsala, Sweden), and the antiserum against Raf-1 was from U. Rapp (Würzburg, Germany). Polyclonal antibodies against ERK2 (C-14), G_i proteins (C-10), G_q/11 proteins (C-19), and Src (N-16) were from Santa Cruz. The monoclonal pan-Ras antibody (R02120) was purchased from Transduction, and the phospho-specific antibodies against MEK (9121/9122) were from New England BioLabs. [³H]Palmitate and Rainbow protein marker were from Amersham, bradykinin was from Bachem; aprotinin (Trasylol©) was from Bayer, AG-X8 anion-exchanger resin was from Bio-Rad; leupeptin was from Boehringer Mannheim; AG1478, GF109203X, Genistein, Gö6976, Gö6983, LY294002, PD98059, and PP1 were from Calbiochem; Pefabloc was from Fluka; Lipofectamine and protein ladder markers (10 to 200 kDa) were from Life Technologies; the luciferase assay system kit was from Promega; ATP, carbachol, glutathione agarose, myelin basic protein (MBP), and all tissue culture reagents were from Sigma; thin-layer chromatography (TLC) plates LK5 D were from Whatman; and protein A agarose was from Zymed.

Plasmids. The following constructs were used: ARK-ct in pRK5 (from R. J. Lefkowitz); CD8-ARK1 in pcDNA3 (from S. Gutkind); human B₂R in pcDNA3 (from A. Pizard); Csk in pSV and kinase-inactive Src in pSGT (from S. Courtneidge); pFA-Elk1 and pFR-Luc reporter plasmids of the PathDetect Reporting System (from Stratagene); HA-ERK2 in pRK5 (from C. J. Marshall); G_i1Q204C, G_i2Q205L, and G_i3Q204L in pCDNA1 from (M. Faure); G_qQ209L and G₁₁Q209L in pCIS (from M. Simon); human m₁ muscarinic receptor in pCD-PS (from C. van Koppen); human m₃ muscarinic receptor in pcDNA3 (from A. Tobin); kinase-inactive Raf-1 and Raf1-RBD in pCMV5 (from W. Kolch); GST-Raf-1(1-149)-RBD in pGEX2 (from S. Taylor); pTKCIII and -galactosidase in pCMV5 (from J. Ericsson); and dominant-negative Ras (N17-Ras) in pZipNeo (from M. Karin).

Cell culture and transfections. Human embryonic kidney cells HEK293T were grown to about 50 to 70% confluence in Dulbecco modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS). Transfections were done in serum-free medium with the indicated cDNAs using 0.2 to 0.4 µg/well of a 24-well plate, 0.4 to 1 µg/well of a 12-well plate, 1 to 2 µg/well of a 6-well plate, or 10 µg/10-cm dish by the Lipofectamine method according to the supplier's manual. About 24 h after transfection cells were serum starved in DMEM containing 0.1% (wt/vol) bovine serum albumin (BSA) for another 24 h and then used for experiments. HF-15 human foreskin fibroblasts (provided by W. Müller-Esterl) were grown in DMEM containing 10% FBS. Mouse embryonic fibroblasts were kept in the same medium supplemented with 1 mM sodium pyruvate and nonessential amino acids. These cells were transfected using a modified calcium phosphate method according to the supplier's manual (MBS; Stratagene) with 1.2 to 1.6 µg cDNA/well of a 12-well plate.

PLC and PLD assays. Phospholipase C (PLC) activity was measured by analyzing cellular inositol phosphate accumulation (11). Cells grown on 24-well plates were labeled with 1 µCi of myo-[³H]inositol per ml for 24 h in inositol-free Ham's F-12 including 0.1% (wt/vol) BSA, treated for 10 min with 10 mM LiCl, and then challenged with 1 µM bradykinin for 10 min. Reactions were stopped by addition of 1 ml of ice-cold 10 mM formic acid. Water-soluble inositols were extracted for 2 to 12 h at 4°C and separated by anion-exchange chromatography using AG-X8 as a resin. Finally, isolated inositol phosphates were quantified by liquid scintillation counting.

ERK in vitro kinase reactions. ERK activity was analyzed as previously described (4). Briefly, serum-starved cells on six-well plates were stimulated with 1 µM bradykinin at 37°C, followed by lysis in 0.5 ml of ice-cold buffer containing 50 mM HEPES (pH 7.2), 150 mM NaCl, 1 mM EDTA, 20 mM NaF, 2 mM sodium orthovanadate, 1% (wt/vol) Triton X-100, 10% (wt/vol) glycerol, and protease inhibitors (1 mM Pefabloc, 10 µg of leupeptin per ml, and 1% Trasylol). Equal amounts of soluble fractions were subjected to immunoprecipitation with 5 µl of a polyclonal antiserum against the HA tag or ERK2 and 35 µl of protein A-agarose slurry for 3 h at 4°C. Resultant precipitates were washed three times with lysis buffer, followed by two washes with kinase buffer (10 mM Tris, pH 7.5; 10 mM MgCl₂). Kinase reactions were started by the addition of 30 µl of kinase buffer including 200 µM ATP, 1 µCi of [-³²P]ATP, and 10 µg of MBP. After 20 min at room temperature, reactions were stopped by the addition of 20 µl of sodium dodecyl sulfate (SDS) sample buffer and boiling for 2 min. Proteins were separated by SDS-12% polyacrylamide gel electrophoresis (PAGE), and gels were cut at the 30-kDa marker band. Upper parts were transferred onto nitrocellulose membranes and probed with 0.5 µg of anti-ERK2 antibodies per ml in 5% BSA in TBS (50 mM Tris, pH 7.6; 150 mM NaCl) to check for equal immunoprecipitation of HA-ERK2. Lower parts were stained with Coomassie brilliant blue R250 to monitor the amounts of MBP. Substrate phosphorylation was analysed using a phosphorimager (Fuji BAS2000). When dominant-negative constructs were used, Western blotting of total cell lysates with respective antibodies was performed to prove their expression as well as the equal HA-ERK2 levels. For statistic analysis, Student t tests for independent samples were performed using the QuickTTest program from S. Ashcroft.

Ras pulldown. The activation status of Ras was assayed using the glutathione S-transferase (GST) fusion protein of the Ras-binding domain (RBD) of Raf-1 (positions 1 to 149) which has a high affinity for GTP-loaded, active Ras (53). Prior to the experiment, baits were prepared by incubating GST-Raf-1-RBD-containing bacterial lysates with glutathione agarose for 1 h at 4°C. Beads were washed three times with lysis buffer (20 mM Tris, pH 7.5; 100 mM NaCl; 10 mM NaF; 10 mM MgCl₂; 1 mM sodium orthovanadate; 1% [wt/vol] Triton X-100; 10% [wt/vol] glycerol; and protease inhibitors [see above]) and stored on ice. Starved and bradykinin-treated cells were lysed in 500 µl of lysis buffer, and soluble fractions (1 to 2 mg of protein) were incubated with RBD baits for 1 h at 4°C. After three washing steps (lysis buffer with 0.5% Triton X-100), 30 µl of SDS sample buffer was added and samples were boiled for 2 min. Precipitated Ras was analyzed by SDS-15% PAGE, followed by Western blotting using 1 µg of a monoclonal pan-Ras antibody per ml.

Elk1 reporter gene assay. Luciferase assays were done using pFA-Elk1 and pFR-Luc reporter plasmids of the PathDetect Reporting System (Stratagene) and the Luciferase Assay System kit (Promega). HEK293T cells grown on 24-well plates to 50 to 70% confluence were transfected using the Lipofectamine method, with the following amounts of cDNA per well: 100 ng of B₂R, 50 ng of -galactosidase (pCMV5-Gal), 25 ng of luciferase (pFR-Luc), and 25 ng Elk1 (pFA-Elk1). Mouse embryonic fibroblasts grown on 12-well plates were transfected using a modified calcium phosphate method (MBS; Stratagene) with the following amounts of cDNA per well: 250 ng of B₂R, 500 ng of pCMV5-Gal, 125 ng of pFR-Luc, 60 ng of pFA-Elk1, and 565 ng of pTKCIII. After 4 to 6 h of incubation, transfection medium was replaced with regular culture medium, and cells were allowed to regenerate for 12 h. Thereafter, cells were serum starved for 24 h, stimulated with 1 µM bradykinin for 12 h, and lysed in 100 µl of 1% Triton X-100-10% glycerol-25 mM Tris phosphate (pH 7.8)-2 mM dithiothreitol-2 mM CDTA per well. Luciferase activity in cleared lysates was measured using the Luciferase Assay System from Promega and a Wallac 1420 multilabel counter. To determine transfection efficiency, parts of the lysates were analyzed for -galactosidase activity.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

The B₂R-mediated ERK2 and Elk1 activation in HEK293T cells involves pertussis toxin (Ptx)-sensitive G proteins and PKC. For biochemical studies on the mechanisms of GPCR-mediated ERK activation, we transiently coexpressed the B₂R with HA-tagged ERK2 and dominant-interfering constructs in HEK293T cells. B₂R expression was confirmed by [³H]bradykinin binding assays, demonstrating a single population of binding sites with an apparent K_d of 2 nM, a value which is similar to values obtained with cells with endogenous receptors (28, 48). Functional coupling to PLC was observed with a 50% effective concentration value for bradykinin of about 5 nM, which is close to the affinity of the B₂R for this agonist (data not shown). Furthermore, we have demonstrated in former studies that the B₂R is phosphorylated and sequestrated upon bradykinin treatment in HEK293T cells and that it is linked to the ERK/MAPK cascade (4, 47). Thus, the transiently expressed B₂R in HEK293T cells is functionally coupled to major signaling pathways and is pharmacologically similar to endogenous receptors on native cells.

View larger version (27K): [in this window] [in a new window]   FIG. 1.   Effects of Ptx and PKC inhibition on the B2R-mediated signal transduction in HEK293T cells. (A) HEK293T cells grown on six-well plates were transiently transfected with B2R (1 µg/well) and HA-ERK2 (0.5 µg/well) using the Lipofectamine method (6 µl/well). After 24 h of serum starvation and 16 h of Ptx or 30 min of GF109203X pretreatment with the indicated concentrations, 1 µM bradykinin (Bk) was applied for 5 min. Cells were lysed, and ERK2 activity was determined after immunoprecipitation (IP) with a polyclonal anti-HA antibody using MBP as a substrate. Western blots (WB) with polyclonal ERK2 antibodies (0.5 µg/ml) were performed to confirm that equal amounts of ERK2 were present in the precipitates, and phosphorylated MBP was detected by autoradiography and quantified by phosphorimager analysis (Fuji BAS2000). MBP phosphorylation is expressed as a percentage of the signal obtained in the particular experiment in the absence of any inhibitor. In general, the background of basal ERK activity was about 2 to 10% of the signal seen with agonist stimulation. Representative autoradiograms and blots from three experiments with identical results are shown. (B) To analyze Elk1 activation, a reporter gene assay was used. Luciferase activities in cells stimulated with 1 µM bradykinin for 12 h were measured and compared with those obtained after pretreatment with Ptx (50 ng/ml, 16 h) or GF109203X (5 µM, 30 min) prior to agonist challenge. Means ± the standard deviation (SD) of three independent experiments performed in triplicates are shown. (C and D) In B2R-transfected HEK293T cells grown on 12-well plates, the increase of total inositol phosphates (PLC activation) and the production of phosphatidic acid, trapped as [3H]phosphatidylbutanol (PLD activation), were measured without or with 10 min of 1 µM bradykinin stimulation. Results were compared with those obtained under identical conditions but with inhibitor pretreatment prior to bradykinin challenge. Means ± the SD of triplicates from typical experiments are shown.

The B₂R-mediated ERK2 activation in HEK293T cells is independent of tyrosine kinases and G signaling. The inhibitory effect of Ptx on the B₂R-mediated ERK2 and Elk1 activation points to an involvement of G_i proteins. It is well established that mitogenic signaling of G_i is primarily mediated by subunits, which can associate with and activate PI 3-kinase  (12, 37). Subsequently, Src-family tyrosine kinases become involved, phosphorylating the adapter protein Shc that recruits Grb2-Sos complexes to the membrane, enhancing the GDP-GTP exchange of Ras, and finally linking to the ERK/MAPK cascade (18, 26). To elucidate the role of G subunits in B₂R signaling, we coexpressed increasing amounts of two sequestrating constructs, a minigene coding for the C terminus of -adrenergic receptor kinase 1 (ARK1), together with constant amounts of B₂R and HA-ERK2. In vitro kinase reactions after bradykinin challenge revealed a very small inhibitory effect of these constructs on ERK2, which was only observed with the highest expression levels (Fig. 2A). Cellular levels of the dominant-interfering proteins and HA-tagged ERK2 were monitored by Western blotting with respective antibodies. Since PI 3-kinase  is involved in B₂R signaling in human colon carcinoma cells (21) and to further exclude an involvement of G pathways, we blocked PI 3-kinase activity that is downstream of G subunits (37). Application of a concentration of up to 50 µM of the potent and specific PI 3-kinase inhibitor LY294002 did not decrease bradykinin-induced ERK2 activation (Fig. 2B), thus excluding a role of PI 3-kinase in bradykinin signaling to ERK/MAPK in HEK293T cells. Therefore, we considered it unlikely that G proteins or PI 3-kinase play a major role in the B₂R-mediated ERK activation, as was convincingly demonstrated for other GPCRs such as the LPA and the m₂ muscarinic receptor (26, 37).

View larger version (39K): [in this window] [in a new window]   FIG. 2.   The B2R-mediated ERK2 activation in HEK293T cells is independent of G, PI 3-kinase and tyrosine kinases. HEK293T cells grown on six-well plates were cotransfected with B2R (0.25 µg/well), HA-tagged ERK2 (0.1 µg/well), and increasing amounts of dominant-interfering constructs (0, 0.25, 0.5, 0.75, 1, and 1.25 µg of ARK-ct, CD8-ARK1, Src-K, or Csk per well). The total amount of cDNA was adjusted to 1.6 µg/well with respective empty expression vectors. After 24 h of serum starvation cells were stimulated with 1 µM bradykinin for 5 min and the ERK2 activity was analyzed by in vitro kinase reactions using MBP as a substrate. Levels of precipitated HA-ERK2 and expression of dominant-interfering constructs in total cell lysates (TCL) were controlled by Western blotting, with the respective antibodies diluted 1:400 to 1:2,000 (0.2 to 1 µg/ml) in 5% BSA-TBS. For inhibitor experiments, cells were pretreated with the indicated concentrations of LY294002 and genistein 30 min before bradykinin stimulation. Assays were repeated at least three times, and representative autoradiograms and blots are shown.

Ras and Raf are intermediates in the B₂R-induced ERK2 activation in HEK293T cells. The small G protein Ras is a common intermediate in GPCR-initiated mitogenic signaling. In particular, the link between Ptx-sensitive G proteins and ERK/MAPK was shown to be Ras dependent in several cell types. G_q/11-PKC can target the ERK/MAPK cascade via Ras as well, but also a direct activation of Raf-1 and MEK has been observed (17, 26, 33, 38, 50). To investigate the role of Ras and Raf in B₂R signaling, we coexpressed increasing amounts of dominant-negative RasN17 and two inhibitory Raf-1 constructs, a kinase-inactive mutant (Raf-K) and the Ras-binding domain of Raf (Raf-RBD), together with the B₂R and HA-tagged ERK2. All three constructs significantly decreased the bradykinin-mediated ERK2 activation in correlation with their expression levels (Fig. 3A). Thus, the B₂R utilizes Ras and its effectors, such as Raf, to transmit signals to the ERK/MAPK cascade in HEK293T cells.

View larger version (26K): [in this window] [in a new window]   FIG. 3.   The bradykinin-induced ERK2 activation is Ras and Raf dependent. (A) HEK293T cells grown on six-well plates were cotransfected with B2R (0.25 µg/well), HA-tagged ERK2 (0.1 µg/well), and increasing amounts of dominant-interfering Ras and Raf constructs (0, 0.25, 0.5, 0.75, 1, and 1.25 µg of RasN17, Raf-K, or Raf-RBD per well). The total amount of cDNA was adjusted to 1.6 µg/well with respective empty expression vectors. After serum starvation, cells were challenged with 1 µM bradykinin for 5 min and ERK2 activity was analyzed by in vitro kinase reactions after immunoprecipitation. Levels of HA-ERK2 and expression of dominant-interfering constructs were controlled by Western blotting, with the respective antibodies diluted 1:400 to 1:2,000 (0.2 to 1 µg/ml) in 5% BSA-TBS. We could not prove Raf-RBD expression since the protein does not cross-react with our Raf antibodies. (B) B2R-transfected HEK293T cells grown on 10-cm plates were starved for 24 h and stimulated for 1 min with 1 µM bradykinin. After lysis, Ras activation was analyzed by GST-Raf-1-RBD pulldown experiments using 1 to 2 mg of cell lysate and ca. 5 µg of GST-Raf-1-RBD bound to glutathione agarose. Bound Ras was eluted and analyzed by SDS-15% PAGE and Western blotting using a monoclonal Ras antibody (1 µg/ml). After antibody detection, the use of equal amounts of bait was checked by staining membranes with amido black. To indirectly monitor Raf activity, MEK phosphorylation was analyzed in total cell lysates using a phospho-specific antibody (pMEK; 0.2 µg/ml). To prove equal MEK levels, the membranes were stripped and blots were reprobed with a MEK antibody (MEK; 0.2 µg/ml). Experiments were repeated at least three times, and representative autoradiograms and blots are shown. (C) MEK phosphorylation and ERK2 in vitro kinase activity were measured after pretreatment of B2R- or B2R-HA-ERK2-expressing cells with 0.2 µM PMA for 20 h and 5 µM Gö6983 or 1 µM Gö6976 for 15 min. A representative experiment, including the control of MEK and HA-ERK2 levels, is shown.

Cooperation of G proteins leads to ERK2 activation in HEK293T cells. Our data obtained with the B₂R in HEK293T cells suggest a cooperation of signals derived from G_q/11 and G_i proteins in ERK/MAPK activation. To verify this hypothesis we cotransfected HEK293T cells with constitutive active mutants of heterotrimeric G proteins G_q, G₁₁, G_i1, G_i2, and G_i3, either alone or in combination with HA-ERK2. In every experiment HA-ERK2 and G protein expression were confirmed by Western blotting with the respective antibodies (Fig. 4A and data not shown). In Fig. 4A a representative experiment is shown, and Figure 4B displays a quantitative analysis of a range of independent experiments. Whereas a transfection with activated G_q alone was ineffective, mutant G₁₁ increased ERK2 activity 2.1 ± 0.6-fold, a result which is slightly above the levels of the G_i variants (Fig. 4). Coexpression of G_q with G_i2 and G₁₁ with G_i1, G_i2, or G_i3 amplified the signal seen with either of the G proteins alone. In most experiments the G₁₁-G_i2 combination was more potent than all others, resulting in a 4.9 ± 0.8-fold increase in ERK2 activity. These receptor-independent experiments indicate that a cooperation of G_q/11- and G_i-derived signals could lead to a more efficient activation of ERK/MAPK in HEK293T cells.

View larger version (20K): [in this window] [in a new window]   FIG. 4.   Cooperation of activated G proteins leads to a stimulation of ERK2 in HEK293T cells. To study G protein cooperation HEK293T cells grown on six-well plates were transfected with 1.4 µg of constitutively activated G mutants and 0.2 µg of HA-ERK2 per well. After 24 h of serum starvation, ERK2 activity was measured by in vitro kinase reactions and compared with cells transfected with empty expression vector (mock). (A) A representative experiment, including the control of HA-ERK2 levels in the kinase reactions, is shown. (B) Quantitative evaluation of data (means ± the SD) from up to 17 independent experiments. The increased activation of ERK2 by G11, Gi1, Gi2, and Gi3 and all G protein combinations was statistically significant as judged by Student t tests for independent samples (P < 0.001 to P < 0.05). The P values of all G11-Gi combinations were significantly above those of the individual G proteins (P < 0.001 to P < 0.05), Gq-Gi1 and Gq-Gi3 (P < 0.001 to P < 0.01).

G_q/11-G_i cooperation is observed in different cell types with other GPCRs. The preceding experiments with constitutively active G protein mutants suggest that the G protein cooperation observed for the bradykinin-induced ERK activation might have more general implications. Therefore, we tested additional G_i-G_q-coupled GPCRs, such as m₁ and m₃ muscarinic receptors, for their ability to activate ERK in HEK293T cells. We coexpressed these receptors with HA-ERK2 and challenged them with 1 mM carbachol after pretreatment with Ptx and GF109203X, respectively. Both inhibitors largely abolished the carbachol-induced ERK2 activation (Fig. 5A), suggesting a cooperative mechanism similar to that observed with the B₂R (cf. Fig. 1 to 3).

View larger version (24K): [in this window] [in a new window]   FIG. 5.   Cooperative G protein signaling by muscarinic receptors in HEK293T cells and the endogenous B2R in human fibroblasts. (A) HEK293T cells were cotransfected with m1 or m3 muscarinic receptors (m1R and m3R) and HA-ERK2. After serum starvation, control and inhibitor-treated cells (16 h, 50 ng of Ptx per ml, and 30 min, 5 µM GF109203X) were stimulated with 1 mM carbachol, and ERK2 activity was analyzed by in vitro kinase reactions. HA-ERK2 levels were checked by Western blots, and kinase activities expressed as the fold increase of unstimulated cells of a representative experiment are shown. (B) Serum-starved HF-15 cells grown on 10-cm plates were pretreated with Ptx (16 h, 50 ng/ml) or GF109203X (30 min, 5 µM). B2R-induced Ras activation was analyzed by GST-Raf-1-RBD pulldown after challenge with 1 µM bradykinin for 1 min. Results were compared with those obtained without inhibitor application. In the same lysates, Raf activation was monitored by analyzing MEK phosphorylation with phospho-specific antibodies. In addition, ERK activity after 5 min of 1 µM bradykinin stimulation was measured by in vitro kinase reactions. A representative result from three independent experiments, including quantitative analysis of the increase of kinase activity compared to unstimulated cells, is shown.

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View larger version (22K): [in this window] [in a new window]   FIG. 6.   B2R signaling in Gq/11-deficient cells. (A) Stable B2R-expressing clones of wild-type mouse embryonic fibroblasts (MEF/B2) and Gq/11-deficient (q/11/B2) cells (200 to 600 fmol of B2R/mg of protein) were analyzed for the Gi and PKC dependence of bradykinin-induced ERK activation by performing in vitro kinase reactions. Means ± the SD from 6 to 10 independent experiments are presented. Furthermore, a typical set of in vitro kinase reactions, including blots of precipitated ERK is shown. (B) To analyze Elk1 transcriptional activation in parental (MEF) and Gq/11-deficient cells (q/11/), a reporter gene assay was used. Cells grown on 12-well plates were cotransfected with B2R and respective reporter constructs. Luciferase activities in cells stimulated with 1 µM bradykinin for 12 h were measured and compared with those obtained after pretreatment with Ptx (16 h, 50 ng/ml) or GF109203X (30 min, 5 µM) prior to agonist challenge. Means ± the SD of three independent experiments performed in triplicates are presented.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Many GPCRs can simultaneously couple to members of various G protein families. Furthermore, several cells express more than one subtype of a particular receptor, each potentially able to stimulate different G proteins (22, 59). Therefore, the activation of several G protein subtypes within a single cell at the same time reflects a physiological relevant situation. Mutual interactions between the signal transduction pathways initiated by different G protein subtypes are well established (22). One such example is the m₃ muscarinic receptor for which a G_i-mediated potentiation of the G_q-dependent PLC activation has been described (49). The aim of our study was to investigate how dually G_i-G_q/11-coupled receptors activate the ERK/MAPK cascade, one of the major pathways that mediates mitogenic effects of GPCRs. The majority of the mechanisms of GPCR-induced ERK/MAPK activation has been considered as linear, initiated either by subunits of Ptx-sensitive G proteins or by G_q (26). By coexpression studies in HEK293T cells and using inhibitory compounds for key elements of signal transduction pathways, we have obtained evidence for a cooperation of G_i and G_q/11 proteins in ERK/MAPK activation by the B₂R. Both, Ptx-sensitive G proteins and PKC were identified as being indispensable for a significant stimulation of ERK2 and transcriptional activity of Elk1. A similar situation has been reported for the ₂-adrenergic receptor expressed in CHO cells, where PKC was found to be downstream of Ptx-sensitive G_o proteins (56). However, we failed to detect G_o expression in HEK293T cells, thus excluding such a pathway as an explanation for our findings (data not shown and reference 58). Della Rocca et al. have suggested PLC as a common intermediate in the signal transduction of G_q-coupled _1B-adrenergic and G_i-coupled _2A-adrenergic receptors toward ERK/MAPK in HEK293 cells (14). We observed only a marginal effect of Ptx on the B₂R-induced inositol phosphate accumulation, indicating only a minor contribution of G_i to PLC activity which is unlikely to account for the strong inhibition of ERK by Ptx. In addition, PLD stimulation was not modified by G_i inhibition, whereas it was efficiently blocked by GF109203X. Therefore, we consider G_i-mediated signals to be independent from those carried by the G_q/PLC/PKC cascade induced through the B₂R. However, both pathways are necessary for efficient activation of the ERK/MAPK cascade.

Dual regulation of the ERK/MAPK module has only been observed for a limited number of GPCRs. Chen et al. have revealed that G_i and G_q-PKC signals mediate ERK1/2 activation by the calcitonin receptor expressed in HEK293 cells. They found that both pathways contribute equally to ERK activation and suggest a convergence at the level of the adapter protein Shc (8). Coexpression of the human anaphylatoxin C5a receptor together with the G_q family member G₁₆ has been shown to result in a PKC-dependent amplification of the otherwise-moderate and solely G_i-mediated ERK activation in HEK293 cells (6). Recently, the B₂R exogenously expressed in COS-7 cells has been reported to activate ERK1 via parallel signals from PKC and transactivated EGF receptors (1). A common feature of these findings is the generation of two independent signals, which contribute in an additive manner to ERK/MAPK activation. In contrast, we have observed that inhibition of one signaling pathway activated by the B₂R is sufficient to completely block ERK and Elk1 activation, suggesting a cooperative interaction. Similar to Adomeit et al., we propose the Ras-Raf complex as a point of convergence for the two signaling branches (1). Bradykinin caused a weak activation of Ras in HEK293T and HF-15 cells, which was blocked by Ptx but only slightly affected by PKC inhibitors. In contrast, MEK phosphorylation that is presumably catalyzed by Raf decreased after both treatments. It has previously been shown that in some cells Ras is involved in the PKC-mediated stimulation of Raf (38, 44). Marais et al. found that phorbol ester treatment of COS cells results in a Ras-dependent Raf activation (38). Surprisingly, RasN17, which is thought to inhibit guanine nucleotide exchange factors, did not affect Raf-1 functions under these conditions. Furthermore, Marais et al. showed that PKC inhibitors block the m₁ muscarinic receptor-mediated Ras and Raf-1 stimulation. We failed to detect any Ras-GTP loading after phorbol ester treatment in HEK293T cells. Furthermore, Ptx but not GF109203X abolished the B₂R-mediated Ras activation in HEK293T and HF-15 cells. Therefore, the Ras activation we measured most likely results from G_i signaling and is independent of PKC. In addition, we have observed an inhibitory effect of RasN17 and of the Ras-binding domain of Raf-1, indicating a different mechanism in B₂R-expressing HEK293T cells. We propose that two signals are required for efficient ERK activation by the B₂R: a G_i-mediated Ras stimulation and the action of PKC on the Ras-Raf complex. This is similar to the model recently suggested by Chiloeches et al., who analyzed several GPCRs in rat ventricular myocytes and proposed a G_i-mediated priming of Ras-Raf for activation by PKC (9). The exact mechanism of the signal integration at the Ras-Raf levels remains to be further investigated.

It is well established that subunits of Ptx-sensitive G proteins play a major role in mediating signal transmission to Ras and MAPK cascades (12, 19, 55). We found that G-sequestrating constructs were largely ineffective in inhibiting ERK activation by the B₂R in HEK293T cells, suggesting instead an important function of G_i subunits. Furthermore, the Ptx-sensitive branch of B₂R signaling to the MAPK cascade involves neither protein tyrosine kinases nor PI 3-kinase, which is in contrast to results found for the majority of typical G_i-coupled receptors (26, 37). Recently, the -opioid receptor expressed in Jurkat T cells has also been shown to utilize a Ptx-sensitive but G- and PI 3-kinase-independent pathway to activate ERK and mobilize AP-1 transcription factors (29). Currently, it is unclear which intermediates connect G_i subunits with Ras/MAPK signaling, but the search for novel G protein interactions partners might give an answer. Recently, GTPase-activating proteins for Rap1 have been identified in yeast two-hybrid screenings as potential links between G_i/o and the regulation of MAPK cascades (32, 40). Therefore, GTPase-activating proteins or guanine nucleotide exchange factors might be potential candidates that could establish a junction between heterotrimeric G proteins and small GTPases such as Ras and Rap.

By coexpressing mutationally activated G subunits and HA-ERK2, we studied G protein cross-talk in a receptor-independent approach. Contradicting data have been published as to whether or not G protein  subunits can increase ERK activity (12, 19, 24, 31). The specific cell type and experimental conditions seem to have an important impact on the results obtained. We found in HEK293T cells that activated G_i subunits caused a rather weak ERK stimulation, whereas G_q was almost ineffective. Surprisingly, G₁₁, even though expressed at levels similar to those of other G proteins, was most potent in stimulating the ERK cascade. Considering the high degree of redundancy of G_q and G₁₁ in signal transduction (23, 46), further studies are needed to judge whether the apparently stronger ERK activation by G₁₁ reflects a property that distinguishes it from G_q in vivo. However, we cannot entirely exclude that slight differences in the expression levels of G proteins or an increased apoptosis induced by some constitutive active mutants (15) might also have an impact on their ability to activate ERK in these assays. In contrast to the rather weak effect of the different G protein subtypes when expressed alone, several combinations of G_q and G₁₁ with G_i proteins led to a considerable overadditive ERK activation. In agreement with a former study performed in COS cells (12), coexpression of G_q and G_i2 had a rather moderate effect compared to the G₁₁-G_i2 combination, which caused a four- to five-fold increase in ERK activity. Perhaps the most important observation is that the synergistic effects, at least for the G₁₁-G_i2 combination, were more than additive, indicating cooperative rather than parallel mechanisms in signaling. The fact that ERK activation measured with this approach was much lower than that obtained with stimulation of dually coupled GPCRs might reflect cellular desensitization and downregulation in response to chronic stimulation by constitutively activated G protein  subunits.

Using fibroblasts from G_q/11-deficient mice, we confirmed a requirement for dual G protein coupling in signaling by the B₂R. In different stable B₂R-expressing clones of control cells, bradykinin-induced ERK and Elk1 activation was sensitive to Ptx and GF109203X. In G_q/11-deficient cells, signaling of the B₂R to ERK and Elk1 was in general reduced and not affected by PKC inhibition, though still sensitive to Ptx treatment. Though Elk1 transcriptional activity was not quantitatively blocked by Ptx and GF109203X, the application of these substances led to a reduction to the same level that seen with the specific MEK inhibitor PD98059. These findings suggest a contribution of additional MAPK, such as the G_12/13-activated c-Jun N-terminal kinase to Elk1 regulation in mouse fibroblasts. The fact that there was still a considerable B₂R-mediated ERK stimulation detectable in G_q/11 knockout cells might reflect the redundancy of intracellular signaling and adaptation processes occurring during the embryonic development of G_q/11-deficient mice.

We have also provided evidence that dual G_q/11-G_i signaling is a more general mechanism implicated in GPCR signaling to ERK/MAPK cascades. ERK activities induced by m₁ and m₃ muscarinic receptors expressed in HEK293T and bombesin receptors in mouse embryonic fibroblasts were also sensitive to Ptx and GF109203X treatment. Identical observations have previously been reported for m₁ and m₃ receptors expressed in CHO cells (56, 60). Furthermore, we have observed a dual B₂R signaling in human fibroblasts (HF-15) and in H-69 small cell lung cancer cells (unpublished results), for which bradykinin acts as a potent mitogen (51). Given the fact that several G protein family members are commonly expressed in the same cell, their cooperation might contribute to the careful control of strength and specificity in signal transduction. We provide here a detailed molecular characterization of the pathways involved in the quantitative regulation of the ERK/MAPK pathway through cooperation of G_q/11 and G_i signals. In a recent study, Stam et al. suggested a requirement for signals from small G proteins of the Rho family, which are presumably activated by G_12/13 and G_q/11 PLC pathways in regulating the invasion of T-lymphoma cells into monolayers of rat embryo fibroblasts (52). Therefore, cooperative signaling by multiple G proteins might represent a novel concept implicated in the regulation of different cellular responses induced by GPCRs.