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Molecular Endocrinology, doi:10.1210/me.2001-0220
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Molecular Endocrinology 16 (10): 2393-2404
Copyright © 2002 by The Endocrine Society

Dopamine-D2S Receptor Inhibition of Calcium Influx, Adenylyl Cyclase, and Mitogen-Activated Protein Kinase in Pituitary Cells: Distinct G{alpha} and Gß{gamma} Requirements

Behzad Banihashemi and Paul R. Albert

Ottawa Health Research Institute, Neuroscience, Departments of Medicine and Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario, Canada K1H-8M5

Address all correspondence and requests for reprints to: Paul R. Albert, University of Ottawa, 451 Smyth Road, Ottawa, Ontario, Canada K1H-8M5. E-mail: palbert{at}uottawa.ca.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The G protein specificity of multiple signaling pathways of the dopamine-D2S (short form) receptor was investigated in GH4ZR7 lactotroph cells. Activation of the dopamine-D2S receptor inhibited forskolin-induced cAMP production, reduced BayK8644- activated calcium influx, and blocked TRH-mediated p42/p44 MAPK phosphorylation. These actions were blocked by pretreatment with pertussis toxin (PTX), indicating mediation by Gi/o proteins. D2S stimulation also decreased TRH-induced MAPK/ERK kinase phosphorylation. TRH induced c-Raf but not B-Raf activation, and the D2S receptor inhibited both TRH-induced c-Raf and basal B-Raf kinase activity. After PTX treatment, D2S receptor signaling was rescued in cells stably transfected with individual PTX-insensitive G{alpha} mutants. Inhibition of adenylyl cyclase was partly rescued by G{alpha}i2 or G{alpha}i3, but G{alpha}o alone completely reconstituted D2S-mediated inhibition of BayK8644-induced L-type calcium channel activation. G{alpha}o and G{alpha}i3 were the main components involved in D2S-mediated p42/44 MAPK inhibition. In cells transfected with the carboxyl-terminal domain of G protein receptor kinase to inhibit Gß{gamma} signaling, only D2S-mediated inhibition of calcium influx was blocked, but not inhibition of adenylyl cyclase or MAPK. These results indicate that the dopamine-D2S receptor couples to distinct Gi/o proteins, depending on the pathway addressed, and suggest a novel G{alpha}i3/G{alpha}o-dependent inhibition of MAPK mediated by c-Raf and B-Raf-dependent inhibition of MAPK/ERK kinase.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
G PROTEIN-COUPLED RECEPTORS comprise a large superfamily of receptors that is critical for signaling of a diverse group of ligands to heterotrimeric G proteins (1). Activation of these receptors results in dissociation of G{alpha}- and Gß{gamma}-subunits, which couple to different effectors in the cell. Both the G{alpha}- and Gß{gamma}-subunits are capable of transferring receptor signals to effectors. The inhibitory Gi and Go proteins (Gi/o proteins) couple to adenylyl cyclase (AC) and inhibit production of cAMP (2, 3). Pertussis toxin (PTX) selectively blocks Gi/o proteins by ADP ribosylating the G{alpha}i/G{alpha}o-subunit (4). Different receptors utilize specific combinations of G protein subunits to elicit distinct responses in different cells (5, 6, 7). Understanding the roles of individual G proteins to couple receptors to distinct signaling pathways remains one of the central issues in receptor research (8).

The dopamine D2 receptor belongs to the Gi/o- coupled family of receptors and inhibits pituitary cell proliferation, transformation, and hormone production, and is implicated in neurobiological control of movement and behavior (9). The D2 receptor contains an alternately spliced exon encoding 29 amino acids to generate short (D2S) and long (D2L) forms of the receptor that are pharmacologically and functionally similar. By coupling to PTX-sensitive Gi/o proteins, the D2S receptor mediates inhibitory or stimulatory cellular responses, depending on the cell type (10, 11). In mesenchymal cells such as BALB/c-3T3, Chinese hamster ovary, or C6 glioma cells, the D2 receptor stimulates phospholipase C activity to induce calcium mobilization and activates the MAPK cascade. These actions correlate with enhanced gene transcription, cell proliferation, and oncogenic transformation (6, 12, 13, 14). In lactotrophs, D2S receptor activation opens potassium channels to hyperpolarize the cell membrane, blocks dihydropyridine-sensitive L-type calcium channels, inhibits cAMP production, and inhibits MAPK activation. These inhibitory actions correlate with D2-mediated inhibition of hormone secretion, gene transcription, and cell proliferation in lactotrophs (15, 16, 17, 18, 19, 20). In light of the cell type-specific coupling of D2S receptors, we hypothesize that different subunits of Gi/o proteins may mediate distinct D2S receptor signaling pathways.

As a model of D2 receptor signaling, we have used rat pituitary GH4C1 cells stably transfected with the D2S receptor (GH4ZR7 clone), a receptor absent in GH4C1 cells but present in normal lactotrophs (10, 15). GH4ZR7 cells express all three known G{alpha}i- subunits and two types of G{alpha}o, as well as various effectors that are regulated by PTX-sensitive G proteins (10, 17, 21). Thus, this cell system provides an excellent model to study the specificity of PTX-sensitive G proteins in coupling of D2S receptors to effectors. To address the contribution of specific G{alpha}-subunits in the D2S signaling pathway, PTX- insensitive mutants of G{alpha}i2, G{alpha}i3, and G{alpha}o, in which the ribosyl acceptor cysteine in the carboxyl-terminal region was changed to a nonaccepting serine, were stably transfected into GH4ZR7 cells. The Cys-to-Ser mutation is a structurally conservative change, and the mutant G proteins remain functional after PTX pretreatment (6, 7, 22). The role of Gß{gamma}-subunits in D2S signaling was evaluated by using the G protein- coupled receptor kinase C terminal (GRK-ct) as a selective Gß{gamma} scavenger (23). In this study, we focused on the role of G{alpha}i2-, G{alpha}i3-, G{alpha}o-, and Gß{gamma}-subunits in the inhibition of AC, L-type calcium channels, and MAPK pathway in GH4ZR7 cells. Our results indicate that each of these D2S-induced pathways has distinct requirements for different G{alpha}- or Gß{gamma}-subunits.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Expression of PTX-Insensitive G{alpha}i/o-Subtypes and GRK-ct in GH4ZR7 Cells
G{alpha}i2 and G{alpha}i3 PTX-insensitive mutants were Flag tagged in the N-terminal region, and these mutants, G{alpha}o-PTX, and GRK-ct cDNA were transfected individually in GH4ZR7 cells. Cell extracts from stably transfected clones and wild-type GH4ZR7 cells were analyzed for G protein expression by Western blot analysis (Fig. 1Go). Expression of G{alpha}i2-PTX and G{alpha}i3-PTX was confirmed by using anti-Flag antibody. The Gi2Z23 clone displayed a 2.9-fold increase in G{alpha}i2 immunoreactivity vs. GH4ZR7 cells (Fig. 1AGo). Since G{alpha}i3-specific antibodies were not available, anti-Flag staining revealed that G{alpha}i3 was expressed in two clones at levels similar to the G{alpha}i2 clone (Fig. 1BGo). Staining with anti-G{alpha}o antibody indicated that the G{alpha}oZ7 and G{alpha}oZ15 clones expressed G{alpha}o approximately 3.1- and 1.9-fold, respectively, compared with GH4ZR7 cells (Fig. 1CGo). Thus, the level of mutant G{alpha}i/o proteins was approximately equivalent to that of endogenous G{alpha}i2 or G{alpha}o. The expression of GRK-ct was confirmed using an antibody directed against full-length GRK2 (24). GRK2 was detected at the expected molecular size (70 kDa) in all cell lines, whereas GRK-ct was detected at 28 kDa only in the transfected clones. Expression of GRK-ct was about 40% and 50% of the GRK2 level for GRKZ16 and GRKZ17 clones, respectively (Fig. 1DGo).



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  		Figure 1. Protein Level of Stably Transfected PTX-Insensitive G{alpha}i/o Mutants and GRK-ct

Equal amounts of cell extracts (100 µg/lane) were subjected to Western blot analysis as described in Materials and Methods. GH4ZR7 cells (wild type) were compared with: A, GH4ZR7 cells expressing Flag-G{alpha}i2-PTX (Gi2Z23); B, Flag-G{alpha}i3-PTX (Gi3Z6 and Gi3Z15); C, G{alpha}o-PTX (G{alpha}oZ7 and G{alpha}oZ15); and D, GRK-ct (GRKZ16 and GRKZ17). The blots were probed with anti-Flag (A and B), anti-G{alpha}i2 (A), anti-G{alpha}o (C), anti-ß-actin (A–D), and anti-GRK-ct (D).

 
D2S-Induced Inhibition of Forskolin-Stimulated AC via G{alpha}i2
Under the present experimental conditions, the basal cAMP level in GH4ZR7 was very low and D2S receptor activation did not alter basal cAMP production (data not shown). Forskolin (1 µM) increased cAMP levels by 10-fold, and this was almost completely blocked by treatment with the D2 receptor agonist apomorphine (1 µM). Apomorphine-induced inhibition was reversed by pretreatment with PTX, indicating the role of Gi/o proteins in this signaling pathway. In GH4ZR7 cells stably expressing the Gß{gamma} blocker GRK-ct (GRKZ16 and GRKZ17), apomorphine-induced inhibition of forskolin-stimulated AC was not significantly different from the effect in GH4ZR7 cells, indicating no apparent role for Gß{gamma}-subunits in this pathway. The role of specific G{alpha}i/o proteins in D2S-induced inhibition of AC was evaluated in cells expressing specific PTX-insensitive G{alpha}-subunits (Fig. 2Go). In all clones apomorphine inhibited forskolin action to increase cAMP level, comparable to original GH4ZR7 cells. PTX pretreatment completely blocked D2S-mediated inhibition of cAMP in both G{alpha}o-PTX clones (G{alpha}oZ7 and G{alpha}oZ15) as observed in GH4ZR7 cells. However, in multiple experiments the G{alpha}i3-PTX (Gi3Z6 and Gi3Z15) and G{alpha}i2-PTX (Gi2Z23) clones were significantly resistant to PTX by 20 ± 1% and 65%, respectively. These results indicate that G{alpha}i2 and, to a lesser extent, G{alpha}i3 mediate D2S-induced inhibition of forskolin-stimulated cAMP production in GH4ZR7 cells, consistent with previous results (18).



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  		Figure 2. D2S-Induced Inhibition of Forskolin-Stimulated cAMP Requires G{alpha}i2

Cells were incubated with no drug, forskolin (1 µM), apomorphine (1 µM), or both, with or without pretreatment with PTX (20 ng/ml, 12 h) as indicated. Percent inhibition of apomorphine action was calculated as described in Materials and Methods and normalized to the value for GH4ZR7 (100%). The data are expressed as mean ± SEM of three independent experiments. In all clones, basal and forskolin-stimulated cAMP levels were not significantly different from corresponding levels in GH4ZR7 cells. GH4ZR7 clones are labeled as shown in Fig. 1Go.

 
G{alpha}o and Gß{gamma} Transduce Inhibition of Calcium Entry by D2S Receptors
Dopamine (10 µM) had no detectable effect on basal [Ca2+]i in GH4ZR7 cells but induced an immediate decrease in [Ca2+]i level after stimulation by the dihydropyridine BayK8644 (an L-type calcium channel agonist) (Fig. 3AGo), as observed previously (17). As an indicator of responsiveness, cells were challenged with TRH, which induced a PTX-insensitive Gq-mediated mobilization of calcium stores (25). Dopamine-induced inhibition of [Ca2+]i was completely blocked by PTX pretreatment, indicating signaling through Gi/o proteins. Blockade of dopamine action by PTX was not reversed in at least three independent experiments with G{alpha}i2-PTX and G{alpha}i3-PTX clones (Fig. 3Go, B–D), indicating that G{alpha}i2- and G{alpha}i3-subunits have a negligible role in mediating D2S-induced calcium channel inhibition. By contrast, dopamine-induced inhibition of BayK8644-stimulated [Ca2+]i level was completely rescued in both G{alpha}o-PTX-expressing clones after PTX pretreatment (Fig. 3Go, E and F), indicating a critical role for Go in D2S-mediated inhibition of L-type calcium channels. The role of Gß{gamma}-subunits in dopamine- mediated inhibition of [Ca2+]i was investigated in GRK-ct-expressing GH4ZR7 clones in which dopamine failed to decrease [Ca2+]i (Fig. 4Go). These results indicate that the D2S-induced decrease in [Ca2+]i level in GH4ZR7 cells depends on mobilization of Gß{gamma}- subunits from functional Go heterotrimers.



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  		Figure 3. G{alpha}o-Subunits Mediate Inhibition of Dihydropyridine-Induced [Ca2+]i

A, [Ca2+]i was measured in GH4ZR7 cells and GH4ZR7 cells expressing: B, G{alpha}i2-PTX (Gi2Z23); C and D, G{alpha}i3-PTX (Gi3Z6 and Gi3Z15); and E and F, G{alpha}o-PTX (G{alpha}oZ7 and G{alpha}oZ15). The cells were not treated (solid line) or treated (dashed line) with PTX (20 ng/ml, 12 h), and the change in [Ca2+]i level in response to BayK8644 (BayK, 100 nM), dopamine (10 µM), or TRH (100 nM) was measured (Materials and Methods). Arrows show the time of addition of drugs. Similar results were obtained in at least three independent assays for each clone.

 


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  		Figure 4. Expression of GRK-ct Blocks D2S-Mediated Inhibition of Calcium Entry

A, Change in [Ca 2+]i was measured in GH4ZR7 cells (wild type) and GH4ZR7 cells expressing GRK-ct protein (panel B, GRKZ16; and panel C, GRKZ17). Arrows indicate the addition of drugs as explained in Fig. 3Go. These results were reproduced in three independent assays.

 
Inhibition of TRH-Induced p42/44 Phospho-MAPK upon D2S Receptor Activation
Activation of MAPK was evaluated by measuring phosphorylation of MAPK, detected by Western blotting using an antibody specific for dual phosphorylated (activated) MAPK. In GH4ZR7 cells no basal phosphorylation of MAPK was detected after a 1-h incubation in fetal bovine serum-free medium (Fig. 5Go). Activation of the D2S receptor had no detectable effect on basal MAPK phosphorylation. TRH is known to activate MAPK in rat pituitary GH3 cells (26, 27, 28). In GH4ZR7 cells, TRH stimulated MAPK phosphorylation (Fig. 5Go), which was maximal within 7 min (data not shown). Upon activation of the D2S receptor by apomorphine, TRH-induced MAPK activation was almost completely blocked. This action of the D2S receptor was completely reversed by PTX pretreatment, implicating Gi/o proteins. PTX treatment had no detectable effect on basal (data not shown) or TRH-induced level of phospho-MAPK.



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  		Figure 5. TRH-Induced MAPK Activation Is Blocked by Apomorphine via Gi/o Proteins

GH4ZR7 cells were treated or not with PTX (20 ng/ml, 12 h) and incubated for 1 h in serum-free medium. For assay, cells were incubated with no drug (control), or pretreated with apomorphine (1 µM) for 15 min, after which TRH (1 µM) was added to wells for 7 min followed by cell lysis (Materials and Methods). Western blot analysis of lysates was done using specific antibody against phospho-p42/44 MAPK. Membranes were reprobed with ß-actin antibody as a loading control.

 
G{alpha}i3 and G{alpha}o Mediate D2S Inhibition of TRH-Induced Phospho-MAPK
The role of different Gi/o-subunits in D2S inhibition of the MAPK pathway was evaluated in different GH4ZR7 clones expressing PTX-insensitive G{alpha}-subunits (Fig. 6Go). Although apomorphine almost completely inhibited TRH-induced MAPK activation in GH4ZR7 or G{alpha}oZ7 cells, pretreatment with PTX revealed a partial (50%) rescue of the inhibitory response in the G{alpha}oZ7 cells but not in GH4ZR7 cells (Fig. 6AGo). Apomorphine almost completely inhibited TRH-induced MAPK activation in all clones compared with GH4ZR7 cells (Fig. 6Go, B and C), but upon pretreatment with PTX, differences among the clones were observed. In the G{alpha}i2-PTX-expressing clone (Gi2Z23), PTX pretreatment completely blocked this inhibitory effect, indicating that the G{alpha}i2-subunit is not involved in D2S-mediated inhibition of MAPK phosphorylation. Interestingly, PTX pretreatment failed to completely reverse the inhibitory action of apomorphine in both G{alpha}o-PTX- and G{alpha}i3-PTX-expressing clones, suggesting a role for Go and Gi3 in D2S-induced inhibition of MAPK activity. The importance of Gß{gamma}-subunits in D2S-induced inhibition of MAPK was evaluated using GRK-ct-expressing GH4ZR7 clones, GRKZ16 and GRKZ17 (Fig. 7Go). D2S-mediated inhibition of TRH-stimulated MAPK was identical to that observed in GH4ZR7 cells. To address whether GRK-ct influenced the sensitivity of D2S receptor action, MAPK inhibition was quantitated at a range of apomorphine concentrations (Fig. 8Go). Apomorphine inhibited TRH-induced MAPK with an EC50 of less than 1 nM. Concentration dependencies of apomorphine were not significantly different in GH4ZR7 cells and both GRK-ct clones, indicating that GRK-ct did not alter the sensitivity of TRH-induced MAPK phosphorylation to D2S receptor activation, suggesting that Gß{gamma}-subunits are unlikely to mediate inhibition of MAPK phosphorylation.



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  		Figure 6. G{alpha}o and G{alpha}i3 Mediate D2S Inhibition of TRH-Activated MAPK

A, Western blot showing an example of apomorphine-induced MAPK inhibition in G{alpha}oZ7 cells compared with original GH4ZR7 cells. Cells were pretreated with or without PTX (20 ng/ml for 12 h). Experimental compounds were added as indicated, and the level of phosphorylated MAPK was measured. B and C, D2S-induced MAPK inhibition was calculated as percent inhibition of TRH (1 µM)-induced MAPK activity produced by apomorphine (1 µM) compared with control, based on densitometric analysis of phospho-p44 (panel B) and phospho-p42 (panel C) MAPK bands. Data are expressed as mean ± SEM of at least three independent experiments.

 


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  		Figure 7. D2S-Induced MAPK Inhibition in GH4ZR7 Cells Expressing GRK-ct Protein

MAPK phosphorylation was measured in GRKZ16 and GRKZ17 cells, after addition of TRH and apomorphine as in Fig. 5Go. Densitometric analysis data are expressed as mean ± range (n = 2) of the percent inhibition by apomorphine of TRH-stimulated phospho-MAPK (p44 or p42, as indicated) compared with control GH4ZR7 cells (=100%).

 


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  		Figure 8. Concentration Dependence of Apomorphine Inhibition of TRH-Induced MAPK Phosphorylation Is Similar in GH4ZR7 Cells and GRK-ct-Expressing Clones

A, Western blots illustrating a sample of apomorphine (10-9 to 10-6 M) inhibition of TRH-induced MAPK activation in GH4ZR7 and GRKZ17 clones. B, D2S-induced MAPK inhibition was calculated as percent inhibition of TRH (1 µM)-induced MAPK activity produced by different concentrations of apomorphine compared with control, and plotted as a concentration-dependence curve for GH4ZR7 cells (wild type) and GRKZ16 and GRKZ17 clones. Each value represents the mean ± SD of three independent experiments.

 
Apomorphine Decreases Phospho-MAPK/ERK Kinase (MEK)1/2, c-Raf, and B-Raf Kinase Activity
To further investigate upstream protein kinases involved in D2S inhibition of MAPK phosphorylation, regulation of MEK1/2 phosphorylation was examined (Fig. 9Go). Apomorphine induced a complete inhibition of TRH-stimulated MEK1/2 phosphorylation with a concentration dependence identical to that for inhibition of p42/44 MAPK, indicating that apomorphine inhibits TRH-stimulated MAPK via inhibition of MEK1/2 in rat pituitary cells. Next, actions of the D2 receptor on c-Raf and B-Raf, which phosphorylate MEK1/2, were examined (Fig. 10Go). Each kinase was separately immunoprecipitated from cell lysates and assayed in vitro using a coupled assay involving sequential phosphorylation of recombinant MEK and MAPK. Using this immunoprecipitation/kinase assay, we detected basal activity of both c-Raf and B-Raf after 1 h serum starvation of GH4ZR7 cells. TRH selectively increased c-Raf kinase activity (164 ± 11% of basal) but had no effect on B-Raf kinase activity (104 ± 16% of basal). Apomorphine inhibited TRH-induced c-Raf kinase activity back to the basal level (84 ± 12% of basal), while apomorphine alone inhibited c-Raf activity to 70% of basal activity (data not shown). Thus, D2S receptor mediated a complete inhibition of TRH-induced c-Raf activation. In contrast, B-Raf kinase activity was not significantly induced by TRH treatment in GH4ZR7 cells, but was decreased by apomorphine in the presence (42 ± 9% of basal) or absence (45%, not shown) of TRH. These results indicate that both c-Raf and B-Raf are involved in D2S action, but that reduction of c-Raf activity is more important for inhibition of TRH action, while inhibition of B-Raf appears to be more important to regulate basal MAPK activity.



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  		Figure 9. Dopamine D2 Stimulation by Apomorphine Inhibits TRH-Induced Phosphorylation of MEK1/2 in GH4ZR7 Cells

Western blot showing the effect of different concentration of apomorphine on GH4ZR7 cells on MEK1/2 phosphorylation. Cells were pretreated with the indicated concentration of apomorphine for 15 min and then subjected to TRH stimulation for 7 min at 37 C. Each figure is representative of three independent sets of experiments.

 


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  		Figure 10. D2S Stimulation Decreases c-Raf and B-Raf Kinase Activity

Cells were treated with TRH (1 µM) and/or apomorphine (Apo, 1 µM) or untreated (Basal) as explained in Fig. 5Go. Equal amounts of lysate protein were immunoprecipated using anti c-Raf or anti-B-Raf. Kinase activity in the precipitate was measured by kinase cascade assay (see Materials and Methods). Buffer alone (CTL) or constitutively active B-Raf protein (B-Raf*) was used in the assay as negative or positive controls, respectively. Above, Kinase activity was assayed by phosphorylation of GST-ERK2 detected by Western blot analysis. Total c-Raf and B-Raf in the immunoprecipitate were determined by reprobing with anti-c-Raf or anti-B-Raf antibody. Below, c-Raf or B-Raf kinase activity was calculated based on densitometric analysis of phospho-ERK2 bands compared with basal level. Data are shown as mean ± SEM of three independent experiments. *, P < 0.05 vs. Basal.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The dopamine-D2 receptor signals through multiple pathways in lactotrophs to regulate prolactin (PRL) secretion, synthesis, and cell proliferation (9, 29, 30). Dopamine-D2 agonists, such as bromocriptine, have been used in the clinic for many years to inhibit the growth of PRL-secreting pituitary adenomas (31, 32). A crucial role for the dopamine-D2 receptor in the antiproliferative action of dopamine in pituitary cells was identified in homozygous mice deficient in the gene encoding the D2 receptor, which develop pituitary adenomas (33, 34, 35). In contrast, mice that lack the gene encoding the dopamine transporter hypersecrete dopamine, resulting in pituitary hypotrophy due to a paucity of lactotrophs (36). Although of great importance in vivo, the detailed signaling pathways mediating the antiproliferative actions of D2 receptors in pituitary cells remain to be characterized. In contrast to the numerous stimulatory actions in mesenchymal cells, including MAPK activation and enhanced cell proliferation (6, 9), D2S activation causes inhibitory effects in pituitary cells (9, 10, 37). We expressed PTX-insensitive G{alpha}i/o protein mutants to evaluate the G protein specificity of D2S-induced inhibition of AC, calcium influx, and MAPK activity in GH4ZR7 cells.

Modulation of AC via Distinct G Proteins
Inhibition of AC by receptors that couple to Gi/o appears to be a ubiquitous pathway (3, 38) and is mediated by dopamine-D2S receptor activation in a wide variety of cell types (15, 17, 39). Using PTX-insensitive mutants in GH4ZR7 cells, G{alpha}i2 (and to a lesser extent G{alpha}i3) rescued inhibition of forskolin-stimulated cAMP production by D2S receptor activation after PTX treatment, whereas G{alpha}o was not involved in this pathway. These findings are consistent with previous studies in pituitary and fibroblast cells (6, 7, 18) that implicate G{alpha}i2 in regulation of forskolin-stimulated AC activity. Interestingly, selective depletion of G{alpha}i2 by stable expression of antisense G{alpha}i2 RNA in GH4ZR7 cells had little influence on D2S inhibition of Gs-stimulated AC (17). The different Gi specificity in coupling to Gs- or forskolin-induced AC in these cells could result from different G{alpha}i specificity for different subtypes of AC, or different states of activation (e.g. by Gs or forskolin) of a single AC subtype. Expression of GRK-ct in GH4ZR7 cells blocked D2S-mediated inhibition of calcium influx but did not change D2S-mediated inhibition of cAMP levels, indicating that mobilization of Gß{gamma}-subunits is not necessary for the latter signaling pathway (7). Inhibitory regulation of cAMP signaling via G{alpha}i2 could play a role in cAMP-dependent MAPK activation and PRL gene transcription and secretion (40, 41).

G Protein Specificity for Inhibition of Calcium Influx
In GH4ZR7 cells, D2S receptors mediated inhibition of L-type calcium channels via a PTX-sensitive pathway (17, 19, 42), as observed in lactotrophs (43). G{alpha}o-PTX, but not G{alpha}i2-PTX or G{alpha}i3-PTX, rescued D2S-mediated inhibition of dihydropyridine-sensitive calcium influx, indicating that the Go protein has the prominent role in L-type calcium channel inhibition. In agreement with these results, antisense depletion of G{alpha}o-, but not other G{alpha}i-subunits, reduced coupling of multiple receptors (including the D2S receptor) to inhibit BayK8644-stimulated calcium influx and PRL secretion (17, 21, 44). Inhibition of L-type calcium influx was also blocked by expression of GRK-ct in GH4ZR7 cells, indicating a prominent role for Gß{gamma}-subunits in this pathway. Although Go plays a crucial role in coupling to N-type calcium channels, a direct interaction between mobilized Gß{gamma}-subunits and the channel {alpha}1B-subunit actually transduces the receptor signal (45, 46). How Go regulates L-type channels remains unclear because both L-type channel {alpha}1C- and {alpha}1D- subunits fail to bind Gß{gamma} (47, 48). The L-type channel in GH3 cells may actually be heteromeric including at least one {alpha}1A-subunit to confer Gß{gamma} sensitivity, since expression studies have identified {alpha}1A, {alpha}1C, and {alpha}1D RNA in these cells (48). Activation of L-type calcium channels contributes, in part, to multiple stimulatory actions of TRH, including TRH-induced sustained calcium entry, MAPK activation, PRL secretion, and gene transcription (25, 28, 44). Hence, Go-mediated inhibition of L-type channel opening could play an important role in dopamine-induced inhibition of TRH action.

D2S-Induced Inhibition of TRH-Stimulated MAPK Activation
Our results demonstrate that among PTX-insensitive G{alpha} mutants, G{alpha}i3-PTX and G{alpha}o-PTX could partially rescue D2S inhibition of TRH-induced MAPK phosphorylation, indicating the crucial role of G{alpha}i3 and G{alpha}o in this pathway. Although activation of MAPK mediated by Gi-coupled receptors in mesenchymal cells is transmitted via Gß{gamma}-subunits (49, 50, 51), our results suggest that Gß{gamma}-subunits were not involved in D2S-induced MAPK inhibition in GH4ZR7 cells. Although GRK-ct blocked D2-induced inhibition of BayK8644-induced calcium entry in these cells, we cannot rule out the possibility that Gß{gamma}-subunits not blocked by GRK-ct expression are mediating D2S receptor action to inhibit MAPK phosphorylation.

The signaling pathway from G{alpha}i/o proteins to inhibition of MAPK phosphorylation is not yet known. In neuronal cells, activation of a Gi/o-coupled receptor may inhibit MAPK phosphorylation by lowering cellular cAMP levels (52, 53). However, TRH has no effect on cAMP formation in these cells, and D2S receptor activation did not affect basal cAMP levels under our conditions. TRH-induced MAPK activation is complex and appears to involve calcium- and protein kinase C-dependent signaling to endocytosis, epidermal growth factor receptor activation, and initiation of the ras-c-Raf-MAPK pathway (26, 27, 54, 55) (Fig. 11Go). Consistent with this, in our experiments TRH induced c-Raf kinase and MEK1/2 activity but not B-Raf kinase activity. D2S activation completely blocked TRH- induced c-Raf activation and MEK1/2 phosphorylation, suggesting that TRH-induced signaling to MAPK converges at c-Raf activation. However, D2S receptor activation does not inhibit early upstream events such as TRH-induced phosphatidyl inositol turnover or calcium mobilization (56, 57). Consistent with this, TRH-induced Ser259 phosphorylation to desensitize c-Raf (58) was not inhibited by D2 receptor activation (data not shown). Hence, the most important site of D2S action to inhibit TRH-induced MAPK activation appears to be c-Raf activation, but not earlier Gq-mediated signaling events. D2S-mediated MAPK inhibition could, in turn, inhibit TRH-induced PRL gene transcription and PRL synthesis, which is mediated by a ras-Raf-MAPK-Ets pathway (28, 41, 59, 60).



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  		Figure 11. Pathways of D2S-Induced Inhibition of MAPK in GH4ZR7 Cells

A summary of D2S receptor actions on basal and TRH-induced MAPK is presented. TRH (via Gq) stimulates MAPK via Shc/GRB2-dependent and protein kinase C-dependent pathways. Our data indicate that both G{alpha}i3 and G{alpha}o mediate D2S inhibition of TRH-induced MAPK via inhibition of the c-Raf pathway perhaps by activating tyrosine phosphatase SHP-1, leading to decreased phosphorylation of MEK1/2 and MAPK. The D2S may utilize G{alpha}o-induced activation of RapGAP (GTPase activating protein) to inhibit rap and decrease B-Raf activity, as reported for other systems. See Discussion for further details.

 
Based on our results, the D2S receptor inhibits MAPK phosphorylation in at least two ways (Fig. 11Go). Apomorphine pretreatment completely blocked TRH-induced c-Raf activation, suggesting that the D2 receptor, via activation of G{alpha}i3 or G{alpha}o, may utilize a novel signaling pathway to inhibit TRH-induced MAPK activation via block of c-Raf-dependent MEK1/2 activation. Dopamine-D2S-induced phosphotyrosine phosphatase activity in GH4ZR7 cells is blocked by PTX (37) and could mediate Gi-induced inhibition of TRH signaling via tyrosine dephosphorylation of downstream signaling components such as the epidermal growth factor receptor to block ras-c-Raf activation (61). Consistent with this, Gi-induced activation of various phosphotyrosine phosphatase subtypes [Src homology 2 domain-containing protein tyrosine phosphatase (SHP-1), rat protein tyrosine phosphatase-{eta}] has been implicated in inhibition of cell proliferation by somatostatin (62, 63). Interestingly, G{alpha}i3 was specifically associated with SHP-1 after somatostatin treatment, suggesting a specific role for SHP-1 in G{alpha}i3-induced MAPK inhibition (63, 64). Additionally, the D2S receptor could act via G{alpha}o to inhibit the Rap1-GTP/B-Raf/MEK/MAPK cascade. Activation of G{alpha}o releases Rap1-GTPase activating protein to inhibit Rap1- induced B-Raf activation (65), providing an appealing mechanism for G{alpha}o-mediated D2S action. Although basal MAPK phosphorylation was not detectable (see also Ref. 20), a basal level of endogenous phospho-MAPK was present in B-Raf immunoprecipitates and was inhibited by apomorphine treatment (data not shown). A B-Raf-dependent mechanism could be important for D2-induced inhibition of basal MAPK activity or stimulation by cAMP or calcium, both of which activate B-Raf kinase activity (52, 66). These findings suggest that one possible pathway for D2S-induced MAPK dephosphorylation in rat pituitary cells is by activation of G{alpha}o-subunit and inhibition of B-Raf kinase activity that probably involves Rap-GTPase activating protein.

By mapping Gi/Go signaling pathways using PTX-insensitive or antisense approaches, we have defined differences in G protein specificity for particular actions, such as inhibition of cAMP, MAPK, or calcium channel activation. Using the antisense approach, we recently found that, in contrast to D2S inhibition of BayK8644-stimulated PRL secretion, which required Go primarily, inhibition of TRH-stimulated secretion required Gi2, Gi3, and Go (44). This suggests that recruitment of known Go- or Gi3-induced pathways, as well as Gi2-dependent signaling mediates inhibition of secretion. Utilization of PTX-insensitive or antisense G protein expression will provide a relatively nonperturbing method by which to identify G protein-induced signaling networks and their functional roles (8).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Materials
Apomorphine, dopamine, EGTA, forskolin, 3-isobutyl-1-methyl xanthine, PTX, puromycin, TRH, (+/-)-Bay K8644, vasoactive intestinal peptide (VIP), Sepharose G protein beads, anti-ß-actin, and anti-Flag antibody were from Sigma (St. Louis, MO); Fura-2 AM was purchased from Molecular Probes, Inc. (Eugene, OR); [125I]succinyl cAMP (2200 Ci/mmol) and polyvinylidene difluoride membrane were from NEN Life Science Products (Boston, MA); enhanced chemiluminescence detection kits were from Amersham Pharmacia Biotech (Arlington Heights, IL); sera and media were obtained from Life Technologies, Inc. Endonucleases and DNA polymerase were purchased from New England Biolabs, Inc. (Boston, MA); anti-G{alpha}o was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); anti-G{alpha}i1/2 was obtained from Calbiochem (San Diego, CA); anti-phospho-p42/44 MAPK antibody (T202/Y204) and anti phospho-MEK 1/2 (Ser 217/221) antibodies were from New England Biolabs, Inc. Anti-B-Raf, anti-c-Raf and Raf kinase cascade assay kit were from Upstate Biotechnology, Inc. (Lake Placid, NY). Polyclonal antibody against recombinant bovine GRK2 was kindly provided by Dr. J. L. Benovic (24).

Cell Culture and Transfection
GH4ZR7 cells and derivative clones were maintained in Ham’s F10 medium with 8% fetal bovine serum at 37 C, 5% CO2. PTX-insensitive G{alpha}i/o mutants and His-GRK-ct were constructed previously (7). G{alpha}i2-PTX and G{alpha}i3-PTX were Flag tagged at the initiator ATG codon and subcloned in KpnI/EcoRI-cut pcDNA3 (Invitrogen, San Diego, CA) to generate Flag-G{alpha}i2-PTX and Flag-G{alpha}i3-PTX, and their sequences were confirmed by DNA sequencing. These constructs were cotransfected individually (20 µg) with pGK-puro (2 µg) into GH4ZR7 cells using calcium phosphate coprecipitation. The transfected cells were cultured in F10 + 8% fetal bovine serum containing puromycin (20 µg/ml) for 3–4 wk. Antibiotic-resistant clones were picked (24 clones per transfection) and tested for expression of the corresponding G{alpha}i/o proteins by Western blot analysis.

Western Blot Analysis
Cells (3 x 105 cells per well) were harvested and resuspended by pipetting in 50 µl of RIPA-L buffer [10 mM Tris (pH 8), 1.5 mM MgCl2, 5 mM KCl, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate (SDS), 0.5% sodium deoxycholate, 5 µg/ml leupeptin] and incubated on ice for 30 min. The lysate was centrifuged (12,000 x g, 10 min, 4 C), and the supernatant was recovered and measured for protein content by the bicinchoninic acid protein assay kit (Pierce Chemical Co., Rockford, IL). Lysate was equally loaded and separated on 12% polyacrylamide gel and transferred onto polyvinylidene difluoride membrane. Blots were blocked overnight at 4 C, and then incubated at 4 C with primary antibody for 24 h, followed by a 45-min incubation with horseradish peroxidase-conjugated secondary antibody; the peroxidase product was developed using the enhanced chemiluminescence protocol.

cAMP Measurement
Equal numbers of cells were plated in six-well plates and grown to 70–80% confluence. The cells were incubated at 37 C in 1 ml/well of serum-free Ham’s F-10 medium-20 mM HEPES, pH 7.0–100 µM isobutylmethylxanthine, with or without experimental compounds. After 20 min the media were recovered and centrifuged at 12,000 x g for 2 min at 4 C. The supernatant was stored at -20 C for further analysis. Samples then were analyzed by a specific RIA to measure cAMP level. Percent inhibition was calculated as 100 - [100(D - C)/(S - C)], where C is cAMP level in nontreated cells, S is stimulated cAMP in forskolin-treated cells, and D is cAMP level in apomorphine/forskolin-treated cells. These values were normalized to control GH4ZR7 cells (=100%).

Measurement of [Ca2+]i
Cells were grown to 80% confluence in 15-cm plates and harvested with HEPES-buffered salt solution (HBSS) + EDTA. The cells were resuspended in 2 ml of HBSS + Ca2+ with 2.5 µM Fura-2 AM and incubated at 37 C for 30 min with gentle shaking (100 rpm) (7). Cells were washed twice and resuspended in 2 ml of HBSS + Ca2+ and subjected to fluorometric measurement of [Ca2+]i as described (7). Experimental compounds were added directly to cuvettes at times indicated in the figures. Because of fluorescent interference of the Fura-2 signal by apomorphine autofluorescence, dopamine was used in these experiments.

Measurement of Phospho-MAPK and -MEK1/2
Equal number of cells (3x105 cells per well) were plated in six-well plates. At 80% confluence, the cells were placed in serum-free Ham’s F-10 medium (1 h, 37 C). Cells were treated with the indicated drugs at 37 C, and after the indicated time the plates were transferred on ice and washed two times with cold PBS. Cells were lysed in 50 µl of 5x SDS loading buffer (500 mM Tris, pH 6.8; 2% SDS; 40 µl/ml 2- mercaptoethanol; 0.1% bromophenol blue; 10% glycerol), stored on ice, sonicated for 10 sec, and centrifuged at 12,000 x g for 5 min at 4 C. The supernatant (30 µl) was heated (100 C, 2 min) and rapidly cooled on ice. Samples were centrifuged 30 sec and were separated by SDS-PAGE, blotted on polyvinylidene difluoride membrane, and subjected to Western blot analysis. Phosphorylation was detected using (1:1000) anti-phospho-p42/44 MAPK or -phospho-MEK1/2 antibody. The corresponding band for p42 MAPK and p44 MAPK (collectively referred to as p42/44 MAPK) was digitally quantified using UN-SCAN-IT program (Silk Scientific Inc., Orem, UT). The results were normalized to the control.

Immunoprecipitation/Kinase Assay
The ability of c-Raf and B-Raf to activate MEK was measured by an immune complex-coupled assay in which recombinant glutathione-S-transferase (GST)-MEK1 activates and phosphorylates GST-p42 MAPK. Equal number of cells in six-well plates were serum starved for 1 h and then treated by indicated drugs and were lysed in buffer containing 1% Nonidet P-40; 50 mM Tris-Cl, pH 7.5; 150 mM NaCl; plus protease and phosphatase inhibitors. Lysates with an equal amount of protein were incubated with antibodies against c-Raf or B-Raf for 2 h at 4 C while rotating. Immune complex was collected with protein G-sepharose for 1 h at 4 C, centrifuged, and washed three times. The pellet was used for the following kinase assay. For each reaction, 20 µl of assay dilution buffer I [20 mM 3-(N-morpholino)propanesulfonic acid, pH 7.2; 25 mM ß-glycerophosphate; 5 mM EGTA; 1 mM sodium orthovanadate; 1 mM dithiothreitol], and 10 µl of Mg/ATP cocktail (75 mM MgCl2 and 500 µM ATP in assay dilution buffer I) were added to dephosphorylated GST-MEK1 and GST-MAPK2 plus immunoprecipitate or active B-Raf as a positive control. After 30 min shaking at 30 C, the reaction was terminated by adding SDS-loading dye, boiled for 2 min, and loaded on SDS-PAGE. Specific phospho-GST-MAPK2 bands were detected with anti-phospho-MAPK to assay kinase activity.


   ACKNOWLEDGMENTS
 


   FOOTNOTES
 
This work was supported by the National Cancer Institute, Canada. P.R.A. holds the Novartis/CIHR Michael Smith Chair in Neurosciences.

Abbreviations: AC, Adenylyl cyclase; GRK-ct, G protein-coupled receptor kinase C terminal; GST, glutathione-S-transferase; HBSS, HEPES-buffered salt solution; MEK, MAPK/ERK kinase; PRL, prolactin; PTX, pertussis toxin; SDS, sodium dodecyl sulfate; SHP-1, Src homology 2 domain-containing protein tyrosine phosphatase.

Received for publication September 4, 2001. Accepted for publication July 10, 2002.


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G. Liu, L. Robillard, B. Banihashemi, and P. R. Albert
Growth Hormone-induced Diacylglycerol and Ceramide Formation via Galpha i3 and Gbeta gamma in GH4 Pituitary Cells. POTENTIATION BY DOPAMINE-D2 RECEPTOR ACTIVATION
J. Biol. Chem., December 6, 2002; 277(50): 48427 - 48433.
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