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Medical Research Service, Veterans Affairs Medical Center and Department of Behavioral Neuroscience, Oregon Health Sciences University, Portland, Oregon 97201
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Summary |
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Summary
Introduction
Procedures
Results & Discussion
References
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The D2-like dopamine receptors couple to a variety of
signal transduction pathways, including inhibition of adenylate
cyclase, mitogenesis, and activation of potassium channels. Although
these effects are mediated via pertussis toxin-sensitive G proteins, Gi/o, it is likely that some of these effects are
influenced by the release of G protein 
subunits. Type II
adenylate cyclase (ACII) is highly regulated by multiple biochemical
stimuli, including protein kinase C, forskolin, G protein 
subunits,
and G protein subunits. The ability of subunits to
activate this enzyme in the presence of activated s has
been particularly well characterized. Although stimulation by
subunits has been described as conditional on the presence of activated
s, subunits also potentiate ACII activity after
activation of protein kinase C. We created stable cell lines expressing
ACII and the D2L receptor, the D3 receptor, or
the D4.4 receptor. Activation of D2L or
D4.4 receptors, but not D3 receptors,
potentiated -adrenergic receptor/Gs-stimulated activity
of ACII, as measured by the intracellular accumulation of cAMP.
Similarly, stimulation of D2L or D4.4 receptors
potentiated phorbol ester-stimulated ACII activity in the absence of
activated s, whereas stimulation of D3
receptors did not. The effect of D2-like receptor
stimulation was blocked by pretreatment with pertussis toxin and by
inhibition of protein kinase C. We propose that activation of both
D2L and D4.4 dopamine receptors potentiated phorbol-12-myristate-13-acetate-stimulated ACII activity through the
release of subunits from pertussis toxin-sensitive G proteins. In contrast, the lack of D3 receptor-mediated effects
suggests that stimulation of D3 receptors does not result
in an appreciable release of subunits.
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Introduction |
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Summary
Introduction
Procedures
Results & Discussion
References
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The D2-like receptor family is composed of D2, D3,and D4 dopamine receptors, which share considerable amino acid homology and generally have high affinity for butyrophenone and benzamide ligands. The most striking differences observed among these receptors are found in their ability to activate pertussis toxin-sensitive signaling events. For example, D2 and D3 receptors inhibit dopamine synthesis in a dopamine-producing cell line, whereas D4 receptors do not (1, 2), and D2 and D4 receptors mediate robust inhibition of cAMP accumulation in a variety of cell lines, whereas inhibition of cAMP accumulation by the D3 receptor is modest or absent (2-5).1 On the other hand, D2, D3, and D4 receptors all activate K+ channels in Xenopus laevis oocytes and stimulate mitogenesis in Chinese hamster ovary cells in a pertussis toxin-sensitive manner (3, 6). Thus, all D2-like receptors couple to pertussis toxin-sensitive pathways, but the efficiency and specificity of coupling are not identical for all subtypes.
ACII is widely expressed in the central nervous system, and its
activity is regulated by a variety of biochemical signals (7-9).
Although ACII is not inhibited by Gi (10), it is
stimulated by s (11), phorbol esters (12), and G protein
subunits (13) in reconstituted systems. Additionally, ACII is
synergistically activated by s and PMA (8, 12) as well
as by s and subunits (11, 13, 14). In intact
cells, ACII is activated by subunits in combination with
activated s, whereas stimulation alone has no
detectable effect on ACII activity (7, 9, 15-17). In those studies
subunits were supplied by stimulating Gi/o-coupled receptors (e.g., the D2 dopamine receptor), and activated
s was provided by stimulating Gs-coupled
receptors or by co-transfection with a constitutively active mutant of
s, s-Q227L. The synergy between activated
s and either PMA or G protein subunits has led to
ACII being described as a coincidence detector that integrates multiple
signals (8, 18). Further, liberation of subunits via
activation of Gi/o-coupled receptors enhances ACII
stimulation by Gq-coupled receptors or phorbol esters (19).
The divergent signaling pathways of the D2-like dopamine
receptors and the unique regulatory properties of ACII provide the basis for the current study. We examined and compared the ability of
D2, D3, and D4 dopamine receptors
to potentiate (presumably via subunits)
isoproterenol- and PMA-induced activation of ACII. To this end, we
created cells stably expressing ACII and the D2L dopamine
receptor (ACII/D2L), ACII and the D3 dopamine receptor
(ACII/D3), or ACII and the D4.4 dopamine receptor
(ACII/D4). We now report that D2 agonists potentiated
isoproterenol-stimulated cAMP accumulation in HEK293 cells expressing
the D2L or the D4.4 dopamine receptor together
with ACII, whereas the D3 receptor did not. Consistent with
the hypothesis that subunits enhance the responsiveness of ACII
to a variety of stimuli, we also found that activation of
D2L and D4.4 receptors potentiated protein kinase C-activated ACII activity.
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Experimental Procedures |
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Summary
Introduction
Procedures
Results & Discussion
References
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Materials. [3H]cAMP was purchased from Dupont NEN. Spiperone, quinpirole, and forskolin were purchased from Research Biochemicals International. HEK 293 cells expressing ACII (HEK-ACII) were obtained from Dr. Daniel Storm and Mark Nielsen (University of Washington, Seattle). Rat D2L (Dr. Olivier Civelli, University of California at Irvine) and human D4.4 cDNAs (Dr. Hubert Van Tol, University of Toronto, and Dr. David Grandy, Oregon Health Sciences University, Portland, OR) were generous gifts. Dopamine (3-hydroxytyramine) and most other reagents were purchased from Sigma Chemical (St. Louis, MO).
Production of cell lines. Creation of HEK-D2L, HEK-D3, and HEK-D4.4 cells was carried out by electroporation (0.17 kV, 950 µF, 0.4-cm cuvette gap). HEK 293 cells (8 × 106) were resuspended in DMEM supplemented with 10% FBS and 5 mM N,N-bis-(2-hydroxyethyl)-2-aminoethanesulfonic acid in a total volume of 400 µl, including pcDNA1-D2L cDNA (15 µg), pcDNA1-D3 cDNA (15 µg), or pcDNA1-D4.4 cDNA (15 µg) with pBabe Puro (2 µg), to confer resistance to puromycin (20). Transfectants were isolated and screened by radioligand binding as described previously (21). Creation of ACII/D2L, ACII/D3, and ACII/D4 cells, was carried out by transfection of HEK-ACII cells with pcDNA1-D2L, pcDNA1-D3, or pcDNA1-D4.4 as described above.
Cell culture. HEK 293 cells expressing D2-like receptors were maintained in DMEM supplemented with 5% FBS and 5% CBS, penicillin/streptomycin, and puromycin (2 µg/ml). ACII/D2L, ACII/D3, and ACII/D4 cells were maintained in DMEM supplemented with 5% FBS and 5% CBS, penicillin/streptomycin, puromycin (2 µg/ml), and hygromycin (460 units/ml). HEK-ACII cells were maintained in DMEM supplemented with 5% FBS and 5% CBS, penicillin/streptomycin, and hygromycin (460 units/ml). Cells were grown in a humidified incubator at 37° in the presence of 10% CO2.
cAMP accumulation assays. Cells were plated at densities between 100,000 and 150,000 cells/well in 48-well tissue culture clusters. Confluent cells were preincubated with 200 µl of assay buffer (Earle's balanced salt solution, containing 0.02% ascorbic acid and 2% CBS) for 10 min, then placed on ice. All drugs were added at 4°, then each cluster was transferred to a 37° water bath. After 15 min, the medium was decanted, and the cells were placed on ice and lysed with 3% trichloroacetic acid. The 48-well plates were then stored at 4° for at least 1 hr and centrifuged at 1000 × g for 15 min before quantification of cAMP. For pertussis toxin experiments, the toxin was added to the growth medium (25 ng/ml) 18 hr before the cAMP accumulation assay. This treatment has been determined to eliminate detectable coupling of D2 dopamine receptors to inhibition of cAMP accumulation (22).
Quantification of cAMP.
cAMP was quantified using a
competitive binding assay adapted with minor modifications from
Nordstedt and Fredholm (23). Duplicate samples of the cell lysate
(10-20 µl) were added to reaction tubes containing cAMP assay buffer
(100 mM Tris/HCl, pH, 7.4, 100 mM NaCl, 5 mM EDTA). [3H]cAMP (1 nM final
concentration) was added to each tube, followed by cAMP-binding protein
( 
100 µg of crude extract from bovine adrenal cortex in 200 µl
of cAMP buffer). The reaction tubes were incubated on ice for 3 hr. The
tubes were then harvested by filtration (Whatman GF/C filters) using a
96-well Tomtec cell harvester. Filters were allowed to dry, and
BetaPlate scintillation fluid (50 µl) was added to each sample.
Radioactivity on the filters was determined using an Wallac BetaPlate
scintillation counter. The concentration of cAMP in each sample was
estimated in duplicate assays from a standard curve ranging from 0.1 to
100 pmol cAMP/assay.
Data analysis. Dose-response curves for cAMP were analyzed by nonlinear regression using the program Prism 2.0 (GraphPad Software, San Diego, CA). Statistical comparisons were made using ANOVA followed by Dunnett's post hoc t test comparing control with drug groups, except where indicated in the figure legends.
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Results and Discussion |
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Summary
Introduction
Procedures
Results & Discussion
References
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We examined cAMP accumulation in HEK 293 cells expressing only the
D2L receptor. HEK-D2L cells were treated with
isoproterenol (100 nM) or PMA (100 nM) in the
absence or presence of dopamine (1 µM) or quinpirole (1 µM), and cAMP accumulation was determined. There was no
significant stimulation of cAMP accumulation above basal levels by
isoproterenol (100 nM) or PMA (100 nM),
reflecting the low level of adenylate cyclase activity that has made
the HEK 293 cell line valuable for the characterization of recombinant adenylate cyclases (24) (data not shown). We also examined cAMP accumulation in HEK 293 cells expressing ACII (HEK-ACII). We found that
isoproterenol acting via endogenously expressed
-adrenergic receptors stimulated cAMP accumulation 3-fold above
basal levels in HEK-ACII cells (data not shown). PMA, which is thought
to bypass Gs and activate ACII by protein kinase
C-dependent phosphorylation of the enzyme (12), stimulated cAMP
accumulation 40-fold above basal levels in HEK-ACII cells (data not
shown). The D2 agonists, quinpirole or dopamine, had no
effect on isoproterenol- or PMA-stimulated cAMP accumulation in
HEK-ACII cells (data not shown). However, when cAMP accumulation is
stimulated by forskolin in HEK-D2L cells, activation of D2L
receptors inhibits cAMP accumulation (22).
Potentiation of s-stimulated ACII by
D2L, D3, and D4.4 receptors.
To study the effects of subunits on s-stimulated
ACII activity we created cells stably expressing ACII and the
Gi/o-coupled dopamine receptors, D2L (ACII/D2L
cells), D3 (ACII/D3), or D4.4 (ACII/D4 cells).
In ACII/D2L cells, D2 agonists alone did not alter cAMP
accumulation (data not shown). In contrast, when dopamine (1 µM) or quinpirole (1 µM) was added in
combination with an activator of s (100 nM
isoproterenol), there was marked potentiation of isoproterenol-stimulated cAMP accumulation (Fig. 1 and
Table 1). This potentiation seemed to be mediated
via D2L receptors acting through a
Gi/o protein because it was blocked by the D2
antagonist, spiperone, and by pretreatment of the cells with pertussis
toxin (25 ng/ml for 18 hr; Table 1). The effect of dopamine on
isoproterenol-stimulated cAMP accumulation was dose-dependent, with an
EC50 value of 32 nM (Fig. 2).
Like the D2L receptor, the D4.4 receptor
inhibits the activity of endogenous adenylate cyclases in a variety of cell lines (e.g., see Ref. 5); recent work, however, has suggested that
the D2 and D4 receptors may act through
different pertussis toxin-sensitive G proteins (1). In light of this,
we examined whether the D4.4 receptor could stimulate ACII
activity. As we observed with the D2L receptor, activation
of the D4.4 receptor potentiated isoproterenol activation
of ACII in ACII/D4 cells (Fig. 1), and the effects were blocked by
spiperone or by pretreatment with pertussis toxin (Table 1).
Staurosporine did not prevent the D2-like receptor
potentiation of isoproterenol-stimulated activity, indicating that the
potentiation is not mediated by dopamine receptor activation of protein
kinase C (Table 1). In contrast to the effects of D2L and
D4.4 receptors, activation of D3 receptors was
without effect on cAMP accumulation (Fig. 1). The lack of a
D3-mediated effect does not seem to be due to low receptor
density. Although the ACII/D2L and ACII/D4 cells had receptor densities
of 600 and 1500 fmol/mg of membrane protein, respectively, we tested
nine ACII/D3 clones ranging in receptor density from 200-1300 fmol/mg
of protein, and none produced significant potentiation of
isoproterenol-stimulated cAMP accumulation in the presence of dopamine
agonists (data not shown). The data presented in Figs. 1 and
3 are from clone ACII/D3-17, which expressed the D3 receptor at a density of approximately 1100 fmol/mg of
membrane protein. Additionally, increasing the concentration of
dopamine agonists to 10 µM failed to result in
significant potentiation of isoproterenol-stimulated ACII activity in
ACII/D3 cells (data not shown).
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s (via Gs-coupled receptors or
co-transfection with a constitutively active s, s-Q227L), stimulates ACII (7, 9, 15, 16). The activation of Gi/o-coupled receptors releases subunits, which
in turn potentiate the activation of ACII by s. The role
of subunits is supported by the observation that co-expression
of t, which presumably sequesters released
subunits, blocks activation of ACII by Gi/o-coupled
receptors (7, 9, 17, 19). Moreover, reconstitution studies indicate
that ACII is directly stimulated by subunits in combination with
activated s (11, 13) and also by subunits alone,
albeit to a lesser extent (13). The subunits of Gi/o do
not directly modulate ACII, because transfection of constitutively
active mutants of these subunits has little effect on basal or
receptor-stimulated activity of ACII in HEK 293 cells (9).
D2 dopaminergic and 2-adrenergic receptors
are among the Gi/o-coupled receptors that activate ACII,
but they differ in their mode of activation (7). Unlike the
D2 receptor, the 2-adrenergic receptor
stimulates ACII in the absence of constitutively active s-Q227L. It has been suggested that the ability of the
2-adrenergic receptor to couple to Gs is
responsible for this difference between the two receptors (7).
Consistent with this suggestion, the activation of ACII by
D2 receptors was abolished by pretreatment with pertussis
toxin, whereas the activation by 2-adrenergic receptors
was not (7). In the present study, we have confirmed the finding that
potentiation of Gs-stimulated cAMP accumulation by
D2L receptors is blocked by pretreatment with pertussis
toxin and have extended this observation to the D4.4
receptor.
Potentiation of PMA-stimulated ACII by D2L,
D3, and D4.4 receptors.
Results from
reconstitution studies have demonstrated that protein kinase C
phosphorylates and activates ACII independently of s
(12). Furthermore, short term PMA treatment of intact cells does not
activate s, as assessed by reconstituted adenylate cyclase activity in the membranes of S49 cyc
cells (25).
To examine the hypothesis that subunits released by the
activation of Gi/o-coupled receptors can potentiate the actions of protein kinase C on ACII, we assessed the ability of dopamine agonists to enhance PMA-stimulated cAMP accumulation in
ACII/D2L, ACII/D3, and ACII/D4 cells. When stimulation by PMA (100 nM) was conducted in the presence of dopamine (1 µM) or quinpirole (1 µM), cAMP accumulation
was 2-4-fold greater, compared with PMA alone, indicating that
D2 agonists potentiated the actions of PMA in ACII/D2L
cells (Fig. 3 and Table 2). The effects of D2 agonists on PMA-stimulated cAMP accumulation were
blocked by co-incubation with spiperone (1 µM) and by
overnight treatment with pertussis toxin (Table 2). Analysis of dose
response curves revealed that the potentiation by dopamine was
dose-dependent, with an EC50 value of 132 nM
(Fig. 2). Similarly, activation of D4.4 receptors
potentiated PMA-stimulated cAMP accumulation in ACII/D4 cells, and this
effect was blocked by spiperone and by pretreatment with pertussis
toxin (Fig. 3 and Table 2). The effect of D4.4 receptor
activation on PMA-stimulated cAMP accumulation was also blocked by the
D4 antagonist clozapine (data not shown). We also observed
that pertussis toxin-treatment significantly reduced PMA-stimulated
cAMP accumulation in ACII/D2L and ACII/D4 cells. There was also a trend
for spiperone to decrease PMA-stimulated activity in both cell lines
(Table 2), and to decrease isoproterenol-stimulated activity in ACII/D4
cells (Table 1). These results may reflect constitutive activity of the
D2-like receptors and further suggest that spiperone is an
inverse agonist at these receptors.
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s (i.e.,
isoproterenol) or protein kinase C (PMA) in a pertussis toxin-sensitive
manner strongly suggests that activated s is not an
absolute requirement for stimulation of ACII by subunits, but
that the binding of subunits to ACII enhances the effects of
other activators. In studies with ACII/D2L and ACII/D4 cells, the
protein kinase C inhibitor, staurosporine, abolished both
PMA-stimulated ACII activity and the potentiation of that pathway by
dopamine agonists (Table 2), further indicating that the activation of
ACII by subunits requires co-activation of the protein kinase C
pathway. PMA activation of ACII was enhanced by subunits in both
ACII/D2L and ACII/D4 cells, but not in ACII/D3 cells.
The current study adds another divergent signaling pathway to the
D2-like dopamine receptor family. The results of these
studies are similar to those examining D2, D3,
and D4-mediated inhibition of cAMP accumulation in which
D2 and D4 receptors display robust inhibition
and D3 receptors show little or no effect (2-4). Thus, it
seems that the inefficient coupling of D3 receptors to
Gi/o proteins provides a concentration of subunits
that is not sufficient to potentiate s- or
PMA-stimulated ACII activity. Although D3 receptors couple
to several pertussis toxin-sensitive signaling events including
K+ channel conductance, mitogenesis, neurite outgrowth,
dopamine synthesis, and dopamine release, in many instances the
functional response to D3 receptor activation is reduced
compared with the response that is mediated by D2 and
D4 receptors (1, 2, 6, 26, 27). Specifically, the
muscarinic receptor-gated atrial potassium channel, Girk1, is activated
by D2, D3, and D4 dopamine
receptors, but the maximal current is 3-fold larger for D2
and D4 receptors than for D3 receptors (6).
Because Girk1 is also activated by subunits (28), the results of
the present study support the hypothesis that smaller current induced
by D3 receptor activation could be due to diminished
release of subunits. The reasons for the functional differences
among D2, D3, and D4 receptors are
largely unknown, but most likely reflect differences among the receptor
subtypes in the efficiency of activation of various G proteins.
The observation that D2L receptors potentiate the actions
of PMA on ACII is important considering the evidence that has linked D2 dopamine receptors and the protein kinase C pathway. For
example, D2 dopamine receptors have been shown to stimulate
phosphoinositide hydrolysis in Ltk fibroblasts expressing
D2 dopamine receptors (29). Thus, in Ltk
fibroblasts expressing ACII and D2 receptors, it is
possible that D2 agonists could stimulate cAMP accumulation
due to increased protein kinase C activity in combination with the
release of subunits from Gi/o. The protein kinase C
pathway has also been implicated in D2 receptor-potentiated
arachadonic acid release in Chinese hamster ovary cells and in
inhibition of cell proliferation in GH4ZR7
cells, because inhibitors of protein kinase C block both of these
D2 receptor effects (30, 31). Taken together, these
observations and the current study suggest that the interactions between the protein kinase C pathway and D2 dopamine
receptors are important in modulating neurotransmission in a variety of cell types.
In summary, we have demonstrated that activation of D2L and
D4.4 receptors potentiated isoproterenol- and
PMA-stimulated cAMP synthesis by ACII, whereas D3 receptors
did not. Furthermore, our data confirm that ACII can be synergistically
activated by multiple signals, including PMA and subunits,
s and subunits, or PMA and s.
However, the activation of ACII by subunits is conditional,
requiring co-activation by either protein kinase C or s,
suggesting that the binding of to ACII results in an enhancement
of responsiveness of the enzyme to other activators. Potentiation of
PMA-stimulated ACII activity by subunits represents another
example of coincident signal detection and may influence the
interactions between D2L and D4.4 dopamine
receptors and the protein kinase C pathway.
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Acknowledgments |
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We acknowledge Drs. Aaron Janowsky and Amy Eshleman for careful reading of the manuscript, and Dr. Brenda Wiens and Mr. Minh Vu for assistance with the D3 and D4.4 receptor cDNAs. We would also like to thank Drs. Hubert Van Tol and David Grandy for providing us with the cDNA for the human D4.4 receptor and Dr. Daniel Storm and Mark Nielsen for providing us with HEK-ACII cells.
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Footnotes |
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Received March 3, 1997; Accepted April 17, 1997
1 V. J. Watts and K. A. Neve, unpublished observations.
This work was supported by the Johnson and Johnson Focused Giving Program, the Veterans Affairs Merit Review and Research Career Scientist Programs, a Young Investigator Award from the National Alliance for Research on Schizophrenia and Depression, and T32 DA07262.
Send reprint requests to: Val J. Watts, Medical Research Service (151LL), VA Medical Center, 3710 SW US Veterans Hospital Road, Portland, OR 97201. E-mail: wattsv{at}ohsu.edu
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Abbreviations |
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ACII, type II adenylate cyclase; CBS, calf bovine serum; D2L, long (444-amino acid) form of D2 receptors; D4.4., a variant of the D4 dopamine receptor with four copies of a direct imperfect repeat in the third cytoplasmic loop ; DMEM, Dulbecco's modified Eagle's media; FBS, fetal bovine serum; HEK, human embryonic kidney; PMA, phorbol-12-myristate-13-acetate; ANOVA, analysis of variance.
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References |
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Summary
Introduction
Procedures
Results & Discussion
References
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) was 132 nM and for dopamine + isoproterenol (
) was 32 nM.
