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Activation of adenosine A1 receptor modulates dopamine D1 receptor activity in stably cotransfected human embryonic kidney 293 cells
European Journal of Pharmacology 548 (2006) 29 – 35 www.elsevier.com/locate/ejphar
Activation of adenosine A1 receptor modulates dopamine D1 receptor activity in stably cotransfected human embryonic kidney 293 cells Yan Cao, Wan-Chun Sun, Lei Jin, Ke-Qiang Xie, Xing-Zu Zhu ⁎ Department of Pharmacology, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203, China Received 27 April 2006; received in revised form 13 July 2006; accepted 17 July 2006 Available online 3 August 2006
Abstract The antagonistic interactions between adenosine A1 receptors and dopamine D1 receptors were studied in a human embryonic kidney 293 cell line stably cotransfected with human adenosine A1 receptor and dopamine D1 receptor cDNAs. In the cotransfected cells, but not in control cells only transfected with dopamine D1 receptors, adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA, 10 μM) increased the Kd of dopamine D1 receptor antagonist [N-methyl-3H]R(+)-7-Chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine ([3H] SCH23390) without affecting the Bmax. Moreover, CPA induced a concentration-dependent decrease in the affinity of dopamine D1 receptors for the agonist (±)-1-Phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol hydrochloride (SKF38393) and inhibited dopamine D1 receptormediated cyclic AMP response element recruitment. Furthermore, pertussis toxin treatment completely counteracted the effects of low concentrations of CPA but only partially counteracted the effects of high concentrations of CPA. These results suggest that adenosine A1 receptors antagonistically modulate dopamine D1 receptors at the level of receptor binding and the second messenger generation. Furthermore, the antagonistic interactions between these two receptors induced by low concentrations of CPA might have a different manner with those induced by high concentrations of CPA. © 2006 Elsevier B.V. All rights reserved. Keywords: Dopamine D1 receptor; Adenosine A1 receptor; Modulation; CRE-SEAP reporter gene
1. Introduction A general property of adenosine is to modulate neuronal responses and the efficiency of synaptic transmission in specific brain areas, in response to the metabolic status of the nervous system (Brundege and Dunwiddie, 1997). In particular, adenosine has been reported to reinforce the action of GABAB receptors (Sodickson and Bean, 1998) or to inhibit several effects of dopamine in the cortex and basal ganglia (Ferre et al., 1997). There is increasing evidence suggesting that antagonistic intramembrane interactions between specific subtypes of adenosine and dopamine receptors constitute an important integrative mechanism in the basal ganglia (Ferre et al., 1997; Kull et al., 1999). The modulatory effects of adenosine on dopamine systems have been investigated for
⁎ Corresponding author. Tel./fax: +86 21 50806096. E-mail address: [email protected] (X.-Z. Zhu). 0014-2999/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2006.07.051
their relevance to human pathology such as schizophrenia and Parkinson’s disease. Furthermore, in animal models, adenosine agonists and antagonists are potent atypical neuroleptics and antiparkinsonian drugs, respectively (Ferre et al., 1997; Kanda et al., 1998; Rimondini et al., 1997). Thus, adenosine agonists inhibit and adenosine antagonists, such as caffeine, potentiate the behavioral effects induced by dopamine agonists. The evidence suggests that this antagonism is at least in part caused by an intramembrane interaction between specific subtypes of dopamine and adenosine receptors, namely, between adenosine A1 receptors and dopamine D1 receptors and between adenosine A2A receptors and dopamine D2 receptors (Ferre et al., 1997; Kull et al., 1999). This antagonism is evident in crude membrane preparations from cell lines expressing the two receptors and from rat striatum in which, for instance, activation of adenosine A1 receptors reduces the proportion of dopamine D1 receptors in the high-affinity state without changing the dissociation constants of the high- and the low-affinity binding sites (Ferre et al., 1994; Ferre et al., 1998). Gines et al. gave the
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first evidence that this receptor/receptor interaction could involve A1/D1 heteromeric receptor complexes since such complexes could be demonstrated in cotransfected A1/D1 fibroblast Ltk-cells by means of coimmunoprecipitation (Gines et al., 2000). Therefore the antagonistic interaction of these two receptors could be the result of a physical interaction of adenosine A1 receptor with dopamine D1 receptor in this heteromeric complex. This antagonistic mechanism may contribute to the adenosine A1 receptor and dopamine D1 receptor functional antagonism found in the brain and offers a basis for the design of novel agents to treat Parkinson’s disease and neuropsychiatric disorders, based on the pharmacological properties of the A1/D1 heteromeric complex (Gines et al., 2000). In the present studies, an additional mechanism of adenosine-mediated modulation of dopamine D1 receptor activity is presented. We demonstrated the existence of an antagonistic A1–D1 intramembrane interaction in the presence of adenosine A1 receptor agonist in human embryonic kidney 293 (HEK293) cells stably cotransfected with human adenosine A1 receptor and dopamine D1 receptor cDNAs. The functional antagonistic interaction between adenosine A1 receptors and dopamine D1 receptors in the cotransfected cells was studied by means of cyclic AMP response element (CRE)-secreted alkaline phosphatase (SEAP) activity assay. Furthermore, we investigated G protein involvement by pertussis toxin treatment, and found that the antagonistic interactions between these two receptors induced by low concentrations of adenosine A1 receptor agonist CPA might have a different manner with those induced by high concentrations of CPA. 2. Materials and methods 2.1. Materials Geneticin and hygromycin were obtained from Gibco. N6cyclopentyladenosine (CPA) and (±)-1-Phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol hydrochloride (SKF38393) were obtained from RBI (Natick, MA, USA). 4-methylumbelliferyl phosphate (4-MUP), leupeptin, pepstatin A, aprotinin, phenylmethylsulphonylfluoride (PMSF) and pertussis toxin were purchased from Sigma (USA). [N-methyl- 3 H]R(+)-7-Chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine ([3H]SCH23390) and [propyl-3H]8-Cyclopentyl-1,3-dipropylxanthine ([3H]DPCPX) were purchased from Amersham (USA). Plasmids pcDNA3.1/ Hygro(+) and Lipofectamine2000 were purchased from Invitrogen (USA), and pCRE-SEAP was from BD Biosciences Clontech (USA).
GAGGACTCTGAACACCTCTGCCA-3) and dopamine D1 receptor reverse (5-GCGCCTCGAGTCAGGTTGGGTGCTGACCGTTTTGT-3) as primers. The PCR product was cloned into the BamI/XhoI site of pcDNA3.1/Hygro (+). 2.3. Generation of stably transfected HEK293/A1D1 cells Cells from the HEK 293 cell line previously transfected with the human adenosine A1 receptor cDNA (Sun et al., 2005a) were used and grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum, penicillin (100 units/ml), streptomycin (100 μg/ml), and geneticin (200 μg/ml). For the generation of HEK293/A1D1 cell line, dopamine D1 receptor cDNA (cloned into pcDNA3.1/Hygro (+)) was transfected into HEK293/A1 cells by the Lipofectamine2000 reagent. These transfected HEK293/A1 cells were selected with hygromycin (250 μg/ml) in DMEM medium to generate a single clone of HEK293/A1D1 cell line (A1D1 cells) which stably expressed both adenosine A1 receptors and dopamine D1 receptors. Cells were incubated in a humid atmosphere of 5% CO2 and 95% air at 37 °C. 2.4. Membrane preparation The A1D1 cells were lifted from Petri dishes with a cell scraper. Harvested cells were washed twice with ice-cold PBS and centrifuged at 420 ×g for 5 min at 4 °C. The cell pellet was resuspended with hyponic buffer (Tris–HCl 5 mM, EDTA 2 mM, leupeptin 1 μg/ml, pepstatin A1 μg/ml, aprotinin 1 μg/ ml, PMSF 1 mM, pH 7.4) and sonicated (18 s) three times on ice. The homogenate was centrifuged at 960× g for 10 min at 4 °C. The precipitated nucleic fraction was discarded and the supernatant was centrifuged at 40,000 ×g for 30 min at 4 °C. The pellet was washed with 50 mM Tris–HCl buffer (pH 7.4) and centrifuged again under the same conditions. Finally, the pellet was resuspended in the incubation buffer. In the experiment with [3 H]DPCPX, the incubation buffer was 50 mM Tris–HCl (pH 7.4). In the experiment with [3H] SCH23390, the incubation buffer was 50 mM Tris–HCl (pH 7.4) containing 120 mM NaCl, 5 mM KCl, 1 mM MgCl2, and 2 mM CaCl2. Protein concentration was determined by the BCA Kit (Pierce) as described previously (Hsia et al., 1984). 2.5. Pertussis toxin pretreatment Experiments with pertussis toxin were performed with A1D1 cells exposed to pertussis toxin (100 ng/ml) overnight before radioligand binding experiments or CRE-SEAP activity assay.
2.2. Cloning of human dopamine D1 receptor cDNA
2.6. Radioligand binding experiments
The full-length gene for human dopamine D1 receptor cDNA was amplified from the expression vector dopamine D1 receptor-pcDNA3 (Sun et al., 2005b) containing the full coding sequence of the human dopamine D1 receptor with dopamine D1 receptor forward (5-GCGCGGATCCGCCGCCACCAT-
Saturation experiments with adenosine A1 receptor antagonist [3H]DPCPX were carried out with 8 concentrations (0.05– 10.0 nM) of [3H]DPCPX (128.0 Ci/mmol) by incubation in 50 mM Tris–HCl buffer (pH 7.4) for 1 h at 37 °C. Nonspecific binding was defined as the binding in the presence of 10 μM
Y. Cao et al. / European Journal of Pharmacology 548 (2006) 29–35
Fig. 1. Representative saturation curve of specific binding of adenosine A1 receptor antagonist [3H]DPCPX in membrane preparations from A1D1 cells. The Bmax and Kd values were obtained by Scatchard analysis.
unlabled DPCPX. Saturation experiments with dopamine D1 receptor antagonist [3H]SCH23390 were carried out with 8 concentrations (0.1–10.0 nM) of [3H]SCH23390 (79.0 Ci/ mmol) by incubation in Tris–HCl buffer (Tris–HCl 50 mM, NaCl 120 mM, KCl 5 mM, MgCl2 1 mM, CaCl2 2 mM, pH 7.4) for 30 min at 37 °C. Nonspecific binding was defined as the binding in the presence of 10 μM butaclamol, a dopamine receptor antagonist. Competition experiments of SKF38393 (50 pM to 0.5 mM) versus dopamine D1 receptor antagonist [3H]SCH23390 (0.5 nM) were performed in intact cells at a density of 200,000 cells/tube by incubation in Tris–HCl buffer (Tris–HCl 50 mM, NaCl 120 mM, KCl 5 mM, MgCl2 1 mM, CaCl2 2 mM, pH 7.4) for 30 min at 37 °C in the presence or absence of CPA. The incubation was stopped by fast filtration through glass-fiber filters (GF/B, Whatman) by washing three times with 5 ml of 50 mM ice-cold Tris–HCl buffer (pH 7.4) with an automatic cell harvester (Brandel). The radioactivity was counted with Beckman LS6500 liquid scintillation analyzer (Sun et al., 2005a). 2.7. CRE-SEAP activity assay pCRE-SEAP plasmid was transfected into the cells by means of the calcium phosphate precipitation method (Sun et al., 2005b). Cells were seeded in 100 mm dishes, and transfection was performed when cells were 50% confluent. After 12 h, the transfection medium was replaced with fresh medium, and the transfected cells were seeded into 24-well plates (150,000 cells/ well) and cultured overnight (Durocher et al., 2000). Medium was then replaced by 500 μl of free serum DMEM containing
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30 μM rolipram and the different concentrations of SKF38393 in the absence or presence of CPA. Then cells were incubated for 8 h. The CRE-SEAP assay was subsequently performed. Following the incubation period of transiently transfected cells, culture medium was inactivated for 30 min at 65 °C and centrifuged at 12,000 rpm at 4 °C. The supernatant (100 μl) was transferred to a new 96-well plate and mixed with 100 μl CRESEAP assay buffer (50 mM Tris/0.1% bovine serum albumin buffer, pH 8.0) containing 36 μM 4-methyl-umbelliferyl phosphate (4-MUP). The mixture was incubated for 1 h at 37 °C, and the fluorescence intensity was measured at 460 nm by the FLUOstar plate reader (BMG labtechnologies, Offenburg, Germany). Excitation wavelength was 355 nm. 2.8. Data analysis Experiments were performed in triplicate. All data were analyzed with the GraphPad Prism 4.0 program (GraphPad Software, San Diego). IC50 values obtained from competition curves were converted to Ki values using the Cheng Prusoff equation (Cheng and Prusoff, 1973). Results were expressed as mean ± S.E.M. and were analyzed using a one-way analysis of variance (ANOVA) followed by the Student–Newman–Keuls test. The level of significance was set at P < 0.05. 3. Results 3.1. Saturation experiments with adenosine A1 receptor antagonist [3H]DPCPX [3H]DPCPX saturation experiments demonstrated the existence of a moderate density of adenosine A1 receptor in the selected A1D1 cell clone. The naive HEK293 cells and D1 cells showed no significant [3H]DPCPX-specific binding (data not shown). The Bmax and Kd values for [3H]DPCPX binding in the A1D1 cells were, respectively (Fig. 1). 1992 ± 130.7 fmol/mg of protein and 1.3 ± 0.2 nM (means ± S.E.M., n = 3). 3.2. Saturation experiments with dopamine D1 receptor antagonist [3H]SCH23390 The naive HEK293 cells showed no significant [3 H] SCH23390-specific binding (data not shown). In the D1 cells,
Table 1 Saturation experiments with dopamine D1 receptor antagonist [3H]SCH23390 in membrane preparations from D1 and A1D1 cells
D1 cells A1D1 cells
Pretreatment
Bmax (fmol/mg)
Kd (nM)
Control CPA, 10 μM Control CPA, 10 nM CPA, 10 μM
2373 ± 67 2377 ± 13 1821 ± 78 1844 ± 21 1916 ± 64
0.70 ± 0.06 0.60 ± 0.10 0.84 ± 0.15 0.91 ± 0.02 1.30 ± 0.20 a
Bmax and Kd values are expressed as mean ± S.E.M. n = 3. a P < 0.05 compared with control by one-way ANOVA followed by the Student–Newman–Keuls test.
Fig. 2. Representative saturation curves of specific binding of dopamine D1 receptor antagonist [3H]SCH23390 in membrane preparations from A1D1 cells in the absence and presence of adenosine A1 receptor agonist CPA. The Bmax and Kd values were obtained by Scatchard analysis.
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no significant differences were obtained in the absence or presence of adenosine A1 receptor agonist CPA (10 μM) regarding the Bmax and Kd values for the dopamine D1 receptor binding sites labeled with [3H]SCH23390 (Table 1). In the A1D1 cells, CPA (10 nM) had no effect on [H]SCH23390 binding in cell membranes (Fig. 2, Table 1). However, CPA (10 μM) significantly increased the Kd value of [H]SCH23390, without affecting the Bmax value. The Kd values in the absence and presence of CPA (10 μM) were 0.84 ± 0.15 and 1.3 ± 0.2 nM (means ± S.E.M., n = 3), respectively (Fig. 2, Table 1). 3.3. Competition experiments of SKF38393 versus dopamine D1 receptor antagonist [3H]SCH23390 Competition experiments of SKF38393 versus dopamine D1 receptor antagonist [3H]SCH23390 in intact A1D1 cells showed a significant difference in the absence or presence of adenosine A1 receptor agonist CPA. CPA produced a concentrationdependent increase in the Ki value of SKF38393 (Fig. 3A, Table 2). However, in the D1 cells, no significant differences were obtained between in the absence and presence of adenosine A1
Table 2 Competition experiments of SKF38393 versus dopamine D1 receptor antagonist [3H]SCH23390
D1 cells A1D1 cells
Pretreatment
Ki (μM)
Control CPA, 10 μM Control CPA, 10 nM CPA, 10 μM PTX PTX + CPA, 10 nM PTX + CPA, 10 μM DPCPX, 10 μM
3.15 ± 0.15 3.43 ± 0.13 1.89 ± 0.09 6.49 ± 0.50 a 8.82 ± 1.43 a 1.82 ± 0.06 1.94 ± 0.25 4.99 ± 0.22 a 1.17 ± 0.03 b
Ki values are expressed as mean ± S.E.M. n = 3. PTX, pertusis toxin. a P < 0.01 compared with control by one-way ANOVA followed by the Student–Newman–Keuls test. b P < 0.05 compared with control by one-way ANOVA followed by the Student–Newman–Keuls test.
receptor agonist CPA for the Ki values (Table 2). Pretreatment of the A1D1 cells with pertussis toxin counteracted the effect of a low concentration of CPA (10 nM), and only slightly counteracted the effect of 10 μM CPA (Fig. 3B, Table 2). The adenosine A1 receptor antagonist DPCPX (10 μM) significantly decreased the Ki value of SKF38393 in A1D1 cells (Fig. 3C, Table 2). All competition curves were best fitted if a single binding site was assumed. 3.4. Dopamine D1 receptor agonist SKF38393 induced CRESEAP activity increase In D1 and A1D1 cells which were transiently transfected with pCRE-SEAP reporter vector, SKF38393 induced a
Fig. 3. Representative competitive inhibition curves of dopamine D1 receptor agonist SKF38393 versus dopamine D1 receptor antagonist [3H]SCH23390 in A1D1 cells. Competition assays were done at 0.5 nM [3H]SCH23390 and increasing concentrations of SKF38393. PTX, pertussis toxin. Ki values are means (95% confidence intervals) obtained from three independent experiments performed in triplicate using GraphPad Prism 4.0 program.
Fig. 4. CRE-SEAP reporter gene activity induced by dopamine D1 receptor agonist SKF38393 in the absence and presence of adenosine A1 receptor agonist CPA in D1 (A) and A1D1 cells (B) transiently transfected with pCRE-SEAP reporter gene. SEAP activity is expressed as a percentage of the maximal activity obtained for each curve. PTX, pertussis toxin. EC50 values are expressed as median (95% confidence intervals) obtained from three independent experiments performed in triplicate using GraphPad Prism 4.0 program.
Y. Cao et al. / European Journal of Pharmacology 548 (2006) 29–35 Table 3 Dopamine D1 receptor agonist SKF38393 induced CRE-SEAP activity
D1 cells A1D1 cells
Pretreatment
EC50 (×10− 8 M)
Control CPA, 10 μM Control CPA, 10 nM CPA, 10 μM PTX + CPA, 10 nM PTX + CPA, 10 μM
5.79 ± 0.58 6.17 ± 1.54 6.80 ± 1.04 8.68 ± 1.28 12.70 ± 1.55 a 6.88 ± 1.34 10.28 ± 1.19
EC50 values are expressed as mean ± S.E.M. n = 3. PTX, pertussis toxin. a P < 0.05 compared with control by one-way ANOVA followed by the Student–Newman–Keuls test.
concentration-dependent increase in the CRE-SEAP activity (Fig. 4). adenosine A1 receptor agonist CPA (10 μM) significantly decreased the EC50 value of SKF38393 in A1D1 cells but not in D1 cells (Fig. 4, Table 3). The CRE-SEAP activity basal levels were not modified by CPA (Fig. 4). Thus, the decrease in CRE-SEAP activity by activation of adenosine A1 receptors could be ignored. In contrast, pertussis toxin treatment counteracted the effect of a low concentration of CPA (10 nM) on dopamine D1 receptor-mediated CRE recruitment, and only slightly counteracted the effect of 10 μM CPA (Fig. 4, Table 3). 4. Discussion For G protein-coupled receptors, it is widely believed that the critical event in agonist action is the stabilization of the ternary complex of agonist/receptor/G-protein (Lefkowitz et al., 1993), and the better this ternary complex is stabilized, the more efficacious the agonist is. Both adenosine A1 receptor and dopamine D1 receptor belong to the G protein-coupled receptor family. Coupling to Gs and Gi proteins, stimulation of dopamine D1 receptor and adenosine A1 receptor activates and inhibits adenylyl cyclase, respectively. It has been shown that the binding characteristics of one type of G protein-coupled receptor can be altered by the stimulation of another type of G protein-coupled receptor in crude membrane preparations (Zoli et al., 1993). Such intramembrane interactions have been postulated to represent direct interactions between the receptor molecules and/or to involve G proteins or other mobile molecules associated with the membrane (Zoli et al., 1993). In the present studies, the radioligand binding experiments carried out from the cotransfected A1D1 cells showed that the adenosine A1 receptor-mediated modulation of dopamine D1 receptor induced a concentration-dependent decrease in the affinity of dopamine D1 receptor for agonist SKF38393. These results were similar to those obtained with the cotransfected A1/ D1 fibroblast Ltk-cells (Ferre et al., 1998). The only difference was that the inhibition of binding to dopamine D1 receptor by SKF38393 was best described as occurring to a single type of site in our results. Previous evidence has demonstrated that adenosine A1 receptor and dopamine D1 receptor heteromers were presented in the membrane of cotransfected fibroblast cells (Gines et al., 2000). Thus, we speculated that the decrease in the affinity of dopamine D1 receptor for SKF38393 induced by
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adenosine A1 receptor stimulation could be the result of a physical interaction of adenosine A1 receptor with dopamine D1 receptor in the heteromeric complex, leading to an uncoupling of dopamine D1 receptor from its Gs-like protein in this functionally interacting heteromeric complex. The effects of adenosine A1 receptor antagonist DPCPX on the Ki value of SKF38393 in A1D1 cells also suggest that adenosine released by these cells exerts a tonic inhibition of dopamine D1 receptormediated function through the A1–D1 interaction.(Le Crom et al., 2002). Pertussis toxin is a good tool to investigate G protein involvement because of its selectivity of action by inducing an ADP-ribosylation of the Gá subunit of the Gi (and Go) protein family (Gilman, 1987). A1D1 cells were exposed to pertussis toxin to study the possible involvement of Gi protein in the adenosine A1 receptor-mediated uncoupling of the dopamine D1 receptor from the Gs protein. It was found that pertussis toxin counteracted the effect of CPA (10 nM) on dopamine D1 receptor binding characteristics, suggesting that the Gi protein was necessary for the intramembrane A1–D1 interaction in the presence of a low concentration of CPA. However, a higher concentration of CPA (10 μM) could still uncouple dopamine D1 receptor from the Gs protein after pertussis toxin pretreatment. These results suggest that the effect of adenosine A1 receptor activated by a high concentration of CPA might be mediated by a different mechanism than the effect of adenosine A1 receptor activated by a low concentration of CPA. The density of two interacting receptors and the number of receptors activated by the agonist in each population would determine the proportion of monomers and homodimers and heterodimers and thus the overall action on target cell function (Zoli et al., 1993). It is highly probable that clustering occurs for several G proteincoupled receptors when they are activated by their ligands. Occurrence of clustering clearly reflects that G protein-coupled receptors form high molecular order structures in which multimers of the receptors and, probably, other interacting proteins form functional complexes. It has been reported that exposure to adenosine A1 receptor agonist R(−)N6-(2-phenylisopropyl)adenosine (R-PIA, 100 nM) induced the formation of clusters (aggregations) containing both adenosine A1 receptor and dopamine D1 receptor immunoreactivities (Gines et al., 2000). Thus, a high concentration of CPA (10 μM) treatment might induce the formation of adenosine A1 receptor/dopamine D1 receptor heteromers leading to conformational changes in their respective binding pockets. However, a low concentration of CPA (10 nM) could not induce enough clustering resulting in the conformational changes in the binding pockets and might only lead to an uncoupling of dopamine D1 receptor from its Gs protein by a manner similar to the dopamine D1 receptordopamine D2 receptor interaction which seems to involve G protein (Seeman et al., 1989). The subunits of the stimulatory G protein, Gs (serving dopamine D1 receptor), may be in equilibrium with those of the inhibitory G protein, Gi (serving adenosine A1 receptor). In the presence of CPA (to activate adenosine A1 receptor), the subunits of the G protein may rearrange. This rearrangement converts dopamine D1 receptor to low affinity for SKF38393. Exposure to a high concentration of CPA might
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induce the formation of adenosine A1 receptor/dopamine D1 receptor heteromers. In this case, besides the G proteindependent manner as in the presence of a low concentration of CPA, the physical interaction is maintained when the adenosine A1 receptor and dopamine D1 receptor binding pockets are simultaneously activated by agonists allowing the antagonistic intramembrane receptor/receptor interaction to take place by a G protein-independent manner. Taken together, the functional meaning of this intramembrane receptor/receptor interaction in the presence of a high concentration of CPA is therefore to decrease the affinity of dopamine D1 receptor for SKF38393 in G protein-dependent and -independent manners. A well recognized characteristic of agonist binding to receptors such as the β2-adrenergic receptor is the increase in high affinity binding states, indicative of receptor/G-protein interactions (Costa et al., 1992; De Lean et al., 1980; Pin et al., 2005; Samama et al., 1993). Similarly, the high affinity binding state of dopamine D1 receptor requires the receptor coupling with Gs protein. Thus, adenosine A1 receptor stimulation leads to the uncoupling of dopamine D1 receptor from its Gs protein so as to decrease the affinity of dopamine D1 receptor for agonist. However, antagonists are drugs that bind to the receptor without activating the effector system for that receptor. Therefore in the low concentration of CPA treatment, adenosine A1 receptor stimulation involves no changes in the affinity of dopamine D1 receptor for the antagonist [3H]SCH23390 in the saturation experiments (Fig. 2, Table 1), which were consistent with the results reported by Ferre et al. (1998). Nevertheless, exposure to the high concentration of CPA might induce the formation of adenosine A1 receptor/dopamine D1 receptor heteromers so as to decrease the affinity of dopamine D1 receptor for the antagonist [3H]SCH23390 in a G proteinindependent manner. Dopamine D1 receptor can activate adenylyl cyclase, resulting in cAMP production. Cell based assays relying on transcriptionally controlled reporter genes have been developed and are suited to monitor and are signaled the cellular responses induced by G protein-coupled receptors. Both Gs- and Gqcoupled receptors are signaled through CREs (Chen et al., 1995; Durocher et al., 2000; Stratowa et al., 1995). Since CRE is a pivotal target in G protein-coupled receptor signaling pathways, it has become one of the most widely used response elements in reporter gene assays. Since the transcription of CRE-SEAP reporter genes is controlled by CRE-containing promoters, CRE-SEAP activity can be used to monitor the activation of receptors. Previously we found that the CRE-SEAP activity assay could substitute the traditional radioimmunoassay for the measurement of cAMP accumulation (Sun et al., 2005b). In the present experiments, dopamine D1 receptor agonist-induced CRE-SEAP activity increase was demonstrated in the D1 and A1D1 cells. Furthermore, in A1D1 cells, adenosine A1 receptor agonist CPA counteracted the CRE-SEAP activity increase induced by SKF38393 in a concentration-dependent manner. Pertussis toxin counteracted the effect of the low concentration of CPA on the D1 receptor-mediated CRE recruitment, and only slightly affected the inhibitory effect of the high concentration of CPA. These results suggest that adenosine A1 receptor
activation can inhibit the effect induced by dopamine D1 receptor activation at the second messenger level. Thus, the clear correlation between the results obtained with the radioligand binding and functional interaction experiments suggests that the intramembrane A1–D1 interaction involved in the binding experiments is related to the A1–D1 interaction found at the second messenger level. In summary, two main findings have been obtained in this study. First, in A1D1 cells, adenosine A1 receptor activation can decrease the binding affinity of dopamine D1 receptor for agonists and inhibit the effect induced by dopamine D1 receptor activation at the second messenger level. Secondly, the mechanisms of the antagonistic interaction between adenosine A1 receptor and dopamine D1 receptor are different in the presence of a high concentration or a low concentration of CPA. When adenosine A 1 receptors are activated by a low concentration of CPA, Gi proteins are necessary for the antagonistic effect. However, in the presence of a high concentration of CPA, besides the G protein involved manner, the conformational changes induced by the A1/D1 heteromers result in the antagonistic intramembrane receptor/receptor interaction to take place by a G protein-independent manner. Such concerted interactions between functionally distinct neurotransmitter receptors increasingly appear to be part of a general overall mechanism by which neurons are able to receive and integrate diverse extracellular signals (Le Crom et al., 2002; Maggio et al., 2005). Acknowledgements This work was supported by a research grant from the Ministry of Science and Technology of China (2004CB720305) and the Shanghai Metropolitan Fund for Research and Development (04DZ14005). We thank Dr. Lin-lin Yin, Dr. Zhong-hua Liu and Dr. Wei-yu Zhang for their expert advice and assistance. We also acknowledge Dr. Lin-yin Feng for revision of the manuscript. References Brundege, J.M., Dunwiddie, T.V., 1997. Role of adenosine as a modulator of synaptic activity in the central nervous system. Adv. Pharmacol. 39, 353–391. Chen, W., Shields, T.S., Stork, P.J., Cone, R.D., 1995. A colorimetric assay for measuring activation of Gs- and Gq-coupled signaling pathways. Anal. Biochem. 226, 349–354. Cheng, Y., Prusoff, W.H., 1973. Relationship between the inhibition constant (Ki) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem. Pharmacol. 22, 3099–3108. Costa, T., Ogino, Y., Munson, P.J., Onaran, H.O., Rodbard, D., 1992. Drug efficacy at guanine nucleotide-binding regulatory protein-linked receptors: thermodynamic interpretation of negative antagonism and of receptor activity in the absence of ligand. Mol. Pharmacol. 41, 549–560. De Lean, A., Stadel, J.M., Lefkowitz, R.J., 1980. A ternary complex model explains the agonist-specific binding properties of the adenylate cyclasecoupled beta-adrenergic receptor. J. Biol. Chem. 255, 7108–7117. Durocher, Y., Perret, S., Thibaudeau, E., Gaumond, M.H., Kamen, A., Stocco, R., Abramovitz, M., 2000. A reporter gene assay for high-throughput screening of G-protein-coupled receptors stably or transiently expressed in HEK293 EBNA cells grown in suspension culture. Anal. Biochem. 284, 316–326.
Y. Cao et al. / European Journal of Pharmacology 548 (2006) 29–35 Ferre, S., Popoli, P., Gimenez-Llort, L., Finnman, U.B., Martinez, E., Scotti de Carolis, A., Fuxe, K., 1994. Postsynaptic antagonistic interaction between adenosine A1 and dopamine D1 receptors. Neuroreport 6, 73–76. Ferre, S., Fredholm, B.B., Morelli, M., Popoli, P., Fuxe, K., 1997. Adenosinedopamine receptor-receptor interactions as an integrative mechanism in the basal ganglia. Trends Neurosci. 20, 482–487. Ferre, S., Torvinen, M., Antoniou, K., Irenius, E., Civelli, O., Arenas, E., Fredholm, B.B., Fuxe, K., 1998. Adenosine A1 receptor-mediated modulation of dopamine D1 receptors in stably cotransfected fibroblast cells. J. Biol. Chem. 273, 4718–4724. Gilman, A.G., 1987. G proteins: transducers of receptor-generated signals. Annu. Rev. Biochem. 56, 615–649. Gines, S., Hillion, J., Torvinen, M., Le Crom, S., Casado, V., Canela, E.I., Rondin, S., Lew, J.Y., Watson, S., Zoli, M., Agnati, L.F., Verniera, P., Lluis, C., Ferre, S., Fuxe, K., Franco, R., 2000. Dopamine D1 and adenosine A1 receptors form functionally interacting heteromeric complexes. Proc. Natl. Acad. Sci. U. S. A. 97, 8606–8611. Hsia, J.A., Moss, J., Hewlett, E.L., Vaughan, M., 1984. ADP-ribosylation of adenylate cyclase by pertussis toxin. Effects on inhibitory agonist binding. J. Biol. Chem. 259, 1086–1090. Kanda, T., Jackson, M.J., Smith, L.A., Pearce, R.K., Nakamura, J., Kase, H., Kuwana, Y., Jenner, P., 1998. Adenosine A2A antagonist: a novel antiparkinsonian agent that does not provoke dyskinesia in parkinsonian monkeys. Ann. Neurol. 43, 507–513. Kull, B., Ferre, S., Arslan, G., Svenningsson, P., Fuxe, K., Owman, C., Fredholm, B.B., 1999. Reciprocal interactions between adenosine A2A and dopamine D2 receptors in Chinese hamster ovary cells co-transfected with the two receptors. Biochem. Pharmacol. 58, 1035–1045. Le Crom, S., Prou, D., Vernier, P., 2002. Autocrine activation of adenosine A1 receptors blocks D1A but not D1B dopamine receptor desensitization. J. Neurochem. 82, 1549–1552. Lefkowitz, R.J., Cotecchia, S., Samama, P., Costa, T., 1993. Constitutive activity of receptors coupled to guanine nucleotide regulatory proteins. Trends Pharmacol. Sci. 14, 303–307.
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Maggio, R., Novi, F., Scarselli, M., Corsini, G.U., 2005. The impact of Gprotein-coupled receptor hetero-oligomerization on function and pharmacology. FEBS J. 272, 2939–2946. Pin, J.P., Kniazeff, J., Liu, J., Binet, V., Goudet, C., Rondard, P., Prezeau, L., 2005. Allosteric functioning of dimeric class C G-protein-coupled receptors. FEBS J. 272, 2947–2955. Rimondini, R., Ferre, S., Ogren, S.O., Fuxe, K., 1997. Adenosine A2A agonists: a potential new type of atypical antipsychotic. Neuropsychopharmacology 17, 82–91. Samama, P., Cotecchia, S., Costa, T., Lefkowitz, R.J., 1993. A mutation-induced activated state of the beta 2-adrenergic receptor. Extending the ternary complex model. J. Biol. Chem. 268, 4625–4636. Seeman, P., Niznik, H.B., Guan, H.C., Booth, G., Ulpian, C., 1989. Link between D1 and D2 dopamine receptors is reduced in schizophrenia and Huntington diseased brain. Proc. Natl. Acad. Sci. U. S. A. 86, 10156–10160. Sodickson, D.L., Bean, B.P., 1998. Neurotransmitter activation of inwardly rectifying potassium current in dissociated hippocampal CA3 neurons: interactions among multiple receptors. J. Neurosci. 18, 8153–8162. Stratowa, C., Himmler, A., Czernilofsky, A.P., 1995. Use of a luciferase reporter system for characterizing G-protein-linked receptors. Curr. Opin. Biotechnol. 6, 574–581. Sun, W.C., Cao, Y., Jin, L., Wang, L.Z., Meng, F., Zhu, X.Z., 2005a. Modulating effect of adenosine deaminase on function of adenosine A1 receptors. Acta Pharmacol. Sin. 26, 160–165. Sun, W.C., Jin, L., Cao, Y., Wang, L.Z., Meng, F., Zhu, X.Z., 2005b. Cloning, expression, and functional analysis of human dopamine D1 receptors. Acta Pharmacol. Sin. 26, 27–32. Zoli, M., Agnati, L.F., Hedlund, P.B., Li, X.M., Ferre, S., Fuxe, K., 1993. Receptor-receptor interactions as an integrative mechanism in nerve cells. Mol. Neurobiol. 7, 293–334.
Activation of adenosine A1 receptor modulates dopamine D1 receptor activity in stably cotransfected human embryonic kidney 293 cells Yan Cao, Wan-Chun Sun, Lei Jin, Ke-Qiang Xie, Xing-Zu Zhu ⁎ Department of Pharmacology, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203, China Received 27 April 2006; received in revised form 13 July 2006; accepted 17 July 2006 Available online 3 August 2006
Abstract The antagonistic interactions between adenosine A1 receptors and dopamine D1 receptors were studied in a human embryonic kidney 293 cell line stably cotransfected with human adenosine A1 receptor and dopamine D1 receptor cDNAs. In the cotransfected cells, but not in control cells only transfected with dopamine D1 receptors, adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA, 10 μM) increased the Kd of dopamine D1 receptor antagonist [N-methyl-3H]R(+)-7-Chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine ([3H] SCH23390) without affecting the Bmax. Moreover, CPA induced a concentration-dependent decrease in the affinity of dopamine D1 receptors for the agonist (±)-1-Phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol hydrochloride (SKF38393) and inhibited dopamine D1 receptormediated cyclic AMP response element recruitment. Furthermore, pertussis toxin treatment completely counteracted the effects of low concentrations of CPA but only partially counteracted the effects of high concentrations of CPA. These results suggest that adenosine A1 receptors antagonistically modulate dopamine D1 receptors at the level of receptor binding and the second messenger generation. Furthermore, the antagonistic interactions between these two receptors induced by low concentrations of CPA might have a different manner with those induced by high concentrations of CPA. © 2006 Elsevier B.V. All rights reserved. Keywords: Dopamine D1 receptor; Adenosine A1 receptor; Modulation; CRE-SEAP reporter gene
1. Introduction A general property of adenosine is to modulate neuronal responses and the efficiency of synaptic transmission in specific brain areas, in response to the metabolic status of the nervous system (Brundege and Dunwiddie, 1997). In particular, adenosine has been reported to reinforce the action of GABAB receptors (Sodickson and Bean, 1998) or to inhibit several effects of dopamine in the cortex and basal ganglia (Ferre et al., 1997). There is increasing evidence suggesting that antagonistic intramembrane interactions between specific subtypes of adenosine and dopamine receptors constitute an important integrative mechanism in the basal ganglia (Ferre et al., 1997; Kull et al., 1999). The modulatory effects of adenosine on dopamine systems have been investigated for
⁎ Corresponding author. Tel./fax: +86 21 50806096. E-mail address: [email protected] (X.-Z. Zhu). 0014-2999/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2006.07.051
their relevance to human pathology such as schizophrenia and Parkinson’s disease. Furthermore, in animal models, adenosine agonists and antagonists are potent atypical neuroleptics and antiparkinsonian drugs, respectively (Ferre et al., 1997; Kanda et al., 1998; Rimondini et al., 1997). Thus, adenosine agonists inhibit and adenosine antagonists, such as caffeine, potentiate the behavioral effects induced by dopamine agonists. The evidence suggests that this antagonism is at least in part caused by an intramembrane interaction between specific subtypes of dopamine and adenosine receptors, namely, between adenosine A1 receptors and dopamine D1 receptors and between adenosine A2A receptors and dopamine D2 receptors (Ferre et al., 1997; Kull et al., 1999). This antagonism is evident in crude membrane preparations from cell lines expressing the two receptors and from rat striatum in which, for instance, activation of adenosine A1 receptors reduces the proportion of dopamine D1 receptors in the high-affinity state without changing the dissociation constants of the high- and the low-affinity binding sites (Ferre et al., 1994; Ferre et al., 1998). Gines et al. gave the
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first evidence that this receptor/receptor interaction could involve A1/D1 heteromeric receptor complexes since such complexes could be demonstrated in cotransfected A1/D1 fibroblast Ltk-cells by means of coimmunoprecipitation (Gines et al., 2000). Therefore the antagonistic interaction of these two receptors could be the result of a physical interaction of adenosine A1 receptor with dopamine D1 receptor in this heteromeric complex. This antagonistic mechanism may contribute to the adenosine A1 receptor and dopamine D1 receptor functional antagonism found in the brain and offers a basis for the design of novel agents to treat Parkinson’s disease and neuropsychiatric disorders, based on the pharmacological properties of the A1/D1 heteromeric complex (Gines et al., 2000). In the present studies, an additional mechanism of adenosine-mediated modulation of dopamine D1 receptor activity is presented. We demonstrated the existence of an antagonistic A1–D1 intramembrane interaction in the presence of adenosine A1 receptor agonist in human embryonic kidney 293 (HEK293) cells stably cotransfected with human adenosine A1 receptor and dopamine D1 receptor cDNAs. The functional antagonistic interaction between adenosine A1 receptors and dopamine D1 receptors in the cotransfected cells was studied by means of cyclic AMP response element (CRE)-secreted alkaline phosphatase (SEAP) activity assay. Furthermore, we investigated G protein involvement by pertussis toxin treatment, and found that the antagonistic interactions between these two receptors induced by low concentrations of adenosine A1 receptor agonist CPA might have a different manner with those induced by high concentrations of CPA. 2. Materials and methods 2.1. Materials Geneticin and hygromycin were obtained from Gibco. N6cyclopentyladenosine (CPA) and (±)-1-Phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol hydrochloride (SKF38393) were obtained from RBI (Natick, MA, USA). 4-methylumbelliferyl phosphate (4-MUP), leupeptin, pepstatin A, aprotinin, phenylmethylsulphonylfluoride (PMSF) and pertussis toxin were purchased from Sigma (USA). [N-methyl- 3 H]R(+)-7-Chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine ([3H]SCH23390) and [propyl-3H]8-Cyclopentyl-1,3-dipropylxanthine ([3H]DPCPX) were purchased from Amersham (USA). Plasmids pcDNA3.1/ Hygro(+) and Lipofectamine2000 were purchased from Invitrogen (USA), and pCRE-SEAP was from BD Biosciences Clontech (USA).
GAGGACTCTGAACACCTCTGCCA-3) and dopamine D1 receptor reverse (5-GCGCCTCGAGTCAGGTTGGGTGCTGACCGTTTTGT-3) as primers. The PCR product was cloned into the BamI/XhoI site of pcDNA3.1/Hygro (+). 2.3. Generation of stably transfected HEK293/A1D1 cells Cells from the HEK 293 cell line previously transfected with the human adenosine A1 receptor cDNA (Sun et al., 2005a) were used and grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum, penicillin (100 units/ml), streptomycin (100 μg/ml), and geneticin (200 μg/ml). For the generation of HEK293/A1D1 cell line, dopamine D1 receptor cDNA (cloned into pcDNA3.1/Hygro (+)) was transfected into HEK293/A1 cells by the Lipofectamine2000 reagent. These transfected HEK293/A1 cells were selected with hygromycin (250 μg/ml) in DMEM medium to generate a single clone of HEK293/A1D1 cell line (A1D1 cells) which stably expressed both adenosine A1 receptors and dopamine D1 receptors. Cells were incubated in a humid atmosphere of 5% CO2 and 95% air at 37 °C. 2.4. Membrane preparation The A1D1 cells were lifted from Petri dishes with a cell scraper. Harvested cells were washed twice with ice-cold PBS and centrifuged at 420 ×g for 5 min at 4 °C. The cell pellet was resuspended with hyponic buffer (Tris–HCl 5 mM, EDTA 2 mM, leupeptin 1 μg/ml, pepstatin A1 μg/ml, aprotinin 1 μg/ ml, PMSF 1 mM, pH 7.4) and sonicated (18 s) three times on ice. The homogenate was centrifuged at 960× g for 10 min at 4 °C. The precipitated nucleic fraction was discarded and the supernatant was centrifuged at 40,000 ×g for 30 min at 4 °C. The pellet was washed with 50 mM Tris–HCl buffer (pH 7.4) and centrifuged again under the same conditions. Finally, the pellet was resuspended in the incubation buffer. In the experiment with [3 H]DPCPX, the incubation buffer was 50 mM Tris–HCl (pH 7.4). In the experiment with [3H] SCH23390, the incubation buffer was 50 mM Tris–HCl (pH 7.4) containing 120 mM NaCl, 5 mM KCl, 1 mM MgCl2, and 2 mM CaCl2. Protein concentration was determined by the BCA Kit (Pierce) as described previously (Hsia et al., 1984). 2.5. Pertussis toxin pretreatment Experiments with pertussis toxin were performed with A1D1 cells exposed to pertussis toxin (100 ng/ml) overnight before radioligand binding experiments or CRE-SEAP activity assay.
2.2. Cloning of human dopamine D1 receptor cDNA
2.6. Radioligand binding experiments
The full-length gene for human dopamine D1 receptor cDNA was amplified from the expression vector dopamine D1 receptor-pcDNA3 (Sun et al., 2005b) containing the full coding sequence of the human dopamine D1 receptor with dopamine D1 receptor forward (5-GCGCGGATCCGCCGCCACCAT-
Saturation experiments with adenosine A1 receptor antagonist [3H]DPCPX were carried out with 8 concentrations (0.05– 10.0 nM) of [3H]DPCPX (128.0 Ci/mmol) by incubation in 50 mM Tris–HCl buffer (pH 7.4) for 1 h at 37 °C. Nonspecific binding was defined as the binding in the presence of 10 μM
Y. Cao et al. / European Journal of Pharmacology 548 (2006) 29–35
Fig. 1. Representative saturation curve of specific binding of adenosine A1 receptor antagonist [3H]DPCPX in membrane preparations from A1D1 cells. The Bmax and Kd values were obtained by Scatchard analysis.
unlabled DPCPX. Saturation experiments with dopamine D1 receptor antagonist [3H]SCH23390 were carried out with 8 concentrations (0.1–10.0 nM) of [3H]SCH23390 (79.0 Ci/ mmol) by incubation in Tris–HCl buffer (Tris–HCl 50 mM, NaCl 120 mM, KCl 5 mM, MgCl2 1 mM, CaCl2 2 mM, pH 7.4) for 30 min at 37 °C. Nonspecific binding was defined as the binding in the presence of 10 μM butaclamol, a dopamine receptor antagonist. Competition experiments of SKF38393 (50 pM to 0.5 mM) versus dopamine D1 receptor antagonist [3H]SCH23390 (0.5 nM) were performed in intact cells at a density of 200,000 cells/tube by incubation in Tris–HCl buffer (Tris–HCl 50 mM, NaCl 120 mM, KCl 5 mM, MgCl2 1 mM, CaCl2 2 mM, pH 7.4) for 30 min at 37 °C in the presence or absence of CPA. The incubation was stopped by fast filtration through glass-fiber filters (GF/B, Whatman) by washing three times with 5 ml of 50 mM ice-cold Tris–HCl buffer (pH 7.4) with an automatic cell harvester (Brandel). The radioactivity was counted with Beckman LS6500 liquid scintillation analyzer (Sun et al., 2005a). 2.7. CRE-SEAP activity assay pCRE-SEAP plasmid was transfected into the cells by means of the calcium phosphate precipitation method (Sun et al., 2005b). Cells were seeded in 100 mm dishes, and transfection was performed when cells were 50% confluent. After 12 h, the transfection medium was replaced with fresh medium, and the transfected cells were seeded into 24-well plates (150,000 cells/ well) and cultured overnight (Durocher et al., 2000). Medium was then replaced by 500 μl of free serum DMEM containing
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30 μM rolipram and the different concentrations of SKF38393 in the absence or presence of CPA. Then cells were incubated for 8 h. The CRE-SEAP assay was subsequently performed. Following the incubation period of transiently transfected cells, culture medium was inactivated for 30 min at 65 °C and centrifuged at 12,000 rpm at 4 °C. The supernatant (100 μl) was transferred to a new 96-well plate and mixed with 100 μl CRESEAP assay buffer (50 mM Tris/0.1% bovine serum albumin buffer, pH 8.0) containing 36 μM 4-methyl-umbelliferyl phosphate (4-MUP). The mixture was incubated for 1 h at 37 °C, and the fluorescence intensity was measured at 460 nm by the FLUOstar plate reader (BMG labtechnologies, Offenburg, Germany). Excitation wavelength was 355 nm. 2.8. Data analysis Experiments were performed in triplicate. All data were analyzed with the GraphPad Prism 4.0 program (GraphPad Software, San Diego). IC50 values obtained from competition curves were converted to Ki values using the Cheng Prusoff equation (Cheng and Prusoff, 1973). Results were expressed as mean ± S.E.M. and were analyzed using a one-way analysis of variance (ANOVA) followed by the Student–Newman–Keuls test. The level of significance was set at P < 0.05. 3. Results 3.1. Saturation experiments with adenosine A1 receptor antagonist [3H]DPCPX [3H]DPCPX saturation experiments demonstrated the existence of a moderate density of adenosine A1 receptor in the selected A1D1 cell clone. The naive HEK293 cells and D1 cells showed no significant [3H]DPCPX-specific binding (data not shown). The Bmax and Kd values for [3H]DPCPX binding in the A1D1 cells were, respectively (Fig. 1). 1992 ± 130.7 fmol/mg of protein and 1.3 ± 0.2 nM (means ± S.E.M., n = 3). 3.2. Saturation experiments with dopamine D1 receptor antagonist [3H]SCH23390 The naive HEK293 cells showed no significant [3 H] SCH23390-specific binding (data not shown). In the D1 cells,
Table 1 Saturation experiments with dopamine D1 receptor antagonist [3H]SCH23390 in membrane preparations from D1 and A1D1 cells
D1 cells A1D1 cells
Pretreatment
Bmax (fmol/mg)
Kd (nM)
Control CPA, 10 μM Control CPA, 10 nM CPA, 10 μM
2373 ± 67 2377 ± 13 1821 ± 78 1844 ± 21 1916 ± 64
0.70 ± 0.06 0.60 ± 0.10 0.84 ± 0.15 0.91 ± 0.02 1.30 ± 0.20 a
Bmax and Kd values are expressed as mean ± S.E.M. n = 3. a P < 0.05 compared with control by one-way ANOVA followed by the Student–Newman–Keuls test.
Fig. 2. Representative saturation curves of specific binding of dopamine D1 receptor antagonist [3H]SCH23390 in membrane preparations from A1D1 cells in the absence and presence of adenosine A1 receptor agonist CPA. The Bmax and Kd values were obtained by Scatchard analysis.
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no significant differences were obtained in the absence or presence of adenosine A1 receptor agonist CPA (10 μM) regarding the Bmax and Kd values for the dopamine D1 receptor binding sites labeled with [3H]SCH23390 (Table 1). In the A1D1 cells, CPA (10 nM) had no effect on [H]SCH23390 binding in cell membranes (Fig. 2, Table 1). However, CPA (10 μM) significantly increased the Kd value of [H]SCH23390, without affecting the Bmax value. The Kd values in the absence and presence of CPA (10 μM) were 0.84 ± 0.15 and 1.3 ± 0.2 nM (means ± S.E.M., n = 3), respectively (Fig. 2, Table 1). 3.3. Competition experiments of SKF38393 versus dopamine D1 receptor antagonist [3H]SCH23390 Competition experiments of SKF38393 versus dopamine D1 receptor antagonist [3H]SCH23390 in intact A1D1 cells showed a significant difference in the absence or presence of adenosine A1 receptor agonist CPA. CPA produced a concentrationdependent increase in the Ki value of SKF38393 (Fig. 3A, Table 2). However, in the D1 cells, no significant differences were obtained between in the absence and presence of adenosine A1
Table 2 Competition experiments of SKF38393 versus dopamine D1 receptor antagonist [3H]SCH23390
D1 cells A1D1 cells
Pretreatment
Ki (μM)
Control CPA, 10 μM Control CPA, 10 nM CPA, 10 μM PTX PTX + CPA, 10 nM PTX + CPA, 10 μM DPCPX, 10 μM
3.15 ± 0.15 3.43 ± 0.13 1.89 ± 0.09 6.49 ± 0.50 a 8.82 ± 1.43 a 1.82 ± 0.06 1.94 ± 0.25 4.99 ± 0.22 a 1.17 ± 0.03 b
Ki values are expressed as mean ± S.E.M. n = 3. PTX, pertusis toxin. a P < 0.01 compared with control by one-way ANOVA followed by the Student–Newman–Keuls test. b P < 0.05 compared with control by one-way ANOVA followed by the Student–Newman–Keuls test.
receptor agonist CPA for the Ki values (Table 2). Pretreatment of the A1D1 cells with pertussis toxin counteracted the effect of a low concentration of CPA (10 nM), and only slightly counteracted the effect of 10 μM CPA (Fig. 3B, Table 2). The adenosine A1 receptor antagonist DPCPX (10 μM) significantly decreased the Ki value of SKF38393 in A1D1 cells (Fig. 3C, Table 2). All competition curves were best fitted if a single binding site was assumed. 3.4. Dopamine D1 receptor agonist SKF38393 induced CRESEAP activity increase In D1 and A1D1 cells which were transiently transfected with pCRE-SEAP reporter vector, SKF38393 induced a
Fig. 3. Representative competitive inhibition curves of dopamine D1 receptor agonist SKF38393 versus dopamine D1 receptor antagonist [3H]SCH23390 in A1D1 cells. Competition assays were done at 0.5 nM [3H]SCH23390 and increasing concentrations of SKF38393. PTX, pertussis toxin. Ki values are means (95% confidence intervals) obtained from three independent experiments performed in triplicate using GraphPad Prism 4.0 program.
Fig. 4. CRE-SEAP reporter gene activity induced by dopamine D1 receptor agonist SKF38393 in the absence and presence of adenosine A1 receptor agonist CPA in D1 (A) and A1D1 cells (B) transiently transfected with pCRE-SEAP reporter gene. SEAP activity is expressed as a percentage of the maximal activity obtained for each curve. PTX, pertussis toxin. EC50 values are expressed as median (95% confidence intervals) obtained from three independent experiments performed in triplicate using GraphPad Prism 4.0 program.
Y. Cao et al. / European Journal of Pharmacology 548 (2006) 29–35 Table 3 Dopamine D1 receptor agonist SKF38393 induced CRE-SEAP activity
D1 cells A1D1 cells
Pretreatment
EC50 (×10− 8 M)
Control CPA, 10 μM Control CPA, 10 nM CPA, 10 μM PTX + CPA, 10 nM PTX + CPA, 10 μM
5.79 ± 0.58 6.17 ± 1.54 6.80 ± 1.04 8.68 ± 1.28 12.70 ± 1.55 a 6.88 ± 1.34 10.28 ± 1.19
EC50 values are expressed as mean ± S.E.M. n = 3. PTX, pertussis toxin. a P < 0.05 compared with control by one-way ANOVA followed by the Student–Newman–Keuls test.
concentration-dependent increase in the CRE-SEAP activity (Fig. 4). adenosine A1 receptor agonist CPA (10 μM) significantly decreased the EC50 value of SKF38393 in A1D1 cells but not in D1 cells (Fig. 4, Table 3). The CRE-SEAP activity basal levels were not modified by CPA (Fig. 4). Thus, the decrease in CRE-SEAP activity by activation of adenosine A1 receptors could be ignored. In contrast, pertussis toxin treatment counteracted the effect of a low concentration of CPA (10 nM) on dopamine D1 receptor-mediated CRE recruitment, and only slightly counteracted the effect of 10 μM CPA (Fig. 4, Table 3). 4. Discussion For G protein-coupled receptors, it is widely believed that the critical event in agonist action is the stabilization of the ternary complex of agonist/receptor/G-protein (Lefkowitz et al., 1993), and the better this ternary complex is stabilized, the more efficacious the agonist is. Both adenosine A1 receptor and dopamine D1 receptor belong to the G protein-coupled receptor family. Coupling to Gs and Gi proteins, stimulation of dopamine D1 receptor and adenosine A1 receptor activates and inhibits adenylyl cyclase, respectively. It has been shown that the binding characteristics of one type of G protein-coupled receptor can be altered by the stimulation of another type of G protein-coupled receptor in crude membrane preparations (Zoli et al., 1993). Such intramembrane interactions have been postulated to represent direct interactions between the receptor molecules and/or to involve G proteins or other mobile molecules associated with the membrane (Zoli et al., 1993). In the present studies, the radioligand binding experiments carried out from the cotransfected A1D1 cells showed that the adenosine A1 receptor-mediated modulation of dopamine D1 receptor induced a concentration-dependent decrease in the affinity of dopamine D1 receptor for agonist SKF38393. These results were similar to those obtained with the cotransfected A1/ D1 fibroblast Ltk-cells (Ferre et al., 1998). The only difference was that the inhibition of binding to dopamine D1 receptor by SKF38393 was best described as occurring to a single type of site in our results. Previous evidence has demonstrated that adenosine A1 receptor and dopamine D1 receptor heteromers were presented in the membrane of cotransfected fibroblast cells (Gines et al., 2000). Thus, we speculated that the decrease in the affinity of dopamine D1 receptor for SKF38393 induced by
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adenosine A1 receptor stimulation could be the result of a physical interaction of adenosine A1 receptor with dopamine D1 receptor in the heteromeric complex, leading to an uncoupling of dopamine D1 receptor from its Gs-like protein in this functionally interacting heteromeric complex. The effects of adenosine A1 receptor antagonist DPCPX on the Ki value of SKF38393 in A1D1 cells also suggest that adenosine released by these cells exerts a tonic inhibition of dopamine D1 receptormediated function through the A1–D1 interaction.(Le Crom et al., 2002). Pertussis toxin is a good tool to investigate G protein involvement because of its selectivity of action by inducing an ADP-ribosylation of the Gá subunit of the Gi (and Go) protein family (Gilman, 1987). A1D1 cells were exposed to pertussis toxin to study the possible involvement of Gi protein in the adenosine A1 receptor-mediated uncoupling of the dopamine D1 receptor from the Gs protein. It was found that pertussis toxin counteracted the effect of CPA (10 nM) on dopamine D1 receptor binding characteristics, suggesting that the Gi protein was necessary for the intramembrane A1–D1 interaction in the presence of a low concentration of CPA. However, a higher concentration of CPA (10 μM) could still uncouple dopamine D1 receptor from the Gs protein after pertussis toxin pretreatment. These results suggest that the effect of adenosine A1 receptor activated by a high concentration of CPA might be mediated by a different mechanism than the effect of adenosine A1 receptor activated by a low concentration of CPA. The density of two interacting receptors and the number of receptors activated by the agonist in each population would determine the proportion of monomers and homodimers and heterodimers and thus the overall action on target cell function (Zoli et al., 1993). It is highly probable that clustering occurs for several G proteincoupled receptors when they are activated by their ligands. Occurrence of clustering clearly reflects that G protein-coupled receptors form high molecular order structures in which multimers of the receptors and, probably, other interacting proteins form functional complexes. It has been reported that exposure to adenosine A1 receptor agonist R(−)N6-(2-phenylisopropyl)adenosine (R-PIA, 100 nM) induced the formation of clusters (aggregations) containing both adenosine A1 receptor and dopamine D1 receptor immunoreactivities (Gines et al., 2000). Thus, a high concentration of CPA (10 μM) treatment might induce the formation of adenosine A1 receptor/dopamine D1 receptor heteromers leading to conformational changes in their respective binding pockets. However, a low concentration of CPA (10 nM) could not induce enough clustering resulting in the conformational changes in the binding pockets and might only lead to an uncoupling of dopamine D1 receptor from its Gs protein by a manner similar to the dopamine D1 receptordopamine D2 receptor interaction which seems to involve G protein (Seeman et al., 1989). The subunits of the stimulatory G protein, Gs (serving dopamine D1 receptor), may be in equilibrium with those of the inhibitory G protein, Gi (serving adenosine A1 receptor). In the presence of CPA (to activate adenosine A1 receptor), the subunits of the G protein may rearrange. This rearrangement converts dopamine D1 receptor to low affinity for SKF38393. Exposure to a high concentration of CPA might
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induce the formation of adenosine A1 receptor/dopamine D1 receptor heteromers. In this case, besides the G proteindependent manner as in the presence of a low concentration of CPA, the physical interaction is maintained when the adenosine A1 receptor and dopamine D1 receptor binding pockets are simultaneously activated by agonists allowing the antagonistic intramembrane receptor/receptor interaction to take place by a G protein-independent manner. Taken together, the functional meaning of this intramembrane receptor/receptor interaction in the presence of a high concentration of CPA is therefore to decrease the affinity of dopamine D1 receptor for SKF38393 in G protein-dependent and -independent manners. A well recognized characteristic of agonist binding to receptors such as the β2-adrenergic receptor is the increase in high affinity binding states, indicative of receptor/G-protein interactions (Costa et al., 1992; De Lean et al., 1980; Pin et al., 2005; Samama et al., 1993). Similarly, the high affinity binding state of dopamine D1 receptor requires the receptor coupling with Gs protein. Thus, adenosine A1 receptor stimulation leads to the uncoupling of dopamine D1 receptor from its Gs protein so as to decrease the affinity of dopamine D1 receptor for agonist. However, antagonists are drugs that bind to the receptor without activating the effector system for that receptor. Therefore in the low concentration of CPA treatment, adenosine A1 receptor stimulation involves no changes in the affinity of dopamine D1 receptor for the antagonist [3H]SCH23390 in the saturation experiments (Fig. 2, Table 1), which were consistent with the results reported by Ferre et al. (1998). Nevertheless, exposure to the high concentration of CPA might induce the formation of adenosine A1 receptor/dopamine D1 receptor heteromers so as to decrease the affinity of dopamine D1 receptor for the antagonist [3H]SCH23390 in a G proteinindependent manner. Dopamine D1 receptor can activate adenylyl cyclase, resulting in cAMP production. Cell based assays relying on transcriptionally controlled reporter genes have been developed and are suited to monitor and are signaled the cellular responses induced by G protein-coupled receptors. Both Gs- and Gqcoupled receptors are signaled through CREs (Chen et al., 1995; Durocher et al., 2000; Stratowa et al., 1995). Since CRE is a pivotal target in G protein-coupled receptor signaling pathways, it has become one of the most widely used response elements in reporter gene assays. Since the transcription of CRE-SEAP reporter genes is controlled by CRE-containing promoters, CRE-SEAP activity can be used to monitor the activation of receptors. Previously we found that the CRE-SEAP activity assay could substitute the traditional radioimmunoassay for the measurement of cAMP accumulation (Sun et al., 2005b). In the present experiments, dopamine D1 receptor agonist-induced CRE-SEAP activity increase was demonstrated in the D1 and A1D1 cells. Furthermore, in A1D1 cells, adenosine A1 receptor agonist CPA counteracted the CRE-SEAP activity increase induced by SKF38393 in a concentration-dependent manner. Pertussis toxin counteracted the effect of the low concentration of CPA on the D1 receptor-mediated CRE recruitment, and only slightly affected the inhibitory effect of the high concentration of CPA. These results suggest that adenosine A1 receptor
activation can inhibit the effect induced by dopamine D1 receptor activation at the second messenger level. Thus, the clear correlation between the results obtained with the radioligand binding and functional interaction experiments suggests that the intramembrane A1–D1 interaction involved in the binding experiments is related to the A1–D1 interaction found at the second messenger level. In summary, two main findings have been obtained in this study. First, in A1D1 cells, adenosine A1 receptor activation can decrease the binding affinity of dopamine D1 receptor for agonists and inhibit the effect induced by dopamine D1 receptor activation at the second messenger level. Secondly, the mechanisms of the antagonistic interaction between adenosine A1 receptor and dopamine D1 receptor are different in the presence of a high concentration or a low concentration of CPA. When adenosine A 1 receptors are activated by a low concentration of CPA, Gi proteins are necessary for the antagonistic effect. However, in the presence of a high concentration of CPA, besides the G protein involved manner, the conformational changes induced by the A1/D1 heteromers result in the antagonistic intramembrane receptor/receptor interaction to take place by a G protein-independent manner. Such concerted interactions between functionally distinct neurotransmitter receptors increasingly appear to be part of a general overall mechanism by which neurons are able to receive and integrate diverse extracellular signals (Le Crom et al., 2002; Maggio et al., 2005). Acknowledgements This work was supported by a research grant from the Ministry of Science and Technology of China (2004CB720305) and the Shanghai Metropolitan Fund for Research and Development (04DZ14005). We thank Dr. Lin-lin Yin, Dr. Zhong-hua Liu and Dr. Wei-yu Zhang for their expert advice and assistance. We also acknowledge Dr. Lin-yin Feng for revision of the manuscript. References Brundege, J.M., Dunwiddie, T.V., 1997. Role of adenosine as a modulator of synaptic activity in the central nervous system. Adv. Pharmacol. 39, 353–391. Chen, W., Shields, T.S., Stork, P.J., Cone, R.D., 1995. A colorimetric assay for measuring activation of Gs- and Gq-coupled signaling pathways. Anal. Biochem. 226, 349–354. Cheng, Y., Prusoff, W.H., 1973. Relationship between the inhibition constant (Ki) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem. Pharmacol. 22, 3099–3108. Costa, T., Ogino, Y., Munson, P.J., Onaran, H.O., Rodbard, D., 1992. Drug efficacy at guanine nucleotide-binding regulatory protein-linked receptors: thermodynamic interpretation of negative antagonism and of receptor activity in the absence of ligand. Mol. Pharmacol. 41, 549–560. De Lean, A., Stadel, J.M., Lefkowitz, R.J., 1980. A ternary complex model explains the agonist-specific binding properties of the adenylate cyclasecoupled beta-adrenergic receptor. J. Biol. Chem. 255, 7108–7117. Durocher, Y., Perret, S., Thibaudeau, E., Gaumond, M.H., Kamen, A., Stocco, R., Abramovitz, M., 2000. A reporter gene assay for high-throughput screening of G-protein-coupled receptors stably or transiently expressed in HEK293 EBNA cells grown in suspension culture. Anal. Biochem. 284, 316–326.
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