Introduction

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Dopaminergic transmission is involved in the control of locomotor activity and cognitive and neuroendocrine functions (Dolan et al., 1995; Williams and Goldman-Rakic, 1995). Dysfunction of the dopaminergic system, due to degeneration of the nigrostriatal dopaminergic pathway, leads to Parkinson's disease, whereas increased mesolimbic dopaminergic activity is believed to be involved in schizophrenia (Heimer, 1994).

The action of dopamine is mediated by receptors that belong to the superfamily of G protein-coupled receptors. Originally, dopamine receptors were classified into two families (D₁ and D₂) based on pharmacology and signal transduction (Kebabian and Calne, 1979). The cloning of various dopamine receptor genes led to the description of the D₁-like family (D₁ and D₅ receptors) and the D₂-like family (D₂, D₃, and D₄ receptors) (Missale et al., 1998).

Human dopamine D₂ (hD₂) and D₃ (hD₃) receptors display considerable amino acid sequence similarity. All known antipsychotics bind to D₂ receptors, and most also bind to D₃ receptors (Leysen et al., 1998). In general, dopamine and several dopaminergic agonists have a higher affinity for hD₃ than for hD₂ receptors, whereas the affinity of antagonists is usually slightly higher for hD₂ receptors (Sokoloff et al., 1992). The distribution of hD₃ receptors in the brain seems to be confined to mesolimbic areas, whereas hD₂ receptors are found in all dopaminergic brain areas (De Keyser, 1993; Landwehrmeyer et al., 1993). Both hD₂ and hD₃ receptors possess a large third intracytoplasmic loop and a short carboxyl-terminal tail, a characteristic of receptors that couple to the G_i/o subfamily of G proteins (Dohlman et al., 1991). Modulation of agonist binding at hD₂ receptors by guanine nucleotides and of hD₂ receptor-activated signaling pathways has been clearly demonstrated. In contrast, conflicting results have been reported regarding the G protein-coupling and -signaling properties of hD₃ receptors. First, several groups failed to find modulation of agonist binding at hD₃ receptors by guanine nucleotides (Freedman et al., 1994; Tang et al., 1994a; Akunne et al., 1995; McAllister et al., 1995). However, other groups have reported a small rightward shift of the dopamine inhibition binding curve by guanine nucleotides (Sokoloff et al., 1992; MacKenzie et al., 1994). Second, there are several reports of negative findings on hD₃-mediated inhibition of adenylyl cyclase activity, stimulation of arachidonic acid release, or phospholipase C activity (Freedman et al., 1994; MacKenzie et al., 1994; Tang et al., 1994a). Also, stimulation of adenylyl cyclase subtype II by hD₃ receptors could not be demonstrated (Watts and Neve, 1997). In contrast, a slight inhibition of adenylyl cyclase activity was shown by McAllister et al. (1995) and Griffon et al. (1996). Third, a slight activation of G proteins measured as [³⁵S]guanosine-5'-O-(3-thio)triphosphate (GTPS) binding by hD₃ receptors in transfected mammalian cell lines has been reported (Gardner et al., 1996; Malmberg et al., 1998). However, further downstream effects of hD₃ receptor signaling, such as inhibition of dopamine synthesis and release or stimulation of neurite outgrowth, have been clearly demonstrated in neuronal cells, indicating a functional role for hD₃ receptors (Tang et al., 1994b; O'Hara et al., 1996; Swarzenski et al., 1996). Most surprisingly, some reports have described coupling to signal transduction pathways but no effect of guanine nucleotides on agonist binding, and vice versa (MacKenzie et al., 1994; Tang et al., 1994a,b; McAllister et al., 1995; O'Hara et al., 1996).

From these conflicting findings, it is clear that the hD₃ receptor-signaling mechanism has not yet been fully established. We report an extensive study of the signaling of hD₃ receptors expressed in Chinese hamster ovary (CHO) cells, investigated in parallel with hD_2L receptors expressed in CHO cells. We studied the ligand-binding and -signaling properties of the same type of parent CHO cells with the same high expression levels of hD_2L and hD₃ receptors. In addition, we investigated a hD₃-CHO cell clone with an 8-fold lower expression level. The effect of receptor-G protein uncoupling was investigated by examination of the effect of GTPS on the dopamine-binding curve. Using the [³⁵S]GTPS binding assay, we compared the activation of G proteins by hD_2L and hD₃ receptors on binding of dopamine, (+)-7-(dipropylamino)-5,6,7,8-tetrahydro-2-naphthalenol (7-OH-DPAT), and PD128907. Haloperidol, risperidone, raclopride, and nemonapride were tested for their ability to inhibit dopamine-stimulated [³⁵S]GTPS binding. We investigated the inhibition by dopamine of forskolin-stimulated cyclic AMP (cAMP) formation in CHO cells stably expressing cloned human D_2L or D₃ receptors (hD_2L-CHO; D₃-CHO). In addition, we compared extracellular acidification rates on dopamine stimulation of hD_2L-CHO and hD₃-CHO cells and the effect of pertussis toxin pretreatment thereupon. The various investigations revealed clear signal transduction phenomena for both dopamine receptor subtypes, indicating that both receptors couple to G_i/o proteins.

	Experimental Procedures

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Cell Culture and Membrane Preparation. The cDNA clones of hD_2L and hD₃ receptors were purchased, corrected by polymerase chain reaction techniques, and inserted into the pKCRE expression vector (Stam et al., 1992). The sequence was verified by DNA sequencing. The expression constructs were stably transfected into CHO cells by the calcium phosphate method, and clonal cell colonies were isolated in medium containing 800 µg/ml G418. Clones expressing high levels of receptor were selected (hD_2L-CHO and hD₃-CHO-high; Schotte et al., 1996); for hD₃-CHO, we also selected a clone with a lower expression level. CHO cells expressing hD_2L or hD₃ receptors were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% fetal calf serum (FCS), in a humidified atmosphere of 5% CO₂ at 37°C. Cells were subcultured at 80 to 90% confluence.

Radioligand-Binding Assays. hD_2L-CHO and hD₃-CHO cell membranes were thawed on ice and suspended in 50 mM Tris-HCl buffer, pH 7.4, with 120 mM NaCl. For the radioligand binding assay, 5 to 10 µg membrane protein/assay was used. For [³H]spiperone binding, the incubation volume was 0.5 ml and incubation was performed for 30 min at 37°C. For [¹²⁵I]iodosulpride binding, the incubation mixture contained 0.1% BSA, the assay volume was 0.25 ml, and incubation was performed for 30 min at 25°C. Nonspecific binding was estimated in the presence of 10 µM haloperidol for both hD_2L and hD₃ receptors. The reaction was terminated by filtration through Whatman GF/B filters presoaked in 0.1% polyethyleneimine. Filters were rinsed twice with 5 ml of ice-cold incubation buffer. The filter-bound radioactivity was measured in a liquid scintillation spectrometer (Tricarb; Packard, Meriden, CT) with 3 ml of scintillation fluid. Specific binding was calculated as the difference between total binding and nonspecific binding. For ligand concentration binding isotherms, [³H]spiperone was used at 10 to 12 concentrations in the range 0.01 to 1 nM, and [¹²⁵I]iodosulpride was used at 10 concentrations in the range 0.1 to 3 nM. Ligand concentration binding isotherms were fitted to a rectangular hyperbola by nonlinear regression analysis in which the K_d and B_max values were free parameters.

[³⁵S]GTPS-Binding Assays. The [³⁵S]GTPS-binding assay was performed essentially as described by Gardner et al. (1996). Briefly, the [³⁵S]GTPS-binding assay was carried out in a final volume of 0.5 ml containing 50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 5 mM MgCl₂, 1 mM EGTA, 0.1 mM dithiothreitol, 1 µM guanosine diphosphate, and 0.2 nM [³⁵S]GTPS. Membranes (5-10 µg of membrane protein) and ligands were preincubated without [³⁵S]GTPS for 30 min at 30°C to obtain steady-state receptor occupation. After the addition of [³⁵S]GTPS, membranes were further incubated for 30 min. Basal [³⁵S]GTPS binding was measured in the absence of ligands. Reactions were terminated by rapid filtration (see above) through Whatman GF/B filters soaked in incubation buffer. The amount of radioactivity collected on the filter was determined by liquid scintillation counting. The maximum amount of [³⁵S]GTPS binding was always less than 10% of [³⁵S]GTPS added. In preliminary experiments, nonspecific binding was measured in the presence of 100 µM GTPS; this never exceeded 10% of basal binding. Nonspecific binding was not subtracted; all values represent the total levels of [³⁵S]GTPS binding.

Measurement of cAMP Content. Cells were grown overnight in a Falcon multiwell 96-plate (Becton Dickinson Labware Ekembodegem, Belgium) at 10,000 cells/well. Sodium butyrate (5 mM) was added 24 h before the experiment. On the day of the experiment, cells were washed with controlled salt solution (CSS; 120 mM NaCl, 5 mM KCl, 0.8 mM MgCl₂, 1.8 mM CaCl₂, 15 mM glucose, 0.04 mM phenol red in 25 mM Tris-HCl, pH 7.4) at 37°C. After removal of CSS, the cells were preincubated for 20 min at 37°C in 60 µl of CSS containing 0.1% BSA. Dopamine was not included during the preincubation. Then, the cells were further incubated for 20 min at 37°C by the addition of 60 µl of CSS supplemented with 0.1% BSA, 2 mM 3-isobutyl-1-methylxanthine, 2 µM pargyline (a monoamine oxidase inhibitor), 2 µM cocaine (a dopamine uptake inhibitor), 100 µM forskolin, and the appropriate concentration of dopamine. After the addition of 20 µl of 1 M HClO₄ (ice-cold) to terminate the reaction, the plates were frozen and thawed, and 20 µl of ice-cold KOH/K₃PO₄ (0.5 M, pH 13.5) was added to neutralize the samples (final pH 7.4). After formation of the KClO₄ precipitate (30 min at 4°C), the plates were centrifuged (10 min at 650g, 4°C). The amount of cAMP in each well was determined with a commercial ¹²⁵I-labeled cAMP radioimmunoassay kit according to the procedure recommended by the manufacturer. Results are calculated as percentages of forskolin levels. GraphPad Prism was used to fit dose-response curves to a sigmoid in which the pEC₅₀ value and the Hill coefficient were free parameters.

Measurement of Extracellular Acidification Rate. Microphysiometry was performed as described elsewhere (Owicki et al., 1990). Briefly, cells in DMEM containing 10% FCS were seeded onto Transwell capsules at 24 h before the experiment. The capsules were briefly rinsed in running medium (DMEM without serum and without NaHCO₃) and set up in the Cytosensor (Molecular Devices, Munchen, Germany). The superfusion speed was 100 µl/min, and a cycle was 120 s, composed of 90 s of pumping, 3 s of rest, 25 s of measurement, and 2 s of rest. Results are expressed as percentages of basal value; basal acidification rates were measured between 50 and 70 µV/s. Cells were stimulated each half hour for 4 min with increasing concentrations of dopamine. In one series of experiments, hD_2L-CHO and hD₃-CHO cells were pretreated in the Transwell capsules with pertussis toxin for 6 h at 100 ng/ml. Subsequently, the cells were washed in toxin-free medium and used for microphysiometry.

Materials. [³H]Spiperone (3.5 TBq/mmol), [¹²⁵I]iodosulpride (74.1 TBq/mmol), and [³⁵S]GTPS (±40.7 TBq/mmol) were purchased from Amersham Pharmacia Biotech (Little Chalfont, UK). Pargyline and forskolin were obtained from Sigma-Aldrich (St. Louis, MO). 3-Isobutyl-1-methylxanthine was obtained from Fluka (Buchs, Switzerland). DMEM and FCS were purchased from Life Technologies (Gaithersburg, MD). The protein assay kit was obtained from Bio-Rad (Hercules, CA). The ¹²⁵I-labeled cAMP radioimmunoassay kit (SMP001J) was obtained from Dupont-NEN (Boston, MA). PD128907, lisuride, and dopamine were purchased from Research Biochemicals, Inc. (Natick, MA). Raclopride and nemonapride were purchased from Astra Arcus (Stockholm, Sweden) and Yamanouchi (Tokyo, Japan), respectively. TL99 was obtained from ICN Pharmaceuticals (Costa Mesa, CA). Haloperidol, domperidone, risperidone, and spiperone are original products of Janssen Pharmaceutica (Beerse, Belgium). 7-OH-DPAT was synthesized in-house. Guanosine diphosphate, GTPS, and 4-(2-aminoethyl)-benzenesulfonylfluoride hydrochloride were purchased from Boehringer Mannheim (Mannheim, Germany). All other reagents were of analytical grade and obtained from Merck (Haasrode, Belgium) or Sigma Chemical Co. (St. Louis, MO). GF/B glass-fiber filters were purchased from Whatman (Kent, UK). The scintillation fluid (Ultima Gold MV) was purchased from Packard (Meriden, CT). Transwell capsules (Costar) were obtained from Elscolab (Kruibeke, Belgium). Dopamine, 7-OH-DPAT, and PD128907 were dissolved and diluted in assay buffer. Lisuride and TL99 were dissolved and diluted in ethanol. Haloperidol, spiperone, domperidone, risperidone, raclopride, and nemonapride were dissolved and diluted in dimethyl sulfoxide (DMSO). For compounds that were dissolved and diluted in DMSO or ethanol, the final, 20-fold dilution step in assay buffer was performed just before addition to the assay mixture, in which the dilution was 10-fold. In control assays, ethanol or DMSO was added to a final concentration of 0.5%.

	Results

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Characterization of hD_2L and hD₃ Receptor Binding in Transfected CHO Cell Membranes. The hD_2L and hD₃ receptor binding was investigated by use of [³H]spiperone and [¹²⁵I]iodosulpride; K_d and B_max values are listed in Table 1. No binding was detectable in wild-type CHO cell membranes (results not shown).

View larger version (29K): [in this window] [in a new window]   Fig. 1.   Inhibition of [3H]spiperone binding to hD2L-CHO cell membranes by various dopaminergic agonists (A) or antagonists (B) or of [125I]iodosulpride binding to hD3-CHO-high cell membranes by various dopaminergic agonists (C) or antagonists (D). Total binding was determined in the absence of compounds; nonspecific binding was measured in the presence of 10 µM haloperidol. The data represent a typical experiment. For each experiment, the pIC50 value was derived by nonlinear regression analysis. Mean pIC50 and Ki values and the number of independent experiments are presented in Table 2.

Effect of GTPS on Dopamine Binding. The effect of G protein uncoupling on dopamine binding to hD_2L and hD₃ receptors was investigated by measurement of the inhibition of [³H]spiperone binding by dopamine in the absence and presence of GTPS (100 µM; Table 3; curves shown in Fig. 2). For both hD_2L and hD₃ receptors, GTPS induced a steepening (hD_2L, p < .01; hD₃, p < .05) and a rightward shift (hD_2L, 0.73 log unit, p < .005; hD₃, 0.33 log unit, p < .005) of the dopamine inhibition curve when fitted to a single binding site (paired two-tailed Student's t test).

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Inhibition of [3H]spiperone binding by dopamine in the presence and absence of 100 µM GTPS at hD2L-CHO (A) and hD3-CHO-high (B). The data represent the mean ± S.D. of five independent experiments. The pIC50 values and Hill coefficients are presented in Table 3. In the absence of GTPS, a two-site model better described the data for hD2L-CHO (p < .005) but not for hD3-CHO-high (p > .05). In the presence of GTPS, no significant improvement for a two-site model could be observed for hD2L-CHO (p > .05) and hD3-CHO-high (p > .05).

hD_2L and hD₃ Receptor-Mediated Modulation of [³⁵S]GTPS Binding. The effects of dopamine, 7-OH-DPAT, and PD128907 on [³⁵S]GTPS binding to hD_2L-CHO, hD₃-CHO-high, and hD₃-CHO-low cell membranes are shown in Fig. 3; pEC₅₀ values and the levels of stimulation are listed in Table 4. We used dopamine as a reference agonist. 7-OH-DPAT and PD128907 did not produce the maximum stimulation compared with dopamine and hence seem to be partial agonists (see Table 4). 7-OH-DPAT inhibited dopamine-stimulated [³⁵S]GTPS binding to the level of its partial agonistic effect in hD_2L-CHO and in both hD₃-CHO clones, but PD128907 did not show any antagonistic effect at either receptor (results not shown).

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Dopamine- (A), 7-OH-DPAT- (B), and PD128907- (C) stimulated [35S]GTPS binding to hD2L-CHO, hD3-CHO-low, and hD3-CHO-high cell membranes. After preincubation with these compounds for 30 min at 30°C, membranes were further incubated with 0.2 nM [35S]GTPS for 30 min. Results are expressed as the percentage of maximum dopamine stimulation and are the mean ± S.D. of three or four experiments. The amount of stimulation and the pEC50 values are listed in Table 4.

View this table: [in this window] [in a new window]   TABLE 4 Agonist stimulation of [35S]GTPS binding (30°C) to hD2L-CHO and hD3-CHO cell membranes: pEC50 values and percentages of stimulation For hD3-CHO, clones with low (hD3-CHO-low) and high (hD3-CHO-high) expression levels were used. Experiments were performed as described in the text. Values represent mean ± S.D. of n experiments, in which each point was determined in duplicate.

View larger version (26K): [in this window] [in a new window]   Fig. 4.   Inhibition of dopamine-stimulated (10 µM) [35S]GTPS binding to hD2L-CHO (A) and hD3-CHO-high (B) cell membranes by nemonapride, haloperidol, risperidone, and raclopride. Curves are mean ± S.D. of three to five experiments with duplicate determinations and are shown as the percentages of maximum dopamine stimulation. Mean pIC50 values are listed in Table 5.

View this table: [in this window] [in a new window]   TABLE 5 Potencies of antagonists for inhibition of dopamine-stimulated (10 µM) [125S]GTPS binding to hD2L-CHO and hD3-CHO-high cell membranes Experiments were performed as described in the text. Values represent mean ± S.D. of n experiments, in which each point was determined in duplicate. Corrected IC50 values (cIC50) were calculated as described in the text.

hD_2L and hD₃ Receptor-Mediated Inhibition of cAMP. Forskolin-stimulated (50 µM) cAMP levels were 4.5 ± 0.7 pmol/well for hD_2L-CHO (n = 5), 14.0 ± 3.4 pmol/well for hD₃-CHO-high (n = 7), and 10.0 ± 4.2 pmol/well for untransfected CHO cells (n = 2). Basal cAMP levels never exceeded 5% of the forskolin-stimulated level. Wild-type CHO cells did not show any response on the addition of dopamine.

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Dopamine inhibition of forskolin-stimulated adenylyl cyclase activity in hD2L-CHO and hD3-CHO-high cells. Experiments were performed as described in Experimental Procedures. Data are shown as the percentage of forskolin-stimulated cAMP level ± S.D. Curves represent the mean of four experiments for hD2L-CHO and seven experiments for hD3-CHO-high, in which each point is determined in duplicate. The pEC50 values and inhibition levels are discussed in the text.

hD_2L and hD₃ Receptor-Mediated Acidification of Cell Culture Medium. The dose-response curves for the acidification rate of the cell culture medium after the application of dopamine to hD_2L-CHO cells and hD₃-CHO-high cells, and the effect of pertussis toxin pretreatment thereupon, are shown in Fig. 6. Dopamine stimulated the extracellular acidification rates up to 15 and 45% over basal levels in hD₃-CHO and hD_2L-CHO cells, respectively. In the absence of pertussis toxin, derived pEC₅₀ values were 7.87 ± 0.16 (n = 4) and 8.40 ± 0.22 (n = 6) for hD_2L-CHO and hD₃-CHO cells, respectively. Pertussis toxin treatment almost completely abolished hD_2L and hD₃ receptor-stimulated extracellular acidification.

View larger version (23K): [in this window] [in a new window]   Fig. 6.   Dopamine-stimulated extracellular acidification rates for hD2L-CHO and hD3-CHO-high cells measured with the aid of a microphysiometer and inhibition by pertussis toxin. Experiments were performed as described in Experimental Procedures. Data were normalized to 0 and 100% for the basal and the maximum extracellular acidification rate, respectively. Dopamine stimulated the extracellular acidification rates up to 15 and 45% over basal levels in hD3-CHO-high and hD2L-CHO cells, respectively. Curves show the mean points of four (hD2L-CHO) or six (hD3-CHO) experiments.

	Discussion

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In this study, we compared the binding and signaling properties of hD_2L and hD₃ receptors in the same background of CHO cells. Discrepancies between previous reports regarding hD₃ receptor coupling to G proteins and/or the generation of second messengers necessitated clear demonstration of these mechanisms in parallel with hD_2L receptors.

Therefore, we performed experiments with CHO cells expressing approximately equal, high levels of hD_2L and hD₃ receptors. Both receptors bound [³H]spiperone and [¹²⁵I]iodosulpride with high affinity. The pharmacological properties of the receptors corresponded well with previously reported data (Sokoloff et al., 1990). Like dopamine, the agonists tested had an apparent preference for hD₃ over hD_2L receptors in ligand-binding assays with CHO cell membranes. PD128907 was the most selective ligand for hD₃ receptors, and lisuride was the least selective. The antagonists investigated showed nanomolar binding affinities for both hD_2L and hD₃ receptors. We observed a 20- to 30-fold higher binding affinity of haloperidol and domperidone for hD_2L over hD₃ receptors, which is in agreement with data reported by Sokoloff et al. (1990).

GTPS was used to modulate the dopamine inhibition of [³H]spiperone binding. In the absence of GTPS, the data for hD_2L-CHO were best explained by a two-site competition curve with a high- and a low-affinity site. In contrast, the hD₃-CHO-high dopamine inhibition curve was best fitted to a one-site model. GTPS induced a significant rightward shift and steepening of the dopamine inhibition curve for both hD_2L-CHO and hD₃-CHO-high, although the effect was less pronounced for hD₃-CHO-high. However, the latter was observed consistently and was statistically significant. Similar experiments with another hD₃ clone (hD₃-CHO-low), expressing about 8-fold fewer receptors, also yielded a slight rightward shift and steepening of the dopamine inhibition curve. Our observations suggest that the hD₃ receptor exists in different conformational states, but dopamine does not seem to distinguish much between the G protein-coupled conformation and the uncoupled receptor conformation, as suggested by the small shift in the dopamine inhibition curve in the presence of GTPS. Observations on dopamine binding to hD₃ receptors, expressed in Escherichia coli providing the receptor in the complete absence of G proteins, support this hypothesis (unpublished observation). In contrast, the hD_2L receptor displays a low- and high-affinity conformation for agonist binding, presumably representing the uncoupled and G protein-coupled receptor conformation, respectively.

In the present study, the [³⁵S]GTPS binding assay was used to investigate G protein activation by agonist stimulation of hD_2L and hD₃ receptors in CHO cell membranes. The effects of GDP concentration and incubation time on agonist-stimulated [³⁵S]GTPS binding at hD_2L-CHO have already been described (Gardner et al., 1996). In preliminary experiments, these results were confirmed (results not shown). We have demonstrated that dopamine, PD128907, and 7-OH-DPAT concentration-dependently stimulated [³⁵S]GTPS binding in hD_2L-CHO and hD₃-CHO membranes. Dopamine stimulated [³⁵S]GTPS binding 5-fold more in hD_2L-CHO cell membranes than in hD₃-CHO-high cell membranes, but with a 40-fold lower potency. Maximum stimulation of [³⁵S]GTPS binding was 2-fold higher in hD₃-CHO-high than in hD₃-CHO-low, suggesting an increase in G protein coupling due to the higher number of receptors in the membranes (i.e., higher [receptor]/[G protein] ratio). This could mean that the receptor density drives this reaction. The amount of stimulation at hD₃-CHO-high in this study was more than 2-fold higher than that recently reported (Malmberg et al., 1998). Based on the partial agonism of 7-OH-DPAT and PD128907, we tried to inhibit dopamine-stimulated [³⁵S]GTPS binding by using these compounds. 7-OH-DPAT inhibited dopamine stimulation whereas PD128907 did not, indicating that 7-OH-DPAT is a partial agonist with antagonist properties and that PD128907 is a partial agonist that lacks antagonist properties. In addition, we used a number of structurally different antagonists to inhibit dopamine-stimulated [³⁵S]GTPS binding. Haloperidol is a butyrophenone, risperidone is a benzisoxazole, and raclopride and nemonapride are benzamides. All compounds inhibited dopamine-stimulated [³⁵S]GTPS binding with potencies similar to those found in radioligand-binding experiments (see Tables 2 and 5). We could not observe inverse agonism under the conditions used (i.e., curves did not level out below basal [³⁵S]GTPS binding), although haloperidol and raclopride have been reported to be inverse agonists (Hall and Strange, 1997; Malmberg et al., 1998). The concentration of GDP that we used (1 µM) probably decreased basal levels of [³⁵S]GTPS binding to such an extent that inverse agonism became hard to detect. So far, inverse agonism at hD₂ receptors has been clearly shown only on second messenger formation in whole cells, which represents a more physiological condition than [³⁵S]GTPS binding in membrane preparations.

In this study, we were able to demonstrate that dopamine inhibited forskolin-stimulated cAMP formation in a dose-dependent way in both hD_2L-CHO and hD₃-CHO-high cells. Surprisingly, a similar, strong dopamine inhibition of cAMP formation (up to 70%) was apparent in both types of cell, whereas earlier reports on hD₃ receptors have shown only 30 to 40% inhibition of forskolin levels by dopamine (McAllister et al., 1995; Griffon et al., 1996) or even no inhibition at all (Freedman et al., 1994; MacKenzie et al., 1994; Tang et al., 1994). However, it should be noted that forskolin-stimulated cAMP levels were 2- to 3-fold lower in hD_2L-CHO cells than in hD₃-CHO-high or in wild-type cells. This suggests a tonic inhibition of cAMP formation by hD_2L receptors. Interestingly, we found nearly the same potency of dopamine for stimulating hD_2L-CHO and hD₃-CHO-high cells in cAMP assays, which contrasts with our results from [³⁵S]GTPS-binding experiments involving cell membrane preparations, where dopamine was at least 10-fold less potent at hD_2L receptors. Because the slopes of the dopamine curves are near unity in the [³⁵S]GTPS binding and cAMP experiments, the higher shift in potency at hD_2L-CHO compared with hD₃-CHO cannot be attributed to promiscuous coupling to a wide variety of G proteins with different affinities. The shift in potency between these assays could be attributed to the different assay conditions (e.g., temperature, buffer composition). The most striking difference in assay conditions is the use of membranes instead of intact cells. Indeed, comparison of the potencies of dopamine in membranes (radioligand- and [³⁵S]GTPS-binding experiments) and in intact CHO cells (cAMP content measurement and microphysiometry experiments) indicates that dopamine is more potent in intact CHO cells. This suggests that during the membrane preparation, receptor-G protein interaction may be disturbed, leading to lower potencies of dopamine; the hD_2L receptor would be more susceptible to this perturbation than the hD₃ receptor (see Fig. 2).

In microphysiometry experiments, dopamine stimulated extracellular acidification rates for hD_2L-CHO and hD₃-CHO-high cells. As in [³⁵S]GTPS-binding experiments, we found a higher efficacy of dopamine stimulation of hD_2L receptors than of hD₃ receptors but with a higher potency at the latter. Also, dopamine was more potent at hD_2L and hD₃ receptors in microphysiometry experiments than in cAMP assays, which might indicate a different level of signal amplification. The effect of dopamine could be blocked almost completely by pertussis toxin pretreatment of the cells, indicating involvement of G proteins from the G_i/o family in the signaling of hD_2L and hD₃ receptors.

We demonstrated clear activation of hD_2L and hD₃ receptors in CHO cells by using various signal transduction assays. The high levels of stimulation, compared with previous reports (which have 2- to 40-fold lower expression levels), could be ascribed to the high expression level. Hence, it can be supposed that for G protein coupling of the D₃ receptor in vivo, high expression levels in specific cells may be required.

In conclusion, we compared the signal transductions of hD_2L-CHO and hD₃-CHO using [³⁵S]GTPS-binding experiments, dopamine inhibition of forskolin-stimulated cAMP formation, and microphysiometry experiments. In all cases, we found stimulation of hD_2L and hD₃ receptors. In [³⁵S]GTPS-binding and microphysiometry experiments, stimulation of hD₃-CHO was much less pronounced than stimulation of hD_2L-CHO. Interestingly, we found dopamine inhibition of forskolin-stimulated adenylyl cyclase activity to be almost equally high in hD₃-CHO and hD_2L-CHO cells. These data suggest that functional effects of hD₃ receptors can be measured reliably in CHO cells when expressed at high levels.