Introduction

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Dopaminergic neurotransmission is mediated by five receptor subtypes (D₁ to D₅) which can be grouped into two receptor families. D₁-like receptors include the D₁ and D₅ subtypes, whereas D₂-like receptors include the D₂, D₃, and D₄ subtypes. D₂ and D₃ receptors, in particular, display marked sequence homology and pharmacological similarity in their in vitro ligand binding profiles (Levant, 1997; Missale et al., 1998). However, D₃ receptors may be distinguished from D₂ receptors by several factors. D₃ receptors are concentrated in limbic rather than striatal brain regions (Liu et al., 1996; Hall et al., 1996). Furthermore, they mediate stimulation, rather than inhibition, of c-fos expression in striatal neurones (Pilon et al., 1994; Morris et al., 1997), and inhibition, rather than stimulation, of locomotor activity in rats (Svensson et al., 1994; Starr and Starr, 1995). In addition, whereas D₂ receptors couple efficiently to second-messenger systems, markedly inhibiting adenylyl cyclase activity, such responses have proved elusive and complex for D₃ receptors (e.g., Freedman et al., 1994; MacKenzie et al., 1994; Tang et al., 1994; Griffon et al., 1997). Indeed, D₃ receptors couple selectively to inhibition of adenylyl cyclase type V, but not type I or VI, and only weakly to type II (Robinson and Caron, 1997; Watts and Neve, 1997). In vitro studies of agonist efficacy have employed other measures of receptor activation, including medium acification (Cox et al., 1995), and stimulation of mitogenesis (Pilon et al., 1994; Svensson et al., 1994; Sautel et al., 1995). However, these approaches measure responses "downstream" of the receptor in the intracellular activation cascade and the relevance of an increase in mitogenesis for postmitotic central nervous system neurones is unclear. A more promising approach may be to measure receptor-mediated G protein activation by stimulation of guanosine-5'-O-(3-[³⁵S]thio)-triphosphate ([³⁵S]GTPS) binding: this corresponds to the first step of the intracellular activation cascade and directly reflects ligand binding events at the receptor itself (Pregenzer et al., 1997; Malmberg et al., 1998). Thus, the present study adopted this strategy to address several questions concerning, principally, the functional properties of human (h) D₃ receptors. In addition, in some tests results at hD₃ receptors were compared with those at hD₂ receptors. First, differences in the second-messenger actions of D₃ and D₂ receptors may be related to differing capacities for stimulation of G proteins. We addressed this issue by investigating the ability of hD₃ receptors to mediate dopamine-stimulated [³⁵S]GTPS binding. Second, the relationship between binding affinity and functional potency of dopaminergic agonists and antagonists was investigated using the most potent and selective D₃ receptor ligands reported to date: the agonists (+)-7-OH-DPAT (7-hydroxy-2-(di-n-propylamino)tetralin) and PD 128,907 [(+)-(4aR,10bR)-3,4,4a,10b-tetrahydro-4-propyl-2H,5H- [1]benzopyrano[4,3-b]-1,4-oxazin-9-ol] (Pugsley et al., 1995) and the antagonists, S 14297 ((+)-[7-(N, N-dipropylamino)-5,6,7,8-tetrahydro-naphtho(2,3b)dihydro-2,3-furane]) and GR 218,231 (2(R,S)-(dipropylamino)-6-(4-methoxyphenylsulfonylmethyl)-1,2,3,4-tetrahydronaphtalene) (Millan et al., 1995b; Murray et al., 1996). The hD₃/hD₂ selectivities based on K_i ratios were compared with those based on EC₅₀ and K_B ratios (Burris et al., 1995; Levant, 1997). Third, the signal transduction differences between D₃ and D₂ receptors, such as the differential coupling to adenylyl cyclase isoforms, could be due to receptor interactions with different G protein populations. Indeed, at least 16 distinct G protein  subunits have been identified, divided into four families: G_i, G_S, G_q/11, and G_12/13 (Simon et al., 1991). Although a previous study suggested differences in coupling profiles of D₂ and D₃ receptors for modulation of outward K⁺ currents (Liu et al., 1996), no information is available from a functional test more proximal to the receptor and the G protein subtypes involved in D₃ coupling are unclear (cf. Tang et al., 1994). The present study, therefore, examined G protein coupling specificity directly at the G protein activation level by challenging the receptor-mediated stimulation of [³⁵S]GTPS binding with specific antisera raised against different G subunits. In fact, antibodies raised against the COOH terminal part of G subunits have proved useful to determine the G protein specificity of several other 7-transmembrane domain receptors (Harris-Warrick et al., 1988; McFadzean et al., 1989; Lledo et al., 1992; Izenwasser and Côté, 1995).

	Materials and Methods

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Membrane Preparations of Chinese Hamster Ovary (CHO)-hD₃ and CHO-hD₂ Cells. CHO cells expressing hD₃ receptors were grown as described previously (Sokoloff et al., 1992). Cells were harvested from adherent culture and homogenized using a Kinematica Polytron (Kinematica GmBH, Littau, Switzerland) in a buffer containing 50 mM Tris (pH 7.4), 5 mM MgCl₂. The suspension was then centrifuged at 20,000g for 15 min at 4°C and the pellet was resuspended in the appropriate binding buffer (see below) and stored at 80°C. CHO-hD_2(short) cell membranes were purchased from Receptor Biology (Baltimore, MD). The "short" hD₂ isoform, which lacks a 29-amino acid insert in the putative third intracellular loop, is processed faster to mature receptors at the cell surface than the "long" form and may couple more efficiently to certain G protein subtypes (Fishburn et al., 1995; Boundy et al., 1996).

[¹²⁵I]Iodosulpride Binding to hD₃ and hD₂ Receptors. Saturation binding at hD₂ and hD₃ receptors was carried out with 12 concentrations of [¹²⁵I]iodosulpride (1000 Ci/mmol; Amersham, Les Ulis, France). For competition binding experiments, membranes (10 to 20 µg protein) of CHO-hD₂ or CHO-hD₃ cells were incubated with [¹²⁵I]iodosulpride (0.1 nM for hD₂ and 0.2 nM for hD₃) at 30°C for 30 min in a buffer containing 50 mM Tris (pH 7.4), 120 mM NaCl, 5 mM KCl, 1 mM EDTA, and 5 mM MgCl₂. Nonspecific binding was defined with raclopride (10 µM). Isotherms were analyzed by nonlinear regression, using the computer program PRISM (Graphpad Software Inc., San Diego, CA) to yield IC₅₀ values. Inhibition constants (K_i values) were derived from IC₅₀ values according to the Cheng-Prusoff equation. The goodness of fit was tested by runs test. For compounds that yielded P < .05 in the runs test and/or shallow inhibition isotherms (nH values markedly inferior to unity), 14-point competition binding experiments were carried out and one- and two-site fits were compared by F test.

Measurement of Agonist Efficacy and Antagonist Potency at hD₃ and hD₂ Receptors. Receptor-linked G protein activation by dopamine at hD₂ and hD₃ receptors was determined by measuring the stimulation of [³⁵S]GTPS (1332 Ci/mmol; NEN, Les Ulis, France) binding induced by dopamine. CHO-hD₂ membranes (30-40 µg protein) were incubated (60 min, 22°C) with agonists and/or antagonists in a buffer containing 20 mM HEPES (pH 7.4), 3 µM GDP, 10 mM MgCl₂, 150 mM NaCl, and 0.1 nM [³⁵S]GTPS. CHO-hD₃ membranes (30-50 µg protein) were incubated (40 min, 22°C) with agonists and/or antagonists in a buffer containing 20 mM HEPES (pH 7.4), 3 µM GDP, 3 mM MgCl₂, 100 mM NaCl, and 1.0 nM [³⁵S]GTPS. Nonspecific binding was defined with GTPS (10 µM). Agonist efficacy is expressed relative to that of dopamine (100%), which was tested at a maximally effective concentration (10 µM) in each experiment. For all tests, membranes were preincubated with agonist and/or antagonist for 15 min before the addition of [³⁵S]GTPS. K_B values for inhibition of dopamine (1 and 3 µM for hD₃ and hD₂ respectively)-stimulated [³⁵S]GTPS binding were calculated according to Lazareno and Birdsall (1993):

nH¹

hD₃ Receptor Alkylation with N-Ethoxycarbonyl-2-Ethoxy-1,2-Dihydroquinoline (EEDQ). CHO-hD₃ cell membranes were treated by EEDQ at a final concentration between 0 and 300 µM (0, 10, 33, 100, and 300 µM). The membrane suspension (3 ml final volume) was vortexed and immediately centrifuged at 4°C for 15 min at 20,000g. The supernatant was discarded and the membrane pellet was resuspended in the appropriate buffer and [³⁵S]GTPS or [¹²⁵I]iodosulpride binding was performed as described above. K_A values were determined by Furchgott analysis, as described by Atkinson and Minneman (1992) and Adham et al. (1993), with CHO-hD₃ membranes treated with 33 µM EEDQ. Plots were derived of 1/[A] versus 1/[A']; where [A] and [A'] are equiactive concentrations for stimulation of [³⁵S]GTPS binding before and after receptor alkylation, respectively. K_A was calculated from

Characterization of G proteins by Immunoblotting and ADP-Ribosylation. Immunoblotting of G subunits was performed using antisera purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA) raised against G_i/O (C10), G_S (C18), and G_q/11 (C19). Approximately 2 µg protein from CHO-hD₂ and CHO-hD₃ membrane preparation was separated on 10% polyacrylamide gel and transferred onto nitrocellulose. Antisera were incubated at 1/1000 followed by enhanced chemiluminescence detection with horseradish peroxidase as secondary antibody (Amersham, Buckinghamshire, UK).

Antiserum Treatment of CHO-hD₃ or -hD₂ Membranes. CHO-hD₃ and -hD₂ membranes (30-50 µg protein) were preincubated at 4°C for 5 h with 3.3 µg of antisera against different G proteins. [³⁵S]GTPS binding was then performed in absence and in presence of dopamine (10 µM) as described above. The antisera used were the same as described above and were chosen for their capability to recognize the COOH terminal part of G subunits involved in receptor interactions. Another antiserum (C17) against c-Jun NH₂-terminal kinase (JNK1), a target unrelated to G proteins, was also tested as a control to exclude nonspecific antibody effects.

Compounds. (+)7-OH-DPAT was obtained from CNRS (Paris, France). PD 128,907 was purchased from RBI (Natick, MA); dopamine, haloperidol, EEDQ, and cholera and PTXs were purchased from Sigma. GR 218,231 and S 14297 were synthesized by J.-L. Peglion, Servier.

	Results

Top Summary Introduction Materials and Methods Results Discussion References

Saturation Binding Experiments. The receptor expression level of hD₃ receptors, determined in [¹²⁵I]iodosulpride saturation binding experiments, was almost 11-fold higher than that of hD₂ receptors (Table 1 and Fig. 1).

View this table: [in this window] [in a new window]   TABLE 1 Densities of recombinant receptors and agonist-activated G proteins in CHO cells stably expressing hD2 and hD3 receptors Receptor expression levels (Bmax) of hD3 and hD2 receptors stably expressed in CHO cell membranes were determined by saturation binding experiments with [125I]iodosulpride. Treatment of CHO-hD3 membranes with EEDQ (33 µM) significantly reduced hD3 receptor expression (p < 0.05, 2-tailed t test). Number of dopamine-activated G proteins was determined by [35S]GTPS isotopic dilution saturation binding, as described in Materials and Methods. Apparent KD for [35S]GTPS saturation binding is denoted KAPP. EEDQ (33 µM) treatment of CHO-hD3 membranes did not significantly alter Bmax or KAPP for [35S]GTPS saturation.

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Saturation binding of [125I]iodosulpride and [35S]GTPS to CHO-hD3 and CHO-hD2 cell membranes. A, representative saturation binding isotherms of [125I]iodosulpride to CHO-hD2 and CHO-hD3 membranes. B, representative saturation binding isotherms of [35S]GTPS to CHO-hD2 and CHO-hD3 membranes. Basal and dopamine (10 µM)-stimulated [35S]GTPS binding were determined in the presence of increasing concentrations of GTPS. These data were transformed as described in Materials and Methods to generate a saturation binding isotherm for net agonist-dependent [35S]GTPS binding. Points shown are means of duplicate determinations from representative experiments repeated on at least four occasions. Bmax and KD/apparent KD (Kapp) data from these experiments are shown in Table 1.

hD₃ Receptor Alkylation with EEDQ. CHO-hD₃ membranes were sensitive to EEDQ treatment. Addition of EEDQ to ice-cold membranes and immediate centrifugation (15 min, 4°C, 20,000g) was sufficient to reduce the number of hD₃ binding sites in a concentration-dependent manner without a change in affinity of the radioligand (Table 2 and Fig. 2). EEDQ also concentration dependently reduced the stimulation of [³⁵S]GTPS binding induced by dopamine (Table 2 and Fig. 3). At the maximal EEDQ concentration tested (300 µM), the density of hD₃ receptors was reduced by over 80%, whereas dopamine-induced [³⁵S]GTPS binding was reduced by 64%. Subsequent experiments were carried out with an EEDQ concentration of 33 µM, which reduced dopamine-induced [³⁵S]GTPS binding by about 50% (Tables 1 and 2). Under these conditions, the pEC₅₀ (log EC₅₀) of dopamine was slightly reduced to 7.87 ± 0.09 (compared with 8.00 for control membranes, Table 4), without alteration of basal [³⁵S]GTPS binding. The K_A value determined by Furchgott analysis was 53 ± 23 nM, which corresponded closely to the affinity of dopamine at hD₃ receptors determined in competition binding experiments (54.9 nM, Table 3). The resulting K_A/EC₅₀ ratio for dopamine was 5.3, indicating the presence of receptor reserve. This was confirmed in occupancy/response plots, which yielded hyperbolic curves, with the mean half-maximal response to dopamine being observed at 11.8 ± 2.3% occupation of hD₃ binding sites (Fig. 3).

View this table: [in this window] [in a new window]   TABLE 2 Concentration-dependent actions of EEDQ on receptor density and function in CHO-hD3 membranes CHO-hD3 cell membranes were treated with different concentrations of EEDQ. Receptor density was determined in [125I]iodosulpiride saturation binding experiments. Percentage of remaining hD3 binding sites is calculated as a percentage of mean Bmax determined in absence of EEDQ (15.43 pmol/mg, Table 1). [35S]GTPS binding is expressed as percentage of dopamine (10 µM)-dependent binding observed under control conditions in parallel experiments.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Concentration-dependent reduction of hD3 receptor density by EEDQ. A, representative saturation binding isotherms of [125I]iodosulpride to CHO-hD3 membranes pretreated with different concentrations of EEDQ. B, Scatchard representation of data from A. Points shown are means of duplicate determinations from representative experiments repeated on at least three occasions. Bmax and KD data from these experiments are shown in Table 2.

View larger version (36K): [in this window] [in a new window]   Fig. 3.   hD3 receptor inactivation with EEDQ reveals receptor reserve for dopamine-stimulated [35S]GTPS binding to CHO-hD3 membranes. A, concentration-dependent reduction of dopamine-stimulated [35S]GTPS binding by pretreatment with EEDQ (0 to 300 µM). Columns represent mean ± S.E.M. from at least three experiments carried out in triplicate. B, stimulation by dopamine of [35S]GTPS binding to control or EEDQ (33 µM)-pretreated CHO-hD3 membranes. C, double-reciprocal plot of 1/[A] versus 1/[A'], where [A] and [A'] are equiactive concentrations for stimulation of [35S]GTPS binding with and without EEDQ treatment, respectively. D, dopamine occupancy/response relationship, derived using the value of KA from C. Hyperbolic isotherm indicates the presence of receptor reserve. For B, C, and D, points shown are means of triplicate determinations from a representative experiment repeated on at least three occasions. Mean KA value was 53 ± 23 nM. Mean half-maximal response to dopamine was observed at at 11.8 ± 2.3% occupation of hD3 binding sites.

View this table: [in this window] [in a new window]   TABLE 3 Competition binding of dopaminergic ligands at hD2 and hD3 receptors Affinities (pKi values) at hD3 and hD2 receptors stably expressed in CHO cells were determined by competition binding experiments with [125I]iodosulpride. Two-site analysis of isotherms at hD2 receptors is shown if this was significantly superior to a one-site fit (P  0.05, F test). Percentage of high-affinity binding sites is denoted % high. Isotherms at hD3 receptors fitted best to a single binding site model. Results are means ± S.E. of mean of at least four independent experiments. Ki/H/L values were calculated from respective mean pKi/H/L values. hD2/hD3 affinity ratio was obtained by dividing Ki/H/L value at hD2 receptors by Ki value at hD3: for agonist ligands ratios show a wide variation depending on whether Ki value at hD3 is compared with KH or KL value at hD2.

[¹²⁵I]Iodosulpride Competition Binding hD₂ and hD₃ At hD₃ receptors, agonist competition binding isotherms were monophasic, although in some experiments with dopamine a small (~10% of binding sites), high-affinity (pK_H, log K_H, ~ 9) component was apparent (data not shown). At hD₂ receptors, agonist competition isotherms were biphasic and fitted better to a two-site model (p < .05, F test; Fig. 4), yielding estimates of affinity for the high- and low-affinity components (Table 3), presumably reflecting binding to G protein-coupled and -uncoupled states of the receptor, respectively. Selectivity ratios of affinity at hD₂/hD₃ receptors were calculated by comparing the K_i at hD₃ receptors with the K_H and the K_L at hD₂ receptors. Competition binding curves with antagonist ligands, haloperidol, S 14297, and GR 218,231, were monophasic for both hD₂ and hD₃ sites (Table 3).

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Competition binding of dopaminergic agonists at hD3 and hD2 receptors. Representative [125I]iodosulpride competition binding isotherms for dopamine, (+)-7-OH-DPAT and PD 128,907 at hD2 and at hD3 receptors. At hD2 receptors the data fitted better to a two-site model (P < .05, F test). Points shown are means of triplicate determinations from representative experiments repeated on at least three occasions. pKi, pKH, and pKL data from these experiments are shown in Table 3.

[³⁵S]GTPS Binding Conditions at CHO-hD₃ and CHO-hD₂ Cell Membranes. In preliminary experiments (not shown), conditions were defined that yielded optimal dopamine-induced stimulation of [³⁵S]GTPS binding. 1) Optimal stimulation was observed at NaCl concentrations of 100 and 150 mM for hD₂ and hD₃ membranes, respectively. 2) GDP concentration dependently reduced basal binding of [³⁵S]GTPS to both hD₂ and hD₃ cell membranes. 3) MgCl₂ increased dopamine-dependent [³⁵S]GTPS binding to a maximum at around 3 to 10 mM for both receptor subtypes. 4) Stimulation of [³⁵S]GTPS binding was linear with time over the period of the incubations. In view of the lower stimulation of [³⁵S]GTPS binding by agonists at hD₃ receptors, a higher concentration of [³⁵S]GTPS was used (1.0 nM) than with hD₂ (0.1 nM) to provide a stronger signal. Typical binding of [³⁵S]GTPS (0.1 nM) to CHO-hD₂ membranes was 90 to 100 fmol/mg basal and 230 to 250 fmol/mg in the presence of dopamine (10 µM). Typical binding of [³⁵S]GTPS (1 nM) to CHO-hD₃ membranes was 1000 to 1100 fmol/mg basal and 1500 to 1600 fmol/mg with dopamine (10 µM). In control experiments in which the concentration of [³⁵S]GTPS used for hD₃ receptors was 0.1 nM (as for hD₂ receptors), the pEC₅₀ for dopamine was 8.04 ± 0.03 (n = 3) nM, not significantly different from the pEC₅₀ observed with a [³⁵S]GTPS concentration of 1 nM (8.00 ± 0.07, Table 4). Dopamine-induced stimulation was 45.8 ± 2.8% (n = 3).

View this table: [in this window] [in a new window]   TABLE 4 Stimulation of [35S]GTPS binding by dopaminergic ligands at hD2 and hD3 receptors Agonist efficacies were determined by [35S]GTPS binding. Results are means ± S.E. of mean of at least three independent experiments. EC50 values were calculated from mean pEC50 values. Haloperidol and GR 218,231 did not induce any alteration of [35S]GTPS binding to either hD2 or hD3 membranes. S 14297 did not alter [35S]GTPS binding to hD3 membranes.

[³⁵S]GTPS Binding at CHO-hD₃ and CHO-hD₂ Cell Membranes: Agonist Actions. Dopamine, PD 128,907, and (+)-7-OH-DPAT increased [³⁵S]GTPS binding to CHO-hD₃ and CHO-hD₂ membranes in a concentration-dependent manner, with EC₅₀, E_max, and nH values shown in Table 4. S 14297 exhibited slight agonist actions at hD₂ receptors (E_max = 20.6%) but no agonist activity was detected at hD₃ receptors (Fig. 5). PD 128,907 was almost twice as efficacious at hD₂ as at hD₃ receptors, whereas (+)-7-OH-DPAT was a partial agonist at both receptor subtypes. The ratios of EC₅₀ values at hD₂/hD₃ were intermediate between the K_H(hD₂)/K_i(hD₃) and the K_L(hD₂)/K_i(hD₃) ratios in Table 3. In control experiments in which the concentrations of NaCl and MgCl₂ were inverted between hD₂ and hD₃, we did not observe marked changes in EC₅₀ and E_max values of (+)-7-OH-DPAT and PD 128,907 (data not shown), but a slight decrease in percentage of stimulation by agonists was noted.

View larger version (13K): [in this window] [in a new window]   Fig. 5.   Agonist stimulation of hD3 and hD2 receptor-mediated G protein activation. [35S]GTPS binding is expressed as a percentage of maximal stimulation given by dopamine. A, fold stimulation of [35S]GTPS binding by dopamine at hD2 and hD3 receptors. B, agonist concentration-response curves at hD2 receptors. C, agonist concentration-response curves at hD3 receptors ( dopamine;  PD 128,907;  (+)-7-OH-DPAT; and  S 14297). Points shown are means of triplicate determinations from representative experiments repeated on at least three occasions. Emax, pEC50, and nH data from these experiments are shown in Table 4.

[³⁵S]GTPS Binding at CHO-hD₃ and CHO-hD₂ Cell Membranes: Antagonist Actions. Haloperidol, GR 218,231, and S 14297 did not alter [³⁵S]GTPS binding from basal levels at hD₃ receptors or, except S 14297 (as described above) at hD₂ receptors. The inhibition of dopamine-stimulated [³⁵S]GTPS binding (Table 5 and Fig. 6), yielded antagonist potencies (K_B values), which conserved the same order of potency as the K_i values shown for these compounds in Table 3. The novel ligand GR 218,231 was shown to behave as a competitive antagonist, inducing a rightward parallel shift of the dopamine stimulation curve without loss of maximal efficacy (Fig. 7), yielding a linear Schild plot (r = 0.96, slope = 1.06 ± 0.10) and a pA₂ value of 9.34 similar to its affinity calculated by competition binding (pK_i = 8.95, Table 3).

View this table: [in this window] [in a new window]   TABLE 5 Antagonism of dopamine-stimulated [35S]GTPS binding to CHO-hD2 and -hD3 membranes Antagonist potencies (pKB values) were calculated from IC50 values for inhibition of dopamine (3 µM for hD2, 1 µM for hD3)-stimulated [35S]GTPS binding. pKB values are expressed as means ± S.E. of mean of at least three independent experiments. KB values were calculated from mean pKB values.

View larger version (17K): [in this window] [in a new window]   Fig. 6.   Antagonism of hD3 and hD2 receptor-mediated G protein activation. Antagonism of dopamine (3 µM)-stimulated [35S]GTPS binding at hD2 receptors and of dopamine (1 µM)-stimulated [35S]GTPS binding at hD3 receptors ( haloperidol,  GR 218,231, and  S 14297). Points shown are means of triplicate determinations from representative experiments repeated on at least three occasions. pIC50 and pKB data from these experiments are shown in Table 4.

View larger version (18K): [in this window] [in a new window]   Fig. 7.   Competitive antagonism of hD3 receptor-mediated G protein activation by GR 218,231. A, concentration-response isotherms for stimulation of [35S]GTPS binding by dopamine at hD3 receptors in the presence of increasing concentrations of GR 218,231 ( 3 nM,  10 nM,  30 nM, and  100 nM). Points shown are means of triplicate determinations from representative experiments repeated on at least three occasions. B, Schild plot of the hD3 dopamine concentration-response experiments. Points shown are mean values from at least three independent experiments performed in triplicate.

Effect of Pertussis and Cholera Toxins on hD₃ Receptor Coupling. Membranes were prepared from CHO-hD₃ cells treated with PTX (100 ng/ml) or cholera toxin (1 µg/ml) for 6 h. Pretreatment with PTX reduced, but did not totally suppress, dopamine-dependent [³⁵S]GTPS binding: it was attenuated by about 80% (81 ± 16 fmol/mg versus 430 ± 26 fmol/mg in control), without changes in basal [³⁵S]GTPS binding (Fig. 8). The incomplete suppression of dopamine-stimulated [³⁵S]GTPS binding was not due to an insufficiently long incubation of CHO-hD₃ cells with PTX. Indeed, when membranes were prepared from CHO-hD₃ cells after the 6-h incubation, no subsequent incorporation of [³²P]ADP-ribose was observed, indicating that all the G_i/o proteins present had already been ADP-ribosylated (Fig. 8).

View larger version (39K): [in this window] [in a new window]   Fig. 8.   Partial attenuation of dopamine-stimulated [35S]GTPS binding by PTX, but not cholera toxin. A, [32P]ADP-ribose incorporation catalyzed by PTX. ADP-ribosylation of CHO-hD3 cell membranes preincubated with or without PTX or cholera toxin (CT) for 6 h was carried out as described in Materials and Methods. [32P]ADP-ribosylated Gi/O proteins were revealed by a 8-h exposure to Hyperfilm. The data shows that PTX pretreatment abolished subsequent [32P]ADP-ribose incorporation. The same result was obtained in a second, independent experiment. B, Effect of PTX and CT on dopamine-stimulated [35S]GTPS binding. CHO-hD3 cell were incubated for 6 h with PTX or CT and stimulation of [35S]GTPS binding was determined with dopamine (10 µM). Bars represent mean ± S.E.M. values from at least three independent experiments performed in triplicate and are expressed in fentomoles per milligram of dopamine-induced [35S]GTPS binding.

Effect of Antibodies on hD₃ and hD₂ Receptor Coupling. The presence of G_i/o, G_S, and G_q/11 in both CHO-hD₃ and CHO-hD₂ cell membranes was demonstrated by immunodetection with specific antibodies (Fig. 9). Preincubation of hD3 and hD₂ cell membranes with anti-G_i/_o subunit antiserum significantly (P < .05, Student's paired t test) attenuated dopamine-dependent [³⁵S]GTPS binding, at both hD₃ and hD₂ receptors (Fig. 9). Anti-G_q/₁₁ antiserum significantly attenuated dopamine-dependent [³⁵S]GTPS binding to CHO-hD₃ but not CHO-hD₂ membranes (P < .05, Student's paired t test). Antisera directed against G_S and an unrelated target, JNK1, did not affect [³⁵S]GTPS binding at either receptor (data not shown).

View larger version (50K): [in this window] [in a new window]   Fig. 9.   Inhibition by anti-G protein antibodies of dopamine-stimulated [35S]GTPS binding to CHO-hD3 and CHO-hD2 cell membranes. A, immunodetection of Gi/o, GS, and Gq/11 subunits in both CHO-hD3 and CHO-hD2 cell membranes was performed as described in Materials and Methods. B, CHO-hD2 and CHO-hD3 cell membranes were incubated for 5 h at 4°C in the presence of antibodies used in A. Stimulation of [35S]GTPS binding was determined with dopamine (10 µM). Bars represent mean ± S.E.M. values from at least three independent experiments performed in triplicate and are expressed as percentage of dopamine-dependent [35S]GTPS binding observed in control (untreated) samples. *P < .05; **P < .01 versus control (2-tailed, paired t test).

	Discussion

Top Summary Introduction Materials and Methods Results Discussion References

The primary purpose of the present study was to investigate the G protein coupling of dopamine hD₃ receptors. The results demonstrate that hD₃ (and hD₂) receptors mediate stimulation of [³⁵S]GTPS binding when expressed in mammalian CHO cells, indicating that they are capable of activating intracellular G proteins. A robust degree of stimulation was observed (Fig. 5), enabling a detailed investigation of the coupling of these receptor subtypes and the identification of some marked differences between hD₃ and hD₂ sites.

First, despite the 11-fold higher hD₃ receptor expression level (15 pmol/mg), the dopamine-elicited increase in [³⁵S]GTPS binding (up to 1.6-fold) was less than that at hD₂ receptors. Partial inactivation of hD₃ receptors using the alkylating agent EEDQ showed that high hD₃ receptor expression levels are necessary for stimulation of G protein activation, because EEDQ treatment reduced the stimulation of [³⁵S]GTPS binding induced by dopamine (Table 2). Nevertheless, Furchgott analysis yielded a hyperbolic occupancy/response plot (Fig. 3), indicating the presence of marked receptor reserve for half-maximal stimulation of [³⁵S]GTPS binding by dopamine and is consistent with the 5-fold K_A/EC₅₀ ratio for activation of hD₃ receptors (see Results). Thus the limited stimulation of [³⁵S]GTPS binding to CHO-hD₃ membranes appears to be a property of hD₃ receptors themselves and not due to insufficient intrinsic efficacy of dopamine. It should be noted that the modest stimulation at hD₃ receptors is not a consequence of augmented basal [³⁵S]GTPS binding, because basal [³⁵S]GTPS binding was unaffected by receptor inactivation (not shown). Furthermore, the low-fold stimulation in CHO-hD₃ membranes is unlikely to be due to a global lack of activatable G proteins: the amount (B_max) of dopamine-activated G proteins in CHO-hD₃ cell membranes is about 3-fold higher than that in CHO-hD₂ cell membranes (Table 1 and Fig. 1). Taken together, the present data suggest that stimulation of hD₃ receptors less effectively induces the conformational changes necessary for G protein activation than at hD₂ receptors (Chio et al., 1994), perhaps due to a slower rate of G protein coupling/uncoupling at hD₃ receptors or, alternatively, to interaction with different G protein subtypes at hD₃ versus hD₂ receptors, a possibility discussed below.

Second, agonist efficacy varied between hD₂ and hD₃ receptors. S 14297, previously characterized as a D₃ receptor antagonist in vivo (Millan et al., 1995a,b) exhibited residual intrinsic activity at hD₂ receptors (Table 4) but no detectable agonist activity at hD₃ receptors. In the present high-expressing CHO-hD₃ cell membranes, it might have been expected that partial agonist actions at hD₃ receptors would be "amplified" to yield maximal activation of [³⁵S]GTPS binding, like dopamine. Nevertheless, both (+)-7-OH-DPAT and PD 128,907 behaved as partial agonists at hD₃ receptors (Table 4 and Fig. 5). It therefore appears that despite the high levels of hD₃ receptors, the high R/G ratio in CHO-hD₃ membranes, and, as discussed above, receptor reserve for activation by dopamine, the present [³⁵S]GTPS binding methodology more readily differentiates partial agonist efficacies at hD₃ receptors than certain downstream models of hD₃ (or hD₂) receptor activation, such as mitogenesis (Sautel et al., 1995), where dopamine, (+)-7-OH-DPAT and PD 128,907 all behaved as full agonists. Furthermore, in the present study, the degree of selectivity of (+)-7-OH-DPAT and PD 128,907 for activation of hD₃ versus hD₂ receptors was greater than previously reported (Levant, 1997). (+)-7-OH-DPAT displayed an hD₃/hD₂ EC₅₀ ratio of 77 (Table 4) compared with 14 and 7 for inhibition of adenylyl cyclase and mitogenesis experiments respectively (Chio et al., 1994; Sautel et al., 1995). PD 128,907 was 382-fold as selective in this study compared with only 6-fold as selective in mitogenesis experiments (Pugsley et al., 1995). The source of these differences is unclear but probably relates to the fact that mitogenesis and extracellular acidification measure responses that are distal to agonist-induced receptor/G protein conformational changes. In contrast, [³⁵S]GTPS binding measures G protein activation, which provides a more proximal indication of agonist/antagonist actions at the receptor itself.

Third, the antagonist rank order of potency of haloperidol, S 14297, and the novel selective antagonist, GR 218,231 (Murray et al., 1996; Figs. 6 and 7) at hD₃ and hD₂ receptors corresponded to the order of affinity determined in competition binding experiments, although a reduced preference for hD₃ sites was observed in functional tests (Table 5). It is noteworthy that, whereas the pK_B values of the antagonists resembled their respective pK_i values, the antagonists did not exhibit negative efficacy at either hD₃ or hD₂ receptors at concentrations up to 10⁵ M. At higher concentrations, [³⁵S]GTPS binding was somewhat reduced below basal levels in some experiments but this was taken to be a nonspecific effect, because it occurred at concentrations >1000-fold greater than their binding affinity, the effects did not show a discernible correlation with the order of potency, and a similar trend was observed in untransfected CHO cell membranes (D. Cussac, unpublished observations). A recent study using hD₃ receptors expressed in CHO cells (Malmberg et al., 1998) reported that basal [³⁵S]GTPS binding could be increased by dopaminergic agonists and decreased by antagonists. However, in that study dopamine-induced stimulation was very low (only ~1.2-fold), negative efficacy was only observed at very high drug concentrations (10⁶ M), and control untransfected CHO cells were not examined. Nevertheless, further investigation of the issue of negative efficacy is desirable, because the conditions used for [³⁵S]GTPS binding both in the present study and that of Malmberg et al. (1998) (high concentrations of GDP and NaCl) favor suppression of constitutive hD₃ and hD₂ receptor activation (Gardner et al., 1996). Indeed, some studies reported that haloperidol shows negative efficacy in models of D₃ and D₂ receptor activation (mitogenesis, Griffon et al., 1996; prolactin secretion, Nilsson et al., 1996).

Fourth, G protein activation by dopamine at hD₃ receptors is PTX sensitive (Fig. 8), implicating G_i/o G proteins. This is analogous to the known PTX sensitivity of D₂ receptors (Neve et al., 1989; Lajiness et al., 1993; Seabrook et al., 1994; Swarzenski et al., 1996; Hall and Strange, 1997). However, marked differences were observed between hD₃ and hD₂ receptors in antibody tests. In the present study, dopamine-stimulated [³⁵S]GTPS binding at hD₃ and hD₂ receptors was inhibited by an antiserum that recognizes the three _i subunits (_i1/i2/i3) and, more weakly, _O subunits (Cussac et al., 1996). This antiserum inhibited dopamine-stimulated [³⁵S]GTPS binding to CHO-hD₃ membranes more strongly than to CHO-hD₂ membranes (67% versus 40% inhibition, Fig. 9). The greater effect at hD₃ receptors may be due to a coupling by hD₃ to both G_i and G_O proteins, whereas hD₂ receptors may couple only to members of the G_i protein family. This would be consistent with a study that found that an attenuation of D₂ receptor-mediated inhibition of adenylyl cyclase activity was achieved by pretreatment with anti-G_i1/i2 but not by anti-G_O antibodies (Izenwasser and Côté, 1995). Alternatively, hD₃ receptor functional coupling to G proteins may be more labile than that at hD₂ receptors. Thus, the hD₃ receptor/G protein interaction may be more susceptible to the steric hindrance of antibody binding to G subunits, in accordance with the apparently less "efficient" G protein coupling of hD₃ receptors discussed above.

Fifth, an interaction of hD₃ receptors with a G protein other than G_i or G_O is suggested by the observation that, when CHO-hD₃ cells were treated with PTX, there remained a residual capacity of dopamine to stimulate [³⁵S]GTPS binding. This was not due to an insufficient incubation period with PTX, because control experiments (Fig. 8) indicated that 6 h were sufficient to completely ADP-ribosylate G_i/o proteins in CHO-hD₃ cells, in agreement with previous studies in pituitary cells (Cussac et al., 1996). Thus, a component of dopamine-dependent [³⁵S]GTPS binding at hD₃ receptors may be mediated by a G protein that is not PTX sensitive. This possibility is supported by the inhibition of dopamine-dependent [³⁵S]GTPS binding at hD₃ receptors by anti-G_q/11 antibodies (Fig. 9). Such an effect was not observed at hD₂ receptors, although _q/11 (as well as _i/O and _S) subunits are expressed in both cell lines (Fig. 9). Furthermore, the inhibition by anti-G_q/11 antibodies is not simply due to nonspecific Ig interactions, because the same concentration of antisera against an unrelated target (JNK1) did not affect dopamine-dependent [³⁵S]GTPS binding (not shown). Thus, these data suggest that hD₃ receptors in the present CHO-hD₃ cell line may interact with G_q/11 and, potentially, modulate phosphatidyl inositol turnover. Although a previous study of hD₃ receptors expressed in CHO cells did not find such an effect (Freedman et al., 1994), that may have been due to a 50-fold lower hD₃ expression level (0.3 versus 15 pmol/mg in the cells used in this study).

In conclusion, the [³⁵S]GTPS binding strategy employed in this study enabled the characterization of G protein coupling at hD₃ receptors. The data suggest that the coupling of hD₃ receptors to G proteins is less efficacious than that at hD₂ receptors, yielding a less pronounced stimulation of [³⁵S]GTPS binding despite the high expression levels of receptors and G proteins, and the presence of receptor reserve for dopamine. In addition, unlike hD₂ receptors, hD₃ receptors may couple to G proteins other than G_i, such as G_O and/or G_q/11 proteins. The precise G protein subtypes involved in hD₃ receptor coupling and their relevance to physiological actions require further investigation.