Introduction

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Molecular biology techniques have enabled the cloning and pharmacological characterization of multiple dopamine receptor subtypes belonging to two families. D₁ and D₅ receptors exhibit high structural homology and similar pharmacological profiles (Sunahara et al., 1991). Likewise, D₂, D₃ and D₄ receptors exhibit similarities in both their pharmacological profiles and their coupling to G proteins and signal transduction pathways (Levesque et al., 1992; O'Hara et al., 1996; Tang et al., 1994; Werner et al., 1996). The D₄ receptor is of particular interest for several reasons. First, in situ hybridization, autoradiographic and immunohistochemical studies indicate the existence of D₄-like receptor sites in limbic structures, such as cerebral cortex and hippocampus, associated with regulation of mood and cognition (Lahti et al., 1995; Matsumoto et al., 1996; Meador-Woodruff et al., 1996; Mrzljak et al., 1996). In contrast, only low levels of D₄ receptors are detected in regions associated with control of locomotor activity, such as the striatum (Meador-Woodruff et al., 1996; Seeman et al., 1993b). Furthermore, the atypical antipsychotic clozapine, which is known to act as an antagonist at dopamine D₂ and 5-HT_2A receptors (Canton et al., 1994; Meltzer, 1996), an inverse agonist at 5-HT_2C receptors (Labrecque et al., 1995) and a partial agonist at 5-HT_1A receptors (Newman-Tancredi et al., 1996a), also has significant affinity at dopamine D₄ receptors (Van Tol et al., 1991). This suggests that these sites may mediate some of the therapeutic actions of atypical antipsychotics. In fact, D₄-like receptor up-regulation in postmortem schizophrenic brain has been observed using indirect binding techniques (Murray et al., 1995a; Seeman et al., 1993a). Second, D₄ receptors have recently been discovered to display a "promiscuous" pharmacological profile, binding epinephrine and norepinephrine with high affinity, similar to that of dopamine (Lanau et al., 1997; Newman-Tancredi et al., 1997a). Hence, D₄ receptors may play a role in integrating dopaminergic and adrenergic transmission. Furthermore, D₄ receptor activation by norepinephrine is blocked by clozapine (Lanau et al., 1997; Newman-Tancredi et al., 1997a), suggesting that some of its clinical effects may be mediated by the antagonism of noradrenergic activity at D₄ receptors. Indeed, it has been suggested that noradrenergic overactivity may contribute to acute exacerbation of psychosis (Hornykiewicz, 1982; Van Kammen et al., 1990). Third, D₄ receptors may be implicated in the secondary action of antiparkinsonian drugs because clozapine, which has high affinity at D₄ receptors, is effective in the treatment of L-DOPA-induced psychoses (Factor et al., 1995; Meltzer et al., 1995). In fact, although dopaminergic agonists such as bromocriptine are known to be effective in alleviating the symptoms of PD (Weddell and Weiser, 1995), the dopaminergic receptor subtypes involved remain to be further defined (De Keyser et al., 1995; Jenner, 1995).

In view of the above considerations, the efficacy and potency of a range of agonists and antagonists at dopamine D₄ receptors were investigated. Previous studies have revealed the existence of D₄ receptor alleles, differing in the number of a 16-amino acid repeat sequence found in the putative third intracellular loop of the receptor (Van Tol et al., 1992). However, all of these alleles are negatively coupled to adenylyl cyclase activity (Asghari et al., 1995; McHale et al., 1994) and have similar binding and G protein interaction profiles (Asghari et al., 1994), although they may differ in their sensitivity to monovalent cations (Van Tol et al., 1992). Previous studies have determined the affinities of some dopaminergic ligands at D₄ receptors by radioligand competition binding (Lawson et al., 1994; Roth et al., 1995). This technique has yielded estimates of agonist efficacies at D_4.4 receptors by comparing their affinities at different receptor states (Lahti et al., 1996). Other studies have investigated the agonist/antagonist activity at D₄ receptors of a limited number of compounds (e.g., dopamine and quinpirole as agonists and/or clozapine and haloperidol as antagonists) by adenylyl cyclase determinations (Asghari et al., 1995; Bouvier et al., 1995; Tang et al., 1994). Hence, to date, no study of a broad range of agonist efficacies and antagonist potencies in a functional test has been conducted. The present study addressed this issue by evaluation of [³⁵S]GTPS binding (Chabert et al., 1994) to membranes of mammalian (CHO) cells transfected with the D_4.4 receptor (four repeat sequence), the most common allele in humans (Chang et al., 1996; Lichter et al., 1993). Agonist stimulation of [³⁵S]GTPS binding, a nonhydrolyzable analog of GTP, provides a measure of receptor-mediated G protein activation (Hilf et al., 1989; Lazareno et al., 1993). An initial characterization defined the experimental conditions under which optimum agonist stimulation of [³⁵S]GTPS binding was observed. Previous studies in other receptor systems (Gierschik et al., 1991; Hilf et al., 1989; Lazareno et al., 1993; Lorenzen et al., 1993) have highlighted the importance of monovalent and divalent cations (particularly Na⁺ and Mg⁺⁺) and of GDP as being critical for modulation of agonist activation of [³⁵S]GTPS binding. Furthermore, given the importance of receptor density and/or receptor reserve on functional responses, the number of receptor-coupled G proteins activated by the endogenous agonist dopamine was determined in relation to the density of receptors present in the cell line. Indeed, the stoichimetric relationship between receptors and G proteins can significantly affect the definition of agonist efficacies (Adham et al., 1993; Kenakin, 1996; Newman-Tancredi et al., 1997c). The potency and efficacy for stimulation of [³⁵S]GTPS binding of 12 dopaminergic agonists, including several antiparkinsonian drugs currently in development, were determined. Finally, in view of the potential use of D₄ receptors as a target for antipsychotic activity, the potency of a large series of antipsychotics for blocking dopamine-induced [³⁵S]GTPS binding was determined and compared with the action of reference dopaminergic antagonists. In addition to the neuroleptic haloperidol and the "atypical" antipsychotic clozapine, we examined the action of ziprasidone, olanzapine, sertindole, seroquel and other putatively atypical antipsychotics in late-stage development (Goldstein, 1995). Furthermore, the action of the novel selective D₄ receptor antagonist L 745,870 (Kulagowski et al., 1996), was investigated.

	Methods

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[³H]Spiperone binding to CHO-D_4.4 cell membranes. Saturation binding at D_4.4 receptors was carried out with 8 concentrations of [³H]spiperone (100 Ci/mmol; Amersham, Les Ulis, France) from 0.02 to 2.5 nM. For competition binding experiments, the concentration of [³H]spiperone was 0.5 nM. Membranes (10-20 µg of protein) from transfected CHO cells stably expressing the human dopamine D_4.4 receptor (Receptor Biology, Baltimore, MD) were incubated with [³H]spiperone at 25°C for 60 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 haloperidol (10 µM). Affinity (inhibition constants, K_i) at hD_4.4 receptors was determined in [³H]spiperone competition binding experiments. Isotherms were analyzed by nonlinear regression using the program Prism (GraphPAD Software, San Diego, CA) to yield IC₅₀ values. Inhibition constants (K_i) were derived from IC₅₀ values according to the Cheng-Prusoff equation: K_i = IC₅₀/(1 + L/K_d), where L is the concentration of radioligand and K_d is the dissociation constant of [³H]spiperone at D_4.4 receptors (0.37 nM).

Isotopic dilution [³⁵S]GTPS saturation binding to CHO-D_4.4 cell membranes. Receptor-linked G protein activation at D_4.4 receptors was determined by measuring the stimulation of [³⁵S]GTPS (1332 Ci/mmol; New England Nuclear, Les Ulis, France) binding. Except where stated otherwise, CHO-D_4.4 membranes (50 µg of protein) were incubated (20 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 0.1 nM [³⁵S]GTPS. Nonspecific binding was defined with GTPS (10 µM). In isotopic dilution experiments, the basal and dopamine (10 µM)-stimulated binding of radiolabeled [³⁵S]GTPS was inhibited with unlabeled GTPS. Two concentration ranges of GTPS were tested: 0 to 10 µM and 0 to 45 nM. For the former, IC₅₀ values were derived by nonlinear regression. For the latter, saturation binding curves were derived to estimate the number of G proteins activated by dopamine. The total amount of ligand bound to G protein (BOUND_TOT) was calculated by equation 1: BOUND_TOT = [³⁵S]GTPS_BOUND × GTPS_TOT/[³⁵S]GTPS_CONC, where [³⁵S]GTPS_BOUND is observed dopamine-dependent binding in the tubes (fmol/mg), [³⁵S]GTPS_CONC is [³⁵S]GTPS concentration in the tubes (0.1 mM) and GTPS_TOT is [³⁵S]GTPS_CONC plus GTPS concentration.

Measurement of agonist efficacy and antagonist potency at D_4.4 receptors. Agonist efficacy is expressed relative to that of DA (100%), which was tested at a maximally effective concentration (10 µM) in each experiment. For antagonist tests, membranes were preincubated with dopamine and a single concentration of antagonist for 30 min before the addition of [³⁵S]GTPS. For concentration-response curves of the inhibition of dopamine-stimulated [³⁵S]GTPS binding, K_b values were calculated by equation 2: K_b = IC₅₀/{([agonist]/EC₅₀) + 1}, where [agonist] is agonist concentration.

Compounds. Fananserin (RP 62203) was obtained from Rhone-Poulenc Rorer (Vitry-sur-Seine, France). Lisuride and terguride were from Schering (Berlin, Germany). Ocaperidone was from Janssen (Beerse, Belgium). ORG 5222 (trans-5-chloro-2-methyl-2,3,3a,12b-tetrahydro-1H-dibenz[2,3:6,7]oxepino-[4,5-c]pyrrole) was from Organon (Oss, Netherlands). Olanzapine and quinerolane were from Eli Lilly (Indianapolis, IN). Raclopride was from Astra (Sodertalje, Sweden). Seroquel was from Zeneca (Macclesfield, UK). Sertindole was from Lundbeck (Copenhagen, Denmark). Tiaspirone and BMY 14802 (1-[4-(4-fluorophenyl)-4-hydroxybutyl]-4-(5-fluoropyrimidin-2-yl)-piperazine) was from Bristol-Myers (Wallingford, CT). (+)-7-OH-DPAT [7-hydroxy-2-(di-n-propylamino)tetralin] was from CNRS (Paris, France). dp-ADTN (5,6-dihydroxy-2-di-n-propylamino-1,2,3,4-tetrahydronaphthalene) was kindly donated by Dr. Ann Mills-Duggan (Glaxo-Wellcome, Stevenage, UK). Ropinirole, piribedil, FG 5893 (2-[4-[4,4-bis(4-fluorophenyl)butyl]-1-piperazinyl]pyridine-3-carboxylic acid), GR 103,691 (4-acetyl-N-{4-[(2-methoxyphenyl)-piperazin-1-yl] butyl-biphenyl-4-carboxamide), risperidone, ziprasidone and L 745,870 (3-(4-[4-chlorophenyl]piperazin-1-yl)methyl-1H-pyrrolo-[2,3b]pyridine) were synthesized by J.-L. Peglion and G. Lavielle (Servier). Clozapine, bromocriptine, ()-quinpirole and spiperone were purchased from RBI (Natick, MA). Haloperidol, ()-apomorphine and L-DOPA were purchased from Sigma.

	Results

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[³H]Spiperone competition binding at D_4.4 receptors. D_4.4 receptor density in CHO-D_4.4 membranes was determined by [³H]spiperone saturation binding. The isotherms were monophasic, with a dissociation constant (K_d) of 0.37 ± 0.05 nM (n = 4) and a B_max value of 1.40 ± 0.12 pmol/mg protein (4) (fig. 1). In [³H]spiperone competition binding experiments, agonist isotherms were shallow, with pseudo-Hill coefficients of <0.8 (table 1). In contrast, the antagonist competition isotherms were steeper and exhibited pseudo-Hill coefficients close to unity.

View larger version (K): [in this window] [in a new window]   Fig. 1.   Saturation binding of [3H]spiperone to CHO-D4.4 cell membranes. Saturation binding was carried out by incubating [3H]spiperone (0.02-2.5 nM) with CHO-D4.4 membranes. Analysis was by nonlinear regression using the program Prism. A, Representative saturation binding isotherm. Points shown are mean of triplicate determinations from an experiment repeated on at least three occasions. B, Scatchard transformation of specific binding data from A.

View this table: [in this window] [in a new window]   TABLE 1 Action of dopaminergic agonists at cloned human dopamine D4.4 receptors Affinities (Ki) at human dopamine D4.4 receptors stably expressed in CHO cells were determined from competition binding experiments with [3H]spiperone. Agonist potencies (EC50) and efficacies were determined by [35S]GTPS binding. Efficacy is expressed relative to that of dopamine (100%) that stimulated specific [35S]GTPS binding 2.2-fold from basal values of 3950 ± 440 dpm to a maximum of 8810 ± 480 dpm. Nonspecific [35S]GTPS binding (defined with 10 µM GTPS) amounted to 1030 ± 130 dpm. Results are expressed as mean ± S.E.M. of at least three independent experiments.

Definition of [³⁵S]GTPS binding conditions. Dopamine (10 µM) stimulated [³⁵S]GTPS binding to CHO-D_4.4 membranes in a linear manner over the first 20 min (3) of time course experiments, and a standard incubation time of 20 min was therefore used. In contrast, no stimulation of [³⁵S]GTPS binding was observed in membranes of untransfected CHO cells (results not shown). Basal (nonagonist-stimulated) binding of [³⁵S]GTPS to CHO-D_4.4 membranes was dependent on the concentration of GDP present in the buffer (fig. 2B) and was reduced from ~90,000 dpm in the absence of GDP to ~4000 dpm at a GDP concentration of 3 µM. In contrast, agonist-dependent [³⁵S]GTPS binding (i.e., the difference between agonist-stimulated and basal binding) amounted to ~5000 dpm (see legend to table 1) and was not decreased by GDP concentrations of 3 µM. The decrease in basal binding augmented the ratio of agonist-stimulated to basal [³⁵S]GTPS binding to 2.2-fold at GDP concentrations of 3 µM (fig. 2B, inset). Like GDP, NaCl reduced basal [³⁵S]GTPS binding, from 13,000 dpm in the absence of NaCl to 4000 dpm at a concentration of 100 mM (fig. 2C). However, high concentrations of NaCl (200 mM) also decreased agonist-dependent [³⁵S]GTPS binding. Although the latter was observed in both the presence and absence of GDP and NaCl, it exhibited an absolute dependence on the presence of magnesium in the incubation medium (fig. 2D). Agonist stimulation of [³⁵S]GTPS binding was observed over a wide range of MgCl₂ concentrations, from 0.1 to 30 mM. The effect of MgCl₂ on both basal and agonist-dependent [³⁵S]GTPS binding was biphasic, increasing to a first maximum at ~0.1 mM and then to a second, higher, maximum at 3 to 10 mM. A set of standard experimental conditions was defined (3 µM GDP, 3 mM MgCl₂, 100 mM NaCl, 20-min incubation) that yielded the highest agonist stimulation of [³⁵S]GTPS binding and was used in all subsequent experiments.

View larger version (K): [in this window] [in a new window]   Fig. 2.   Effect of (A) time, (B) GDP, (C) NaCl and (D) MgCl2 on [35S]GTPS binding to membranes of CHO cells stably expressing cloned human D4.4 receptors. Except where shown, experiments were carried out in a buffer containing HEPES (20 mM, pH 7.4), GDP (3 µM), MgCl2 (3 mM) and [35S]GTPS (0.1 nM) for 20 min at 22°C. Nonspecific binding was defined with GTPS (10 µM). Points shown are mean of triplicate determinations from representative experiments repeated on at least three independent occasions. DA, dopamine. , Basal [35S]GTPS binding (no dopamine). , Agonist-stimulated [35S]GTPS binding (with 10 µM dopamine). A, Specific basal and dopamine-stimulated [35S]GTPS binding determined over time points ranging from 1 to 60 min. B, Specific basal and dopamine-stimulated [35S]GTPS binding determined in the presence of GDP concentrations between 0 and 100 µM. Inset, effect of GDP concentration on agonist stimulation ratio. The stimulation ratio was calculated as specific dopamine-stimulated [35S]GTPS binding divided by specific basal [35S]GTPS binding. C, Specific basal and dopamine-stimulated [35S]GTPS binding determined in the presence of concentrations of NaCl between 0 and 200 mM. D, Specific basal and dopamine-stimulated [35S]GTPS binding determined in the presence of concentrations of MgCl2 between 0 and 100 mM. Inset, effect of MgCl2 concentration on agonist stimulation ratio.

Isotopic dilution [³⁵S]GTPS saturation binding. Inhibition of basal [³⁵S]GTPS binding to CHO-D_4.4 membranes with GTPS (0.1 nM to 10 µM) exhibited a low affinity component (IC₅₀ = 110 ± 18 nM). In contrast, inhibition of dopamine (10 µM)-stimulated [³⁵S]GTPS binding produced biphasic isotherms with IC_50(high) = 4.4 ± 1.9 nM (4) and IC_50(low) = 257 ± 67 nM (4) (fig. 3A). [³⁵S]GTPS saturation binding isotherms were derived for the high-affinity binding component by isotopic dilution with GTPS (0-45 nM; fig. 3B). These yielded an apparent K_d for [³⁵S]GTPS binding to the high-affinity (agonist-dependent) binding site of 15.0 ± 4.2 nM and a B_max of 2.29 ± 0.44 pmol/mg (4) (fig. 3B). The K_d value did not differ significantly from the IC_50(high) value above (P > .05, two-tailed t test).

View larger version (K): [in this window] [in a new window]   Fig. 3.   Effect of GTPS on [35S]GTPS binding to membranes of CHO stably expressing cloned human D4.4 receptors. Experiments were carried out in a buffer containing HEPES (20 mM, pH 7.4), GDP (3 µM), MgCl2 (3 mM) and [35S]GTPS (0.1 nM) for 20 min at 22°C. Representative curves are shown in which each point is the mean of duplicate determinations. Similar results were obtained in at least three independent experiments. A, Basal and dopamine (10 µM)-stimulated [35S]GTPS binding determined in the presence of concentrations of GTPS between 0 and 10 µM. B, Basal and dopamine (10 µM)-stimulated [35S]GTPS binding determined in the presence of concentrations of GTPS between 0 and 45 nM. These data were transformed as described in the text to generate a saturation binding isotherm for agonist-dependent [35S]GTPS binding. Inset, Scatchard plot of the saturation binding isotherm.

Agonist and antagonist action at D_4.4 receptors. The EC₅₀ values for stimulation of [³⁵S]GTPS binding by agonists, including the antiparkinsonian drugs quinerolane, ()-apomorphine, (+)7-OH-DPAT and lisuride, correlated (r = .99, P < .01) with their binding affinity (K_i; table 1; see fig. 5B). In contrast, other clinically used antiparkinsonian drugs (e.g., bromocriptine, piribedil) had low affinity and/or efficacy at D_4.4 receptors. None of the compounds, other than dopamine, acted as full agonists for stimulation of [³⁵S]GTPS binding.

View larger version (K): [in this window] [in a new window]   Fig. 5.   Agonism and antagonism at cloned human D4.4 receptors defined by [35S]GTPS binding. Experiments were carried out in a buffer containing HEPES (20 mM, pH 7.4), GDP (3 µM), MgCl2 (3 mM) and [35S]GTPS (0.1 nM) for 20 min at 22°C. Nonspecific binding was defined with GTPS (10 µM). Points shown are mean of triplicate determinations from representative experiments repeated on at least three independent occasions. [35S]GTPS binding is expressed as a percentage of the maximal stimulation given by dopamine. A, Agonist concentration-response curves. B, Correlation of minus log EC50 values (pEC50) for agonist stimulation of [35S]GTPS binding with minus log Ki values (pKi; table 1) for inhibition of [3H]spiperone binding. The correlation coefficient was .99 (P < .001). C, Shift of dopamine concentration-response [35S]GTPS binding curve in the presence of fixed concentrations of antagonists (table 2). D, Correlation of minus log Kb values (pKb) for antagonist potency with minus log Ki values (pKi, table 2) for inhibition of [3H]spiperone binding. The correlation coefficient was .99 (P < .001). The point corresponding to FG 5893 was not included in the calculation of the correlation.

View larger version (K): [in this window] [in a new window]   Fig. 4.   Competition binding and antagonism at cloned human D4.4 receptors by dopaminergic antagonists. A, Representative [3H]spiperone competition binding isotherms. B, Antagonism of dopamine (1 µM)-stimulated [35S]GTPS binding. Points shown are mean of triplicate determinations from representative experiments repeated on at least three occasions.

View this table: [in this window] [in a new window]   TABLE 2 Antagonism of dopamine-stimulated [35S]GTPS binding to CHO-D4.4 membranes Antagonist potencies (Kb) were calculated from IC50 values for the inhibition of dopamine (1 µM)-stimulated [35S]GTPS binding. Results are expressed as mean ± S.E.M. of at least three independent experiments.

View this table: [in this window] [in a new window]   TABLE 3 Action of dopaminergic antagonists at cloned human D4.4 receptors Affinities (Ki) at human dopamine D4.4 receptors stably expressed in CHO cells were determined from competition binding experiments with [3H]spiperone. Antagonist potencies (Kb) were determined by the shift in the dopamine stimulation curve of [35S]GTPS binding to a higher concentration (EC50) in the presence of a fixed concentration of antagonist. Dopamine alone yielded an EC50 of 100.6 ± 13.7 nM (table 1). Results are expressed as mean ± S.E.M. of at least three independent experiments.

	Discussion

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Effects of GDP, NaCl and MgCl₂ on [³⁵S]GTPS binding to CHO-D_4.4 membranes. [³⁵S]GTPS binding affords a measure of receptor-mediated G protein activation (the first step of the signal transduction pathway) and is applicable regardless of the second-messenger system(s) involved. In CHO-D_4.4 cell membranes, [³⁵S]GTPS binding was modulated by buffer concentrations of GDP. The latter reduced the level of basal (non-agonist-stimulated) binding, without affecting agonist-dependent binding. Hence, as GDP concentration increased, the ratio of agonist-stimulated to basal [³⁵S]GTPS binding increased to 2.2-fold at a GDP concentration of 3 µM (fig. 2B, inset). This compares with stimulation ratios of 1.4-, 2.2-, 2.5- and 3-fold for 5-HT_1D, 5-HT_1D, muscarinic and 5-HT_1A receptors, respectively (Hilf et al., 1989; Newman-Tancredi et al., 1996b; Thomas et al., 1995). Like GDP, NaCl reduced basal binding of [³⁵S]GTPS but reduced dopamine-stimulated binding only at concentrations of >100 mM. Similar data have been reported for agonist stimulation of [³⁵S]GTPS binding at alpha-2 adrenoceptors (Tian et al., 1994).

Determination of G protein number in CHO-D_4.4 membranes by [³⁵S]GTPS isotopic dilution. Unlabeled GTPS inhibited basal [³⁵S]GTPS binding to CHO-D_4.4 monophasically and with low affinity [IC_50(low) = 110 nM]. In contrast, GTPS inhibited agonist-stimulated [³⁵S]GTPS binding biphasically, with an additional high-affinity site [IC_50(high) = 4.4 nM] as well as a low-affinity binding component. Two points should be made regarding these data. First, the IC₅₀ values are a function of the GDP concentration in the assays, because GTPS in effect competes with GDP for binding to G proteins. Second, whereas the low-affinity binding component reflects inhibition of the endogenous GDP/GTP exchange rate of all CHO-D_4.4 G proteins, the high-affinity binding component reflects only inhibition of agonist-stimulated GDP/GTP exchange at D_4.4 receptor-linked G proteins (fig. 3A; Tian et al., 1994). The K_d value for this high-affinity component (15 ± 4.2 nM) was not significantly different from the IC_50(high) above, but serotonin-activated recombinant human 5-HT_1A receptors, also expressed in CHO cells, display a K_d value for [³⁵S]-GTPS of only 1.29 ± 0.13 nM (Newman-Tancredi et al., 1977c P < .01, two-tailed t test, compared with the K_d value for D_4.4 receptors). This suggests that D_4.4 and 5-HT_1A receptors may differently activate G proteins in the same host cell line. In fact, CHO-K1 cells express G_i2 and G_i3, both of which can couple to 5-HT_1A receptors (Raymond et al., 1993), whereas D_4.4 receptor coupling to G_i2 may be a cell-dependent property because it is observed in mouse fibroblast CCL1.3 cells but not MN9D mesencephalic cells (Tang et al., 1994). Additional studies are therefore required to determine which G protein subtype or subtypes are activated by D₄ receptors in CHO cells.

Agonist stimulation of [³⁵S]GTPS binding to CHO-D_4.4 membranes. The action at D_4.4 receptors of 12 dopaminergic agonists was characterized. Their EC₅₀ values for activation of [³⁵S]GTPS binding correlated closely with the K_i values obtained for inhibition of [³H]spiperone binding, and their order of potency agrees with that previously reported for D₄ receptors (Chabert et al., 1994; Tang et al., 1994; Van Tol et al., 1991). In the case of agonist competition binding isotherms, the presence of more than one receptor affinity state was suggested by the low (<.8) pseudo-Hill coefficients (table 1), probably reflecting ligand binding to G protein-coupled and -uncoupled forms of the receptor.

Antagonism of dopamine-stimulated [³⁵S]GTPS binding to CHO-D_4.4 membranes. In agreement with previous reports, clozapine showed marked affinity (K_i = 38 nM) at D_4.4 receptors (table 3). This indicates that in our hands, clozapine is ~2-fold selective for D_4.4 compared with D₂ receptors (K_i = 76 nM) (Millan et al., 1995), although using different radioligands and experimental conditions, other authors have reported selectivities of 17-fold (Durcan et al., 1995; Lawson et al., 1994; Van Tol et al., 1991). In contrast, the novel D₄ receptor antagonist L 745,870 has negligible affinity at D₂ receptors (K_i = 890 nM)² but high affinity at D_4.4 receptors. Its K_i value (1.99 nM) is in the same range as that (0.4 nM) reported by Kulagowski et al. (1996). Although it has no effect alone, L 745,870 completely antagonized dopamine-stimulated [³⁵S]GTPS binding (fig. 4B), with a K_b value of 1.07 nM, and shifted the dopamine concentration-response curve to the right with a K_b value of 1.19 nM (table 2), confirming its high potency at D_4.4 receptors.

Conclusions. [³⁵S]GTPS binding methodology has been applied to recombinant human dopamine D_4.4 receptors expressed in a mammalian (CHO) cell line. In this system, high experimental concentrations of GDP (3 µM), NaCl (100 mM) and MgCl₂ (3 mM) are necessary to obtain optimum ratios of agonist-induced over basal [³⁵S]GTPS binding. CHO-D_4.4 cells display a high ratio of dopamine-activated G proteins to receptors, indicating the absence of spare receptors and enabling partial agonist activity to be defined. A range of antiparkinsonian drugs exhibited widely varying affinities and efficacies at D_4.4 receptors, suggesting that activity at this site is not an important factor in their clinical effectiveness, although D_4.4 receptor agonism may be associated with side effects on mood. In contrast, a range of neuroleptic and atypical antipsychotics antagonized the dopamine-induced stimulation of [³⁵S]GTPS binding, suggesting that D₄ receptor antagonism may be a potentially clinically important feature of many antipsychotic drugs.