0022-3565/97/2821-0181$03.00/0
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics
JPET 282:181–191, 1997
Vol. 282, No. 1
Printed in U.S.A.
[35S]Guanosine-59-O-(3-thio)triphosphate Binding as a Measure
of Efficacy at Human Recombinant Dopamine D4.4 Receptors:
Actions of Antiparkinsonian and Antipsychotic Agents
A. NEWMAN-TANCREDI, V. AUDINOT, C. CHAPUT, L.VERRIÈLE and M. J. MILLAN
Department of Psychopharmacology, Institut de Recherches Servier, 78290 Croissy-sur-Seine, France
Accepted for publication March 17, 1997
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
Received for publication September 23, 1996
D4.4 receptors. The potency at D4.4 receptors of the novel,
selective D4.4 receptor antagonist L 745,870 was determined,
indicating that it has high affinity (Ki 5 1.99 nM) without detectable agonist activity. Furthermore, L 745,870 completely inhibited dopamine-stimulated [35S]GTPgS binding with a Kb value
of 1.07 nM. The action of an additional 20 chemically diverse
dopaminergic ligands, including clozapine, ziprasidone, sertindole, olanzapine and several other “atypical” antipsychotics, in
advanced development was investigated. Each of these ligands
shifted the dopamine stimulation curve to the right in a parallel
manner consistent with competitive antagonism at this site and
yielding Kb values (32.6, 22.4, 17.2 and 26.5 nM, respectively)
that agreed closely with their Ki values (38.0, 14.9, 18.5 and
26.1 nM). In contrast, raclopride and seroquel exhibited low
affinity at D4.4 receptors (Ki . 1000 nM). Other compounds that
showed antagonist activity at D4.4 receptors included the
5-hydroxytryptamine2A receptor antagonist fananserin (RP
62203), the sigma ligand BMY 14,802 and the D3 receptor
antagonist GR 103,691. In conclusion, dopamine D4.4 receptor
activity is unlikely to be an important factor in the clinical
effectiveness of antiparkinsonian drugs, although low agonist
efficacy at D4.4 receptors might be associated with a lesser
incidence of side effects. Furthermore, antagonist activity at
D4.4 receptors is a common property of many typical and
atypical antipsychotic agents.
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
ABBREVIATIONS: 5-HT, 5-hydroxytryptamine; CHO, Chinese hamster ovary; CHO-D4.4 , Chinese hamster ovary cells expressing dopamine D4.4
receptors, GTPgS, guanosine-59-O-(3-thio)triphosphate; PD, Parkinson’s disease; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid.
181
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ABSTRACT
Recombinant human dopamine D4.4 receptor-mediated G protein activation was characterized in membranes of transfected
mammalian (Chinese hamster ovary) cells by the use of
[35S]guanosine-59-O-(3-thio)triphosphate ([35S]GTPgS) binding.
An initial series of experiments defined the conditions (3 mM
GDP, 100 mM NaCl, 3 mM MgCl2) under which optimal stimulation (2.2-fold increase in specific [35S]GTPgS binding) was
achieved with the endogenous agonist dopamine. The number
of dopamine-activated G proteins in Chinese hamster ovaryD4.4 membranes was determined through [35S]GTPgS isotopic
dilution saturation binding, yielding a Bmax value of 2.29 pmol/
mg. This compared with a D4.4 receptor Bmax value of 1.40
pmol/mg determined by [3H]spiperone saturation binding, indicating that 1 or 2 G proteins were activated per D4.4 receptor
and that there were few or no “spare receptors” in this cell line.
Under these conditions, the efficacy for stimulation of
[35S]GTPgS binding at D4.4 receptors of 12 dopaminergic agonists was determined. Several antiparkinsonian drugs, including ropinirole, quinerolane and lisuride, exhibited agonist activity at D4.4 receptors (Emax 5 74.3%, 72.4% and 32.2%,
respectively, compared with dopamine 5 100%). The EC50
values for agonist stimulation of [35S]GTPgS binding correlated
well with the inhibition constants derived from competition
binding with [3H]spiperone (r 5 1.99). However, other antiparkinsonian drugs (bromocriptine, L-DOPA and terguride)
showed low affinity and/or were devoid of agonist activity at
182
Newman-Tancredi et al.
al., 1993). An initial characterization defined the experimental conditions under which optimum agonist stimulation of
[35S]GTPgS 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 Na1 and Mg11) and of GDP as being critical for modulation of agonist activation of [35S]GTPgS 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]GTPgS 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]GTPgS
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
3
[ H]Spiperone binding to CHO-D4.4 cell membranes. Saturation binding at D4.4 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 mg of protein)
from transfected CHO cells stably expressing the human dopamine
D4.4 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 mM). Affinity
(inhibition constants, Ki) at hD4.4 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
(Ki) were derived from IC50 values according to the Cheng-Prusoff
equation: Ki 5 IC50/(1 1 L/Kd), where L is the concentration of
radioligand and Kd is the dissociation constant of [3H]spiperone at
D4.4 receptors (0.37 nM).
Isotopic dilution [35S]GTPgS saturation binding to CHOD4.4 cell membranes. Receptor-linked G protein activation at D4.4
receptors was determined by measuring the stimulation of
[35S]GTPgS (1332 Ci/mmol; New England Nuclear, Les Ulis, France)
binding. Except where stated otherwise, CHO-D4.4 membranes (50
mg of protein) were incubated (20 min, 22°C) with agonists and/or
antagonists in a buffer containing 20 mM HEPES, pH 7.4, 3 mM
GDP, 3 mM MgCl2, 100 mM NaCl and 0.1 nM [35S]GTPgS. Nonspecific binding was defined with GTPgS (10 mM). In isotopic dilution
experiments, the basal and dopamine (10 mM)-stimulated binding of
radiolabeled [35S]GTPgS was inhibited with unlabeled GTPgS. Two
concentration ranges of GTPgS were tested: 0 to 10 mM and 0 to 45
nM. For the former, IC50 values were derived by nonlinear regression. For the latter, saturation binding curves were derived to esti-
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dopamine D2 and 5-HT2A receptors (Canton et al., 1994;
Meltzer, 1996), an inverse agonist at 5-HT2C receptors (Labrecque et al., 1995) and a partial agonist at 5-HT1A 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 D4.4 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]GTPgS binding (Chabert et al.,
1994) to membranes of mammalian (CHO) cells transfected
with the D4.4 receptor (four repeat sequence), the most common allele in humans (Chang et al., 1996; Lichter et al.,
1993). Agonist stimulation of [35S]GTPgS binding, a nonhydrolyzable analog of GTP, provides a measure of receptormediated G protein activation (Hilf et al., 1989; Lazareno et
Vol. 282
D4 Receptors and [35S]GTPgS Binding
1997
Results
3
[ H]Spiperone competition binding at D4.4 receptors. D4.4 receptor density in CHO-D4.4 membranes was determined by [3H]spiperone saturation binding. The isotherms were monophasic, with a dissociation constant (Kd) of
0.37 6 0.05 nM (n 5 4) and a Bmax value of 1.40 6 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.
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.
Definition of [35S]GTPgS binding conditions. Dopamine (10 mM) stimulated [35S]GTPgS binding to CHO-D4.4
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]GTPgS binding was observed in membranes of untransfected CHO cells (results not shown). Basal (nonagoniststimulated) binding of [35S]GTPgS to CHO-D4.4 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
mM. In contrast, agonist-dependent [35S]GTPgS 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 mM. The decrease in
basal binding augmented the ratio of agonist-stimulated to
basal [35S]GTPgS binding to 2.2-fold at GDP concentrations
of 3 mM (fig. 2B, inset). Like GDP, NaCl reduced basal
[35S]GTPgS 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]GTPgS 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]GTPgS 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]GTPgS 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 mM GDP, 3
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mate the number of G proteins activated by dopamine. The total
amount of ligand bound to G protein (BOUNDTOT) was calculated
by equation 1: BOUNDTOT 5 [35S]GTPgSBOUND 3 GTPgSTOT/
[35S]GTPgSCONC, where [35S]GTPgSBOUND is observed dopaminedependent binding in the tubes (fmol/mg), [35S]GTPgSCONC is
[35S]GTPgS concentration in the tubes (0.1 mM) and GTPgSTOT is
[35S]GTPgSCONC plus GTPgS concentration.
Measurement of agonist efficacy and antagonist potency at
D4.4 receptors. Agonist efficacy is expressed relative to that of DA
(100%), which was tested at a maximally effective concentration (10
mM) 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]GTPgS. For concentrationresponse curves of the inhibition of dopamine-stimulated
[35S]GTPgS binding, Kb values were calculated by equation 2: Kb 5
IC50/{([agonist]/EC50) 1 1}, where [agonist] is agonist concentration.
For the dopamine concentration-response curves determined in
the presence of a fixed concentration of antagonist, antagonist potency values (Kb) were calculated by equation 3: Kb 5 [antagonist]/
[(EC509/EC50) 21], where [antagonist] is antagonist concentration,
EC509 was determined in the presence of antagonist and EC50 was
determined in the absence of antagonist (dopamine alone).
Experiments were terminated by rapid filtration through Whatman GF/B filters (pretreated with 0.1% polyethyleneimine in the
case of [3H]spiperone binding) using a Brandel cell harvester. Radioactivity retained on the filters was determined by liquid scintillation
counting. Protein concentration was determined colorimetrically using a bicinchonic acid assay kit (Sigma, S. Quentin Fallavier,
France). All results are expressed as mean 6 S.E.M. of three or more
independent determinations.
Compounds. Fananserin (RP 62203) was obtained from RhonePoulenc 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-methyl2,3,3a,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).
(1)-7-OH-DPAT [7-hydroxy-2-(di-n-propylamino)tetralin] was from
CNRS (Paris, France). dp-ADTN (5,6-dihydroxy-2-di-n-propylamino1,2,3,4-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 (49-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, (2)-quinpirole
and spiperone were purchased from RBI (Natick, MA). Haloperidol,
(2)-apomorphine and L-DOPA were purchased from Sigma.
183
184
Newman-Tancredi et al.
Vol. 282
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]GTPgS binding. Efficacy is expressed relative to that of dopamine (100%) that stimulated specific [35S]GTPgS
binding 2.2-fold from basal values of 3950 6 440 dpm to a maximum of 8810 6 480 dpm. Nonspecific [35S]GTPgS binding (defined with 10 mM GTPgS) amounted
to 1030 6 130 dpm. Results are expressed as mean 6 S.E.M. of at least three independent experiments.
Ligand
EC50 (pEC50)
Ki (pKi)
%
nM
nM
100
74.3 6 2.4
72.4 6 2.0
69.4 6 4.1
59.2 6 1.6
57.6 6 6.3
56.1 6 4.0
39.8 6 0.8
32.2 6 2.9
N.D.
N.D.
N.D.
100.6 6 13.7 (7.00)
2910 6 410 (5.54)
30.3 6 7.3 (7.52)
48.5 6 13.6 (7.31)
7.68 6 2.5 (8.11)
369 6 112 (6.43)
60.4 6 33.5 (7.21)
3.85 6 1.59 (8.41)
6.77 6 2.97 (8.17)
.10,000 (,5)
.10,000 (,5)
.10,000 (,5)
42.9 6 9.6 (7.37)
873 6 123 (6.06)
21.7 6 7.5 (7.66)
37.3 6 4.7 (7.43)
8.80 6 1.62 (8.06)
140 6 19 (6.85)
53.2 6 12.1 (7.27)
3.47 6 1.40 (8.46)
4.74 6 0.82 (8.32)
315 6 77 (6.50)
.1000 (,6)
.10,000 (,5)
nH
0.41 6 0.01
0.52 6 0.09
0.77 6 0.16
0.59 6 0.04
0.75 6 0.02
0.62 6 0.04
0.61 6 0.06
0.57 6 0.02
0.60 6 0.05
0.82 6 0.08
N.D.
N.D.
N.D., not determined.
mM MgCl2, 100 mM NaCl, 20-min incubation) that yielded
the highest agonist stimulation of [35S]GTPgS binding and
was used in all subsequent experiments.
Isotopic dilution [35S]GTPgS saturation binding. Inhibition of basal [35S]GTPgS binding to CHO-D4.4 membranes with GTPgS (0.1 nM to 10 mM) exhibited a low affinity
component (IC50 5 110 6 18 nM). In contrast, inhibition of
dopamine (10 mM)-stimulated [35S]GTPgS binding produced
biphasic isotherms with IC50(high) 5 4.4 6 1.9 nM (4) and
IC50(low) 5 257 6 67 nM (4) (fig. 3A). [35S]GTPgS saturation
binding isotherms were derived for the high-affinity binding
component by isotopic dilution with GTPgS (0–45 nM; fig.
3B). These yielded an apparent Kd for [35S]GTPgS binding to
the high-affinity (agonist-dependent) binding site of 15.0 6
4.2 nM and a Bmax of 2.29 6 0.44 pmol/mg (4) (fig. 3B). The
Kd value did not differ significantly from the IC50(high) value
above (P . .05, two-tailed t test).
Agonist and antagonist action at D4.4 receptors. The
EC50 values for stimulation of [35S]GTPgS binding by agonists, including the antiparkinsonian drugs quinerolane, (2)apomorphine, (1)7-OH-DPAT and lisuride, correlated (r 5
.99, P , .01) with their binding affinity (Ki; table 1; see fig.
5B). In contrast, other clinically used antiparkinsonian drugs
(e.g., bromocriptine, piribedil) had low affinity and/or efficacy
at D4.4 receptors. None of the compounds, other than dopamine, acted as full agonists for stimulation of [35S]GTPgS
binding.
The antagonist activity of a range of dopaminergic ligands
was tested, including the novel, selective D4 receptor ligand L
745,870 (Ki 5 1.99 nM). Like spiperone, haloperidol and
clozapine, L 745,870 concentration-dependently and completely blocked the stimulation of [35S]GTPgS binding induced by 1 mM dopamine (Kb 5 1.07 nM; fig. 4 and table 2).
L 745,870 (100 nM) also shifted the dopamine concentrationresponse curve to the right, with an 89-fold increase in EC50
(8910 6 1250 nM), yielding a Kb value of 1.19 nM (table 3 and
fig. 5C). Other compounds that showed antagonist activity
were the dopamine D3 receptor ligand GR 103,691, the serotonin 5-HT2A receptor antagonist fananserin (RP 62,203), the
sigma ligand BMY 14,802 and the antiparkinsonian drug
terguride.
The effect on [35S]GTPgS binding of a range of antipsychot-
ics was tested, including clozapine, olanzapine, risperidone
and ziprasidone, none of which altered [35S]GTPgS binding
from basal levels when tested alone. However, fixed concentrations of antagonist shifted the dopamine concentrationresponse curve to the right in a parallel manner, consistent
with competitive antagonism at D4.4 receptors (table 3). For
all the compounds tested, the Kb values calculated from these
shifts agreed closely with their respective Ki values (r 5 .99,
P , .01; fig. 5D), except FG 5893, which showed a 6-fold lower
Kb value than Ki value (P , .05, table 3) and was not included
in the calculation of correlation coefficient.
Discussion
Effects of GDP, NaCl and MgCl2 on [35S]GTPgS binding to CHO-D4.4 membranes. [35S]GTPgS 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-D4.4 cell membranes, [35S]GTPgS 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]GTPgS binding increased to 2.2-fold at a GDP concentration of 3 mM (fig. 2B, inset). This compares with stimulation ratios of 1.4-, 2.2-, 2.5- and 3-fold for 5-HT1Da, 5-HT1Db,
muscarinic and 5-HT1A receptors, respectively (Hilf et al.,
1989; Newman-Tancredi et al., 1996b; Thomas et al., 1995).
Like GDP, NaCl reduced basal binding of [35S]GTPgS but
reduced dopamine-stimulated binding only at concentrations
of .100 mM. Similar data have been reported for agonist
stimulation of [35S]GTPgS binding at alpha-2 adrenoceptors
(Tian et al., 1994).
[35S]GTPgS binding to CHO-D4.4 membranes has an absolute requirement for magnesium, similar to that observed for
A1 adenosine receptors (Lorenzen et al., 1993). In the present
study, the modulatory effects of magnesium were complex,
exhibiting a biphasic action on agonist-dependent
[35S]GTPgS binding (fig. 2D). The [35S]GTPgS binding peak
at a MgCl2 concentration of 3 to 10 mM probably reflects
conditions that favor the formation of a ternary complex of
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Dopamine
Ropinirole
Quinerolane
(2)-Quinpirole
dp-ADTN
(1)-7-OH-DPAT
Pergolide
(2)-Apomorphine
Lisuride
Piribedil
Bromocriptine
L-DOPA
Efficacy
1997
D4 Receptors and [35S]GTPgS Binding
185
agonist/receptor/G protein. In contrast, agonist stimulation
of [35S]GTPgS binding at a MgCl2 concentration of 0.1 mM is
low, because these conditions may be less favorable for the
formation of the ternary complex. However, MgCl2 also had a
biphasic effect on basal [35S]GTPgS binding, suggesting that
Mg11 ions may have modulatory effects on G proteins themselves. These may reflect altered levels of G protein attachment to cell membranes. For example, transducin solubility
increases at Mg11 concentrations of ,0.1 mM (Bornancin et
al., 1989), suggesting that the association of G proteins to
membrane-bound receptors would be favored by higher
Mg11 concentrations. Although the exact mechanistic basis
for the biphasic action of magnesium is unclear, the present
observations agree with similar biphasic effects reported for
muscarinic acetylcholine receptors (Hilf et al., 1989) and
fMet-Leu-Phe (fMet) chemotactic receptors (Gierschik et al.,
1991). For subsequent experiments, a magnesium concentration of 3 mM was selected, providing the highest ratio of
dopamine-stimulated over basal [35S]GTPgS binding (fig.
2D).
The buffer composition chosen was similar to that of
Chabert et al. (1994) for [35S]GTPgS binding to D4.4 -transfected Sf9 insect cells and for the other receptors mentioned
above. This suggests that buffer conditions that yield optimal
agonist stimulation of [35S]GTPgS binding may be similar for
many receptor and cell types. Furthermore, the present data
show that there is no necessity for agonist to be present for G
protein activation to occur. Rather, G protein activation can
be induced, in the absence of agonist, by selecting conditions
that favor coupling of G protein to receptor (millimolar magnesium, low sodium and low GDP). These factors do not,
however, appear to influence the ability of dopamine to further stimulate [35S]GTPgS binding, suggesting that an active
receptor conformation is induced by agonists that is not
achieved by merely manipulating buffer conditions. Thus,
basal and agonist-stimulated [35S]GTPgS binding may reflect different activation states of the D4.4 receptor. In addition, the presence of a basal level of receptor-mediated G
protein activation enables, in principle, the identification of
compounds that inhibit G protein activation (inverse ago-
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Fig. 2. Effect of (A) time, (B) GDP, (C) NaCl and (D) MgCl2 on [35S]GTPgS 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 mM), MgCl2 (3 mM) and
[35S]GTPgS (0.1 nM) for 20 min at 22°C. Nonspecific binding was defined with GTPgS (10 mM). Points shown are mean of triplicate determinations
from representative experiments repeated on at least three independent occasions. DA, dopamine. M, Basal [35S]GTPgS binding (no dopamine).
f, Agonist-stimulated [35S]GTPgS binding (with 10 mM dopamine). A, Specific basal and dopamine-stimulated [35S]GTPgS binding determined
over time points ranging from 1 to 60 min. B, Specific basal and dopamine-stimulated [35S]GTPgS binding determined in the presence of GDP
concentrations between 0 and 100 mM. Inset, effect of GDP concentration on agonist stimulation ratio. The stimulation ratio was calculated as
specific dopamine-stimulated [35S]GTPgS binding divided by specific basal [35S]GTPgS binding. C, Specific basal and dopamine-stimulated
[35S]GTPgS binding determined in the presence of concentrations of NaCl between 0 and 200 mM. D, Specific basal and dopamine-stimulated
[35S]GTPgS binding determined in the presence of concentrations of MgCl2 between 0 and 100 mM. Inset, effect of MgCl2 concentration on
agonist stimulation ratio.
186
Newman-Tancredi et al.
nists). Xanthine amine congener, for example, lowers basal G
protein activation at A1 adenosine receptors (Freissmuth et
al., 1991), whereas methiothepin and ketanserin inhibit
[35S]GTPgS binding to membranes of CHO cells stably expressing 5-HT1Da and 5-HT1Db receptors (Thomas et al.,
1995). In the present system, however, the level of basal
[35S]GTPgS binding was minimized to facilitate the definition of agonist effects. Hence, reduction of basal binding by
inverse agonists, may be relatively small. Furthermore, the
ability to detect inverse agonist activity may depend on the
presence of a high receptor to G protein ratio. Indeed, in a
CHO cell line manipulated to express a high level of 5-HT1A
receptors (without a change in G protein number), the inverse agonist spiperone exhibited increased negative efficacy
(Newman-Tancredi et al., 1997b, 1997c).
Determination of G protein number in CHO-D4.4
membranes by [35S]GTPgS isotopic dilution. Unlabeled
GTPgS inhibited basal [35S]GTPgS binding to CHO-D4.4
monophasically and with low affinity [IC50(low) 5 110 nM]. In
contrast, GTPgS inhibited agonist-stimulated [35S]GTPgS
binding biphasically, with an additional high-affinity site
[IC50(high) 5 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 GTPgS in effect competes with GDP for
binding to G proteins. Second, whereas the low-affinity bind-
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
mM)-stimulated [35S]GTPgS binding. Points shown are mean of triplicate determinations from representative experiments repeated on at
least three occasions.
TABLE 2
Antagonism of dopamine-stimulated [35S]GTPgS binding to CHOD4.4 membranes
Antagonist potencies (Kb) were calculated from IC50 values for the inhibition of
dopamine (1 mM)-stimulated [35S]GTPgS binding. Results are expressed as
mean 6 S.E.M. of at least three independent experiments.
Antagonist
IC50
Kb (pKb)
nM
Spiperone
L 745,870
Haloperidol
Clozapine
Raclopride
1.48 6 0.13
11.7 6 0.7
15.0 6 2.0
533 6 40
.10,000
0.13 6 0.01 (9.89)
1.07 6 0.06 (8.97)
1.37 6 0.18 (8.86)
48.4 6 3.7 (7.32)
.10,000 (,5)
ing component reflects inhibition of the endogenous GDP/
GTP exchange rate of all CHO-D4.4 G proteins, the highaffinity binding component reflects only inhibition of agoniststimulated GDP/GTP exchange at D4.4 receptor-linked G
proteins (fig. 3A; Tian et al., 1994). The Kd value for this
high-affinity component (15 6 4.2 nM) was not significantly
different from the IC50(high) above, but serotonin-activated
recombinant human 5-HT1A receptors, also expressed in
CHO cells, display a Kd value for [35S]-GTPgS of only 1.29 6
0.13 nM (Newman-Tancredi et al., 1977c P , .01, two-tailed
t test, compared with the Kd value for D4.4 receptors). This
suggests that D4.4 and 5-HT1A receptors may differently activate G proteins in the same host cell line. In fact, CHO-K1
cells express Gia2 and Gia3, both of which can couple to
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Fig. 3. Effect of GTPgS on [35S]GTPgS 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 mM),
MgCl2 (3 mM) and [35S]GTPgS (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 mM)-stimulated
[35S]GTPgS binding determined in the presence of concentrations of
GTPgS between 0 and 10 mM. B, Basal and dopamine (10 mM)stimulated [35S]GTPgS binding determined in the presence of concentrations of GTPgS between 0 and 45 nM. These data were transformed
as described in the text to generate a saturation binding isotherm for
agonist-dependent [35S]GTPgS binding. Inset, Scatchard plot of the
saturation binding isotherm.
Vol. 282
D4 Receptors and [35S]GTPgS Binding
1997
187
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]GTPgS binding to a higher concentration (EC509) in the presence of a fixed
concentration of antagonist. Dopamine alone yielded an EC50 of 100.6 6 13.7 nM (table 1). Results are expressed as mean 6 S.E.M. of at least three independent
experiments.
Antagonist (nM)
EC509
Kb (pKb)
Ki (pKi)
nM
4850 6 50
4390 6 1050
8910 6 1250
6770 6 480
6460 6 2420
2860 6 480
3460 6 780
2880 6 590
2200 6 340
700 6 76
6290 6 1033
589 6 111
488 6 50
381 6 49
827 6 67
919 6 76
N.D.
N.D.
N.D.
N.D.
N.D.
0.21 6 0.01 (9.68)
0.78 6 0.16 (9.11)
1.19 6 0.18 (8.92)
1.52 6 0.12 (8.82)
2.22 6 0.92 (8.65)
3.26 6 0.62 (8.49)
3.83 6 0.62 (8.41)
4.03 6 1.04 (8.39)
4.98 6 0.70 (8.30)
17.2 6 2.0 (7.76)
17.3 6 3.4 (7.76)
22.4 6 4.4 (7.65)
26.5 6 3.0 (7.58)
32.6 6 3.0 (7.49)
42.3 6 4.3 (7.37)
124 6 12 (6.91)
N.D.
N.D.
N.D.
N.D.
N.D.
0.33 6 0.02 (9.48)
1.05 6 0.08 (8.98)
1.99 6 0.61 (8.70)
1.87 6 0.05 (8.73)
3.29 6 1.08 (8.48)
5.57 6 0.58 (8.25)
4.99 6 1.85 (8.30)
5.64 6 1.77 (8.25)
8.40 6 1.61 (8.08)
18.5 6 0.2 (7.73)
106 6 32 (6.97)
14.9 6 2.7 (7.83)
26.1 6 3.2 (7.58)
38.0 6 2.8 (7.42)
24.5 6 2.5 (7.61)
83.6 6 5.1 (7.08)
795 6 165 (6.10)
2290 6 190 (5.64)
3080 6 980 (5.51)
5070 6 730 (5.30)
.10,000 (,5)
N.D., not determined.
5-HT1A receptors (Raymond et al., 1993), whereas D4.4 receptor coupling to Gia2 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.
Information regarding the receptor/G protein stoichiometry in CHO-D4.4 cells can be obtained from the Bmax values
for dopamine-stimulated [35S]GTPgS binding in CHO-D4.4
cells (2.29 pmol/mg) and the Bmax value for D4.4 receptor
expression (1.40 pmol/mg). These data indicate that 1 or 2
dopamine-activated G proteins are labeled in CHO-D4.4
membranes per D4.4 receptor. This is similar to that seen for
atrial natriuretic factor receptors (1 G protein/receptor;
Khurana and Pandey, 1995) and for mu opioid receptors (2 G
proteins/receptor; Traynor and Nahorski, 1995). However,
much higher degrees of amplification have been observed for
fMet chemotactic receptors (20 G proteins/receptor; Gierschik et al., 1991) and for human cardiac muscarinic acetylcholine receptors (50–80 G proteins/receptor; Böhm et al.,
1994). Further investigation is required to elucidate the origin of these widely varying ratios, but they may be a function
of the rapidity of cycling between different receptor subtypes
and their respective G proteins. Indeed, in a comparative
study in rat striatal membranes, mu and sigma opioid receptors activated 20 G proteins/receptor, whereas cannabinoid
receptors only activated 3 G proteins/receptor (Sim et al.,
1996).
Agonist stimulation of [35S]GTPgS binding to CHOD4.4 membranes. The action at D4.4 receptors of 12 dopaminergic agonists was characterized. Their EC50 values for
activation of [35S]GTPgS binding correlated closely with the
Ki 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.
In measurements of [35S]GTPgS binding, all the agonists
exhibited efficacies (relative to dopamine) of markedly
,100%, with the highest (;74%) observed with ropinirole
and quinerolane. In contrast to the present data, Lahti et al.
(1996) reported that the efficacy of (2)-apomorphine at D4.4
receptors, estimated by the ratio of ligand affinities for G
protein-coupled and -uncoupled receptor states, was about
double (80%) that seen here ('40%; table 1). Similarly,
Chabert et al. (1994) found that (2)-apomorphine exhibited
an efficacy of 85%, whereas two further partial agonists
tested here (dp-ADTN and (2)-quinpirole) were full agonists
at D4.4 receptors expressed in insect Sf9 cells. At least two
possibilities may account for these differences. First, although buffer conditions were similar, the incubation temperature in the present study was lower (22°C) than that
used by Chabert et al. (1994) (30°C). A temperature rise may
augment the apparent efficacy of partial agonists through
thermodynamic facilitation of GDP release from G proteins
(Lorenzen et al., 1996). However, in control experiments incubated at 30°C, the efficacy of (2)-apomorphine was increased by only 5% to 10%,1 which is insufficient to account
for the 45% difference in efficacies. Second, the D4.4 receptor
Bmax value in the CHO cells used here (1.40 pmol/mg) was
4-fold lower than that in the Sf9 cells used by Mills et al.
(1993) (5–6 pmol/mg). This suggests that apparent agonist
efficacies in Sf9 cells may be higher due to the presence of
“spare” receptors. Indeed, a high ratio of receptor to G protein
1
A. Newman-Tancredi and C. Chaput, unpublished observations.
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Spiperone (10)
ORG 5222 (30)
L 745,870 (100)
Haloperidol (100)
Ocaperidone (100)
Risperidone (100)
Fananserin (100)
Tiaspirone (100)
Terguride (100)
Sertindole (100)
FG 5893 (1000)
Ziprasidone (100)
Olanzapine (100)
Clozapine (100)
BMY 14,802 (300)
GR 103,691 (1000)
SCH 23,390
Seroquel
DUP 734
Raclopride
Rimcazole
188
Newman-Tancredi et al.
Vol. 282
in Sf9 cells would be expected to increase agonist efficacy, as
has been shown for weak partial agonists such as pindolol at
5-HT1B receptors and eltoprazine at 5-HT1A receptors
(Adham et al., 1993; Newman-Tancredi et al., 1997c).
The agonists tested in the present study include several
compounds that are in development for the treatment of PD,
such as quinerolane, bromocriptine and ropinirole. Dopaminergic agonists are known to alleviate the symptoms of PD
(De Keyser et al., 1995; Wolters et al., 1995), but their exact
mechanism of action is unclear, and may involve multiple
dopamine receptor subtypes (Jenner, 1995). Quinerolane,
(2)-apomorphine and lisuride displayed high affinity for D4.4
receptors, but bromocriptine and piribedil displayed low or
negligible affinity at this site. Furthermore, although quinerolane was an efficacious agonist (Emax 5 72.4%), lisuride
had only weak partial agonist activity (Emax 5 32.2%), and
terguride is an antagonist at D4.4 receptors (see tables 1 and
2). It is concluded that no correlation exists between the
antiparkinsonian effects of these drugs and their activity at
D4.4 receptors. However, given that dopaminergic agonists
can have propsychotic actions (Factor et al., 1995) and the
possible importance of D4.4 receptors in mediating mood dys-
functions, an antiparkinsonian drug with antagonist activity
at D4.4 , such as the ergot terguride (trans-dihydrolisuride),
may present a therapeutic advantage. Indeed, terguride attenuates parkinsonian symptoms in 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine-lesioned monkeys and suppresses
hyperactivity induced by apomorphine treatment (Akai et al.,
1993), whereas in clinical trials, terguride was effective
against PD with only a low incidence of psychotic side effects
(Filipova et al., 1988). Thus, it may be hypothesized that
antiparkinsonian drugs with low efficacy at D4.4 receptors
may induce less-pronounced psychotic side effects. This is
consistent with the observed effectiveness of clozapine, which
has significant affinity and antagonist activity at D4 receptors, in countering L-DOPA-induced psychosis in PD patients
(Factor et al., 1995; Meltzer et al., 1995).
Antagonism of dopamine-stimulated [35S]GTPgS
binding to CHO-D4.4 membranes. In agreement with previous reports, clozapine showed marked affinity (Ki 5 38 nM)
at D4.4 receptors (table 3). This indicates that in our hands,
clozapine is ;2-fold selective for D4.4 compared with D2 receptors (Ki 5 76 nM) (Millan et al., 1995), although using
different radioligands and experimental conditions, other au-
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Fig. 5. Agonism and antagonism at cloned human D4.4 receptors defined by [35S]GTPgS binding. Experiments were carried out in a buffer
containing HEPES (20 mM, pH 7.4), GDP (3 mM), MgCl2 (3 mM) and [35S]GTPgS (0.1 nM) for 20 min at 22°C. Nonspecific binding was defined with
GTPgS (10 mM). Points shown are mean of triplicate determinations from representative experiments repeated on at least three independent
occasions. [35S]GTPgS 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]GTPgS 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]GTPgS
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.
D4 Receptors and [35S]GTPgS Binding
1997
[35S]GTPgS binding agreed closely with those calculated for
the shift in the dopamine concentration-response curves (tables 2 and 3). None of the drugs tested, including L 745,870,
terguride and clozapine, exhibited any intrinsic agonist activity. This indicates that they act as “neutral” or “silent”
antagonists in this system, although experiments in a cell
line with a high receptor expression level might reveal weak
agonist activity (Newman-Tancredi et al., 1997c). Taken together, the present data found no distinction in (lack of)
intrinsic activity at D4.4 receptors between typical antipsychotics such as haloperidol and atypical antipsychotics such
as clozapine, risperidone and olanzapine.
The physiological significance of D4 receptors is unclear,
but the controversial up-regulation of D4-like receptors in
postmortem schizophrenic brain tissue (Murray et al., 1995;
//Reynolds, 1996; Seeman et al., 1993a) suggests that an
interaction at these sites may be involved in the etiology of
the disease. However, the benzamide antipsychotic raclopride, which is effective in countering productive schizophrenic symptoms, has low affinity at D4.4 receptors (table 3),
whereas the potent and selective D4 receptor antagonist L
745,870 is ineffective in treating acutely psychotic patients
(Kramer et al., 1996). Nevertheless, raclopride induces a high
incidence of extrapyramidal symptoms and poorly treats negative schizophrenic symptoms, whereas L 745,870 did not
induce extrapyramidal symptoms, and its therapeutic interest in treating negative and cognitive symptoms is as yet
unknown. These observations, together with the known distribution of D4-like receptors in frontal cortex and hippocampus and on GABAergic neurons (Matsumoto et al., 1996;
Meador-Woodruff et al., 1996; Mrzljak et al., 1996), suggest
that the functional significance of D4 receptors may be related to deficit symptoms, cognitive dysfunction or anxiodepressive states. Indeed, the D4 receptor antagonist (and antiparkinsonian drug) terguride significantly attenuated
negative, but not positive, schizophrenic symptoms in clinical
trials (Olbrich and Schanz, 1988, 1991).
Conclusions. [35S]GTPgS binding methodology has been
applied to recombinant human dopamine D4.4 receptors expressed in a mammalian (CHO) cell line. In this system, high
experimental concentrations of GDP (3 mM), NaCl (100 mM)
and MgCl2 (3 mM) are necessary to obtain optimum ratios of
agonist-induced over basal [35S]GTPgS binding. CHO-D4.4
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 D4.4 receptors, suggesting that activity at this
site is not an important factor in their clinical effectiveness,
although D4.4 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]GTPgS binding, suggesting that D4 receptor
antagonism may be a potentially clinically important feature
of many antipsychotic drugs.
Acknowledgments
We thank Paul Chazot, Chris Breivogel, Frederic Bornancin and
Ann Mills-Duggan for helpful discussions.
References
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