Dopamine D2 Receptor Dimer Formation
EVIDENCE FROM LIGAND BINDING*
Duncan
Armstrong and
Philip G.
Strange
From the School of Animal and Microbial Sciences, University of
Reading, Whiteknights, Reading RG6 6AJ, United Kingdom
Received for publication, August 2, 2000, and in revised form, February 22, 2001
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ABSTRACT |
We have examined the binding of two radioligands
([3H]spiperone and [3H]raclopride) to
D2 dopamine receptors expressed in Chinese hamster ovary cells. In saturation binding experiments in the presence of sodium ions, both radioligands labeled a similar number of sites,
whereas in the absence of sodium ions [3H]raclopride
labeled about half the number of sites labeled by [3H]spiperone. In competition experiments in the absence
of sodium ions, however, raclopride was able to inhibit
[3H]spiperone binding fully. In saturation analyses with
[3H]spiperone in the absence of sodium ions raclopride
exerted noncompetitive effects, decreasing the number of sites labeled
by the radioligand. These data are interpreted in terms of a
model where the receptor exists as a dimer, and in the absence of
sodium ions, raclopride exerts negative cooperativity across the dimer
both for its own binding and the binding of spiperone. A model of the
receptor has been produced that provides a good description of the
experimental phenomena described here.
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INTRODUCTION |
The G-protein-coupled receptors
(GPCRs)1 constitute a large
family of proteins responsible for the transduction of a wide range of
signals (e.g. hormones, neurotransmitters, odorants, light, etc.) via G-proteins (1). GPCRs possess a common structural motif of
seven
-helical membrane-spanning domains, and it is often assumed
that the functional unit (i.e. the ligand binding and
G-protein interaction domains) of the GPCR is wholly contained in a
single polypeptide. Indeed, most models of GPCR function assume a
monomeric receptor interacting with the G-protein (see, for example,
Ref. 2). Several lines of evidence, however, suggest that the some
GPCRs may exist in dimeric or oligomeric forms.
Immunoblotting has in several cases revealed species corresponding not
only to the predicted molecular weight of the receptor but also to
multiples of the molecular weight. Bands corresponding to approximately
twice the predicted molecular weight of the receptor have been
interpreted as homodimers for several receptors including D2 dopamine (3, 4), D3 dopamine (5),
2-adrenergic (6), substance P (7), opiate (8) and
M1 and M2 muscarinic acetylcholine receptors
(9). Co-immunoprecipitation has also been used to demonstrate homodimer
formation for the
2-adrenergic receptor (6), opiate
receptor (8), and somatostatin SSTR5 receptor (10). In some cases,
formation of heterodimers of GPCRs has been reported with differences
in the pharmacological properties of the receptors in the heterodimer
(e.g. GABAB receptor isoforms (11-13),
and
opiate receptors (14), dopamine, and somatostatin receptors
(10)).
Further evidence for interaction of GPCRs was provided by Maggio
et al. (15), who created two chimeric receptors
2/M3 and M3/
2, in
which the C-terminal regions (transmembrane domains VI and VII) were
exchanged between the
2C adrenergic and M3
muscarinic receptors. Expression of either chimera alone did not result
in any detectable binding of typical radiolabeled muscarinic or
adrenergic ligands. However, cotransfection of COS7 cells with both
chimeras resulted in the appearance of binding activity corresponding
to both native receptors. This has lead to the proposal that some GPCRs
might form domain-swapped dimers (16). Evidence for GPCR interaction in
cells has been obtained by expressing GPCRs fused to different
chromophores. Transfer of energy between the chromophores has been
shown for the
2-adrenergic receptor (17) and
somatostatin SSTR5 receptor (10) and provides good evidence for the
close proximity of the two molecules of GPCRs.
Some radioligand binding studies suggest differences in the number of
binding sites labeled by different radioligands. At M2
muscarinic receptors, the antagonist [3H]QNB labeled
twice as many sites as did [3H]AF-DX 384 or
N-[3H]methylscopolamine under certain
conditions (18). These data were interpreted in terms of a model where
the receptor exists as a tetramer. The D2 dopamine receptor
is of interest in this regard. Several studies suggest that the
substituted benzamide radioligand [3H]nemonapride can
label more D2 receptor sites in radioligand binding studies
than the butyrophenone [3H]spiperone (3, 19-21),
although this was not seen in all reports (22, 23). For D2
dopamine receptors expressed in recombinant cells, Seeman et
al. (21) reported that [3H]raclopride labeled more
D2 dopamine receptor sites than did [3H]spiperone, although Malmberg et al. (23)
were unable to replicate these findings. Interestingly, Hall et
al. (24) found that the number of sites labeled by
[3H]raclopride in rat striatal membranes was dependent on
the conditions used. [3H]Raclopride labeled more sites in
the presence of sodium ions than in their absence. The number of sites
labeled by [3H]spiperone was, however, unaffected by
sodium ions and was similar to the number of sites labeled by
[3H]raclopride in the presence of sodium ions. Theodorou
et al. (25) found that another substituted benzamide,
[3H]sulpiride, also detected more D2 receptor
binding sites in rat striatum in the presence of sodium ions than it
did in their absence, and similar observations have been made for
[3H]raclopride binding to the related D3
dopamine receptor expressed in recombinant cells (26).
These observations are not consistent with the labeling by these
radioligands of single populations of independent D2
dopamine receptors. They are more consistent with the labeling of
oligomeric arrays with different degrees of cooperativity between the
monomeric units, depending on assay conditions (18, 27). Because of the
importance of the D2 receptor in the actions of the
antipsychotic drugs, we have examined this phenomenon in more detail.
In this paper, therefore, we have studied the binding of two
radioligands ([3H]spiperone and
[3H]raclopride) to D2 dopamine receptors
expressed in CHO cells and provide evidence for the formation of
homodimers for this receptor.
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EXPERIMENTAL PROCEDURES |
Materials--
[3H]Spiperone (15-30 Ci/mmol) was
purchased from Amersham Pharmacia Biotech, and
[3H]raclopride (60-86 Ci/mmol) was purchased from
PerkinElmer Life Sciences. S-(
)-Sulpiride, haloperidol,
and butaclamol were purchased from RBI (Natick, MA). All other
materials were obtained from commercial sources and were of the highest
available purity.
Cell Growth and Membrane Preparation--
CHO cells expressing
the human D2(short) dopamine receptor (28) were grown as
monolayers in RPMI medium supplemented with 2 mM glutamine,
200 µg/ml active Geneticin, and 5% fetal calf serum at 37 °C in a
moist, 5% CO2 atmosphere. Cells were washed with 5 ml of
ice-cold buffer A (20 mM HEPES, 1 mM EDTA (free
acid) 1 mM EGTA, pH 7.4, with KOH), removed from the flask
by gentle shaking with 2-mm diameter glass beads in 5 ml of buffer A,
and homogenized with 30 strokes of a Dounce homogenizer. The homogenate was centrifuged at 260 × g for 10 min, and the
resulting supernatant centrifuged at 48,000 × g for
1 h at 4 °C. The pellet was resuspended in ice-cold buffer A to
~5-10 mg/ml, and aliquots were stored at
70 °C. Protein
concentration was determined by the method of Lowry et al.
(29).
Saturation Radioligand Binding Experiments--
Control
experiments were performed in buffer A, while 100 mM NaCl
or 100 mM N-methyl-D-glucamine
(NMDG) was included in the buffer in order to determine the effects of
sodium ions or ionic strength. Total and nonspecific binding were
defined in the presence of 3 µM (
)-butaclamol and 3 µM (+)-butaclamol, respectively. [3H]raclopride saturation binding experiments were
performed in a total volume of 0.5 ml using 3-15 µg of membrane
protein per tube and 10 concentrations of [3H]raclopride
typically ranging between 20 pM and 10 nM.
[3H]Spiperone saturation binding experiments were
performed in total volumes of either 1 or 10 ml, both using 10-30 µg
of membrane protein/tube and 10 concentrations of the radioligand,
typically between 10 pM and 5 nM for 1-ml
saturations, or 16 concentrations between 1 pM and 1 nM for 10-ml experiments. In experiments that included
raclopride or haloperidol, the range of [3H]spiperone
concentrations was varied according to the apparent Kd values obtained in initial experiments. Each
experiment was performed in triplicate and incubated at 25 °C for
3 h ([3H]raclopride and 1-ml
[3H]spiperone experiments) or 7 h (10-ml
[3H]spiperone experiments), by which time the
radioligands had reached equilibrium. Experiments were terminated by
rapid filtration through Whatman GF/C glass fiber filters using a
Brandel cell harvester followed with four washes of 3 ml of ice-cold
phosphate-buffered saline (140 mM NaCl, 10 mM
KCl, 1.5 mM KH2PO4, 8 mM Na2HPO4). Filter discs were
soaked in 2 ml each of Optiphase Hi-Safe 3 (Wallac) for at least 6 h before radioactivity was determined by liquid scintillation spectroscopy.
Inhibition of Equilibrium [3H]Spiperone
Binding--
A range of concentrations of the competing ligand were
incubated with 10-30 µg of membranes and a fixed concentration of
[3H]spiperone in triplicate for 3 h at 25 °C
before harvesting as described above. Total and nonspecific binding
were defined in the presence of 3 µM (
)-butaclamol and
3 µM (+)-butaclamol, respectively.
Data Analysis--
Data were analyzed using Prizm (GraphPad, San
Diego CA). In saturation experiments, specifically and nonspecifically
bound [3H]spiperone were calculated from saturation data
using the method of Golds et al. (30), which makes a
correction for the depletion of the radioligand. Data were fitted to
equations describing one- or two-site binding models, and the best fit
was determined using an F-test. Competition experiments were fitted to
four-parameter logistic equations, and the best fit between a variable
Hill coefficient and a Hill coefficient fixed to unity was determined
using an F-test. In the analysis of the competition data, the free
radioligand concentration was taken as the added minus total bound in
the absence of competitor. The amount bound will be in fact be
different at the top and bottom of the competition curve. The total
bound was, however, ~12 and ~1% of the added radioligand in the
absence and presence, respectively, of saturating concentrations
of competitor ([3H]spiperone, ~0.25 nM).
The total bound radioligand in the absence of competitor was <10% for
the higher radioligand concentrations used. The effect of this
correction on estimates of Ki is slight (~5% at
0.25 nM radioligand).
The statistical significance of difference between data was determined
at the 0.05 level, using ANOVA or Student's t test, as
appropriate. Ki and Kd values
were first converted to the respective normally distributed negative
logarithm (pKi or pKd). Mean
values are quoted with the respective S.E.
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RESULTS |
[3H]Raclopride Labels Two Different Receptor
Populations, Depending on the Presence of Sodium Ions--
Saturation
binding studies were performed on human D2(short) dopamine
receptors expressed in membrane preparations from recombinant CHO
cells. [3H]raclopride binding in the absence of sodium
ions ("control" conditions) revealed a single population of binding
sites with a Kd of 1.1 nM and Bmax of 0.84 pmol/mg of
membrane protein (Fig. 1 and Table I). In
the presence of 100 mM NaCl, the data were also best
described by a single population of binding sites in which the
Kd for [3H]raclopride was decreased
significantly to 0.23 nM and Bmax
significantly increased to 1.63 pmol/mg. 100 mM NMDG
was used as a control for changes in ionic strength, and experiments
performed under these conditions gave values for Kd
of 1.2 nM and Bmax of 0.89 pmol/mg,
which did not differ significantly from the control values (ANOVA,
p > 0.05).

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Fig. 1.
Saturation binding of
[3H]raclopride to human D2(short) dopamine
receptors expressed in CHO cells. [3H]Raclopride was
incubated for 3 h at 25°C in a total volume of 0.5 ml with
membranes expressing D2(short) receptors in the presence of
100 mM NaCl ( ) or 100 mM NMDG ( ) or in
the absence of either ion ( ) as described under "Experimental
Procedures." The data were fitted best by a one binding site model,
and Kd and Bmax values are
given in Table I. Data shown are from a single experiment performed in
triplicate and are representative of at least three other
experiments.
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Table I
The binding of [3H]spiperone and [3H]raclopride to
D2 dopamine receptors
The binding of [3H]spiperone and [3H]raclopride to
D2(short) dopamine receptors expressed in CHO cells was
determined as described under "Experimental Procedures."
Kd and Bmax values were
determined for both radioligands, and pKd and
Bmax values are given as the mean ± S.E. with
the number of experiments in parentheses. The corresponding
Kd value is given as nM.
[3H]Spiperone saturation assays were performed in a total
volume of 10 ml in the presence of 100 mM NaCl or 100 mM NMDG or in the absence of either ion and were also
performed in a total volume of 1 ml in the absence of ions.
[3H]Raclopride saturation assays were performed in 0.5 ml
volumes under the same ionic conditions as [3H]spiperone.
Bmax determinations were all performed on the same
preparation of membranes in order to allow comparisons, whereas
Kd values are from different preparations. The
concentration of receptor binding sites in the assays is given in
pM.
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Saturation binding experiments with [3H]spiperone were
performed under two conditions: large volume (10-ml) assays to minimize the extent of ligand depletion and, more routinely, smaller volume (1-ml) assays. In control conditions, in both assay volumes a single
population of binding sites was found, with Bmax
values of 1.2 pmol/mg and 1.6 pmol/mg for 10-ml and 1-ml volume assays, respectively (Table I). These values are not significantly different (ANOVA, p > 0.05). The dissociation constant of
[3H]spiperone, 15 pM, was significantly lower
when determined in 10-ml assays, compared with the value, 63 pM, found in 1-ml assays (Table I).
In [3H]spiperone saturation assays performed in a 10-ml
volume, the Bmax value found in the presence of
100 mM NaCl, 2 pmol/mg, was not significantly different
from that found in the presence of 100 mM NMDG, 1.6 pmol/mg
(Table I) (ANOVA, p > 0.05). In the absence of
monovalent cations (control conditions), the
Bmax value of [3H]spiperone was
1.2 pmol/mg, which, while significantly less than the value in the
presence of sodium ions, was not different from the value in the
presence of NMDG (ANOVA). The dissociation constant of
[3H]spiperone was unaffected by either sodium ions or
NMDG (Table I).
Raclopride Decreases the Number of Receptors Labeled by
[3H]Spiperone--
Parallel [3H]spiperone
saturation binding experiments (1-ml volume) were performed in the
absence of sodium ions and in the absence and presence of raclopride
(10 µM). In the absence of raclopride, the
Bmax value for [3H]spiperone was
1.7 ± 0.1 pmol/mg (n = 3). Including 10 µM raclopride in the [3H]spiperone
saturation assays increased the apparent Kd value
~116-fold and significantly reduced the Bmax
to 1.3 ± 0.1 pmol/mg (n = 3) (Fig.
2A).

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Fig. 2.
A, the Bmax of
[3H]spiperone is decreased by raclopride in the absence
of sodium ions. [3H]Spiperone saturation binding assays
were performed in a total volume of 1 ml in the presence of raclopride
and the absence of sodium ions as described under "Experimental
Procedures." Data were fitted best to a one-binding site model, and
Kd and Bmax values were
determined. One representative example of three similar experiments is
shown. , absence of raclopride, Bmax = 1.71 pmol/mg, Kd = 0.02 nM. , 1 µM raclopride, Bmax = 1.57 pmol/mg, Kd = 0.79 nM. , 3 µM raclopride, Bmax = 1.29 pmol/mg, Kd = 1.3 nM. , 10 µM raclopride, Bmax = 1.28 pmol/mg, Kd = 4.26 nM.
B, Schild plot of raclopride effect on
[3H]spiperone affinity in the absence of sodium ions.
[3H]spiperone saturation binding assays were performed in
a 1-ml volume in the absence of sodium ions and in the presence of a
range of concentrations of raclopride as described under
"Experimental Procedures." Data were fitted to a one-binding site
model, and the Kd was obtained. Data from each of
four or five experiments were transformed according to the method of
Schild (44), and the mean ± S.E. was determined for each
raclopride concentration. Data plotted above were subjected to linear
regression (slope = 0.98), and the pA2 value was
determined as the intercept. , experimental data, pA2 = 7.12; dashed line, values predicted by a simple
competitive model, pA2 = 8.97.
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A range of raclopride concentrations was included in 1-ml volume
[3H]spiperone saturation binding experiments. Analysis of
the data according to the method of Schild gave rise to the plot in
Fig. 2b. The pA2 value for raclopride derived
from these data was 7.12, corresponding to 76 nM. The
pA2 value predicted from a simple competitive model, using
the affinity of [3H]raclopride given in Table I, was
8.97, corresponding to 1.1 nM, a difference of 71-fold from
the measured value.
Similar experiments were performed, in the absence of sodium ions or
NMDG, using haloperidol as the competing ligand. Fig. 3A shows that haloperidol had
no significant effect on the Bmax of
[3H]spiperone, while the pA2 value of 9.32 (0.48 nM) from the Schild plot (Fig. 3B) agreed
very well with the value predicted by a simple competitive model:
pA2 = 9.45 (0.36 nM)
(31).2

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Fig. 3.
A, the Bmax of
[3H]spiperone is unaffected by haloperidol.
[3H]Spiperone saturation binding assays were performed in
a total volume of 1 ml in the presence of haloperidol and the absence
of sodium ions as described under "Experimental Procedures." Data
were fitted best to a one binding site model, and Kd
and Bmax values were determined. One
representative example of three similar experiments is shown. ,
absence of haloperidol, Bmax = 0.81 pmol/mg,
Kd = 0.01 nM. , 3.2 nM
haloperidol, Bmax = 0.85 pmol/mg,
Kd = 0.09 nM. , 10 nM
haloperidol, Bmax = 0.83 pmol/mg,
Kd = 0.42 nM. , 32 nM
haloperidol, Bmax = 0.89 pmol/mg,
Kd = 1.34 nM. B, Schild plot
of haloperidol effect on [3H]spiperone affinity in the
absence of sodium ions. [3H]spiperone saturation binding
assays were performed in a 1-ml volume in the absence of sodium ions
and in the presence of a range of concentrations of haloperidol as
described under "Experimental Procedures." Data were fitted to a
one-binding site model, and the Kd was obtained.
Data from each of three experiments were transformed according to the
method of Schild (44), and the mean ± S.E. was determined for
each haloperidol concentration. Data plotted above were subjected to
linear regression (slope = 1.1), and the pA2 value was
determined as the intercept. , experimental data, pA2 = 9.32; dashed line, values predicted by a simple
competitive model, pA2 = 9.45.
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In the presence of 100 mM NaCl, no significant effect of
raclopride on [3H]spiperone Bmax
was found (Fig. 4A). Analysis
of the data according to the method of Schild (Fig. 4B)
resulted in a pA2 value for raclopride in the presence of
sodium ions of 8.79 (1.6 nM). This is 6.9-fold greater than
the value of 9.63 (0.23 nM) predicted by a simple
competitive model.

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Fig. 4.
A, the Bmax of
[3H]spiperone is unaffected by raclopride in the presence
of sodium ions. [3H]spiperone saturation binding assays
were performed in a total volume of 1 ml in the presence of raclopride
and 100 mM NaCl as described under "Experimental
Procedures." Data were fitted to a one-binding site model, and
Bmax values were determined. Data were expressed
as a proportion of the Bmax determined in the
absence of raclopride for each experiment, and the means with S.E.
error bars are shown for four experiments at each raclopride
concentration. B, Schild plot of raclopride effect on
[3H]spiperone affinity in the presence of sodium ions.
[3H]spiperone saturation binding assays were performed in
a 1-ml volume in the presence of 100 mM NaCl and in the
presence of a range of concentrations of raclopride as described under
"Experimental Procedures." Data were fitted to a one-binding site
model and the Kd obtained. Data from each of four
experiments were transformed according to the method of Schild (44),
and the mean ± S.E. was determined for each raclopride
concentration. Data plotted above were subjected to linear regression,
and the pA2 value was determined as the intercept. ,
experimental data, pA2 = 8.79; dashed
line, values predicted by a simple competitive model,
pA2 = 9.63.
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Raclopride Fully Inhibits [3H]Spiperone Binding in
Competition Experiments--
Raclopride was competed against different
[3H]spiperone concentrations between 0.2 and 4.3 nM in the absence of sodium ions, and representative
competition curves are shown in Fig. 5.
Raclopride inhibited [3H]spiperone binding to within 5%
of the level of nonspecific binding defined in the presence of 3 µM (+)-butaclamol. The data from 14 experiments (at
different [3H]spiperone concentrations) were fitted well
by a sigmoidal competition curve with a Hill coefficient of unity. The
mean Ki of raclopride derived from all of these data
was 19.5 nM (7.71 ± 0.06, pKi ± S.E., n = 14). Competition binding experiments with
raclopride and 0.25 nM [3H]spiperone were
also performed in the presence of 100 mM NaCl, yielding a
Ki of 0.95 nM (9.02 ± 0.05, pKi ± S.E., n = 3) or in the
presence of 100 mM NMDG, resulting in a Ki of 11.3 nM (7.95 ± 0.08, pKi ± S.E., n = 3).
[3H]spiperone binding was inhibited by raclopride to the
level of nonspecific binding under both of these conditions.

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Fig. 5.
Raclopride inhibition of
[3H]spiperone binding.
Raclopride/[3H]spiperone competition experiments were
performed with 0.22 ( ), 0.66 ( ), or 3.46 nM ( )
[3H]spiperone as described under "Experimental
Procedures." Specific [3H]spiperone binding was
determined as that inhibited by 3 µM (+)-butaclamol. The
curves shown are representative curves from single experiments with
data points determined in triplicate and are best described by a
one-binding site model. The IC50 values from all
experiments at all [3H]spiperone concentrations were
converted to Ki values by the method of Cheng and
Prusoff (49), and the mean was determined to be 19.5 nM (7.71 ± 0.06, pKi ± S.E.,
n = 14).
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DISCUSSION |
In this study, we have examined the binding of two ligands
(raclopride and spiperone) to human D2(short) dopamine
receptors expressed in CHO cells. Saturation and competition
experiments have been used to show that the D2 receptor
functions as an oligomer and that the properties of this oligomeric
receptor may be modulated by the ionic conditions.
In saturation binding assays, sodium ions were found to exert
allosteric effects on [3H]raclopride binding to
recombinant human D2(short) dopamine receptors. The
presence of sodium ions was found to increase the affinity of
[3H]raclopride for the D2 receptor, and this
phenomenon has been described extensively before for drugs of the
substituted benzamide class (25, 32-38). In the present study, the
dissociation constant decreased from 1.1 nM approximately
5-fold to 0.23 nM. The effect was specific to sodium ions,
since the presence of an equal concentration (100 mM) of
NMDG as a control for changes in ionic strength had no appreciable
effect on the Kd of
[3H]raclopride.
Sodium ions also exerted a second effect on
[3H]raclopride binding to D2 receptors by
changing the number of binding sites that were labeled
(Bmax). A 2-fold increase in
Bmax was found in the presence of sodium ions as
compared with the absence of sodium ions. Again, there was no
appreciable effect of NMDG compared with the absence of ions. This
effect of sodium ions on the number of sites labeled by
[3H]raclopride has been noticed before in studies of
D2 dopamine receptors in the brain and in recombinant cells
(24, 38) but not analyzed further.
In contrast to [3H]raclopride, the Kd
of [3H]spiperone for binding to D2 receptors
was unaffected by the presence or absence of sodium ions, in agreement
with many other reports (19, 24, 35, 39). It should be noted that
experimental design can be important in determining the affinity of
some high affinity radioligands. Here an assay volume of 1 ml gave a
value for the Kd of [3H]spiperone of
~63 pM. When assays were performed to minimize ligand
depletion via binding to receptor and nonspecifically to tissue
(i.e. use of a 10-ml total volume, in which there is 10-fold more radioligand present but the same amount of receptor), this experimental protocol provided an estimate of Kd for [3H]spiperone of 15 pM. Even under these
conditions, there is some depletion at the lower concentrations of
radioligand, but the estimate of Kd agrees well with
determinations of Kd for spiperone designed to
eliminate depletion artifacts (23). The effects of such artifacts on
determination of Kd values have been discussed in
detail elsewhere (23, 40-43).
The effect of sodium ions on the number of binding sites labeled by
[3H]spiperone was less than for
[3H]raclopride. Bmax values for
[3H]spiperone were similar in the presence of sodium
ions, in the presence of NMDG, and in the absence of monovalent cations
(in 1-ml assays) and similar to the Bmax seen
for [3H]raclopride in the presence of sodium ions. In
10-ml assays in the absence of monovalent cations, some reduction in
Bmax was seen for [3H]spiperone.
These data suggest that the Bmax of
[3H]spiperone is less sensitive to monovalent cations
than the Bmax of [3H]raclopride,
but from the data of Table I it appears that the binding of
[3H]spiperone is not completely insensitive to the
effects of sodium ions. An important comparison can be made between the
Bmax values for the two radioligands in the
presence of sodium ions and in the presence of NMDG, since this
comparison takes account of the effects of ionic strength. In this
comparison, [3H]spiperone labels similar numbers of sites
under both conditions and similar to the Bmax
for [3H]raclopride in the presence of sodium ions. In the
presence of NMDG, [3H]raclopride labels about half the
number of sites. Previous reports would suggest that the binding of
both [3H]spiperone and
N-[3H]-methylspiperone are largely insensitive
to sodium ions (19, 24, 25, 35). Discrepancies between the number of
D2 receptors labeled by different radioligands in
vitro have been reported extensively and for several ligands,
e.g. [3H]spiperone (or
N-[3H]methylspiperone) compared with either
[3H]raclopride (21, 24), [3H]sulpiride
(42), or [3H]nemonapride (3, 19, 21), although this was
not seen in all reports (22, 23).
In the present study, therefore, in the presence of sodium ions,
[3H]raclopride and [3H]spiperone label
similar numbers of sites, whereas in the absence of sodium ions
[3H]raclopride labels fewer sites than are labeled by
[3H]spiperone (69% under control conditions and 55% in
the presence of NMDG). If all of these sites are independent and the
interaction of spiperone and raclopride is competitive, then it would
be expected that in the absence of sodium ions raclopride would not
fully compete with a high concentration of [3H]spiperone
for binding to the receptors. It was found, however, that raclopride
inhibited all of the specific [3H]spiperone binding (to
within 5% of the level of nonspecific binding defined by 3 µM (+)-butaclamol) over a range of
[3H]spiperone concentrations between 0.2 and 4.3 nM, suggesting that raclopride may exhibit some negative
cooperativity with [3H]spiperone at the D2
dopamine receptor.
In order to examine this further, we performed saturation binding
assays with [3H]spiperone in the presence of different
concentrations of raclopride. First, however, this experimental design
was employed with a ligand that has been shown to act in a purely
competitive manner at D2 receptors, haloperidol (31).
Inclusion of haloperidol in [3H]spiperone saturation
binding assays reduced the apparent affinity of the radioligand in a
concentration-dependent manner, as seen in Fig.
3B. The Kd value derived from this
analysis was in excellent agreement with values derived using assay
conditions defined to avoid artifacts (23, 31, 41) The
Bmax of the radioligand was unaffected by
haloperidol, as seen in Fig. 3A. These data are in agreement
with a simple competitive model of two ligands competing for a single
population of identical binding sites.
In a similar experimental design, raclopride (in the absence of sodium
ions) also decreased the apparent affinity of
[3H]spiperone, but the observed decrease in affinity was
smaller than predicted assuming competitive inhibition and using the
Kd of raclopride derived from saturation analyses.
Similarly, in competition experiments versus
[3H]spiperone in the absence of sodium ions, the
Ki for raclopride implied a lower affinity than
suggested from saturation analyses. These differences in estimates of
ligand affinity suggest that there is negative cooperativity between
the two ligands. In addition, however, raclopride lowered the apparent
Bmax of [3H]spiperone in the
saturation experiments. None of these observations is predicted by a
simple competitive model. The observations are similar to those of Hall
et al. (24), who tested a single concentration of raclopride
(30 nM) in the absence of sodium ions and found a 40%
decrease in the Bmax of
[3H]spiperone and a 4-fold decrease in apparent affinity
of this radioligand for binding to rat striatal membranes.
When the present experiments were performed in the presence of sodium
ions, raclopride decreased the apparent affinity of [3H]spiperone in a manner closer, but not identical, to
that predicted from the competitive model. The
Bmax of [3H]spiperone was not
affected by raclopride in the presence of sodium ions in these studies.
Similarly, in competition studies with raclopride versus
[3H]spiperone in the presence of sodium ions, the
Ki was very similar to the Kd
derived from saturation analyses. These data show that sodium ions
affect the interaction between [3H]spiperone and
raclopride at the D2 dopamine receptor and that the
interaction is largely competitive in the presence of sodium ions.
It should be noted that we analyzed some of the data obtained from
saturation analyses in the present study using the method of Schild
(44). This method is strictly applicable only when there is no change
in the Bmax of the radioligand. It is, therefore suitable for analyses of the effects of haloperidol (without
Na+) and raclopride (with Na+) on saturation
analyses of [3H]spiperone binding. Saturation analyses of
[3H]spiperone binding in the presence of raclopride (no
Na+) do show a change in Bmax. The
application of this technique in this case does, however, allow us to
compare the pA2 value of raclopride, based on its effects
on saturation analyses of [3H]spiperone, with the value
of log Kd for raclopride determined in saturation analyses.
The data described above, particularly those in the absence of sodium
ions, cannot be described by a simple competitive model, so other
models must be considered. A model in which the D2 dopamine receptor is able to form a dimeric unit is described under
"Appendix," and simulations of the experiments according to the
model have been compared with the data. In this model, two receptor
monomers are able to interact, providing two identical ligand binding
sites per dimer, which allows the binding of one equivalent of a ligand to affect the binding of a second equivalent of the same ligand or of a
different ligand in a cooperative manner. In this model, in the
presence of sodium ions, the binding of the first and second equivalents of either [3H]raclopride or
[3H]spiperone exert little cooperativity with each other,
so both halves of each dimeric receptor are occupied by each
radioligand, and the affinities of the first and second equivalents of
ligand are similar.
In the absence of sodium ions, the binding of one equivalent of
[3H]raclopride to one half of the dimeric receptor exerts
a strong negative cooperativity on the binding of the second equivalent such that the affinity of the second site of the dimer for
[3H]raclopride is greatly reduced, and little binding at
this site is detected for the range of radioligand concentrations used
in the present study. The number of sites labeled under the conditions used in Fig. 1 (highest [3H]raclopride concentration ~7
nM) is then about 60% of that found in the presence of
sodium ions. The observed Bmax of
[3H]spiperone is, however, much less affected by the
absence of sodium ions, and the total number of sites occupied is
similar to that occupied by [3H]raclopride in the
presence of sodium ions (i.e. both halves of the
dimer are occupied over the range of radioligand
concentrations used in the present study). The model suggests that the
first and second equivalents of [3H]spiperone exert
little cooperativity across the dimer in the absence or presence of
sodium ions.
In saturation binding assays with [3H]spiperone in the
presence of raclopride and the absence of sodium ions, there is an
apparent reduction in the Bmax of the
radioligand, and the concentrations of raclopride required to affect
[3H]spiperone binding are higher than predicted from
saturation analyses. These observations may be explained in the model
if raclopride exerts homotropic negative cooperativity with itself and
heterotropic negative cooperativity with [3H]spiperone
across the dimer.
The negative cooperativity between raclopride and
[3H]spiperone means that for moderate concentrations of
[3H]spiperone not all of the binding sites are occupied
by [3H]spiperone. This leads to an apparent reduction in
Bmax dependent on the conditions of the
experiment (range of radioligand concentrations) and the analysis of
the data according to a single saturation. If it were possible to use
an extended range of concentrations of radioligand, then all of the
sites would be occupied. The combination of homotropic negative
cooperativity between two molecules of raclopride and the heterotropic
negative cooperativity between raclopride and spiperone leads to the
need for higher concentrations of raclopride (than predicted from
simple competition) to prevent [3H]spiperone binding. In
the presence of sodium ions, the strength of cooperativity is reduced,
and the ligands behave more competitively. These effects are also seen
where the same experiment is performed but in a standard competition
format where a range of concentrations of raclopride is used to inhibit
the binding of [3H]spiperone. In the absence of sodium
ions, the Ki for raclopride derived from these
experiments is higher than the Kd derived from
saturation analyses, whereas in the presence of sodium ions the
Ki for raclopride is very similar to the
Kd derived from saturation analyses.
The simulations of experiments using the model described under
"Appendix" are for the most part in good agreement with the data obtained. This indicates that the model (Scheme 1) proposed here provides a first approximation to describing the experimental phenomena
described here. An alternative possible model that could explain some
of the behavior seen in the present study would be one where raclopride
binds to two sets of independent sites of higher and lower affinity in
the absence of sodium ions. This might explain some of the experimental
observations such as the effects of raclopride on the
Bmax of [3H]spiperone. For such a
model, however, if spiperone has equal affinities for the two putative
sets of sites, it would be expected that raclopride inhibition of
[3H]spiperone binding would be described by inhibition
curves with Hill coefficients substantially less than 1. In the present
study, raclopride/[3H]spiperone inhibition curves do not
exhibit such behavior, and this observation seems to rule out the
"two-site" model.
The data described in the present report can, therefore, be
approximated in terms of a scheme in which there are two interacting sites in a dimer for the D2 dopamine receptor. Some of the
observations reported here have been reported for D2
receptors in the brain (24) so that this phenomenon is not confined to
receptors expressed in recombinant cells. The present data complement
data obtained on this receptor using protein chemical methods under
denaturing conditions, where the existence of dimers was inferred (3). The model may also explain some other phenomena that have been observed
for the D2 dopamine receptor such as the observation of two
rates of radioligand dissociation (31, 45), the complex pH dependence
of the binding of some ligands (46), and the pseudocompetition of
amiloride analogues versus [3H]spiperone
binding with Hill coefficients close to 2 (31, 45).
The dimeric model may be an oversimplification, since there are reports
that other radioligands (e.g. [3H]nemonapride)
are able to label more receptors than [3H]spiperone (19,
21), suggesting that higher order species than dimers may exist. The
present data are, however, consistent with homodimers. There are also
some similarities between the present observations with the
D2 dopamine receptor and the model proposed by Wreggett and
Wells (18) for the muscarinic acetylcholine receptor, suggesting that
the observations may have some generality.
 |
FOOTNOTES |
*
This work was supported by the Wellcome Trust and the
University of Reading.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: School of Animal and
Microbial Sciences, University of Reading, Whiteknights, Reading RG6
6AJ, United Kingdom.
Published, JBC Papers in Press, February 23, 2001, DOI 10.1074/jbc.M006936200
2
D. Armstrong and P. G. Strange, unpublished results.
 |
ABBREVIATIONS |
The abbreviations used are:
GPCR, G-protein-coupled receptor;
NMDG, N-methyl-D-glucamine;
ANOVA, analysis of
variance;
CHO, Chinese hamster ovary.
 |
APPENDIX |
A Model Describing the Interaction of Two Competing Ligands with a
Dimeric Receptor
In Scheme 1, R is a divalent receptor oligomer with binding sites
for competitive ligands A and B for which equilibrium association constants are KA and KB,
respectively. The allosteric constants
and
govern the effect of
the presence of a first equivalent of ligand on the formation of a
homo-bi-liganded species for ligands A and B, respectively. The
constant
governs formation of the hetero-bi-liganded species.
Stoichiometrically equivalent species (e.g. monoliganded species
where the ligand is bound to either half of the dimer) are assumed to
be functionally indistinguishable so the equilibrium constant is taken
as the same. Association constants describing the scheme above are
listed in Scheme 2.
The total number of ligand binding sites is twice the number of
receptor dimers.
|
(Eq. 1)
|
|
(Eq. 2)
|
The amount of ligand B which is bound is defined as follows.
|
(Eq. 3)
|
|
(Eq. 4)
|
Thus fractional occupancy of ligand binding sites by ligand B is
described as follows.
|
(Eq. 5)
|
Similarly, for ligand A, fractional occupancy of binding sites is
described as follows.
|
(Eq. 6)
|
Equations 5 and 6 were then used to simulate the experimental data
reported above. It was important that the model described three
experimental observations presented above: first, the reduction in
[3H]raclopride Bmax in the absence
of sodium ions; second, the reduction of [3H]spiperone
Bmax and affinity by raclopride; and third, full
competition for [3H]spiperone binding by raclopride in
competition experiments. Values of the parameters
,
, and
(0.015, 1, and 0.2, respectively) were found that when used in the
model provided simulations that described each of these experimental
observations well.
First, considering the change in the Bmax for
[3H]raclopride in the presence and absence of sodium
ions, [3H]raclopride was represented by ligand A in the
model, and it was assumed that the apparent differential binding
capacities for the radioligand were due to the occupancy of
approximately one-half of the sites in the dimer in the absence of
sodium ions and both sites in the presence of sodium ions for the range
of radioligand concentrations used in the experiments described above. In the presence of sodium ions, where the radioligand occupies both
sites with a single apparent affinity, the microscopic association constant (KA) can be equated to the reciprocal of
the macroscopic dissociation constant Kd (0.23 nM, Table I). This conclusion may be derived using Equation 6 in the absence of ligand B and with
= 1.
In the absence of sodium ions, the microscopic and macroscopic
dissociation constants (K(micro) and
K(macro) respectively) for occupancy of the
ith site on a protein with n sites (47, 48) are
given by Equation 7.
|
(Eq. 7)
|
For the first site of a dimer and substituting
K(macro) as the dissociation constant for the
radioligand in a ligand binding assay (Kd (values in
Table I)) and K(micro) as 1/KA in
Equation 5 or 6, then Kd = 1/2KA.
Hence, under these conditions KA = 4.17.108 M
1 in the
presence of NMDG and 4.55 × 108
M
1 in the absence of sodium ions
or NMDG.
Fig. 6 shows simulated
[3H]raclopride saturation data together with the actual
data points as in Fig. 1. The simulations shown have been generated
with no competing ligand and with values of
= 1,
= 0.2, and
set to either 1 or 0.015. When
= 1 (with Na+), the radioligand labels both sites of the dimer, and
the maximum number of binding sites are labeled with a single affinity.
When
= 0.015 (without Na+), the radioligand will
label both sites of the dimer at high concentrations, but there is some
flattening of the saturation curve so that if the range of
[3H]raclopride concentrations is limited to that used in
Figs. 1 and 6 then it appears as if only about half of the sites are
being labeled.

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Fig. 6.
Comparison of simulated and actual raclopride
saturation analysis of a dimeric receptor. Equation 6 was used to
simulate data representing the occupancy of a hypothetical receptor
dimer by ligand A (raclopride) over a range of concentrations from 1 fM to 10 mM , and the simulated curves are
shown together with the real data (from Fig. 1) in the presence of
Na+ ( ) or NMDG ( ) or in the absence of either ion
( ) . In the simulations, the following parameter values were used.
, K A = 4.35 × 109
M-1 (the reciprocal of the K
d for [3H]raclopride determined in the presence
of Na+ , Table 1), = 1, [B] = 0. / ,
K A = 4.55 × 108
M-1 (1/2K d for
[3 H]raclopride determined in the absence of
Na+ or NMDG), = 0.015, [B] = 0.
|
|
Sodium ions had little effect on the Bmax of
[3H]spiperone, which can be represented in the model by
ligand B. If KB is taken to be the reciprocal of the
Kd of [3H]spiperone and the allosteric
constant,
, assumed to be 1, then [3H]spiperone
labels the maximum number of binding sites in a single saturation (Fig.
2).
The second consideration is that in the absence of sodium ions,
inclusion of 10 µM raclopride in
[3H]spiperone saturation experiments reduced the
experimentally measured Bmax to approximately
three-quarters of that determined in the absence of competing ligand
(Fig. 2) and increased the apparent Kd to ~3.5
nM. Intermediate concentrations of raclopride exerted
smaller effects. These experimental data can be simulated in the model
(Fig. 7) by taking KA
as 1/2Kd for [3H]raclopride and
KB as 1/Kd for
[3H]spiperone as outlined above and values of
= 0.015,
= 1,
= 0.2, and [A] = 1, 3, or 10 µM. If very high concentrations of
[3H]spiperone are used, then both sites of the dimer will
be occupied, although the presence of raclopride leads to a flattening
of the saturation curve. If the range of concentrations of
[3H]spiperone (ligand B) is limited to a maximum of 10 nM as used in the experiments described in Fig. 2, then the
presence of raclopride leads to an apparent reduction in the number of
sites labeled by [3H]spiperone.

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Fig. 7.
Comparison of simulated and actual spiperone
saturation analysis of a dimeric receptor in the presence and absence
of raclopride. Equation 5 was used to simulate data representing
the occupancy of a hypothetical receptor dimer by ligand B (spiperone)
over a range of concentrations from 1 fM to 10 nM in the presence or absence of ligand A (raclopride). The
simulated curves are shown together with the real data (taken from Fig.
2A). In the simulations, the following parameter values were
used: K A = 4.55 × 108
M-1 (1/2K d for
[3H]raclopride determined in the absence of sodium ions),
K B = 6.67 × 1010
M-1 (1/K d for
[3H]spiperone determined in the absence of sodium ions),
= 0.015, = 1, = 0.2, and [A] = 0 ( ) , 1 µM ( ), 3 µM ( ), and 10 µM ( ).
|
|
In the presence of sodium ions, the highest concentration of raclopride
tested, 36 nM, had no significant effect on the
experimentally measured Bmax of
[3H]spiperone and reduced the apparent affinity to ~0.5
nM as described above. These data can be simulated by the
model taking KA and KB as
1/Kd for [3H]raclopride and
[3H]spiperone determined in saturation analyses,
respectively, as above, and with each allosteric constant equal to 1. Under these conditions, therefore, [3H]spiperone binding
corresponds to a single saturation, and raclopride serves to decrease
the apparent affinity of the radioligand without altering its
Bmax over the range of radioligand
concentrations used in the experiments described here.
The third observation that the model must explain is that raclopride
fully inhibits [3H]spiperone binding in competition
experiments in both the absence and presence of sodium ions. In the
presence of sodium ions, each ligand can bind to both sites of the
dimer with equal affinity as described above. Under these conditions,
interactions between the two ligands appear competitive. In the absence
of sodium ions, the data may be simulated by using the parameters
= 0.015,
= 1, and
= 0.2 and
KA = 1/Kd for
[3H]spiperone and KB = 1/2Kd for [3H]raclopride (Fig.
8). The simulations of the model are in
good agreement with the data, although there is some deviation at low concentrations of raclopride.

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Fig. 8.
Comparison of simulated and actual data for
inhibition of spiperone binding by raclopride at a dimeric
receptor. Equation 5 was used to simulate data representing the
inhibition of a fixed concentration of ligand B (spiperone) by a range
of concentrations of ligand A (raclopride) between 1 pM and
10 mM. The simulated curves are shown together with the
actual data (taken from Fig. 5). In the simulations the following
parameter values were used: KA = 4.55 × 108 M-1 (1/2Kd
for [3H]raclopride determined in the absence of sodium
ions), KB = 6.67 × 1010
M-1 (1/Kd for
[3H]spiperone determined in the absence of sodium ions),
= 0.015, = 1, = 0.2, and [B] = 0.22 nM ( ), 0.66 nM ( ), and 3.46 nM ( ).
|
|
In the analysis presented above, it has been assumed that sodium ions
had no effect on the Bmax of
[3H]spiperone. Simulations have therefore been performed
assuming that the allosteric constant
was 1. The binding of
[3H]spiperone in the absence of raclopride follows a
single saturation, and both sites of the dimer are labeled. Based on
the data in Table I, however, this is an oversimplification in that the
Bmax of this radioligand is sensitive to sodium
ions but less so than for [3H]raclopride.
It is important to emphasize that, under all experimental conditions,
assuming the model presented here, if a wide enough range of
radioligand concentrations were used then both sites of the dimer would
be occupied. The apparent reduction in the Bmax
for [3H]raclopride in the absence of sodium ions (Table
I) and the apparent reduction in the Bmax for
[3H]spiperone induced by raclopride (Figs. 2 and 7)
result from the shape of the saturation curves and the range of
radioligand concentrations used in the experiments performed. It would
be very interesting to determine, for example, the effects of
raclopride in [3H]spiperone saturation analyses in the
absence of sodium ions with greatly increased concentrations of
[3H]spiperone, although this may not be feasible technically.
The examples above indicate that the model is capable of describing the
interactions of spiperone and raclopride reported above and that
D2 dopamine receptors may therefore exist as dimers. It is
notable that the model presented here is essentially an abbreviation of
the tetrameric receptor model described by Wreggett and Wells (18), in
which radioligand binding to the M2 muscarinic acetylcholine receptor was found to be consistent with the receptor forming tetrameric oligomers. The radioligand [3H]QNB
labeled ~1.5-2-fold more sites than [3H]NMS or
[3H]AFDX. The data from experiments with
[3H]AFDX could be described by a dimeric model, while
data with [3H]NMS required at least a trimer, but data
with all three radioligands required the assumption of at least a
tetrameric arrangement. It is possible then that the D2
dopamine receptor could exist as higher order oligomers, but a dimer is
sufficient to describe the data presented here. Use of other
radioligands and immunological techniques would be required to
demonstrate the existence of dopamine receptor oligomers.
 |
REFERENCES |
1.
|
Ji, T. H.,
Grossmann, M.,
and Ji, I.
(1998)
J. Biol. Chem.
273,
17299-17302[Free Full Text]
|
2.
|
Samama, O.,
Cotecchia, S.,
Costa, T.,
and Lefkowitz, R. J.
(1993)
J. Biol. Chem.
268,
4625-4636[Abstract/Free Full Text]
|
3.
|
Ng, G. Y. K.,
O'Dowd, B. F.,
Lee, S. P.,
Chung, H. T.,
Brann, M. R.,
Seeman, P.,
and George, S. R.
(1996)
Biochem. Biophys. Res. Commun.
227,
200-204[CrossRef][Medline]
[Order article via Infotrieve]
|
4.
|
Zawarynski, P.,
Tallerico, T.,
Seeman, P.,
Lee, S. P.,
O'Dowd, B. F.,
and George, S. R.
(1998)
FEBS Lett.
441,
383-386[CrossRef][Medline]
[Order article via Infotrieve]
|
5.
|
Nimchinsky, E. A.,
Hof, P. R.,
Janssen, W. G. M.,
Morrison, J. H.,
and Schmauss, C.
(1997)
J. Biol. Chem.
272,
29229-29237[Abstract/Free Full Text]
|
6.
|
Hebert, T.,
Moffett, S.,
Morello, J.-P.,
Loisel, T. P.,
Bichet, D. G.,
Barret, C.,
and Bouvier, M.
(1996)
J. Biol. Chem.
271,
16384-16392[Abstract/Free Full Text]
|
7.
|
Schreurs, J.,
Yamamoto, R.,
Lyons, J.,
Munemitsu, S.,
Conroy, L.,
Clark, R.,
Takeda, Y.,
Krause, J. E.,
and Innis, M.
(1995)
J. Neurochem.
64,
1622-1631[Medline]
[Order article via Infotrieve]
|
8.
|
Cvejic, S.,
and Devi, L. A.
(1997)
J. Biol. Chem.
272,
26959-26964[Abstract/Free Full Text]
|
9.
|
Parker, E. M.,
Kameyama, K.,
Higashijima, T.,
and Ross, E. M.
(1991)
J. Biol. Chem.
266,
519-527[Abstract/Free Full Text]
|
10.
|
Rocheville, M.,
Lange, D. C.,
Kumar, U.,
Patel, S. C.,
Patel, R. C.,
and Patel, Y. C.
(2000)
Science
288,
154-157[Abstract/Free Full Text]
|
11.
|
Jones, K. A.,
Borowsky, B.,
Tamm, J. A.,
Craig, D. A.,
Durkin, M. M.,
Dai, M.,
Yao, W. J.,
Johnson, M.,
Gunwaldsen, C.,
Huang, L. Y.,
Tang, C.,
Shen, Q.,
Salon, J. A.,
Morse, K.,
Laz, T.,
Smith, K. E.,
Nagarathnam, D.,
Noble, S. A.,
Branchek, T. A.,
and Gerald, C.
(1998)
Nature
396,
674-679[CrossRef][Medline]
[Order article via Infotrieve]
|
12.
|
Kaupmann, K.,
Malitschek, B.,
Schuler, B.,
Heid, J.,
Froestl, W.,
Beck, P.,
Mosbacher, J.,
Bischoff, S.,
Kulik, A.,
Shiegemoto, R.,
Karschin, A.,
and Bettler, B.
(1998)
Nature
396,
683-687[CrossRef][Medline]
[Order article via Infotrieve]
|
13.
|
White, J. H.,
Wise, A.,
Main, M. J.,
Green, A.,
Fraiser, N. J.,
Disney, G. H.,
Barnes, A. A.,
Emson, P.,
Foord, S. M.,
and Marshall, F. H.
(1998)
Nature
396,
679-682[CrossRef][Medline]
[Order article via Infotrieve]
|
14.
|
Jordan, B. A.,
and Devi, L. A.
(1999)
Nature
396,
697-700
|
15.
|
Maggio, R.,
Vogel, Z.,
and Wess, J.
(1993)
Proc. Natl. Acad. Sci. U. S. A.
90,
3103-3107[Abstract]
|
16.
|
Gouldson, P. R.,
Snell, C. R.,
Bywater, R. P.,
Higgs, C.,
and Reynolds, C. A.
(1998)
Protein Eng.
1,
1181-1193
|
17.
|
Angers, S.,
Salahpour, A.,
Joly, E.,
Hilairet, S.,
Chelsky, D.,
Dennis, M.,
and Bouvier, M.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
3684-3689[Abstract/Free Full Text]
|
18.
|
Wreggett, K. A.,
and Wells, J. W.
(1995)
J. Biol. Chem.
270,
22488-22499[Abstract/Free Full Text]
|
19.
|
Niznik, H. B.,
Grigoriadis, D. E.,
Pri-Bar, I.,
Buchman, O.,
and Seeman, P.
(1985)
Naunyn-Schmiedeberg's Arch. Pharmacol.
329,
333-343[Medline]
[Order article via Infotrieve]
|
20.
|
Terai, M.,
Hidaka, K.,
and Nakamura, Y.
(1989)
Eur. J. Pharmacol.
173,
177-182[CrossRef][Medline]
[Order article via Infotrieve]
|
21.
|
Seeman, P.,
Guan, H.-C.,
Civelli, O.,
van Tol, H. H. M.,
Sunahara, R. K.,
and Niznik, H. B.
(1992)
Eur. J. Pharmacol.
227,
139-146[Medline]
[Order article via Infotrieve]
|
22.
|
Vile, J. M.,
D'Souza, U. M.,
and Strange, P. G.
(1995)
J. Neurochem.
64,
940-943[Medline]
[Order article via Infotrieve]
|
23.
|
Malmberg, A.,
Jerning, E.,
and Mohell, N.
(1996)
Eur. J. Pharmacol.
303,
123-128[CrossRef][Medline]
[Order article via Infotrieve]
|
24.
|
Hall, H.,
Wedel, I.,
Halldin, C.,
Kopp, J.,
and Farde, L.
(1990)
J. Neurochem.
55,
2048-2057[Medline]
[Order article via Infotrieve]
|
25.
|
Theodorou, A. E.,
Jenner, P.,
and Marsden, C. D.
(1983)
Life Sci.
32,
1243-1254[Medline]
[Order article via Infotrieve]
|
26.
|
Malmberg, A.,
Jackson, D. M.,
Eriksson, A.,
and Mohell, N.
(1993)
Mol. Pharmacol.
43,
749-754[Abstract]
|
27.
|
Strange, P. G.
(1994)
Trends Pharmacol. Sci.
15,
317-319[Medline]
[Order article via Infotrieve]
|
28.
|
Gardner, B. R.,
Hall, D. A.,
and Strange, P. G.
(1997)
J. Neurochem.
69,
2589-2598[Medline]
[Order article via Infotrieve]
|
29.
|
Lowry, O. H.,
Rosebrough, N. J.,
Farr, A. L.,
and Randall, R. J.
(1951)
J. Biol. Chem.
193,
263-273
|
30.
|
Golds, P. R.,
Przylo, F. R.,
and Strange, P. G.
(1980)
Br. J. Pharmacol.
68,
541-549[Abstract]
|
31.
|
Hoare, S. R. J.,
and Strange, P. G.
(1996)
Mol. Pharmacol.
50,
1295-1308[Abstract]
|
32.
|
Steffanini, E.,
Marchisio, A. M.,
Devoto, P.,
Vernaleone, F.,
Collu, R.,
and Spano, P. F.
(1980)
Brain Res.
198,
229-233[Medline]
[Order article via Infotrieve]
|
33.
|
Imafuku, J.
(1987)
Brain Res.
402,
331-338[CrossRef][Medline]
[Order article via Infotrieve]
|
34.
|
Reader, T. A.,
Boulianne, S.,
Molina-Holgado, E.,
and Dewar, K. M.
(1990)
Biochem. Pharmacol.
40,
1739-1746[Medline]
[Order article via Infotrieve]
|
35.
|
Neve, K. A.
(1991)
Mol. Pharmacol.
39,
570-578[Abstract]
|
36.
|
Neve, K. A.,
Cox, B. A.,
Henningsen, R. A.,
Spanoyannis, A.,
and Neve, R. L.
(1991)
Mol. Pharmacol.
39,
737-739
|
37.
|
Neve, K. A.,
Henningsen, R. A.,
Kinzie, J. M.,
De Paulis, T.,
Schmidt, D. E.,
Kessler, R. M.,
and Janowsky, A.
(1990)
J. Pharmacol. Exp. Ther.
252,
1108-1116[Abstract]
|
38.
|
Schetz, J. A.,
Chu, A.,
and Sibley, D. R.
(1999)
J. Pharmacol. Exp. Ther.
289,
956-964[Abstract/Free Full Text]
|
39.
|
Schetz, J. A.,
and Sibley, D. R.
(1997)
J. Neurochem.
68,
1990-1997[Medline]
[Order article via Infotrieve]
|
40.
|
Hulme, E. C.,
and Birdsall, N. J. M.
(1992)
in
Receptor-Ligand Interactions: A Practical Approach
(Hulme, E. C., ed)
, pp. 63-176, Oxford University Press, Oxford
|
41.
|
Strange, P. G.
(1997)
Neuropsychopharmacology
16,
166-122
|
42.
|
Zahniser, N. R.,
and Dubocovich, M. L.
(1983)
J. Pharmacol. Exp. Ther.
227,
592-599[Abstract]
|
43.
|
Seeman, P.,
Ulpian, C.,
Wreggett, K. A.,
and Wells, J. A.
(1984)
J. Neurochem.
43,
221-235[Medline]
[Order article via Infotrieve]
|
44.
|
Arunlakshana, O.,
and Schild, H. O.
(1959)
Br. J. Pharmacol.
14,
48-58
|
45.
|
Hoare, S. R. J.,
Armstrong, D.,
Coldwell, M.,
and Strange, P. G.
(2000)
Br. J. Pharmacol.
130,
1045-1059[Abstract/Free Full Text]
|
46.
|
D'Souza, U.,
and Strange, P. G.
(1995)
Biochemistry
34,
13635-13641[Medline]
[Order article via Infotrieve]
|
47.
|
Wells, J. W.
(1992)
in
Receptor-Ligand Interactions: A Practical Approach
(Hulme, E. C., ed)
, pp. 289-395, Oxford University Press, Oxford
|
48.
|
Klotz, I. M.
(1974)
Acc. Chem. Res.
7,
162-168
|
49.
|
Cheng, Y.,
and Prusoff, W. H.
(1973)
Biochem. Pharmacol.
22,
3099-3108[CrossRef][Medline]
[Order article via Infotrieve]
|
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