Constitutive oligomerization of human D
2
dopamine receptors
expressed in
Spodoptera frugiperda
9(
Sf9
) and in HEK293 cells
Analysis using co-immunoprecipitation and time-resolved fluorescence resonance
energy transfer
Lucien Gazi
1,
†, Juan F. Lo
´
pez-Gime
´
nez
1,
*†, Martin P. Ru¨ diger
2
and Philip G. Strange
1
1
School of Animal and Microbial Sciences, University of Reading, Reading, UK;
2
GlaxoSmithKline, New Frontiers Science Park,
Harlow, UK
Human D
2Long
(D
2L

receptors in Sf9 cells. When the FLAG-tagged
D
2S
and HIV-tagged D
2L
receptors were co-expressed,
co-immunoprecipitation showed that the two isoforms can
also form hetero-oligomers in Sf9 cells. Time-resolved
FRET with europium and XL665-labelled antibodies was
appliedtowholeSf9 cellsandtomembranesfromSf9 cells
expressing epitope-tagged D
2
receptors. In both cases, con-
stitutive homo-oligomers were revealed for D
2L
and D
2S
isoforms. Time-resolved FRET also revealed constitutive
homo-oligomers in HEK293 cells expressing FLAG-tagged
D
2S
receptors. The D
2
receptor ligands dopamine,
R-(–)propylnorapomorphine, and raclopride did not affect
oligomerization of D
2L
and D
2S
in Sf9 and HEK293 cells.

2
-adrenoceptor [3,4], the chemokine
receptor CCR5 [5,6], the M
3
muscarinic acetylcholine
receptor [7], the M
2
muscarinic cholinergic receptor [8],
the melatonin MT
1
and MT
2
receptors [9], the V
2
vasopressin receptor [10], the 5-HT
1A
,5-HT
1B
and
5-HT
1D
receptors [11], the d and j opioid receptors [12–14],
the histamine H
2
receptor [15], the somatostatin sst2A and
sst3 receptors [16], the yeast Ste2 receptor [17] and the D
2
dopamine receptor [18,19]. Hetero-oligomerization between
c-aminobutyric acid GABA
B

and GABA
B
R2 receptors [11]. All these data strongly
Correspondence to P. G. Strange, School of Animal and Microbial
Sciences, University of Reading, Whiteknights, Reading, RG6 6AJ,
UK. Fax: + 44 118 378 6537, Tel.: + 44 118 378 8015,
E-mail: [email protected]
Abbreviations: BRET, bioluminescence resonance energy transfer;
D
2L
,D
2Long
;D
2S
,D
2Short
;Eu
3+
, europium; FRET, fluorescence
resonance energy transfer; GPCR, G protein-coupled receptor;
HIV, human immunodeficiency virus; m.o.i., multiplicity of infection;
NPA, R-(–)propylnorapomorphine; Sf9, Spodoptera frugiperda 9.
*Present address: Molecular Pharmacology Group, Division of Bio-
chemistry and Molecular Biology, Institute of Biomedical and Life
Sciences, University of Glasgow, Glasgow G12 8QQ, UK.
Authors who contributed equally to this work.
(Received 9 April 2003, revised 8 July 2003, accepted 30 July 2003)
Eur. J. Biochem. 270, 3928–3938 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03773.x
suggest that oligomerization is a general phenomenon
commontoalltheGPCRs.

family of dopamine receptors (which comprises D
2
,D
3
and D
4
receptors). These receptors are GPCRs that
couple to G proteins of the G
i/o
family. There are two
isoforms of the D
2
receptor, D
2Short
(D
2S
)andD
2Long
(D
2L
), which derive from alternative splicing of the same
mRNA [24,25]. D
2L
differs from D
2S
by an additional 29
amino acids in the putative third intracellular loop.
Oligomerization has been reported for each of the two
isoforms using different approaches, e.g. radioligand
binding [18], energy transfer [19], immunoblot analysis

:enhanced yellow fluorescent
protein [19].
In the present study, we used both co-immunoprecipita-
tion and time-resolved FRET to monitor the oligo-
merization of the D
2L
and D
2S
receptors expressed in
Spodoptera frugiperda 9(Sf9) and HEK293 cells. Our data
show that both D
2L
and D
2S
form constitutive homo-
oligomers in living cells that can be detected by FRET and
constitutive hetero-oligomers that can be detected by
co-immunoprecipitation. We also applied, for the first time,
the FRET approach to membranes prepared from Sf9 cells
expressing D
2L
and D
2S
receptors. Finally, our data show
that oligomerization of D
2L
and D
2S
dopamine receptors is
not regulated by D

ively.Inordertoaddanepitopetagtobothreceptorsat
their N-terminus, complementary synthetic oligonucleotides
encoding an HIV epitope tag sequence [29] were designed as
follows: 5¢-AGTACTAGTATCAGAGGCAAGGTACA
ACATATG-3¢ and 5¢-CATATGTTGTACCTTGCCTCT
GATACTAGTACT-3¢. This introduces a 3¢ NdeI site to the
tag sequence. These oligonucleotides were then annealed
anddigestedwithNdeI. TOPOD2L and TOPOD2S were
digested with EcoRI and NdeI, and the DNA fragments and
the HIV tag were ligated. The ligation mixture was subjected
to PCR to selectively amplify tagged receptor whilst, at the
same time, adding an XhoI site and a start codon to the 5¢
end of the tag. To achieve this, the following primers were
used: 5¢-TTGAATTCTCAGCAGTGGAGGATC-3¢ and
5¢-TTCTCGAGGATGGATAGTACTAGTATCAGAG
GC-3¢. Both PCR products were digested with XhoI and
EcoRI and ligated into the plasmid pBlueBac4.5 (Invitro-
gen), to produce the recombinant plasmids pBBHD2L and
pBBHD2S. These plasmids were then co-transfected with
Bac-N-Blue
TM
DNA (Invitrogen) in Sf9 insect cells, and
underwent recombination to produce recombinant baculo-
viruses. The same strategy was employed for the construc-
tion of recombinant baculoviruses encoding FLAG-tagged
D
2
receptors, using, in this case, the following oliogo-
nucleotide encoding the FLAG epitope sequence:
5¢-GCGGCCGCATGGACTACAAGGACGACGATGA

6
cellsÆmL
)1
. Infections were carried out
with different multiplicities of infection (m.o.i.) of baculo-
viruses in order to reach an optimum expression level, as
described previously [30,31]. Sf9 cells were harvested 48 h
after infection and used directly for FRET experiments on
intact cells or for membrane preparations. HEK293 cells
expressing FLAG-D
2S
were grown in Dulbecco’s modified
Eagle’s medium supplemented with 10% FCS and in the
presence of 600 lgÆmL
)1
geneticin.
Membrane preparation
Cells were collected by centrifugation (1700 g,10 min,4 °C)
and resuspended in 15 mL of buffer (20 m
M
Hepes, 6 m
M
MgCl
2
,1m
M
EDTA, 1 m
M
EGTA, pH 7.4). Cell suspen-
sions were then homogenized using an Ultra-TurraxÒ at

M
. The reaction was started by
the addition of membrane proteins, and was incubated for
3hat25°C. Reactions were terminated by rapid filtration
through Whatman GF/C glass-fibre filters, using a Brandel
cell harvester, followed by four washes of 3 mL of ice-cold
NaCl/P
i
(140 m
M
NaCl, 10 m
M
KCl, 1.5 m
M
KH
2
PO
4
,
8m
M
Na
2
HPO
4
). Filter discs were soaked in 2 mL of
Optiphase Hi-Safe 3 (Wallac) for at least 6 h before the
radioactivity was determined by liquid scintillation spectro-
metry. Non-specific binding was defined in the presence
of 3 l

incubation at 4 °C for 2–4 h before being analysed by
Western blot.
Immunoblotting
For Western blot analysis of non-immunoprecipitated
samples, 25 lg of membrane protein was resuspended in
loading buffer and denatured by incubation at 4 °Cfor
2–4 h before being subjected to immunoblotting.
Samples were resolved by SDS/PAGE (10% gel) and
transferred to nitrocellulose membranes using the BioradÒ
semi-dry transfer system. Prestained protein marker, broad
range (6–175 kDa) (New England Biolabs) was used to
define the molecular mass of the bands. Nitrocellulose
membranes were incubated for 1 h with 5% dried milk
(w/v) in NaCl/Tris (TBS) buffer (150 m
M
NaCl, 50 m
M
Tris/HCl, pH 7.5). Membranes were then incubated over-
night at 4 °C with a single primary antibody or an anti-
FLAGÒ M2-peroxidase conjugate Ig (Sigma). The primary
antibodies used for immunoblotting were as follows: 3 lL
of mouse monoclonal anti-FLAGÒ M2 Ig (4 mgÆmL
)1
;
Sigma) or 30 lL of rat monoclonal anti-gp120 Ig. Immu-
noreactivity was detected with horseradish peroxidase-
conjugated anti-mouse IgG (1 : 5000) for anti-FLAGÒ or
anti-rat IgG (1 : 5000) for anti-gp120 Ig. After four washes
with buffer (150 m
M

M
NaH
2
PO
4
, 150 m
M
NaCl) supplemented with 50% FCS
(HEK293 cells). A 2-h incubation was performed at room
temperature on a rotating wheel before washing the cells
twice with incubation buffer and resuspending the final
pellet in 50 lL of incubation buffer. Cells were then placed
in a 384-well microtitre plate and the fluorescence signal was
monitored using an Analyst
TM
(Molecular Devices) or an
Ultra-384 (Tecan) fluorimeter configured for time-resolved
fluorescence. The Eu
3+
-labelled anti-FLAG Ig was excited
at 320 nm and the emission monitored at 620 nm. A 500-ls
reading was taken after a delay of 100 ls. For experiments
conducted on membranes, preliminary experiments were
performed to determine the optimal conditions for
3930 L. Gazi et al. (Eur. J. Biochem. 270) Ó FEBS 2003
observation of signal. Membranes containing the equivalent
of 100 fmol of receptors (as labelled with [
3
H]spiperone)
were incubated with 2.5 n

Analysis of data
Data were analysed using the computer program
GRAPHPAD
PRISM
(GraphPad Software Inc.). [
3
H]Spiperone saturation
binding experiments were fitted to a one binding-site model
(which provided the best fit to the data) to define the B
max
(receptor expression level) and K
d
(dissociation constant for
[
3
H]spiperone). Statistical comparisons were performed
using an unpaired Student’s t-testoranalysisofvariance
(
ANOVA
), where appropriate. A P-value of <0.05 was
considered significant.
Results
Expression of epitope-tagged D
2
receptors
in
Sf9
and HEK293 cells
The expression of differentially epitope-tagged dopamine
D

2L
receptors (B
max
 1.5 pmolÆmg
)1
of protein, Table 1). As expected,
[
3
H]spiperone showed a high affinity (pK
d
 10) for the
epitope-tagged dopamine D
2
receptors expressed in Sf9 cells
(Table 1), with no difference in the affinity observed
between the differentially tagged receptors (one-way
ANOVA
, P >0.05).
When other preparations were used in this study (in
particular, when HIV- and FLAG-tagged receptors were
co-expressed in the same Sf9 host cells), the expression
levels varied between 1 and 4 pmolÆmg
)1
of protein and
the pK
d
value for [
3
H]spiperone was  10 (data not
shown).

were reversed, thus confirming their specificity (Fig. 1).
Co-immunoprecipitation experiments were conducted in
order to investigate further the nature of these bands.
Solubilized membranes from Sf9 cells expressing both
epitope-tagged receptors for a given isoform (D
2L
or
D
2S
), as well as a combination of both isoforms tagged
with two different epitopes (D
2L
and D
2S
), were immu-
noprecipitated with anti-gp120 Ig, resolved subsequently
by SDS/PAGE and immunoblotted with anti-FLAG Ig.
We first sought to analyse different conditions for
separation of the samples. As shown in Fig. 2, samples
were separated by centrifugation at 4500 g for 5 min,
centrifugation at 12 000 g for10min,orbyusing
filtration (0.2-lm filter). Immunoblots corresponding to
D
2S
receptors revealed two bands with molecular masses
equivalent to those observed previously (39 and 80 kDa)
in all three conditions (Fig. 2). The two bands were
visible, even after filtration, showing that they probably
derive from soluble receptors. In the subsequent experi-
ments, all the samples were separated by centrifugation at

B
max
(mean ± SEM,
fmolÆmg
)1
of protein)
pK
d
(mean ± SEM,
K
d
,p
M
)
Sf9-HIV-D
2L
1445 ± 217 10.14 ± 0.06 (72)
Sf9-FLAG-D
2L
728 ± 88 10.01 ± 0.08 (100)
Sf9-HIV-D
2S
1785 ± 357 10.14 ± 0.03 (72)
Sf9-FLAG-D
2S
941 ± 105 9.98 ± 0.03 (100)
Ó FEBS 2003 D
2
dopamine receptor oligomerization (Eur. J. Biochem. 270) 3931
subjected to the same co-immunoprecipitation experi-

2L
and FLAG-D
2S
receptors. Fluorescence signal was also present in cells
expressing HIV-tagged receptors. However, this latter
fluorescence represented an average of 4–6% of the
fluorescence observed with corresponding FLAG-tagged
receptors, and corresponded to background fluorescence
(Fig. 4A).
Similar experiments were conducted on membranes
prepared from Sf9 cells expressing the differentially tagged
dopamine D
2L
and D
2S
receptors, as shown in Fig. 4A. As
for the living cells, the Eu
3+
-anti FLAG bound specifically
to membranes of Sf9 cells expressing the FLAG-tagged
receptors (as compared with HIV-tagged receptors). On
membranes, the background fluorescence (HIV-tagged
receptors) represented 8–10% of the fluorescence at
FLAG-tagged receptors (Fig. 4A). The fluorescence signal
(in countsÆs
)1
), obtained on whole Sf9 cells, was higher than
that obtained with membranes (4–6 · 10
6
vs. 3 · 10

receptors were
expressed in Sf9 cells using the baculovirus expression
system. A combination of Eu
3+
- and XL665-labelled anti-
FLAG Ig (2.5 n
M
each) was then used as energy donor and
acceptor, respectively. The time-resolved FRET was mon-
itored by light emission at 665 nm (XL665) following
excitation at 320 nm (Eu
3+
). The specific FRET signal was
obtained by subtracting the fluorescence observed with
Eu
3+
-anti-FLAG alone from that observed with both
Eu
3+
-anti-FLAG and XL665-anti-FLAG Ig. FRET signal
was observed on Sf9 cells expressing FLAG-tagged dop-
amine D
2L
or D
2S
receptors (Fig. 5A). The specific fluor-
escence values obtained amounted to 32 112 ± 5871
countsÆs
)1
and 40 209 ± 5670 countsÆs

2S
;3,FLAG-D
2L
;4,HIV-D
2L
] were immunoblotted using
anti-FLAG Ig (upper panel) or anti-gp120 Ig (lower panel), as des-
cribed in the Experimental procedures. Molecular mass markers are
indicated in kDa. The immunoblots shown are representative of at
least three independent experiments. A multiplicity of infection (m.o.i.)
of 10 was used for infection with each baculovirus.
3932 L. Gazi et al. (Eur. J. Biochem. 270) Ó FEBS 2003
D
2L
and D
2S
receptors, respectively. These fluorescence
signals were not significantly different (Student’s t-test,
P > 0.05). When the cells were incubated with Eu
3+
-anti-
FLAG Ig and XL665-anti-FLAG Ig separately, and then
mixed before the fluorescence was monitored, no FRET
was detected (Fig. 5A, ÔmixÕ).
We also analysed D
2
dopamine receptor homo-oligome-
rization on membranes prepared from Sf9 cells expressing
FLAG-tagged D
2L

was not significant (Student’s t-test, P >0.05)
(Fig. 5B). The overall FRET signal was higher for both
receptors when experiments were carried out on mem-
branes, with a marked difference observed for dopamine
D
2S
receptor and only a minor increase for dopamine D
2L
receptor.
FRET experiments were also carried out on HEK293
cells expressing FLAG-D
2S
receptor. The specific fluores-
cence value observed in mammalian cells was 13 898 ± 297
countsÆs
)1
(Fig. 5C). When these same cells were incubated
separately with the two fluorescent-labelled antibodies and
mixed just before reading, no FRET signal was observed
(Fig. 5C, ÔmixÕ).
Fig. 3. Co-immunoprecipitation of differentially epitope-tagged dopamine D
2
receptor isoforms. Solubilized membranes from Spodoptera frugiperda 9
(Sf9) cells co-expressing FLAG-D
2S
and human immunodeficiency virus (HIV)-D
2S
(lane 1), FLAG-D
2L
and HIV-D

HEK293 cells. Binding of Eu
3+
-anti-FLAG Ig (2.5 n
M
) was carried out on whole Sf9 cellsoronSf9 cell membranes expressing different D
2
receptor isoforms (A), or on whole HEK293 cells (control and those expressing FLAG-D
2S
) (B). The Eu
3+
was excited at 320 nm and the
fluorescence measured at 620 nm, as described in the Experimental procedures. Data shown represent the mean ± SEM from six to eight
experiments.
Ó FEBS 2003 D
2
dopamine receptor oligomerization (Eur. J. Biochem. 270) 3933
Lack of regulation of D
2
receptor oligomerization
by the ligands selective for D
2
receptor
To investigate the effect of D
2
receptor ligands on the
oligomerization phenomenon, Sf9 and HEK293 cells were
preincubated with saturating concentrations of dopamine
(10
)3
M

2S
receptor
expressed in HEK293 cells. Both immunological and
fluorescence-based approaches provide evidence that the
two isoforms of D
2
dopamine receptors can display
constitutive homo- and hetero-oligomerization when
expressed in Sf9 cells. In HEK293 cells expressing FLAG-
D
2S
, our fluorescence-based approach also revealed a
constitutive oligomerization for the D
2S
receptor, in agree-
ment with recent data reported by Guo et al.[33].
The Sf9 cells expressed the D
2
dopamine receptors with
fidelity, as shown by the high-affinity binding of [
3
H]spip-
erone (Table 1). Indeed, this system has been used widely to
express heterologous receptors, including, for example, the
M
2
muscarinic receptor [34,35], the human serotonin
5-HT
5A
receptor [36], the b

Figure. In the ÔmixÕ conditions, the samples were incubated with either
antibody separately and mixed just before the reading was taken. (B)
Membranes prepared from Sf9 cells expressing FLAG-tagged dop-
amine D
2L
receptors (black bars) or FLAG-tagged dopamine D
2S
receptors (white bars) were incubated for 1 h with 2.5 n
M
fluorescent-
labelled antibodies, as indicated on the Figure. The ÔmixÕ condition was
as described above. (C) Intact HEK293 cells expressing FLAG-D
2S
receptor were incubated for 2 h with fluorescent-labelled antibodies, as
indicatedontheFigure.TheÔmixÕ condition was as described above.
After washing with incubation buffer, time-resolved fluorescence res-
onance energy transfer (FRET) was monitored by measuring the light
emission at 665 nm, following excitation at 320 nm. The FRET signal
was obtained by subtracting the fluorescence observed with Eu
3+
-anti-
FLAG Ig alone from that observed with both Eu
3+
-anti-FLAG Ig
and XL665-anti-FLAG Ig. Data shown represent the mean
results ± SEM from seven to 10 experiments.
3934 L. Gazi et al. (Eur. J. Biochem. 270) Ó FEBS 2003
might lead to an artefactual protein–protein interaction.
However, in the system used here, the receptor expression
level assessed by [

could also be demonstrated (Fig. 3). Several
controls were applied in order to verify the specificity of
these interactions: (a) we used different procedures to
separate solubilized from non-solubilized membranes,
including filtration of the samples (0.2-lmfilter)and(b)
we mixed cell membranes expressing differentially epitope-
tagged receptors and subjected the mixture to co-immuno-
precipitation. The results obtained clearly showed a specific
signal in the different separation conditions, but no signal
for the mixed samples. The mixing experiments establish the
specificity of the observations and the filtration experiment
shows that the signals derive from solubilized receptors.
This is the first study to successfully apply the co-immuno-
precipitation approach to study D
2L
and D
2S
receptor
hetero-oligomerization. This approach also revealed some
differences between the two isoforms of D
2
dopamine
receptor regarding the oligomerization process. Indeed,
co-immunoprecipitation experiments revealed two bands
(at 39 and 80 kDa) for D
2S
, but only one band (85 kDa) for
D
2L
receptors (Fig. 3, lane 3). The smaller band (39 kDa)

method to the study of oligomerization of d-opioid recep-
tors [14]. However, our present approach for FRET analysis
used a single antibody (anti-FLAG Ig) derivatized with
both energy donor (Eu
3+
) and energy acceptor (XL665).
This differs from others in the literature [14], where donor
and acceptor are on two different antibodies. The present
method is based on that described by Farrar et al.[41].
These authors studied FRET between epitope (c-myc)-
tagged subunits of the GABA
A
receptor, and found that
these subunits assemble with a stoichiometry of (a
1
)
2
(b
2
)
2
c
2
,
validating the use of a single derivatized antibody for the
analysis of protein–protein interaction. In the present study,
we also applied these technologies to study receptor–
receptor interaction in cell membranes. Thus, a strong
FRET signal could be detected on whole Sf9 cells and cell
membranes, as well as on HEK293 cells expressing FLAG-

and the incubation was continued for 2 h. The measurements were
performed as described in the legend to Fig. 5 and the data shown
represent the mean results ± SEM from seven experiments. The data
were normalized as a percentage of control [i.e. fluorescence resonance
energy transfer (FRET) in the absence of ligand].
Ó FEBS 2003 D
2
dopamine receptor oligomerization (Eur. J. Biochem. 270) 3935
observed between the two D
2
receptor isoforms, despite an
apparently lower signal for D
2L
on membranes (Fig. 5B).
This contrasts with the clear difference we observed between
D
2S
and D
2L
while using co-immunoprecipitation (see
above). It seems probable that the data obtained using the
FRET approach are more reliable as they are determined on
intact cells and membranes. The co-immunoprecipitation
experiments depend on detergent solubilization and are thus
more prone to artefacts. Nevertheless, the two approaches
can provide complementary information if taken together,
as in the present study. The overall FRET signal was higher
for both receptors when experiments were performed on
membranes. As the amount of antibodies used to analyse
the oligomerization on whole cells and on cell membranes is

form will be favoured, but Ramsey et al. [44] have recently
reported that the formation of hetero-oligomers by d and j
opioid receptors is as efficient as the formation of j receptor
homo-oligomers. These data suggest that for closely related
GPCRs (such as the two isoforms of the D
2
receptor)
hetero-oligomerization may occur as efficiently as homo-
oligomerization. It is possible that hetero-oligomerization of
D
2L
and D
2S
plays an important role in the trafficking and/
or the function of these receptors. Such observations were
made recently for opioid receptors [45]. Indeed, He et al.
[45] demonstrated that oligomerization of opioid receptors
was important for their trafficking. Interestingly, this study
also demonstrated the regulation of morphine tolerance in
animal models by receptor oligomerization, providing
evidence for possible physiological and therapeutic roles
for receptor oligomerization.
Another way to approach the physiological importance
of receptor oligomerization is to analyse the effect of
ligands on the oligomerization process. Thus, several
studies have addressed this question and the results have
shown that agonist ligands can increase, decrease or have
no effect on receptor oligomerization [1,2]. In the present
study we used two agonists (dopamine and NPA) and one
inverse agonist (raclopride), and demonstrated that none

It was also shown that these ligands did not alter the
oligomerization state of the receptors [9]. These results
suggest that the ligand-induced changes in the BRET
signal, as observed for melatonin receptors, are probably
reflecting conformational changes of these proteins rather
than changes in their oligomerization state, and that the
conformational change is unrelated to receptor activation.
Despite the lack of effect of ligands on D
2
receptor
oligomerization in the present report, the presence of
oligomers may have functional consequences. For exam-
ple, we have shown previously [18,46] that the binding of
ligands to the D
2
dopamine receptor may exhibit
co-operativity, which can be accounted for in terms of
interactions between binding sites in an oligomer.
There is also the question of the effects of G proteins on
the oligomerization process. In the insect cell system used in
this report there is little interaction between the exogenously
expressed D
2
receptor and the endogenous insect cell G
proteins [30,31]. It will be important, in the future, to
examine the oligomerization process in the presence of G
proteins, either expressed exogenously [30,31] or as a fusion
protein [47].
In conclusion, we have demonstrated that the dopamine
D

Nickolls for preparation of the baculoviruses containing epitope-tagged
D
2
receptors.
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