Tài liệu Báo cáo khoa học: Constitutive oligomerization of human D2 dopamine receptors expressed in Spodoptera frugiperda 9 (Sf9 ) and in HEK293 cells Analysis using co-immunoprecipitation and time-resolved fluorescence resonance energy transfer pdf

Eur. J. Biochem. 270, 3928–3938 (2003) Ó FEBS 2003

doi:10.1046/j.1432-1033.2003.03773.x

Constitutive oligomerization of human D2 dopamine receptors
expressed in Spodoptera frugiperda 9 (Sf9 ) and in HEK293 cells
Analysis using co-immunoprecipitation and time-resolved fluorescence resonance
energy transfer
´
´
Lucien Gazi1,†, Juan F. Lopez-Gimenez1,*†, Martin P. Rudiger2 and Philip G. Strange1
ă
1

School of Animal and Microbial Sciences, University of Reading, Reading, UK; 2GlaxoSmithKline, New Frontiers Science Park,
Harlow, UK

Human D2Long (D2L) and D2Short (D2S) dopamine receptor
isoforms were modified at their N-terminus by the addition
of a human immunodeficiency virus (HIV) or a FLAG
epitope tag. The receptors were then expressed in Spodoptera frugiperda 9 (Sf9) cells using the baculovirus system,
and their oligomerization was investigated by means of
co-immunoprecipitation and time-resolved fluorescence
resonance energy transfer (FRET). [3H]Spiperone labelled
D2 receptors in membranes prepared from Sf9 cells expressing epitope-tagged D2L or D2S receptors, with a pKd value
of  10. Co-immunoprecipitation using antibodies specific
for the tags showed constitutive homo-oligomerization of
D2L and D2S receptors in Sf9 cells. When the FLAG-tagged
D2S and HIV-tagged D2L 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
applied to whole Sf9 cells and to membranes from Sf9 cells
expressing epitope-tagged D2 receptors. In both cases, constitutive homo-oligomers were revealed for D2L and D2S
isoforms. Time-resolved FRET also revealed constitutive
homo-oligomers in HEK293 cells expressing FLAG-tagged
D2S receptors. The D2 receptor ligands dopamine,
R-(–)propylnorapomorphine, and raclopride did not affect
oligomerization of D2L and D2S in Sf9 and HEK293 cells.
Human D2 dopamine receptors can therefore form constitutive oligomers in Sf9 cells and in HEK293 cells that can be
detected by different approaches, and D2 oligomerization in
these cells is not regulated by ligands.

The G protein-coupled receptors (GPCR) represent one of
the largest families of genes in the human genome. They are
responsible for the detection of a large variety of stimuli and
control many physiological processes, including neurotransmission, cellular metabolism, secretion, differentiation, and
inflammatory and immune responses. Consequently, many
existing therapeutic agents act by either activating or
blocking GPCRs. There is now an increasing body of
evidence showing that GPCRs can form oligomers and that,
in some cases, oligomerization of the receptors is required

for their function [1,2]. The diversity of the receptors
described suggests that the phenomenon of oligomerization
may be general to the whole GPCR family, rather than
being restricted to some subgroups of receptors. Hence,
oligomerization of receptors belonging to the same family
(homo-oligomerization) or between receptors belonging to
different families (hetero-oligomerization) has been reported. These include the b2-adrenoceptor [3,4], the chemokine

receptor CCR5 [5,6], the M3 muscarinic acetylcholine
receptor [7], the M2 muscarinic cholinergic receptor [8],
the melatonin MT1 and MT2 receptors [9], the V2
vasopressin receptor [10], the 5-HT1A, 5-HT1B and
5-HT1D receptors [11], the d and j opioid receptors [12–14],
the histamine H2 receptor [15], the somatostatin sst2A and
sst3 receptors [16], the yeast Ste2 receptor [17] and the D2
dopamine receptor [18,19]. Hetero-oligomerization between
c-aminobutyric acid GABABR1 and GABABR2 receptors
was shown to be a requirement for the expression of
functional receptors at the cell surface [20]. Other examples
of hetero-oligomerization include b2-adrenoceptor and the d
or j opioid receptors [13], dopamine D2 receptor and
somatostatin sst5 receptor [21], a2-adrenoceptor and M3
muscarinic receptors [22], dopamine D2 and D3 receptors
[23]. More recently, Salim et al. described the heterooligomerization of 5HT1A receptors with a large number of
diverse receptor subtypes, including EDG1, EDG3, GPR26
and GABABR2 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:
Abbreviations: BRET, bioluminescence resonance energy transfer;
D2L, D2Long; D2S, D2Short; Eu3+, 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 Biochemistry 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)

Keywords: G protein-coupled receptors; D2 dopamine
receptor; oligomerization; Sf9 cells; HEK293 cells.


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D2 dopamine receptor oligomerization (Eur. J. Biochem. 270) 3929

suggest that oligomerization is a general phenomenon
common to all the GPCRs.
One major question that remains unanswered is the effect
of receptor ligands on the phenomenon of oligomerization.
For different GPCRs, ligands have been reported to
increase, decrease or have no effect on the oligomerization
process, which for many GPCRs, seems to be constitutive
[1,2]. These apparently contradictory reports may be
explained, at least in part, by the different methodologies
used to monitor GPCR oligomerization. Early studies used
either functional complementation of chimeric mutants or
co-immunoprecipitation of differentially epitope-tagged
receptors [1,2]. The functional complementation approach,
however, does not demonstrate a direct interaction between
the two pairs, and co-immunoprecipitation data may lead
to misinterpretation, owing to interactions resulting from
detergent dissolution of cellular membranes. Recent studies
have applied biophysical approaches, such as fluorescence
resonance energy transfer (FRET) or bioluminescence

resonance energy transfer (BRET), to describe GPCR
homo- and hetero-oligomerization [1,2]. However these
methods also have some limitations, for example changes in
energy transfer observed could be caused by conformational
changes in the proteins rather than changes in protein–
protein interaction. A combination of several methods
seems therefore important for the demonstration of GPCR
oligomerization.
The D2 dopamine receptor is a member of the D2-like
family of dopamine receptors (which comprises D2, D3
and D4 receptors). These receptors are GPCRs that
couple to G proteins of the Gi/o family. There are two
isoforms of the D2 receptor, D2Short (D2S) and D2Long
(D2L), which derive from alternative splicing of the same
mRNA [24,25]. D2L differs from D2S 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
or photolabelling, as well as inhibition of cell-surface
expression by mutant receptors [26–28]. In a recent report,
Wurch et al. [19] used a biophysical approach to analyse
the oligomerization of D2L and D2S expressed in COS-7
cells. Their study suggested a possible difference between
D2L and D2S isoforms in their ability to form oligomers,
with D2S appearing more efficient than D2L. However, the
method used by these authors, i.e. the fusion of
the receptor to a fluorescent protein, may have affected
the conformation of these receptors. Indeed agonist
dose–response curves for the stimulation of [35S]GTPcS

binding could not be performed at D2L:enhanced cyan
fluorescent protein and D2L:enhanced yellow fluorescent
protein [19].
In the present study, we used both co-immunoprecipitation and time-resolved FRET to monitor the oligomerization of the D2L and D2S receptors expressed in
Spodoptera frugiperda 9 (Sf9) and HEK293 cells. Our data
show that both D2L and D2S form constitutive homooligomers 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 D2L and D2S receptors. Finally, our data show

that oligomerization of D2L and D2S dopamine receptors is
not regulated by D2 receptor ligands.

Experimental procedures
Materials
Antisera for immunoprecipitation and immunoblotting
studies were obtained from Sigma (Gillingham, Dorset,
UK). Europium (Eu3+)- and allophycocyanin XL665labelled antibodies for time-resolved FRET were obtained
from Perkin-Elmer Life Sciences (Cambridge, UK) and CIS
bio international (West Sussex, UK), respectively. The
antibody directed against the human immunodeficiency
virus (HIV) epitope tag (ARP3035) was a monoclonal antigp120 Ig (clone 11/4C) from the National Institute for
Biological Standards and Controls (NIBSC, London, UK).
[3H]Spiperone was from Amersham International (Bucks.,
UK). All the other reagents were obtained as indicated.
Construction of recombinant baculoviruses
cDNAs encoding human D2L and D2S dopamine receptors
were subcloned into the vector TOPOÒ (Invitrogen)
between an NdeI site at the 5¢ end of the insert and an

EcoRI site at the 3¢ end of the insert, to produce the
recombinant plasmids TOPOD2L and TOPOD2S, respectively. In order to add an epitope tag to both receptors at
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
and digested with NdeI. 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 (Invitrogen), to produce the recombinant plasmids pBBHD2L and
pBBHD2S. These plasmids were then co-transfected with
Bac-N-BlueTM DNA (Invitrogen) in Sf9 insect cells, and
underwent recombination to produce recombinant baculoviruses. The same strategy was employed for the construction of recombinant baculoviruses encoding FLAG-tagged
D2 receptors, using, in this case, the following oliogonucleotide encoding the FLAG epitope sequence:
5¢-GCGGCCGCATGGACTACAAGGACGACGATGA
CAAGGATCCACTGAATCTGTCCTGG-3¢.
This
sequence contains, in addition to nucleotides corresponding
to the FLAG sequence, a NotI site and a start codon in its 5¢
end. Other modifications in comparison with HIV epitopetagged receptors are that we used a pGem-T EasyÒ
(Promega) plasmid instead of TOPO, and BaculogoldTM
DNA (Pharmingen) instead of Bac-N-BlueTM DNA. All the

viruses were purified using plaque assay purification and
amplified by serial infection of Sf9 cells.


Ó FEBS 2003

3930 L. Gazi et al. (Eur. J. Biochem. 270)

Cell culture
Sf9 insect cells were grown in suspension in TC-100 medium
supplemented with 10% FCS and 0.1% pluronic F-68Ò.
The cells were maintained at a density of 0.5–2.5 · 106
cellsỈmL)1 and passaged every 2–3 days. For infections, cells
were seeded at a density of 0.3–0.6 · 106 cellsỈmL)1 and
infected when they reached log-phase growth, i.e. at a
density of  1 · 106 cellsỈmL)1. Infections were carried out
with different multiplicities of infection (m.o.i.) of baculoviruses 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-D2S were grown in Dulbecco’s modified
Eagle’s medium supplemented with 10% FCS and in the
presence of 600 lgỈmL)1 geneticin.
Membrane preparation
Cells were collected by centrifugation (1700 g, 10 min, 4 °C)
and resuspended in 15 mL of buffer (20 mM Hepes, 6 mM
MgCl2, 1 mM EDTA, 1 mM EGTA, pH 7.4). Cell suspensions were then homogenized using an Ultra-TurraxÒ at
19 000–22 000 r.p.m. for 20 s. The homogenate was centrifuged at 1700 g for 10 min and the supernatant was
collected and centrifuged at 48 000 g for 1 h at 4 °C. The
resulting pellet was resuspended in buffer and stored at

)80 °C in aliquots of 500 lL. The protein concentration
was determined by the method of Lowry et al. [32], using
BSA as the standard.
Radioligand-binding assay
[3H]Spiperone (15–30 CiỈmmol)1, Amersham) saturation
binding experiments were performed in a final volume of
1 mL of buffer (20 mM Hepes, 6 mM MgCl2, 1 mM EDTA,
1 mM EGTA, pH 7.4) and 15–25 lg of membrane protein
per tube. Eight concentrations of radioligand were used,
ranging from  10 pM to 2 nM. The reaction was started by
the addition of membrane proteins, and was incubated for
3 h at 25 °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/Pi (140 mM NaCl, 10 mM KCl, 1.5 mM KH2PO4,
8 mM Na2HPO4). 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 spectrometry. Non-specific binding was defined in the presence
of 3 lM (+)-butaclamol. Assays were performed in
triplicate.
Co-immunoprecipitation experiments
For immunoprecipitation experiments, membrane proteins
(500 lg) were solubilized by incubation in lysis buffer
(100 mM Tris/HCl, 200 mM NaCl, 1 mM EDTA, 0.2%
SDS, 1% cholate, 1% Igepal Ca630 and protease inhibitors;
Complete, Roche) for 1 h at 4 °C on a rotating wheel.
Samples were centrifuged at 4500 g for 5 min, or at
12 000 g for 10 min, or were filtered through a 0.2-lm
filter; the supernatant was then collected and incubated with

immunoprecipitating antibody (50 lL of rat monoclonal
anti-gp120 Ig; NIBSC) for 1 h at 4 °C on a rotating wheel.
Then, 25–50 lg of protein G–sepharose (Sigma) was added
and incubation was carried out at 4 °C overnight on a
rotating wheel. Samples were then washed five times with
lysis buffer and the final pellets were resuspended in 25 lL
of loading buffer (100 mM Tris/HCl, pH 6.8, 200 mM
dithiothreitol, 4% SDS, 0.2% bromophenol blue, 20%
glycerol, 8% urea). The proteins were denatured by
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 °C for
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 mM NaCl, 50 mM
Tris/HCl, pH 7.5). Membranes were then incubated overnight at 4 °C with a single primary antibody or an antiFLAGÒ M2-peroxidase conjugate Ig (Sigma). The primary
antibodies used for immunoblotting were as follows: 3 lL
of mouse monoclonal anti-FLAGỊ M2 Ig (4 mgỈmL)1;
Sigma) or 30 lL of rat monoclonal anti-gp120 Ig. Immunoreactivity was detected with horseradish peroxidaseconjugated anti-mouse IgG (1 : 5000) for anti-FLAGÒ or
anti-rat IgG (1 : 5000) for anti-gp120 Ig. After four washes
with buffer (150 mM NaCl, 50 mM Tris/HCl, 0.1% Tween,
pH 7.5), membranes were exposed to equal volumes of

enhanced chemiluminescence (ECL) detection reagents
(Amersham) and bands were visualized after exposure of
the membranes to Hybond-ECL X-ray film (Amersham).
Binding of Eu3+ chelate-labelled antibodies
Experiments were conducted using whole Sf9 and HEK293
cells or by using membranes prepared from Sf9 cells
expressing epitope-tagged D2 receptors. Sf9 cells (500 000)
or HEK293 cells (1 · 106) were incubated with 2.5 nM
Eu3+-labelled anti-FLAG Ig (Perkin-Elmer Life Sciences),
in a total volume of 500 lL of cell culture medium (Sf9 cells)
or in 100 lL of incubation buffer (16 mM Na2HPO4, 5 mM
NaH2PO4, 150 mM 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 AnalystTM (Molecular Devices) or an
Ultra-384 (Tecan) fluorimeter configured for time-resolved
fluorescence. The Eu3+-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


Ó FEBS 2003

D2 dopamine receptor oligomerization (Eur. J. Biochem. 270) 3931

observation of signal. Membranes containing the equivalent
of 100 fmol of receptors (as labelled with [3H]spiperone)
were incubated with 2.5 nM Eu3+-labelled anti-FLAG Ig, in
a total volume of 500 lL of incubation buffer, for 1 h at
room temperature on a rotating wheel. The samples were
then centrifuged at 19 000 g for 5 min using a microcentrifuge and the pellet was washed twice with incubation buffer.
The experiments were stopped as described above for
whole cells.

Table 1. Saturation analysis of [3H]spiperone binding to membranes
prepared from Spodoptera frugiperda 9 (Sf9) cells expressing differentially epitope-tagged dopamine D2 receptors. [3H]Spiperone saturationbinding analyses were conducted as described in the Experimental
procedures. Saturation curves were fitted best by a one-binding-site
model. The data correspond to the mean results ± SEM from four to
12 experiments. A multiplicity of infection (m.o.i.) of 10 was used for
each infection with the different baculoviruses.

Preparation

Time-resolved FRET
Whole cells (500 000 Sf9; 1 · 106 HEK293) or Sf9 cell
membranes (containing the equivalent of 100 fmol of
receptors) were incubated with a mixture of Eu3+-labelled
anti-FLAG Ig and XL665-labelled anti-FLAG Ig (CIS bio
international) antibodies (2.5 nM each). The experiments
were conducted as described above for whole cells and cell
membranes. When the effects of ligands were analysed, they
were preincubated with the cells for 15 min prior to the
addition of the antibodies. The energy transfer was assessed
by exciting the Eu3+ at 320 nm and monitoring the XL665
emission at 665 nm.

Bmax (mean ± SEM,
fmolỈmg)1of protein)

pKd (mean ± SEM,
Kd, pM)

Sf9-HIV-D2L
Sf9-FLAG-D2L
Sf9-HIV-D2S
Sf9-FLAG-D2S

1445
728
1785
941

10.14
10.01
10.14
9.98

±
±
±
±

217
88
357

105

±
±
±
±

0.06
0.08
0.03
0.03

(72)
(100)
(72)
(100)

the pKd value for [3H]spiperone was  10 (data not
shown).
[3H]Spiperone saturation-binding experiments, performed
on membranes prepared from HEK293 cells expressing
FLAG-D2S, revealed a Bmax of 14.52 ± 2.99 pmolỈmg)1
and a pKd of 9.79 ± 0.05 (mean ± SEM, n ¼ 4).

Analysis of data
Data were analysed using the computer program GRAPHPAD
3
PRISM (GraphPad Software Inc.). [ H]Spiperone saturation
binding experiments were fitted to a one binding-site model
(which provided the best fit to the data) to define the Bmax

(receptor expression level) and Kd (dissociation constant for
[3H]spiperone). Statistical comparisons were performed
using an unpaired Student’s t-test or analysis of variance
(ANOVA), where appropriate. A P-value of <0.05 was
considered significant.

Results
Expression of epitope-tagged D2 receptors
in Sf9 and HEK293 cells
The expression of differentially epitope-tagged dopamine
D2 receptors in Sf9 cells was assessed by [3H]spiperone
saturation binding to membranes prepared from infected
cells. The Bmax (receptor expression level) and the Kd
(dissociation constant) values for [3H]spiperone are summarized in Table 1. The results demonstrated that when
FLAG-tagged dopamine D2S or D2L receptors were
expressed in Sf9 cells, the Bmax of [3H]spiperone was
 700 and 900 fmolỈmg)1 of protein, respectively. These
expression levels were lower than those obtained with the
HIV-tagged dopamine D2S and D2L receptors (Bmax
 1.5 pmolỈmg)1 of protein, Table 1). As expected,
[3H]spiperone showed a high affinity (pKd  10) for the
epitope-tagged dopamine D2 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 pmolỈmg)1 of protein and

Western blot and co-immunoprecipitation experiments
Western blot assays were carried out to assess the
expression of HIV- and FLAG-tagged D2L and D2S
receptors in Sf9 cells. To achieve this, mAbs directed
against gp120 (clone 11/4C) and the FLAG sequence
were used, and Fig. 1 shows the band pattern visualized
by means of secondary conjugated antibodies. Anti-gp120
Ig and anti-FLAG Ig identified bands corresponding to
proteins with a molecular mass equivalent to  43 kDa
and 85 kDa for D2L and  39 kDa and 80 kDa for D2S
(Fig. 1). No bands were detected when the antibodies
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 (D2L or
D2S), as well as a combination of both isoforms tagged
with two different epitopes (D2L and D2S), were immunoprecipitated 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 for 10 min, or by using
filtration (0.2-lm filter). Immunoblots corresponding to
D2S 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 experiments, all the samples were separated by centrifugation at
4500 g for 5 min. Figure 3 shows the results obtained for

both isoforms of the D2 receptor. Thus, for D2L, and in
contrast to the results obtained with D2S, only one band
was identified at  85 kDa (Fig. 3, lane 3). When cell
membranes co-expressing FLAG-D2S and HIV-D2L were


3932 L. Gazi et al. (Eur. J. Biochem. 270)

Ó FEBS 2003

No specific immunoreactivity was observed when
membranes from Sf9 cells, differentially expressing each
epitope-tagged receptor, were mixed before being immunoprecipitated and immunoblotted (Fig. 3, lanes 2, 4 and 6),
demonstrating that the receptors need to be expressed in the
same cell membranes to interact.
Detection of FLAG-tagged receptors by Eu3+-anti-FLAG
Ig at the cell surface and on cell membranes

Fig. 1. Expression of differentially epitope-tagged D2 dopamine receptor
isoforms, as visualized by Western blot. Membranes from Spodoptera frugiperda 9 (Sf9) cells expressing differentially epitope-tagged D2
receptor isoforms [1, FLAG-D2S; 2, human immunodeficiency virus
(HIV)-D2S; 3, FLAG-D2L; 4, HIV-D2L] were immunoblotted using
anti-FLAG Ig (upper panel) or anti-gp120 Ig (lower panel), as described 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.

Fig. 2. Co-immunoprecipitation of differentially epitope-tagged dopamine D2 receptors: effect of varying separation procedures. Solubilized
membranes from Spodoptera frugiperda 9 (Sf9) cells co-expressing
FLAG-D2S and human immunodeficiency virus (HIV)-D2S were

centrifuged at 4500 g for 5 min (lane 1) or 12 000 g for 10 min (lane 2),
or filtered through a 0.2-lm filter (lane 3). Subsequently, the samples
were immunoprecipitated with anti-gp120 Ig, resolved by SDS/PAGE
and then immunoblotted with an anti-FLAG Ig. Molecular mass
markers are indicated in kDa. The immunoblots shown are representative of at least three independent experiments. For co-expression of
the differentially tagged receptors, a multiplicity of infection (m.o.i.) of
7 was used for each baculovirus.

subjected to the same co-immunoprecipitation experiments, two bands were obtained at  42 kDa and
84 kDa (Fig. 3, lane 5).

In order to verify the specific recognition of FLAG-tagged
dopamine D2 receptors by the Eu3+-derivatized anti-FLAG
Ig, Sf9 cells expressing FLAG-tagged or HIV-tagged
receptors were probed with 2.5 nM Eu3+-anti-FLAG Ig.
Figure 4A shows the results obtained on whole Sf9 cells.
The Eu3+-anti-FLAG Ig bound specifically to the FLAGtagged receptors, as shown by the high fluorescence
observed with cells expressing FLAG-D2L and FLAG-D2S
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 D2L and D2S receptors, as shown in Fig. 4A. As
for the living cells, the Eu3+-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 countsỈs)1), obtained on whole Sf9 cells, was higher than
that obtained with membranes (4–6 · 106 vs. 3 · 106
countsỈs)1). There was no significant difference (Student’s
t-test, P > 0.05) in the fluorescence signal obtained with
dopamine D2L receptor and dopamine D2S receptor, despite
an apparently lower signal for the former isoform on whole
cells (Fig. 4A).
Eu3+-anti-FLAG Ig also bound specifically to HEK293
cells expressing FLAG-D2S receptor, as compared to nontransfected HEK293 cells (Fig. 4B). In mammalian cells the
non-specific fluorescence represented 37% of the total signal.
FRET studies of D2 dopamine receptor oligomerization
To analyse homo-oligomerization of D2 dopamine receptors, FLAG-tagged dopamine D2L and D2S receptors were
expressed in Sf9 cells using the baculovirus expression
system. A combination of Eu3+- and XL665-labelled antiFLAG Ig (2.5 nM each) was then used as energy donor and
acceptor, respectively. The time-resolved FRET was monitored by light emission at 665 nm (XL665) following
excitation at 320 nm (Eu3+). The specific FRET signal was
obtained by subtracting the fluorescence observed with
Eu3+-anti-FLAG alone from that observed with both
Eu3+-anti-FLAG and XL665-anti-FLAG Ig. FRET signal
was observed on Sf9 cells expressing FLAG-tagged dopamine D2L or D2S receptors (Fig. 5A). The specific fluorescence values obtained amounted to 32 112 ± 5871
countsỈs)1 and 40 209 ± 5670 countsỈs)1 for dopamine


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D2 dopamine receptor oligomerization (Eur. J. Biochem. 270) 3933

Fig. 3. Co-immunoprecipitation of differentially epitope-tagged dopamine D2 receptor isoforms. Solubilized membranes from Spodoptera frugiperda 9
(Sf9) cells co-expressing FLAG-D2S and human immunodeficiency virus (HIV)-D2S (lane 1), FLAG-D2L and HIV-D2L (lane 3) or FLAG-D2S and
HIV-D2L (lane 5) were immunoprecipitated with anti-gp120 Ig, the samples resolved by SDS/PAGE and then immunoblotted with anti-FLAG Ig.
Lanes 2, 4 and 6 correspond to membranes from Sf9 cells expressing epitope-tagged dopamine D2 receptors that were mixed and then submitted to
co-immunopecipitation assay. The different combinations were as follows: FLAG-D2S + HIV-D2S (lane 2), FLAG-D2L + HIV-D2L (lane 4),
FLAG-D2S + HIV-D2L (lane 6). Molecular mass markers are indicated in kDa. The immunoblots shown are representative of at least five
independent experiments. A multiplicity of infection (m.o.i.) of 7 was used for each baculovirus.

Fig. 4. The Eu3+-anti-FLAG Ig recognizes specifically the FLAG-tagged dopamine D2 receptor expressed in Spodoptera frugiperda 9 (Sf9) and
HEK293 cells. Binding of Eu3+-anti-FLAG Ig (2.5 nM) was carried out on whole Sf9 cells or on Sf9 cell membranes expressing different D2
receptor isoforms (A), or on whole HEK293 cells (control and those expressing FLAG-D2S) (B). The Eu3+ 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.

D2L and D2S receptors, respectively. These fluorescence
signals were not significantly different (Student’s t-test,
P > 0.05). When the cells were incubated with Eu3+-antiFLAG 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 D2 dopamine receptor homo-oligomerization on membranes prepared from Sf9 cells expressing
FLAG-tagged D2L or D2S dopamine receptors, using timeresolved FRET. As shown in Fig. 5B, a strong FRET signal
was observed on membranes containing either dopamine
D2L or D2S receptors. On membranes, the specific fluorescence values were 53 187 ± 14 906 countsỈs)1 and
75 943 ± 8015 countsỈs)1 for dopamine D2L and D2S
receptors, respectively. Again, when the membranes were
incubated with Eu3+-anti-FLAG Ig and XL665-anti-

FLAG Ig separately, and then mixed, no FRET was
detected (Fig. 5B, ÔmixÕ). Despite an apparently lower
FRET signal for D2L on membranes, the difference with

D2S 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 membranes, with a marked difference observed for dopamine
D2S receptor and only a minor increase for dopamine D2L
receptor.
FRET experiments were also carried out on HEK293
cells expressing FLAG-D2S receptor. The specific fluorescence value observed in mammalian cells was 13 898 ± 297
countsỈs)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).


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3934 L. Gazi et al. (Eur. J. Biochem. 270)

Fig. 5. Homo-oligomerization of D2 dopamine receptors expressed in
Spodoptera frugiperda 9 (Sf9) and HEK293 cells. (A) Intact Sf9 cells
expressing FLAG-tagged dopamine D2L receptors (black bars) or
FLAG-tagged dopamine D2S receptors (white bars) were incubated for
2 h with 2.5 nM fluorescent-labelled antibodies, as indicated on the
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 dopamine D2L receptors (black bars) or FLAG-tagged dopamine D2S
receptors (white bars) were incubated for 1 h with 2.5 nM fluorescentlabelled antibodies, as indicated on the Figure. The ÔmixÕ condition was
as described above. (C) Intact HEK293 cells expressing FLAG-D2S
receptor were incubated for 2 h with fluorescent-labelled antibodies, as
indicated on the Figure. The ÔmixÕ condition was as described above.
After washing with incubation buffer, time-resolved fluorescence resonance 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 Eu3+-antiFLAG Ig alone from that observed with both Eu3+-anti-FLAG Ig
and XL665-anti-FLAG Ig. Data shown represent the mean
results ± SEM from seven to 10 experiments.

the three ligands are reported in Fig. 6. At D2L, dopamine
and raclopride had no effect on the FRET signal (Fig. 6A).
NPA showed a tendency to decrease the FRET signal;
however, this decrease was not significant (one-way ANOVA,
P > 0.05) (Fig. 6A). For D2S expressed in Sf9 cells and
HEK293 cells, the three ligands tested had no effect on the
FRET signal observed, as shown in Fig. 6B,C.

Discussion

Lack of regulation of D2 receptor oligomerization
by the ligands selective for D2 receptor
To investigate the effect of D2 receptor ligands on the
oligomerization phenomenon, Sf9 and HEK293 cells were
preincubated with saturating concentrations of dopamine
(10)3 M), R-(–)propylnorapomorphine (NPA) (10)6 M), or
raclopride (10)4 M). A 15-min preincubation period was
applied to allow the binding of the ligand to the receptor
before addition of the antibodies. The results obtained with

In the present study we used a combination of different
approaches to demonstrate oligomerization of D2L and D2S
dopamine receptors expressed in Sf9 cells and D2S receptor
expressed in HEK293 cells. Both immunological and
fluorescence-based approaches provide evidence that the

two isoforms of D2 dopamine receptors can display
constitutive homo- and hetero-oligomerization when
expressed in Sf9 cells. In HEK293 cells expressing FLAGD2S, our fluorescence-based approach also revealed a
constitutive oligomerization for the D2S receptor, in agreement with recent data reported by Guo et al. [33].
The Sf9 cells expressed the D2 dopamine receptors with
fidelity, as shown by the high-affinity binding of [3H]spiperone (Table 1). Indeed, this system has been used widely to
express heterologous receptors, including, for example, the
M2 muscarinic receptor [34,35], the human serotonin
5-HT5A receptor [36], the b2-adrenergic receptors [37], and
the D2 dopamine receptor [38–40]. Hence, heterologous
receptors expressed in Sf9 cells showed pharmacological
properties similar to those expressed in mammalian cell
systems. Several studies have also reported the oligomerization of some GPCRs expressed in Sf9 cells [26,34,40].
Thus, the baculovirus expression system using Sf9 cells can
be used to analyse both the pharmacology and the
oligomerization of the D2 dopamine receptors. One of the
major characteristics of the baculovirus expression system is
that the insect cells tend to overexpress exogenous proteins
[36–38]. Overexpression of receptors can be a factor that


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D2 dopamine receptor oligomerization (Eur. J. Biochem. 270) 3935

Fig. 6. Effect of receptor ligands on D2 receptor oligomerization. Intact
Spodoptera frugiperda 9 (Sf9) cells expressing FLAG-tagged dopamine
D2L receptors (A) or FLAG-tagged dopamine D2S receptors (B), and
intact HEK293 cells expressing FLAG-tagged dopamine D2S receptors
(C), were preincubated for 15 min with or without ligands. Eu3+-antiFLAG Ig and XL665-anti-FLAG Ig (2.5 nM each) were then added

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].

might lead to an artefactual protein–protein interaction.
However, in the system used here, the receptor expression
level assessed by [3H]spiperone saturation binding (Table 1)
was lower for FLAG-tagged than for HIV-tagged receptors.
Despite this lower expression level, derivatized anti-FLAG
Ig specifically bound to the corresponding FLAG-tagged
receptor (Fig. 4, see below).
Western blot analysis of Sf9 membranes expressing D2
receptors demonstrated the presence of two species with
molecular masses of 39/43 kDa and 80/85 kDa, repectively,
which might correspond to monomer and dimer forms of
both D2S and D2L. Others [40] reported similar results for
the D2L dopamine receptor expressed in Sf9 cells. In the
present study we applied the co-immunoprecipitation
approach and showed that both D2L and D2S form

homo-oligomers in Sf9 cells and, when the two receptor
isoforms were co-expressed, hetero-oligomerization of D2L
and D2S 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-lm filter) and (b)
we mixed cell membranes expressing differentially epitopetagged receptors and subjected the mixture to co-immunoprecipitation. 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-immunoprecipitation approach to study D2L and D2S receptor
hetero-oligomerization. This approach also revealed some
differences between the two isoforms of D2 dopamine
receptor regarding the oligomerization process. Indeed,
co-immunoprecipitation experiments revealed two bands
(at 39 and 80 kDa) for D2S, but only one band (85 kDa) for
D2L receptors (Fig. 3, lane 3). The smaller band (39 kDa)
observed for D2S may correspond to a disruption of an
oligomeric form of the receptor. It is possible that oligomers
formed by D2L are more resistant to stringent conditions
than those formed by D2S receptors. Others have reported
differences between the two isoforms of D2 receptors
regarding the oligomerization process [19]. In fact, Wurch
et al. [19] found a significant difference between D2L and
D2S in their ability to form oligomers, when both receptors
were fused to fluorescent proteins and the receptor
oligomerization was analysed by FRET. However, differences in the approaches used (immunological or fluorescence-based assays), or the expression systems used, may
also affect the results.
We then used time-resolved FRET to analyse the
oligomerization of D2 dopamine receptors in Sf9 and
HEK293 cells. Others have previously applied a similar
method to the study of oligomerization of d-opioid receptors [14]. However, our present approach for FRET analysis
used a single antibody (anti-FLAG Ig) derivatized with
both energy donor (Eu3+) 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 GABAA receptor, and found that
these subunits assemble with a stoichiometry of (a1)2(b2)2c2,
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 FLAGtagged D2 receptors. The specificity of the signal was
confirmed by incubating the samples (cells or membranes)
with the energy donor and acceptor separately. When such
samples were mixed, no energy transfer could be monitored
(Fig. 5). This suggests that the D2 receptor oligomers preexist on cell membranes and that the energy transfer
observed is not the result of artefactual aggregation of
proteins. No significant difference in the FRET signal was


Ó FEBS 2003

3936 L. Gazi et al. (Eur. J. Biochem. 270)

observed between the two D2 receptor isoforms, despite an
apparently lower signal for D2L on membranes (Fig. 5B).
This contrasts with the clear difference we observed between
D2S and D2L 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
identical, the difference observed in the FRET signal
probably reflects a difference in the number of receptors
used. In fact, we used 100 fmol of receptors in the
membranes, which is probably higher than the number of
receptors present on 500 000 cells.
Despite the extensive research carried out in recent years,
which clearly demonstrate that oligomerization is a phenomenon common to all the GPCRs, the physiological
significance of receptor oligomers has yet to be precisely
demonstrated. We have demonstrated herein that the two
isoforms of D2 dopamine receptors can form heterooligomers when expressed in the same cell. Under
physiological conditions, one of the prerequisites for the
oligomerization is the co-localization (in the same cell) of the
different entities under study. Immunohistochemistry and
in situ hybridization approaches have shown that D2L and
D2S are co-localized in several brain areas, including the
interneurons of the prefrontal cortex and the anterior lobe
of the pituitary gland [25,42,43]. Based on the data of the
present report, it seems that both homo-oligomers and
hetero-oligomers of the D2 receptor isoforms could occur in
these brain regions. It is difficult to know which oligomeric
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 D2 receptor)

hetero-oligomerization may occur as efficiently as homooligomerization. It is possible that hetero-oligomerization of
D2L and D2S 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
of these ligands affects the oligomerization process for the
D2 receptor. These results contrast with data reported by

Wurch et al. [19], who found a concentration-dependent
increase in the energy transfer signal at D2S, expressed in
COS-7 cells, for both dopamine and NPA. This difference
could reflect the placement of the tags in the present study
at the N-terminus of the receptor, whereas Wurch et al.
[19] used C-terminally placed fluorescent probes. The
N-terminus may be less prone to undergo conformational
changes upon ligand activation. We further analysed the
effect of the D2 ligands on the FRET signal observed in

HEK293 cells expressing D2S receptor. Interestingly, no
modulation was observed, suggesting that the lack of
effect observed in Sf9 cells is not a consequence of the
expression system used. It is noteworthy that in a recent
study, it was shown that agonists, neutral antagonists and
inverse agonists all increased the BRET signal for
melatonin MT2R receptor homo-oligomers, but not for
MT1R homo-oligomers [9]. The similar effects of ligands
with different efficacies on the BRET signal for MT2R
receptors suggest a lack of correlation between the
receptor activation state and the increase in BRET signal.
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 D2 receptor
oligomerization in the present report, the presence of
oligomers may have functional consequences. For example, we have shown previously [18,46] that the binding of
ligands to the D2 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 D2 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
D2L and D2S receptors can form constitutive homo- and
hetero-oligomers in two expression systems (Sf9 and
HEK293 cells) and these are not regulated by receptor
ligands. Our study applied, for the first time, time-resolved
FRET to membranes and showed that similar results may
be obtained when the same method is applied to whole cells.
The hetero-oligomerization of D2L and D2S is of particular
interest as it may affect the function of the two isoforms of
the receptors, with possible direct consequences on the effect
of antipsychotic drugs.

Acknowledgements
This work was supported by the BBSRC and the Wellcome Trust. We
sincerely thank Molecular Devices (Winnersh Triangle, Reading) for
providing us with the AnalystTM for time-resolved FRET studies. We
also thank Dr C. Dean and C. Shotton and the NIBSC for the
preparation of anti-gp120 Ig. We are grateful to Dr J. A. Javitch
(Columbia University, New York) for kindly providing us with


Ó FEBS 2003

D2 dopamine receptor oligomerization (Eur. J. Biochem. 270) 3937

HEK293 cells expressing the FLAG-D2S receptor. We thank Dr Sarah
Nickolls for preparation of the baculoviruses containing epitope-tagged
D2 receptors.

16.

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