The receptors were then expressed in Spodop-tera frugiperda9 Sf9 cells using the baculovirus system, and their oligomerization was investigated by means of co-immunoprecipitation and tim
Trang 1Constitutive oligomerization of human D2 dopamine receptors
Analysis using co-immunoprecipitation and time-resolved fluorescence resonance energy transfer
Lucien Gazi1,†, Juan F Lo´pez-Gime´nez1,*†, Martin P Ru¨diger2and Philip G Strange1
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
Spodop-tera frugiperda9 (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
D2receptors in membranes prepared from Sf9 cells
expres-sing epitope-tagged D2Lor D2Sreceptors, with a pKdvalue
of 10 Co-immunoprecipitation using antibodies specific
for the tags showed constitutive homo-oligomerization of
D2Land D2Sreceptors 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 D2receptors In both cases, con-stitutive 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 D2Land D2Sin Sf9 and HEK293 cells Human D2dopamine receptors can therefore form consti-tutive oligomers in Sf9 cells and in HEK293 cells that can be detected by different approaches, and D2oligomerization in these cells is not regulated by ligands
Keywords: G protein-coupled receptors; D2 dopamine receptor; oligomerization; Sf9 cells; HEK293 cells
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
neurotrans-mission, 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 repor-ted 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-HT1Dreceptors [11], the d and j opioid receptors [12–14], the histamine H2receptor [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 hetero-oligomerization of 5HT1Areceptors with a large number of diverse receptor subtypes, including EDG1, EDG3, GPR26 and GABA 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: p.g.strange@rdg.ac.uk
Abbreviations: BRET, bioluminescence resonance energy transfer;
D 2L , D 2Long ; D 2S , D 2Short ; 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
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)
Trang 2suggest 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] D2Ldiffers from D2Sby 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
D2Land D2Sisoforms in their ability to form oligomers,
with D2Sappearing 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-immunoprecipita-tion and time-resolved FRET to monitor the
oligo-merization of the D2L and D2S receptors expressed in
Spodoptera frugiperda9 (Sf9) and HEK293 cells Our data
show that both D2L and D2S 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 and D receptors Finally, our data show
that oligomerization of D2Land D2Sdopamine receptors is not regulated by D2receptor ligands
Experimental procedures
Materials Antisera for immunoprecipitation and immunoblotting studies were obtained from Sigma (Gillingham, Dorset, UK) Europium (Eu3+)- and allophycocyanin XL665-labelled 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 anti-gp120 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 D2Land D2Sdopamine receptors were subcloned into the vector TOPO (Invitrogen) between an NdeIsite at the 5¢ end of the insert and an EcoRIsite at the 3¢ end of the insert, to produce the recombinant plasmids TOPOD2L and TOPOD2S, respect-ively I n 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¢ NdeIsite to the tag sequence These oligonucleotides were then annealed and digested with NdeI TOPOD2L and TOPOD2S were digested with EcoRIand 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 XhoIsite 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 XhoIand EcoRIand 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-BlueTM 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
D2 receptors, using, in this case, the following oliogo-nucleotide encoding the FLAG epitope sequence: 5¢-GCGGCCGCATGGACTACAAGGACGACGATGA
sequence contains, in addition to nucleotides corresponding
to the FLAG sequence, a NotIsite and a start codon in its 5¢ end Other modifications in comparison with HIV epitope-tagged receptors are that we used a pGem-T Easy (Promega) plasmid instead of TOPO, and BaculogoldTM
DNA (Pharmingen) instead of Bac-N-BlueTMDNA All the viruses were purified using plaque assay purification and amplified by serial infection of Sf9 cells
Trang 3Cell culture
Sf9insect cells were grown in suspension in TC-100 medium
supplemented with 10% FCS and 0.1% pluronic F-68
The cells were maintained at a density of 0.5–2.5· 106
cellsÆmL)1and passaged every 2–3 days For infections, cells
were seeded at a density of 0.3–0.6· 106 cellsÆmL)1 and
infected when they reached log-phase growth, i.e at a
density of 1 · 106cellsÆmL)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-D2Swere grown in Dulbecco’s modified
Eagle’s medium supplemented with 10% FCS and in the
presence of 600 lgÆmL)1geneticin
Membrane preparation
Cells were collected by centrifugation (1700 g, 10 min, 4C)
and resuspended in 15 mL of buffer (20 mMHepes, 6 mM
MgCl2, 1 mMEDTA, 1 mMEGTA, pH 7.4) Cell
suspen-sions were then homogenized using an Ultra-Turrax at
19 000–22 000 r.p.m for 20 s The homogenate was
cen-trifuged at 1700 g for 10 min and the supernatant was
collected and centrifuged at 48 000 g for 1 h at 4C 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 CiÆmmol)1, Amersham) saturation
binding experiments were performed in a final volume of
1 mL of buffer (20 mMHepes, 6 mMMgCl2, 1 mMEDTA,
1 mMEGTA, pH 7.4) and 15–25 lg of membrane protein
per tube Eight concentrations of radioligand were used,
ranging from 10 pMto 2 nM The reaction was started by
the addition of membrane proteins, and was incubated for
3 h at 25C 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
spectro-metry 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 4C 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 4C on a rotating wheel Then, 25–50 lg of protein G–sepharose (Sigma) was added and incubation was carried out at 4C 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 4C 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 4C for 2–4 h before being subjected to immunoblotting
Samples were resolved by SDS/PAGE (10% gel) and transferred to nitrocellulose membranes using the Biorad 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 over-night at 4C with a single primary antibody or an anti-FLAG M2-peroxidase conjugate Ig (Sigma) The primary antibodies used for immunoblotting were as follows: 3 lL
of mouse monoclonal anti-FLAG 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-FLAG or anti-rat IgG (1 : 5000) for anti-gp120 Ig After four washes with buffer (150 mMNaCl, 50 mMTris/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 D2receptors 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 mMNa2HPO4, 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
Trang 4observation of signal Membranes containing the equivalent
of 100 fmol of receptors (as labelled with [3H]spiperone)
were incubated with 2.5 nMEu3+-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
microcentri-fuge and the pellet was washed twice with incubation buffer
The experiments were stopped as described above for
whole cells
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
Analysis of data
Data were analysed using the computer programGRAPHPAD
PRISM(GraphPad Software Inc.) [3H]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 D2receptors
inSf9 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
sum-marized 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 fmolÆmg)1 of protein, respectively These
expression levels were lower than those obtained with the
HIV-tagged dopamine D2S and D2L receptors (Bmax
1.5 pmolÆmg)1 of protein, Table 1) As expected,
[3H]spiperone showed a high affinity (pKd 10) for the
epitope-tagged dopamine D2receptors 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)1of protein and
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 pmolÆmg)1 and a pKdof 9.79 ± 0.05 (mean ± SEM, n¼ 4) 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 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 for 10 min, or by using filtration (0.2-lm filter) Immunoblots corresponding to
D2Sreceptors 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
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-D and HIV-D were
Table 1 Saturation analysis of [3H]spiperone binding to membranes prepared from Spodoptera frugiperda 9 (Sf9) cells expressing differen-tially epitope-tagged dopamine D 2 receptors [ 3 H]Spiperone saturation-binding 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
B max (mean ± SEM, fmolÆmg)1of 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)
Trang 5subjected to the same co-immunoprecipitation
experi-ments, two bands were obtained at 42 kDa and
84 kDa (Fig 3, lane 5)
No specific immunoreactivity was observed when membranes from Sf9 cells, differentially expressing each epitope-tagged receptor, were mixed before being immuno-precipitated 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
In order to verify the specific recognition of FLAG-tagged dopamine D2receptors 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 FLAG-tagged receptors, as shown by the high fluorescence observed with cells expressing FLAG-D2Land 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 D2Land D2Sreceptors, 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 countsÆs)1), obtained on whole Sf9 cells, was higher than that obtained with membranes (4–6· 106 vs 3· 106
countsÆs)1) There was no significant difference (Student’s t-test, P > 0.05) in the fluorescence signal obtained with dopamine D2Lreceptor and dopamine D2Sreceptor, 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-D2Sreceptor, as compared to non-transfected HEK293 cells (Fig 4B) In mammalian cells the non-specific fluorescence represented 37% of the total signal FRET studies of D2dopamine receptor oligomerization
To analyse homo-oligomerization of D2 dopamine recep-tors, FLAG-tagged dopamine D2Land D2Sreceptors were expressed in Sf9 cells using the baculovirus expression system A combination of Eu3+- and XL665-labelled anti-FLAG Ig (2.5 nMeach) 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 (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 dop-amine D2Lor D2Sreceptors (Fig 5A) The specific fluor-escence values obtained amounted to 32 112 ± 5871 countsÆs)1 and 40 209 ± 5670 countsÆs)1 for dopamine
Fig 2 Co-immunoprecipitation of differentially epitope-tagged
dop-amine D 2 receptors: effect of varying separation procedures Solubilized
membranes from Spodoptera frugiperda 9 (Sf9) cells co-expressing
FLAG-D 2S and human immunodeficiency virus (HIV)-D 2S 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 I g Molecular mass
markers are indicated in kDa The immunoblots shown are
represen-tative 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.
Fig 1 Expression of differentially epitope-tagged D 2 dopamine receptor
isoforms, as visualized by Western blot Membranes from
Spodop-tera frugiperda 9 (Sf9) cells expressing differentially epitope-tagged D 2
receptor isoforms [1, FLAG-D 2S ; 2, human immunodeficiency virus
(HIV)-D 2S ; 3, FLAG-D 2L ; 4, HI V-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.
Trang 6D2L 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+
-anti-FLAG Ig and XL665-anti anti-FLAG Ig separately, and then
mixed before the fluorescence was monitored, no FRET
was detected (Fig 5A, mix)
We also analysed D2dopamine receptor
homo-oligome-rization on membranes prepared from Sf9 cells expressing
FLAG-tagged D2Lor D2Sdopamine receptors, using
time-resolved FRET As shown in Fig 5B, a strong FRET signal
was observed on membranes containing either dopamine
D2Lor D2Sreceptors On membranes, the specific
fluores-cence values were 53 187 ± 14 906 countsÆs)1 and
75 943 ± 8015 countsÆs)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, mix) Despite an apparently lower FRET signal for D2Lon 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 mem-branes, with a marked difference observed for dopamine
D2Sreceptor and only a minor increase for dopamine D2L receptor
FRET experiments were also carried out on HEK293 cells expressing FLAG-D2Sreceptor 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, mix)
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 2L (lane 3) or FLAG-D 2S and HIV-D 2L (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 D 2 receptors that were mixed and then submitted to co-immunopecipitation assay The different combinations were as follows: FLAG-D 2S + HI V-D 2S (lane 2), FLAG-D 2L + HI V-D 2L (lane 4), FLAG-D 2S + HI V-D 2L (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 D 2 receptor expressed in Spodoptera frugiperda 9 (Sf9) and HEK293 cells Binding of Eu3+-anti-FLAG Ig (2.5 n M ) was carried out on whole Sf9 cells or on Sf9 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.
Trang 7Lack of regulation of D2receptor oligomerization
by the ligands selective for D2receptor
To investigate the effect of D2 receptor ligands on the
oligomerization phenomenon, Sf9 and HEK293 cells were
preincubated with saturating concentrations of dopamine
(10)3M), R-(–)propylnorapomorphine (NPA) (10)6M), or
raclopride (10)4M) 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
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-wayANOVA,
P> 0.05) (Fig 6A) For D2Sexpressed 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
In the present study we used a combination of different approaches to demonstrate oligomerization of D2Land D2S dopamine receptors expressed in Sf9 cells and D2Sreceptor 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
FLAG-D2S, our fluorescence-based approach also revealed a constitutive oligomerization for the D2Sreceptor, in agree-ment with recent data reported by Guo et al [33]
The Sf9 cells expressed the D2dopamine 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
M2 muscarinic receptor [34,35], the human serotonin 5-HT5Areceptor [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 oligomeri-zation 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 D2dopamine 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
Fig 5 Homo-oligomerization of D 2 dopamine receptors expressed in Spodoptera frugiperda 9 (Sf9) and HEK293 cells (A) Intact Sf9 cells expressing FLAG-tagged dopamine D 2L receptors (black bars) or FLAG-tagged dopamine D 2S receptors (white bars) were incubated for
2 h with 2.5 n M fluorescent-labelled antibodies, as indicated on the Figure In the mix 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 mix condition was
as described above (C) Intact HEK293 cells expressing FLAG-D 2S
receptor were incubated for 2 h with fluorescent-labelled antibodies, as indicated on the Figure The mix 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.
Trang 8might 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 D2Sand 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 D and D 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 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 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
D2Lreceptors (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 D2Lare more resistant to stringent conditions than those formed by D2Sreceptors 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 D2Land
D2Sin their ability to form oligomers, when both receptors were fused to fluorescent proteins and the receptor oligomerization was analysed by FRET However, differ-ences in the approaches used (immunological or fluores-cence-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 recep-tors [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 FLAG-tagged 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 D2receptor oligomers pre-exist 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
Fig 6 Effect of receptor ligands on D 2 receptor oligomerization Intact
Spodoptera frugiperda9 (Sf9) cells expressing FLAG-tagged dopamine
D 2L receptors (A) or FLAG-tagged dopamine D 2S receptors (B), and
intact HEK293 cells expressing FLAG-tagged dopamine D 2S receptors
(C), were preincubated for 15 min with or without ligands Eu 3+
-anti-FLAG Ig and XL665-anti anti-FLAG Ig (2.5 n M 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].
Trang 9observed between the two D2receptor isoforms, despite an
apparently lower signal for D2Lon 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
pheno-menon 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
hetero-oligomers 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 situhybridization approaches have shown that D2Land
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 D2receptor 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
homo-oligomerization It is possible that hetero-oligomerization of
D2Land D2Splays 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
D 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 exam-ple, 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
D2Land 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 D2Land D2Sis 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 Analyst TM
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
Trang 10HEK293 cells expressing the FLAG-D 2S receptor We thank Dr Sarah
Nickolls for preparation of the baculoviruses containing epitope-tagged
D 2 receptors.
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