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 (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 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 (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: p.g.strange@rdg.ac.uk 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 *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 April 2003, revised July 2003, accepted 30 July 2003) Keywords: G protein-coupled receptors; D2 dopamine receptor; oligomerization; Sf9 cells; HEK293 cells Ó FEBS 2003 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 (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  · 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, °C) and resuspended in 15 mL of buffer (20 mM Hepes, mM MgCl2, mM EDTA, 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 and the supernatant was collected and centrifuged at 48 000 g for h at °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 mL of buffer (20 mM Hepes, mM MgCl2, mM EDTA, 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 nM The reaction was started by the addition of membrane proteins, and was incubated for 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 mL of ice-cold NaCl/Pi (140 mM NaCl, 10 mM KCl, 1.5 mM KH2PO4, mM Na2HPO4) Filter discs were soaked in mL of Optiphase Hi-Safe (Wallac) for at least h before the radioactivity was determined by liquid scintillation spectrometry Non-specific binding was defined in the presence of 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, mM EDTA, 0.2% SDS, 1% cholate, 1% Igepal Ca630 and protease inhibitors; Complete, Roche) for h at °C on a rotating wheel Samples were centrifuged at 4500 g for 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 h at °C on a rotating wheel Then, 25–50 lg of protein G–sepharose (Sigma) was added and incubation was carried out at °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 °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 °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 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 °C with a single primary antibody or an antiFLAGÒ M2-peroxidase conjugate Ig (Sigma) The primary antibodies used for immunoblotting were as follows: 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, 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 h at room temperature on a rotating wheel The samples were then centrifuged at 19 000 g for using a microcentrifuge and the pellet was washed twice with incubation buffer The experiments were stopped as described above for whole cells Table Saturation analysis of [3H]spiperone binding to membranes prepared from Spodoptera frugiperda (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; · 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 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 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) 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 and 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 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 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 Figure 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, 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 Expression of differentially epitope-tagged D2 dopamine receptor isoforms, as visualized by Western blot Membranes from Spodoptera frugiperda (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 Co-immunoprecipitation of differentially epitope-tagged dopamine D2 receptors: effect of varying separation procedures Solubilized membranes from Spodoptera frugiperda (Sf9) cells co-expressing FLAG-D2S and human immunodeficiency virus (HIV)-D2S were centrifuged at 4500 g for (lane 1) or 12 000 g for 10 (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 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 · 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 Ó FEBS 2003 D2 dopamine receptor oligomerization (Eur J Biochem 270) 3933 Fig Co-immunoprecipitation of differentially epitope-tagged dopamine D2 receptor isoforms Solubilized membranes from Spodoptera frugiperda (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, and 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 was used for each baculovirus Fig The Eu3+-anti-FLAG Ig recognizes specifically the FLAG-tagged dopamine D2 receptor expressed in Spodoptera frugiperda (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, ƠmixÕ) Ĩ FEBS 2003 3934 L Gazi et al (Eur J Biochem 270) Fig Homo-oligomerization of D2 dopamine receptors expressed in Spodoptera frugiperda (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 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 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 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 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 Ó FEBS 2003 D2 dopamine receptor oligomerization (Eur J Biochem 270) 3935 Fig Effect of receptor ligands on D2 receptor oligomerization Intact Spodoptera frugiperda (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 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 h The measurements were performed as described in the legend to Fig 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 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receptor oligomerization Cell 108, 271–282 46 Hoare, S.R & Strange, P.G (1996) Regulation of D2 dopamine receptors by amiloride and amiloride analogs Mol Pharmacol 50, 1295–1308 47 Gazi, L., Wurch, T., Lopez-Gimenez, J.F., Pauwels, P.J & Strange, P.G (2003) Pharmacological analysis of a dopamine D (2Short): G (alphao) fusion protein expressed in Sf9 cells FEBS Lett 545, 155–160  ... 27 0) Fig Homo -oligomerization of D2 dopamine receptors expressed in Spodoptera frugiperda (Sf 9) and HEK 293 cells (A) Intact Sf9 cells expressing FLAG-tagged dopamine D2L receptors (black bars)... co-immunoprecipitation and time-resolved FRET to monitor the oligomerization of the D2L and D2S receptors expressed in Spodoptera frugiperda (Sf 9) and HEK 293 cells Our data show that both D2L and D2S form constitutive. .. FLAG-tagged dopamine D2 receptor expressed in Spodoptera frugiperda (Sf 9) and HEK 293 cells Binding of Eu3+-anti-FLAG Ig (2.5 nM) was carried out on whole Sf9 cells or on Sf9 cell membranes expressing