Dopamine D 4 receptor oligomerization – contribution to receptor biogenesis Kathleen Van Craenenbroeck 1 , Dasiel O. Borroto-Escuela 2 , Wilber Romero-Fernandez 2 , Kamila Skieterska 1 , Pieter Rondou 1, *, Be ´ atrice Lintermans 1 , Peter Vanhoenacker 1, , Kjell Fuxe 2 , Francisco Ciruela 3 and Guy Haegeman 1 1 Laboratory of Eukaryotic Gene Expression and Signal Transduction (LEGEST), Ghent University Hospital, UZ Gent, Belgium 2 Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden 3 Departament Patologia i Terape ` utica Experimental, Facultat de Medicina, Unitat de Farmacologia, IDIBELL-Universitat de Barcelona, L’Hospitalet de Llobregat, Barcelona, Spain Keywords bioluminescence resonance energy transfer; dimerization; dopamine D 4 receptor; G protein-coupled receptors; receptor biogenesis Correspondence K. Van Craenenbroeck, Laboratory of Eukaryotic Gene Expression and Signal Transduction (LEGEST), Ghent University- UGent, KL Ledeganckstraat 35, 9000 Gent, Belgium Fax: +32 (0)9 264 53 04 Tel: +32 (0)9 264 51 35 E-mail: kathleen.vancraenenbroeck@ugent.be Present addresses *Center for Medical Genetics Ghent (CMGG), Ghent University Hospital – UZ Gent, Belgium ActoGeniX, Technologiepark 4, Zwijnaarde, Belgium (Received 4 August 2010, revised 23 January 2011, accepted 10 February 2011) doi:10.1111/j.1742-4658.2011.08052.x Dopamine D 4 receptors (D 4 Rs) are G protein-coupled receptors that play a role in attention and cognition. In the present study, we investigated the dimerization properties of this receptor. Western blot analysis of the human D 4.2 R, D 4.4 R and D 4.7 R revealed the presence of higher molecular weight immunoreactive bands, which might indicate the formation of receptor dimers and multimers. Homo- and heterodimerization of the receptors was confirmed by co-immunoprecipitation and bioluminescence resonance energy transfer studies. Although dimerization of a large number of G protein-coupled receptors has been described, the functional impor- tance often remains to be elucidated. Folding efficiency is rate-limiting for D 4 R biogenesis and quality control in the endoplasmic reticulum plays an important role for D 4 R maturation. Co-immunoprecipitation and immuno- fluorescence microscopy studies using wild-type and a nonfunctional D 4.4 R folding mutant show that oligomerization occurs in the endoplasmic reticu- lum and that this plays a role in the biogenesis and cell surface targeting of the D 4 R. The different polymorphic repeat variants of the D 4 R display dif- ferential sensitivity to the chaperone effect. In the present study, we show that this is also reflected by bioluminescence resonance energy transfer sat- uration assays, suggesting that the polymorphic repeat variants have differ- ent relative affinities to form homo- and heterodimers. In summary, we conclude that D 4 Rs form oligomers with different affinities and that dimer- ization plays a role in receptor biogenesis. Structured digital abstract l D4.4R physically interacts with D4.2R by anti tag coimmunoprecipitation (View interaction) l D4.xR physically interacts with D4.4R by anti tag coimmunoprecipitation (View interaction) l D4.xR and D4.xR physically interact by bioluminescence resonance energy transfer (View interaction) Abbreviations BRET, bioluminescence resonance energy transfer; CHO, Chinese hamster ovary; DAPI, 4¢,6-diamidino-2-phenylindole; D n R, dopamine D n receptor; ER, endoplasmic reticulum; GPCR, G protein-coupled receptor; HRP, horseradish peroxidase; MP, milk powder; YFP, yellow fluorescent protein. FEBS Journal 278 (2011) 1333–1344 ª 2011 The Authors Journal compilation ª 2011 FEBS 1333 Introduction Although the existence of homo- and ⁄ or hetero-oligo- meric complexes of G protein-coupled receptors (GPCRs) is generally accepted, their functional impor- tance often remains to be elucidated [1]. Evidence sug- gests that dimerization or oligomerization is required for signal transduction by several GPCRs in a fashion similar to the non-GPCR receptor families, such as receptor tyrosine kinases. In addition, di- or oligomeri- zation might also be important in biosynthesis. Biosyn- thesis and transport of GPCRs towards the plasma membrane is a multistep process in which the exit of GPCRs from the endoplasmic reticulum (ER) repre- sents a crucial step in the control of their expression at the cell surface. Incompletely folded or misfolded pro- teins are retained in the ER and targeted for proteaso- mal degradation; thus, only correctly-folded proteins are allowed to leave the ER in the direction of the cell surface. The formation of oligomeric complexes repre- sents an important step in ER quality control because it may mask retention sequences or hydrophobic domains that would otherwise result in protein reten- tion in the ER. Several studies, especially of class C receptors, have shown that GPCR dimerization occurs in the ER. The heterodimerization of c-aminobutyric acid receptors GABA B1 R and GABA B2 R is an obli- gate step in the formation of a functional GABA B receptor, and masking the retention signal RXR(R) in the C-terminal of the receptor plays an important role in intracellular transport [2,3]. A role for receptor dimerization in receptor biogenesis has been reported for the b 2 -adrenergic receptor. b 2 -adrenergic receptor mutants lacking an ER-export motif or receptors fused to an ER-retention motif still dimerize with the wild-type b 2 -adrenergic receptor but trafficking to the plasma membrane is inhibited [4]. Another study with class A receptors revealed that a truncated dopamine D 3 R (D3nf, missing the third intracellular and sub- sequent regions) [5] prevents cell surface expression of wild-type dopamine D 3 Rs upon heteromerization [6]. These data strengthen the hypothesis that early hetero- merization of wild-type and mutant receptors influ- ences receptor biogenesis expression. The dopamine receptor family can be subdivided into the dopamine D 1 -like receptor subfamily compris- ing the dopamine D 1 receptor (D 1 R) and the D 5 R, which mainly couples to Gs ⁄ olf and transduces the sig- nal via activation of adenylyl cyclase, and the dopa- mine D 2 -like receptor subfamily, containing D 2 R, D 3 R and D 4 R, which couples to Gi ⁄ o, thus resulting in the inhibition of adenylyl cyclase. Dopamine receptors have also been reported to associate with themselves, as well as with other receptors, and form multireceptor networks that may have unique functional properties. Several studies suggest the dimerization of D 2 Rs [7,8] and even show a potential role for D 2 R dimerization in the pathology of schizophrenia [9]. Ligand-binding studies confirm D 2 R oligomer formation and indicate its functional relevance [10]. Searching for the func- tional role of GPCR oligomerization is more conve- nient when studying heteromerization. For example, it has been demonstrated that D 2 R and D 3 R form hete- rodimers that possess a reduced affinity for agonists, and an increased potency for coupling with adenylyl cyclase [11,12]. Another example of dopamine receptor association is the D 1 R ⁄ D 2 R heterodimer: both recep- tors are present in the striatum where they are known to colocalize and show functional synergy in the mod- ulation of striatal activity [13]. In addition, the D 1 R ⁄ D 2 R complex requires agonist binding to both receptors for G protein activation and intracellular cal- cium release. Therefore, D 1 R ⁄ D 2 R association could be important in dopamine-mediated synaptic plasticity in the brain [14,15]. Finally, D 1 R ⁄ D 2 R heterodimers are able to co-internalize upon selective activation of either receptor [16]. Furthermore, heterodimerization between the D 1 R and D 3 R in the striatum was reported to be involved in receptor internalization [17] and can also enhance the dopaminergic response in striatal neurons co-expressing both receptors [18]. Besides associating with themselves, dopamine recep- tors are also able to associate with other receptors, such as the adenosine 2A receptor [19,20] and the can- nabinoid CB1 receptor [21,22]. Furthermore, a combi- nation of bimolecular fluorescence complementation and bioluminescence energy transfer techniques was used to identify the occurrence of D 2 R–cannabinoid CB1 receptor–adenosine 2A receptor hetero-oligomers in living cells [23,24]. Recently, it was shown that D 2 R and adenosine 2A receptor can form oligomers with the metabotropic glutamate type 5 receptor and that they co-distribute in the extrasynaptic plasma mem- brane of the same dendritic spines of striatal synapses [25]. Other examples of dopamine receptor heterodi- merization occur with the somatostatin-2 [26] and -5 [27] receptors. In the present study, we investigated the human D 4 R oligomerization capability. This receptor contains a variable number of tandem repeats in its third intra- cellular loop, denoted as D 4.x R, where x is the number of repeats [28]. Most common are the D 4.2 R, D 4.4 R and D 4.7 R, and we show that all these polymorphic variants can form homo- and heterodimers. We have Dopamine D 4 receptor oligomerization K. Van Craenenbroeck et al. 1334 FEBS Journal 278 (2011) 1333–1344 ª 2011 The Authors Journal compilation ª 2011 FEBS previously reported [29,30] that the folding efficiency is rate-limiting in the biogenesis of D 4 R and the addition of chemical and pharmacological chaperones can up-regulate receptor expression and even rescue a non- functional D 4 R folding mutant (D 4.4 M345 R). This chap- erone-mediated effect involves the stabilization of newly-synthesized receptor in the ER [29,30]. In addi- tion, we also reported that different repeat variants of D 4 R display differential sensitivity to the chaperone effect, namely the D 4.2 R (with only two repeats) is less up-regulated compared to the D 4.4 (with four repeats) [29]. Accordingly, in the present study, using biolumi- nescence resonance energy transfer (BRET) saturation studies, we show that D 4.4 R homodimerization is more efficient compared to the formation of D 4.2 R homodi- mers. We also provide evidence that oligomerization of the dopamine D 4 R plays a role in receptor biogenesis and, more particularly, in trafficking of the receptor to the plasma membrane. Results Investigation of human D 4.x R oligomerization by classical biochemical approaches We have previously shown by immunoblot experiments that, in lysates from cells expressing the D 4.2 R, several bands for the receptor can be detected [29,31]. Briefly, the band with a molecular mass of 51–54 kDa (Fig.1, lane 3, open arrow) represents mature, fully processed receptor, whereas the lower band(s) with a molecular mass of 46–49 kDa represents immature, ER-retained receptor of which the N-linked glycosylation is shorter than the fully processed glycosylation tree on plasma membrane-expressed D 4 R (Fig. 1, lane 3, black arrow). Similar results are detected for the D 4.0 R, D 4.4 R and D 4.7 R (Fig. 1). Besides these expected bands of the D 4 R monomer, immunoblot analysis of HEK293T transiently transfected with pHA-D 4.x R polymorphic variants also revealed bands at a higher molecular weight, probably representing D 4 R dimers (Fig. 1, indicated by *). The specific smear at the top of the blot can represent receptor multimers, receptor forms with different post-translational modification patterns (e.g. D 4 R ubiquitination) [31] or receptor aggregates formed during the denaturation step inherent to this kind of approach. It is worth noting that similar results were obtained after receptor immunoprecipita- tion (Fig. 1, right); thus, although the concentration in the lysates of ER-retained D 4 R is comparable to the amount of plasma membrane-expressed D 4 R (Fig. 1, left) in the immunoprecipitates, the ER-retained recep- tor is more efficiently immunoprecipitated compared to the fully glycosylated D 4 R (Fig. 1, right). We tested whether D 4 R oligomerization occurred in HEK293T cells transiently expressing these receptors by performing a co-immunoprecipitation assay. First, we demonstrated heterodimerization of FLAG-D 4.4 R and HA-D 4.2 R(Fig. 2A) and homodimerization of FLAG-D 4.4 R and HA-D 4.4 R (Fig. S1). These experi- ments indicate that D 4 R dimerization already occurs in the ER because the ER-retained D 4 R (lower-molec- ular weight band) is clearly isolated. To confirm this finding, the same experiment was performed in the presence of brefeldin A, a drug that disrupts the struc- ture and function of the Golgi, preventing protein transport from the ER to the Golgi and thus transport to the plasma membrane of fully processed receptors. Fig. 1. Western blot analysis of HA-tagged D 4 R polymorphic variants. HEK293T cells were transiently transfected with pcDNA3(–), pHA- D 4.0 R, pHA-D 4.2 R, pHA-D 4.4 R and pHA-D 4.7 R. Forty-eight hours after transfection, cells were lysed; 20 lL of lysate was loaded on an 8% gel (left). Immunoprecipitation of the receptor was performed with mouse anti-HA (16B12) (2 lg) and receptor expression was analyzed by 8% SDS ⁄ PAGE and western blotting (right). Next, the blots were probed with rabbit anti-HA sera (dilution 1 : 1000) to detect receptor mono- mers and dimers. *D 4.x dimers. Open arrows indicate mature fully glycosylated D 4.x R; black arrows indicate immature ER-retained D 4.x R. The experiment was repeated three times. K. Van Craenenbroeck et al. Dopamine D 4 receptor oligomerization FEBS Journal 278 (2011) 1333–1344 ª 2011 The Authors Journal compilation ª 2011 FEBS 1335 Figure 2B shows that the ER-retained D 4 Rs form oligomers; both homodimerization of HA-D 4.4 R and FLAG-D 4.4 R and heterodimerization of HA-D 4.2 R and FLAG-D 4.4 R is demonstrated. To further investigate whether heterodimerization of D 4.2 R and D 4.4 R occurs at the plasma membrane, a specific membrane co-immunoprecipitation assay was performed. Accordingly, HEK293T cells were tran- siently transfected with plasmids encoding HA- and FLAG-tagged D 4 Rs. Forty-eight hours post-transfec- tion, the cells were first incubated with a primary anti- HA serum to target specifically plasma membrane- expressed D 4 Rs. Next, cell lysates were made and immunoprecipitation of the FLAG-D 4 R was per- formed. By immunoblotting with anti-HA and anti-FLAG sera, the presence of HA-D 4.2 and FLAG- D 4.4 R, respectively, was visualized in the immuno- precipitates, indicating D 4 R heterodimerization at the plasma membrane (Fig. 2C). To discriminate between oligomerization in living cells and experimental oligomerization that might occur during lysis, HEK293T cells independently A B C Fig. 2. Dimerization of D 4 Rs studied by co-immunoprecipitation. (A) Dimerization of the D 4 R in total cell lysates. Co-immunoprecipitation studies of FLAG-D 4.4 R and HA-D 4.2 R were performed in HEK293T cells. Immunoprecipitation (IP) was performed with mouse anti-HA (16B12) serum (2 lg). After western blot analysis, proteins were visualized with HRP-coupled anti-FLAG M2 or mouse anti-HA (16B12) sera (dilution 1 : 1000) and HRP-coupled anti-mouse (dilution 1 : 3000). Mature, fully processed, plasma membrane (PM) and immature ER- retained (ER) D 4 R are indicated by an arrow. Signal denoting the association of two heavy chains (2 · 50 kDa) (*) or one light chain (25 kDa) (**) of anti-HA sera. The same experiment was performed with cells treated for 24 h with brefeldin A (BFA) (B). (C) Dimerization of the D 4 R at the plasma membrane. Immunoprecipitation of membrane D 4 Rs was performed in HEK293T cells, transiently expressing FLAG-D 4.4 R and HA-D 4.2 R, by adding 2 lg mouse anti-HA to the living cells. Subsequently, cell lysates were made and membrane-labeled receptors immuno- precipitated, followed by denaturation and SDS ⁄ PAGE. Immunoblotting was performed with HRP-coupled anti-FLAG or mouse anti-HA (16B12) sera (dilution 1 : 1000) and HRP-coupled anti-mouse (dilution 1 : 3000). °Samples in which cells were independently transfected and mixed post-transfection. All experiments were repeated at least three times. Dopamine D 4 receptor oligomerization K. Van Craenenbroeck et al. 1336 FEBS Journal 278 (2011) 1333–1344 ª 2011 The Authors Journal compilation ª 2011 FEBS expressing FLAG-D 4.4 R or HA-D 4.2 R were mixed post-transfection and immunoprecipitated under identi- cal conditions. As shown in Figs 2C and S1 (lanes marked°), we only obtained a specific signal when both receptors were co-expressed in the same cells, indicat- ing that the human D 4 R does form dimers in living cells. Study of D 4.x R oligomerization by BRET assays In HEK293 cells, we examined the possibility of direct receptor–receptor interaction by constructing quantita- tive BRET 1 saturation curves upon co-transfection of a constant amount of receptor-Rluc construct and increasing concentrations of the receptor-yellow fluo- rescent protein (YFP) plasmids. Although the curves generated by fluorescence- and luminescence-directed measurements provide the theoretical behavior suffi- cient to predict receptor oligomerization complexes, they do not provide sufficient information on the bind- ing parameters required for proper quantitative analy- sis of receptor–receptor interactions. Accordingly, we decided to perform BRET analysis in a quantitative fashion. To complete this analysis, we conducted saturation experiments in which the amount of each receptor effectively expressed in transfected cells was monitored for each individual experiment by correlat- ing both total luminescence and total fluorescence with the number of [ 3 H]-spiperone-binding sites in permea- bilized cells. Total luminescence and total fluorescence emitted by the Rluc and YFP fusion proteins were measured after the addition of the Rluc substrate h-coelenterazine and direct excitation of the YFP at 485 nm. Correlation obtained between receptor density (the number of total binding sites) and either the lumi- nescence or fluorescence emitted by each of the recep- tor fusion molecules was linear (Fig. S2). The linear regression equations derived from these data were used to transform the luminescence and fluorescence values to the receptor number. BRET 1 signals were plotted as a function of the ratio between the receptor- YFP ⁄ receptor-Rluc numbers. As shown in Fig. 3A, significant quantitative BRET 1 signals were observed for each D 4.x R homodimer pair, confirming the co-immunoprecipitation experiments displayed in Fig. 1. In all cases, BRET 1 signals increased as a hyperbolic function of the increased concentration of the YFP fusion construct, reaching an asymptote at the highest concentrations used. How- ever, when comparing the BRET 1 signals, it is clear that, at the concentration of the acceptor correspond- ing to 50% of the maximum energy transfer (BRET 50 ), the ability to interact is not the same for the different homodimer pairs (Table S1). These results suggest that D 4.7 R homodimer pairs present the highest affinity fol- lowed by increased reduction of affinity by D 4.4 R and D 4.2 R homodimer pairs, respectively. In addition, the BRET max value for each donor–acceptor pair was found to be lower for the D 4.7 R homodimer. This could suggest that the total number of D 4.7 R homodi- mers is lower than the total number of the other homodimers under the same experimental conditions or that the relative position between Rluc and eYFP within the D 4.7 R donor–acceptor pair was less favor- able for energy transfer. The only difference between each isoform is the number of repeat sequences in the third cytoplasmic loop. The difference in BRET 50 val- ues strengthens the hypothesis that the polymorphic repeat region in the third cytoplasmic loop is involved in folding efficiency. Cells co-expressing D 2 R Rluc and D 2 R YFP were used as a positive control, in view of previous studies reporting D 2 R dimerization [7,8]. In these cells, a BRET signal was detected that was higher than that for the other D 4.x R isoforms. In addi- tion, as a negative control, we used cells co-expressing D 2L R Rluc with soluble YFP, leading to marginal sig- nals that increased linearly with increasing amounts of YFP added. When comparing the BRET 1 saturation curves obtained for the D 4.x R homo- and heterodimers (Fig. 3A,B and Table S1), different BRET 50 values were obtained, indicating that the receptors had differ- ent relative affinities for one another. However, BRET 50 values of D 4.2 R–D 4.4 R heterodimers showed similar affinity with respect to D 4.4 R homodimers. This has important implications because it suggests that, under basal conditions, D 4.2 R and D 4.4 R homo- and heterodimers have a similar probability of forming when the two receptors are heterologously expressed. Previous studies indicate that heterotrimeric formation between homologous receptors is highly probable [32]. On the other hand, it is very likely that D 4.7 R sub- types, when co-expressed with other D 4.x R variants, will preferably form D 4.7 R homodimers because the BRET 50 values for homo- versus heterodimers are significantly lower. To test whether the BRET signal was indeed a result of a specific protein–protein interaction, we performed two essential control experiments. First, we co- expressed the D 4.x R Rluc with an increasing concentra- tion of D 4.x R YFP in the presence or absence of a fixed and saturated concentration of D 4.x R. Comparing the saturation curves generated in both cases, we can con- clude that the overexpression of a fixed concentration of the receptor significantly shifted the saturation curves of the D 4.x R Rluc –D 4.x R YFP pair to the right, K. Van Craenenbroeck et al. Dopamine D 4 receptor oligomerization FEBS Journal 278 (2011) 1333–1344 ª 2011 The Authors Journal compilation ª 2011 FEBS 1337 resulting in an increase BRET 50 value (Fig. S3A shows an example for the D 4.2 R homodimer and Fig. S3B shows an example for the D 4.2 R–D 4.4 R heterodimer). Second, we overexpressed increasing concentrations of the D 4.x R in combination with the protomers of the BRET pair (constant ratio 1 : 1) and investigated whether the wild-type receptor could reduce the BRET signal. Over-expression of D 4.x R significantly reduced the BRET ratio, as shown by the BRET competition curves (Fig. S3). Finally, to examine the effect of ligands on D 4.x R oligomerization, cells co-expressing D 4.2 R Rluc and D 4.2 R YFP were incubated with 10 lm of the full D 4 R agonist WAY-100635, the D 4 R antagonist A-381393 or the inverse D 4 R agonist FAUC F41 for 10 min. Stimulation with any of these ligands failed to promote any consistent change in the BRET 1 ratio, indicating that the dimers form constitutively, and that agonist- mediated receptor activation does not affect their oligomerization state (Fig. 4). Functional consequences of D 4 R oligomerization The data from the BRET and co-immunoprecipitation experiments clearly show that oligomerization already occurs in the ER. Therefore, we hypothesized that 0.10 A 120 90 60 30 0.05 0.06 0.04 0.02 0.00 0.04 0.03 0.02 0.01 0.00 0 0.08 0.06 0.04 BRET 1 ratio BRET 1 ratio BRET 1 ratio BRET 1 ratio [% of BRET max] [Receptor-YFP]/[Receptor-Rluc] 0.02 0.00 05 D 2L R-Rluc/eYFP D 2L R-Rluc/D 2L R-YFP D 4.7 R-Rluc/D 4.7 R-YFP D 4.4 R-Rluc/D 4.4 R-YFP D 4.2 R-Rluc/D 4.2 R-YFP D 4.2 R-Rluc/YFP D 4.2 R-Rluc/D 4.2 R-YFP D 4.2 R-Rluc/D 4.7 R-YFP D 4.2 R-Rluc/D 4.4 R-YFP D 4.4 R-Rluc/D 4.7 R-YFP D 4.2 R-Rluc/D 4.7 R-YFP D 4.7 R-Rluc/eYFP D 4.7 R-Rluc/D 4.7 R-YFP D 2L R-Rluc/eYFP D 2L R-Rluc/D 2L R-eYFP D 4.7 R-Rluc/D 4.7 R-eYFP D 4.4 R-Rluc/D 4.4 R-eYFP D 4.2 R-Rluc/D 4.2 R-eYFP 10 15 20 25 [Receptor-YFP]/[Receptor-Rluc] 0 5 10 15 20 25 [Receptor-YFP]/[Receptor-Rluc] 0 5 10 15 20 25 [Receptor-YFP]/[Receptor-Rluc] 0 5 10 15 20 25 B Fig. 3. Quantitative analysis of D 4.x R homodimerization (A) and heterodimerization (B). BRET 1 donor saturation curves were performed by transfecting HEK293T cells with a constant DNA concentration of acceptor receptor-Rluc and increasing concentrations of donor receptor- YFP constructs. BRET 1 ratio, total fluorescence and total luminescence, as well as transformed values into receptor numbers, were deter- mined as described in the Materials and methods. The curves represent ten saturation curves that were fitted using a nonlinear regression equation assuming a single binding site. Dopamine D 4 receptor oligomerization K. Van Craenenbroeck et al. 1338 FEBS Journal 278 (2011) 1333–1344 ª 2011 The Authors Journal compilation ª 2011 FEBS oligomerization could play a functional role in D 4 R maturation. We have shown previously that chemical (dimethylsulfoxide, glycerol) and pharmacological (receptor ligands) chaperones can help in the folding procedure of the receptor in the ER, thereby decreas- ing receptor degradation in the proteasome and enhancing expression on the plasma membrane. The pharmacological chaperone quinpirole (a D 2 -like receptor agonist) clearly enhances the expression not only of wild-type D 4.4 R, but also of the folding mutant D 4.4 M345 R. This folding mutant D 4.4 M345 R does not meet the quality control of the ER and is routed to the proteasome for degradation [29,30]. We used this folding mutant to investigate the role of oligomeriza- tion in D 4.4 M345 R folding and subsequent plasma membrane expression. Therefore, Chinese hamster ovary (CHO) cells stably expressing the folding mutant FLAG-D 4.4 M345 R [29] were transiently co-transfected with the control vector pcDNA3 or the vector encod- ing the wild-type HA-D 4.2 R(Fig. 5). Untreated CHO FLAG-D 4.4 M345 R cells transfected with the back bone vector pcDNA3 do not show clear FLAG-D 4.4 M345 R expression (Fig. 5, left). Upon treatment of these cells with the pharmacological chaperone (quinpirole, 10 lm, 16 h), the receptor is expressed (Fig. 5, middle). This is in agreement with our previous data [29]. Note that not all cells show a clear expression of the recep- tor, which could be the result of a loss of receptor expression (e.g. silencing of the constitutive FLAG- D 4.4 M345 R gene transcription) [33]. When this CHO FLAG-D 4.4 M345 R cell line was transiently transfected with the plasmid coding for a wild-type D 4.2 R, namely pHA-D 4.2 R (Fig. 5, right, red), the mutant FLAG- D 4.4 M345 R is expressed in the CHO cell line (Fig. 5, right, green). In these experiments, receptors on the membrane were first labeled with anti-FLAG and anti- HA sera. Then cells were fixed, labeled with secondary antibodies and, finally, DNA was stained with 4¢,6-di- amidino-2-phenylindole (DAPI) to visualize the nuclei (blue). These results indicate that receptor oligomeriza- tion can have a chaperone effect. In Fig. S3, we also included the BRET data of the folding mutant FLAG D 4.4 M345 R. The results confirm the interaction between the mutant D 4.4 M345 R and both the wild-type D 4.2 R and D 4.4 R. Discussion During recent years, the number of studies reporting GPCR dimerization has increased greatly and it is now well accepted that most GPCRs are able to form homodimers. The data obtained in the present study demonstrate that the dopamine D 4 R is no exception. Western blot analysis already indicated that the differ- ent polymorphic variants of the dopamine D 4 R (D 4.2 R, D 4.4 R and D 4.7 R) are interacting with them- selves. The present study is an extra element in the research on dopamine receptor oligomerization that, until now, has focused on the dopamine receptors D 1 , D 2 ,D 3 and D 5 . By performing traditional co-immuno- precipitation assays, we confirmed both D 4 receptor homodimerization (HA-D 4.4 R and FLAG-D 4.4 R) and heterodimerization (HA-D 4.2 R and FLAG-D 4.4 R). Although some criticisms suggest that GPCR dimeriza- tion might be promoted at relatively high receptor expression levels and hence potentially be at least Fig. 4. Ligand binding effect on D 4.2 R homodimerization. Effects of 10 min of stimulation of 10 l M full agonist WAY-100635, antagonist A-381393 and the inverse agonist FAUC F41 on the BRET 1 ratios for the human D 4.2 R homodimers. Ratios are expressed as the mean ± SEM from at least six experiments. Fig. 5. Role for dimerization of D 4 R in receptor biogenesis. CHO cells, stably transfected with pFLAG-D 4.4 M345 R, were grown on coverslips in six-well plates and transiently transfected with pcDNA3 or a plasmid encoding the wild-type HA-D 4.2 R. Thirty-six hours post-transfection, cells were left untreated or treated for 16 h with the D 2 -like agonist quinpirole (Q, 10 lM). Membrane- expressed D 4.4 R was first recognized by rabbit anti-FLAG serum (for the mutant FLAG-D 4.4 M345 R) and mouse anti-HA serum (for the wild-type HA-D 4.2 R). Subsequently, cells were fixed and samples were incubated with anti-rabbit Alexa 488 (green) and anti-mouse Alexa 594 (red) and the cell nuclei were stained with DAPI (blue). The images shown are representative of the whole experiment (performed in triplicate). K. Van Craenenbroeck et al. Dopamine D 4 receptor oligomerization FEBS Journal 278 (2011) 1333–1344 ª 2011 The Authors Journal compilation ª 2011 FEBS 1339 partially an artifact of overexpression, studies of b 2 -adrenergic receptor dimerization have indicated that dimerization is unaltered over a wide range of expres- sion levels [34]. We also obtained evidence with the quantitative BRET 1 technique (keeping receptor expression near physiological level) indicating that D 4.x Rs can form homo- and heteromers, although with different degrees of efficiency and affinity, with the D 4.7 R being the least capable to form heteromers, as seen from the reduced BRET max and increased BRET 50 values compared to those obtained with the D 4.2 R and D 4.4 R protomers. From these experiments, we can conclude that the human D 4 R does form hetero- and homodimers in liv- ing cells. It is noteworthy that, for the D 2 R, the mini- mal signaling unit is suggested to be two receptors and one G protein [32]. A model developed to study D 2 R dimerization suggests that the way in which the two protomers contribute to the active complex with the G protein is not symmetric and that activation requires different conformational changes in each protomer [32]. On the other hand, the results of the present study show only a weak D 4 R oligomerization at the plasma membrane, although membrane immunofluo- rescence studies (Fig. 5), whole cell binding assays (data not shown) and functionality studies (data not shown) confirm that the D 4 R is functionally expressed on the plasma membrane. The low amount of receptor dimerization upon immunoprecipitation of the D 4 Rat the cell surface can be the result of a transient interac- tion of the D 4 R protomers at the plasma membrane, as recently suggested for several GPCRs [35,36]. The band pattern of the co-immunoprecipitation specified that receptor dimerization already occurred in the ER. We have studied D 4 R biogenesis intensively [29,30] and shown that folding of the D 4 R in the ER forms the bottle neck of receptor biogenesis. Several drugs, both chemical and pharmacological chaperones, can help to enhance folding efficiency in the ER. As soon as membrane proteins are correctly folded, they can proceed to the Golgi and to the plasma mem- brane. Slow folding of receptors in the ER enhances ER-associated degradation by the proteasome and leads to a decrease of mature receptor on the plasma membrane. Because the data from the present study indicate that receptor dimerization starts in the ER, it was tempting to assume that this process could influ- ence receptor biogenesis. We obtained data to strengthen this hypothesis from two independent experiments: (a) expression of the D 4 R folding mutant (D 4.4 M345 R) upon co-expression of wild-type receptor as visualized with a specific membrane labeling immu- nofluorescence technique and (b) BRET analysis indicating that homodimerization of D 4.7 Rs is more efficient compared to homodimerization of D 4.4 Rs and D 4.2 Rs. We do not know whether D 4 R dimerization involves the masking of a retention signal (as discussed in the Introduction) because the mutant D 4.4 M345 Risa folding mutant, although we can conclude that dimer- ization helps with D 4 R biogenesis. The acquisition of this role for D 4 R dimerization does not rule out the possibility that oligomeric D 4 Rs may have additional functions, once they are brought to the cell surface. In summary, we conclude that the D 4 R forms hetero- and homodimers. This dimerization already occurs in the ER and the quaternary structure enhances the fold- ing process of the receptor, which is linked to receptor ER export and cell surface trafficking. Materials and methods Plasmids The plasmids pFLAG-D 4.4 R, pFLAG-D 4.4 M345T R (folding mutant), pHA-D 4.0 R, pHA-D 4.2 R, pHA-D 4.4 R and pHA- D 4.7 R have been described previously [29,31] and were kindly provided by Dr H. Van Tol (University of Toronto, Ontario, Canada). An N-terminal myc (EQKLISEED) epitope-tagged D 4 R was created by PCR in three steps and plasmids for BRET were made using standard PCR and fragment replacement strategies (Fig. S4). The reading frame and PCR integrity of all cloned constructs were confirmed by DNA sequencing. Cell culture, transfection and western blot analysis Development of the CHO cell line stably transfected with the D 4 R folding mutant (CHO FLAG-D 4.4 M345T R), as well as the transient transfection method using lipofectamine (Invitrogen, Carlsbad, CA, USA), has been described previously [29]. HEK293T cells were transiently transfected with 10 lg of plasmid DNA using the poly(ethylenimine) transfection method. Therefore, cells were grown in 10 cm dishes until subconfluency in DMEM (Invitrogen) with 10% fetal bovine serum. Before transfection, the medium was refreshed with 9 mL of DMEM, supplemented with 2% fetal bovine serum. A mixture of 475 lL of serum-free medium and 25 lL(1lgÆlL )1 ) of poly(ethylenimine) (Sigma Aldrich, St Louis, MO, USA) was added to a solu- tion of 500 lL of serum-free medium containing 10 lgof DNA. Upon mixing thoroughly and incubation for 10 min at room temperature, the DNA ⁄ poly(ethylenimine) mixture was added to the cells. Six hours later, the medium was refreshed with DMEM, supplemented with 10% fetal bovine serum. Forty-eight hours after transfection, the cells were washed twice with NaCl ⁄ Pi, collected by scraping, Dopamine D 4 receptor oligomerization K. Van Craenenbroeck et al. 1340 FEBS Journal 278 (2011) 1333–1344 ª 2011 The Authors Journal compilation ª 2011 FEBS centrifuged and frozen at )70 °C for at least 1 h. RIPA ly- sates were performed as described previously [31] and loaded on a 10% SDS ⁄ PAGE gel. Proteins were transferred onto a nitrocellulose membrane (Schleicher & Schuell Bio- sciences, Dassel, Germany). Subsequently, membranes were blocked with 5% nonfat dry milk powder (MP) ⁄ NaCl ⁄ - Tris ⁄ Tween 20 (20 mm Tris ⁄ HCl, 137 mm NaCl, 0.05% Tween 20, pH 7.6) overnight, after which the membranes were incubated for 1 h with primary antibodies (dilution 1 : 1000) mouse anti-HA (clone 16B12; Covance Research Products, Berkley, CA, USA), rabbit anti-HA (Genetex, Irvine, CA, USA) or mouse anti-FLAG (clone M2; Sigma Aldrich) in 5% MP ⁄ NaCl ⁄ Tris ⁄ Tween 20. Thereafter, the blots were incubated with secondary antibodies (dilution 1 : 2000) anti-mouse or anti-rabbit, horseradish peroxidase (HRP)-linked (Amersham Biosciences, Piscataway, NI, USA) for 1 h at room temperature in 5% MP ⁄ NaCl ⁄ - Tris ⁄ Tween 20. The membranes were developed using the Western LightningÔ Cheminuluminescence Reagent Plus (PerkinElmer Life Sciences, Wellesley, MA, USA) detection system. Co-immunoprecipitation Co-immunoprecipitation studies were performed as previ- ously described [37]. In short, HEK293T cells were grown in 10 cm dishes and transiently transfected as described above. For control experiments, cells were independently transfected with plasmids encoding only one receptor type and mixed after transfection. Cells were collected and fro- zen at )70 °C after which the cells were lysed in 400 lLof RIPA buffer (50 mm Tris ⁄ HCl, pH 7.4, 100 mm NaCl, 1% Triton-X100, 0.5% sodium deoxycholate, 0.2% SDS and 1mm EDTA) for 1 h at 4 °C. A 40 lL sample of lysate was used for immediate testing of protein expression by western blot analysis. To the rest of the lysate, 2 lg of either primary antibody mouse anti-HA 16B12 or mouse anti- FLAG M2 was added. After rotation for 4 h at 4 °C, 20 lL of protein A TrisacrylÒ beads (Pierce, Rockford, IL, USA) were added and further rotated overnight at 4 °C. After washing the beads three times with RIPA buffer, the beads were denatured at 37 °C for 10 min in SDS-loading buffer (62 mm Tris ⁄ HCl, pH 6.8, 4% SDS, 20% glycerol, 0.01 bromophenol blue) + 20 mm dithiothreitol (freshly added). Proteins were separated on a 10% SDS ⁄ PAGE gel and trans- ferred onto a nitrocellulose membrane. Dilutions of 1 : 1000 mouse anti-HA 16B12 and 1 : 1000 mouse anti-FLAG M2 HRP (Sigma) were used as primary antibodies and 1 : 2000 HRP-linked anti-mouse (Amersham Biosciences) as secon- dary antibody. For isolation of the receptor at the plasma membrane, cells, transiently transfected with dopamine receptor-encod- ing plasmids, were incubated with 2 lg of antibody for 1 h at 37 °C in serum-free medium before lysis. The remainder of the protocol is similar to that described above. Immunofluorescence microscopy CHO FLAG-D 4.4 M345 R cells were seeded in wells with cov- erslips and transfected using lipofectamine. Thirty-six hours later, cells were treated with quinpirole (10 lm, 16 h). Membrane receptors were labeled by adding primary anti- body [rabbit anti-FLAG (Sigma) and mouse anti-HA 16B12], diluted 1 : 500 in serum-free medium supplemented with Hepes, to the cells for 1 h at 37 °C. After labeling, cells were fixed (150 mm NaCl, 10 mm sodium phosphate, pH 7.4, 3.7% formaldehyde) for 15 min at room tempera- ture. After washing, cells were quenched in 50 mm glycine for 15 min and washed again. Cells were permeabilized with Blotto ⁄ Triton (3% MP, 1 mm CaCl 2 , 0.1% Triton X-100, 50 mm Tris HCl, pH 7.5) for 20 min at room temperature. After washing, cells were incubated for 5 min with Blotto (3% MP, 1 mm CaCl 2 ,50mm Tris HCl, pH 7.5) and then with secondary antibody (anti-rabbit Alexa Fluor 488 and anti-mouse Alexa Fluor 594; Invitrogen) diluted 1 : 500 in Blotto for 20 min. Nuclei were visualized by incubating cells for 5 min with DAPI. Samples were analyzed using the Axiocam 200 microscope (Zeiss, Thornwood, NY, USA). BRET 1 assays HEK293T cells were transiently transfected with a constant (1 lg) amount of cDNA encoding D 4.2 R Rluc ,D 4.4 R Rluc or D 4.7 R Rluc and an increasing (0.25–5 lg) amount of D 4.2 R YFP ,D 4.4 R YFP or D 4.7 R YFP cDNA’s. Forty-eight hours after transfection, HEK293T cells were rapidly washed twice in NaCl ⁄ Pi, detached, and resuspended in the same buffer. Cell suspensions (20 lg of protein) were dis- tributed in duplicate into 96-well microplates (either black clear-bottomed or white opaque, Corning 3651 or 3600; Corning Inc., Lowell, MA, USA) for fluorescence and luminescence determinations. The total fluorescence of cell suspensions was quantified and then divided by the back- ground (mock-transfected cells) in a POLARstar Optima plate-reader (BMG Lab-Technologies, Offenburg, Ger- many) equipped with a high-energy xenon flash lamp, using a 10 nm bandwidth excitation filter at 485 nm, and a 10 nm bandwidth emission filter corresponding to 535 nm. Total bioluminescence was determined on samples incu- bated for 10 min with 5 lm h-coelenterazine (Molecular Probes, Eugene, OR, USA). The background values for total luminescence were negligible and subtracted from sample values. For BRET 1 measurement, h-coelenterazine substrate was added at a final concentration of 5 lm, and readings were performed 1 min later using the POLARstar Optima plate-reader, which allows the sequential integra- tion of the signals detected with two filter settings [485 nm (440–500 nm) and 530 nm (510–560 nm)]. The BRET ratio is defined as described previously [38]. Ligands-promoted BRET 1 was calculated by subtracting the BRET 1 ratio K. Van Craenenbroeck et al. Dopamine D 4 receptor oligomerization FEBS Journal 278 (2011) 1333–1344 ª 2011 The Authors Journal compilation ª 2011 FEBS 1341 obtained in the absence of ligand (agonist or antagonist) addition from that obtained in the presence of the ligands. BRET 1 measurements were always performed after 10 min of ligand incubation. Luminescence and fluorescence levels of several receptor- RLuc and receptor-YFP fusion proteins have been found to be linearly correlated with receptor numbers [33]. Because this correlation is an intrinsic characteristic of each fusion protein, correlation curves have to be estab- lished for each construct. HEK293 cells were transfected with increasing cDNA concentrations of the Receptor-Rluc or YFP fusion protein constructs. Maximal luminescence and fluorescence was determined as described above and correlated with relative receptor number determined in the same cells as described in the radioligand binding experi- ments (Table S2). Luminescence and fluorescence were both plotted against binding sites, and linear regression curves were generated. The standard curves generated for each single experiment were used to transform fluorescence and luminescence values into fmol of receptor. Thus, the fluorescence ⁄ luminescence ratios were transformed into (receptor-YFP) ⁄ (receptor-RLuc) ratios, which allowed us to determine accurate BRET max and BRET 50 values. To control the number of cells and also to express receptor numbers in fmolÆmg )1 of total cell protein, protein concen- tration was determined using a Bradford assay kit (Bio- Rad, Hercules, CA, USA). Data analysis All binding data were analyzed using graphpad prism, ver- sion 4.0 (GraphPad Prism, San Diego, CA, USA). BRET saturation curves were analyzed using graphpad prism. Isotherms were fitted using a nonlinear regression equation assuming a single binding site, which provided BRET max and BRET 50 values. The correlation between fluorescence or luminescence and receptor density was analyzed by a linear regression curve fitting with the same software. For statistical evaluation, and unless otherwise specified, one- way analysis of variance was used. Acknowledgements K.V.C. has a postdoctoral fellowship from FWO (Fonds voor Wetenschappelijk Onderzoek). This work was supported by grants SAF2008-01462 and Con- solider-Ingenio CSD2008-00005 from Ministerio de Ciencia e Innovacio ´ n to F.C.; by European Social Foundation and Gobierno de Catalunya FI2004- BE2006 to D.O.B E.; and from the Swedish Research Council (04X-715), Torsten and Ragnar So ¨ derberg Foundation to. KF. The authors would like to thank Hubert Van Tol for helpful discussion at the beginning of the study. 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Mol Pharmacol 67, 169 7–1 704 22 Marcellino D et al (2008) Antagonistic cannabinoid CB1 ⁄ dopamine D2 receptor interactions in striatal CB1... 81 3–8 25 Supporting information The following supplementary material is available: Doc S1 Supplementary materials and methods FEBS Journal 278 (2011) 133 3–1 344 ª 2011 The Authors Journal compilation ª 2011 FEBS 1343 Dopamine D4 receptor oligomerization K Van Craenenbroeck et al Fig S1 Homodimerization of D4. 4Rs in total cell lysates studied by co-immunoprecipitation Fig S2 Titration of donor and acceptor... BRET competition assay to study D4. 2R homodimerization and D4. 2R D4. 4R heterodimerization Fig S4 Schematic overview of the cloning strategy for pRluc-N1-myc -D4. 2R Table S1 Parameters from BRET1 saturation curves Table S2 Ligand binding properties of D4. xR constructs 1344 This supplementary material can be found in the online version of this article Please note: As a service to our authors and readers, . max] [Receptor- YFP]/ [Receptor- Rluc] 0.02 0.00 05 D 2L R-Rluc/eYFP D 2L R-Rluc/D 2L R-YFP D 4.7 R-Rluc/D 4.7 R-YFP D 4.4 R-Rluc/D 4.4 R-YFP D 4.2 R-Rluc/D 4.2 R-YFP D 4.2 R-Rluc/YFP D 4.2 R-Rluc/D 4.2 R-YFP D 4.2 R-Rluc/D 4.7 R-YFP D 4.2 R-Rluc/D 4.4 R-YFP D 4.4 R-Rluc/D 4.7 R-YFP D 4.2 R-Rluc/D 4.7 R-YFP D 4.7 R-Rluc/eYFP D 4.7 R-Rluc/D 4.7 R-YFP D 2L R-Rluc/eYFP D 2L R-Rluc/D 2L R-eYFP D 4.7 R-Rluc/D 4.7 R-eYFP D 4.4 R-Rluc/D 4.4 R-eYFP D 4.2 R-Rluc/D 4.2 R-eYFP 10 15 20 25 [Receptor- YFP]/ [Receptor- Rluc] 0 5 10 15 20 25 [Receptor- YFP]/ [Receptor- Rluc] 0 5 10 15 20 25 [Receptor- YFP]/ [Receptor- Rluc] 0 5 10 15. influ- ences receptor biogenesis expression. The dopamine receptor family can be subdivided into the dopamine D 1 -like receptor subfamily compris- ing the dopamine