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Keywords:

  • bioluminescence resonance energy transfer;
  • dimerization;
  • dopamine D4 receptor;
  • G protein-coupled receptors;
  • receptor biogenesis

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Dopamine D4 receptors (D4Rs) 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 D4.2R, D4.4R and D4.7R 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 importance often remains to be elucidated. Folding efficiency is rate-limiting for D4R biogenesis and quality control in the endoplasmic reticulum plays an important role for D4R maturation. Co-immunoprecipitation and immunofluorescence microscopy studies using wild-type and a nonfunctional D4.4R folding mutant show that oligomerization occurs in the endoplasmic reticulum and that this plays a role in the biogenesis and cell surface targeting of the D4R. The different polymorphic repeat variants of the D4R display differential sensitivity to the chaperone effect. In the present study, we show that this is also reflected by bioluminescence resonance energy transfer saturation assays, suggesting that the polymorphic repeat variants have different relative affinities to form homo- and heterodimers. In summary, we conclude that D4Rs form oligomers with different affinities and that dimerization plays a role in receptor biogenesis.

Structured digital abstract


Abbreviations
BRET

bioluminescence resonance energy transfer

CHO

Chinese hamster ovary

DAPI

4′,6-diamidino-2-phenylindole

DnR

dopamine Dn receptor

ER

endoplasmic reticulum

GPCR

G protein-coupled receptor

HRP

horseradish peroxidase

MP

milk powder

YFP

yellow fluorescent protein

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Although the existence of homo- and/or hetero-oligomeric complexes of G protein-coupled receptors (GPCRs) is generally accepted, their functional importance often remains to be elucidated [1]. Evidence suggests 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 oligomerization might also be important in biosynthesis. Biosynthesis and transport of GPCRs towards the plasma membrane is a multistep process in which the exit of GPCRs from the endoplasmic reticulum (ER) represents a crucial step in the control of their expression at the cell surface. Incompletely folded or misfolded proteins are retained in the ER and targeted for proteasomal degradation; thus, only correctly-folded proteins are allowed to leave the ER in the direction of the cell surface. The formation of oligomeric complexes represents an important step in ER quality control because it may mask retention sequences or hydrophobic domains that would otherwise result in protein retention in the ER. Several studies, especially of class C receptors, have shown that GPCR dimerization occurs in the ER. The heterodimerization of γ-aminobutyric acid receptors GABAB1R and GABAB2R is an obligate step in the formation of a functional GABAB 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 β2-adrenergic receptor. β2-adrenergic receptor mutants lacking an ER-export motif or receptors fused to an ER-retention motif still dimerize with the wild-type β2-adrenergic receptor but trafficking to the plasma membrane is inhibited [4]. Another study with class A receptors revealed that a truncated dopamine D3R (D3nf, missing the third intracellular and subsequent regions) [5] prevents cell surface expression of wild-type dopamine D3Rs upon heteromerization [6]. These data strengthen the hypothesis that early heteromerization of wild-type and mutant receptors influences receptor biogenesis expression.

The dopamine receptor family can be subdivided into the dopamine D1-like receptor subfamily comprising the dopamine D1 receptor (D1R) and the D5R, which mainly couples to Gs/olf and transduces the signal via activation of adenylyl cyclase, and the dopamine D2-like receptor subfamily, containing D2R, D3R and D4R, 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 D2Rs [7,8] and even show a potential role for D2R dimerization in the pathology of schizophrenia [9]. Ligand-binding studies confirm D2R oligomer formation and indicate its functional relevance [10]. Searching for the functional role of GPCR oligomerization is more convenient when studying heteromerization. For example, it has been demonstrated that D2R and D3R form heterodimers 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 D1R/D2R heterodimer: both receptors are present in the striatum where they are known to colocalize and show functional synergy in the modulation of striatal activity [13]. In addition, the D1R/D2R complex requires agonist binding to both receptors for G protein activation and intracellular calcium release. Therefore, D1R/D2R association could be important in dopamine-mediated synaptic plasticity in the brain [14,15]. Finally, D1R/D2R heterodimers are able to co-internalize upon selective activation of either receptor [16]. Furthermore, heterodimerization between the D1R and D3R 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 receptors are also able to associate with other receptors, such as the adenosine 2A receptor [19,20] and the cannabinoid CB1 receptor [21,22]. Furthermore, a combination of bimolecular fluorescence complementation and bioluminescence energy transfer techniques was used to identify the occurrence of D2R–cannabinoid CB1 receptor–adenosine 2A receptor hetero-oligomers in living cells [23,24]. Recently, it was shown that D2R and adenosine 2A receptor can form oligomers with the metabotropic glutamate type 5 receptor and that they co-distribute in the extrasynaptic plasma membrane of the same dendritic spines of striatal synapses [25]. Other examples of dopamine receptor heterodimerization occur with the somatostatin-2 [26] and -5 [27] receptors.

In the present study, we investigated the human D4R oligomerization capability. This receptor contains a variable number of tandem repeats in its third intracellular loop, denoted as D4.xR, where x is the number of repeats [28]. Most common are the D4.2R, D4.4R and D4.7R, and we show that all these polymorphic variants can form homo- and heterodimers. We have previously reported [29,30] that the folding efficiency is rate-limiting in the biogenesis of D4R and the addition of chemical and pharmacological chaperones can up-regulate receptor expression and even rescue a nonfunctional D4R folding mutant (D4.4M345R). This chaperone-mediated effect involves the stabilization of newly-synthesized receptor in the ER [29,30]. In addition, we also reported that different repeat variants of D4R display differential sensitivity to the chaperone effect, namely the D4.2R (with only two repeats) is less up-regulated compared to the D4.4 (with four repeats) [29]. Accordingly, in the present study, using bioluminescence resonance energy transfer (BRET) saturation studies, we show that D4.4R homodimerization is more efficient compared to the formation of D4.2R homodimers. We also provide evidence that oligomerization of the dopamine D4R plays a role in receptor biogenesis and, more particularly, in trafficking of the receptor to the plasma membrane.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Investigation of human D4.xR oligomerization by classical biochemical approaches

We have previously shown by immunoblot experiments that, in lysates from cells expressing the D4.2R, 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 D4R (Fig. 1, lane 3, black arrow). Similar results are detected for the D4.0R, D4.4R and D4.7R (Fig. 1). Besides these expected bands of the D4R monomer, immunoblot analysis of HEK293T transiently transfected with pHA-D4.xR polymorphic variants also revealed bands at a higher molecular weight, probably representing D4R 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. D4R 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 immunoprecipitation (Fig. 1, right); thus, although the concentration in the lysates of ER-retained D4R is comparable to the amount of plasma membrane-expressed D4R (Fig. 1, left) in the immunoprecipitates, the ER-retained receptor is more efficiently immunoprecipitated compared to the fully glycosylated D4R (Fig. 1, right).

image

Figure 1.  Western blot analysis of HA-tagged D4R polymorphic variants. HEK293T cells were transiently transfected with pcDNA3(–), pHA-D4.0R, pHA-D4.2R, pHA-D4.4R and pHA-D4.7R. Forty-eight hours after transfection, cells were lysed; 20 μL of lysate was loaded on an 8% gel (left). Immunoprecipitation of the receptor was performed with mouse anti-HA (16B12) (2 μg) 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 monomers and dimers. *D4.x dimers. Open arrows indicate mature fully glycosylated D4.xR; black arrows indicate immature ER-retained D4.xR. The experiment was repeated three times.

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We tested whether D4R oligomerization occurred in HEK293T cells transiently expressing these receptors by performing a co-immunoprecipitation assay. First, we demonstrated heterodimerization of FLAG-D4.4R and HA-D4.2R (Fig. 2A) and homodimerization of FLAG-D4.4R and HA-D4.4R (Fig. S1). These experiments indicate that D4R dimerization already occurs in the ER because the ER-retained D4R (lower-molecular 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 structure 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. Figure 2B shows that the ER-retained D4Rs form oligomers; both homodimerization of HA-D4.4R and FLAG-D4.4R and heterodimerization of HA-D4.2R and FLAG-D4.4R is demonstrated.

image

Figure 2.  Dimerization of D4Rs studied by co-immunoprecipitation. (A) Dimerization of the D4R in total cell lysates. Co-immunoprecipitation studies of FLAG-D4.4R and HA-D4.2R were performed in HEK293T cells. Immunoprecipitation (IP) was performed with mouse anti-HA (16B12) serum (2 μg). 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) D4R 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 D4R at the plasma membrane. Immunoprecipitation of membrane D4Rs was performed in HEK293T cells, transiently expressing FLAG-D4.4R and HA-D4.2R, by adding 2 μg mouse anti-HA to the living cells. Subsequently, cell lysates were made and membrane-labeled receptors immunoprecipitated, 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.

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To further investigate whether heterodimerization of D4.2R and D4.4R occurs at the plasma membrane, a specific membrane co-immunoprecipitation assay was performed. Accordingly, HEK293T cells were transiently transfected with plasmids encoding HA- and FLAG-tagged D4Rs. Forty-eight hours post-transfection, the cells were first incubated with a primary anti-HA serum to target specifically plasma membrane-expressed D4Rs. Next, cell lysates were made and immunoprecipitation of the FLAG-D4R was performed. By immunoblotting with anti-HA and anti-FLAG sera, the presence of HA-D4.2 and FLAG-D4.4R, respectively, was visualized in the immunoprecipitates, indicating D4R 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 expressing FLAG-D4.4R or HA-D4.2R were mixed post-transfection and immunoprecipitated under identical 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, indicating that the human D4R does form dimers in living cells.

Study of D4.xR oligomerization by BRET assays

In HEK293 cells, we examined the possibility of direct receptor–receptor interaction by constructing quantitative BRET1 saturation curves upon co-transfection of a constant amount of receptor-Rluc construct and increasing concentrations of the receptor-yellow fluorescent protein (YFP) plasmids. Although the curves generated by fluorescence- and luminescence-directed measurements provide the theoretical behavior sufficient to predict receptor oligomerization complexes, they do not provide sufficient information on the binding parameters required for proper quantitative analysis 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 correlating both total luminescence and total fluorescence with the number of [3H]-spiperone-binding sites in permeabilized 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 luminescence or fluorescence emitted by each of the receptor 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. BRET1 signals were plotted as a function of the ratio between the receptor-YFP/receptor-Rluc numbers.

As shown in Fig. 3A, significant quantitative BRET1 signals were observed for each D4.xR homodimer pair, confirming the co-immunoprecipitation experiments displayed in Fig. 1. In all cases, BRET1 signals increased as a hyperbolic function of the increased concentration of the YFP fusion construct, reaching an asymptote at the highest concentrations used. However, when comparing the BRET1 signals, it is clear that, at the concentration of the acceptor corresponding to 50% of the maximum energy transfer (BRET50), the ability to interact is not the same for the different homodimer pairs (Table S1). These results suggest that D4.7R homodimer pairs present the highest affinity followed by increased reduction of affinity by D4.4R and D4.2R homodimer pairs, respectively. In addition, the BRETmax value for each donor–acceptor pair was found to be lower for the D4.7R homodimer. This could suggest that the total number of D4.7R homodimers 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 D4.7R donor–acceptor pair was less favorable for energy transfer. The only difference between each isoform is the number of repeat sequences in the third cytoplasmic loop. The difference in BRET50 values strengthens the hypothesis that the polymorphic repeat region in the third cytoplasmic loop is involved in folding efficiency. Cells co-expressing D2RRluc and D2RYFP were used as a positive control, in view of previous studies reporting D2R dimerization [7,8]. In these cells, a BRET signal was detected that was higher than that for the other D4.xR isoforms. In addition, as a negative control, we used cells co-expressing D2LRRluc with soluble YFP, leading to marginal signals that increased linearly with increasing amounts of YFP added.

image

Figure 3.  Quantitative analysis of D4.xR homodimerization (A) and heterodimerization (B). BRET1 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. BRET1 ratio, total fluorescence and total luminescence, as well as transformed values into receptor numbers, were determined 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.

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When comparing the BRET1 saturation curves obtained for the D4.xR homo- and heterodimers (Fig. 3A,B and Table S1), different BRET50 values were obtained, indicating that the receptors had different relative affinities for one another. However, BRET50 values of D4.2R–D4.4R heterodimers showed similar affinity with respect to D4.4R homodimers. This has important implications because it suggests that, under basal conditions, D4.2R and D4.4R 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 D4.7R subtypes, when co-expressed with other D4.xR variants, will preferably form D4.7R homodimers because the BRET50 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 D4.xRRluc with an increasing concentration of D4.xRYFP in the presence or absence of a fixed and saturated concentration of D4.xR. Comparing the saturation curves generated in both cases, we can conclude that the overexpression of a fixed concentration of the receptor significantly shifted the saturation curves of the D4.xRRluc–D4.xRYFP pair to the right, resulting in an increase BRET50 value (Fig. S3A shows an example for the D4.2R homodimer and Fig. S3B shows an example for the D4.2R–D4.4R heterodimer). Second, we overexpressed increasing concentrations of the D4.xR 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 D4.xR significantly reduced the BRET ratio, as shown by the BRET competition curves (Fig. S3).

Finally, to examine the effect of ligands on D4.xR oligomerization, cells co-expressing D4.2RRluc and D4.2RYFP were incubated with 10 μm of the full D4R agonist WAY-100635, the D4R antagonist A-381393 or the inverse D4R agonist FAUC F41 for 10 min. Stimulation with any of these ligands failed to promote any consistent change in the BRET1 ratio, indicating that the dimers form constitutively, and that agonist-mediated receptor activation does not affect their oligomerization state (Fig. 4).

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Figure 4.  Ligand binding effect on D4.2R homodimerization. Effects of 10 min of stimulation of 10 μm full agonist WAY-100635, antagonist A-381393 and the inverse agonist FAUC F41 on the BRET1 ratios for the human D4.2R homodimers. Ratios are expressed as the mean ± SEM from at least six experiments.

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Functional consequences of D4R oligomerization

The data from the BRET and co-immunoprecipitation experiments clearly show that oligomerization already occurs in the ER. Therefore, we hypothesized that oligomerization could play a functional role in D4R 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 decreasing receptor degradation in the proteasome and enhancing expression on the plasma membrane. The pharmacological chaperone quinpirole (a D2-like receptor agonist) clearly enhances the expression not only of wild-type D4.4R, but also of the folding mutant D4.4M345R. This folding mutant D4.4M345R 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 oligomerization in D4.4M345R folding and subsequent plasma membrane expression. Therefore, Chinese hamster ovary (CHO) cells stably expressing the folding mutant FLAG-D4.4M345R [29] were transiently co-transfected with the control vector pcDNA3 or the vector encoding the wild-type HA-D4.2R (Fig. 5). Untreated CHO FLAG-D4.4M345R cells transfected with the back bone vector pcDNA3 do not show clear FLAG-D4.4M345R expression (Fig. 5, left). Upon treatment of these cells with the pharmacological chaperone (quinpirole, 10 μm, 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 receptor, which could be the result of a loss of receptor expression (e.g. silencing of the constitutive FLAG-D4.4M345R gene transcription) [33]. When this CHO FLAG-D4.4M345R cell line was transiently transfected with the plasmid coding for a wild-type D4.2R, namely pHA-D4.2R (Fig. 5, right, red), the mutant FLAG-D4.4M345R 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-diamidino-2-phenylindole (DAPI) to visualize the nuclei (blue). These results indicate that receptor oligomerization can have a chaperone effect. In Fig. S3, we also included the BRET data of the folding mutant FLAG D4.4M345R. The results confirm the interaction between the mutant D4.4M345R and both the wild-type D4.2R and D4.4R.

image

Figure 5.  Role for dimerization of D4R in receptor biogenesis. CHO cells, stably transfected with pFLAG-D4.4M345R, were grown on coverslips in six-well plates and transiently transfected with pcDNA3 or a plasmid encoding the wild-type HA-D4.2R. Thirty-six hours post-transfection, cells were left untreated or treated for 16 h with the D2-like agonist quinpirole (Q, 10 μm). Membrane-expressed D4.4R was first recognized by rabbit anti-FLAG serum (for the mutant FLAG-D4.4M345R) and mouse anti-HA serum (for the wild-type HA-D4.2R). 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).

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

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 D4R is no exception. Western blot analysis already indicated that the different polymorphic variants of the dopamine D4R (D4.2R, D4.4R and D4.7R) are interacting with themselves. The present study is an extra element in the research on dopamine receptor oligomerization that, until now, has focused on the dopamine receptors D1, D2, D3 and D5. By performing traditional co-immunoprecipitation assays, we confirmed both D4 receptor homodimerization (HA-D4.4R and FLAG-D4.4R) and heterodimerization (HA-D4.2R and FLAG-D4.4R). Although some criticisms suggest that GPCR dimerization might be promoted at relatively high receptor expression levels and hence potentially be at least partially an artifact of overexpression, studies of β2-adrenergic receptor dimerization have indicated that dimerization is unaltered over a wide range of expression levels [34]. We also obtained evidence with the quantitative BRET1 technique (keeping receptor expression near physiological level) indicating that D4.xRs can form homo- and heteromers, although with different degrees of efficiency and affinity, with the D4.7R being the least capable to form heteromers, as seen from the reduced BRETmax and increased BRET50 values compared to those obtained with the D4.2R and D4.4R protomers.

From these experiments, we can conclude that the human D4R does form hetero- and homodimers in living cells. It is noteworthy that, for the D2R, the minimal signaling unit is suggested to be two receptors and one G protein [32]. A model developed to study D2R 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 D4R oligomerization at the plasma membrane, although membrane immunofluorescence studies (Fig. 5), whole cell binding assays (data not shown) and functionality studies (data not shown) confirm that the D4R is functionally expressed on the plasma membrane. The low amount of receptor dimerization upon immunoprecipitation of the D4R at the cell surface can be the result of a transient interaction of the D4R 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 D4R biogenesis intensively [29,30] and shown that folding of the D4R 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 membrane. 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 influence receptor biogenesis. We obtained data to strengthen this hypothesis from two independent experiments: (a) expression of the D4R folding mutant (D4.4M345R) upon co-expression of wild-type receptor as visualized with a specific membrane labeling immunofluorescence technique and (b) BRET analysis indicating that homodimerization of D4.7Rs is more efficient compared to homodimerization of D4.4Rs and D4.2Rs. We do not know whether D4R dimerization involves the masking of a retention signal (as discussed in the Introduction) because the mutant D4.4M345R is a folding mutant, although we can conclude that dimerization helps with D4R biogenesis. The acquisition of this role for D4R dimerization does not rule out the possibility that oligomeric D4Rs may have additional functions, once they are brought to the cell surface.

In summary, we conclude that the D4R forms hetero- and homodimers. This dimerization already occurs in the ER and the quaternary structure enhances the folding process of the receptor, which is linked to receptor ER export and cell surface trafficking.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Plasmids

The plasmids pFLAG-D4.4R, pFLAG-D4.4M345TR (folding mutant), pHA-D4.0R, pHA-D4.2R, pHA-D4.4R and pHA-D4.7R 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 D4R 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 D4R folding mutant (CHO FLAG-D4.4M345TR), 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 μg 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 μL of serum-free medium and 25 μL (1 μg·μL−1) of poly(ethylenimine) (Sigma Aldrich, St Louis, MO, USA) was added to a solution of 500 μL of serum-free medium containing 10 μg of 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, centrifuged and frozen at −70 °C for at least 1 h. RIPA lysates were performed as described previously [31] and loaded on a 10% SDS/PAGE gel. Proteins were transferred onto a nitrocellulose membrane (Schleicher & Schuell Biosciences, 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 Lightning™ Cheminuluminescence Reagent Plus (PerkinElmer Life Sciences, Wellesley, MA, USA) detection system.

Co-immunoprecipitation

Co-immunoprecipitation studies were performed as previously 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 frozen at −70 °C after which the cells were lysed in 400 μL of RIPA buffer (50 mm Tris/HCl, pH 7.4, 100 mm NaCl, 1% Triton-X100, 0.5% sodium deoxycholate, 0.2% SDS and 1 mm EDTA) for 1 h at 4 °C. A 40 μL sample of lysate was used for immediate testing of protein expression by western blot analysis. To the rest of the lysate, 2 μg of either primary antibody mouse anti-HA 16B12 or mouse anti-FLAG M2 was added. After rotation for 4 h at 4 °C, 20 μL of protein A Trisacryl® 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 transferred 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 secondary antibody.

For isolation of the receptor at the plasma membrane, cells, transiently transfected with dopamine receptor-encoding plasmids, were incubated with 2 μg 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-D4.4M345R cells were seeded in wells with coverslips and transfected using lipofectamine. Thirty-six hours later, cells were treated with quinpirole (10 μm, 16 h). Membrane receptors were labeled by adding primary antibody [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 temperature. 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 CaCl2, 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 CaCl2, 50 mm 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).

BRET1 assays

HEK293T cells were transiently transfected with a constant (1 μg) amount of cDNA encoding D4.2RRluc, D4.4RRluc or D4.7RRluc and an increasing (0.25–5 μg) amount of D4.2RYFP, D4.4RYFP or D4.7RYFP 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 μg of protein) were distributed 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 background (mock-transfected cells) in a POLARstar Optima plate-reader (BMG Lab-Technologies, Offenburg, Germany) 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 incubated for 10 min with 5 μmh-coelenterazine (Molecular Probes, Eugene, OR, USA). The background values for total luminescence were negligible and subtracted from sample values. For BRET1 measurement, h-coelenterazine substrate was added at a final concentration of 5 μm, and readings were performed 1 min later using the POLARstar Optima plate-reader, which allows the sequential integration 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 BRET1 was calculated by subtracting the BRET1 ratio obtained in the absence of ligand (agonist or antagonist) addition from that obtained in the presence of the ligands. BRET1 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 established 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 experiments (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 BRETmax and BRET50 values. To control the number of cells and also to express receptor numbers in fmol·mg−1 of total cell protein, protein concentration was determined using a Bradford assay kit (Bio-Rad, Hercules, CA, USA).

Data analysis

All binding data were analyzed using graphpad prism, version 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 BRETmax and BRET50 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

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

K.V.C. has a postdoctoral fellowship from FWO (Fonds voor Wetenschappelijk Onderzoek). This work was supported by grants SAF2008-01462 and Consolider-Ingenio CSD2008-00005 from Ministerio de Ciencia e Innovación 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 Söderberg Foundation to. KF. The authors would like to thank Hubert Van Tol for helpful discussion at the beginning of the study.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Doc. S1. Supplementary materials and methods.

Fig. S1. Homodimerization of D4.4Rs in total cell lysates studied by co-immunoprecipitation.

Fig. S2. Titration of donor and acceptor fusion proteins.

Fig. S3. 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.

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