Role of a Gα_i2 protein splice variant in the formation of an intracellular dopamine D₂ receptor pool

Drug-induced augmentation of D₂ receptor on plasma membrane has been reported in a cell line that expresses endogenous D₂ receptors (Ivins et al., 1991), as well as in HEK-293 cells (Boundy et al., 1995; Filtz et al., 1993), CHO cells (Itokawa et al., 1996; Zhang et al., 1994), C₆ glioma cells and Ltk^– cells (Starr et al., 1995), and Sf9 cells (Ng et al., 1997) when expressing recombinant D₂ receptors. This upregulation was not affected by the inhibition of protein synthesis (Filtz et al., 1993; Starr et al., 1995; Ng et al., 1997) and, therefore, it was proposed that this increase of receptors was owing to their recruitment from existing intracellular reservoir(s) (Ng et al., 1997). Increase in receptor numbers following drug treatments has also been observed in other transmitter-receptor systems (Creese and Sibley, 1981). The most recent work describing agonist exposure of cells that express D₂ receptors found translocation of receptor from cytoplasm to plasma membrane (Ng et al., 1997), providing direct evidence of functional receptor pool in the cytoplasm. It is well known that the interaction of plasma-membrane-bound dopamine D₂ receptor with Gα_i2 protein is fundamental for signal transmission. However, we and others have shown previously that, in contrast to Gα_i2 protein – which is localized at the cell surface, sG_i2 is an intracellular protein and not found at plasma membrane (Khan and Gutierrez, 2004; Montmayeur and Borrelli, 1994). A Proline-rich motif at the C-terminus is thought to be crucial for the intracellular translocation of this protein (Picetti and Borrelli, 2000). The sG_i2 transcript encodes a protein with a different C-terminus, in which a 24-amino acid (aa) strech is replaced by a 35-aa sequence. Like sG_i2, other G-proteins have also been identified on intracellular membranes (Audigier et al., 1988; Weiss et al., 2001), where they participate in different functions, such as protein transport (Bomsel and Mostov, 1992; Erkolani et al., 1990; Helms, 1995; Pimplikar and Simons, 1993). On the basis of the interaction between non-spliced Gα_i2 protein and dopamine D₂ receptor (Gazi et al., 2003; Senogles, 1994), and the localization of sG_i2 protein in brain dopaminergic cells (Khan and Gutierrez, 2004) where prominent expression of D₂ receptors was also found (Khan et al., 1998a), we hypothesized that the sG_i2 protein takes part in the translocation of dopamine D₂ receptors to cell surface. To our surprise, sGi2 protein not only regulated the density of D₂ receptor at cell surface but also participated in the formation of an intracellular reservoir of this receptor.

In intact BHK cells, co-expression of the long isoform of the D₂ receptor (D_2L) and the sG_i2 protein led to a 31% decrease in the number (B_max) of plasma-membrane-bound receptor (5.25±0.41 fmol per 10⁶ cells of D_2L alone versus 3.65±0.35 fmol per 10⁶ cells of D_2L with sG_i2). This loss in D_2L receptor was dependent on the amount of sG_i2 protein co-expression (Fig. 1A). As the level of expression of sG_i2 increased, more reduction in B_max of D_2L receptor was observed. However, no further reduction was observed at 30 μl of pseudovirions (not shown). The substitution of sG_i2 with Gα_i2 had no effect (Fig. 1A). This finding suggests that the receptor loss seen is specifically associated to sG_i2 protein. To exclude the possibility that sG_i2 inhibits protein expression, we checked the expression levels of both D_2L and sG_i2 proteins in these experiments by immunoblotting, by using affinity-purified antibodies [for data on specificity tests for D_2L and D_2S antibodies see Khan et al. (Khan et al., 1998a) and for sG_i2 antibodies see supplementary material Fig. S1]. In fact, we found that the D_2L receptor concentration was unchanged, whereas expression of sG_i2 protein was increased as expected (Fig. 1B), indicating that reduction of D_2L was not due to lower expression of this receptor. Next, we analyzed the binding affinity (K_d) of D₂ receptor that might have been compromised during co-expression of both proteins, but no discernable change was observed (K_d=80±15 pM without sG_i2 and 73±16 pM with sG_i2). A similar effect of sG_i2 protein on D_2L as well as D_2S receptors was also observed in other cell lines, including JEG-3 (Fig. 1C) and NG108-15 (Fig. 3B). These results not only confirm the observations made in BHK cells but also implicate that the regulation of the density of cell surface D_2S and D_2L receptors by sG_i2 protein is a common characteristic among various cell types. The use of human carcinoma JEG-3 cells that lack D₂ receptor and Gα_i2 (Guiramand et al., 1995) also showed effects similar to other cell lines. It was observed that co-expression of either D_2L or D_2S receptors with sG_i2 protein produced a loss of 32% or 35%, respectively (Fig. 1C). Furthermore, evidence from immunoblot analysis (Fig. 1B) suggests that, even though the total expression of D_2L receptors (cell-surface-bound plus intracellular) was unchanged, localization of D_2L receptor on the cell surface was reduced. Therefore, we further examined the localization of D₂ receptors in these cells by double immunofluorescence labeling (Fig. 2) and found that, when D_2S or D_2L receptors were expressed alone, they mainly localized at plasma membrane (Fig. 2A), co-expression with sG_i2, membrane localization of the receptor was noticeably reduced. This reduction in cell surface localization was probably due to accumulation of D_2S-sG_i2 protein complex seen in the intracellular membranes (Fig. 2B,C,D).

To further demonstrate that the loss of D₂ receptor activity in intact cells was due to a reduction in functional cell surface receptor population, we performed in-vivo Ca²⁺-transient studies in these cells by Ca²⁺ imaging. It is known that antagonist-mediated blockade of D₂ receptor augments intracellular Ca²⁺ flow via membrane-bound voltage-gated Ca²⁺ channels (Chronwall et al., 1995; Pauwels et al., 2001). Therefore, we used this paradigm expecting that a decrease in the cell-surface-associated D₂ receptor after co-expression of sG_i2 proportionally reduces the D₂-antagonist-mediated Ca²⁺ rise. Because of downstream D₂-signaling pathways, NG108-15 – a neuroblastoma/glioma cell line of neural origin (Pilon et al., 1994) – was also included in this study. Indeed, application of 15 μM raclopride, a D₂ receptor antagonist, produced a transient increase in the intracellular Ca²⁺ concentration, which was significantly reduced (30-35%) in cells that co-expressed sG_i2, similar to the observation made in ligand-binding experiments (D_2L 2.1±0.1 versus D_2L+sG_i2 1.53±0.1 and D_2S 2.2±0.2 versus D_2S+sG_i2 1.44±0.1; values are F/F_o ratios of Ca²⁺ changes after drug application from the baseline) (Fig. 3A-C). In control experiments, the use of neither Gα_i2 protein in place of sG_i2 nor D₁ in place of D_2S or D_2L receptor produced any such effect (Fig. 3C). In contrast to antagonist, D₂ receptor activation by agonist reduces Ca²⁺ flow (Lledo et al., 1990; Wolfe and Morris, 1999). Consistent with this, agonist treatment led to a decrease in Ca²⁺ levels in cells expressing D₂ receptor; however, co-expression of sG_i2 reduced this decrease (see supplementary material Fig. S2). These results suggest that a reduced efficiency of Ca²⁺ flow is associated with a lower number of D₂ receptors at the plasma membrane. To rule out the participation of intracellular Ca²⁺ stores in our experiments, we used 2-aminoethoxydiphenyl borate (APB), an inhibitor of the IP3 receptor. Treatment of cells with APB did not influence the Ca²⁺ transients, whereas the thapsigargin-stimulated Ca²⁺ release from intracellular stores could still be observed. This observation suggested that intracellular Ca²⁺ stores were intact but did not participate in D₂-modulated Ca²⁺ increase. In addition, the use of Ca²⁺-free medium or medium containing EGTA and CoCl₂ (blockers of membrane Ca²⁺ channels) and APB yielded the same results, further supporting this notion (see supplementary material Fig. S3).

Direct evidence of interaction between D₂ receptor and sG_i2 protein came from the co-elution of a D₂-sG_i2 complex using affinity-columns (Fig. 4A). The columns were prepared with affinity-purified specific antibodies against D_2L, D_2S (Khan et al., 1998a; Khan et al., 2001) or sG_i2 protein (see supplementary material Fig. S1 for evidence on antibody specificity). Both affinity-columns that were immobilized with antibodies against D_2L and D_2S co-eluted sG_i2 protein (Fig. 4A). Using the sG_i2 antibody affinity-column, we observed co-elution of D_2S and D_2L receptors but not of D₁ receptor (Fig. 4A). To further demonstrate the functional interaction between sG_i2 protein and active D₂ receptors, solubilized proteins from cells were incubated with antiserum against sG_i2 and presence of co-imunoprecipitated D₂ receptor was determined. We observed 24.6±4.9% co-precipitation of D₂ receptor with sG_i2 antibodies (Fig. 4B). The fact that sG_i2 antibody did not bind D₁ receptor suggests again that the D₂-sG_i2 interaction is specific.

To find out whether the complex of D₂-sG_i2 also exists in brain tissues, we used extracts from substantia nigra, a region where most neurons express high number of dopamine D_2S receptors (Khan et al., 1998a) and where we have also observed a strong immunolabeling of sG_i2 protein (Khan and Gutierrez, 2004). Immunoaffinity co-elution experiments similar to those described above confirmed the existence of a D_2S-sG_i2 protein complex in this tissue (Fig. 5).

Furthermore, we performed deletion experiments with cDNA of sG_i2 to dissect the site involved in their intracellular interaction. As indicated in Fig. 6A, deletion constructs of sG_i2 were co-expressed with D_2S receptor in BHK cells and D₂ receptor density at cell surface was determined by whole-cell binding assays. Truncated protein constructs lacking 108 bp of the extreme 3′-terminal end (sG_i2-N1, sG_i2-N2 and sG_i2-C2) lost the capability to interact (Fig. 6B); in contrast to sG_i2 and its deletion construct (sG_i2-C1) that both contain this extreme 3′-terminal, which retained this activity. These results suggest that the 36 amino acids C-terminal of sG_i2 are essential for the binding with D₂ receptor and necessary to invoke the effect of complex formation.

To further test the effect of D₂ drugs on cell surface receptor density, we treated cells that expressed both D_2S and sG_i2 protein with D₂-agonists for 30 minutes. Exposure with 10 μM of dopamine or 5 μM quinpirole led to an increase in the density of cell-surface-bound D_2S receptors (Fig. 7A). These results are in agreement with earlier reports, in which an increase in the cell-surface-bound D₂ receptor was observed after exposure to dopamine D₂ drugs (Filtz et al., 1993; Starr et al., 1995; Ng et al., 1997). Treatment of the same cells with 5 μM raclopride, a D₂ antagonist, had no effect (Fig. 7A). Next, we used the cells from the experiment described in Fig. 7A to isolate the sG_i2-D₂ complex with sG_i2-immunoaffinity columns and to determine the concentration of D_2S receptor bound to sG_i2. The combination of immunoblots and optical-density measurements showed that dopamine and quinpirole treatment reduced the amount of D₂ bound to sG_i2, whereas levels of D₂ in both raclopride-treated and untreated control cells was unchanged (Fig. 7B,C). The levels of sG_i2 under these conditions were unchanged (Fig. 7B). Our findings suggest that D₂-agonist-mediated activity has freed D₂ receptors from sG_i2-containing intracellular complexes, and that these D₂ receptors then localized to the plasma membrane. Values obtained in the experiments using D2L or D2S were not significantly different from each other.

Here, we have presented evidence that the sG_i2 protein participates in the formation of an intracellular D₂ receptor pool by specific protein-protein interaction and in the regulation of the density of these receptors at the cell membrane. To our knowledge, this constitutes the first description of how the D₂ receptor pool system functions within the cell. Our results of intracellular D_2S- and D_2L-receptor sequestration by sG_i2 protein, and their translocation to cell surface after D₂-agonist treatment explain how an increase in numbers of D₂ receptor is possible while protein synthesis is blocked. An increase in the number of D_2L receptors on the cell surface and a proportional decrease in the number of the same receptors in the cytoplasm were observed when D_2L-expressing cells were exposed to agonist (Ng et al., 1997). In rats treated with haloperidol, dopamine challenge led to upregulation of the dopamine D₂ receptor (Creese et al., 1976; Severson et al., 1984). However, the prevalence of steady-state D₂ receptor RNA was unaffected (Goss et al., 1991). It was therefore, suggested that an intracellular D₂ receptor pool is needed to upregulate D₂ during dopamine challenge. It is likely that this pool supplies receptor to plasma membrane in conditions such as those described by `the law of denervation' and when normal protein synthesis is not capable to fulfill the requirement. Furthermore, the maintenance of the D₂ receptor reservoir while protein synthesis is active (Starr et al., 1995), suggests that the machinery synthesizing dopamine D₂ receptor is not a substitute for the D₂ receptor pool. However, it remains to be explored whether, after synthesis, dopamine D₂ receptors are first localized to the intracellular reservoir before being translocated to the cell surface, or whether they are transported directly to the plasma membrane when the reservoir is saturated. It is also reasonable to argue that this sG_i2-driven reservoir might not only function as a stock room for D₂ receptor but might also control the amount of D₂-mediated signal to be transmitted inside the cell by regulating the cell surface density of this receptor.

The presence of sG_i2-D₂ receptor complex in brain tissues suggests their physiological importance in brain function. Apart from expression in neuronal intracellular compartments (Khan and Gutierrez, 2004), sG_i2 proteins were also found in abundance in axons and spines, where the most nota observation was its frequent localization to or near to the neck of spines (70% of 41 labeled spines observed) (see supplementary material Fig. S4). In addition, these proteins are often localized not at, but in close proximity to, the synapse. Given the binding capability of sG_i2 with D₂ receptor, this strategic extra-synaptic presence of sG_i2 proteins suggests that these proteins bind D₂ receptors and prevent their localization at the synapse and, as a result, this binding can interrupt the full participation of D₂ receptors in synaptic neurotransmission events. Therefore, this mechanism might fine-tune the D₂-mediated synaptic transmission, depending on the requirement of local circuits. Although we have confirmed the non-binding to other dopamine, glycine and GABA_A receptors, it remains to be determined whether sG_i2 protein binds to other receptors and synaptic proteins to participate in similar processes.

The C-terminal of the Gα_i2 protein is known to interact with the dopamine D₂ receptor in order to transmit signals at the plasma membrane (Boundy et al., 1993; Damaj et al., 1996; Senogles et al., 2004); the third cytoplasmic loop of dopamine D₂ receptor was found to be crucial for this G_i protein interaction (Malek et al., 1993). Though, in sG_i2, this terminal end is replaced, our results using deletion constructs demonstrate that 36 amino acids of the C-terminal end are essential for the intracellular interaction with the D₂ receptor. In contrast to sG_i2, proteins that interact with the third intracellular loop of the dopamine D₂ receptor, such as spinophilin (Smith et al., 1999), filamin A (Li et al., 2000; Lin et al., 2001) and heart fatty-acid-binding protein (Takeuchi and Fukunaga, 2003), have also been identified. In addition, the dimerization of D₂ receptors through interaction has also been shown (Lee et al., 2003). Thus, these evidences point to the ability of dopamine D₂ receptor to participate in protein-protein interaction with various cellular proteins and not only with sG_i2 protein, as reported here.

In the central nervous system, cell surface dopamine D₂ receptors are the major target of all effective antipsychotic drugs. Their interaction is considered to be the key event associated with improvements in patients (Kapur and Remington, 2001; Seeman and Kapur, 2000) and also in the generation of extrapyramidal side effects (Strange, 2001). Usually, antipsychotic drugs block dopamine D₂ receptor signalling; therefore, intracellular sequestration of D₂ receptor may offer an alternative in reducing the D₂-mediated signaling without blocking its function. The advantage of this approach is that intracellular sequestration of D₂ receptor might reduce inhibitory components of D₂ signaling, one of the main causes for side effects in patients using antipsychotics.

In conclusion, our results demonstrate that the sGi₂ protein and the dopamine D₂ receptor form intracellular complexes that serve as of D₂ receptor reservoir. Treatment with D₂-specific drugs break down this protein complex and free D2 receptor can translocate to cell surface. We postulate that, in contrast to a long-term strategy where protein synthesis is essential, this mechanism is a short-term cellular strategy to cope with the demand for D2 receptor while the protein synthesis machinery is unable to respond.

cDNA clone of Gα_i2 (GenBank accession number, M17528) and Gα_i3 (GenBank accession number M20713) was provided by Randall R. Reed (Johns Hopkins University, Baltimore, MD). Human D₁, D_2S and D_2L cDNA clones were from Olivier Civelli (University of California, Irvine, CA). The full-length cDNA of sG_i2 was obtained from human brain poly A⁺ RNA (Clontech) and was submitted to GenBank at the accession number AY677118.

Genes were amplified by PCR using primers containing MluI and ApaI or MluI and XbaI restriction sites at their 5′ and 3′ ends, respectively. For the deletion constructs, PCR primers were designed to amplify the sG_i2 DNA sequences containing restriction sites like those described above. The digestions were performed by incubating 3 μg of DNA with 40 units of MluI and ApaI or MluI and XbaI at 37° C for 4 hours. Digested DNA was separated on 1.4% agarose gels and recovered with Gel Extraction Kit (Qiagen). Digestion of pSinRep5 plasmid vector (Sindbis Expression System from Invitrogen) was also performed as above and was used for ligation. Gene DNA (0.5 μg) was added to 0.5 μg of digested plasmid DNA, ligated in the presence of 2 Weiss units of bacteriophage T4 DNA ligase and incubated for 1 hour at room temperature. The ligation mixture was directly transformed into a competent TOP10 One Shot cells (Invitrogen). Colonies were selected on LB agar plates containing 100 μg/ml ampicillin. After isolation of plasmid DNA with Wizard Plus Minipreps DNA Purification System (Promega) from several colonies, they were analyzed for the presence of gene by restriction digestion with MluI and ApaI and then by PCR using a combination of primers from both plasmid and gene. Usually, two to three colonies that showed correct size gene insert were then processed for large-quantity DNA isolation using Wizard Maxi-Plasmid Preparation System (Promega). The isolated DNA was quantified, aliquoted and stored at –20° C. These recombinant samples, including deletion constructs, were sequenced to confirm the DNA sequence.

The above described recombinant genes were used as template to produce recombinant RNA with the InvitroScript Cap SP6 in vitro Transcription Kit (Invitrogen). Briefly, 5 μg of recombinant DNA was linearized with 50 units of NotI. The digest was extracted once with phenol-chloroform and 0.1 volumes of 5 M ammonium acetate and 2 volumes of ethanol were added and the mix was incubated at –20°C for 1 hour. After centrifugation, DNA pellet was suspended in RNase-free water to 0.5 μg/μl. The in vitro transcription reaction was set up at room temperature by mixing 1 μg of the linearized recombinant DNA with the SP6 transcription reagents as indicated by the Invitrogen protocol. The reaction was mixed gently and incubated for 2 hours at 37°C. A typical reaction yielded 10-20 μg of RNA. The RNA product was purified with phenol-chloroform extraction, quantified with spectrophotometer, aliquoted into 10 μl samples and stored at –80°C.

2×10⁵ baby hamster kidney (BHK) cells were seeded into six-well culture plates in 2 ml of growth medium and incubated at 37° C in 5% CO₂ for 12-18 hours until 80% confluency. Cells of each well were then washed with 2 ml OPTI-MEM I reduced-serum medium at room temperature. For liposome-mediated transfection, DMRIE-C reagent from Gibco was used. RNA-lipid complexes were prepared by adding 9 μl of liposome reagent (DMRIE-C), 9 μg of recombinant RNA and 9 μg of helper RNA to 1 ml of OPTI-MEM I in polystyrene tubes and were mixed briefly by vortexing. The lipid-RNA complexes were immediately added to the washed BHK cells and incubated for 4 hours at 37°C. Following the incubation, transfection medium was replaced with complete growth medium containing αMEM medium supplemented with 2 mM L-glutamine and 5% fetal bovine serum and the cells were incubated for an additional 36 hours. During this period, recombinant RNA are packaged into pseudovirion particles and then released into the medium. The medium from the cells was collected, aliquoted into 1 ml samples and stored at –80°C.

Cell lines used in this study were obtained from the American Type Culture Collection and they were cultured at 37°C in a 5% CO₂ atmosphere. BHK cells were grown in αMEM medium supplemented with 2 mM L-glutamine and 5% fetal bovine serum. JEG-3 human carcinoma cells were cultured in Eagle's MEM with 2 mM L-glutamine and Earle's BSS containing 1.5 g/l sodium bicarbonate, 0.1 mM non-essential amino acids, 1.0 mM sodium pyruvate, and 10% fetal bovine serum. NG108-15 neuroblastoma/glioma cells were grown in DMEM with 4 mM L-glutamine without sodium pyruvate and modified to contain 4.5 g/l glucose, 1.5 g/l sodium bicarbonate, 0.005 mM pyridoxin-HCl, HAT supplement, and 10% fetal bovine serum. Cells for infection were grown to approximately 70-80% confluency in 60-mm tissue culture plates, and pseudovirions (0-30 μl) diluted to 450 μl was added to each well. After incubation at room temperature for 1 hour, 4 ml of medium were added and cells were incubated for 30-34 hours for expression of functional protein. The optimal amount of pseudovirions needed for maximal protein expression was determined by making serial dilutions of the stock.

After infection with recombinant pseudovirions and protein expression, intact cells were harvested, counted and processed for the binding assays. As described previously (Khan et al., 1998b; Khan et al., 2001), the binding of [³H]sulpiride (NEN-PerkinElmer) to 10⁵ cells was done by incubation with 0-1 nM of the radioligand for 1 hour at 24°C in a total volume of 0.5 ml. The reaction was terminated by rapid filtration through glass filters and counted for the retained radioactivity. This value was considered as total binding. Non-specific binding was determined with 1 μM of (+)-butaclamol-HCl or fluphenazine (RBI-Sigma). Specific binding was calculated by subtracting non-specific binding from total binding. B_max and K_d values were calculated with Prism program (GraphPad Software). Data are presented as the mean ± s.d. from six to eight independent experiments.

To control the amount of receptor expression, cells were homogenized in parallel experiments and used for receptor binding, similar as described for whole-cells receptor binding, to determine the total number of receptors in each condition. Variations in the total number of receptor within the same experiment and also between experiments were below 3%. Furthermore, the amount of pseudovirions and number of cells used in experiments were kept constant throughout the study.

A peptide corresponding to sG_i2 protein residues 343-354, LSGPDQHPHPSP (GenBank accession number AY677118), was synthesized and coupled to keyhole limpet hemocyanin (KLH). Peptide conjugation and rabbit immunizations were performed as described previously (Khan and Gutierrez, 2004; Khan et al., 1998a; Khan et al., 1998b). Development of immune response was monitored by ELISA using immobilized synthetic peptides. Antibodies against sG_i2 were affinity-purified on the corresponding immobilized peptide as described in detail elsewhere (Khan et al., 1993). Briefly, peptide (5 mg) was coupled to 1 g of activated thiopropyl-Sepharose 6B (Pharmacia LKB). One milliliter of antiserum diluted fivefold in 10 mM phosphate-buffered saline (PBS) (10 mM Na₂PO₄, 0.14 M NaCl, 0.01 M KCl, pH 7.4) was circulated through the peptide column. After washing, the antibody was eluted with 50 mM glycine-HCl pH 2.3, and collected in 1-ml fractions. OD₂₈₀ was determined for each fraction and fractions containing antibodies were pooled and dialyzed in PBS. Antibodies were stored as 50-μl aliquots at –20°C. Specificity of affinity-purified antibody was then determined (Khan and Gutierrez, 2004) (supplementary material Fig. S1). The isoform-specific D_2S and D_2L antibodies had been prepared earlier by us and their specificity have already been demonstrated (Khan et al., 1998a; Khan et al., 2001).

Immunoblots were done as described previously (Khan et al., 1998a; Khan et al., 1993). Solubilization of proteins from harvested intact cells was done with solubilization buffer provided in Seize X Mammalian Immunoprecipitation kit (Pierce). These solubilized proteins were separated by 10% SDS-PAGE and transblotted to nitrocellulose membranes. Membranes containing proteins were incubated with 5 μg/ml antibodies to sG_i2, D_2S or D_2L, followed by incubation with anti-rabbit IgG-HRP (1:2000; Amersham). Bands were visualized using an ECL kit (Amersham).

The concentration of bands in blot experiment (Fig. 7B) was obtained by OD measurements (Fig. 7C). These results suggest that approximately 75% of D₂ receptor was uncoupled from the D2-sGi2 complex after drug treatment. This calculation is based on the assumption that D₂ receptor population bound to sGi2-D2 complex is 100% (Fig. 7B, C5) under normal conditions when D₂ receptor and sGi2 are co-expressed. However, when comparing data of blots with binding experiments, this 100% of D₂ receptor population of the D2-sGi2 complex represent approximately 30% in binding experiments (see Fig. 1). Therefore, normalizing the results of both blots and binding experiments to the same level (30%), the 75% value of blots comes down to 22%. A slightly lower value in the binding experiments might reflect the population of unbound receptor still in transit.

After infection with recombinant pseudovirions and after protein expression, cells grown on Flask-style glass slides (Nunc) were fixed with 4% paraformaldehyde and 0.2% glutaraldehyde for 10 minutes and permeabilized with 0.3% Triton X-100. Immunofluorescence staining of cells was performed as described earlier (Khan et al., 1998a; Khan et al., 1998b; Khan et al., 2001; Khan and Gutierrez, 2004; Lopez-Aranda et al., 2006). Briefly, after incubation with sG_i2 antibody (1:500), cells were incubated with FITC (green) coupled to anti-rabbit Fab2 fragment (1:100; Jackson), followed by incubation with D_2S antibody (1:200) and Cy3-conjugated secondary antibody(red) (1:200; Jackson). Images were taken with Zeiss confocal microscope.

Cells infected with recombinant pseudovirions were grown on glass coverslips and incubated with 2 μM fluo-4-AM (fluo-4 acetoxymethyl ester from Molecular Probes) for 15-30 minutes. The fluorescence change in cells after application of 15 μM raclopride was measured with a Zeiss LSM 410 confocal laser scanning microscope system as described previously (Koulen et al., 1999). Images were acquired every 500 mseconds. Changes in fluorescence intensity were calculated by dividing the fluorescence intensity during drug application (F) by the average baseline fluorescence intensity (F₀). Non-stimulus-related spontaneous changes in fluorescence were 1-3%. Data are presented as the mean ± s.d. of four independent experiments.

Protein solubilization, antibody immobilized affinity column preparation and protein elution was performed as described in Seize X Mammalian Immunoprecipitation Kit (Pierce). In brief, 0.8 ml of gel-immobilized protein G was washed with PBS buffer (10 mM Na₂PO₄, 0.14 M NaCl, 0.01 M KCl, pH 7.4) by centrifugation and incubated with 2 mg of affinity-purified antibodies for 1 hour. The mixture was then transferred to spin cups and centrifuged. Flow-through was collected to determine the amount of antibody bound to resin. Approximately 80-90% of antibodies were bound; 1.3 mg of DSS crosslinker in DMSO was added to the resin and gently mixed by inversion for 1 hour. Resin was washed with Tris buffer (25 mM Tris, 0.15 M NaCl, pH 7.2) in spin cups and stored in 1 ml of PBS buffer containing 0.01% sodium azide.

After infection with recombinant pseudovirions, whole cells (2×10⁶) were harvested and lysed with 2 ml of M-PER Mammalian Protein Extraction reagent (Pierce) for 10 minutes. After removing cell debris by centrifugation, clear supernatant was diluted 1:1 with PBS buffer and added to spin columns with resin bound to antibody. The samples were incubated for 2 hour at 4°C and eluted with 400 μl of elution buffer (pH 2.8). Immunoaffinity eluted proteins were then analyzed by immunoblots.

For the brain tissues, membrane was prepared as described earlier (Khan et al., 1998a; Khan et al., 1998b; Khan et al., 2001; Khan et al., 1993). The prepared membrane was then used for the protein solubilization, binding with affinity-column and elution as explained above.

Cells were infected with recombinant pseudovirions, harvested and their proteins were solubilized with 1% digitonin (Khan et al., 1998a; Khan et al., 1998b). After centrifugation, the supernatant was used for incubation with 20 μl of affinity-purified sG_i2 antibody. The protein-antibody complexes were separated by incubation with 80 μl of proteinA-agarose (Sigma) followed by centrifugation. The non-immunoprecipitated supernatant was used for the binding assay using D₂-specific ([³H]raclopride) and D₁-specific ([³H]SCH 23390) radioligands as described elsewhere (Khan et al., 1993; Khan et al., 1998a; Khan et al., 1998b; Khan et al., 2001). For the binding assay, supernatant was incubated with 1 nM radioligand in total of 0.5 ml. Reaction was terminated by rapid filtration and retained radioactivity was counted as described above in detail in whole-cell binding assays. The amount of co-immunoprecipitated receptors was calculated by subtracting the binding values of supernatant from the total (100%) binding of radioligands. Incubation without addition of antiserum represented 100% binding.

For calculation and deduction of immunoprecipitation values as in Fig. 4, proteins extract of cells was incubated with pre-immune serum or antiserum against sGi2 protein after termination of the experiment. The immunocomplex (sGi2-D2 complex) was precipitated using proteinA-agarose. The supernatant portion of this reaction was used for binding assays and cpm values were obtained after counting in scintillation counter. A value of 100% was assigned to the cpm values obtained from the extract treated with preimmune serum. The immunoprecipitation values were calculated by subtracting the values of supernatant originated from extract treated with antiserum to the 100% value, meaning amount of supernatant derived from extract treated with preimmune serum minus amount of supernatant derived from extract treated with antiserum equal the amount of immunoprecipitated receptor.

Following infection and protein expression, harvested intact cells were incubated with agonists (10 μM dopamine and 5 μM quinpirole, both from Sigma/RBI) and antagonist (5 μM raclopride from Sigma/RBI) for 30 minutes. After washing, cells were processed for whole-cell binding assays and immunoblots as described above.

We thank O. Civelli for cDNA of dopamine receptors; R. Reed for cDNA of G-proteins. This work was supported by BFI2003-03464, BFU2006-0306 and Ramón y Cajal program grants from MEC (to Z.U.K.), FIS PI060556 grant from MSC (to A.G.) and a Young Investigator Award from NARSAD (to P.K.).