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CELLULAR AND MOLECULAR

G Protein Coupling and Ligand Selectivity of the D_2L and D₃ Dopamine Receptors

Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, United Kingdom (J.R.L., G.M.); and Screening and Compound Profiling, GlaxoSmithKline Research and Development, New Frontiers Science Park, Harlow, Essex, United Kingdom (B.P., A.W., S.R.)

Received for publication November 14, 2007
Accepted January 23, 2008.

	Abstract

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The human dopamine D_2L receptor couples promiscuously to multiple members of the G_i/o subfamily. Despite the high homology of the D_2L and D₃ receptors, the G protein coupling specificity of the human D₃ receptor is less clearly characterized. The primary aim of this study, then, was the parallel characterization of the G protein coupling specificity of the D_2L and D₃ receptors. By using both receptor-G protein fusion proteins and stable cell lines in which pertussis toxin-resistant mutants of individual G_i-family G proteins were expressed in an inducible fashion, we demonstrated highly selective coupling of the D₃ receptor to G_o1. Furthermore, by using the fusion proteins to ensure identical stoichiometry of receptor to G protein for each pairing, a range of ligands displayed higher potency and, for partial agonists, higher efficacy at the D₃ receptor when coupled to G_o1 compared with the D_2L receptor. The second aim of this study was to investigate the molecular basis of the above differential G protein coupling specificity. The importance of a 12-amino acid sequence from the C-terminal end of the third intracellular loop of the D_2L receptor in providing promiscuity in G protein coupling was demonstrated using a chimeric D₃/D₂ receptor in which the equivalent region of the D₃ receptor was exchanged for this sequence. This chimera displayed D₃-like affinity for [³H]spiperone and potency for agonists but gained D₂-like ability to couple to each of G_i1–3 as well as G_o1.

The second aim of this study was to investigate the molecular basis of the differential G protein-coupling specificities of the dopamine D_2L and D₃ receptors. To investigate regions within receptors that are important in directing potential differences in G protein interaction selectivity, an accepted approach is the use of chimeric constructs in which one region of a donor receptor is replaced with the equivalent region of a homologous, acceptor receptor. We used a previously studied D₃/D₂ receptor chimera (Filteau et al., 1999), here termed D_3/2, that has the backbone of the D₃ dopamine receptor but with a 12-amino acid section of D₂ at the end of the third intracellular loop replacing the equivalent D₃ sequence. This chimeric D_3/2 receptor had the capacity to couple to each of G_i1, G_i2, G_i3, and G_o1. Pharmacological analysis indicated that the D_3/2 receptor displayed D₃-like affinity for [³H]spiperone (Robinson and Caron, 1996) but D₂-like ability to couple to each of G_i1–3 as well as G_o1 in response to dopamine. However, detailed pharmacological studies indicated that the swapped segment of the third intracellular loop was insufficient to fully define the capacity of agonists other than dopamine to activate multiple G proteins.

	Materials and Methods

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[³H]Spiperone (65–140 Ci/mmol) was from GE Healthcare (Little Chalfont, Buckinghamshire, UK), and [³⁵S]GTPS (1250 Ci/mmol) was from PerkinElmer Life and Analytical Sciences (Waltham, MA). (+)-Butaclamol, dopamine, (–)-quinpirole, m-tyramine, p-tyramine, S-(–)-3-PPP, R-(+)-3-PPP, R-(–)-10,11-dihydroxy-N-n-propylnorapomorphine (NPA), 7-OH-DPAT, and GTPS were purchased from Sigma Chemical (Poole, Dorset, UK). Spiperone hydrochloride was from Tocris Cookson Inc. (Bristol, UK). Oligonucleotides were obtained from Thermo Electron GmbH (Ulm, Germany), and all materials for tissue culture were from Invitrogen (Paisley, UK). All other reagents were obtained as indicated.

D₃ and D_3/2 Receptor Subcloning into pcDNA3. cDNA of the human D₃ dopamine receptor and the chimeric D_3/2 dopamine receptor were initially in the vectors pRC and pcDNA3.1, respectively. The two cDNAs were amplified by PCR using the following primers: sense, CCC AAG CTT ATG GCA TCT CTG AGT CAG CTG; and antisense, AAA AAA AAC CAT GGA AGA CAG GAT CTT GAG GAA GGC. Underlined bases indicate the restriction sites HindIII (sense) and XhoI (antisense). The resulting PCR fragments were digested with HindIII and XhoI and inserted into pcDNA3.

D_2L Receptor Subcloning into pcDNA3. D_2L was initially in the vector pDEST12.2. D_2L cDNA was amplified by PCR using the following primers: sense, AAA AGA ATC CGC CAC CAT GGA TCC ACT GAA TCT GTC C; and antisense, AAA ACT CGA GTC AGC AGT GGA GGA TCT TCA GGA AGG. Underlined bases indicate the restriction sites EcoRI (sense) and XhoI (antisense). The resulting PCR fragment was digested with EcoRI and XhoI and inserted into pcDNA3.

Construction of the Myc-D_2L-G-Protein Subunit Fusion Proteins. The construction of Myc-D_2L-G protein subunit fusion proteins have been described previously (Lane et al., 2007). Pertussis toxin-resistant _2A-adrenoceptor-G protein fusion proteins were prepared as described previously (Wise and Milligan, 1997). In brief, Cys351 of rat G_i1, G_i3, and G_o1 (or Cys352 in G_i2) was mutated to isoleucine by site-directed mutagenesis and then used to create the _2A-adrenoceptor-G fusion proteins using the porcine _2A-adrenoceptor in pcDNA3. These constructs were cloned into pcDNA3 using created 5' KpnI and 3' EcoRI sites, with an NcoI site between receptor and G protein subunit cDNAs. To create D_2L-G protein subunit proteins, the _2A-adrenoceptor-G fusion proteins (NcoI) were excised from pcDNA3 using KpnI and EcoRI, digested with NcoI, and the G subunit cDNA was purified. The G_i subunit cDNAs were then cloned into pcDNA3 with the Myc-D_2L PCR fragment to create the four D_2L-G protein subunit fusion proteins.

Construction of the Myc-D₃/D_3/2-G-Protein Subunit Fusion Proteins. Primers encoding the Myc epitope sequence were used to generate N-terminally tagged Myc-D₃ and Myc-D_3/2 and remove the stop codon: sense, 5'-AAA AAA AG GTA CCA TGG AAC AAA AAC TTA TTT CTG AAG AAG ATC TGG CAT CTC TGA GTC AGC TGA GTA GC-3'; and antisense, 5'-AAA AAA AAC CAT GGA AGA CAG GAT CTT GAG GAA GGC-3'. Underlined bases indicate introduced restriction sites (sense, KpnI; antisense, NcoI), and bases in bold indicate introduced N-terminal Myc tag. The PCR fragment was digested using KpnI and NcoI. As for D_2L, G_i subunit cDNAs were purified and cloned into pcDNA3 with the Myc-D₃ or Myc-D_3/2 PCR fragment to create four D₃-G protein subunit fusion proteins and four D_3/2-G protein subunit fusion proteins. All PCR fragments were sequenced in their entirety.

Flp-In Constructs. The previously described pertussis toxin-resistant mutants of rat G_i1, G_i2, G_i3, and G_o1 were cloned into pcDNA3. cDNAs were excised using KpnI and ApaI (G_i1–3) or ApaI (G_o1) and subcloned into the pcDNA5/FRT/TO vector (Invitrogen).

Cell Culture and Transfection. HEK293 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 0.292 g/l L-glutamine and 10% (v/v) newborn calf serum at 37°C in a 5% CO₂ humidified atmosphere. Cells were grown to 60 to 80% confluence before transient transfection. Transfection was performed using Lipofectamine transfection reagent (Invitrogen) according to the manufacturer's instructions.

Generation of Stable Flp-In T-REx HEK293 Cells. To generate Flp-In T-REx HEK293 cells able to inducibly express the G protein subunit of interest, the cells were transfected with a mixture containing the desired G protein subunit cDNA in the pcDNA5/FRT/TO vector and pOG44 vectors (1:9) using Lipofectamine according to the manufacturer's instructions. Cell maintenance and selection were as described previously (Milasta et al., 2006). Clones were screened for G protein expression by Western blotting. To constitutively stably coexpress D_2L, D₃, or D_3/2 in G protein-inducible cell lines, the appropriate cells were further transfected with D_2L, D₃, or D_3/2 cDNA in pcDNA3 as described above, and resistant cells were selected in the presence of 1 mg/ml G418. Resistant clones were screened for receptor expression using specific [³H]spiperone binding. Cells were treated with 1 µg/ml tetracycline 24 to 48 h before assay to induce expression of the G protein subunit cloned into the Flp-In locus.

Membrane Preparation. Cells were collected by centrifugation (1700g, 5 min, 4°C), frozen at –80°C for at least 1 h, and resuspended in 15 ml of buffer (10 mM Tris, 0.1 mM EDTA, pH 7.4). Cell suspensions were then homogenized using an Ultra Turrax three times for 20 s. The homogenate was centrifuged at 1700g for 10 min, and the supernatant was collected and centrifuged at 48,000g for 45 min at 4°C. The resulting pellet was resuspended in buffer and stored at –80°C in aliquots of 1 ml.

Saturation Binding Assays Using [³H]Spiperone. Cell membranes (10 µg of protein) were incubated in triplicate with [³H]spiperone (0.001–2 nM) in a total volume of 1 ml of buffer (20 mM HEPES, 6 mM MgCl₂, 1 mM EDTA, 1 mM EGTA, pH 7.4). Nonspecific binding was determined by the inclusion of 10 µM(+)-butaclamol. The reaction was initiated by the addition of membranes, and the tubes were incubated at 25°C for 3 h. The reaction was terminated by rapid filtration using a Brandel cell harvester with three 5-ml washes of ice-cold phosphate-buffered saline (140 mM NaCl, 10 mM KCl, 1.5 mM KH₂PO₄, 8 mM Na₂HPO₄). The filters were soaked in 3 ml of scintillation fluid, and radioactivity present was determined by liquid scintillation spectrometry.

[³⁵S]GTPS Binding Assays. Cell membranes (10 µg) were incubated in 900 µl of buffer (20 mM HEPES, 100 mM NaCl, 6 mM MgCl₂, 40 µM ascorbic acid, pH 7.4) containing 10 µM GDP and various concentrations of ligands. All experiments were performed in triplicate. The reaction was initiated by the addition of cell membranes and incubated at 30°C for 30 min. A 100-µl volume of [³⁵S]GTPS (0.1 nM final concentration) was then added, and the incubation was continued for a further 30 min. The reaction was terminated by rapid filtration with a Brandel cell harvester and three 4-ml washes with ice-cold phosphate-buffered saline. Radioactivity was determined as described for saturation analysis.

[³⁵S]GTPS Binding Assay with Immunoprecipitation Step. [³⁵S]GTPS-binding experiments were initiated by the addition of cell membranes containing 10 fmol of the various fusion constructs to an assay buffer (20 mM HEPES, pH 7.4, 3 mM MgCl₂, 100 nM NaCl, 10 µM GDP, 0.2 mM ascorbic acid, 50 nCi of [³⁵S]GTPS) in the presence or absence of dopamine (10 µM). Nonspecific binding was determined in the same conditions but with the addition of 100 µM GTPS. Reactions were incubated for 30 min at 30°C and were terminated by the addition of 0.5 ml of ice-cold buffer, containing 20 mM HEPES, pH 7.4, 3 mM MgCl₂, and 100 mM NaCl. The samples were centrifuged at 16,000g for 15 min at 4°C, and the resulting pellets were resuspended in solubilization buffer (100 mM Tris, 200 mM NaCl, 1 mM EDTA, 1.25% Nonidet P-40) plus 0.2% SDS. Samples were precleared with Pansorbin, followed by immunoprecipitation with in-house antisera raised against either the C terminus of either G_i2 or G_o1. Finally, the immunocomplexes were washed twice with solubilization buffer, and bound [³⁵S]GTPS was measured by liquid scintillation spectrometry.

[³⁵S]GTPS Binding Assay: Agonist Stimulation of [³⁵S]GTPS Binding by Fusion Proteins. [³⁵S]GTPS binding assays were performed at room temperature in a 384-well format. Membranes (10 µg/point) were diluted to 0.4 mg/ml in assay buffer (20 mM HEPES, 100 mM NaCl, 10 mM MgCl₂, pH 7.4) supplemented with saponin (10 mg/l), preincubated with 10 µM GDP and wheat germ agglutinin SPA beads (GE Healthcare) (0.5 mg), and incubated at room temperature for 45 min with agitation. Various concentrations of D₂ dopamine receptor agonists were added, followed by [³⁵S]GTPS (1170 Ci/mmol; GE Healthcare) at 0.3 nM (total volume of 46 µl), and binding was allowed to proceed at room temperature for 4 h. Bound [³⁵S]GTPS was determined by scintillation counting on a ViewLux ultraHTS Microplate Imager (PerkinElmer Life and Analytical Sciences).

Data Analysis. Data were analyzed using Prism (GraphPad Software Inc., San Diego, CA), and statistical significance was determined using either Student's t test or ANOVA followed by the Bonferroni post-hoc analysis as appropriate. p < 0.05 Determined statistical significance. [³⁵S]GTPS binding was analyzed using nonlinear regression with variable slope, in which neither top nor bottom values were constrained. No outlying values were excluded.

	Results

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The Use of a Chimeric D_3/2 Receptor to Investigate a Region of D_2L and D₃ Important for G Protein Coupling Selectivity. Such a chimera was generated, based on the human D₃ receptor sequence, in which a 12-amino acid segment of the C terminus of the third intracellular loop was replaced with the equivalent region of the human D_2L (Fig. 1). The 12 amino acids are entirely different but are flanked by identical sequences to their C-terminal side, close to transmembrane domain (TM) VI, and N-terminal side. Signal transduction from both D₂ and D₃ dopamine receptors is generally pertussis toxin-sensitive and, therefore, mediated by one, or more, G_i subclass G protein(s). There is significant endogenous G_i subunit expression in HEK293T cells. Consequently, to allow the investigation of coupling between specific receptor and G protein pairs, HEK293T cells were cotransfected with a dopamine receptor (D_2L, D₃, or D_3/2) and either C352I G_i2 or C351I G_o1, which are pertussis toxin-insensitive, and treated with or without pertussis toxin (25 ng/ml) for the final 16 h before cell harvest. To determine the number of appropriately folded receptors in membranes of these cells, the high-affinity dopaminergic antagonist/inverse agonist, [³H]spiperone, was used in saturation binding studies. In D_2L cotransfections, a single population of high-affinity sites for [³H]spiperone was observed with K_d of approximately 0.04 nM (Table 1). Receptor density, as indicated by the B_max value, was approximately 3 to 4 pmol/mg membrane protein in all transfections. For both C352I G_i2 and C351I G_o1 cotransfections, there were no significant differences in K_d or B_max for membranes from the same transfection with or without pertussis toxin treatment. However, although no difference in K_d was observed, a significantly higher receptor density was observed in C351I G_o1 cotransfections compared with C352I G_i2 transfections. [³H]Spiperone displayed a lower affinity for the D₃ receptor, with a K_d of 0.1 to 0.15 nM but with receptor density similar to that for the D_2L cotransfections. For the chimeric D_3/2 receptor cotransfections, affinity for [³H]spiperone was similar to that observed for the D₃ receptor. However, B_max values, at 0.5 to 1 pmol/mg membrane protein were approximately 4-fold lower than for either D_2L or D₃ (Table 1). As for D_2L cotransfections, a significantly higher value of B_max was observed for both D₃ and D_3/2 with C351I G_o1 compared with the equivalent C352I G_i2 cotransfection. Again, no significant difference in K_d or B_max was observed for membranes from the same transfection with or without pertussis toxin treatment. However, it was noted that only in the case of the D₃ receptor, a significant difference in K_d for [³H]spiperone was dependent on whether C352I G_i2 or C351I G_o1 was coexpressed.

View larger version (28K): [in this window] [in a new window]   Fig. 1. The D3/2 receptor consists of the D3 receptor with 12 amino acids in the C terminus of intracellular loop 3 exchanged by the equivalent region from D2. Sections of the sequence of the human D2 and D3 receptors are shown, highlighting the C-terminal region of the third intracellular loop and TMVI. The D3/2 chimera was generated by replacing the highlighted sequence of D3 with the equivalent region of D2. The exchanged 12 amino acids are flanked by identical proximal and distal regions.

View this table: [in this window] [in a new window]   TABLE 1 [3H]Spiperone binds with similar affinity to D3 and D3/2 when coexpressed with either C352I Gi2 or C351I Go1 Experiments were performed in triplicate to n = 3. Differences in Kd and Bmax were analyzed using an unpaired Student's t test. No significant differences in Kd or Bmax were seen for the same transfection with or without pertussis toxin treatment. Bmax and Kd values were compared between Gi2 and Go1 cotransfections with the same receptor and pertussis toxin pretreatment condition.

G protein expression was measured for both C352I G_i2 and C351I G_o1 cotransfections by Western blotting using antisera raised against the C-terminal decapeptide of the respective G protein subunit (Fig. 2A). HEK293T cell membranes transfected with equivalent amounts of pcDNA3 vector were included as a control. These demonstrated that both C352I G_i2 and C351I G_o1 were significantly overexpressed in all membrane preparations (Fig. 2A). Although pertussis toxin treatment did not affect levels of either C352I G_i2 or C351I G_o1 (Fig. 2A), there was significant variation in the levels of G protein subunit detected between different transfections (Fig. 2A).

View larger version (26K): [in this window] [in a new window]   Fig. 2. Characterization of HEK293T cells transiently cotransfected with pertussis toxin-insensitive C352I Gi2 or C351I Go1 and D2L,D3,or D3/2. A, HEK293T cells were transiently cotransfected with a pertussis toxin-insensitive G protein (C352I Gi2 or C351I Go1) and a dopamine receptor (D2L,D3,or D3/2). These cells were treated with or without pertussis toxin (PTX) (25 ng/ml) for the last 16 h before cell harvest. Cell membranes were prepared, resolved by SDS-polyacrylamide gel electrophoresis, and immunoblotted to detect either Gi2 or Go1. Receptor expression levels were estimated in [3H]spiperone saturation binding studies (Table 1). Membranes as above, expressing the same number of [3H]spiperone binding sites, were used to measure basal [35S]GTPS binding (open bars) and the effect of 100 µM dopamine on this (filled bars). Assays were terminated by an immunoprecipitation step using antisera raised against the C-terminal decapeptide of Go1 (B) or Gi2 (C). Significant differences (Student's t test; ***, p < 0.001; **, 0.001 < p < 0.01; *, 0.01 < p < 0.05) between both basal and dopamine-stimulated [35S]GTPS binding are indicated. An identical test was applied to identify significant differences between basal levels of [35S]GTPS binding for the pertussis toxin-treated conditions for each receptor cotransfection compared with other cotransfections of the same G subunit.

Alterations in GPCR/G protein ratio can result in substantial differences in agonist potency and apparent efficacy. Therefore, for any direct and detailed comparisons of pharmacological variation between different GPCRs, this must be controlled. To achieve this, two approaches were used. One approach was the generation of receptor-G protein fusion proteins. The fusion proteins have a major advantage in defining and equalizing the receptor/G protein ratio for each GPCR and G protein, but they are an inherently artificial system. Therefore, it was important to also examine the G protein coupling selectivity of D₂, D₃, and D_3/2 in a system expressing receptor and G protein as separate polypeptides. With this in mind, the first approach was the generation of cell lines that express the pertussis toxin-insensitive forms of G_i1, G_i2, G_i3, or G_o1 in an inducible fashion.

Cell Lines That Can Express Pertussis Toxin-Insensitive Forms of G_i1, G_i2, G_i3, or G_o1 in an Inducible Fashion. A series of HEK293 cell lines based on the Flp-In T-REx system (Milasta et al., 2006; Lane et al., 2007) were generated. In these, one of D_2L, D₃, or D_3/2 was expressed stably and constitutively, whereas either C351I G_o1 or C352I G_i2 was cloned into the Flp-In locus, allowing their expression in an inducible fashion. In concert with pertussis toxin pretreatment, this provided an alternative system in which the dopamine receptor-mediated stimulation of [³⁵S]GTPS binding must reflect activation of only a single, defined G protein. Tetracycline-dependent expression of the G protein of interest was confirmed in all cases (Fig. 3). In support of the earlier studies, [³H]spiperone saturation binding demonstrated lower affinity for this ligand in membranes expressing D₃ and D_3/2 compared with the equivalent D_2L Flp-In T-REx cell lines. Levels of D_2L, D₃, and D_3/2 were unaffected by tetracycline-induced turn-on of C352I G_i2 or C351I G_o1 (Table 2). Dopamine stimulated [³⁵S]GTPS binding in the C351I G_o1-inducible cells via D_2L, D₃, and D_3/2 only after induction of C351I G_o1 expression, whereas this was also observed after induction of expression of C352I G_i2 in cells constitutively expressing D_2L and D_3/2 but not D₃ (Fig. 4). Although entirely consistent with the data from the transient cotransfections, the markedly different levels of receptor expression in the various lines we were able to isolate indicated that these cells would not be appropriate for detailed studies on the pharmacology of biased agonism and relative efficacy.

View larger version (35K): [in this window] [in a new window]   Fig. 3. Characterization of Flp-In T-REx cells harboring pertussis toxin-insensitive mutant G proteins at the Flp-In locus and constitutively expressing D2L,D3,or D3/2. Flp-In T-REx HEK293 cell lines were established with either C351I Gi2 (A) or C351I Go1 (B) cloned into the Flp-In locus. These cells were further transfected to constitutively and stably express D2L,D3,or D3/2 (for details, see Table 2). These cells were treated with or without tetracycline (Tet.) (1.0 µg/ml for 24 h). Cell membranes were prepared, resolved by SDS-polyacrylamide gel electrophoresis and immunoblotted to detect either Gi2 (A) or Go1 (B).

View this table: [in this window] [in a new window]   TABLE 2 Expression levels of constitutively expressed dopamine receptors are unaffected by tetracycline-induced expression of C352I Gi2 or C351I Go1 Experiments were performed in triplicate to n = 3. Although expression levels of the dopamine receptors varied markedly between lines, no significant differences in Bmax values were observed in receptor expression with and without induction of G protein expression, unpaired Student's t test.

View larger version (29K): [in this window] [in a new window]   Fig. 4. Dopamine-stimulated binding of [35S]GTPS in membranes of Flp-In T-REx HEK293 cells constitutively expressing D3 requires expression of C351I Go1. Membranes of cells expressing D2L or D3/2 require expression of either C352I Gi2 or C351I Go1. Flp-In T-REx HEK293 cells stably expressing the D2L (A), D3 (B), or D3/2 (C) and harboring either C352I Gi2 (i) or C351I Go1 (ii) at the Flp-In locus were treated with or without 1 µg/ml tetracycline for 24 h. Both sets of cells were also treated with pertussis toxin. Membranes from these cells were used to measure basal [35S]GTPS binding (open bars) and the effect of 10–5 M dopamine (filled bars) on this. Significant effects of dopamine were observed upon induction of C352I Gi2 and C351I Go1 for membranes expressing D2L and D3/2 but, for D3, only after expression of C351I Go1 (Student's t test; ***, p < 0.001; **, 0.001 < p < 0.01; *, 0.01 < p < 0.05). An identical test was applied to identify significant differences between basal levels of [35S]GTPS binding with and without the induction of expression of the same pertussis toxin G subunit variant.

Studies with Dopamine Receptor-Pertussis Toxin-Insensitive CysIle Variant G Protein Fusions. To control relative levels of G protein and receptor expression, a series of dopamine receptor-G protein fusion proteins were generated. Constraining GPCR and G protein within a fusion protein defines the stoichiometry of expression of the two polypeptides as 1:1 and ensures their colocalization after expression (Milligan et al., 2007). D_2L receptor-CysIle pertussis toxin-insensitive G subunit fusion proteins have been described previously for each of G_i1, G_i2, G_i3, and G_o1 (Lane et al., 2007). Using the same methodology, eight further dopamine receptor-CysIle G subunit fusion proteins were generated (Fig. 5A) by fusing both D₃ and D_3/2 to each of the CysIle pertussis toxin-insensitive variants of G_i1, G_i2, G_i3, and G_o1. After transient transfection of these constructs and before cell harvest, HEK293T cells were treated with pertussis toxin to cause ADP-ribosylation of endogenous forms of G_i and hence allow interaction between the receptor and G protein within the fusion protein to be studied in isolation. [³H]Spiperone displayed a similar and high affinity for each of the D_2L-G protein fusion proteins with K_d of some 0.04 nM, whereas receptor binding site density was approximately 1.5 pmol/mg membrane protein for each (Table 3). The D₃ receptor fusion proteins displayed a lower affinity for [³H]spiperone, with K_d approximately 0.12 nM (Table 3). Receptor binding site density was between 0.5 and 2 pmol/mg membrane protein for the D₃ receptor fusion proteins, with the observed B_max significantly higher at the D₃-C351I G_o1 fusion protein. The D_3/2 receptor fusion proteins displayed "D₃-like" affinity for [³H]spiperone (Table 3), with B_max values between 0.5 and 1 pmol/mg membrane protein. Significantly higher B_max values were obtained for D_3/2-C351I G_i1 and D_3/2-C351I G_o1 than the other D_3/2 receptor fusion proteins.

View larger version (27K): [in this window] [in a new window]   Fig. 5. Generation and characterization of dopamine D2 receptor, D3 receptor, or chimeric D3/2 receptor-CysIle variant G protein subunit fusion proteins. A, series of fusion proteins were generated by linking CysIle mutant, pertussis toxin-insensitive variants of Gi1,Gi2,Gi3, and Go1 in frame with the C-terminal tail of D3 or D3/2. Equivalent fusions incorporating D2 have previously been reported (Lane et al., 2007). B, HEK293T cells were transiently transfected with dopamine (D2L,D3,or D3/2) receptor-CysIle G subunit fusion protein cDNAs; i, dopamine receptor-C351I Gi1; ii, dopamine receptor-C352I Gi2; iii, dopamine receptor-C351I Gi3; iv, dopamine receptor-C351I Go1. Cells were treated with pertussis toxin (25 ng/ml) 16 h before cell harvest. Membranes from these cells were used to measure basal [35S]GTPS binding (open bars) and the effect of 10–4 M dopamine (filled bars) on this. At the end of assay, an immunoprecipitation step was incorporated using antisera raised against the C-terminal decapeptide of Gi1/2 (i and ii), Gi3 (iii), or Go1 (iv). The significance of differences between basal and dopamine-stimulated [35S]GTPS binding was determined using an unpaired Student's t test. Significant effects of dopamine: ***, p < 0.001; **, 0.001 < p < 0.01; and *, 0.01<p < 0.05. An identical test was applied to identify significant differences between basal levels of [35S]GTPS binding for the pertussis toxin-treated conditions for each receptor cotransfection compared with other cotransfections of the same G subunit.

[³⁵S]GTPS assays were performed with immunocapture by appropriate antisera to establish whether the coupling selectivity of the D_2L, D₃, and D_3/2 receptors observed in the transient cotransfection studies was preserved after G protein fusion. It was; for the C351I G_i1 fusions, significant stimulation by dopamine was produced by D_2L-C351I G_i1 and D_3/2-C351I G_i1, but not by D₃-C351I G_i1 (Fig. 3B, i). This pattern was repeated for C352I G_i2 and C351I G_i3 (Fig. 3B, ii and iii), whereas dopamine greatly enhanced binding of [³⁵S]GTPS to the fused C351I G_o1 via all three receptors (Fig. 3B, iv). It should be noted, then, that the pattern of G protein selectivity observed for the dopamine receptor-G protein fusion proteins was entirely consistent with that of both the transient transfection and inducible cell line approaches described above. To explore the detailed pharmacology of G protein activation by ligands at D_2L, D₃, and D_3/2, membranes of HEK293T cells transfected with each of the 12 fusion proteins and pretreated with pertussis toxin were used in high-throughput, filtration-based [³⁵S]GTPS assays. A complex pattern of responses was produced. As reported previously for D_2L (Lane et al., 2007), both p-tyramine and S-(–)-3-PPP displayed agonism only for activation of C351I G_o1, whereas 7-OH-DPAT displayed partial agonism at D_2L for each of C351I G_i1, C351I G_i3, and C351I G_o1 but not for C352I G_i2 (Fig. 6; Table 3). For D₃, each of the ligands tested displayed agonism only for C351I G_o1 (Fig. 7; Table 4). However, for all of the agonists examined, with the exception of NPA, the measured potency at D₃-C351I G_o1 was significantly higher than at D₂-C351I G_o1, and, in general, partial agonists displayed significantly higher efficacy compared with dopamine at D₃-C351I G_o1 than D₂-C351I G_o1 (Tables 4 and 5). Indeed, although as demonstrated previously (Lane et al., 2007), S-(–)-3-PPP was a weak partial agonist at D₂-C351I G_o1 (21 ± 2% efficacy), at D₃-C351I G_o1, it displayed 63 ± 2% efficacy relative to dopamine (Table 4). The D_3/2 chimera retained the characteristic D₃-like high potency of the ligands to stimulate binding of [³⁵S]GTPS to C351I G_o1 (Fig. 8; Table 6) but did not acquire all of the signal transduction characteristics of the D_2L receptor. Although dopamine could stimulate [³⁵S]GTPS binding to all of the G proteins fused to D_3/2, and all of the ligands tested also displayed agonism at D_3/2-C351I G_i3 (Table 6), this was not true for D_3/2-C351I G_i1 (Table 6), where neither m-tyramine nor R-(+)-3-PPP displayed agonism.

View larger version (37K): [in this window] [in a new window]   Fig. 6. G protein fusion proteins identify agonism via the D2L receptor. Each of the fusion proteins D2L-C351I Gi1 (A), D2L-C352I Gi2 (B), D2L-C351I Gi3 (C), and D2L-C351I Go1 (D) were expressed transiently in HEK293T cells. After pertussis toxin treatment (25 ng/ml, 24 h), cells were harvested, membranes were generated, and [35S]GTPS binding studies performed in the absence and presence of varying concentrations of a variety of ligands [dopamine, ; m-tyramine, ; p-tyramine, ; R-(+)-3-PPP, ; S-(–)-3-PPP, ; quinpirole, ; NPA, ; 7-OH-DPAT, ] reported to have affinity and efficacy at D2. Data are representative, and full details are provided in Table 4.

View larger version (34K): [in this window] [in a new window]   Fig. 7. Agonists are identified at D3 only when the receptor is fused to C351I Go1. Each of D3-C351I Gi1 (A), D3-C352I Gi2 (B), D3-C351I Gi3 (C), and D3-C351I Go1 (D) was expressed transiently in HEK293 cells. After pertussis toxin treatment (25 ng/ml, 24 h) cells were harvested, membranes were generated, and [35S]GTPS binding studies were performed as in Fig. 4 in the absence and presence of varying concentrations of a variety of ligands [dopamine, ; m-tyramine, ; p-tyramine, ; R-(+)-3-PPP, ; S-(–)-3-PPP, ; quinpirole, ; NPA, ; 7-OH-DPAT, ] reported to have affinity and efficacy at dopamine D2-like receptors. Data are representative, and full details are provided in Table 5.

View larger version (42K): [in this window] [in a new window]   Fig. 8. G protein fusion proteins identify agonists at D3/2 when coupled to all four pertussis-toxin resistant Gi/o subunits. Each of D3/2-C351I Gi1 (A), D3/2-C352I Gi2 (B), D3/2-C351I Gi3 (C), and D3/2-C351I Go1 (D) was expressed transiently in HEK293 cells. After pertussis toxin treatment (25 ng/ml, 24 h), cells were harvested, membranes were generated, and [35S]GTPS binding studies were performed in the absence and presence of varying concentrations of a variety of ligands [dopamine, ; m-tyramine, ; p-tyramine, R-(+)-3-PPP, ; S-(–)-3-PPP, ; quinpirole, ; NPA, ; 7-OH-DPAT, ] reported to have affinity and efficacy at dopamine D2-like receptors. Data are representative, and full details are provided in Table 6.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
The D₂ and D₃ receptors exhibit high amino acid homology, particularly within the transmembrane domains, and consequently have similar pharmacology (Sokoloff et al., 1990). There has been considerable interest in the contribution of the D₃ receptor to the action of antipsychotic drugs, particularly after the observation that schizophrenia is associated with a marked elevation of mesolimbic D₃ receptors (Gurevich et al., 1997; Joyce, 2001). The G protein coupling of D₂ has been well characterized with a general consensus for promiscuous coupling to each of G_i1,G_i2,G_i3, and G_o1 (Senogles, 1994; Watts et al., 1998; Ghahremani et al., 1999; Jiang et al., 2001; Gazi et al., 2003). However, the G protein coupling profile of D₃ is poorly characterized (Ahlgren-Beckendorf and Levant, 2004). The present study was based on the expression of D₂ and D₃ receptors along with pertussis toxin-resistant CysIle G_i/o subunits in heterologous expression models based on HEK293 cells. This allowed the characterization and direct comparison of the G protein coupling specificities of D₂ and D₃. Several studies using [³⁵S]GTPS binding have implicated coupling of D₃ to pertussis toxin-sensitive G_i/o subunits, with two studies in particular showing efficient, selective coupling to G_o1 (Zaworski et al., 1999; Beom et al., 2004), although Zaworski et al. postulated that efficient coupling to G_o1 required the expression of adenyl cyclase subtype V. However, this study is the first to assess coupling to each of the four G_i/o subunits directly and, in parallel, to explore pharmacological consequences of attempting to expand the G protein coupling profile of the D₃ receptor. Key to this process was maintaining a clearly defined receptor to G protein expression stoichiometry, and, despite using both a series of transient cotransfection studies and establishing cells lines stably expressing dopamine receptors that allowed regulated expression of a G protein, this was only possible by using GPCR-G protein fusion proteins. The defined 1:1 ratio of receptor and G protein allowed direct comparisons between coupling of the D₂ and D₃ receptors to each G_i/o subunit and detailed analysis of pharmacological characteristics of a previously studied D_3/2 chimera. The highly selective coupling of D₃ to G_o1 may reflect the high amino acid homology among G_i1, ₂, and ₃ (85–90%), whereas G_o1 has lower identity (between 70 and 73%) with each of these. Furthermore, because the C-terminal decapeptide of G protein subunits plays a key role in receptor selectivity, the closer relationship in this region between G_o1 and G_i3 is probably relevant to why G_i3 was activated by all of the ligands examined when fused to the D_3/2 chimera, whereas this was not true for G_i1 or G_i2.

A comparison of the potencies and relative efficacies of various agonists at D_2L and D₃ generally indicated higher potency and, for partial agonists, higher efficacy at the D₃ receptor. Although the GPCR-G protein proteins were used to generate these data, it is important to highlight that G protein selectivity measures were in complete agreement when separate GPCRs and G proteins were either transiently cotransfected or when the Flp-In T-REx cell lines were used. However, because of the inability to define GPCR/G protein ratios adequately, these systems were not deemed as appropriate for the detailed pharmacological comparisons.

In addition to serving in a classic postsynaptic sensory role, D₃ is also a presynaptic autoreceptor that is expressed predominantly in parts of the brain involved with controlling emotion and movement (Joseph et al., 2002). The higher potency of dopamine for D₃ relative to D₂ shown in this study is consistent with its action as a presynaptic autoreceptor. The D₃ receptor may therefore be anticipated to regulate dopamine release at lower dopamine concentrations than D₂. Consistent with presynaptic D₃ autoreceptors signaling through G_o1, the localization of G_i/o proteins has been shown to be different at post- and presynaptic densities, with G_o1 more prevalent at presynaptic densities (Aoki et al., 1992). With the presynaptic role of the dopamine D₃ receptor in mind, it is interesting to consider the potential role D₃ may have in the action of S-(–)-3-PPP, an interesting antipsychotic drug. In a previous study, we demonstrated that S-(–)-3-PPP is a functionally selective ligand at the D_2L receptor, displaying agonism for activation of G_o1, but antagonism for activation of G_i1–3 (Lane et al., 2007). Furthermore, S-(–)-3-PPP has been shown to be an agonist at presynaptic dopamine receptors but an antagonist at postsynaptic dopamine receptors (Arnt et al., 1983; Hjorth et al., 1983). It is possible that S-(–)-3-PPP acts both through presynaptic dopamine D₂ and D₃ receptors coupled to G_o1. It is noteworthy that the current studies demonstrate that S-(–)-3-PPP has significantly higher relative efficacy to activate G_o1 as well as potency at D₃ compared with D_2L and therefore that D₃ may be the key target of S-(–)-3-PPP.

The molecular basis of G protein activation selectivity has been the focus of many studies (Wess, 1998; Wong, 2003). However, despite the wealth of information regarding the coupling specificities of a multitude of GPCRs, no sequence homology has been obvious, even for GPCRs that couple to the same G protein subtype, and consensus motifs predictive of G protein coupling have been difficult to identify (Möller et al., 2001). With the exchange of a 12-amino acid section of D₃ with the equivalent region from D₂ the D_3/2, chimera stimulated [³⁵S]GTPS binding to each of G_i1,G_i2,G_i3, and G_o1 in response to dopamine as opposed to the specific coupling of D₃ to G_o1. Previous studies using this construct reported gain of adenylate cyclase inhibition in a system in which D₃ was unable to produce this effect (Filteau et al., 1999). It would seem that this observed gain in the ability to inhibit adenylate cyclase lies at the level of the improved ability to couple to one, or more, of G_i1,G_i2, and G_i3. Because all the ligands tested allowed coupling of D_3/2 to both G_o1 and G_i3, whereas various ligands were unable to activate either G_i1 or G_i2 via the chimeric receptor, it may now be possible to assess the contribution of different, coexpressed G_i proteins to this endpoint in a direct and pharmacologically defined manner. A characteristic of the 12-amino acid segment that was swapped into D₃ is that nine aliphatic or hydrophobic amino acid residues in D₃ are replaced by charged or polar groups from the D₂ sequence. In contrast, the flanking sequences to this region (EKKATQ, close to TMVI, and the N-terminal TSL) are fully conserved between D₃ and D₂, so exchange of this region is unlikely to disrupt overall receptor structure. It has been suggested that the C-terminal portion of the third intracellular loop of GPCRs forms a short -helix that is extremely important for the interaction with heterotrimeric G proteins (Burstein et al., 1995; Wade et al., 1996). It is possible that proline residues present in this portion of the third intracellular loop of D₃ disrupt such helical structure.

The affinity for the antagonist/inverse agonist [³H]spiperone at D_3/2 and the potency of ligands when the chimera was linked to G_o1 were not significantly different from that of D₃ coupled to G_o1, again in agreement with previous work (McAllister et al., 1993; Filteau et al., 1999). However, the potency of dopamine was markedly lower when the D_3/2 chimera was coupled to G_i1 or G_i2 compared with G_i3 or G_o1.G_i3 has greater sequence similarity to G_o1 compared with that of G_i1 or G_i2, in the extreme C terminus, a region long appreciated to be important in receptor-G protein coupling (Wess, 1998; Slessareva et al., 2003). The different rank order of dopamine potency observed at the D₂ receptor (G_o1 > G_i1 = G_i2 = G_i3) versus D_3/2 (G_i3 = G_o1 >> G_i1 or G_i2) could point toward a difference in coupling specificity. This perhaps indicates that additional sites within the D₂ receptor contribute to G protein coupling specificity, particularly in relation to coupling to G_i1 and G_i2. In support of this, both N- and C-terminal portions of the third intracellular loop are capable of supporting functional coupling of D₂ to adenylate cyclase, but both are required for maximal coupling (Lachowicz and Sibley, 1997). A reciprocal chimera to the one used in this study showed only a reduction in the potency of coupling to adenylate cyclase compared with D₂ rather than a complete abolition of coupling, indicating the presence of additional G protein coupling domains within the D₂ receptor (Filteau et al., 1999). This is further supported by recent studies in which a peptide corresponding to the N-terminal region of the third intracellular loop of D_2L was able to activate G_i1 (Nanoff et al., 2006).

Overall, these studies illustrate ways in which GPCR-G proteins fusions can be used to tease out and explore differences in pharmacology and function of closely related receptors and indicate that many prototypic D₂-like agonist ligands display a degree of selectivity for D₃ over D₂. However, because D₃ couples exclusively to G_o, the range of signaling pathways likely to be activated by such ligands in vivo will be greater via the D₂ receptor.

	Footnotes

 
J.R.L. was supported by a Biotechnology and Biosciences Research Council Collaborative Award in Science and Engineering studentship.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.134296.

ABBREVIATIONS: D_2L, long isoform of the human dopamine D₂ receptor; GTPS, guanosine 5'-O-(thiotriphosphate); D₃, human dopamine D₃ receptor; HEK, human embryonic kidney; R-(+)-3-PPP, R (+) 3-(3-hydroxyphenyl)-N-propylpiperidine; NPA, R-(–)-10,11-dihydroxy-N-n-propylnorapomorphine; 7-OH-DPAT, R(+)-7-hydroxy-dipropylaminotetralin hydrobromide; PCR, polymerase chain reaction; ANOVA, analysis of variance; TM, transmembrane domain; GPCR, G protein-coupled receptor; S-(–)-3-PPP, S-(–)-3-(3-hydroxyphenyl)-N-propylpiperidine; [³⁵S]GTPS, guanosine 5'-O-(3-[³⁵S]thio)triphosphate.

Address correspondence to: Dr. Graeme Milligan, Davidson Building, University of Glasgow, Glasgow G12 8QQ, Scotland, UK. E-mail: g.milligan{at}bio.gla.ac.uk

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