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Protean Agonism at the Dopamine D₂ Receptor: (S)-3-(3-Hydroxyphenyl)-N-propylpiperidine Is an Agonist for Activation of G_o1 but an Antagonist/Inverse Agonist for G_i1,G_i2, and G_i3

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

Received November 15, 2006; accepted February 7, 2007

	Abstract

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A range of ligands displayed agonism at the long isoform of the human dopamine D₂ receptor, whether using receptor-G protein fusions or membranes of cells in which pertussis toxin-resistant mutants of individual G_i-family G proteins could be expressed in an inducible fashion. Varying degrees of efficacy were observed for individual ligands as monitored by their capacity to load [³⁵S]GTPS onto each of G_i1,G_i2,G_i3, and G_o1. By contrast, (S)-(–)-3-(3-hydroxyphenyl)-N-propylpiperidine was a partial agonist when G_o1 was the target G protein but an antagonist/inverse agonist at G_i1,G_i2, and G_i3. In ligand binding assays, dopamine identified both high- and low-affinity states at each of the dopamine D₂ receptor-G protein fusion proteins, and the high-affinity state was eliminated by guanine nucleotide. (S)-(–)-3-(3-Hydroxyphenyl)-N-propylpiperidine bound to an apparent single state of the constructs in which the D₂ receptor was fused to G_i1,G_i2, or G_i3. However, it bound to distinct high- and low-affinity states of the D₂ receptor-G_o1 fusion, with the high-affinity state being eliminated by guanine nucleotide. Likewise, although dopamine identified guanine nucleotide-sensitive high-affinity states of the D₂ receptor when expression of pertussis toxin-resistant forms of each of G_i1, G_i2, G_i3, and G_o1 was induced, (S)-(–)-3-(3-hydroxyphenyl)-N-propylpiperidine identified a high-affinity site only in the presence of G_o1. p-Tyramine displayed a protean ligand profile similar to that of (S)-(–)-3-(3-hydroxyphenyl)-N-propylpiperidine but with lower potency. These results demonstrate (S)-(–)-3-(3-hydroxyphenyl)-N-propylpiperidine to be a protean agonist at the D₂ receptor and may explain in vivo actions of this ligand.

The dopamine D₂ receptor has been one of the most studied monoaminergic GPCRs, not least because of the affinity of a wide range of antipsychotic agents for this receptor (Akam and Strange, 2004). As with a series of GPCRs that interact with members of the pertussis toxin-sensitive subgroup of G proteins, this receptor is able to initiate signals via each of G_i1, G_i2, G_i3, and G_o1 (Gazi et al., 2003). However, ligand pharmacology can be influenced greatly by the ratio of GPCR to G protein expression (Milligan, 2000), and this can be difficult to define in cells, particularly in studies designed to compare activation and function of different G proteins. One means to overcome this issue is to use GPCR-G protein fusions that ensure a fixed 1:1 stoichiometry of GPCR and G protein (Milligan et al., 2004). Because pertussis toxin-sensitive G proteins are endogenously expressed by all cells, we have also used previously variants of each of G_i1,G_i2,G_i3, and G_o1 that have been rendered insensitive to the ADP-ribosyltransferase activity of pertussis toxin by mutation of the cysteine, four amino acids from the C terminus that is the site of modification, to isoleucine (Bahia et al., 1998; Wise et al., 1999).

By using both fusions of the long isoform of the human dopamine D₂ (D2L) receptor with pertussis toxin-resistant cysteine-isoleucine variants of each of G_i1,G_i2,G_i3, and G_o1 and cell lines stably expressing the D2L receptor in which varying amounts of each G protein can be expressed in an entirely tetracycline-dependent manner, we now demonstrate that both (S)-(–)-3-(3-hydroxyphenyl)-N-propylpiperidine [S-(–)-3-PPP] and p-tyramine are protean ligands at the D2L receptor, being agonists for activation of G_o1 but antagonists/inverse agonists at G_i1,G_i2, and G_i3. Such observations provide further evidence for the concept that the dopamine D₂ receptor can exist in multiple conformational states and indicate that it is possible to selectively control the nature of signals generated by D₂ receptor "agonists." Because individual pertussis toxin-sensitive G proteins are expressed differentially pre- and postsynaptically (Aoki et al., 1992), these observations may be relevant to the reported in vivo actions of S-(–)-3-PPP (Arnt et al., 1983; Hjorth et al., 1983).

	Materials and Methods

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Materials. [³H]Spiperone (65–140 Ci/mmol) was from GE Healthcare (Chalfont St. Giles, Buckinghamshire, UK), and guanosine 5'-O-(3-[³⁵S]thio)triphosphate ([³⁵S]GTPS; 1250 Ci/mmol) was from PerkinElmer Life and Analytical Sciences (Boston, MA). (+)-Butaclamol, dopamine, (–)-quinpirole, m-tyramine, p-tyramine, S-(–)-PPP, (R)-(+)-3-(3-hydroxyphenyl)-N-propylpiperidine [R-(+)-3-PPP], (R)-(–)-10,11-dihydroxy-N-n-propylnorapomorphine (NPA), (R)-(+)-7-hydroxy-DPAT hydrobromide (7-OH-DPAT), and GTPS were purchased from Sigma (Gillingham, Dorset, UK). Spiperone hydrochloride was from Tocris (Bristol, UK). Oligonucleotides were from ThermoElectron (Ulm, Germany), and all materials for tissue culture were from Invitrogen (Paisley, UK). All other reagents were obtained as indicated.

D₂ Dopamine Receptor Subcloning into pcDNA3. D2L was initially in the vector pDEST12.2. D2L cDNA was amplified by PCR using the following primers: sense, AAA AGA ATC CGC CAC CAT GGA TCC ACT GAA TCT GTC C; 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 EcoR I and XhoI and inserted into pcDNA3.

Construction of the Myc-D2L-G-Protein Subunit Fusion Proteins. Pertussis toxin-resistant _2A-adrenoceptor-G-protein fusion proteins had been prepared as described previously (Wise and Milligan, 1997; Cavalli et al., 2000). In brief, Cys³⁵¹ of rat G_i1,G_i3, and G_o1 (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 a created 5' KpnI site and 3' EcoRI site with a NcoI site between receptor and G-protein subunit cDNAs. To create D2L:G protein subunit proteins, the first step was to remove the NcoI site from within the D2L cDNA by site-directed mutagenesis using a QuikChange Mutagenesis kit (Stratagene, La Jolla, CA) and the following primers: sense, 5'-CC GAC CCG TCC CAT CAT GGT CTC CAC AG-3'; antisense, 5'-CT GTG GAG ACC ATG ATG GGA CGG GTC GG-3'. Boldface letters indicate altered bases. The PCR product was then digested with DpnI and transformed into bacteria. In a similar manner, NcoI sites were removed from both the G_i1 and G_o1 cDNAs in the respective _2A-adrenoceptor-G fusion protein cDNAs using the following primers: G_i1 sense, 5'-TT GCC ATC ATT AGA GCG ATG GGG AGA TTG AAA ATC G-3'; antisense, 5'-C GAT TTT CAA TCT CCC CAT CGC TCT AAT GAT GGC AA-3'; and G_o1 sense, 5'-CC ATT GTG CGG GCG ATG GAT ACT CTG GG-3'; antisense, 5'-CC CAG AGT ATC CAT CGC CCG CAC AAT GG-3'.

Myc-D2L (NcoI-) was amplified by PCR using the following primers: sense, 5'-AGA ACG GGG TAC CTT ATG GAA CAA CAA AAA CTT ATT TCT GAA GAA GAT CTG GAT CCA CTG AAT CTG TCC TGG TAT GAT G-3'; antisense, 5'-AAAAAAAACCAT GGAGTGGAGGATCTTCAGGAAGGC-3'. Underlined bases indicate introduced restriction sites (sense, KpnI; antisense, NcoI), bases in bold-face type indicate introduced N-terminal Myc tag. The PCR fragment was digested using KpnI and NcoI.

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 Flag-D2L PCR fragment to create the four D2L:G-protein subunit fusion proteins.

Flp-In Constructs. Previously, pertussis toxin-resistant mutants of rat G_i1,G_i3, and G_o1 were created by mutation of Cys³⁵¹ to isoleucine (Cys³⁵² for G_i2) by site-directed mutagenesis. These 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 the D2L receptor in inducible cell lines, the appropriate cells were further transfected with the D2L receptor 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 doxycycline 24 to 48 h before assay to induce the expression of G protein subunits 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 and 0.1 mM EDTA, pH 7.4). Cell suspensions were then homogenized using an Ultra Turrax for 3 x 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, and 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, and 8 mM Na₂HPO₄). The filters were soaked in 3 ml of scintillation fluid, and radioactivity present was determined by liquid scintillation spectrometry.

View larger version (32K): [in this window] [in a new window]   Fig. 1. G protein fusion proteins identify agonists and protean ligands at the D2L receptor. A, a series of fusion proteins was generated by linking the cysteine-isoleucine (C-I) mutant, pertussis toxin-insensitive variants of Gi1,Gi2,Gi3, and Go1 in frame with the C-terminal tail of the human D2L receptor. B, HEK293 cells were mock-transfected or transfected to express D2L-Go1 transiently and treated (+) or not (–) with pertussis toxin (PTX) (25 ng/ml, 24 h). Membranes of these cells were used in [35S]GTPS binding studies in the absence () or presence () of 100 µM dopamine. C, each of the fusion proteins D2L-Gi1 (i), D2L-Gi2 (ii), D2L-Gi3 (iii), and D2L-Go1 (iv) 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-(–)-3PPP (), quinpirole (), NPA (), and 7-OH DPAT ()] reported to have affinity and efficacy at dopamine D2 receptors. Data are representative, and full details are provided in Table 2.

[³⁵S]GTPS Binding Assays. Cell membranes (10 µg) were incubated in 900 µl of buffer (20 mM HEPES, 100 mM NaCl, 6 mM MgCl₂, and 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 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. For antagonist dose-response assays, an EC₅₀ concentration of dopamine was added along with various concentrations of antagonist.

[³⁵S]GTPS Binding Assay: Agonist Stimulation of [³⁵S]GTPS Binding by Fusion Proteins. [³⁵S]GTPS binding assays were performed at room temperature in 384-well format. Membranes (10 µg/point) were diluted to 0.4 mg/ml in assay buffer (20 mM HEPES, 100 mM NaCl, and 10 mM MgCl₂, pH 7.4) supplemented with saponin (10 mg/l) and 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 hours. Bound [³⁵S]GTPS was determined by scintillation counting on a ViewLux ultraHTS Microplate Imager (PerkinElmer).

Data Analysis. Data were analyzed using Prism software (GraphPad Software Inc., San Diego, CA).

	Results

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Studies with Dopamine D₂ Receptor-G Protein Fusions. It has been established previously that the dopamine D₂ receptor is able to interact with and activate each of the pertussis toxin-sensitive G proteins G_i1, G_i2, G_i3, and G_o1 (Gazi et al., 2003). Because particularly G_i2 and G_i3 are expressed endogenously by virtually all cells and we wished to examine potential variations in the ability of ligands at the D2L receptor to activate the different G proteins, cysteine to isoleucine, pertussis toxin-insensitive mutants of the subunit of each of G_i1,G_i2,G_i3, and G_o1 (Wise et al., 1999) were linked in-frame with the C-terminal tail of the human D2L (Fig. 1A). This ensured that the stoichiometry of receptor to G protein would be identical for each G protein to be studied and that the relative cellular distribution and orientation of receptor and G protein would be uniform. Each fusion protein was expressed transiently in HEK293 cells. After pertussis toxin treatment (25 ng/ml, 24 h) of these cells to cause ADP-ribosylation of the endogenously expressed forms of "G_i" and membrane preparation, saturation binding assays using [³H]spiperone indicated each fusion protein to be expressed to similar levels and to bind this ligand with similar and high affinity (Table 1). Membranes of HEK293 cells mock-transfected or transfected to express D2L-Cys³⁵¹Ile G_o1 and treated or not with pertussis toxin were used in [³⁵S]GTPS binding studies. In the absence of D2L-Cys³⁵¹Ile G_o1, binding of the nucleotide was low, essentially unaffected by pertussis toxin treatment, and not modulated by the addition of dopamine (Fig. 1B). With expression of D2L-Cys³⁵¹Ile G_o1, [³⁵S]GTPS binding in the absence of dopamine was increased, and this level was increased substantially further in the presence of dopamine (Fig. 1B). Pertussis toxin treatment produced a small decline in dopamine-stimulated [³⁵S]GTPS binding, consistent with the D2L receptor within the fusion being able to access endogenously expressed G proteins, but after pertussis toxin treatment, the elevation of [³⁵S]GTPS binding by dopamine remained robust (Fig. 1B), indicating direct activation of the fused G protein by the D2L receptor. Equivalent [³⁵S]GTPS binding assays demonstrated all of the fusion proteins to be activated in a pertussis toxin-insensitive manner by dopamine, NPA, quinpirole, m-tyramine, and R-(+)-3-PPP (Fig. 1C), although compared with dopamine, only NPA was a full agonist at each construct, and potency of the individual ligands varied significantly at the various fusion constructs (Table 2). With the exception of NPA and 7-OH DPAT, potency of the ligands was greatest for D2L-Cys³⁵¹Ile G_o1 (Table 2), whereas the potency of quinpirole was particularly low at D2L-Cys³⁵²Ile G_i2 (Table 2). Unlike the ligands mentioned above, although both p-tyramine and S-(–)-3-PPP displayed agonism at the D2L-Cys³⁵¹Ile G_o1 fusion (Table 2), they did not enhance [³⁵S]GTPS binding to any of the other fusions. Although p-tyramine displayed greater agonist efficacy than S-(–)-3-PPP at the D2L-Cys³⁵¹Ile G_o1 fusion, subsequent detailed studies used S-(–)-3-PPP because its potency as an agonist at D2L-Cys³⁵¹Ile G_o1 was 300-fold greater than p-tyramine (Table 2).

The ability of dopamine to compete with [³H]spiperone to bind to the various D2L-G protein fusions (Fig. 2) was best fit by a two-site model in which between 30 and 50% of the sites displayed higher affinity (pK_h = 7.1–7.7) and the remainder lower affinity (pK_l = 5.6–5.8) for dopamine (Table 3). The presence of 100 µM GTP in such assays resulted in this competition becoming monophasic (pK = 5.5–5.9) in each case (Table 3). By contrast, the ability of S-(–)-3-PPP to compete with [³H]spiperone (Fig. 2) was monophasic in the absence of GTP and essentially unaffected by the presence of GTP (pK = 6.2–6.5) for each of the fusions except for D2L-Cys³⁵¹Ile G_o1 in which a biphasic competition curve (pK_h = 8.4, pK_l = 6.2) was converted to a monophasic curve (pK = 6.3) in the presence of GTP (Table 3).

View larger version (34K): [in this window] [in a new window]   Fig. 2. In ligand binding studies, S-(–)-3PPP displays both low- and high-affinity states only at the D2L-Go1 fusion protein. Membranes of pertussis toxin-treated HEK293 cells expressing D2L-Gi1 (A), D2L-Gi2 (B), D2L-Gi3 (C), and D2L-Go1 (D) were used in competition binding studies using 0.1 nM [3H]spiperone and varying concentrations of either dopamine (i) or S-(–)-3PPP (ii). The assays were performed in the absence () or presence () of 100 µM GTP. In the absence of GTP, both high- and low-affinity states for dopamine were observed for each fusion protein, whereas this was only true for S-(–)-3PPP at the D2L-Go1 fusion protein. The presence of GTP converted each competition curve to a single monophasic state displaying low affinity for either dopamine or S-(–)-3PPP.

To explore the details of this apparent protean (Kenakin, 2001) characteristic of S-(–)-3-PPP at the D2L, we compared effects at D2L-Cys³⁵¹Ile G_o1 and D2L-Cys³⁵²Ile G_i2 because G_i2 and G_o1 have the lowest sequence identity among the four G proteins studied, and the observed variation in ligand potency for activation of G_o1 and the other G proteins was most consistent for G_i2. Increasing concentrations of S-(–)-3-PPP inhibited the capacity of an EC₅₀ concentration of dopamine to enhance binding of [³⁵S]GTPSto both fusion constructs with pIC₅₀ = 4.68 ± 0.07 (D2L-Cys³⁵²Ile G_i2) and 5.09 ± 0.12 (D2L-Cys³⁵¹Ile G_o1) (Fig. 3A). However, the maximal effect of S-(–)-3-PPP at D2L-Cys³⁵¹Ile G_o1 in this assay confirmed its partial agonist action at G_o1 because it failed to reduce binding of [³⁵S]GTPS to the level observed in the absence of dopamine. By contrast, at D2L-Cys³⁵²Ile G_i2, S-(–)-3-PPP completely blocked dopamine stimulation of [³⁵S]GTPS and, indeed, acted as an efficacious inverse agonist (Fig. 3A). Spiperone is frequently described as an inverse agonist at the dopamine D₂ receptor, and accordingly, spiperone also acted as an effective inverse agonist at D2L-Cys³⁵²Ile G_i2 (Fig. 3B). Furthermore, this ligand also completely reversed the effect of dopamine at D2L-Cys³⁵¹Ile G_o1 (Fig. 3B). Further studies demonstrated that both spiperone and S-(–)-3-PPP also acted as antagonists/inverse agonists at D2L-Cys³⁵¹Ile G_i1 and D2L-Cys³⁵¹Ile G_i3 (Fig. 3, C and D).

View larger version (30K): [in this window] [in a new window]   Fig. 3. S-(–)-3PPP is an agonist at D2L-Go1 but an antagonist/inverse agonist for the other D2L-G protein fusion proteins. A and B, membranes from pertussis toxin-treated HEK293 cells expressing either D2L-Go1 () or D2L-Gi2 () were used in [35S]GTPS binding studies. Dopamine at an EC50 concentration for each fusion (0.3 µM D2L-Go1,3 µM D2L-Gi2) along with varying concentration of either S-(–)-3PPP (A) or spiperone (B) were used. On the y-axis, 100 is the stimulation produced by dopamine in the absence of a second ligand, and 0 the basal activity in the absence of ligands. C and D, experiments equivalent to those of A and B were performed with membranes expressing D2L-Gi1 () or D2L-Gi3 (). C, studies with S-(–)-3PPP. D, studies with spiperone.

View larger version (29K): [in this window] [in a new window]   Fig. 4. Characterization of Flp-In T-REx cells harboring pertussis toxin-insensitive mutant G proteins at the Flp-In locus. Flp-In T-REx cell lines were established with either Cys351Ile Gi1, Cys352Ile Gi2, Cys351Ile Gi3, or Cys351Ile Go1 cloned into the Flp-In locus. These cells were further transfected to constitutively and stably express the D2L receptor (see Table 4 for details). These cells were treated with concentrations of tetracycline (TET) between 0 and 1.0 µg/ml for 24 h. Cell membranes were then prepared, resolved by SDS-polyacrylamide gel electrophoresis, and immunoblotted to detect each individual G protein (top). Densitometric scans were used to quantitate relative expression levels of the G proteins (bottom).

View larger version (21K): [in this window] [in a new window]   Fig. 5. D2L receptor stimulates [35S]GTPS binding to Cys352Ile Gi2 in membranes of pertussis toxin-treated Flp-In T-REx cells only when G protein expression is induced. Flp-In T-REx HEK293 cells stably expressing the D2L receptor and harboring Cys352Ile Gi2 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 and the effect of 10 µM dopamine on this (A), and to measure the ability of various concentrations of dopamine () or S-(–)-3PPP () to modulate [35S]GTPS binding after induction of Cys352Ile Gi2 (B). ***, dopamine enhanced binding of [35S]GTPS, p < 0.001.

View larger version (19K): [in this window] [in a new window]   Fig. 6. D2L receptor stimulates [35S]GTPS binding to Cys351Ile Go1 in membranes of pertussis toxin-treated Flp-In T-REx cells only when G protein expression is induced. Flp-In T-REx HEK293 cells stably expressing the D2L receptor and harboring Cys351Ile Go1 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 and the effect of 10 µM dopamine on this (A) and to measure the ability of various concentrations of dopamine () or S-(–)-3PPP () to modulate [35S]GTPS binding after induction of Cys351Ile Go1 (B). **, dopamine enhanced binding of [35S]GTPS, p < 0.01.

The ability of dopamine to compete with [³H]spiperone to bind the D2L receptor was monophasic, of low-affinity, and insensitive to GTP in membranes of pertussis toxin-treated cells not induced to express Cys³⁵²Ile G_i2 (Fig. 7A). However, tetracycline induction of Cys³⁵²Ile G_i2 expression resulted in the appearance of a high-affinity site for dopamine that was eliminated in the presence of GTP (Fig. 7A). By contrast, both with and without tetracycline induction of Cys³⁵²Ile G_i2 expression, the capacity of S-(–)-3-PPP to compete with [³H]spiperone was monophasic and unaffected by the presence of GTP (Fig. 7B). In equivalent membranes of pertussis toxin-treated cells that allowed inducible expression of Cys³⁵¹IleG_o1, dopamine again identified both high- and low-affinity sites in [³H]spiperone competition binding studies only after treatment with tetracycline (Fig. 8A). As anticipated, the high-affinity site was absent in the presence of GTP (Fig. 8A). It is interesting that although not as pronounced as with dopamine, S-(–)-3-PPP also identified both high- and low-affinity states in membranes of cells induced to express Cys³⁵¹IleG_o1 (Fig. 8B).

View larger version (25K): [in this window] [in a new window]   Fig. 7. A high-affinity binding site for dopamine but not for S-(–)-3PPP appears with induced expression of Cys352Ile Gi2. Flp-In T-REx HEK293 cells stably expressing the D2L receptor and harboring Cys352Ile Gi2 at the Flp-In locus were treated with (filled symbols) or without (open symbols) tetracycline and pertussis toxin as in Fig. 5. Membranes from these cells were employed in competition binding assays using 0.1 nM [3H]spiperone and varying concentrations on either dopamine (A) or S-(–)-3PPP (B) in the absence (squares) or presence (circles) of 100 µM GTP.

View larger version (26K): [in this window] [in a new window]   Fig. 8. A high affinity binding site for both dopamine and for S-(–)-3PPP appears with induced expression of Cys351Ile Go1. Flp-In T-REx HEK293 cells stably expressing the D2L receptor and harboring Cys351Ile Go1 at the Flp-In locus were treated with (filled symbols) or without (open symbols) tetracycline and pertussis toxin as in Fig. 5. Membranes from these cells were used in competition binding assays using 0.1 nM [3H]spiperone and varying concentrations on either dopamine (A) or S-(–)-3PPP (B) in the absence (squares) or presence (circles) of 100 µM GTP.

Finally, we explored the pharmacology of both spiperone and S-(–)-3-PPP in membranes of cells expressing the D2L receptor and induced to express either Cys³⁵²Ile G_i2 (Fig. 9A) or Cys³⁵¹IleG_o1 (Fig. 9B). Dopamine-mediated stimulation of [³⁵S]GTPS binding was completely reversed by both spiperone and S-(–)-3-PPP when Cys³⁵²Ile G_i2 was the target (Fig. 9A), but whereas spiperone also fully reversed dopamine stimulation of [³⁵S]GTPS binding to Cys³⁵¹IleG_o1 (Fig. 9A), S-(–)-3-PPP produced only partial inhibition (Fig. 9B).

View larger version (24K): [in this window] [in a new window]   Fig. 9. S-(–)-3PPP is an antagonist of D2L-mediated activation of Cys352Ile Gi2 but a partial agonist for Cys351Ile Go1. Flp-In T-REx HEK293 cells stably expressing the D2L receptor and harboring Cys352Ile Gi2 (A) or Cys351Ile Go1 (B) at the Flp-In locus were treated with tetracycline and pertussis toxin as in Fig. 5. Dopamine (0.1 µM for Cys351Ile Go1, 1 µM for Cys352 Ile Gi2) was used to stimulate [35S]GTPS binding, and the effects of varying concentrations of spiperone () or S-(–)-3PPP () on this were assessed. As in Fig. 3, on the y-axis, 100 is the stimulation produced by dopamine in the absence of a second ligand, and 0 the basal activity in the absence of ligands.

	Discussion

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Studies of potential selectivity are hampered by the coexpression of a number of these G proteins in essentially all mammalian cells and tissues. One means to overcome this has been to use insect cell systems (Clawges et al., 1997; Cordeaux et al., 2001) in which levels of expression, or the sequence conservation, of such G proteins is low. When mammalian G proteins are introduced into such cells along with a GPCR of interest, activation is largely restricted to the exogenous G protein. An alternative that allows the use of mammalian cell lines has been to use pertussis toxin-resistant variants (Ghahremani et al., 1999; Wise et al., 1999) in which the cysteine residue that is the target for toxin-mediated ADP-ribosylation is altered to another amino acid but retains the capacity to interact with GPCRs. When such mutants are expressed in mammalian cells, treatment of the cells with pertussis toxin results in ADP-ribosylation of the endogenously expressed forms of G_i and an inability of GPCRs to cause their activation. Studies detailed previously on G_i1 replaced the relevant cysteine with every other natural amino acid and assessed the impact on GPCR-mediated activation (Bahia et al., 1998), and similar studies were subsequently performed with G_i3 (Dupuis et al., 2001).

However, these widely used approaches are still not ideal for detailed studies on differential agonist actions at different G proteins if expression levels are not carefully controlled. Most importantly, although it is well-established that alterations in GPCR-to-G protein ratios can alter ligand function and receptor pharmacology (Milligan, 2000), this can be a challenge to control. For example, in previous studies examining interactions between the D2L and different G protein subunits in insect Sf9 cells, receptor-to-G protein ratios ranging from 1:3 to 1:14 were reported for the different G proteins (Gazi et al., 2003). In the current studies, we therefore combined the use of pertussis toxin-resistant forms of the various G_i-like G proteins with both GPCR-G protein fusion technologies (Milligan et al., 2004) and with the inducible expression of individual G proteins from a single defined site of chromosomal integration in Flp-In T-REx HEK293 cells (Ellis et al., 2006; Milasta et al., 2006).

Although an artificial system, GPCR-G protein fusions can greatly improve signal-to-background in [³⁵S]GTPS binding studies (Milligan, 2003; Milligan et al., 2006), and they ensure the same receptor to G protein stoichiometry for each construct. Although the Flp-In T-REx HEK293 cells cannot ensure exactly the same level of expression of each G protein, each G protein is produced from the same single chromosomal location, and this overcomes the potential for different clones to have more than a single site of integration of the cDNA of interest and that different sites of integration might alter the effectiveness of expression. Furthermore, the inducible nature of expression from this locus, combined with the use of pertussis toxin-insensitive mutants and pertussis toxin pretreatment, provided a null background for expression of each G protein.

As anticipated, the majority of dopamine D₂ receptor agonists stimulated [³⁵S]GTPS binding to all of the four G proteins, although, as reported by others (Gazi et al., 2003), significant variation in potency and efficacy could be observed. However, although both p-tyramine and S-(–)-3PPP were agonists, at Cys³⁵¹IleG_o1, they both failed to act in this manner for the other three G subunits. The potency of p-tyramine was sufficiently low to limit its usefulness for detailed studies. The higher potency of S-(–)-3PPP, however, allowed concentration-response curves to demonstrate that it was able to fully inhibit dopamine-stimulated binding of [³⁵S]GTPS to Cys³⁵²Ile G_i2 and, indeed, acted as an inverse agonist. By contrast, even at maximally effective concentrations, S-(–)-3PPP was unable to fully reverse dopamine-stimulated binding of [³⁵S]GTPS to Cys³⁵¹Ile G_o1, consistent with the direct measures of its partial agonist activity. In competition studies using two agonist ligands of varying efficacy, full receptor occupancy by the ligand with lower efficacy is expected to result in a direct measure of the efficacy of that ligand.

In further support of the protean effect of S-(–)-3PPP at different G proteins, ligand binding studies identified both high- and low-affinity states of the D2L receptor for dopamine with each of the four G proteins but distinct high- and low-affinity states of the receptor for S-(–)-3PPP only for Cys³⁵¹Ile G_o1. Similar results were obtained using both the receptor-G protein fusions and cells able to produce the G protein of choice on demand. Indeed, the Flp-In T-REx cells were particularly useful in this regard because, with pertussis toxin treatment but without tetracycline-induction of expression of an appropriate G protein, all of the [³H]spiperone binding sites displayed monophasic and low-affinity interactions with both dopamine and S-(–)-3PPP, whereas induction of expression resulted in the development of a high-affinity state for dopamine, no matter which of the G proteins was expressed. By contrast, only expression of Cys³⁵¹Ile G_o1 resulted in the appearance of a high-affinity state for S-(–)-3PPP.

In general, the potency of the ligands used was higher for activation of G_o than for G_i2. There were not statistically valid differences between the values obtained for G_i1,G_i2, and G_i3 for enough compounds to allow us to convincingly state rank-order potency differences (which might reflect selective stabilization of distinct states of the receptor) for interactions with G_i1 versus G_i2, for example. This may reflect that the amino acid sequence identities for G_i1,G_i2, and G_i3 are all between 86 and 94%, whereas for each of these against G_o, sequence identity lies between 70 and 73%. It might, therefore, be postulated that greater variation in ligand conformational states could be observed for interactions with G_o versus the others rather than between G_i1, G_i2, and G_i3.

These studies may have implications for the action of S-(–)-3PPP. This ligand has been described to have agonist and antagonist properties in physiologically relevant end-points (Arnt et al., 1983; Hjorth et al., 1983). Of course, one explanation of such observations might relate to its partial agonist function at G_o1 and antagonist/inverse agonist function at G_i1,G_i2, and G_i3. In two studies with S-(–)-3-PPP, patients with schizophrenia showed improvements in both positive and negative symptoms but a limited duration of effectiveness (Lahti et al., 1998). It has been postulated that this is due to the action of S-(–)-3-PPP as a D₂-like dopamine receptor partial agonist and that this would have a "dopamine system stabilization effect," that is, normalization of both dopamine hypo- and hyperactivity in the pathologically affected dopaminergic tracts observed in patients with schizophrenia (Lieberman, 2004). Likewise, the atypical antipsychotic aripiprazole, which now has Food and Drug Administration approval for the treatment of schizophrenia, has also been characterized as a partial agonist at D₂ receptors and again to have a dopamine stabilization effect (Cosi et al., 2006). However, the intrinsic activity and potency of aripiprazole at the D₂ receptor is both cell line- and assay-dependent. For example aripiprazole has been shown to be a partial agonist for inhibition of cAMP accumulation in a Chinese hamster ovary cell line but an antagonist for [³⁵S]GTPS binding. Most significant, however, is the observation that, like S-(–)-3-PPP, this drug is reported to antagonize postsynaptic D₂ receptors but partially activate presynaptic autoreceptors (Kikuchi et al., 1995). In agreement with these findings, a recent study has demonstrated the differential signaling of aripiprazole for several D2L-mediated pathways (Urban et al., 2007). These observations are consistent with aripiprazole, like S-(–)-3-PPP, having differential pharmacology at different signaling pathways, and it will be interesting to ascertain whether aripiprazole has similar protean characteristics in terms of G protein coupling. It will now be interesting to explore the more general contribution of protean effects of clinically relevant ligands that seem to target the same receptor.

	Acknowledgements

 
J. Robert Lane thanks the Biotechnology and Biosciences Research Council for a CASE studentship.

	Footnotes

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

doi:10.1124/mol.106.032722.

ABBREVIATIONS: GPCR, G protein-coupled receptor; 7-OH-DPAT, R-(+)-7-hydroxy-2-dipropylaminotetralin hydrobromide; D2L, long isoform of the human dopamine D₂ receptor; [³⁵S]GTPS, guanosine 5'-O-(3-[³⁵S]thio)triphosphate; NPA, R-(–)-10,11-dihydroxy-N-n-propylnorapomorphine; R-(+)-3-PPP, (R)-(+)-3-(3-hydroxyphenyl)-N-propylpiperidine; S-(–)-3-PPP, (S)-(–)-3-(3-hydroxyphenyl)-N-propylpiperidine; PCR, polymerase chain reaction; HEK, human embryonic kidney.

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

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