Introduction

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5-HT_2C receptors play a major role in the etiology and treatment of affective disorders, anxious states, schizophrenia, and Parkinson's disease (Jenck et al., 1998; Fox and Brotchie, 1999; Meltzer, 1999). They are coupled to phospholipase C (PLC), the activity of which is generally determined by quantification of levels of inositol phosphate and/or intracellular calcium (Conn et al., 1986; Sanders-Bush and Canton, 1995; Porter et al., 1999; Cussac et al., 2000, 2002). In addition to PLC, 5-HT_2C receptors control other transduction systems including cGMP formation, adenylyl cyclase, and phospholipase A₂ (PLA₂) (Kaufman et al., 1995; Lucaites et al., 1996; Berg et al., 1998). Several isoforms of 5-HT_2C receptors, which arise from differential extents of RNA editing, are differentially coupled to PLC as revealed by differing efficacies and potencies of agonists; furthermore, the nonedited INI isoform of 5-HT_2C receptors exhibits constitutive activity (Burns et al, 1997; Backstrom et al., 1999; Herrick-Davis et al., 1999; Wang et al., 2000; Berg et al., 2001).

The efficacy of agonists at 5-HT_2C receptors differs for their stimulation of PLC compared with PLA₂ activity and cGMP production (Berg et al., 1998, 2001; Miller et al., 2000). This suggests that agonists induce different receptor conformations, preferentially engaging specific effector pathways. This property, denoted "agonist-directed trafficking of receptor signaling" (Kenakin, 1995), has been reported for other families of G protein-coupled receptor (GPCR), such as cannabinoid receptors, _2A-adrenoceptors, and adenosine receptors (Bonhaus et al., 1998; Brink et al., 2000; Cordeaux et al., 2000; Kukkonen et al., 2001). Although several studies have investigated agonist-directed trafficking at the G protein level (Gettys et al., 1994a; Yang and Lanier, 1999; Cordeaux et al., 2000; Wenzel-Seifert and Seifert, 2000; Akam et al., 2001), the precise nature of G proteins activated by 5-HT_2C receptors remains to be identified.

In fact, although 5-HT_2C receptors are "classically" considered to couple to Gq, only a few studies have directly demonstrated this. For example, stimulation of [³⁵S]GTPS binding was observed in membranes of Sf9 insect cells expressing high levels of 5-HT_2C receptors reconstituted with exogenous Gq proteins (Hartman and Northup, 1996). More recently, cell-permeable peptides mimicking the C-terminal component of Gq were shown to block activation of native 5-HT_2C receptors in the choroid plexus (Chang et al., 2000). In addition to Gq, when using pertussis toxin (PTX), which uncouples GPCR receptors from Gi/o protein subtypes, it has been shown that 5-HT_2C receptors interact with PTX-sensitive Gi/o proteins controlling 1) DNA synthesis and proliferation in NIH-3T3 cells (Westphal and Sanders-Bush, 1996), 2) adenylyl cyclase activity in an AV12 cell line (Lucaites et al., 1996), and 3) membrane currents in Xenopus laevis oocytes (Quick et al., 1994). Moreover, as with X. laevis oocytes, direct coupling of 5-HT_2C receptors to Gi₁ and Go was shown by an antisense strategy (Chen et al., 1994; Quick et al., 1994). Finally, in human embryonic kidney (HEK) 293 cells expressing high levels of 5-HT_2C receptors, 5-HT was found to stimulate [³⁵S]GTPS binding to Gi proteins (Alberts et al., 1999).

In light of the above observations, the goal of the present study was to characterize the coupling of 5-HT_2C receptors to PTX-insensitive compared with PTX-sensitive G proteins in CHO cell membranes expressing a high level of the VSV (edited) isoform of human 5-HT_2C (h5-HT_2C) receptors, which is preferentially expressed in the central nervous system (Herrick-Davis et al., 1999; Wang et al., 2000). In addition to PTX, selective antibodies directed against different G protein subtypes can be used to identify their roles, for example, in immunoprecipitation assays of [³⁵S]GTPS labeled-G proteins and by uncoupling GPCRs from their respective G proteins (Lledo et al., 1992; Izenwasser and Côté, 1995; Alberts et al., 1999; Newman-Tancredi et al., 1999). However, such techniques are time consuming. Thus, as originally described by DeLapp et al. (1999), we used an antibody capture assay together with a detection technique employing anti-IgG-coated scintillation proximity assay (SPA) beads. This technique permitted measurement of the stimulation of Gq/11 and Gi₃ proteins in response to h5-HT_2C receptor activation induced by different agonists, such as 2(S)-1-(6-chloro-5-fluoro-1H-indol-1-yl)-2-propanamine fumarate (Ro600175) and lisuride, as well as the previously described hallucinogenic compounds LSD and 1-2,5-dimethoxy-4-iodophenyl-2-aminopropane (DOI) (Glennon, 1996).

Differential coupling of GPCRs to distinct G proteins is known to be influenced by the presence of receptor reserve (Brink et al., 2000; Cordeaux et al, 2000), and we have previously shown that h5-HT_2C receptors display a substantial receptor reserve for activation of PLC (Cussac et al., 2002). We therefore examined the influence of receptor reserve at h5-HT_2C receptors on activation of Gq/11 and Gi₃ and agonist-directed trafficking.

	Materials and Methods

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Membrane Preparation of CHO-h5-HT_2C Cells. CHO cells stably expressing ~20 pmol mg¹ of edited h5-HT_2C receptors (VSV isoform) were obtained from Euroscreen (Brussels, Belgium) and grown in adherent culture in 225-cm² flasks with UltraCHO medium (BioWhittaker Europe, Verviers, Belgium) containing sodium pyruvate (1 mM), dialyzed fetal calf serum (0.1%), and geneticin (400 µg/ml). Treatment of cells by pertussis toxin (Sigma-Aldrich, S. Quentin Fallavier, France) was performed overnight at 100 ng/ml. At confluence, cells were washed twice with buffer A containing 20 mM HEPES and 150 mM NaCl, pH 7.4. Cells were harvested from adherent culture and homogenized using a Kinematica Polytron homogenizer (20 s; Basel, Switzerland) in buffer A. The suspension was then centrifuged twice at 20,000g for 20 min at 4°C. The pellet was resuspended in buffer A, and an aliquot (~20 mg of protein per ml) was stored at 80°C until assay.

Competition Binding Assays. Binding affinity at h5-HT_2C receptors was determined essentially as described previously (Cussac et al., 2002) by competition binding with [³H]mesulergine (1 nM; Amersham Biosciences Inc., Saclay, France) in a buffer containing HEPES (20 mM), pH 7.7, EDTA (2 mM), and ascorbic acid (0.1% w/v). Incubations lasted 2 h at 22°C, and nonspecific binding was defined by 5-HT (10 µM). Isotherms were analyzed by nonlinear regression to yield IC₅₀ values. Inhibition constants (K_i values) were derived from IC₅₀ values according to Lazareno and Birdsall (1993).

Measurement of Agonist Efficacy and Antagonist Potency at h5-HT_2C Receptors. Receptor-linked G protein activation by agonists at h5-HT_2C receptors was determined by measuring the stimulation of [³⁵S]GTPS (1332 Ci/mmol; PerkinElmer Life Sciences, Paris, France) binding. CHO-h5-HT_2C membranes (~20-30 µg per well) were preincubated 30 min with agonists and antagonists in a buffer B containing 20 mM HEPES, pH 7.4, 0.1 µM GDP, 50 mM MgCl₂, and 150 mM NaCl, and reaction was started with 0.2 nM [³⁵S]GTPS in a final volume of 200 µl in 96-well plates for 60 min at room temperature. Experiments were terminated by rapid filtration through Unifilter-96 GF/B filters (PerkinElmer) using a Filtermate harvester (PerkinElmer Life Sciences, Boston, MA). Radioactivity retained on the filters was determined by liquid scintillation counting using a TopCount microplate scintillation counter (PerkinElmer Life Sciences). Agonist efficacy is expressed relative to 5-HT, which was tested at a maximal concentration in each experiment. Thus, basal binding (which includes both nonspecific radioactivity detection and endogenous guanine nucleotide turnover) is defined as 0%, whereas 5-HT-stimulated [³⁵S]GTPS binding is defined as 100%. All data are expressed as mean ± S.E.M. of at least three independent determinations.

Characterization of Antibodies Used in SPAs. To verify the specificity of the antibodies used in the SPA procedure, 25 ng of purified recombinant rat Go, Gi₁, Gi₂, Gi₃, Gs, Gq and G13 (Merck Eurolab S.A., Fontenay sous Bois, France) were loaded on 10% polyacrylamide gel and transferred onto nitrocellulose. Immunoblotting of G subunits was performed using the polyclonal anti-Gq/11 (C19) from Santa Cruz Biotechnology (Santa Cruz, CA) (0.4 µg/ml) and the monoclonal antibody anti-Gi₁ from BIOMOL Research Laboratories (Plymouth Meeting, PA) (1 µg/ml), followed by enhanced chemiluminescence detection with horseradish peroxidase as secondary antibody (1/6000) (Amersham Biosciences Inc.).

Scintillation Proximity Assays. Specific activation of different subtypes of G proteins was determined using SPAs essentially as described by DeLapp et al. (1999). [³⁵S]GTPS binding was performed in the same conditions described above but in 96-well optiplates (PerkinElmer Life Sciences). At the end of the incubation period, 20 µl of Nonidet P-40 (0.27% final concentration) was added to each well, and the plates were incubated with gentle agitation for 30 min. Antibodies specific for the G protein -subunit of interest were then added to each well in a volume of 10 µl before 30 min of additional incubation period. The antibodies employed were the polyclonal anti-Gq/11 (1.74 µg/ml final dilution) and the monoclonal antibody anti-Gi₁ (0.87 µg/ml final dilution). SPA beads coated with secondary anti-rabbit or anti-mouse antibodies from Amersham Biosciences UK, Ltd. (Little Chalfont, Buckinghamshire, UK) were added in a volume of 50 µl at a dilution indicated by the manufacturer, and the plates were incubated for 3 h with gentle agitation. The plates were then centrifuged (10 min at 1300g), and radioactivity was detected on a TopCount microplate scintillation counter. Agonist efficacy is expressed relative to that of 5-HT, which was tested at a maximally effective concentration in each experiment (0.1 and 1 µM at Gq/11 and Gi_1,3, respectively). All data are expressed as mean ± S.E.M. of at least three independent determinations.

5-HT_2C Receptor Alkylation with EEDQ. CHO-h5-HT_2C membranes were treated in buffer B with the alkylating agent, EEDQ, at a final concentration of 100 µM for 60 and 90 min at 30°C followed by SPA as described above. The percentage of maximal response as a function of the receptor occupancy (receptor reserve) was determined as described previously (Cussac et al., 2002). Briefly, plots were derived of 1/[A] versus 1/[A']; where [A] and [A'] are equiactive concentrations for stimulation of [³⁵S]GTPS binding before and after receptor alkylation, respectively (90 min of EEDQ treatment for LSD effect at Gq/11 and 60 min of EEDQ treatment for 5-HT, Ro600175, and DOI effect at Gi₃). K_A values were determined by Furchgott analysis; K_A = (slope-1)/y-intercept. Percentage receptor occupancy (O) was calculated by O = 100 × [L/(L + K_A)]; where L is the concentration of agonist. The curve is fitted by a rectangular hyperbola. All data are expressed as means ± S.E. of the mean of three independent determinations performed in triplicate. The level of h5-HT_2C receptor expression after EEDQ treatment was determined by saturation experiments with [³H]mesulergine in parallel with SPA using the same membrane preparation. Protein concentration was determined colorimetrically using a bicinchoninic acid assay kit (Sigma-Aldrich).

Data Analysis. Isotherms were analyzed by nonlinear regression, using GraphPad Prism (GraphPad Software Inc., San Diego, CA) to yield EC₅₀ and IC₅₀ values. K_B values of antagonists for inhibition of 5-HT-stimulated [³⁵S]GTPS binding were calculated according to Lazareno and Birdsall (1993): K_B = IC₅₀/[1 + (Agonist/EC₅₀)], where IC₅₀ = inhibitory concentration₅₀ of antagonist, agonist = concentration of 5-HT, and EC₅₀ = effective concentration₅₀ of 5-HT alone.

Drugs. 5-HT and N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline were purchased from Sigma-Aldrich. Ro600175 and SB242,084 [6-chloro-5-methyl-N-[6-(2-methylpyridin-3-yloxy)pyridin-3-yl]indoline-1-carboxamide] were synthesized by G. Lavielle (Institut de Recherches Servier). DOI and lisuride were purchased from Sigma/RBI (Natick, MA). LSD was supplied by Novartis (Basel, Switzerland).

	Results

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[³⁵S]GTPS Binding at CHO-h5-HT_2C Cell Membranes. At CHO-h5-HT_2C cell membranes, 5-HT elicited an increase in [³⁵S]GTPS binding typically by 1.4- to 1.6-fold with an EC₅₀ of ~10 nM (Fig. 1; Table 1). The 5-HT_2C agonists, Ro600175, DOI, LSD, and lisuride, also stimulated [³⁵S]GTPS binding in a concentration-dependent manner, but only Ro600175 behaved as a full agonist compared with 5-HT (100%) (Fig. 1; Table 1). [³⁵S]GTPS binding induced by these agonists was abolished by the selective 5-HT_2C antagonist, SB242,084 (data not shown).

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Agonist stimulation of h5-HT2C receptor-mediated G protein activation. Agonist concentration-response curves at membrane preparation from CHO-h5-HT2C cells treated () or not treated () with PTX. [35S]GTPS binding is expressed as a percentage of maximal stimulation with 5-HT (100%) obtained in the absence of PTX. Points are means of triplicate determinations from representative experiments repeated on at least three occasions. Emax and pEC50 data from these experiments are shown in Table 1.

View this table: [in this window] [in a new window]   TABLE 1 Stimulation of [35S]GTP S binding by agonist at h5-HT2C receptors Agonist efficacies were determined by [35S]GTP S binding at membrane preparation from CHO-h5-HT2C cells treated or not treated with pertussis toxin. Agonist efficacies are expressed relative to that of 5-HT (1 µ M) determined in the absence of pertussis toxin and are means ± S.E.M. of at least three independent experiments.

Specificity of Antibodies: Scintillation Proximity Assays Coupled to [³⁵S]GTPS Binding. To address the issue of the precise nature of activated G protein in CHO-5-HT_2C cells, we used two antibodies raised against Gq/11 or Gi₁ subunits. Fig. 2 shows that the monoclonal antibody anti-Gi₁ did indeed recognize Gi₁ but that it also cross-reacted with the Gi₃ subunit. However, this antibody did not bind with other G subunits of the Gi family (i.e., Gi₂ and Go) (Fig. 2). Previous studies of immunoreactive G subunits in CHO-K1 cells have detected Gi₂ and Gi₃ together with low levels of Go, whereas Gi₁ is undetectable (Dell'Acqua et al., 1993; Raymond et al., 1993; Gettys et al., 1994b). Thus, we can conclude that the anti-Gi_1,3 antibody recognized Gi₃ in CHO cells. The polyclonal antibodies against Gq/11 have already been well characterized by Western blot and immunoprecipitation assays (DeLapp et al., 1999; Mirotznik et al., 2000; Akam et al., 2001). Indeed, in our hands, the polyclonal anti-Gq/11 antibody recognized the Gq subunit with no affinity for other G subunits tested (Fig. 2). Thus, anti-Gi₃ and anti-Gq/11 antibodies were used in antibody-capture assays with SPA detection. 5-HT and Ro600175 induced robust signals in stimulating [³⁵S]GTPS binding at both Gq/11 (typically ~1.7 to 2.2-fold; Fig. 3A) and Gi₃ (~1.6 to 1.9-fold; Fig. 3B) in CHO-h5-HT_2C cells. The stimulation of Gq/11 and Gi₃ induced by 5-HT and Ro600175 was blocked by SB242,084, a selective h5-HT_2C antagonist (Fig. 3, A and B). SB242,084 also reversed Gq/11 and Gi₃ activation induced by DOI and LSD (data not shown). PTX treatment of CHO-h5-HT_2C cells abolished 5-HT-stimulated [³⁵S]GTPS binding at Gi₃ but not at Gq/11 (Fig. 3C). PTX treatment significantly (P <0.05, unpaired t test) reduced the basal value of [³⁵S]GTPS binding to Gi₃ (2516 ± 13 cpm versus 2958 ± 57 cpm under control conditions) but not to Gq/11 (2061 ± 206 cpm versus 1752 ± 62 cpm), probably reflecting PTX-induced stabilization of i₃ heterotrimeric forms that exhibit a lower basal guanine nucleotide exchange. Although the issue falls outside the scope of the present study, the influence of PTX on basal [³⁵S]GTPS binding may also reflect a disruption of constitutive activity of VSV-h5-HT_2C receptors coupled to Gi₃ proteins.

View larger version (52K): [in this window] [in a new window]   Fig. 2.   Immunodetection of G subunits. Purified Go, Gi1, Gi2, Gi3, Gs, Gq, and G13 subunits were separated on a gel and submitted to immunodetection using polyclonal antibody anti-Gq/11 (A) and the monoclonal antibody anti-Gi1 (B) as described under Materials and Methods.

View larger version (26K): [in this window] [in a new window]   Fig. 3.   Agonist stimulation of h5-HT2C receptor-mediated activation of Gq/11 and Gi3 protein. Activation of G proteins was determined with anti-Gq/11 and anti-Gi3 protein antibodies captured, respectively, via secondary anti-rabbit or anti-mouse antibody-coated SPA beads as described under Materials and Methods. A and B, antagonist action of SB242,084 (1 µM) on 5-HT- (1 µM) and Ro600175-stimulated (1 µM) [35S]GTPS binding at Gq/11 and Gi3 protein, respectively. C, [35S]GTPS binding induced by 5-HT (10 µM) was performed on membranes from CHO-h5-HT2C cells treated or not treated with PTX. Results are expressed relative to basal values (100%). Actual basal values for Gq/11 in cpm were 2061 ± 206 and 1752 ± 62 in control and PTX-treated membranes, respectively, and actual basal values for Gi3 in cpm were 2958 ± 57 and 2516 ± 13 in control and PTX-treated membranes, respectively. Bars represent the mean of triplicate determinations from representative experiments repeated on at least three occasions.

[³⁵S]GTPS Binding at Gq/11 and Gi₃ Coupled to h5-HT_2C Receptors: Concentration-Dependent Agonist and Antagonist Actions. With the exception of lisuride, which behaved as a partial agonist (36%), all agonists were as efficacious as 5-HT in stimulating Gq/11 proteins (Fig. 4; Table 2). These E_max values closely resembled those for [³⁵S]GTPS binding induced by agonists in presence of PTX (Table 1). In contrast, Ro600175 and DOI exhibited lower efficacy than 5-HT at Gi₃ protein (P <0.05, unpaired t test). LSD exhibited partial agonism (~30%) compared with 5-HT, lisuride being inactive at Gi₃ proteins (Fig. 4; Table 2). Agonist potency for stimulation of Gi₃ was about 6- to 8-fold less than for Gq/11 activation, except for LSD, which exhibited similar potency for activation of both G proteins (Table 2). The selective 5-HT_2C antagonist SB242,084 antagonized 5-HT-stimulated [³⁵S]GTPS binding at Gq/11 and Gi₃ proteins with similar potency (pK_B values of 8.64 ± 0.04 and 8.69 ± 0.07, respectively; Fig. 5). LSD and lisuride diminished [³⁵S]GTPS binding at Gi₃ and Gq/11 proteins, respectively, to a similar level as that obtained when the ligand was tested alone (Fig. 5). Antagonist potencies of lisuride at Gq/11 and Gi₃ were 7.76 ± 0.10 and 7.25 ± 0.06, respectively, and LSD exhibited a pK_B of 7.47 ± 0.05 at Gi₃ (Fig. 5).

View larger version (24K): [in this window] [in a new window]   Fig. 4.   Concentration-dependent agonist actions at h5-HT2C receptor-mediated Gq/11 and Gi3 protein activation. Agonist concentration-response curves at Gq/11 () and Gi3 () from membrane preparation of CHO-h5-HT2C cells. [35S]GTPS binding is expressed as a percent of maximal stimulation with 5-HT (100%). Points shown are means of triplicate determinations from representative experiments repeated on at least three occasions. Emax and pEC50 data from these experiments are shown in Table 2.

View this table: [in this window] [in a new window]   TABLE 2 Stimulation of [35S]GTP S binding by agonists at h5-HT2C receptors coupled to Gq/11 and Gi3 and comparison with their respective affinities (pKi values). Agonist efficacies at Gq/11 and Gi3 proteins were determined by [35S]GTP S binding coupled to a scintillation proximity assay. Agonist efficacies are expressed relative to that of 5-HT, which was tested at a maximally effective concentration in each experiment (0.1 and 1 µ M at Gq/11 and Gi3, respectively) and are means ± S.E.M. of at least three independent experiments. pKi values were determined as described under Materials and Methods and are means ± S.E.M. of at least three independent experiments.

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Antagonism of h5-HT2C receptor-mediated Gq/11 and Gi3 protein activation. Antagonist concentration-response curves for SB242,084, LSD, and lisuride against 5-HT-stimulated [35S]GTPS binding coupled to a scintillation proximity assay at Gq/11 (A) and Gi3 (B). Points shown are means of triplicate determinations from representative experiments repeated on at least three occasions. Antagonist potencies (pKB values; see Results) were calculated from IC50 values for the inhibition of 5-HT-stimulated (10 nM for Gq/11 and 100 nM for Gi3) [35S]GTPS binding.

h5-HT_2C Receptor Alkylation with EEDQ. EEDQ treatment of membranes (100 µM, 30°C) time dependently decreased the density of h5-HT_2C sites by 3.4- and 7.4-fold after 60 and 90 min of treatment, respectively, as determined by [³H]mesulergine saturation binding (Fig. 6). The K_D value for [³H]mesulergine binding in control membranes (1.38 ± 0.13 nM) was unchanged by EEDQ treatment (1.37 ± 0.08 nM and 1.59 ± 0.15 nM for 60 and 90 min, respectively).

View larger version (17K): [in this window] [in a new window]   Fig. 6.   Time-dependent reduction of h5-HT2C receptor density by EEDQ. A, representative saturation binding isotherms of [3H]mesulergine to control CHO-h5-HT2C membranes compared with those pretreated with EEDQ (100 µM at 30°C for 60 and 90 min). B, Scatchard representation of data from A. Points shown are means of triplicate determinations from representative experiments repeated on three occasions. The mean Bmax values were 18.5 ± 1.0 pmol mg1 without EEDQ and 5.4 ± 0.4 pmol mg1 and 2.5 ± 0.3 pmol mg1 in the presence of EEDQ for 60 and 90 min, respectively, without significant changes in KD values.

Influence of h5-HT_2C Receptor Alkylation with EEDQ On Gq/11 and Gi₃ Activation. The concentration-response curves of 5-HT-, Ro600175-, and DOI-mediated [³⁵S]GTPS binding to Gq/11 were progressively shifted to the right by treatment of CHO-h5-HT_2C cells with EEDQ without a significant alteration in their relative efficacies, suggesting substantial receptor reserve (Fig. 7; Table 3). In contrast, LSD behaved as a partial agonist after EEDQ treatment, stimulating [³⁵S]GTPS binding to Gq/11 by 38% (EEDQ, 90 min) compared with 5-HT (100%), without modification of its potency (Fig. 7; Table 3). The K_A value for LSD at Gq/11 determined by Furchgott analysis was 12.8 ± 3.6 nM. The derived occupancy/response yielded a linear plot with a half-maximal response to LSD at 48 ± 4.5% occupation of h5-HT_2C binding sites, demonstrating the absence of receptor reserve with this ligand for Gq/11 activation (Fig. 8). In the case of Gi₃ activation, EEDQ treatment reduced both the potency of [³⁵S]GTPS binding by agonists as well as relative efficacies (Fig. 7; Table 3), suggesting a lower level of receptor reserve for Gi₃ activation than Gq/11. The partial agonist, LSD, failed to activate Gi₃ after the diminution of functional h5-HT_2C receptors by EEDQ treatment (Fig. 7). The K_A values for 5-HT (248 ± 102 nM), Ro600175 (110 ± 21 nM), and DOI (249 ± 99 nM) determined by Furchgott analysis yielded hyperbolic curves, with the half-maximal response for Gi₃ activation being observed at h5-HT_2C occupancies of 13.0 ± 3.7% for 5-HT, 21.6 ± 2.7% for Ro600175, and 33.7 ± 2.1% for DOI (Fig. 8). The receptor occupancy by DOI required to yield half-maximal Gi₃ activation was significantly greater (P <0.05, unpaired t test) than for 5-HT or Ro600175.

View larger version (24K): [in this window] [in a new window]   Fig. 7.   Influence of h5-HT2C receptor inactivation by EEDQ on agonist-stimulated [35S]GTPS binding at Gq/11 and Gi3 proteins. Agonist concentration-response curves at membrane preparation from control CHO-h5-HT2C cells compared with those treated by EEDQ (100 µM at 30°C) for 60 and 90 min. [35S]GTPS binding is expressed as a percent of maximal stimulation given by 5-HT (100%) in the absence of EEDQ treatment. Points shown are means of triplicate determinations from representative experiments repeated on at least three occasions. Emax and pEC50 data from these experiments are shown in Table 3. , control; , EEDQ 60 min; , EEDQ 90 min.

View this table: [in this window] [in a new window]   TABLE 3 Effect of receptor alkylation on stimulation of [35S]GTP S binding by agonists at h5-HT2C receptors coupled to Gq/11 and Gi3 Agonist efficacies at Gq/11 and Gi3 proteins were determined by [35S]GTP S binding coupled to a scintillation proximity assay at membrane preparation from control CHO-h5-HT2C cells and cells treated by EEDQ for 60 or 90 min. Agonist efficacies are expressed relative to that of 5-HT, which was tested in the absence of EEDQ treatment at a maximally effective concentration in each experiment (0.1 and 1 µ M at Gq/11 and Gi3, respectively) and are means ± S.E.M. of at least three independent experiments.

View larger version (26K): [in this window] [in a new window]   Fig. 8.   Receptor reserve for agonist-stimulated [35S]GTPS binding to Gq/11 and Gi3 proteins. Agonist occupancy/response relationships derived from data of Fig. 6 using KA values calculated for LSD at Gq/11 (90 min of EEDQ treatment) and for 5-HT, Ro600175, and DOI at Gi3 (60 min of EEDQ treatment). The hyperbolic isotherms and linear plots indicate the presence and absence of receptor reserve, respectively. The mean occupation of h5-HT2C binding sites yielding half-maximal responses of agonists are indicated.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Conclusions References

The key findings of the present study are 1) the detection by [³⁵S]GTPS binding of 5-HT_2C receptor-mediated activation of PTX-sensitive and -insensitive G proteins in CHO cell membranes, 2) the demonstration by an antibody capture/SPA detection strategy that 5-HT_2C receptors coupled more efficiently to Gq/11 than to Gi₃, and 3) as revealed by receptor alkylation, this difference reflects a high versus low level of receptor reserve for 5-HT_2C receptors coupled to Gq/11 and Gi₃, respectively.

[³⁵S]GTPS Binding at Whole-Cell Membrane Preparation: Influence of Pertussis Toxin. In cell membrane preparations of CHO cells expressing the VSV isoform of h5-HT_2C receptors, 5-HT stimulated [³⁵S]GTPS binding. Although the selective 5-HT_2C ligand Ro600175 also displayed high efficacy, DOI, LSD, and lisuride behaved as partial agonists. 5-HT-mediated [³⁵S]GTPS binding was sensitive to pertussis toxin, revealing the involvement of Gi/o proteins. This observation is consistent with reports that Gi/o proteins are involved in 5-HT_2C receptor activation (see Introduction). Moreover, specific [³⁵S]GTPS binding to Gi has been observed in HEK 293 cells expressing 5-HT_2C receptors (Alberts et al., 1999). However, although PTX reduced [³⁵S]GTPS binding, it did not abolish it, suggesting that agonists also stimulated PTX-insensitive G proteins. Agonist potencies and efficacies for stimulation of [³⁵S]GTPS binding in the presence of PTX (Table 1) were very similar to those observed for activation of Gq/11 as determined by SPA detection (see below; Table 2). However some [³⁵S]GTPS binding to G₁₂ and G₁₃ proteins, which can also couple to 5-HT_2C receptors (Berg et al., 1999; Price et al., 2001), cannot be excluded. Thus, although direct labeling of Gq by [³⁵S]GTPS binding is difficult to detect, owing to low GTP-turnover rates (Smrcka et al., 1991), 5-HT_2C receptors are able to activate [³⁵S]GTPS binding to Gq/11 proteins in CHO cells (present study) as well as in nonmammalian (insect Sf9) cells (Hartman and Northup, 1996).

5-HT_2C Receptors Are Coupled to Gq/11 and Gi₃ in CHO Cells: SPA Approach. Antibody capture/SPA detection using specific antibodies (see Results) allowed direct measurement of Gq/11 and Gi₃ activation with a robust signal-to-noise ratio even after extensive receptor inactivation by irreversible alkylation (see below). This SPA approach underpinned the above-discussed results concerning the influence of PTX on total [³⁵S]GTPS binding in directly showing that agonists at h5-HT_2C (VSV) receptors preferentially engaged Gq/11. Activation of G proteins was specifically mediated by h5-HT_2C receptors inasmuch as the selective 5-HT_2C antagonist SB242,084 (Cussac et al., 2002) abolished 5-HT-stimulated [³⁵S]GTPS binding at both Gq/11 and Gi₃ subunits, excluding the involvement of endogenously expressed 5-HT_1B receptors in CHO cells (Giles et al., 1996).

Receptor Reserve of h5-HT_2C Receptor VSV Isoform Coupled to Gq/11 and Gi₃. Recently, Brink et al (2000) reported that agonist-directed trafficking at human _2A-adrenoceptors is dependent on the level of receptor expression and, specifically, the presence of receptor reserve. Indeed, receptor number and receptor/G protein stoichiometry, as well as the specific identity of the G proteins activated, probably influence drug efficacies for activation of differing signaling cascades. For h5-HT_2C receptors, no receptor reserve for PLC activation was demonstrated at edited VSV and nonedited INI isoforms expressed in NIH-3T3 cells, despite the relatively high expression levels (~5 pmol/mg) (Burns et al., 1997). In contrast, the VNV isoform of h5-HT_2C receptors expressed in HEK 293 exhibited receptor reserve for PLC activation, LSD behaving as a full agonist with increasing receptor number (Fitzgerald et al., 1999). Although these observations may reflect differential coupling between h5-HT_2C receptor isoforms, variations in receptor/G protein stoichiometry are likely to be of major importance. Correspondingly, we investigated the influence of reducing receptor number (and therefore receptor/G protein stoichiometry) with the alkylating agent EEDQ.

	Conclusions

Top Abstract Introduction Materials and Methods Results Discussion Conclusions References

To summarize, the present study demonstrates that h5-HT_2C receptors (VSV isoform) couple to both Gq/11 and Gi₃ in CHO cells and that these G protein subtypes are recruited in an agonist- and receptor reserve-dependent manner. The differential influence of agonists on G protein coupling at h5-HT_2C receptors may well be relevant to their functional profiles in vivo. It would be of interest to extend these data in characterizing the significance of receptor reserve, receptor/G protein stoichiometry, and G protein subtypes at other isoforms of h5-HT_2C receptors as well as other classes of GPCR that couple to multiple intracellular signals.