Introduction

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Serotonin 5-HT_1A receptors are therapeutic targets in the management of anxiety, depression, and schizophrenia (Barnes and Sharp, 1999; Millan, 2000). Cloning of human 5-HT_1A receptors and their expression in recombinant cell lines has permitted the elucidation of their signal transduction pathways. They couple, thus, to multiple transduction pathways, including adenylyl cyclase, phospholipase C, sodium-dependent phosphate uptake, protein kinase C, Na⁺/K⁺ ATPase, K⁺ channels, and mitogen-activated protein kinase (Fargin et al., 1989; for reviews, see Gerhardt and Heerikhuizen, 1997; Raymond et al., 1999). These cellular responses are abolished by pretreatment with Bordetella pertussis toxin (PTX), which ADP-ribosylates G subunits of the Gi/o family, indicating the coupling of 5-HT_1A receptors to Gi/o-proteins.

Subtypes of G subunits engaged by 5-HT_1A receptors have been identified by coexpression studies in a variety of bacterial, insect, and mammalian expression systems (for review, see Raymond et al., 1999). Thus, reconstitution of h5-HT_1A receptors expressed in Escherichia coli with recombinant G_i3 subunits most markedly increased affinity for the agonist [³H](+)-8-hydroxy-dipropyl-aminotetralin ([³H]8-OH-DPAT), followed by G_i2 and G_i1 subunits (Bertin et al., 1992). A similar reconstitution strategy in insect Spodoptera frugiperda (Sf9) cells likewise showed coupling of 5-HT_1A receptors to Gi/o G-protein subtypes, and particularly to G_i3 (Butkerait et al., 1995; Clawges et al., 1997). Functional responses of 5-HT_1A receptors have also been shown to be mediated by G_i3 and G_i2 subunits. Thus, microphysiometric measurement of medium acidification showed that PTX-resistant mutants of G_i3 or G_i2 "rescued" agonist-induced Na⁺/H⁺ exchange in h5-HT_1A receptor-expressing Chinese hamster ovary (CHO) cells pretreated with PTX (Garnovskaya et al., 1997). Furthermore, antisera raised against G_i3 and G_i2 subunits attenuated h5-HT_1A receptor-mediated inhibition of adenylyl cyclase activity in CHO and HeLa cells (Raymond et al., 1993). Taken together, these studies converge toward the conclusion that 5-HT_1A receptors couple preferentially to G_i3 subunits, followed by G_i2 and, less strongly, G_o and G_i1 subunits (Clawges et al., 1997). On the other hand, coupling of 5-HT_1A receptors to Gq and Gs is weak or absent (Raymond et al., 1993). Nevertheless, coupling to G-protein subtypes can be investigated when receptor/G-protein stoichiometry is constrained [e.g., in h5-HT_1A-G_i1 fusion proteins (Kellett et al., 1999)], and a recent study reported stimulation of adenylyl cyclase by 5-HT_1A receptors at high agonist concentrations, suggesting that Gs activation is, in fact, possible under certain conditions (Malmberg and Strange, 2000).

The issue of differential coupling to G-proteins is of particular interest in view of accumulating reports describing agonist-directed trafficking of receptor signaling (Kenakin, 1995). Thus G-protein-coupled receptors (GPCRs) may exhibit differential coupling to intracellular G-proteins or second messenger systems depending on the agonist employed. For example, agonists at h5-HT_2C receptors display differential orders of efficacy for activation of phospholipase C versus phospholipase A₂ (Berg et al., 1998; for review, see Clarke and Bond, 1998), whereas agonists differentially induce coupling of h_2A-adrenoceptors to Gi- or Gs-mediated modulation of adenylyl cyclase activity (Brink et al., 2000). In the case of 5-HT_1A receptors, Gettys et al. (1994a) undertook a limited study of the potency of different agonists in activating G_i3 and G_i2 subunits, as detected by 4-azidoanilido-[-³²P]GTP labeling, membrane solubilization, and immunoprecipitation of G subunits. They showed that, whereas some agonists (5-HT and 8-OH-DPAT) more potently activated G_i3 than G_i2, rauwolscine activated both subunits with similar potency (Gettys et al., 1994a). However, this technique is cumbersome and ill-adapted to broad investigation of numerous drugs and assay conditions. Furthermore, the molecular basis for these examples of agonist-directed trafficking are little characterized and are probably influenced by multiple factors, including receptor and G-protein expression levels, ionic conditions, etc.

In view of the above considerations, the present study employed a recently developed antibody-capture technique coupled to scintillation proximity assay (SPA) detection (DeLapp et al., 1999) to characterize G_i3 subunit activation at recombinant human 5-HT_1A receptors stably expressed in Chinese hamster ovary cells (CHO-h5-HT_1A cells; Newman-Tancredi et al., 1998). CHO cells constitute a useful model to investigate 5-HT_1A receptor coupling because they express G_i3 and G_i2 (Gerhardt and Neubig, 1991; Law et al., 1993; Gettys et al., 1994b). CHO cells also express low levels of Go, but G_i1 is undetectable (Gerhardt and Neubig, 1991; Gettys et al., 1994b; Law et al., 1993; present study). Using antibody capture/SPA methods, we show here that high-efficacy agonists, but not partial agonists or inverse agonists, exhibit bell-shaped isotherms for G_i3 activation at h5-HT_1A receptors. The data indicate that low concentrations of high-efficacy agonists direct coupling of the receptor to G_i3, whereas, at higher concentrations, this coupling is suppressed. A preliminary report of the present data was presented in abstract form (Newman-Tancredi et al., 2002).

	Materials and Methods

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Membranes Preparations from CHO-h5-HT_1A cells. Membranes from transfected CHO cells stably expressing the human serotonin 5-HT_1A (h5-HT_1A) receptor (~4 pmol/mg) were prepared as described previously (Newman-Tancredi et al., 1998). Briefly, cells were grown in RPMI 1640 medium containing 10% (v/v) fetal bovine serum, penicillin, and streptomycin until they reached confluence. Cells were harvested by centrifugation and homogenized using a Polytron homogenizer (Kinematica, Basel, Switzerland) in buffer A (20 mM HEPES, pH 7.4, and 3 mM MgCl₂). The homogenate was centrifuged at 50,000g for 30 min, the membrane pellet resuspended in buffer A and stored at 80°C. Protein concentration was determined by use of a bicinchoninic acid kit (Sigma-Aldrich, St. Quentin-Fallavier, France).

[³⁵S]GTPS Binding by Filtration Assays. Unless stated otherwise, [³⁵S]GTPS binding detecting "total" G-protein activation (i.e., without distinguishing between G-protein subtypes) was performed essentially as described previously (Newman-Tancredi et al., 1997, 1998). Briefly, CHO-h5-HT_1A membranes (~25 µg) were incubated (60 min, 22°C) in duplicate in a buffer containing 20 mM HEPES, pH 7.4, 3 µM GDP, 3 mM MgCl₂, 100 mM NaCl and 0.2 nM [³⁵S]GTPS (1300 Ci/mmol; PerkinElmer Life Sciences, Boston, MA). Nonspecific binding was defined with 10 µM GTPS. Agonist efficacy is expressed relative to basal (100%). In antagonist tests, the antagonist was preincubated with cell membranes (30 min, 22°C) before addition of [³⁵S]GTPS. Experiments were terminated by rapid filtration through Whatman GF/B filters using a Packard (PerkinElmer) cell harvester and radioactivity determined by liquid scintillation counting.

[³⁵S]GTPS Binding by Antibody Capture and Scintillation Proximity Assay Detection. To specifically detect [³⁵S]GTPS binding to G_i3 G-protein subunits, an antibody-capture strategy was adopted, coupled to detection by scintillation proximity assay (SPA). Procedures were similar to those described by DeLapp et al. (1999). Unless otherwise indicated, CHO-h5-HT_1A cell membranes (~35 µg) were incubated on 96-well plates with agonists and/or antagonists and 0.2 nM [³⁵S]GTPS for 1 h at 22°C in a buffer containing: 20 mM HEPES, pH 7.4, 3 µM GDP, 3 mM MgCl₂, and 100 mM NaCl (i.e., the same buffer composition as for filtration assays described above). Reaction was stopped by solubilizing cell membranes by addition of detergent [Nonidet P40, 0.3% (v/v) final] and gentle agitation for 30 min. Mouse anti-G_i1/3 monoclonal antibodies (Biomol, Plymouth Meeting, PA) were then added (0.1 µg of IgG per well) and the microwell plates incubated for a further 1 h to allow antibody-G complexes to form. Because CHO cells do not express G_i1 (Gettys et al., 1994b; see Introduction), the assay detects activation of G_i3. The specificity of the anti-G_i1/3 antibody itself was verified by Western blots against a range of purified G subunits, indicating an absence of cross-reactivity with G_i2, G_o, G_S, G_q, and G₁₃ (Newman-Tancredi et al., 2002). At the end of the incubation period, SPA beads coated with anti-mouse second antibody (Amersham Biosciences, Little Chalfont, Buckinghamshire, UK), were added at the manufacturer's recommended concentrations and incubated with gentle agitation overnight before radioactivity counting. All incubation steps were carried out at 22°C. Nonspecific binding was defined with 10 µM GTPS. Results are expressed as the mean ± S.E.M. of n determinations. A monoclonal antibody against phosphorylated extracellular signal regulated kinases (pERK; Nanotools, Germany) was employed to exclude nonspecific effects.

Data Analysis. [³⁵S]GTPS binding data were analyzed by nonlinear regression using the program Prism (GraphPad Software Inc., San Diego, CA). For conditions that produced sigmoidal isotherms, values of potency (pEC₅₀) and pseudo-Hill coefficient (n_H) were determined. The maximal observed stimulation (E_max) was defined as the amount of specific [³⁵S]GTPS binding expressed as a percentage of specific basal (agonist-independent) binding (100%). The "goodness of fit" was tested by "runs" test, and one- and two-site fits were compared by F-test. In cases where a two-site fit was more favorable, potency values are shown for the high- (pEC_50H) and low-affinity (pEC_50L) binding components.

[(c_1/2  x) × b_1/2]

n_H¹

Drugs. Clozapine base, ()-pindolol base, (+)-8-OH-DPAT HBr and 4-iodo-N-[2-[4-(methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinyl-benzamide HCl (p-MPPI), (+)-butaclamol HCl, spiperone HCl, and ()-5-fluoro-8-hydroxy-dipropylaminotetralin HCl were obtained from Sigma/RBI (Natick, MA); 5-HT creatinine sulfate, haloperidol base, and buspirone HCl were obtained from Sigma-Aldrich; S15535 base, eltoprazine HCl, and WAY100,635 were synthesized by J. L. Peglion, Servier (Suresnes, France). 5-Carboxyamidotryptamine (5-CT) maleate, S16924, S14506, S14671, (±)-flesinoxan HCl, and ziprasidone HCl were synthesized by G. Lavielle, Servier (Croissy-sur-Seire, France). Methiothepin maleate was from Tocris Cookson (Southampton, England).

	Results

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Total G-Protein Activation Determined by Filtration Experiments

Disruption of h5-HT_1A receptor-G-protein interactions with anti-G_i1/3 antibody. 5-HT concentration-dependently stimulated [³⁵S]GTPS binding to CHO-h5-HT_1A cell membranes by ~2.2-fold relative to basal values (Table 1). The slope factors (pseudo-Hill coefficients) were less than unity (0.72) and a two-site fit was significantly superior to a single-site fit (P < 0.01; F-test) yielding a pEC_50H of 9.09 and a pEC_50L of 7.70 (Table 1). We have previously shown that 5-HT-stimulated [³⁵S]GTPS binding is totally reversed by the selective 5-HT_1A receptor antagonist, WAY100,635 (Newman-Tancredi et al., 1996). When CHO-h5-HT_1A cell membranes were preincubated (2 h, 22°C) with an anti-G_i1/3 monoclonal antibody (0.2 µg/point), the high potency stimulation component was abolished, yielding a monophasic isotherm: pEC₅₀ = 8.15 (Fig. 1B). Further, basal binding was reduced relative to control basal values, suggesting that constitutive G_i3 activation was attenuated. The partial agonist, pindolol, yielded monophasic stimulation isotherms (Table 1; pEC₅₀ = 7.88). Anti-G_1/3 preincubation did not modify the potency of pindolol (pEC₅₀ = 7.79) but markedly reduced its efficacy (Fig. 1C) suggesting that pindolol preferentially induces h5-HT_1A coupling to G_i3 subunits. A monoclonal antibody against pERK did not markedly modify 5-HT- or pindolol-induced [³⁵S]GTPS binding (Fig. 1, A and C).

View this table: [in this window] [in a new window]   TABLE 1 h5-HT1A receptor-mediated stimulation of [35S]GTP S binding: disruption by anti-Gi1/3 antibodies Membranes were preincubated either with buffer (control conditions) or with an antibody against Gi1/3 subunits or against pERK. The full agonist 5-HT or the partial agonist pindolol and [35S]GTPS were then added and incubated for a further 1 h before filtering the membranes and scintillation counting. Isotherms were analyzed by nonlinear regression. Emin values are the basal [35S]GTP S binding observed in the absence of ligand, and are expressed as a percentage of control basal values (100%). Emax values are the maximal observed stimulation (relative to control basal values = 100%). pEC50H and pEC50L are effective concentration values for the high and low potency components. %High values are the percentage of sites in the high-potency component, and nH is the slope of the isotherms. Data are expressed as mean ± S.E.M. of n independent determinations performed in triplicate. In the case of 5-HT, isotherms were biphasic (P < 0.05, F-test), except when preincubated with anti-Gi1/3. Pindolol yielded monophasic isotherms in all cases. Anti-Gi1/3, but not anti-pERK, antibodies reduced basal [35S]GTP S binding from control values.

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Disruption of receptor/G-protein interaction in CHO-h5-HT1A cell membranes by anti-Gi1/3 antibodies. CHO-h5-HT1A cell membranes were preincubated with anti-Gi1/3 monoclonal antibodies (0.2 µg/well) before carrying out [35S]GTPS binding and filtering cell membranes. The presence of the antibody (but not an anti-pERK IgG) reduced constitutive Gi3 activation and blocked the high-affinity 5-HT stimulation component. Top, influence of antibodies on 5-HT-stimulated [35S]GTPS binding. Anti-Gi1/3 antibodies blocked high-affinity stimulation and lowered basal binding. Middle, the same isotherms from A were normalized to show the loss of the high-affinity stimulation component. Bottom, influence of antibodies on pindolol-stimulated [35S]GTPS binding. Anti-Gi1/3 antibodies suppressed stimulation without a change in potency. Data points are means of triplicate determinations from representative experiments repeated on at least three independent occasions. pEC50 and Emax values from these experiments are shown in Table 1.

Influence of 5-HT and Pindolol on Dissociation Kinetics of [³⁵S]GTPS. We investigated the rates of GTP/GDP exchange at G-proteins in CHO-h5-HT_1A membranes by preincubating them with [³⁵S]GTPS and 5-HT (1 h, 22°C) and then adding an excess of unlabeled GTPS (10 µM final concentration). [³⁵S]GTPS binding was time-dependently inhibited at high concentrations (e.g., 10 µM) but not at lower concentrations (e.g., 30 nM) of 5-HT, which stimulated G_i3 (see above). Thus, after 40 min of incubation, a bell-shaped isotherm was observed, with a peak at about 30 nM of 5-HT (Fig. 2A). In comparison, pindolol-induced [³⁵S]GTPS binding (Fig. 2B) was uniformly and progressively diminished by incubation with GTPS (E_max after 40 min with GTPS = 121 ± 3%). No bell-shaped isotherms appeared and the potency of pindolol was unchanged (pEC₅₀ after 40 min with GTPS = 7.85 ± 0.20). The dissociation kinetics of [³⁵S]GTPS were directly assessed by adding GTPS (10 µM) after the standard 1-h incubation period and determining the remaining bound [³⁵S]GTPS after different dissociation times (Fig. 3). [³⁵S]GTPS binding in the presence of 10 µM 5-HT decreased to basal levels within 60 min (t_1/2 = 6.16 ± 0.69 min for a single-site model). Comparison of single- and two-site fits for 10 µM 5-HT revealed that its dissociation isotherm was biphasic (first component, t_1/2 = 2.4 ± 1.2 min, 51 ± 8% of binding sites; second component, t_1/2 = 24.6 ± 7.0 min; p < 0.05 in F-test) suggesting that [³⁵S]GTPS was dissociating from 2 G-protein populations. In contrast, dissociation isotherms with 30 nM 5-HT or 10 µM pindolol (which preferentially induce coupling to G_i3) decreased monophasically and more slowly (t_1/2 = 21.53 ± 3.05 and 8.99 ± 0.48 min, respectively; Fig. 3).

View larger version (34K): [in this window] [in a new window]   Fig. 2.   Bell-shaped isotherms of G-protein activation in CHO-h5-HT1A cell membranes. [35S]GTPS Binding assays were carried out by classical filtration methods. Top, 5-HT stimulated [35S]GTPS binding in a biphasic manner (P <0.01, F-test). Addition of unlabeled GTPS at the end of the incubation period time-dependently suppressed [35S]GTPS binding at high concentrations of 5-HT but not at 30 nM 5-HT. Bottom, pindolol acted as a partial agonist. Addition of unlabeled GTPS at the end of the incubation period time-dependently suppressed [35S]GTPS binding uniformly at all pindolol concentrations. Data points are means of triplicate determinations from representative experiments repeated on at least three independent occasions with similar results.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Dissociation of [35S]GTPS from CHO-h5-HT1A cell membranes. Top, CHO-h5-HT1A cell membranes were preincubated for 60 min with [35S]GTPS and 5-HT (30 nM) or 5-HT (10µM) or no ligand (basal conditions). Unlabeled GTPS was then added, resulting in a more rapid decrease of binding with 10 µM 5-HT (to basal levels) than with 100 nM 5-HT. Bottom, CHO-h5-HT1A cell membranes were preincubated for 60 min with [35S]GTPS and pindolol (10 µM) or spiperone (10 µM). When unlabeled GTPS was added, the decrease in binding paralleled that under basal conditions. Data points are means of triplicate determinations from representative experiments repeated on at least three independent occasions. t1/2 values from these experiments are described in the text.

G_i3 Subunit Activation

The antibody capture/SPA technique was adopted to specifically detect G_i3 subunit activation. The agonist, 5-HT, yielded bell-shaped [³⁵S]GTPS binding isotherms (see Fig. 4 for analysis parameters), with peak stimulation at a concentration of about 5 nM (Table 2). [³⁵S]GTPS binding then gradually diminished to below baseline values at 5-HT concentrations of about 1 µM (Fig. 5). A series of experiments was carried out to verify the specificity of G_i3 detection by comparing the influence of the full agonist 5-HT with that of an efficacious inverse agonist, spiperone (Newman-Tancredi et al., 1997). First, in G_i3/SPA assays, 5-HT and spiperone did not modify [³⁵S]GTPS binding to membranes of untransfected CHO cells (Fig. 5A). Second, when experiments were carried out in the absence of anti-G_i3 antibodies or in the presence of another IgG (monoclonal anti-pERK), no stimulation of [³⁵S]GTPS binding was detected, although a slight loss of signal was observed at high 5-HT concentrations in some experiments (Fig. 5B). Third, we tested SPA beads coated with anti-rabbit IgG (as opposed to anti-mouse IgG). Once again, no stimulation of [³⁵S]GTPS binding was detected (data not shown). Fourth, we pretreated CHO-h5-HT_1A cells overnight with the ADP-ribosylating agent PTX (100 ng/ml). 5-HT failed to induce G_i3 stimulation in membranes prepared from these cells. Similarly, no inhibition of [³⁵S]GTPS binding by the inverse agonist, spiperone, was detected in PTX-pretreated cell membranes (Fig. 5C). Fifth, we tested SPA beads coated with wheat-germ agglutinin instead of IgG. Wheat-germ agglutinin SPA beads bind cell membranes without distinguishing G-protein subtypes. As expected, the resulting isotherms for 5-HT, (+)8-OH-DPAT, pindolol, and spiperone closely resembled (both in potency and efficacy) those observed in parallel standard filtration assays (data not shown). Sixth, we verified the absence of G_i1 subunits in the present CHO cell membranes. A specific polyclonal anti-G_i1 antibody (I20; Santa Cruz Biotechnology, Santa Cruz, CA) detected recombinant purified G_i1 subunits in Western blot experiments but did not detect a band in cell membranes from CHO-h5-HT_1A cells (data not shown), in agreement with reports from other laboratories (Gettys et al., 1994b; see Introduction).

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Bell-shaped and sigmoidal isotherms: parameters analyzed. Top, a theoretical bell-shaped isotherm is shown to illustrate the various parameters analyzed. a1 = [35S]GTPS binding observed in the absence of ligand (basal conditions). a2 = [35S]GTPS binding observed at maximal ligand concentration. c1 = drug concentration inducing half-maximal stimulation. c2 = drug concentration inducing half of the inhibitory effect. d = maximal stimulation of [35S]GTPS binding. Bottom, theoretical stimulation and inhibition isotherms are shown. pEC50 and pIC50 = drug concentrations inducing half-maximal stimulation or inhibition. Emax and Emin = maximal stimulation induced by agonists and maximal inhibition induced by inverse agonists.

View this table: [in this window] [in a new window]   TABLE 2 Bell-shaped isotherms for h5-HT1A receptor-mediated stimulation of [35S]GTP S binding to Gi3 subunits Activation of Gi3 G-protein subunits in CHO-h5-HT1A cell membranes was determined employing an antibody-capture/SPA detection technique. 5-HT and other high-efficacy agonists yielded bell-shaped isotherms. Parameters analyzed are as shown in Fig. 4. Data are expressed as mean ± s.e.m. of n independent determinations performed in duplicate. Ligands are listed according to their capacity to induce bell-shaped isotherms. For comparison, ligand affinity (pKi) is shown, as determined by competition binding with [3H]8-OH-8DPAT (Newman-Tancredi et al., 2001a,b)

View larger version (17K): [in this window] [in a new window]   Fig. 5.   [35S]GTPS binding to Gi3 subunits: G-protein and receptor specificity. h5-HT1A receptor-mediated Gi3 subunit activation was determined by antibody capture/SPA detection. A, detection of 5-HT-stimulated and spiperone-inhibited [35S]GTPS binding to Gi3 subunits in CHO-h5-HT1A cell membranes but not in membranes of untransfected CHO cells. B, detection of 5-HT-stimulated [35S]GTPS binding to Gi3 subunits in CHO-h5-HT1A cell membranes employing a specific monoclonal anti-Gi3 antibody. No stimulation was observed when an anti-MAPK monoclonal or no antibody are used. C, CHO-h5-HT1A cells were pretreated overnight with pertussis toxin (50 ng/ml) before preparing cell membranes. 5-HT-stimulated and spiperone-inhibited [35S]GTPS binding to Gi3 subunits was abolished in PTX-treated but not control CHO-h5-HT1A cell membranes. Data points are means of duplicate determinations from representative experiments repeated on at least three independent occasions with similar results.

Modulation of G_i3 Activation by Buffer/Incubation Components

We varied the standard incubation buffer (3 µM GDP, 3 mM MgCl₂, and 100 mM NaCl) to examine the influence of buffer components on G_i3 subunit activation by the full agonist (5-HT), a partial agonist (pindolol), and an inverse agonist (spiperone). Each parameter was varied individually while the others were maintained at standard concentrations.

Influence of GDP (0.03, 0.3, 3.0, and 30 µM). GDP concentration influences the rate of GTP/GDP exchange by G subunits. As [GDP] increased, basal binding was suppressed such that, at 30 µM GDP, basal binding was only slightly greater than nonspecific binding determined in the presence of 10 µM GTPS. Under these conditions, very little stimulation of [³⁵S]GTPS binding was induced by 5-HT or pindolol (Fig. 6, A and B). 5-HT yielded bell-shaped isotherms and pindolol yielded sigmoidal isotherms in all cases, but stimulation was greatest when [GDP] = 3 µM. In contrast, spiperone yielded the greatest decrease in dpm at the lowest GDP concentrations [i.e., when high constitutive activity was favored (Fig. 6C)].

View larger version (31K): [in this window] [in a new window]   Fig. 6.   Influence of ionic conditions and guanine nucleotides on [35S]GTPS binding to Gi3 subunits in CHO-h5-HT1A cell membranes. h5-HT1A receptor-mediated Gi3 subunit activation was determined by antibody capture/SPA detection. The influence of four concentrations each of GDP (A, B, and C), MgCl2 (D, E, and F), and NaCl (G, H, and I) on the actions of a full agonist (5-HT), a partial agonist (pindolol), and an inverse agonist (spiperone) are shown. Data points are means of duplicate determinations from representative experiments repeated on at least three independent occasions with similar results.

Influence of MgCl₂ (0.06, 0.33, 3.0, and 30 mM). Mg²⁺ ions are cofactors for GTP binding to G-proteins. Thus, at low [MgCl₂], basal [³⁵S]GTPS binding was only slightly higher than nonspecific binding. As [MgCl₂] increased, basal binding increased but the concentration-response isotherms of 5-HT remained bell-shaped in all cases (Fig. 6D). The peaks shifted to the left as [MgCl₂] increased to 3 mM and then to the right at 30 mM, suggesting that agonist potency (and not just efficacy) is dependent on an optimal Mg²⁺ concentration. Stimulation by pindolol and inhibition by spiperone were most accentuated at 3 mM MgCl₂ (Fig. 6, E and F).

Influence of Sodium Chloride (10, 30, 100, and 300 mM). Monovalent cations, such as Na⁺, modulate receptor interactions with G-proteins and the concentration of NaCl was the only parameter to fundamentally modify the shape of the G_i3 activation isotherms. Thus, at low [NaCl], high basal binding was observed and no stimulation by 5-HT: the only action of 5-HT was to inhibit G_i3 activation (Fig. 6G). Conversely, at high [NaCl], the stimulatory actions of 5-HT were evident, but not the inhibitory actions . Pindolol stimulated G_i3 activation at 100 mM NaCl but inhibited it at 10 mM NaCl (Fig. 6H). Spiperone displayed the greatest inhibition of G_i3 activation at low [NaCl]where basal binding was highest.

Influence of Incubation Time (5, 10, 20, and 60 min). The appearance of bell-shaped G_i3 activation isotherms for 5-HT was time-dependent. At short incubation times, isotherms were essentially sigmoidal, whereas at longer incubation times, bell-shaped isotherms were observed due to stimulation of G_i3 in the presence of nanomolar, but not higher, concentrations of 5-HT (Fig. 7).

View larger version (26K): [in this window] [in a new window]   Fig. 7.   Influence of incubation time on [35S]GTPS binding to Gi3 subunits in CHO-h5-HT1A cell membranes. h5-HT1A receptor-mediated Gi3 subunit activation was determined by antibody capture/SPA detection. Cell membranes were incubated with 5-HT and [35S]GTPS for different incubation times before addition of NP40 detergent to stop the reaction. Gi3 subunit activation was detected by antibody capture/SPA detection. Top, [35S]GTPS binding to Gi3 subunits increased time-dependently at nanomolar, but not micromolar, concentrations of 5-HT. Bottom, [35S]GTPS binding to Gi3 subunits expressed as a percentage of basal values. The bell-shaped isotherms appeared in a time-dependent manner. Data points are means of duplicate determinations from representative experiments repeated on at least three independent occasions with similar results.

Influence of Agonists, Antagonists, and Inverse Agonists

As well as 5-HT (described above), other full agonists (as defined by classical filtration assays, Newman-Tancredi et al., 1998, 2001a,b), including (+)8-OH-DPAT, flesinoxan, and S14506, yielded bell-shaped [³⁵S]GTPS binding isotherms, with peak stimulation at drug concentrations shown in Table 2. Like 5-HT, high concentrations of these ligands reduced [³⁵S]GTPS binding below basal values (Fig. 8). The partial agonists buspirone and S16924 (Newman-Tancredi et al., 2001b), yielded broader bell-shaped isotherms which did not return to baseline values. The isomers (+)-8-OH-DPAT and ()-8-OH-DPAT differed dramatically; the former yielded bell-shaped isotherms but the latter did not (Fig. 8). Two other partial agonists, S15535 and pindolol (Newman-Tancredi et al., 1996, 2001a,b), also induced sigmoidal isotherms. The neutral antagonist WAY100,635 and another antagonist, p-MPPI, modestly stimulated [³⁵S]GTPS binding with sigmoidal concentration-response curves (E_max = 135 and 128%, respectively; Table 3), indicating mild agonist properties for activation of G_i3. Sigmoidal binding curves were also observed with the inverse agonists, spiperone, haloperidol, (+)-butaclamol and methiothepin, which inhibited basal [³⁵S]GTPS binding, consistent with constitutive activation of G_i3 (Table 3).

View larger version (29K): [in this window] [in a new window]   Fig. 8.   Actions of serotonergic ligands and antipsychotic agents on [35S]GTPS binding to Gi3 subunits in CHO-h5-HT1A cell membranes. h5-HT1A receptor-mediated Gi3 subunit activation was determined by antibody capture/SPA detection. High efficacy agonists [e.g., 5-CT, S14671, (+)-8-OH-DPAT (A, D)] yielded bell-shaped isotherms. Partial agonists [e.g., pindolol, S15535, buspirone, S16924 (B, C, and E)] and inverse agonists [(+)-butaclamol, methiothepin, haloperidol (B, C, and F)] yielded concentration-response isotherms for Gi3 activation that were sigmoidal or that did not return to basal binding levels. The antagonist WAY100,635 exhibited slight agonist properties (C). Data points are means of duplicate determinations from representative experiments repeated on at least three independent occasions. Nonlinear regression parameters describing these isotherms are shown in Tables 2 and 3.

View this table: [in this window] [in a new window]   TABLE 3 Sigmoidal isotherms for h5-HT1A receptor-mediated stimulation of [35S]GTP S binding to G i3 subunits Activation of G i3 G-protein subunits in CHO-h5-HT1A cell membranes was determined employing an antibody-capture/SPA detection technique. Data are expressed as mean ± S.E.M. of n independent determinations performed in duplicate. For comparison, ligand affinity (pKi) is shown, as determined by competition binding with [3H]8-OH-8DPAT (Newman-Tancredi et al., 2001a,b).

Antagonist Studies. Two kinds of experiments were carried out with the selective 5-HT_1A receptor antagonist, WAY100,635. i) concentration-response isotherms of 5-HT, (+)8-OH-DPAT, pindolol, and spiperone were shifted in parallel to the right by fixed concentrations (3 or 10 nM) of WAY100,635 without loss of maximal stimulation (Fig. 9), consistent with competitive antagonist actions. pK_B values (shown in Table 4) were similar for both the ascending and descending components of the bell-shape isotherms suggesting that they are mediated by a single binding site. ii) Pindolol (100 nM)-induced stimulation and spiperone (1 µM)-induced inhibition of G_i3 activation were concentration-dependently reversed by WAY100,635. pK_B values resembled the pK_i (9.91) of WAY100,635 at h5-HT_1A receptors (Newman-Tancredi et al., 2001a).

View larger version (17K): [in this window] [in a new window]   Fig. 9.   Action of high-efficacy agonists and the inverse agonist spiperone on [35S]GTPS binding to Gi3 subunits in CHO-h5-HT1A cell membranes: antagonism by WAY100,635. h5-HT1A receptor-mediated Gi3 subunit activation was determined by antibody capture/SPA detection. A, concentration-response isotherms of 5-HT alone or in the presence of WAY100,635 (10 nM). B, concentration-response isotherm of (+)-8-OH-DPAT alone or in the presence of WAY100,635 (10 nM). C, concentration-dependent inhibition by spiperone alone or in the presence of WAY100,635 (3 nM). Data points are means of duplicate determinations from representative experiments repeated on at least three independent occasions. pKB values derived from these experiments are shown in Table 4.

View this table: [in this window] [in a new window]   TABLE 4 Antagonism of h5-HT1A receptor-mediated stimulation of [35S]GTP S binding to Gi3 subunits by WAY100,635 Activation of G i3 G-protein subunits in CHO-h5-HT1A cell membranes was determined employing an antibody-capture/SPA detection technique. Ligands were preincubated with membranes for 30 min before adding [35S]GTP S. Fixed concentrations (3 or 10 nM) of WAY100,635 shifted the isotherms of 5-HT, (+)-8-OH-DPAT, pindolol, and spiperone. For 5-HT and (+)8-OH-DPAT, pKB values were calculated for the ascending and descending components of bell-shaped isotherms. Stimulation induced by pindolol (100 nM) and inhibition induced by spiperone (1 µM) were concentration-dependently reversed by WAY100,635. pKB values resemble the pKi value (9.91) of WAY100,635 at h5-HT1A receptors (Newman-Tancredi et al., 2001a). Data are expressed as mean ± S.E.M. of n independent determinations performed in duplicate.

	Discussion

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Total G-Protein Activation Assays. In studies of total G-protein activation, which do not distinguish between G-protein subtypes, 5-HT stimulated [³⁵S]GTPS binding biphasically, suggesting the presence of multiple h5-HT_1A receptor/G-protein coupling conformations. Indeed, preincubation of CHO-h5-HT_1A cell membranes with an antibody that recognizes G_i3 subunits selectively abolished the high potency 5-HT stimulation component and decreased basal binding (Fig. 1, A and B). Although the G-protein activated at high concentrations of 5-HT remains to be formally identified, it is probably G_i2, a subtype highly expressed in CHO cells and known to interact with h5-HT_1A receptors (see Introduction). Indeed, in preliminary antibody-capture experiments, 5-HT stimulated [³⁵S]GTPS binding to G_i2 (as opposed to G_i3 described below) with a pEC₅₀ of 7.3 (A. Newman-Tancredi and M. J. Millan, unpublished observations), similar to the low potency component observed in filtration experiments (pEC_50L 7.7; Table 1). The involvement of multiple G-protein populations in receptor coupling can be shown by investigation of their contrasting GTP/GDP exchange kinetics (Shea et al., 2000). Indeed, Wenzel-Seifert and Seifert (2000) have shown that Gs, Gq, and G₁₆ differ in their rates of [³⁵S]GTPS dissociation. Herein, when CHO-h5-HT_1A cell membranes were exposed to unlabeled GTPS after the standard incubation period, nanomolar concentrations of 5-HT (or saturating concentrations of the partial agonist, pindolol) stabilized [³⁵S]GTPS binding and slowed its dissociation from CHO-h5-HT_1A cell membranes, whereas dissociation was accelerated by micromolar concentrations of 5-HT (Fig. 3). Taken together, these experiments indicate that pindolol or low concentrations of 5-HT preferentially induce coupling of h5-HT_1A receptors to G_i3 subunits, but that coupling to this G-protein is suppressed at high concentrations of 5-HT, revealing bell-shaped concentration-response isotherms. These observations are reminiscent of a study by van Hooft and Vijverberg (1996) on 5-HT₃ receptors and strongly suggest that full and partial agonists select distinct conformational states of h5-HT_1A receptors.

Bell-Shaped Isotherms for G_i3 Activation: Specific Mediation by 5-HT_1A Receptors. To directly investigate h5-HT_1A receptor coupling to G_i3, we employed an antibody capture/SPA technique similar to that described previously for muscarinic receptors (DeLapp et al., 1999). Initial experiments revealed that 5-HT induced a concentration-dependent rise and then fall in [³⁵S]GTPS labeling of G_i3 subunits, with a striking resemblance to the isotherms obtained from filtration assays after incubation with GTPS (Fig. 2). As discussed previously (Szabadi, 1977; Rovati and Nicosia, 1994; Tucek et al., 2001), several mechanisms may give rise to bell-shaped isotherms in pharmacological systems. First, different signal transduction systems may be activated by ligand binding at two independent receptors (for examples, see Okpako, 1972; Lejeune et al., 1997). In the present system, however, stimulation of G_i3 subunits is entirely attributable to activation via h5-HT_1A receptors. Indeed, there was no influence of 5-HT on G_i3 activation in nontransfected CHO cells and the entire bell-shaped isotherms of 5-HT and the selective 5-HT_1A receptor agonist (+)-8-OH-DPAT were right-shifted by the selective antagonist WAY100,635 (Table 4; Fletcher et al., 1996; Newman-Tancredi et al., 1997). Hence, the presence of multiple receptors can be ruled out, although the possibility remains that agonists may be sequentially acting at multiple binding sites on target receptors, as has been proposed for bell-shaped mobilization of calcium ions by muscarinic agonists in SH-SY5Y neuroblastoma cells (Järv et al., 1995). However, that study did not report whether agonist actions could be blocked by antagonists. On the other hand, a preliminary study by Browning et al. (2000a) showed bell-shaped isotherms for adenosine A₁ receptor-mediated [³⁵S]GTPS binding in CHO cell membranes that were blocked by a selective antagonist (8-cyclopentyl-1,3-dipropylxanthine), although the precise G-protein subtypes involved were not identified.

Influence of Agonists, Partial Agonists, and Inverse Agonists for G_i3 Activation. We investigated the activation of G_i3 subunits by 22 drugs displaying diverse efficacies at 5-HT_1A receptors (Tables 2 and 3). These ligands, which have been previously characterized by binding affinity ratios and classical [³⁵S]GTPS binding (Newman-Tancredi et al., 1996, 1998, 2001a,b), yielded widely differing G_i3 activation patterns. First, spiperone, methiothepin, and the neuroleptics haloperidol and (+)-butaclamol exhibited inverse agonist properties (Figs. 8 and 9), in agreement with previous reports (Newman-Tancredi et al., 1998, 2001a,b; Cosi and Koek, 2001). Second, agonists that display high efficacy in classical filtration experiments (Newman-Tancredi et al., 1998), including 5-CT, (+)flesinoxan, and S14506, exhibited bell-shaped isotherms, generally with narrow peaks. Third, weak partial agonist ligands, including S15535 and pindolol (Newman-Tancredi et al., 1996, 2001a), as well as p-MPPI and WAY100,635, yielded isotherms that were sigmoidal (Fig. 9). In the case of WAY100,635, this ligand is variously reported to behave as a `neutral antagonist' in vivo and at CHO-h5-HT_1A receptors (Fletcher et al., 1996; Newman-Tancredi et al., 1997), or as an inverse agonist at h5-HT_1A receptors expressed in HeLa cells (Cosi and Koek, 2000). Further, a very recent GTPase activity study in human embryonic kidney 293 cells also detected weak partial agonist properties of WAY100,635 for activation of a h5-HT_1A-G_O1 fusion construct in the presence of co-expressed regulators of G-protein signaling-1 (RGS1; Welsby et al., 2002). Thus, WAY100,635 may have differential efficacy at different G-protein subtypes, behaving as a weak partial agonist at G_i3 but as an antagonist or inverse agonist at other G subunits or under the influence of RGS proteins.

Regulatory Mechanisms of G_i3 Activation. Although the precise mechanisms involved in the bell-shaped concentration-response isotherms remains unclear, some tentative hypotheses may be proposed. First, a "strength-of-signal" mechanism may be involved in the present observations (Kenakin, 1995), whereby high-efficacy agonists stimulate multiple pathways, whereas lower efficacy agonists, such as S15535 and pindolol, can stimulate only the most efficiently-coupled pathway (in this case G_i3). However, it is striking that S15535 and pindolol displayed maximal G_i3 stimulation (E_max values 234 and 193%, respectively; Tables 2 and 3) comparable with that of 5-HT (245%), whereas they behaved as partial agonists in classical [³⁵S]GTPS binding experiments (Fig. 1 and Newman-Tancredi et al., 1996). Thus, S15535 and pindolol may be selectively trafficking agonist-directed signaling to G_i3 subunits. This implies in turn that high efficacy for G_i3 activation is not sufficient to induce the descending phase of bell-shaped isotherms. In fact, this downturn strongly suggests that when efficacious agonists attain a high level of occupation of h5-HT_1A receptors, a conformational change may occur that induces coupling to other G subtypes and suppresses signaling through G_i3 subunits. A similar concept has been evoked at muscarinic receptors (Dittman, et al. 1994). A change in receptor conformation is supported by the sensitivity of G_i3 activation to the availability of Na⁺ and Mg²⁺ ions, as well as guanine nucleotides, which are known to influence h5-HT_1A receptor-G-protein coupling (Fig. 6; Pauwels et al., 1997; Cosi and Koek, 2000). Indeed, a distinctive influence of NaCl on G_i3 activation was observed: at high [NaCl], 5-HT stimulated G_i3 activation sigmoidally (Fig. 6), but at low [NaCl], only inhibitory influences were detected. The presumed conformational change may involve formation of h5-HT_1A receptor dimers, as has been reported for numerous GPCRs, including h5-HT_1B and h5-HT_1D receptors (Xie et al., 1999; Marshall, 2001). Such a hypothesis would account for the ability of high concentrations of high-efficacy agonists to decrease [³⁵S]GTPS binding below basal levels (Table 2; Fig. 9). Indeed, if dimers were no longer able to couple to G_i3, constitutive activation of the latter would be suppressed by high concentrations of 5-HT, which would act, therefore, as a pseudo-inverse agonist. Receptor desensitization is another possible mechanism affecting receptor conformation, and its occurrence here is suggested by the time-dependent appearance of the bell-shaped isotherms (Fig. 7). Desensitization may potentially be achieved by receptor phosphorylation although, in preliminary experiments, a G-protein receptor kinase inhibitor (Ro318220, 1 µM) failed to block the descending phase of G_i3 activation by 5-HT (Brzostowski and Kimmel, 2001; A. Newman-Tancredi, unpublished observations). Potential receptor conformational changes could also involve interaction with RGS proteins (for review, see Wieland and Chen, 1999), but regulatory mechanisms such as receptor internalization (Böhm et al., 1997) may be excluded, given that the present study was carried out using membrane preparations.

	Conclusions

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In conclusion, the present data demonstrate that i) h5-HT_1A receptors couple efficiently to G_i3 subunits; ii) GTP/GDP exchange kinetics for G_i3 subunits differ from those of other G subunits coupled to h5-HT_1A receptors; iii) high concentrations of high-efficacy agonists suppress signaling to G_i3. These differences in G_i3 subunit activation constitute compelling evidence of agonist-dependent selection of different h5-HT_1A receptor conformations. Further investigation is necessary to determine whether these phenomena are due to strength-of-signal mechanisms (such as differential efficiency of coupling to specific G-protein subtypes), agonist-directed trafficking, or receptor desensitization (Kenakin, 1995; Clarke and Bond, 1998). In addition, it would be of interest to investigate the consequences of these signaling patterns on downstream responses, such as adenylyl cyclase or MAP kinase, which may be modulated by multiple G-protein subtypes (Raymond et al., 1993; Garnovskaya et al., 1997) and on the profiles of action of therapeutically-employed agents (Li et al., 1997). Finally, studies would be desirable to determine the extent of such G-protein activation patterns for other GPCRs, both in recombinant and native tissue membrane preparations.