Introduction

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In addition to characterization of the pharmacological profiles of cloned, G-protein-coupled receptors, studies of recombinant cell lines have enabled the exploration of cellular parameters that influence diverse signal transduction pathways. For example, in NIH-3T3 fibroblasts, agonist efficacies increased with augmentation of h5-HT_1A receptor expression levels (Varrault et al., 1992), a finding corroborated by studies in other cell lines (Newman-Tancredi et al., 1997; Schoeffter et al., 1997). Conversely, irreversible receptor inactivation by the alkylating agent, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline, reduced agonist efficacies at heterologously expressed h5-HT_1B, h5-HT_1D, and hD₃ receptors (Adham et al., 1993; Zgombick et al., 1996; Newman-Tancredi et al., 1999b). Other factors are also important in determining agonist efficacies at G-protein-coupled receptors. Thus, Fargin et al. (1989) reported that the extent of the 5-HT-induced decrease in forskolin-stimulated cAMP levels in four HeLa cell lines did not correlate with h5-HT_1A receptor expression levels, suggesting that signal transduction characteristics other than receptor density are of importance. Indeed, the efficacy of partial agonists at µ-opioid receptors expressed in Chinese hamster ovary (CHO) cells is related to the stoichiometric ratio of receptors to G-proteins (Selley et al., 1998). In a similar vein, altering receptor/G-protein (R:G) stoichiometry by G-protein coexpression increased the efficacies of agonists at muscarinic receptors (Burnstein et al., 1995) and constitutive activation of alpha-2A adrenoceptors (Pauwels et al., 2000). Moreover, the dopamine D₄ receptor ligand, L745,870, originally characterized as an antagonist (Patel et al., 1997), was later reported to be an agonist in different cellular expression systems, an observation attributed to differences in R:G stoichiometry (Gazi et al., 1999; P. Schoeffter, personal communication).

The above considerations show that quantification of both receptor and G-protein expression levels is important for the appropriate interpretation of drug influences on signal transduction mechanisms (Kenakin, 1997b). However, previous studies have focused on the influence of R:G coupling on agonist effects, whereas the influence of R:G stoichiometry on negative efficacy is relatively uncharacterized. Indeed, to our knowledge, only one study (at 5-HT_1A receptors), has investigated the influence of R:G ratios on the efficacy of inverse agonists at serotonergic receptors (Newman-Tancredi et al., 1997).

To address these issues, we studied G-protein activation at h5-HT_1B receptors stably expressed in CHO cells.¹ 5-HT_1B receptors exhibit marked constitutive activity for G-protein activation, and several inverse agonists have been identified at this site (Thomas et al., 1995; Pauwels et al., 1997; Gaster et al., 1998; Selkirk et al., 1998). Furthermore, inhibitory 5-HT_1B receptors are located as autoreceptors on serotonergic neuronal terminals and are key targets for the modulation of serotonin release, both in the central nervous system (Engel et al., 1986; Bruinvels et al., 1993; Millan et al., 1999; Sari et al., 1999) and in the dura matter. Consequently, an understanding of the mechanisms of signal transduction by 5-HT_1B receptors is relevant to the treatment of affective disorders and to the management of migraine (Hamel, 1996; Millan, 1999).

Herein, using two cell membrane preparations, we characterized the effects of a variation in h5-HT_1B R:G stoichiometry on several parameters. First, receptor expression levels and receptor-activated G-proteins were quantified by saturation-binding experiments, permitting the calculation of R:G ratios. Second, the competition-binding affinities of chemically diverse 5-HT_1B receptor ligands, including several recently described selective compounds, were compared in these cell membranes displaying high versus low R:G ratios. Third, ligand potencies and efficacies were compared by binding of the hydrolysis-resistant GTP analog radioligand, guanosine-5'-O-(3-[³⁵S]thio)-triphosphate ([³⁵S]GTPS) (Lorenzen et al., 1993). This technique affords a measure of the activation of the first step of the intracellular transduction cascade (Birnbaumer and Birnbaumer, 1995; Gudermann et al., 1997). Fourth, the effect of contrasting R:G ratios on constitutive 5-HT_1B receptor-mediated G-protein activation was investigated with a novel procedure using [³⁵S]GTPS versus GTPS homologous inhibition curves. Such binding isotherms allow the detection of high affinity (HA) and low affinity (LA) binding components (Breivogel et al., 1998; Selley et al., 1998), and can be used to directly quantify the amount of agonist-independent constitutive G-protein activation without requiring the use of inverse agonists (Audinot et al., 1999, 2001).

	Materials and Methods

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Binding with [³H]GR125,743 at CHO-h5-HT_1B Membranes. For competition- and saturation-binding experiments, CHO-h5-HT_1B cell membranes (15 µg) were incubated for 60 min at 22°C in buffer A (50 mM Tris-HCl, pH 7.7, 4 mM CaCl₂, 0.1% ascorbic acid) with [³H]GR125,743 (1 nM; 70 Ci/mmol; Amersham, Les Ulis, France; Doménech et al., 1997) and competing ligands. 5-HT (10 µM) was used to define nonspecific binding. Incubations were terminated by rapid filtration through GF/B filters pretreated with polyethylenimine (0.1%, v/v). Data were analyzed by nonlinear regression using the program Prism (Graphpad Software Inc., San Diego, CA), to yield K_D (dissociation of the radioligand) and B_max (maximal binding density) values for saturation experiments, and IC₅₀ values for competition experiments. K_i values were calculated according to the equation: K_i = IC₅₀/(1 + L/K_D), where L is the concentration of radioligand.

Effects of Receptor Ligands on [³⁵S]GTPS Binding at CHO-h5-HT_1B Membranes. Receptor-linked G-protein activation at h5-HT_1B receptors was determined by measuring stimulation of [³⁵S]GTPS (1000 Ci/mmol; New England Nuclear, Paris, France) binding as described previously (Newman-Tancredi et al., 1999a). Briefly, membranes (15 µg of protein/well) were incubated (30 min at 22°C) with ligands in a final volume of 250 µl of buffer B [20 mM N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid, pH 7.4, 3 µM GDP, 3 mM MgCl₂, 100 mM NaCl, and 0.1 nM [³⁵S]GTPS]. Nonspecific binding was defined with GTPS (10 µM).

Inhibition of [³⁵S]GTPS Binding by Unlabeled GTPS at CHO-h5-HT_1B Membranes. Isotopic dilution experiments were carried out in buffer B and incubations lasted 30 min at 22°C. Binding of radiolabeled [³⁵S]GTPS was inhibited with GTPS and the resulting isotherms were best fitted by a two-site nonlinear regression analysis, giving IC₅₀ values for HA and LA binding components. HA binding observed under basal conditions (i.e., not agonist induced) reflects endogenous G-protein activation, providing a direct measure of constitutive activity of G-protein-coupled receptors (Audinot et al., 1999, 2001), whereas LA binding likely reflects endogenous GDP/GTP turnover of CHO cell membrane G-protein G-subunits. Binding data from these experiments expressed in femtomoles per milligram of protein were normalized to account for the concentration of [³⁵S]GTPS present in the assay. Hence, units are denoted fmol/mg/nM [³⁵S]GTPS.

(1)

Membranes and Compounds. CHO-h5-HT_1B cell membranes expressing both high and low levels of h5-HT_1B receptors were purchased from Euroscreen (Brussels, Belgium; Euroscreen only now commercializes the membranes from cells expressing the higher level of h5-HT_1B receptors). 5-HT creatinine sulfate was purchased from Sigma (Saint Quentin Fallavier, France), methiothepin maleate was from Tocris Cookson (Southampton, England). Sumatriptan (GR43,175) was from Glaxo (Greenford, UK). SB224,289 (1'-methyl-5-[[2'-methyl-4'-(5-methyl-1,2,4-oxadiazol-3-yl)biphenyl-4-yl]carbonyl]-2,3,6,7-tetrahydrospiro-[furo-[2,3f]-indole-3,4'-piperidine]-oxalate) was synthesized by Jean-Louis Peglion [Institut de Recherches Servier, Croissy-sur-Seine (Paris), France]. BMS 181,101 (5-fluoro-3-{3-[4-(5-methoxy-pyrimidin-4-yl)-piperazin-1-yl]-propyl}-1H-indole) dihydrochloride, GR 125,743 (N-[4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]-3-methyl-4-(4-pyridyl)benzamide) hydrochloride, and L775,606 (1-[2-(3-fluorophenyl)ethyl]-4-[3-[5-(1,2,4-triazol-4-yl)-1H-indol-3-yl]-propyl]-piperazine) dicitrate were synthesized by Gilbert Lavielle [Institut de Recherches Servier, Croissy-sur-Seine (Paris), France]. Compounds were dissolved in water or in dimethyl sulfoxide and diluted in the appropriate assay buffer to the required experimental concentrations.

	Results

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[³H]GR125,743 and [³⁵S]GTPS Saturation Binding to Cell Membranes from Two CHO Cell Lines Expressing h5-HT_1B Receptors. Saturation binding with [³H]GR125,743 was carried out in CHO cell membranes expressing a high level (RGhigh membranes) and a low level (RGlow membranes) of h5-HT_1B receptors. RGhigh membranes expressed 20 times more h5-HT_1B receptors (B_max = 8.54 pmol/mg) than RGlow cell membranes (B_max = 0.43 pmol/mg) (Table 1 and Fig. 1).

View this table: [in this window] [in a new window]   TABLE 1 [3H]GR125,743 and [35S]GTPS saturation binding to cell membranes from two CHO cell lines expressing h5-HT1B receptors The expression level (Bmax) of h5-HT1B receptors was determined by saturation-binding experiments with [3H]GR125,743. The number of G-proteins activated by h5-HT1B receptors was determined by [35S]GTPS isotopic dilution saturation binding as described under Materials and Methods in the presence or absence of 5-HT (10 µM). The R:G ratio was calculated by dividing the Bmax of [3H]GR125,743 by that of [35S]GTPS. The cell membranes exhibiting a lower Bmax ratio are denoted "RGlow". The cell membranes exhibiting a higher Bmax ratio are denoted "RGhigh". Data are means ± S.E. of at least three independent determinations performed in triplicate.

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Saturation binding at RGlow or RGhigh membranes. Top, h5-HT1B receptor saturation binding with [3H]GR125,743 (insets show Scatchard plots of respective saturation isotherms). Bottom, [35S]GTPS isotopic dilution G-protein saturation binding (as described under Materials and Methods) in the presence or absence of 5-HT (10 µM). Left, isotherms from RGlow cell membranes. Right, isotherms from RGhigh cell membranes (note different y-axes). Points shown are from representative experiments performed in triplicate and repeated on at least three independent occasions. B, bound radioligand; F, free radioligand.

Competition for [³H]GR125,743 Binding to RGlow and RGhigh Cell Membranes. Competition-binding isotherms for [³H]GR125,743 binding were carried out at both RGlow and RGhigh cell membranes with six chemically diverse serotonergic ligands (Table 2). Marked differences in the affinities (pK_i/H/L values) of certain ligands were observed between the two sets of membranes. 5-HT displayed biphasic isotherms with modest pseudo-Hill coefficients (n_H = 0.73) in both RGlow and RGhigh membranes, but the proportion of HA sites was significantly reduced in the latter. Sumatriptan yielded monophasic isotherms in RGlow membranes but biphasic ones in RGhigh membranes, with a significant reduction in n_H value. For BMS181,101 and L775,606, pK_i values were about 0.5 unit higher at RGlow than at RGhigh cell membranes. In contrast, SB224,289 exhibited a pK_i value that was higher at RGhigh membranes. The other inverse agonist, methiothepin, showed a similar tendency, although it did not reach statistical significance (Table 2).

Concentration-Response Effect of 5-HT Receptor Ligands on [³⁵S]GTPS Binding to RGlow and RGhigh Cell Membranes. The influence of 5-HT_1B ligands on G-protein activation was investigated (Table 3 and Fig. 2). The amount of [³⁵S]GTPS binding was markedly higher in RGhigh membranes than in RGlow membranes both in absolute and in relative terms. Thus, basal [³⁵S]GTPS binding was 1222 fmol/mg/nM in RGhigh, but only 477 fmol/mg/nM in RGlow membranes (see Table 4, "Total" column). 5-HT-stimulated [³⁵S]GTPS binding was 2937 fmol/mg/nM in RGhigh membranes (2.4-fold increase) compared with 650 fmol/mg/nM in RGlow membranes (1.4-fold increase) (Table 4; "Total" column).

View this table: [in this window] [in a new window]   TABLE 3 Concentration-response effects of 5-HT receptor ligands on [35S]GTPS binding to RGlow and RGhigh cell membranes The influence of 5-HT1B receptor ligands was compared by [35S]GTPS binding to two preparations of RGlow and RGhigh cell membranes. Concentration-response isotherms were analyzed by nonlinear regression. pEC50 values are shown for agonist effects, whereas pIC50 values are shown for inverse agonists (SB224,289 and methiothepin). Emax is expressed as a percentage of binding observed with a maximally effective (10 µM) concentration of 5-HT (=100%). Data are means ± S.E. of at least three independent experiments.

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Concentration-response isotherms of 5-HT1B receptor ligands for stimulation of [35S]GTPS binding to RGlow () or RGhigh () membranes. 5-HT and sumatriptan exhibited similar efficacy in both membrane preparations. BMS181,101 and L775,606 were more efficacious in RGhigh than in RGlow membranes. The inverse agonists, methiothepin and SB224,289, exhibited greater negative efficacy in RGhigh versus RGlow membranes. Data for the agonists are expressed as percentages of [35S]GTPS binding induced by a maximally effective concentration of 5-HT (10 µM). Data for the inverse agonists are expressed as percentages of [35S]GTPS binding observed under basal conditions (absence of receptor ligand). Points shown are from representative experiments performed in triplicate and repeated on at least three independent occasions.

View this table: [in this window] [in a new window]   TABLE 4 Inhibition by GTPS of [35S]GTPS binding to RGlow and RGhigh cell membranes [35S]GTPS binding to two preparations of RGlow or RGhigh cell membranes, was inhibited with GTPS in the presence or absence of 5-HT (10 µM), SB224,289 (10 µM), or methiothepin (1 µM). Isotherms were analyzed by nonlinear regression yielding an HA and an LA component. In all cases, a two-site fit was statistically superior to a single-site fit (P < .05, F test). The number of HA and LA sites is expressed as femtomoles per milligram per nanomolar [35S]GTPS present in the assay. For Total and HA sites, numbers in parentheses indicate the amount of binding as a fraction of that observed under respective basal conditions. Data are means ± S.E. of at least three independent experiments.

Inhibition by GTPS of [³⁵S]GTPS Binding to RGlow and RGhigh Cell Membranes. Both in the presence and in the absence of receptor ligands, inhibition of [³⁵S]GTPS binding to both RGhigh and RGlow membranes by GTPS produced biphasic isotherms (two-site fit was statistically superior to a single-site fit; P < .05, F test; Table 4 and Fig. 3). The amount of HA [³⁵S]GTPS binding (a measure of constitutive activation) was greater in RGhigh cell membranes (683 fmol/mg/nM) than in RGlow cell membranes (126 fmol/mg/nM). Furthermore, a marked difference was observed in the action of 5-HT (10 µM) on HA sites in the two membrane preparations: 5-HT increased the number of HA sites by 3.7-fold in RGhigh membranes but by only 2.8-fold in RGlow membranes (Table 4 and Fig. 3). Conversely, the number of HA sites was reduced by the inverse agonists, SB224,289 (10 µM) and methiothepin (1 µM), but their actions were more profound in RGhigh than in RGlow membranes (Table 4 and Fig. 3). Indeed, methiothepin reduced HA sites to 70% of their basal level (=100%) in RGlow membranes but to just 29% of basal levels in RGhigh membranes.

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Inhibition by isotopic dilution with GTPS of [35S]GTPS binding to RGlow or RGhigh membranes. Top, [35S]GTPS isotopic dilution in the presence or absence of 5-HT (10 µM). Bottom, [35S]GTPS isotopic dilution in the presence or absence of the inverse agonist methiothepin (1 µM). Left, isotherms from RGlow cell membranes. Right, isotherms from RGhigh cell membranes. The dotted lines indicate the HA binding component of the isotherms that were modulated by receptor ligands. Isotherms determined by nonlinear regression are biphasic (two-site fit was statistically superior to a single-site fit; P < .05, F test). Points shown are from representative experiments performed in triplicate and repeated on at least three independent occasions.

	Discussion

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The key findings of the present study are that an augmentation of h5-HT_1B R:G stoichiometry is associated with changes in ligand-binding affinities, increased relative efficacies of partial agonists, and, notably, increased constitutive G-protein activation and negative efficacy of inverse agonists at h5-HT_1B receptors.

Determination of R:G Stoichiometry. In a comparison of two recombinant CHO cell lines, the first (RGlow) had a low h5-HT_1B receptor expression level, whereas the second (RGhigh) expressed 20-fold more receptors (Table 1 and Fig. 1). Interestingly, in [³⁵S]GTPS saturation-binding experiments, RGhigh membranes exhibited a G-protein B_max 2-fold higher than RGlow, resulting in an R:G ratio of ~3.0 (for RGhigh) versus ~0.3 (for RGlow). The present data therefore indicate that, in studies of receptor density on signal transduction, it is advisable to quantify variations in G-protein levels from one recombinant cell line to another. The R:G ratio determination methodology was similar to that used at some other receptors (Lorenzen et al., 1993; Newman-Tancredi et al., 1997; Breivogel et al., 1998; Pauwels et al., 1998, 2000; Selley et al., 1998). However, a distinctive aspect herein was that the density of receptor-activated G-proteins took into account those G-proteins that are endogenously activated, by calculating the amount of HA [³⁵S]GTPS binding (see under Materials and Methods). In systems that exhibit a marked degree of constitutive G-protein activation, this factor may considerably affect estimates of G-protein density and affinity (Audinot et al., 1999, 2001; Pauwels et al., 2000). Herein, the B_max of [³⁵S]GTPS was not altered by the presence or absence of 5-HT (Table 1). Thus, instead of a change in B_max, 5-HT induced an increase in the apparent binding affinity of [³⁵S]GTPS. These data are consistent with the model of agonist action proposed by Breivogel et al. (1998), in which agonists alter G-protein affinity for guanine nucleotides (see also González-Maeso et al., 2000). Thus, in CHO-h5-HT_1B cell membranes, the total number of activated G-proteins was unchanged, and the effect of the agonist, 5-HT, is to increase their ability to bind low concentrations of [³⁵S]GTPS.

Competition Binding at RGhigh and RGlow Membranes. The present data indicate that RGhigh and RGlow membranes differed in [³H]GR125,743 competition-binding experiments (Table 2) while maintaining a pharmacological profile in general accordance with previous reports (Plosker and McTavish, 1994; Doménech et al., 1997; Pauwels et al., 1998; Selkirk et al., 1998; Longmore et al., 2000). It is noteworthy that the proportion of HA sites detected in RGhigh membranes for 5-HT was significantly lower than in RGlow membranes. Furthermore, the binding isotherms for sumatriptan were biphasic in RGhigh membranes but monophasic in RGlow membranes (Table 2), and the pK_i value of the partial agonist, BMS181,101, was significantly reduced in RGhigh membranes. The simplest interpretation of these observations, in the light of the increased R:G stoichiometry of RGhigh membranes, is that the proportion of G-protein-uncoupled receptors is higher. Indeed, agonists display reduced affinity at receptors that are not coupled to G-proteins (Wregget and De Léan, 1984; Kenakin, 1997a). Conversely, the affinity (pK_i values) of the inverse agonist, SB224,289, showed an increase in RGhigh membranes. Methiothepin showed a similar tendency (Table 2). Given that inverse agonists exhibit higher binding affinity at receptors that exist in inactive conformation(s) (Samama et al., 1994; Leff, 1995), this is also in accordance with the interpretation that attributes this change to an increase in the proportion of G-protein-uncoupled receptors.

Ligand Efficacy at RGhigh and RGlow Membranes. RGhigh and RGlow membranes also differed in their functional responses, as determined by [³⁵S]GTPS binding (Table 3 and Fig. 2). First, the overall degree of stimulation attained above basal binding with 5-HT was markedly higher in RGhigh membranes (2.4-fold) than in RGlow membranes (1.4-fold) (see Table 4; "Total" column), probably reflecting a faster rate of G-protein "cycling" due to the greater availability of h5-HT_1B receptors per G-protein (Birnbaumer and Birnbaumer, 1995; Gudermann et al., 1997; Breivogel et al., 1998). Thus, the relative efficacies of the partial agonists, BMS181,101 and L775,606, were also increased in RGhigh membranes versus RGlow membranes. It is important to note that the present data differ from those for 5-HT_1A receptors expressed in CHO cells (Newman-Tancredi et al., 1997). Therein, the potency of 5-HT, but not its efficacy, was increased by an augmentation of R:G stoichiometry, whereas the reverse was true in the present study. One factor potentially implicated is that the R:G ratio of RGlow membranes herein (~0.3) was substantially less than its counterpart in CHO-h5-HT_1A cell membranes (1.4; Newman-Tancredi et al., 1997). Thus, CHO-h5-HT_1B RGlow membranes may not have attained a "ceiling" whereby, for example, the number of available G-proteins was limiting, as may have been the case for CHO-h5-HT_1A RGlow cell membranes (Newman-Tancredi et al., 1997; Selley et al., 1998). In comparison, when Gq proteins were coexpressed with m₃ receptors, both potency and efficacy were augmented (Burnstein et al., 1995), suggesting that either or both of these parameters may be affected, depending on the R:G stoichiometry and limiting factors in each cellular expression system. These data again highlight the importance of thoroughly characterizing different expression systems at the level of both receptor and coupled G-proteins. Second, inverse agonists displayed increased negative efficacy in RGhigh membranes. Indeed, in RGlow membranes, the inhibitory actions of methiothepin were modest, whereas in RGhigh membranes about 40% of basal [³⁵S]GTPS binding was inhibited by SB224,289 and methiothepin (Table 3 and Fig. 2). These data are reminiscent of observations at CHO-h5-HT_1A cell membranes showing that an increase in R:G ratio augmented the negative efficacy of the inverse agonist, spiperone (Newman-Tancredi et al., 1997). The most likely explanation is that inverse agonists stabilize G-protein-coupled (as well as uncoupled) receptors in inactive conformation(s) (Samama et al., 1994; Leff, 1995; Newman-Tancredi et al., 1997). This would result in a reduction in the pool of G-proteins available for activation by non-inverse agonist-occupied receptors. The present data therefore provide evidence that R:G stoichiometry is an important factor in the detection of inverse agonist actions at h5-HT_1B receptor-coupled G-proteins.

Constitutive Activity at RGhigh and RGlow Membranes. The degree of constitutive h5-HT_1B receptor activation differed markedly between RGhigh and RGlow membranes. As stated in the introduction, the quantitative influence of R:G stoichiometry on constitutive activity is poorly characterized. Herein, constitutive activity was directly quantified by an innovative procedure by which the HA and LA components of homologous inhibition experiments of [³⁵S]GTPS versus GTPS (Audinot et al., 1999, 2000) were analyzed. The stimulatory action of 5-HT on [³⁵S]GTPS binding was due to an action on HA sites, consistent with results at other receptor systems (Breivogel et al., 1998; Pauwels et al., 1998, 2000; Selley et al., 1998), but the key finding of the present study was the increase in the number of HA sites observed in RGhigh cell membranes under basal conditions. Indeed, HA binding in RGhigh membranes was 5-fold greater (683 fmol/mg/nM; Table 4 and Fig. 3) than that observed in RGlow membranes (126 fmol/mg/nM). Given that in both cases no agonists were present, the increase is, most likely, attributable to increased R:G stoichiometry (Kenakin, 1997a). Thus, as R:G stoichiometry increases, the augmented availability of receptors per G-protein favors coupling of the latter to receptors in active conformations, yielding a greater amount of HA binding in the absence of agonist (i.e., constitutive activity). In contrast, LA binding increases by only about 2-fold in RGhigh membranes relative to RGlow membranes, a change similar to the increase in the B_max derived from [³⁵S]GTPS saturation binding (Table 1). Indeed, receptor ligands (whether agonists or inverse agonists) have little, if any, influence on LA sites but exert a major influence on HA binding. Indeed, the present study reveals that methiothepin and SB224,289 reduced the number of HA sites, an effect that was more pronounced in RGhigh membranes than in RGlow membranes. These observations suggest that as R:G stoichiometry increases, the number of constitutively active receptors per G-protein increases, thus providing a greater basal activity on which inverse agonists can exert their inhibitory actions. These data are consistent with a two-state receptor activation model (Leff, 1995; Kenakin, 1997a) but, once again, differ from those obtained for CHO-h5-HT_1A receptors (Newman-Tancredi et al., 1997). For the latter, basal [³⁵S]GTPS binding was not altered by the increase in R:G ratio, whereas for h5-HT_1B receptors it was (Table 4). As discussed above, these data suggest that h5-HT_1B receptor-mediated [³⁵S]GTPS binding in the present CHO cell line is not subject to the same limitation or ceiling as h5-HT_1A receptors in our previous study (Newman-Tancredi et al., 1997; Selley et al., 1998). The limiting factor could be the G-protein expression level, which was ~1 pmol/mg for CHO-h5-HT_1A membranes but ~3 pmol/mg for RGhigh membranes in the present study.

	Conclusions

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The present study provides evidence that R:G stoichiometry is a key factor in the pharmacological profile of h5-HT_1B receptors in CHO cells. Increased R:G ratios are associated with alterations in binding affinity, increased G-protein activation by full agonists, and increased relative efficacy of partial agonists at h5-HT_1B receptors. Importantly, the present study reveals that increased R:G ratios are also associated with increased negative efficacy of inverse agonists and increased constitutive G-protein activation of h5-HT_1B receptors, effects that may be receptor subtype dependent, because they differ from previously reported data at h5-HT_1A receptors. The implications of the present observations for analysis of data obtained from native 5-HT_1B, or other G-protein-coupled receptors, remains to be more fully ascertained but suggest that determination of R:G stoichiometry is an important parameter to consider when interpreting data pertaining to ligand efficacy and receptor constitutive activity.