Introduction

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The neurotransmitter serotonin is involved in the modulation of a wide variety of physiological processes. 5-Hydroxytryptamine (5-HT) acts through a large group of receptors (13 subtypes of which have been identified to date) that were classified into seven distinct families according to their signaling properties and molecular structure (Hoyer and Martin, 1997). All these receptors, with the exception of the 5-HT₃ receptor (R) (a ligand-gated ion channel) are members of the G-protein-coupled receptor (GPCR) superfamily.

Two members of the 5-HT₁ family of serotonin receptors, i.e., the 5-HT_1DR and 5-HT_1BR, were given particular attention because of their apparent role in migraine and depression [see Moskowitz (1992); Halazy et al. (1997); and Pauwels (1997) for reviews]. Even if localization studies gave some indications about the respective physiological role of both receptors, the distinct role of the 5-HT_1DR and 5-HT_1BR often remains unclear. In peripheral tissues, only the 5-HT_1BR could be demonstrated where it mediates vasoconstriction (Hamel et al., 1993). In the central nervous system, the occurrence of the 5-HT_1DR is more restricted than that of the 5-HT_1BR (Bonaventure et al., 1997). However, in the trigeminal system (which is involved in neurogenic pain processes) the 5-HT_1DR mRNA is more abundant than the 5-HT_1BR mRNA (Bonaventure et al., 1998).

Despite the relatively low homology between both receptors at the amino acid level (63%), various ligands bind with similar potencies to both of them (Weinshank et al., 1992). Early 5-HT_1B/_1D receptor studies relied on nonselective compounds, which also acted as antagonists on several 5-HT₁ and 5-HT₂ receptor subtypes. Among these, ketanserin was shown to preferentially bind on the human (h) 5-HT_1D receptor (Peroutka, 1994; Wurch et al., 1998). Ocaperidone, an antagonist that has a higher affinity for h5-HT_1DR than for h5-HT_1BR, is also a 5-HT_2A and D₂ receptor antagonist (Leysen et al., 1992; Leysen et al., 1996; Lesage et al., 1998). The physiological functions that can specifically be attributed to the 5-HT_1BR and to the 5-HT_1DR could not be determined using these compounds because of their broad pharmacological profiles. The first selective h5-HT_1B/_1DR ligands described were a series of benzanilide compounds, of which GR127935 is the prototype (Clitherow et al., 1994). Recently, some specific antagonists have been described for h5-HT_1DR (BRL-15572, Price et al., 1997) and h5-HT_1BR [SB216641 (Price et al., 1997) and SB224289 (Gaster et al., 1998)] [see Halazy et al. (1997) for review].

Besides their distinct localization, differences in signal transduction properties could underlie a distinct function of 5-HT_1BR and 5-HT_1DR. Several studies established that 5-HT_1BR and 5-HT_1DR are negatively coupled to adenylate cyclase through pertussis toxin-sensitive G-proteins in vivo (Hoyer and Schoeffter, 1988) as well as in vitro (Hamblin and Metcalf, 1991; Weinshank et al., 1992). This implies the involvement of members of the G_i/o family of G-proteins. However, to our knowledge, a detailed study of the specific G-protein subtypes that interact with the 5-HT_1DR or 5-HT_1BR has not been reported.

As proved by a growing amount of studies, the baculovirus expression system in insect cells has evolved to an established tool for the study of GPCRs (Butkerait et al., 1995; Wenzel-Seifert et al., 1998). This system offers the possibility to express high amounts of a well-defined receptor in a very low background of potentially interfering GPCR. The receptor can be obtained in a virtually noncoupled form (when highly expressed by itself) and in a highly coupled form (when coexpressed with high amounts of appropriate G-proteins). Thus, the expression of a well-defined combination of G-proteins can be superposed to that of the receptor. As such, the baculovirus-mediated GPCR expression in insect cells represents a unique medium for the study of structural changes induced by the interaction with a G-protein and provides experimental argumentation for the prevailing two-state model for activation of GPCRs. Finally, because it can be used for [³⁵S]GTPS binding experiments and, more specifically for serotonin receptors, for serum-free growth conditions, the Sf9 insect cell expression system is very sensitive at unmasking constitutive activity of GPCRs (Hartman and Northup, 1996; Barr and Manning, 1997b; Wenzel-Seifert et al., 1998). Discerning this agonist-independent receptor activity is crucial because of its implications for therapeutic approaches, e.g., as in the need of inverse agonists for functional blockade of constitutively active receptors.

We aimed at studying h5-HT_1DR by reconstituting its interaction with G-proteins in a baculovirus-based expression system. Specificity of G-protein coupling was analyzed using [³H]5-HT concentration binding experiments. Constitutive receptor activity was evidenced in [³⁵S]GTPS binding assays using the inverse agonist ocaperidone. Further characterization of ocaperidone binding on h5-HT_1DR revealed the profound differences in binding properties of this ligand depending on G-protein coupling, hence providing further evidence for the occurrence of the receptor in different conformations.

	Experimental Procedures

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Materials. Sf-900 II serum-free medium was purchased from Life Technologies (Paisley, UK). The AcGP67 expression vector was from Pharmingen (San Diego, CA). Virions producing the rat G_i1, G_i2, G_i3, and G_o subunits were obtained from Dr. J. Garrisson (Department of Pharmacology, University of Virginia, Health Science Center, Charlottesville, VA), the mouse G_q was kindly provided by Dr. A. Gilman (Department of Pharmacology, University of Texas, Southwestern Medical Center, Dallas, TX), and the bovine G₁₂ helpervector was a gift of Dr. T. Haga (Department of Biochemistry, Institute for Brain Research, Faculty of Medicine, Tokyo University, Hongo, Tokyo). The C6 glioma cell line expressing h5-HT_1DR was obtained from BioMed Consulting (Madrid, Spain). The antisera for h5-HT_1DR, h5-HT_1BR, G_i/o, G₁, and G₂ were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and that for G_q from Chemicon International (Temecula, CA). The peroxidase-conjugated anti-rabbit secondary antibody was obtained from Jackson ImmunoResearch Laboratories Inc. (West Grove, PE). Chemiluminescent Western detection kit (ECL) was from Amersham Pharmacia Biotech (Little Chalfont, UK). Molecular weight markers were from Bio-Rad (Hercules, CA). [³H]5-HT (80 to 130 Ci/mmol) and [³⁵S]GTPS (>1000 Ci/mmol) were obtained from Amersham Pharmacia Biotech. [³H]Ocaperidone (18.3 Ci/mmol) was labeled at the Janssen Research Foundation. Alniditan, ocaperidone, and ketanserin are original products from Janssen Pharmaceutica. 5-HT and dihydroergotamine-mesylate were from Acros Oganics (Geel, Belgium), methiothepin was purchased from Hoffmann-La Roche, GR127935 and pargyline were obtained from Sigma-Aldrich (St. Louis, MO). Sumatriptan was kindly donated by Glaxo. GTPS, GDP, and Gpp(NH)p were from Roche Molecular Biochemicals (Mannheim, Germany). Other chemicals were from Sigma-Aldrich or Merck (Belgium). Stock solutions of compounds were prepared in dimethyl sulfoxide or in assay buffer. Bradford reagent for protein concentration determination was from Bio-Rad. The Ultra Turrax homogenizer was from Janke and Kunkel (Staufen, Germany), the Brandel 96-sample harvester from Brandel (Montreal, Canada). GF/B filters were from Whatman (Kent, UK). The liquid scintillation spectrometer (TriCarb) and the scintillation fluid (Ultima Gold MV) were from Packard (Meriden, CT). The Prism program was purchased from GraphPad Software (San Diego, CA).

Receptor Expression. The reading frame of h5-HT_1DR and h5-HT_1BR cDNAs were joined to the Baculo Ac secretion signal in the AcGP67 vector to improve expression. In this vector, transcription of the cDNA of interest is driven by the polyhedrin promoter. The G-protein cDNAs were expressed from the pVL1393 vector (Pharmingen, San Diego, CA). Sf9 cells were cultured, as described previously (Butkerait et al., 1995), in spinner flasks using serum-free Sf-900 II medium. Exponentially growing Sf9 cells were infected with a multiplicity of infection of 2 for each virus added. Cells were harvested at 48 h postinfection.

Membrane Preparation. For crude membrane preparation, cells were harvested (2,000g, 10 min) and the pellet resuspended in phosphate-buffered saline with subsequent centrifugation (20,000g, 10 min). The procedure was repeated twice. The washed cell pellet was frozen or immediately processed as follows. Cells were resuspended in hypotonic 10 mM Tris-HCl (pH 7.4) buffer, homogenized for 20 s (Ultra-Turrax homogenizer) and the homogenate centrifuged (30,000g, 30 min). The supernatant was discarded, and membranes were resuspended in 50 mM Tris-Cl (pH 7.4) buffer and stored in aliquots at 70°C at a concentration of ~1 mg/ml protein. Finally, after thawing, the protein concentration was determined using the Bradford assay with BSA as standard protein. The membranes were used in ligand concentration-binding, competition binding, [³⁵S]GTPS binding and Western blotting experiments.

Radioligand Binding Assays. Membrane preparations were thawed on ice and diluted in 50 mM Tris-HCl buffer (pH 7.4) containing 10 mM MgCl₂, 1 mM EGTA, and 10 µM pargyline (monoamine oxidase inhibitor). Incubation mixtures of 0.5 ml were composed of 0.4 ml of crude membrane preparation containing approximately 10 µg of membrane protein, 50 µl of radioligand, and 50 µl of solvent or competitor or alniditan (10 µM final concentration, for measuring nonspecific binding of the radioligand). Incubation was performed in the dark for 60 min at 25°C and stopped by rapid filtration under suction over GF/B filters (1.5-cm diameter) using a Brandel 96 sample harvester. Filters were rinsed two times with 3 ml of 50 mM Tris-HCl (pH 7.4) buffer cooled in ice-water. Radioligand concentration-binding experiments were performed with [³H]5-HT (specific activity, 80 to 130 Ci/mmol) using 10 to 12 concentrations ranging from 0.125 to 20 nM or with [³H]ocaperidone (specific activity, 18.3 Ci/mmol), using 8 to 10 concentrations ranging from 0.125 to 4 nM. The same buffer and incubation conditions were used for [³H]5-HT and for [³H]ocaperidone concentration binding experiments. The effect of 100 µM Gpp(NH)p on binding of both [³H]5-HT and [³H]ocaperidone and of various concentrations of ocaperidone on the binding of [³H]5-HT were investigated. In competition binding experiments, the effect of 12 concentrations of the cold competitor (ranging from 10⁴ to 10¹¹ M for agonists and as low as 10¹⁴ M for antagonists) was investigated on the binding of [³H]5-HT (3 nM) or [³H]ocaperidone (1 nM).

[³⁵S]GTPS Binding Assays. Membrane samples were thawed on ice and suspended at a concentration of approximately 15 µg of protein/ml in 50 mM Tris-HCl buffer (pH 7.4), 0.5 mM MgCl₂, 50 mM NaCl, 1 µM GDP, and 10 µM pargyline. Incubation mixtures of 1 ml were preincubated with the test compounds for 5 min at 30°C (compounds were added at six concentrations) before addition of 10 µl of 10 nM [³⁵S]GTPS (>1000 Ci/mmol). The incubation was run for 30 min at 30°C and stopped by rapid filtration over GF/B filters as described above. Filter-bound radioactivity was counted in a liquid scintillation spectrometer after overnight incubation of the filters in 3 ml of scintillation fluid. Basal [³⁵S]GTPS binding was counted in the absence of compounds. Stimulation of [³⁵S]GTPS binding by agonists was presented as a percentage over basal and was calculated as 100 × the difference between stimulated and basal binding (in cpm) divided by the amount of basal binding (in cpm). 5-HT or ocaperidone concentration-response curves for increases/decreases in [³⁵S]GTPS binding were analyzed by nonlinear regression using the Prism software (GraphPad). EC₅₀ values (concentration of compound at which 50% of its own maximal effect is obtained) were derived from the calculated curves.

Western Blotting. Membrane proteins (10 µg for the analysis of h5-HT_1DR, h5-HT_1BR, G and G subunits or 5 µg for analysis of G) were treated at 37°C for 2 h in lysis buffer (62.5 mM Tris-HCl (pH 6.8), 10% glycerol, 5% SDS, 0.01% bromphenol blue, and 1% -mercaptoethanol) and separated by SDS-polyacrylamide gel electrophoresis using standard techniques. Proteins were transferred to nitrocellulose membranes. Immunodetection was performed with a 1000-fold dilution of the antisera for h5-HT_1BR, G_{i/o common}, G_q, G₁, and G₂. The "G_{i/o common}" antibody is cross-reactive for all members of the G_i/o family. The antiserum for h5-HT_1DR was used at a concentration of 3 µg/ml. The peroxidase-coupled anti-rabbit secondary antibody was used in a final dilution of 1:5000. Enhanced chemiluminescence was used to visualize the bands as prescribed by the commercial supplier. Detection of each protein was performed using separate gels. Different exposure times were applied.

	Results

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Immunodetection of h5-HT_1DR, h5-HT_1BR, G-Proteins, and G₁₂ Expressed in Sf9 Cells. Recombinant baculoviruses containing the cDNAs encoding the human h5-HT_1DR and h5-HT_1BR (in fusion with the signal peptide of the baculovirus GP67 protein), diverse G subunits (G_i1, _i2, _i3, _o, and _q) and the G₁₂ subunits were used to perform coexpressions in Sf9 cells. H5-HT_1DR was expressed either alone, together with a mixture of the G_i1, _i2, _i3, and _o proteins (indicated as G_i/o on the figures) or with each of these G subunits individually. Each of these coinfections was performed with and without G₁₂. As a control, h5-HT_1DR was coexpressed with G_q12. For comparison, h5-HT_1BR was also introduced in Sf9 cells, either alone or together with G_i112. Expression of the different proteins was assessed in Western blotting experiments performed on crude membrane preparations issued from the diverse coinfection materials (Fig. 1). For h5-HT_1DR, multiple closely spaced bands around the expected molecular weight (42 kDa) were detected. Multiple immunoreactive bands were also detected for the 5-HT_1A, 1-adrenergic, and N-formyl peptide receptors when expressed in Sf9 cells, and these were postulated to represent biosynthetic receptor precursors or improperly processed receptors (Quehenberger et al., 1992; Butkerait et al., 1995). No immunoreactive band was detected with the same antibody for membrane samples from uninfected Sf9 cells or cells expressing h5-HT_1BR (not shown), which confirms the specificity of the signal. Using an antiserum for h5-HT_1BR, a predominant band could be detected in membrane preparations from Sf9 cells expressing h5-HT_1BR (±40 kDa). This difference (i.e., one predominant band for h5-HT_1BR versus multiple immunoreactive bands for h5-HT_1DR) suggests that these two receptors are processed in a different way or to a different extent in Sf9 cells. No signal was seen for membrane preparations from cells expressing h5-HT_1DR using this anti-h5-HT_1BR antibody (Fig. 1). The diverse G_i/o proteins (±40 kDa) could be detected using an antibody (G_{i common}) cross-reactive for all members of this family of G-proteins. Some variations were apparent in the expression levels of the different G subunits. For G_q, two immunoreactive bands were detected, as described previously (Hepler et al., 1993). The G₁ (36 kDa) and G₂ (6.5 kDa) proteins showed comparable expression levels from one infection to the other. With the antibodies applied, no endogenous cross-reactive G-protein homologues of the mammalian G₁, G₂, or G_i/o proteins were visualized in the membrane samples from uninfected Sf9 cells, despite the broad detection range of the "G_{i common}" antibody. The absence of endogenous cross-reactive G-proteins was also reported by others [Grünewald et al. (1996); Leopoldt et al. (1997) and Wenzel-Seifert et al. (1998)].

View larger version (48K): [in this window] [in a new window]   Fig. 1.   Immunoblot analysis of Gi1, Gi2, Gi3, Go, Gq, G1, G2, and h5-HT1DR and h5-HT1BR expressed in Sf9 cells. The analysis was performed on membranes of uninfected Sf9 cells (Sf9) or Sf9 cells expressing h5-HT1DR alone, together with G12 or together with G12 and each G subtype individually, as indicated at the top. H5-HT1BR was detected in membranes from cells coexpressing this receptor with Gi112. Antisera against h5-HT1DR (a, left panel) or h5-HT1BR (a, right panel), Gi/o or Gq (b), G1 (c), and G2 (d) were used to visualize the proteins expressed. NT: not tested.

Analysis of the Effect of G-Protein Coexpression on the [³H]5-HT Binding Properties of h5-HT_1DR Produced in Sf9 Cells. Membrane material from cells expressing diverse combinations of receptor and G-proteins were used for [³H]5-HT concentration binding experiments to estimate receptor expression levels and agonist affinity. A representative example of the [³H]5-HT concentration binding curves obtained with membrane material from Sf9 cells expressing h5-HT_1DR alone or in combination with G_i112 or G_q12 is shown in Fig. 2. The mean apparent K_d values derived from the computerized curve fitting of the [³H]5-HT binding data for each combination of receptor and G-protein are summarized in Table 1. Oscillations in the B_max values appeared independent from the combination of proteins expressed. The range of B_max values obtained are indicated in Table 1. Because the Sf9 expression system does not allow expression ratios between receptor and G-proteins to remain strictly constant from one infection to the other, experiments were performed on material issued from two to five independent coinfections to evaluate possible effects on apparent K_d values. For [³H]5-HT, an apparent mean K_d of 7 nM was measured for h5-HT_1DR when expressed alone. On average, addition of a single G_i/o subunit or a set of four G_i/o proteins led to an almost 2-fold increase in receptor affinity for [³H]5-HT. Reconstitution of the G-protein trimer by further addition of the G₁₂ subunits switched the receptor population to a high affinity state, increasing the agonist affinity of the receptor almost 5-fold as compared with the receptor expressed alone. For certain membrane preparations from cells overexpressing the receptor without G-protein, a minor high [³H]5-HT affinity component was detected, which probably represents a fraction of h5-HT_1DR coupled to endogenous G-proteins. When detected, this component represented maximally 15% of the total receptor population. The coexpression of G₁₂ or G_q12 with the receptor had no significant effect on agonist affinity of the receptor. For comparison, the [³H]5-HT affinity of h5-HT_1BR expressed in the same expression setup was determined. The relatively low agonist affinity of h5-HT_1BR when expressed alone (22 nM) was increased about 15-fold by the addition of G_i112 (1.5 nM).

View larger version (27K): [in this window] [in a new window]   Fig. 2.   Effect of G-proteins on [3H]5-HT binding at 25°C to h5-HT1DR coexpressed in Sf9 cells. Concentration binding isotherms and Scatchard plots (inset) for [3H]5-HT binding on membranes of Sf9 cells expressing h5-HT1DR alone, together with Gi112 or Gq12, are shown. The figure represents data obtained in one of several experiments. Kd and Bmax values were derived for each individual experiment using nonlinear curve fitting to the rectangular hyperbola, and mean values are summarized in Table 1.

View this table: [in this window] [in a new window]   TABLE 1 Effect of G-protein coexpression and Gpp(NH)p addition on [3H]5-HT binding to 5-HT1DR and 5-HT1BR expressed in Sf9 cells [3H]5-HT concentration binding experiments (0.125 to 20 nM, 10 to 12 points) were performed as described in the text on membrane material from Sf9 cells coexpressing h5-HT1DR or h5-HT1BR with the indicated G-protein combinations. One-site binding analysis was performed on the data. Mean apparent equilibrium dissociation constant (Kd) and range of maximal number of binding sites (Bmax) for [3H]5-HT binding to h5-HT1DR and h5-HT1BR are shown. (Examples of curves are shown in Fig. 1.) Gi/o indicates that the receptor was coexpressed with Gi1, Gi2, Gi3, and Go subunits simultaneously. Experiments were performed with cell membranes from two to five separate infections per case. Because infection efficiency and protein expression can vary with separate infections, the lowest and highest Bmax value is given instead of a mean value.

Pharmacological Profile of h5-HT_1DR. To test the pharmacological robustness of our expression setup for h5-HT_1DR, the capacity of diverse compounds for displacing 3 nM [³H]5-HT was assessed in competition binding experiments. Membrane material of cells expressing the receptor alone, together with G_i112, G_o12, or G_q12 were used. Agonists, a partial agonist and antagonists for h5-HT_1DR [as shown in signal transduction assays (Pauwels, 1997; Lesage et al., 1998)], were selected from diverse major chemical structural classes and tested. Tested compounds, K_i and pK_i values derived from the inhibition curves are listed in Table 2. Comparing pK_i values obtained for h5-HT_1DR expressed in Sf9 and in a stably transformed C6 glioma cell line (Leysen et al., 1996), the order of potency of the compounds appeared to be maintained. The pK_i values obtained for agonists using membrane material containing the receptor coexpressed with G_i112 are similar to the values obtained with C6 glioma cell membranes. For the agonists, the pK_i values obtained with cell membranes containing the receptor alone and the receptor with G_q12 were slightly lower than those obtained with the receptor expressed in combination with G_i112 or G_o12. For the three antagonists, pK_i values obtained for the receptor in noncoupled state (expressed alone or in the presence of G_q12) were up to two orders of magnitude higher than for the receptor in coupled state (expressed in the presence of G_i112 or G_o12). Inhibition curves showing the competition by ocaperidone of [³H]5-HT binding on h5-HT_1DR coexpressed with various G-proteins (in the presence or absence of Gpp(NH)p) are represented in Fig. 3. pIC₅₀ values and the Hill coefficient associated with these curves, when fitting to a one-site model with variable slope, are also shown in Fig. 3. Displacement of [³H]5-HT from the noncoupled h5-HT_1DR did not fit to a one-site competition curve with a Hill coefficient of 1. The fit could be improved by using a one-site model with a shallow slope (pIC₅₀ = 9.1; Hill coefficient = 0.4) or a two-site model, with 42% of the receptors having a high ocaperidone affinity (pIC₅₀ = 10.1) and the remainder a low affinity (pIC₅₀ = 7.1). The two-site model would imply the existence of different binding sites or a nonhomogenous receptor population, the one-site model would imply a mode of inhibition inconsistent with competitive inhibition. Treatment of membranes containing the G_i112- or G_o12-coexpressed receptor with Gpp(NH)p caused a decrease of the Hill coefficient of the inhibition curve for ocaperidone from unity to 0.44 as well as an increase of the potency of ocaperidone to displace [³H]5-HT.

View this table: [in this window] [in a new window]   TABLE 2 Inhibition of [3H]5-HT binding to membranes of Sf9 cells coexpressing the 5-HT1DR and diverse G-protein combinations by 5-HT receptor ligands Radioligand binding was performed with 3 nM [3H]5-HT as described in the text. pIC50 values were derived from individual curves and used to calculate pKi values. Ki values (nM) were added between brackets to facilitate comparison with Table 1. Values for membrane material from a C6-glioma cell line stably expressing the 5-HT1DR were added for comparison and are from Leysen et al. (1996).

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Inhibition at 25oC of [3H]5-HT binding by ocaperidone on h5-HT1DR. Membrane material from Sf9 cells expressing h5-HT1DR with Gi112, Go12, or Gq12 was labeled with 3 nM [3H]5-HT in the presence of increasing amounts of ocaperidone with or without 100 µM Gpp(NH)p. A single experiment (with points determined in duplicate) is shown that is representative of a total of three independent experiments. Curves were generated by fitting to a sigmoidal curve with variable slope. pIC50 values and Hill coefficients derived from this fitting are shown in the insert.

Analysis of [³H]Ocaperidone Binding on h5-HT_1DR. As for [³H]5-HT (see above and Table 1), the effect of G-protein and Gpp(NH)p addition on [³H]ocaperidone binding on h5-HT_1DR was assessed in concentration binding experiments. Representative examples of the [³H]ocaperidone concentration binding curves obtained with membrane material from Sf9 cells expressing h5-HT_1DR alone, in conjunction with G_i112 (with or without Gpp(NH)p) or G_q12 are shown in Fig. 4. The mean apparent K_d and B_max values derived from the computerized curve fitting of the [³H]ocaperidone binding data for each combination of receptor and G-protein are summarized in Table 3. The B_max values obtained with [³H]5-HT concentration binding experiments on the same membrane preparation are shown for comparison. Subnanomolar K_d values were obtained for the three membrane preparations tested, and no effect of Gpp(NH)p addition on the apparent binding affinity of [³H]ocaperidone was seen. For noncoupled receptor material, the B_max values obtained with [³H]ocaperidone were slightly higher as than those detected with [³H]5-HT. This might be due to the existence of an h5-HT_1DR component with low [³H]5-HT affinity, which is not covered by the [³H]5-HT concentration range used. For membranes from cells coexpressing h5-HT_1DR and G_i112, B_max values detected with [³H]ocaperidone were one fourth of those detected with [³H]5-HT. Gpp(NH)p treatment of membrane material containing the coupled receptor led to a 3-fold increase of (high affinity) [³H]ocaperidone binding sites.

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Effect of G-proteins and Gpp(NH)p on [3H]ocaperidone binding (25°C) to h5-HT1DR expressed in Sf9 cells. Concentration binding isotherms and Scatchard plots (inset) for [3H]ocaperidone binding to membranes of Sf9 cells expressing h5-HT1DR alone, together with Gi112 (with or without addition of 100 µM Gpp(NH)p) or Gq12, are shown. The figure represents data obtained in one of three experiments. Kd and Bmax values were derived for each individual experiment using nonlinear curve fitting to the rectangular hyperbola, and mean values are summarized in Table 3.

View this table: [in this window] [in a new window]   TABLE 3 Effect of G-protein coexpression and Gpp(NH)p addition on [3H]ocaperidone binding to the 5-HT1DR [3H]Ocaperidone concentration binding experiments (0.125 to 4 nM, 8 to 10 points) were performed as described in the text on membrane material from Sf9 cells expressing the h5-HT1DR alone, together with Gi112 or Gq12. Mean apparent equilibrium dissociation constant (Kd) and maximal number of binding sites (Bmax) for [3H]ocaperidone binding to the h5-HT1DR are shown. (Examples of binding curves are shown in Fig. 4.) For comparison, the Bmax values obtained with [3H]5-HT for the same membrane preparation are shown.

Effect of 5-HT and Ocaperidone on h5-HT_1DR Activity Assessed by Measurement of [³⁵S]GTPS Binding to G_i1. The agonist and antagonist/inverse agonist activity of 5-HT and ocaperidone, respectively, were investigated in [³⁵S]GTPS binding experiments using membrane preparations of Sf9 cells coexpressing h5-HT_1DR and G_i112. Concentration-effect curves with derived pEC₅₀ values are shown on Fig. 5. Typically, basal [³⁵S]GTPS binding yielded about 4000 cpm. No or weak (20%) enhancement of basal [³⁵S]GTPS binding was obtained on incubation with 5-HT. Addition of ocaperidone, on the contrary, decreased [³⁵S]GTPS binding levels by on average of 40%. The effect of ocaperidone could be competed by 5-HT, returning to the basal [³⁵S]GTPS binding levels. Ocaperidone was found more potent at inhibiting the constitutive h5-HT_1DR-driven [³⁵S]GTPS binding (pEC₅₀ = 7.8) than it was at displacing [³H]5-HT from the coupled receptor (pK_i = 7). For 5-HT, the pEC₅₀ value for activation of [³⁵S]GTPS binding (8.6) is close to the pK_d for the coupled receptor (8.8). Performing the [³⁵S]GTPS binding experiments at higher salt concentrations (100 and 150 mM NaCl) did not significantly influence the basal [³⁵S]GTPS binding levels and did not result in a decrease of the effect of ocaperidone on [³⁵S]GTPS binding levels (data not shown). This indicates that the agonist-independent coupling of h5-HT_1DR can also be seen at physiological salt concentrations.

View larger version (26K): [in this window] [in a new window]   Fig. 5.   Effect of 5-HT and ocaperidone on [35S]GTPS binding (30°C) to the G subunit of Gi112 coexpressed with h5-HT1DR. Membrane material of Sf9 cells coexpressing h5-HT1DR and Gi112 was preincubated with increasing amounts of 5-HT or ocaperidone before the addition of 0.1 nM [35S]GTPS. Basal [35S]GTPS binding was measured in the absence of compound. Competition between 5-HT and ocaperidone was tested by adding [35S]GTPS and 5-HT to membranes preparations preincubated with 1 µM ocaperidone. Results are expressed as a percentage of basal [35S]GTPS binding. Depicted data are mean ± S.D. of four to seven experiments. The mean of the individual pEC50 or pIC50 values ± S.D. as derived from the curve fitting are shown.

Analysis of the Type of Competition between 5-HT and Ocaperidone for Binding on h5-HT_1DR. The type of competition between 5-HT and ocaperidone binding was investigated by the analysis of [³H]5-HT concentration binding curves in the presence and in the absence of different fixed concentrations of ocaperidone performed on membrane preparations from cells expressing h5-HT_1DR alone or together with G_i112. Concentration binding curves and Scatchard plots are shown in Fig. 6.

View larger version (36K): [in this window] [in a new window]   Fig. 6.   Investigation of the competition between [3H]5-HT and ocaperidone in the presence and in the absence of the Gi112 heterotrimer. [3H]5-HT concentration binding experiments (0.0125 to 18 nM, 10 points) were performed on membrane material from cells expressing h5-HT1DR alone or in conjunction with Gi112, in the presence of the indicated ocaperidone concentrations. The concentration binding isotherms (upper panels) and the Scatchard plots (lower panels) are shown. Kd and Bmax values for the different curves are given. A single experiment representative of a total of three is represented.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

We took advantage of the baculovirus expression system in Sf9 cells to study intrinsic properties of h5-HT_1DR. Study of the equilibrium binding of the natural agonist 5-HT and of the inverse agonist ocaperidone on this receptor in coupled or noncoupled state as well as the study of h5-HT_1DR-driven activation of [³⁵S]GTPS binding to G_i1 allowed us to draw conclusions with regard to specificity of coupling, constitutive activity, and conformational changes of this receptor induced by G-protein interaction or ligand binding.

Reconstitution of h5-HT_1DR-G-Protein Coupling. [³H]5-HT concentration binding experiments on membranes from Sf9 cells expressing h5-HT_1DR alone (Table 1) revealed an apparent K_d value in the range of the low affinity form of the receptor expressed in Chinese hamster ovary cells (Hamblin and Metcalf, 1991). In general, no high agonist-affinity receptor population was detectable in these experiments. However, Scatchard analysis of [³H]5-HT concentration binding performed in the presence of 10 nM of the inverse agonist ocaperidone (Fig. 6) resulted in a curved plot. This suggests that, when most of the [³H]5-HT low affinity component of h5-HT_1DR is (preferentially) masked by the inverse agonist ocaperidone, a small high affinity component for [³H]5-HT binding becomes apparent. This might represent the receptor population coupled to endogenous G-proteins. These agonist high affinity sites are not accessible to ocaperidone at a concentration of 10 nM. When detected, these agonist high affinity sites did not exceed 15% of the total receptor population.

Constitutive Activity of h5-HT_1DR. A representative set of h5-HT_1D/1BR agonists and antagonists revealed a similar pharmacological profile for h5-HT_1DR expressed in Sf9 and mammalian cells (Table 2). Agonists showed the tendency to displace [³H]5-HT more efficiently at the coupled receptor, whereas all three antagonists clearly competed with [³H]5-HT binding much more potently at the noncoupled receptor, indicative of an inverse agonistic character. Studies of h5-HT_1DR performed in mammalian cells already suggested this for methiothepin and ketanserin (Thomas et al., 1995; Pauwels, 1997). However, because serum, needed for mammalian cell growth, contains 5-HT levels that might trigger receptor activation, neutral antagonism is difficult to distinguish from inverse agonism in such a system. Furthermore, coupled and noncoupled receptors cannot unambiguously be distinguished in such a mammalian expression system.

Analysis of the Binding of Ocaperidone to h5-HT_1DR. H5-HT_1DR conformations bound by ocaperidone were analyzed in a set of equilibrium binding experiments. Concentration binding experiments showed a saturation of the [³H]ocaperidone binding sites at a concentration of about 3 nM. However, at this concentration, B_max values obtained with [³H]ocaperidone equaled those seen with [³H]5-HT only for the noncoupled receptor preparations. In the case of the coupled receptor, the number of [³H]5-HT binding sites was 4-fold higher (Table 3). The latter discrepancy in B_max values was reduced by Gpp(NH)p addition, showing that uncoupling favors ocaperidone binding. The fact that ocaperidone only binds to a fraction of h5-HT_1DR when it is coexpressed with G_i112 indicates that a substantial amount of h5-HT_1DR couples to G_i112 in the absence of agonist. Thus, the significant level of constitutive activity of h5-HT_1DR was also demonstrated in this experiment. The [³H]ocaperidone concentration binding data, together with the [³H]5-HT competition binding experiments (Fig. 3), suggest the existence of a receptor conformation with high ocaperidone affinity (K_d of 0.5 nM) presumably associated with the noncoupled receptor and a conformation with low ocaperidone affinity (K_d of about 100 nM, not seen in the concentration binding experiment) associated with the coupled receptor.

View larger version (26K): [in this window] [in a new window]   Fig. 7.   h5-HT1DR: hypothesis on the conformational changes upon G-protein coupling or ligand binding. The model presented integrates the observed effects and is based on the two-state model for GPCR activation. The thickness of the arrows indicates the direction toward which the equilibrium is likely to be driven. H5-HT1DR has a high constitutive activity, i.e., it shows a substantial agonist-independent conformational change from the inactive (R) to the active receptor state (R*) in the presence of Gi112. The receptor in this state is able to enhance [35S]GTPS binding to the G-protein, which explains the high basal [35S]GTPS binding levels observed. The natural agonist 5-HT further stabilizes this active receptor conformation (R*.5-HT) in the presence of G-proteins and further increases [35S]GTPS binding to Gi112. Gpp(NH)p diminishes the pool of G-proteins able to interact with h5-HT1DR and enriches the receptor population in the inactive state (R·5-HT) which has a lower 5-HT affinity. The inverse agonist ocaperidone is clearly bound with higher affinity by the receptor expressed alone (R) as compared with the receptor coexpressed with Gi112 (R*). Binding of ocaperidone on the active receptor (R*) diminishes the levels of [35S]GTPS bound. This suggests that ocaperidone induces a receptor state that cannot activate the G-proteins (R·Oca). Apparent equilibrium dissociation constants for 5-HT and ocaperidone for the inactive (R) and active (R*) receptor states, as determined in our experiments, are shown.