Introduction

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5-Hydroxytryptamine (5-HT) is a neurotransmitter that affects diverse physiological processes, including sleep, sexual behavior, food intake, locomotion, and mood. Schizophrenia, depression, and migraine are among the pathological conditions that are associated with a dysfunction of 5-HT transmission. At least 13 different 5-HT receptors have been identified to date. They belong to the superfamily of seven-transmembrane-domain receptors that couple to heterotrimeric guanine nucleotide-binding proteins (G proteins), with the exception of the 5-ht₃ receptor, which forms a 5-HT-gated ion channel (for review, Saudou and Hen, 1994; Hoyer and Martin, 1997).

The 5-ht_5A and 5-ht_5B receptors of the 5-ht₅ receptor subfamily were first identified in mice (Plassat et al., 1992; Matthes et al., 1993) and subsequently in rats (Erlander et al., 1993). Rees et al. (1994) cloned the human 5-ht_5A receptor (h5-ht_5A) homolog, but a 5-ht_5B receptor does not seem to be functionally expressed in humans (Rees et al., 1994). The physiological function of 5-ht₅ receptors is still unclear, partly due to a lack of specific ligands. Recently, results obtained with transgenic mice lacking the 5-ht_5A receptor gene suggested the involvement of the receptor subtype in exploratory behavior (Grailhe et al., 1999). The mouse, rat, and human 5-ht₅ receptors have already been expressed in various cell lines. Initially, no effects on signal transduction systems could be demonstrated (Erlander et al., 1993; Matthes et al., 1993), although agonist binding to the recombinant receptor was found to be guanine nucleotide-sensitive (Plassat et al., 1992). Negative coupling to adenylate cyclase activity was first reported for the rat 5-ht_5A receptor expressed in C6 glioma cells (Carson et al., 1996). Recently, agonist-induced inhibition of adenylate cyclase activity was also demonstrated for the human 5-ht_5A receptor expressed in human embryonic kidney (HEK) 293 cells (Francken et al., 1998; Hurley et al., 1998). In studies of agonistinduced stimulation of [³⁵S]guanosine-5'-O-(3-thio)triphosphate ([³⁵S]GTPS) binding, h5-ht_5A receptors expressed in HEK 293 cells were shown to couple to pertussis toxin-sensitive G proteins (Francken et al., 1998).

The Spodoptera frugiperda 9 (Sf9) insect cell/baculovirus system has already been successfully used to reconstitute the interaction of various G protein-coupled receptors with their cognate G proteins (Butkerait et al., 1995; Grünewald et al., 1996; Barr et al., 1997). When expressed in Sf9 cells at high levels, heterologous receptors display a predominantly uncoupled phenotype in the absence of recombinant G proteins due to the low background of endogenous G proteins (Butkerait et al., 1995; Boundy et al., 1996; Ohtaki et al., 1998). Therefore, receptor-G protein coupling specificity can be examined by coexpression in Sf9 cells of the receptor proteins with a series of G protein subtypes, through simultaneous infection with the appropriate recombinant baculoviruses. Successful receptor-G protein interaction is characterized by high-affinity and guanine nucleotide-sensitive agonist binding and by receptor-mediated activation of G proteins, as measured by agonist-stimulated [³⁵S]GTPS binding or GTPase activity.

To evaluate the G protein-coupling profile of the h5-ht_5A receptor in detail, we coexpressed combinations of receptor, G protein ₁₂ dimer (G₁₂), and various G protein -subunits (G subunits) in Sf9 insect cells. We measured the receptor coupling to members of each of the four families of G proteins using radioligand binding and [³⁵S]GTPS binding to membranes and investigated the pharmacological properties of various 5-HT receptor ligands. It was found that the h5-ht_5A receptor selectively couples to G_i/o proteins and that coexpression of the G₁₂ dimer facilitates receptor-G protein coupling.

	Experimental Procedures

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Materials. Sf9 insect cells were obtained from Invitrogen (Groningen, The Netherlands). The baculovirus transfer vector pAcGP67A and the BaculoGold DNA were purchased from PharMingen (San Diego, CA). The transfer vector pBacPAK9 was obtained from Clontech Laboratories (Palo Alto, CA). [³H]5-Carboxamidotryptamine (5-CT; 50-100 Ci/mmol), [³⁵S]GTPS (>1000 Ci/mmol), and the chemiluminescent Western detection kit (ECL-Plus) were purchased from Amersham Pharmacia Biotech (Little Chalfont, UK). 5-HT, 5-methoxytryptamine (5-MT), and dihydroergotamine (DHE) were purchased from Acros Organics (Geel, Belgium). Lysergic acid diethylamide (LSD) was obtained from Kenija Industriji (Yugoslavia). 5-CT was obtained from Research Biochemicals Inc. (Natick, MA). Methiothepin was purchased from Hoffman-La Roche (Basel, Switzerland). Pargyline was purchased from Sigma-Aldrich (St. Louis, MO). Grace's supplemented insect cell culture medium, Sf-900 II serum-free insect cell culture medium, and antibiotic/antimycotic solution were obtained from Life Technologies (Gaithersburg, MD). Fetal bovine serum was purchased from BioWhittaker (Walkersville, MD). The protein assay kit and the protein molecular weight marker were obtained from Bio-Rad Laboratories (Hercules, CA). Guanosine-5'-(,-imido)triphosphate (Gpp(NH)p) and GDP were obtained from Boehringer-Mannheim (Mannheim, Germany). The anti-G_i/o/t/z/s rabbit antiserum was purchased from Calbiochem (La Jolla, CA). The rabbit antisera for G_q/11, G₁₂, and G₁₃ were obtained from Chemicon International (Temecula, CA). The goat antiserum for G₁₆ was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The peroxidase-conjugated anti-rabbit and anti-goat secondary antibodies were obtained from Jackson ImmunoResearch Laboratories (West Grove, PE).

Cloning of h5-ht_5A Receptor cDNA. The coding region of the human 5-ht_5A receptor was amplified from a QuickScreen cDNA library (Clontech) by polymerase chain reaction using primers 5'-GCGATATGGACCCAGAGATGGATTTACCAGTGAACC-3' and 5'-GCCTCGAGCCTCAGTGTTGCCTAGAAAAGAAGTTCTTG-3'. The inclusion of restriction sites (EcoRV and XhoI) within the oligonucleotide primers allowed cloning of the polymerase chain reaction fragment into the pcDNA3 vector (Invitrogen). The sequence of the insert was identical to that reported by Hurley et al. (1998) and contained a single silent mutation (T to C at nucleotide 300, counting from the A of the start codon), compared with the sequence deposited in the GenBank/EMBL database (accession numbers X81411 and X81412) by Rees et al. (1994).

Construction of Recombinant Transfer Vector. The h5-ht_5A cDNA clone in pcDNA3 was digested with PstI, blunt-ended with Klenow DNA polymerase, and digested with XbaI, yielding a 1145-bp fragment encoding the h5-ht_5A receptor. This fragment was subcloned into the BamHI (filled in with Klenow DNA polymerase) and XbaI positions of the multiple cloning site of the baculovirus transfer vector pAcGP67A, such that the gp67 signal sequence was fused in frame to the N terminus of the h5-ht_5A coding sequence via a nine-amino acid linker sequence (gp67-ADRCDMDPE-h5-ht_5A). For the pBacPAK9-based transfer vector, the h5-ht_5A cDNA was excised from the pcDNA3 clone by digestion with EcoRI and XhoI. The 1142-bp fragment encoding the h5-ht_5A receptor was subcloned into the multiple cloning site of the transfer vector pBacPAK9 that was digested with the same restriction enzymes. Protein expression was under control of the polyhedrin promoter in both transfer vectors. The DNA insert sequences were confirmed by sequencing both strands of the double-stranded DNA.

Generation of Recombinant Baculoviruses. Transfer of the h5-ht_5A receptor cDNA into the wild-type Autographa californica nuclear polyhedrosis virus genome was accomplished by homologous recombination. Sf9 insect cells were cotransfected with linearized modified A. californica nuclear polyhedrosis virus baculovirus DNA (BaculoGold) and the h5-ht_5A-containing recombinant transfer vector using standard techniques (O'Reilly et al., 1992). Purification of recombinant viruses, amplification of purified virus stocks, and determination of virus titers were performed as described by O'Reilly et al. (1992).

Insect Cell Culture and Baculovirus Infection. Sf9 cells were grown at 27°C and at an ambient atmosphere in suspension culture using spinner flasks or in monolayers. For viral stock production, Grace's insect cell culture medium was used supplemented with 10% fetal bovine serum, 0.2 mM L-glutamine, and 1% antibiotic/antimycotic solution, whereas Sf-900 II serum-free insect cell culture medium, supplemented with 0.2 mM L-glutamine and 1% antibiotic/antimycotic solution, was used in recombinant protein expression experiments. Cell viability was determined by trypan blue staining. Cells (50-500 ml) at a density of 1 × 10⁶ cells/ml (log phase growth) were infected with a h5-ht_5A receptor-encoding baculovirus at a multiplicity of infection (m.o.i.) of 2 (unless stated otherwise), with a G₁₂-encoding virus (m.o.i. = 1) and/or with a G-encoding virus (m.o.i. = 2-4). For the expression of single G subunits, the m.o.i. was 4 for any G baculovirus, whereas for the expression of multiple G subunits (G_i1, G_i2, G_i3, and G_o, abbreviated as G_i/o) the m.o.i. was 2 for each virus. At 48 h postinfection, cells were harvested by centrifugation (10 min at 2000g at 4°C), washed with ice-cold PBS, and stored at 80°C or used directly for membrane preparation.

Membrane Preparation and Determination of Protein Content. Harvested Sf9 cells were washed with ice-cold 50 mM Tris-HCl buffer, pH 7.4; resuspended in hypotonic 10 mM Tris-HCl buffer, pH 7.4; and homogenized with an UltraTurrax homogenizer (Janke and Kunkel, Staufen, Germany) for 5 s. The homogenate was centrifuged at 30,000g for 20 min at 4°C. The membrane pellet was resuspended in 50 mM Tris-HCl buffer, pH 7.4, containing 10% glycerol and stored in aliquots at 80°C. Protein content in membrane preparations was estimated with the Bradford protein assay (Bradford, 1976), using the Bio-Rad kit. BSA was used as a standard.

Immunoblot Analysis. Membrane protein (1, 4, or 10 µg) was incubated in 62.5 mM Tris-HCl buffer, pH 6.8, containing 10% glycerol, 5% SDS, and 0.01% bromophenol blue at 37°C for 2 h. Proteins were separated by SDS-polyacrylamide gel electrophoresis and were transferred to polyvinylidene-difluoride membranes, using standard techniques. Immunodetection of G subunits was performed with 1:1000 dilutions of the G_i/o/t/z/s, G_q/11, G₁₆, G₁₂, and G₁₃ antisera. The peroxidase-conjugated anti-rabbit and anti-goat secondary antibodies were diluted 1:5000. Bands were visualized by chemiluminescence using the ECL-Plus detection kit.

Radioligand Binding. [³H]5-CT binding experiments were performed essentially as described previously (Francken et al., 1998). Briefly, 6 µg of membrane protein was diluted in 50 mM Tris-HCl buffer, pH 7.4, containing 10 mM MgCl₂, 1 mM EGTA, and 10 µM pargyline and incubated with [³H]5-CT for 1 h at 25°C in a volume of 0.5 ml. Nonspecific binding was estimated in the presence of 10 µM methiothepin. Reactions were terminated by rapid filtration through glass fiber (GF/B) filters (Whatman, Kent, UK) presoaked in 0.1% polyethyleneimine using a Brandel (Gaithersburg, MD) 96-sample harvester. Filters were washed twice, and filter-bound radioactivity was counted in a liquid scintillation spectrometer (Tricarb) using scintillation fluid (Ultima Gold MV; Packard Instrument Company, Meriden, CT). For radioligand concentration-binding isotherms, 12 concentrations of [³H]5-CT, in a range of 0.1 to 25 nM, were used. Competition binding experiments were performed using 2 nM [³H]5-CT; compounds were added at 7 to 12 concentrations.

[³⁵S]GTPS Binding. [³⁵S]GTPS binding experiments were performed as previously described (Francken et al., 1998). Briefly, 12 µg of membrane protein was diluted in 50 mM Tris-HCl buffer, pH 7.4, containing 50 mM NaCl, 10 mM MgCl₂, 1 mM EGTA, 0.1 mM dithiothreitol, 10 µM pargyline, and 1 µM GDP and preincubated with compound for 30 min at 30°C in a volume of 0.45 ml. Then, 50 µl of [³⁵S]GTPS in assay buffer was added to a final concentration of 0.2 nM, and the assay mixtures were further incubated for 30 min at 30°C. Reactions were terminated by rapid filtration through GF/B filters, presoaked in assay buffer, using a 40-well manual filtration manifold or a Brandel 48-sample harvester. Filters were washed twice, and filter-bound radioactivity was counted in a liquid scintillation spectrometer. Basal [³⁵S]GTPS binding was measured in the absence of compound. Compounds were added at 9 to 11 concentrations. Nonspecific [³⁵S]GTPS binding, as measured in the presence of 100 µM GTPS, did not exceed 10% of basal binding and was never subtracted from experimental data.

Data Analysis. Radioligand concentration-binding isotherms (rectangular hyperbola) were calculated by nonlinear regression analysis according to algorithms described by Oestreicher and Pinto (1987), and sigmoidal inhibition curves were calculated by nonlinear regression using the Prism program (GraphPad Software, San Diego, CA). B_max and K_d values of the radioligand and IC₅₀ values of inhibitors were derived from the curve fitting.

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

Expression of h5-ht_5A Receptors and G Protein Subunits in Sf9 Insect Cells. The h5-ht_5A receptor coding sequence was cloned from a cDNA library, and recombinant baculoviruses were generated and used to infect Sf9 cells. In preliminary [³H]5-CT concentration-binding experiments on membranes of Sf9 cells infected at an m.o.i. of 3, higher expression levels were found for virus generated with the pAcGP67A transfer vector (B_max = 63 ± 11 pmol/mg protein, K_d = 10.1 ± 2.0 nM, mean ± S.D., n = 5) than for pBacPAK9-based virus (B_max = 23 ± 2 pmol/mg protein, K_d = 5.6 ± 1.0 nM, n = 3), probably due to the presence of the gp67 signal sequence. No specific [³H]5-CT binding could be detected to membranes of uninfected or wild-type baculovirus-infected cells (data not shown). Further expression experiments were performed with the pAcGP67A-based virus at an m.o.i. of 2.

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Concentration-binding isotherms and Scatchard plots (insets) of specific [3H]5-CT binding to membranes of baculovirus-infected Sf9 insect cells expressing h5-ht5A receptors alone (A, ) or with G1 and G2 subunits (A, ) or coexpressing h5-ht5A receptors with Gi1 alone (B, ) or Gi1 with G1 and G2 (B, ). The data represent mean values of duplicate determinations from a typical experiment of three to six independent experiments. Sf9 cells were harvested after 48-h infection with a set of baculoviruses encoding the h5-ht5A receptor (m.o.i. = 2), G subunits (m.o.i. = 4), and/or G12 subunits (m.o.i. = 1). Radioligand binding studies were performed on membranes as described in Experimental Procedures. Isotherms were best fitted to a one-binding-site model using nonlinear regression analysis. Bmax and Kd values were derived for each individual experiment, and mean values are summarized in Table 1.

View larger version (41K): [in this window] [in a new window]   Fig. 2.   Immunoblot analysis of baculovirus-infected Sf9 membranes using anti-G subunit antisera. The analysis was performed on membranes of Sf9 cells infected with wild-type baculovirus (pAcNPV) or a combination of recombinant baculoviruses encoding h5-ht5A receptor and/or Gi1, Gi2, Gi3, Go, Gz, Gs, Gq, G11, G16, G12, or G13 heterotrimers (G12), as indicated above each lane. The antisera that were used to visualize expression of the mammalian G proteins are indicated at the right of the bands, whereas the positions of the molecular weight marker proteins are indicated at the left. The anti-Gi/o/t/z/s antiserum was tested on 4 µg of membrane protein, anti-Gq/11 antiserum was tested on 1 µg, and anti-G16, anti-G12, and anti-G13 antisera were tested on 10 µg of membrane protein. Gi/o represents the simultaneous expression of Gi1, Gi2, Gi3, and Go proteins.

Pharmacological Characterization of h5-ht_5A Receptors Expressed Alone or Coexpressed with G Protein Subunits. Various 5-HT receptor ligands were used to inhibit [³H]5-CT binding to membranes of baculovirus-infected Sf9 insect cells. pIC₅₀ values were derived from inhibition curves and are summarized in Table 2. The pharmacological profile of h5-ht_5A receptors expressed alone in Sf9 cells was different from that in stably transfected h5-ht_5A-HEK 293 cells; the agonists 5-CT, 5-HT, and 5-MT had 3.2- to 3.6-fold lower affinities (Student's t test, P < .05), whereas DHE, LSD, and methiothepin had slightly higher affinities for the receptor expressed alone in Sf9 cells. The rank order of potency of the tested compounds was LSD > methiothepin > 5-CT > DHE > 5-HT > 5-MT. This profile did not change on coexpression with G₁₂ subunits, although agonist affinities were slightly, but never significantly, increased.

View this table: [in this window] [in a new window]   TABLE 2 Inhibition by various 5-HT receptor ligands of [3H]5-CT (2 nM) binding to membranes of Sf9 cells coexpressing the h5-ht5A receptor and G protein subunits of the Gi/o and Gs family Radioligand binding studies were performed as described in Experimental Procedures, and pIC50 (logM) values were derived from individual curves. Results are mean pIC50 ± S.D. values from n independent experiments.

View larger version (30K): [in this window] [in a new window]   Fig. 3.   Inhibition of [3H]5-CT (2 nM) binding to membranes of baculovirus-infected Sf9 insect cells expressing h5-ht5A receptors alone (, ) or together with Gi1 and G12 subunits (, ). A, 5-HT (, ) and LSD (, ). B, 5-CT (, ) and DHE (, ). C, 5-MT (, ) and methiothepin (, ). Depicted points are mean ± S.D. values of three to seven independent experiments. Mean values of pIC50 values derived from individual curves are given in Table 2. Baculovirus infection of Sf9 cells and radioligand binding studies were performed as described in Experimental Procedures.

Effect of Gpp(NH)p on [³H]5-CT Binding. The interaction of h5-ht_5A receptors with endogenous or coexpressed G proteins in membranes of baculovirus-infected Sf9 cells was investigated by measuring the sensitivity of agonist binding to the addition of the nonhydrolyzable GTP analog Gpp(NH)p. [³H]5-CT concentration-binding experiments in the presence and absence of 100 µM Gpp(NH)p were performed in parallel, and the K_d values were compared using a paired Student's t test (Table 1). Figure 4 visualizes the ratio of K_d values for [³H]5-CT binding in the presence and absence of Gpp(NH)p. The affinity of [³H]5-CT observed for the h5-ht_5A receptor expressed alone was unaffected by Gpp(NH)p, suggesting the absence of interaction with endogenous G proteins. The small increase in affinity achieved by G₁₂ coexpression was completely reversed by Gpp(NH)p. The affinity of [³H]5-CT significantly decreased on Gpp(NH)p addition to membranes of Sf9 cells coexpressing the h5-HT_5A receptor together with G₁₂ and either the G_i/o mixture, G_i1, G_i2, G_i3, or G_o, whereas no significant decrease in affinity was observed for the other G protein coexpressions (Fig. 4).

View larger version (23K): [in this window] [in a new window]   Fig. 4.   Gpp(NH)p sensitivity of [3H]5-CT binding to membranes of Sf9 cells coexpressing h5-ht5A receptors and diverse G protein subunits. The data represent the mean ratios (±S.D.) of the Kd values for [3H]5-CT in the presence of 100 µM Gpp(NH)p versus the Kd values for [3H]5-CT in the absence of Gpp(NH)p. Concentration-binding experiments were performed in parallel in the absence and presence of 100 µM Gpp(NH)p as described in Experimental Procedures. Bmax and Kd values were derived for each individual experiment, and mean values are summarized in Table 1. For each individual experiment, the ratio was calculated of the Kd value in the presence versus that in the absence of 100 µM Gpp(NH)p. Comparisons were made using the paired two-tailed Student's t test. *Significant (Student's t test, P < .05) difference in Kd value for [3H]5-CT in the presence versus in the absence of 100 µM Gpp(NH)p.

5-HT-Stimulated [³⁵S]GTPS Binding. Receptor-mediated activation of G proteins was examined by concentration-dependent 5-HT-stimulated binding of [³⁵S]GTPS to membranes of baculovirus-infected Sf9 cells. The activation of G proteins involves stimulation of GDP/GTP exchange at the G subunit and can be measured by the incorporation of the nonhydrolyzable GTP analog [³⁵S]GTPS (Wieland and Jakobs, 1994). Figure 5 depicts the dose-dependent increase in 5-HT-stimulated [³⁵S]GTPS binding for Sf9 cells expressing h5-ht_5A receptors alone or together with G₁₂ and/or the G_i/o mixture. In membranes of Sf9 cells expressing only h5-ht_5A receptors without mammalian G protein subunits, stimulation of the receptors with 5-HT resulted in an increase in [³⁵S]GTPS binding to a maximum of 40% over basal, probably due to the activation of endogenous G proteins. Coexpression of G₁₂ resulted in a significant increase of the maximum response (Student's t test, P < .05), up to 110% over basal. When the receptor was coexpressed with the mixture of G_i/o subunits, without or with G₁₂, the maximum stimulation was 330 and 570%, respectively. The effect of 5-HT was specific for the h5-ht_5A receptor, because 5-HT did not affect [³⁵S]GTPS binding to membranes from uninfected or wild-type baculovirus-infected Sf9 cells (data not shown).

View larger version (30K): [in this window] [in a new window]   Fig. 5.   5-HT-stimulated binding of [35S]GTPS to membranes of baculovirus-infected Sf9 cells expressing h5-ht5A receptors alone or in combination with G protein subunits. , h5-ht5A receptors expressed alone. , h5-ht5A receptors coexpressed with the G12 complex. , h5-ht5A receptors coexpressed with the mixture of Gi1, Gi2, Gi3, and Go subunits. , h5-ht5A receptors coexpressed with the G12 complex and the mixture of Gi1, Gi2, Gi3, and Go subunits. Membranes were preincubated with compound for 30 min at 30°C and incubated with 0.2 nM [35S]GTPS for an additional 30 min at 30°C. Basal [35S]GTPS binding was measured in the absence of 5-HT, and the percentage of stimulation was calculated as defined in Experimental Procedure. Depicted points are mean ± S.D. values from two to five independent experiments, each performed in duplicate. Mean pEC50 and maximum stimulation values are summarized in Table 3.

View this table: [in this window] [in a new window]   TABLE 3 Stimulation by 5-HT of [35S]GTPS (0.2 nM) binding to membranes of Sf9 cells coexpressing the h5-ht5A receptor and G protein subunits of the Gi/o, Gs, Gq/11, and G12/13 family [35S]GTPS binding studies were performed as described in Experimental Procedures. The maximum stimulation and pEC50 values were derived from the curves. The results are mean ± S.D. values from n independent experiments.

Modulation of [³⁵S]GTPS Binding by 5-HT Receptor Ligands. Several 5-HT receptor ligands were examined for their ability to modulate [³⁵S]GTPS binding to membranes of Sf9 cells, expressing h5-ht_5A receptors with G₁₂ and either G_i1, G_i2, G_i3, or G_o. Figure 6 shows, as an example, the mean curves for the coexpression of h5-ht_5A receptors with G_i1 and G₁₂. Table 4 summarizes the pEC₅₀, E_max, pIC₅₀-corr, and I_max values from all [³⁵S]GTPS dose-response and inhibition curves.

View larger version (23K): [in this window] [in a new window]   Fig. 6.   [35S]GTPS binding to membranes of baculovirus-infected Sf9 cells coexpressing h5-ht5A receptors with the G12 complex and Gi1 subunits. A, stimulation of [35S]GTPS binding by 5-HT receptor agonists. B, antagonism of 5-HT (10 µM)-stimulated [35S]GTPS binding by 5-HT receptor ligands. Membranes were preincubated with 5-HT for 30 min at 30°C and incubated with 0.2 nM [35S]GTPS for an additional 30 min at 30°C. Basal [35S]GTPS binding was measured in the absence of compounds, and the percentage of stimulation was calculated as defined in Experimental Procedures. Depicted points are mean ± S.D. values from two to seven independent experiments, each performed in duplicate. Mean pEC50, Emax, pIC50-corr, and Imax values are summarized in Table 4.

View this table: [in this window] [in a new window]   TABLE 4 Effect of various 5-HT receptor ligands on [35S]GTPS (0.2 nM) binding to membranes of Sf9 cells coexpressing the h5-ht5A receptor and G protein subunits of the Gi/o family [35S]GTPS binding studies were performed on membranes as described in Experimental Procedures. pEC50 and pIC50 values were derived from the curves. Emax values and Imax values were calculated, and pIC50 values were corrected into pIC50-corr values as described under Experimental Procedures. The results are mean ± S.D. values from n independent experiments.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

Little is known about the pharmacological and functional properties of cloned 5-ht₅ receptors. Recently, h5-ht_5A receptors were shown to mediate inhibition of adenylate cyclase activity in transfected HEK 293 cells (Francken et al., 1998; Hurley et al., 1998). High-affinity agonist binding and agonist-stimulated [³⁵S]GTPS binding to h5-ht_5A-HEK 293 membranes were found to be pertussis toxin-sensitive (Francken et al., 1998), indicating the involvement of G_i/G_o proteins. To provide further insight in its signaling properties, we coexpressed the h5-ht_5A receptor in Sf9 insect cells with a series of 11 mammalian G proteins, from each of the four G families. Using radioligand and [³⁵S]GTPS binding assays, we demonstrated selective coupling of the h5-ht_5A receptor to coexpressed G_i and G_o proteins and the absence of coupling to G_z/G_s/G_q/G₁₁/G₁₆/G₁₂ and G₁₃ proteins. Hence, the h5-HT_5A receptor does not show promiscuous coupling to various G protein families. Although no clear coupling preference to either of the G_i/G_o subtypes was evident, we have observed differences in the coupling behavior of G_o versus G_i.

The overexpression in Sf9 cells of h5-ht_5A receptors alone resulted in a predominantly uncoupled phenotype, as demonstrated by guanine nucleotide-insensitive, low-affinity agonist binding. Although not evident from the binding data, h5-ht_5A receptors coupled to endogenous G proteins to some extent; 5-HT stimulated [³⁵S]GTPS binding to 40% over basal. We conclude that a large excess of uncoupled receptors is present in h5-ht_5A-Sf9 membranes. Although the activation of G proteins by a small fraction of coupled receptors can be detected due to the sensitivity of the [³⁵S]GTPS binding assay, the curve-fitting algorithms for the concentration-binding isotherms cannot reliably detect a high-affinity binding component of less than 10% of the B_max value.

When the h5-ht_5A receptor was coexpressed with G_i1/G_i2/G_i3 or G_o proteins (G₁₂ heterotrimers), the coupled phenotype was achieved, as evident from guanine nucleotide-sensitive, high-affinity agonist binding. In addition, the affinity of methiothepin, which was identified as an inverse agonist at h5-ht_5A-HEK 293 cells (Francken et al., 1998), decreased on coexpression of G_i/G_o proteins. These observations are consistent with two distinct states of the h5-ht_5A receptor, according to the two-state model (Leff, 1995). Receptors are proposed to exist in an active form (R*) that is G protein-coupled and an inactive form (R). Agonists show high affinity for R* and low affinity for R, whereas inverse agonists display the opposite behavior (Milligan et al., 1995). Our binding data suggest that h5-ht_5A receptors expressed in Sf9 cells convert to the active, high agonist affinity state (R*) through interaction with coexpressed G_i/G_o proteins. Remarkably, the affinities of DHE and LSD, which were identified as partial agonists at h5-ht_5A-HEK 293 cells, decreased on coexpression of G_i and/or G_o proteins. This observation might indicate that the two-state model of agonist action is not generally applicable to partial agonists.

Evidence for h5-ht_5A receptor-mediated G_i/G_o protein activation was obtained using [³⁵S]GTPS assays. The maximum level of 5-HT-stimulated [³⁵S]GTPS binding to coexpressed G_o proteins was similar to that found for h5-ht_5A-HEK 293 cells, whereas G_i1/G_i2/G_i3 and the mixture of G_i/o proteins were stimulated by 5-HT with approximately 4-fold higher efficacy. The lower level of 5-HT-mediated stimulation of G_o, compared with G_i, might be explained by the 2.6-fold higher basal [³⁵S]GTPS binding that was found for coexpressed G_o. This high agonist-independent [³⁵S]GTPS binding most probably originates from a larger number of G_o proteins in the Sf9 membranes compared with G_i, as G_o appeared more abundant than the various G_i subunits in immunoblot analysis. Alternatively, the h5-ht_5A receptor may exhibit stronger constitutive activation of G_o, compared with G_i. High agonist-independent binding complicates the detection of agonist-induced increases in [³⁵S]GTPS binding (Wieland and Jakobs, 1994). It could be that the assay conditions (e.g., buffer composition and incubation temperature) optimal for G_o activation differ from the applied conditions, such that the actual maximum stimulation of G_o by 5-HT might well be higher than reported.

Coexpression of the h5-ht_5A receptor with one of the other G proteins tested (G_z/G_s/G_q/G₁₁/G₁₆/G₁₂ or G₁₃) had no effect on agonist binding, and no or only minor 5-HT-induced activation of these G proteins could be detected. The expression of the different heterologous G proteins in the Sf9 membranes was confirmed using immunoblotting. All G proteins were highly expressed, and only G₁₂ showed weak immunoreactivity. Hence, poor subunit expression is not the reason for the absence of effects for the various G proteins, except perhaps for G₁₂. It should be noted that in the [³⁵S]GTPS studies, the assay conditions were not optimized for each individual G protein type. Under the applied conditions, which were optimized for [³⁵S]GTPS binding to h5-ht_5A-HEK 293 membranes, activation of some G protein types may therefore be suboptimal. As the need to optimize assay conditions for individual G proteins has been reported previously (Wieland and Jacobs, 1994), it would be rash to conclude the absolute absence of h5-ht_5A receptor coupling to G_z/G_s/G_q/G₁₁/G₁₆/G₁₂ or G₁₃ proteins based exclusively on the absence of increases in [³⁵S]GTPS binding. The lack of receptor interaction with these G proteins is only suggested by the fact that coexpression of these G proteins did not induce guanine nucleotide-sensitive, high-affinity agonist binding to the h5-ht_5A receptor. It appears that h5-ht_5A receptors selectively couple to G_i/G_o proteins, which is in agreement with the finding that pertussis toxin pretreatment completely abolished high-affinity agonist binding and 5-HT-stimulated [³⁵S]GTPS binding to h5-ht_5A-HEK 293 membranes (Francken et al., 1998).

The G complex has already been shown to be required for optimal receptor-G protein interaction (Fung, 1983; Butkerait et al., 1995). We have used the G₁₂ dimer to enhance G protein coupling to the h5-ht_5A receptor, because this dimer was reported to interact with members of the four G families (Barr et al., 1997). However, the subunit composition of G affects receptor-G protein coupling specificity (Kisselev and Gautam, 1993; Kleuss et al., 1993; Richardson and Robishaw, 1999), such that other G subunit compositions may yield different receptor coupling profiles. Therefore, we also investigated h5-ht_5A receptor-G protein coupling in the absence of the mammalian G₁₂ complex. The interaction of receptor with G_i1, G_i2, and G_i3 could still be detected in agonist binding and [³⁵S]GTPS assays, but it was indeed less effective than that in the presence of G₁₂. Remarkably, coexpression with G_o did not induce high-affinity agonist binding in the absence of G₁₂. Previously, Jockers et al. (1994) found similar results for adenosine A₁ receptors expressed in Escherichia coli; reconstitution of high-affinity agonist binding by purified G proteins was poor in the absence of G for G_o, but not for G_i, whereas in the presence of G, their maximum responses were similar. Despite the lack of effect on agonist affinity of G_o, the activation of h5-ht_5A receptors produced a maximum stimulation of [³⁵S]GTPS binding similar to G_i subunits. Coexpression of h5-ht_5A receptors and either G_z/G_s/G_q/G₁₁/G₁₆/G₁₂ or G₁₃ without G₁₂ did not result in the coupled phenotype, as expected from the lack of effect when G₁₂ heterotrimers were expressed. We conclude that the G₁₂ complex greatly facilitates coupling of G_i/o proteins to the h5-ht_5A receptor when coexpressed in Sf9 cells.

Coexpression of h5-ht_5A receptors and G₁₂ without mammalian G subunits revealed that G₁₂ enhances interaction of heterologous receptor with insect G proteins. Similar results were reported for the serotonin 5-HT_1A and the dopamine D_2S receptor (Butkerait et al., 1995; Boundy et al., 1996). Considering this finding, one should note that an improved interaction of recombinant receptors with endogenous G proteins due to coexpressed G₁₂ subunits may confuse the interpretation of receptor-G protein interaction specificity. Regardless, it is clear that the overexpression of specifically interacting G proteins should yield effects that exceed these observed for the appropriate controls.

For some receptors that couple to pertussis toxin-sensitive G proteins, preferential interaction with one of the G_i/G_o subtypes has been demonstrated (Senogles et al., 1990; Parker et al., 1991; Rubinstein et al., 1991; Grünewald et al., 1996; Clawges et al., 1997; Lorenzen et al., 1998). Our data indicate that the heterotrimeric G_i1, G_i2, G_i3, or G_o proteins interacted equally well with the h5-ht_5A receptor to induce its high-affinity conformation, and no significant differences in the affinities of the tested compounds were observed. However, in contrast to G_i, G_o did not induce high-affinity agonist binding in the absence of G₁₂, suggesting diminished receptor interaction. Furthermore, some striking differences between G_o and G_i proteins appeared from the [³⁵S]GTPS experiments. Maximum stimulation of [³⁵S]GTPS binding by 5-HT was significantly lower at G_o than at G_i, possibly due to the high agonist-independent [³⁵S]GTPS binding to G_o. In addition, the relative efficacies of DHE and LSD were dependent on the G protein type expressed. Both compounds were full agonists at the h5-ht_5A receptor when coexpressed with G_o, whereas coexpression with G_i proteins resulted in partial agonistic behavior, which was also found at the h5-HT_5A-HEK 293 membranes (Francken et al., 1998). These data might be explained by a difference in receptor/G protein stoichiometry, which can influence both agonist potency and efficacy (Hermans et al., 1999). Alternatively, the efficacy of compounds may be determined by the type of G protein interacting with the receptor. In this respect, Yang and Lanier (1999) have reported that recombinant expression of G_o, but not G_i1, increased the relative efficacy of clonidine in NIH-3T3 cells cotransfected with ₂-adrenergic receptor and G subunit, an effect that was not an issue of G protein or receptor levels. Although we cannot exclude that differences in h5-ht_5A receptor-to-G protein ratio cause the distinct behavior of G_o and G_i proteins, it is tempting to speculate that structural differences exist in their interaction with the h5-HT_5A receptor. However, differences in the nucleotide binding properties of the G protein types themselves should also be taken into account; as such, G_o may be easier to activate by receptors than G_i.

In summary, the h5-HT_5A receptor selectively coupled to mammalian G_i1/G_i2/G_i3 and G_o but not to G_z/G_s/G_q/11/16 or G_12/13 proteins, when coexpressed in Sf9 insect cells. Although G_o displayed different receptor coupling characteristics than G_i proteins, no clear coupling preference was evident.