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Regional Differences in the Coupling of 5-Hydroxytryptamine-1A Receptors to G Proteins in the Rat Brain

Unité Mixte de Recherche 677, Institut National de la Santé et de la Recherche Médicale (INSERM)/Université Pierre et Marie Curie, Institut Fédératif 70 des Neurosciences, Facultéde Médecine Pierre et Marie Curie, Paris, France (C.M.L.C., N.H., M.H., L.L.); and INSERM U513, Facultéde Médecine, Créteil, France (S.E.M.)

Received January 19, 2006; accepted June 13, 2006

	Abstract

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Numerous data showed that 5-hydroxytryptamine-1A (5-HT_1A) receptors couple to G_o/_i proteins for signal transduction. However, the subunit isoforms really involved in 5-HT_1A receptor coupling in brain remain to be identified. Moreover, regional differences in the functional characteristics of brain 5-HT_1A receptors have been evidenced repeatedly. Because such differences could be due to variations in G proteins interacting with the same receptor, relevant approaches were used for identifying subunits physically coupled to 5-HT_1A receptors in different regions of the rat brain. Using immunoaffinity chromatography coupled to Western blot detection, 5-HT_1A receptors were found to interact equally with G_o and G_i3 in the cerebral cortex, mainly with G_o and weakly with G_i3 in the hippocampus and exclusively with G_i3 in the anterior raphe area. In the hypothalamus, 5-HT_1A receptors seemed to be coupled to the latter two G proteins plus G_i1 and G_z. Complementary experiments based on an antibody capture technique coupled to both classic radioactivity and scintillation proximity assay detections showed that hippocampal 5-HT_1A receptor stimulation induced 5'-O-(3-[³⁵S]thio)triphosphate binding to immunoprecipitates with G_i3 and G_o antisera. In the anterior raphe, such 5-HT_1A receptor-mediated effect was obtained with G_i3 antiserum only. These results demonstrated the existence of regional differences in the coupling of 5-HT_1A receptors to G proteins in the rat brain. In the anterior raphe, 5-HT_1A receptors seem to interact specifically with G_i3, whereas in the hippocampus, they are mainly coupled to G_o proteins. Such a disparity in G-protein coupling might explain regional differences in adaptive regulations of brain 5-HT_1A receptors.

The development of heterologous recombinant systems expressing the 5-HT_1A receptor, such as COS-7, HeLa, Chinese hamster ovary, Sf9, GH4C1, LLC-PK1, and NIH-3T3 transfected cells, has provided relevant models to study the receptor coupling with different G-protein subtypes and to identify second messengers. These studies led to the conclusion that 5-HT_1A receptors preferentially interact with G_i3 subunits, followed, in decreasing affinity order, by G_i2 and less strongly with G_o, G_i1, and G_z proteins (Fargin et al., 1991; Bertin et al., 1992; Liu et al., 1994; Garnovskaya et al., 1997; Newman-Tancredi et al., 2002). In contrast, 5-HT_1A receptor coupling to G_q and G_s seemed to be weak or absent in such heterologous recombinant systems (Raymond et al., 1993).

The complexity of 5-HT_1A receptor coupling, evidenced in recombinant systems, matches the agonist-directed trafficking of receptor signaling theory (Kenakin, 1995). This concept suggests that, depending on the nature of the agonist used, receptors will selectively activate one specific G-protein subtype and downstream transduction pathway. Moreover, this theory also stresses the influence of the "receptor/G-protein ratio" on both the nature of the G protein involved in receptor signaling and the agonist efficacy (Kenakin, 1995; Newman-Tancredi et al., 1997).

In this context, it is well-established that 5-HT_1A receptor ligands may act as full agonists in the dorsal raphe nucleus (DRN) but only as partial agonists in the hippocampus (Sprouse and Aghajanian, 1988). Another example of 5-HT_1A receptor functional heterogeneity that might also be relevant to this theory concerns the regional differences in 5-HT_1A receptor adaptive changes caused by long-term modifications in central 5-HT neurotransmission. Thus, long-term treatment with selective serotonin reuptake inhibitors (SSRIs) and 5-HT transporter (5-HTT) gene disruption are well-known to induce functional desensitization of 5-HT_1A auto-receptors in the DRN but no changes in postsynaptic 5-HT_1A sites in the hippocampus (Chaput et al., 1986; Le Poul et al., 2000; Mannoury la Cour et al., 2001). In the DRN, this adaptive regulation is associated with a decrease in 5-HT_1A receptor-mediated [³⁵S]GTPS binding, suggesting an alteration of the receptor/G-protein coupling in both SSRI-treated rodents (Hensler, 2002) and 5-HTT knockout mice (Fabre et al., 2000). In the hypothalamus, the 5-HT_1A receptor desensitization that occurs in these two models was reported to be associated with down-regulation of G_o, G_i1, G_i2, G_i3, and G_z proteins (Li et al., 1997; Raap et al., 1999). Taken together, these data support the idea that such regional differences in the functional and adaptive properties of brain 5-HT_1A receptors are probably underlain by variations in receptor coupling to G proteins from one area to another.

To assess this hypothesis, we applied combined immuno-affinity chromatography and immunoblotting approaches. A specific anti-rat 5-HT_1A receptor antiserum was used (El Mestikawy et al., 1990; Riad et al., 1991) to identify the G subunits physically coupled to 5-HT_1A receptors in membrane preparations from different brain regions. Furthermore, G subunits concerned by 5-HT_1A receptor-mediated [³⁵S]GTPS binding were determined using both antibody capture assay with protein A-Sepharose beads and scintillation proximity assay (SPA) (Cussac et al., 2002).

	Materials and Methods

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Experiments were performed using adult male Sprague-Dawley rats (250-400 g body weight; Centre d'Elevage René Janvier, Le Genest-Saint-Isle, France). Animals were maintained under standard laboratory conditions (22 ± 1°C, 60% relative humidity, 12-h light/dark cycle, food and water ad libitum) for 5 to 10 days before the beginning of experiments. All the procedures involving animals and their care were conducted in conformity with the institutional guide-lines that are in compliance with national and international laws and policies (Council directive 87-848, 1987 October 19, Ministère de l'Agriculture et de la Forêt, Service Vétérinaire de la Santé etdela Protection Animale, permissions number 75-116 to M.H. and 75-977 to L.L).

Preparation of Membranes. Rats were killed by decapitation. Their brains were quickly removed, and the cerebral cortex, striatum, hippocampus, cerebellum, hypothalamus, and anterior raphe area were dissected in cold (0-4°C) and stored at -80°C before use. Frozen tissues were homogenized in 10 volumes (v/w) of ice-cold 0.05 M Tris-HCl containing 0.01 mM phenylmethylsulfonyl fluoride and 0.01 mM aprotinin, pH 7.4, with a Polytron tissue disrupter (PT OD; Kinematica, Basel, Switzerland). Homogenates were centrifuged at 40,000g for 20 min at 4°C. The pellets were washed twice by resuspension in 40 volumes of the same ice-cold buffer, followed by centrifugation and homogenization in the same volume of buffer. The resulting membrane suspension was incubated for 10 min at 37°C to allow the dissociation of membrane-bound endogenous 5-HT and then centrifuged and washed three more times as described above. The final pellet was gently homogenized in an appropriate volume of 0.05 M Tris-HCl, pH 7.4, to obtain the membrane suspension (20 mg of membrane proteins per milliliter) to be stored at -80°C until use.

Solubilization Procedure. Thawed membrane suspension was mixed with 0.1 volume (v/v) of 0.1 M [0.6% (w/v)] CHAPS in 0.05 M Tris-HCl, pH 7.4, then briefly sonicated (20 W/5 s) and left for 60 min at 4°C (El Mestikawy et al., 1988). The mixture was then centrifuged at 100,000g for 30 min at 4°C. The clear supernatant was collected and filtered through a 0.22-µm Millex GV filter (Millipore Corporation, Billerica, MA) before its use as the source of solubilized 5-HT_1A binding sites in subsequent assays. The protein concentration in the final soluble extract was 6 mg/ml.

Immunoaffinity Chromatography, Elution, and Concentration Procedures. The anti-rat 5-HT_1A receptor polyclonal antibody was purified (El Mestikawy et al., 1990; Riad et al., 1991) and coupled to an Affigel-10 column (2 cm high, 1.5 cm in diameter) as recommended by the manufacturer (Bio-Rad, Hercules, CA). The filtered supernatant (4 ml) from CHAPS-treated membranes was poured into the affinity column equilibrated with 0.05 M Tris-HCl, pH 7.4. After an overnight incubation at 4°C, the supernatant was removed, and the column was washed four times with 40 ml of 0.05 M Tris-HCl, pH 7.4, containing 0.1 M CHAPS and then with the same volume of the same buffer containing 0.01 M CHAPS.

Elution was performed in two steps. First, the G protein subunits specifically coupled to 5-HT_1A receptors were eluted within 15 min, at room temperature, with 5 ml of 0.05 M Tris-HCl, pH 7.4, supplemented with 1 mM 5-HT and 1 mM GTP. The column was then washed with 4 x 20 ml of 0.05 M Tris-HCl buffer, and the second elution was made at 4°C with 5 ml of 0.01 M glycine-HCl, pH 2, containing 0.01 M CHAPS to collect 5-HT_1A receptors adsorbed onto the column. Eluate was immediately neutralized with 1 M Tris-HCl, pH 7.4, and dialyzed against 2 liters of 0.05 M Tris-HCl, pH 7.4, containing 0.1% SDS, using a MicroProDiCon apparatus (model MPDC-310; Bio Molecular Dynamics, Beaverton, OR). After 3 days at 4°C, the neutralized eluate was concentrated to a final volume of 300 µl. The same dialysis-concentration procedure was applied to the first eluted fraction (5 ml) containing G proteins.

Immunoblot Analysis of Eluted G Proteins and 5-HT_1A Receptors. The solubilized proteins in concentrated dialysates were analyzed by SDS-polyacrylamide gel electrophoresis using 0.5-mm thick 10% acrylamide/bisacrylamide [29:1 (w/w)] gels with 0.1% SDS and 0.375 M Tris, pH 8.8. After migration, the proteins were electrophoretically transferred for 45 min to a nitrocellulose membrane (Hybond ECL; GE Healthcare, Little Chalfont, Buckinghamshire, UK) that was then incubated, at room temperature, in PBS/0.1% Tween (v/v) containing 5% nonfat dry milk for 1 h. The membrane was subsequently incubated overnight with rabbit polyclonal antibodies directed against either G_o, G_i1, G_i2, G_i3, G_z, or G_s (1:200 dilution) at 4°C. Analysis of the specificity of these anti-G antibodies using recombinant G subunits showed that no cross-reactivity occurred at the dilution used in our experiments except for anti-G_i1 and anti-G_i3, which slightly cross-reacted with G_i3 and G_i1 proteins, respectively. The secondary antibody (goat anti-rabbit IgG coupled to horseradish peroxidase conjugate; Sigma, St. Louis, MO) (1:16,000 dilution) was applied to the membrane for 60 min at room temperature. The blot was washed five times with PBS containing 0.1% Tween and once with PBS alone. After a 5-min incubation in ECL Plus chemiluminescence substrate solution (GE Health-care), the membrane was exposed to autoradiography Hyperfilm MP (GE Healthcare) for 15 s or analyzed using filmless autoradio-graphic analysis (FLA2000; Fuji, Tokyo, Japan).

Protein Determination. Proteins were estimated using the Folin phenol procedure (Lowry et al., 1951) with bovine serum albumin as the standard.

[³⁵S]GTPS Binding and G Subunit-Specific Immunoprecipitation. The binding of [³⁵S]GTPS to specific G proteins upon activation of 5-HT_1A receptors was measured using a method adapted from Selkirk et al. (2001). A first series of experiments allowed the determination of the optimal assay conditions leading to the highest ratio of 5-HT_1A-enhanced over basal [³⁵S]GTPS binding when starting with hippocampal membranes. On this basis, brain membranes (0.2-1.0 mg/ml) were incubated for 30 min in assay buffer (67 mM Tris-HCl, 4 mM MgCl₂, 160 mM NaCl, and 0.267 mM EGTA, pH 7.4) containing GDP (1.2 mM), [³⁵S]GTPS (0.4 nM), with or without 5-carboxamidotryptamine (5-CT, 10 µM) and WAY 100635 (10 µM, for the determination of nonspecific [³⁵S]GTPS binding; Fabre et al., 2000) (final volume, 800 µl) in a shaking water bath at 37°C. Incubation was ended by the addition of 500 µl of ice-cold assay buffer and transfer to ice. Membranes were separated from the reaction mix by centrifugation at 20,000g for 6 min, and the supernatant was discarded. Membrane pellets were then solubilized with 500 µl of a solubilization buffer [100 mM Tris-HCl, 1 mM EDTA, 20 mM NaCl, and 0.62% (v/v) Nonidet P-40, pH 7.4] for 1 h at 4°C. Samples were centrifuged (20,000g, 4°C), and 400 µl of the supernatant was incubated with anti-G protein antiserum (1/100) during 90 min at 4°C. Protein A-Sepharose beads [70 µl, 50% (w/v)] were added, and samples were rotated for a further 90 min at 4°C. After centrifugation (20,000g, 4°C), the supernatant was removed by aspiration, and the beads were washed three times with 500 µl of solubilization buffer and then resuspended in the same solubilization buffer (500 µl) and filtered through Whatman GF/B filters. After three washes with ice-cold 67 mM Tris-HCl, pH 7.4, each filter was immersed in 5 ml of scintillation fluid, and the entrapped radioactivity was counted. Data are expressed as mean ± S.E.M. of at least three independent experiments.

Scintillation Proximity Assays. SPAs were used to further determine G-protein subtypes specifically activated by 5-HT_1A receptor stimulation. The procedure described by Cussac et al. (2002) was adapted so as to be used after [³⁵S]GTPS binding and solubilization steps (see above). After solubilization, samples (200 µl) were transferred into a 96-well Opti plate (PerkinElmer Life and Analytical Sciences, Boston, MA) and incubated with 2 µl of specific anti-G polyclonal antibody (1/100) during 1 h at room temperature. SPA beads coated with secondary anti-rabbit antibodies (GE Healthcare) were then added in a volume of 50 µl/well. After overnight incubation under gentle agitation, the plates were centrifuged (10 min at 1300g), and radioactivity entrapped on beads was measured using a TopCount microplate scintillation counter (PerkinElmer Life Sciences). Data are expressed as mean ± S.E.M. of four independent experiments.

Chemicals. The following drugs were used: [³⁵S]GTPS (1000 Ci/mmol), from GE Healthcare; CHAPS, Nonidet P-40, phenylmethylsulfonyl fluoride, aprotinin, 5-HT creatinine sulfate, R-(+)-8-OH-DPAT HBr, and (±)-8-OH-DPAT HBr, from Sigma-Aldrich; 5-CT, from RBI/Sigma (Natick, MA); WAY 100635, from Wyeth-Ayerst (Princeton, NJ); GDP dilithium salt and GTP from Roche (Meylan, France); rabbit polyclonal antibodies directed against rat G_o,G_i1, and G_i3, human G_i2, G_z, and G_s, and mouse G_q (Santa Cruz Biotechnology, Santa Cruz, CA).

Statistical Analysis. All experiments (immunoaffinity chromatography, immunoblot analysis of eluted G proteins and 5-HT_1A receptors, [³⁵S]GTPS binding, and G subunit-specific immunoprecipitation, SPA) have been replicated at least three times (three to eight times) in independent trials.

Data were analyzed using paired Student's t test with the help of Prism 4 software (GraphPad Software Inc., San Diego, CA). Statistical significance was set at p 0.05.

	Results

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Immunoaffinity Chromatography Identification of G Subunits Specifically Coupled to 5-HT_1A Receptors
Regional Distribution of G_o, G_i1, G_i2, G_i3, G_z, and G_s Subunits. The presence of G-protein subtypes in membranes from the different rat brain regions of interest was investigated using an immunoblotting technique. Rabbit polyclonal antiserums used in these experiments were raised against peptide sequence in highly divergent domains of G subunits from rat or human. As shown in Fig. 1, G_o, G_i1, G_i2, G_i3, G_z, and G_s proteins were present in all brain structures studied (hippocampus, anterior raphe area, cortex, and hypothalamus), including striatum and cerebellum, in which 5-HT_1A receptors are not detected (Riad et al., 1991).

View larger version (16K): [in this window] [in a new window]   Fig. 1. Immunoblots of G subunits in membranes from various rat brain regions. Membrane-bound proteins were solubilized by CHAPS, separated by SDS-polyacrylamide gel electrophoresis, and transferred to nitrocellulose membranes as described under Materials and Methods. The latter membranes were incubated overnight with 1:200 dilution of anti-sera against Go (A), Gs (B), Gz (C), Gi1 (D), Gi2 (E), and Gi3 (F). Molecular mass markers, in kilodaltons, are indicated on the left. The first band (from left to right) corresponds to the recombinant protein and the others to various brain structures: Cx, cerebral cortex; Hip, hippocampus; Ra, anterior raphe area; Hy, hypothalamus; Cb, cerebellum; St, striatum.

Evidence for 5-HT_1A Receptor Retention on the Immunoaffinity Column. To validate its capacity to bind 5-HT_1A receptors, the immunoaffinity column was loaded with hippocampal CHAPS-solubilized extracts, and glycine-HCl/CHAPS, pH 2, eluates were analyzed by immunoblotting with anti-5-HT_1A receptor antibodies. As shown in Fig. 2, these antibodies labeled a single diffuse band at 63 kDa in crude hippocampal extracts (Hip), corresponding to the molecular mass of native N-glycosylated 5-HT_1A receptor (Emerit et al., 1987; El Mestikawy et al., 1989; Riad et al., 1991). The same heavily labeled band was found in the glycine-HCl fraction eluted from the immunoaffinity column (Fig. 2).

View larger version (24K): [in this window] [in a new window]   Fig. 2. Immunoaffinity chromatography of hippocampal 5-HT1A receptor. Rat hippocampal 5-HT1A receptor was solubilized and fixed on Affigel-10 immunoaffinity column as described under Materials and Methods. Elution with glycine-HCl (pH 2) allowed the desorption of 5-HT1A receptors which could be visualized on immunoblots with specific polyclonal antibody (left lane, Western blot with 5 µl of glycine-HCl eluate). The right lane (Hip, hippocampus) corresponds to 5-HT1A receptor immunolabeling in soluble extract of rat hippocampal membranes (before loading onto the immunoaffinity column). Molecular mass markers are indicated on the left.

View larger version (40K): [in this window] [in a new window]   Fig. 3. Immunoblots of different G subunits coupled to 5-HT1A receptors in various brain regions. Membrane-bound proteins were solubilized by CHAPS, and 5-HT1A receptor/G protein complexes were adsorbed onto Affigel-10 immunoaffinity column as described under Materials and Methods. G proteins were eluted by a 5-HT-GTP mix and identified by immunoblotting with specific anti-G antibodies (dilution, 1/200). The rp bands correspond to pure recombinant G subunits run in parallel with Affigel-10 immunoaffinity column eluates. Note the presence of Go and Gi3 (equally) in the cerebral cortex (A, Cx), of Go (mainly) and Gi3 (trace) in the hippocampus (B, Hip), of Gi3 (exclusively) in the anterior raphe area (C, Ra), and of Go, Gi1, Gi3, and Gz in the hypothalamus (D, Hy). In contrast, no G protein immunolabeling is detected with eluates from striatum (St) or cerebellum (Cb) extracts.

In contrast, immunoblotting analyses of immunoaffinity column eluates yielded no labeling with anti-G_i2, anti-G_z, and anti-G_s in case of 5-HT_1A receptor-G protein complexes solubilized from cortical (Fig. 3A), hippocampal (Fig. 3B), and anterior raphe (Fig. 3C) membranes. No immunolabeling with anti-G_s and anti-G_i2 was also noted with receptor complexes solubilized from the hypothalamus (Fig. 3D).

As expected from the absence of 5-HT_1A receptors in the striatum and the cerebellum (Hamon, 1997), no G proteins could be detected in eluates from immunoaffinity columns loaded with soluble membrane extracts from these regions (Fig. 3, A-D).

Identification of G-Protein Subtypes Activated by 5-HT_1A Receptor Stimulation in Various Rat Brain Regions
5-HT_1A Receptor Agonist-Induced [³⁵S]GTPS Binding onto Soluble Extracts from Rat Brain Membranes. Under optimal conditions determined from a preliminary series of experiments (see Materials and Methods), [³⁵S]GTPS binding induced by 10 µM 5-CT reached 60% over basal with hippocampal membranes (Fig. 4, A and B). The percentage of increase produced by 5-CT was less with membranes from the cerebral cortex and the anterior raphe area (Fig. 4A), in line with the lower density of 5-HT_1A receptors in these two regions compared with the hippocampus (Fabre et al., 2000).

View larger version (19K): [in this window] [in a new window]   Fig. 4. 5-HT1A receptor-mediated increase in [35S]GTPS binding in soluble extracts from rat brain membranes. A, 5-CT-stimulated [35S]GTPS binding in soluble extracts from hippocampus, cerebral cortex, and anterior raphe area membranes. As expected from their lower density of 5-HT1A receptors, the percentage of stimulation above basal was lower in both cerebral cortex and anterior raphe compared with hippocampus. B, differential efficacy of 5-CT and (+) and (±)-8-OH-DPAT to enhance [35S]GTPS binding in soluble extracts from hippocampal membranes. Each bar corresponds to the percentage increase over basal [35S]GTPS binding. C, concentration-dependent inhibition by WAY 100635 of 5-CT-stimulated [35S]GTPS binding in soluble extracts from hippocampal membranes. Each bar/point is the mean ± S.E.M. of triplicate determinations in three different experiments. **, p < 0.01; ***, p < 0.001 (paired Student's t test).

Immunoprecipitation of G Proteins Labeled with [³⁵S]GTPS in Soluble Extracts from 5-CT-Stimulated Hippocampal Membranes. In a first series of experiments, protein A-Sepharose beads were used to bind immunoprecipitates with various anti-G antibodies of [³⁵S]GTPS-labeled 5-HT_1A receptor-G protein complexes solubilized from hippocampal membranes after incubation with or without 5-CT (10 µM). As shown in Fig. 5, A and B, 5-CT-induced [³⁵S]GTPS labeling of immunoprecipitates was obtained with anti-G_o- and anti-G_i3 antibodies. This effect was mediated by 5-HT_1A receptor activation because it was completely inhibited in the presence of WAY 100635. In contrast, immunoprecipitation with anti-G_q, anti-G_z, and anti-G_s yielded no 5-CT-induced increase in [³⁵S]GTPS labeling of immunoprecipitates adsorbed onto protein A-Sepharose beads (Fig. 5C).

View larger version (22K): [in this window] [in a new window]   Fig. 5. 5-CT-stimulated [35S]GTPS binding onto Go and Gi3 but not Gq, Gz, and Gs subunits in soluble extracts from rat hippocampal membranes: antibody capture assays. [35S]GTPS binding onto Go (A) and Gi3 (B) subunits from 5-HT1A receptor/G protein complexes solubilized from hippocampal membranes after incubation with 10 µM 5-CT and/or 10 µM WAY 100635. 35S-labeled G subunits were immunoprecipitated and then bound onto protein A-Sepharose beads as described under Materials and Methods. In contrast to anti-Go and anti-Gi3 antibodies, anti-Gq, -Gz, and -Gs antibodies (C) did not yield any 5-CT-stimulated 35S-labeling. Results are expressed as the percentage of basal labeling with [35S]GTPS (100% = 2424 ± 29 and 1410 ± 43 dpm with anti-Go and anti-Gi3 antibodies, respectively) in the absence of 5-CT and WAY 100635. Each bar is the mean ± S.E.M. of triplicate determinations in eight independent experiments. ***, p < 0.001 (paired Student's t test), ns, not significant.

SPA Determination of G Proteins Labeled by [³⁵S]GTPS in Soluble Extracts from 5-CT-Stimulated Hippocampal and Raphe Membranes. Additional experiments were performed using an SPA technology with a protocol derived from that used with protein A-Sepharose beads and adapted to 96-well microplates. The high sensitivity of the detection by SPA led us to perform experiments with membranes from the anterior raphe area in addition to hippocampal membranes. A shown in Fig. 6A, 5-CT induced a robust increase in [³⁵S]GTPS binding to both G_o- and G_i3-immunoprecipitated samples, corroborating the results obtained with protein A-Sepharose beads. By contrast, no effect of 5-CT could be detected in assays with anti-G_s antibodies.

View larger version (33K): [in this window] [in a new window]   Fig. 6. Differential 5-CT-stimulated [35S]GTPS binding onto Gi3 and Go in the hippocampus and the anterior raphe area: scintillation proximity assays. [35S]GTPS binding onto Gi3 and Go subunits was measured using SPA after incubation of hippocampal (A) and anterior raphe (B) membranes with 10 µM 5-CT and/or 10 µM WAY 100635. Under similar conditions with hippocampal membranes, no 5-CT-induced [35S]GTPS labeling was found with anti-Gs antibodies (A). Results are expressed as the percentage of basal values in the absence of 5-CT and WAY 100635 (100%, hippocampus: 1080 ± 75 and 938 ± 28 dpm; anterior raphe area: 586 ± 28 and 636 ± 18 dpm, with anti-Go and anti-Gi3 antibodies, respectively). Each bar is the mean ± S.E.M. of triplicate determinations in four independent experiments. **, p < 0.01; ***, p < 0.001; ns, not significant; paired Student's t test.

As expected from its mediation through 5-HT_1A receptors, the 5-CT-induced increase in [³⁵S]GTPS labeling of immunoprecipitates with anti-G_i3 and anti G_o antibodies was not observed with membranes that had been incubated with both 5-CT and WAY 100635 (Fig. 6, A and B).

	Discussion

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Using appropriate immunopurification strategies, we herein report the first direct identification of G proteins coupled to native 5-HT_1A receptors in the rat brain. Our data were obtained using two complementary approaches that have been used already for either identifying the coupling between a receptor and a G protein or estimating the efficacy of this coupling (Shreeve, 2002). The immunoaffinity copurification technique relies on the high specificity of the anti-serum immobilized on the immunoaffinity column. Previous characterizations have shown that our antiserum selectively immunoprecipitates 5-HT_1A but not other 5-HT₁ receptors in solubilized hippocampal membranes and that the receptor/G-protein coupling is preserved in the immune complex (El Mestikawy et al., 1990; Riad et al., 1991). Solubilization of tissue extracts was another critical step, because the detergent could induce the dissociation of the 5-HT_1A receptor/G-protein complexes. However, we found previously that CHAPS enables the solubilization of functional 5-HT_1A receptors physically coupled to G proteins (El Mestikawy et al., 1988; Emerit et al., 1990).

For our immunopurification protocols, we also used immunoprecipitation procedures whose reliability depends on the specificity of anti-G antiserums. This critical point has been evaluated using recombinant G proteins. No cross-reactivity was observed with the used antibodies, except for those directed against G_i1 and G_i3 subunits. Because immunoaffinity chromatography experiments showed that 5-HT_1A receptors did not couple with G_i1 in the hippocampus and the anterior raphe area, it is probable that the radioactivity measured in immunoprecipitates with anti-G_i3 antibodies resulted from the precipitation of G_i3- and not G_i1-[³⁵S]GTPS-labeled complexes. Concerning the SPA approach, the selectivity of the detection was improved by using secondary antibodies coated on beads that recognize the primary antiserum more specifically than the protein A-Sepharose does. The resulting reduction of the background noise makes this technique more sensitive and enabled the detection of low-intensity signals such as those obtained with raphe membranes (Fig. 6B).

One of the most important observations of our study is that the G-protein coupling of 5-HT_1A receptors exhibited clear-cut regional differences in the rat brain. However, all of the G proteins that interact with 5-HT_1A receptors in the cortex, hippocampus, hypothalamus, and anterior raphe area belong to the G_i/G_o family (G_o, G_i1, G_i3, and G_z). All of them have already been identified using heterologous coexpression of 5-HT_1A receptors with various G subunits in recombinant systems (Bertin et al., 1992; Raymond et al., 1993; Garnovskaya et al., 1997).

In vitro data have suggested that 5-HT_1A receptor/G_i2 coupling results in both AC inhibition and increase of intracellular Ca²⁺ concentration (Raymond et al., 1993; Albert et al., 1996). Despite these data, no band corresponding to G_i2 was identified in any tested fractions, indicating that, in the rat brain, native 5-HT_1A receptors do not activate this G-protein subtype.

Although no direct interaction between G_s protein and 5-HT_1A receptor has been observed in transfected Chinese hamster ovary cells (Raymond et al., 1993), mutations in the third intracellular loop of the 5-HT_1A receptor have been shown to induce a weak G_s coupling (Malmberg and Strange, 2000). Furthermore, both microdialysis studies and biochemical experiments performed in rat hippocampus demonstrated an increased cAMP formation in response to 5-HT_1A receptor agonists, such as 8-OH-DPAT and 5-CT, suggesting a positive AC coupling of 5-HT_1A receptors through G_s stimulation (Shenker et al., 1987; Cadogan et al., 1994). However, it has to be stressed that the agonists used in these studies can stimulate other 5-HT receptors in addition to the 5-HT_1A type, notably 5-HT₇ receptors, which are known to activate AC via G_s proteins (Hamon, 1997). In fact, our data clearly showed that 5-HT_1A receptors interact with G_s in neither the hippocampus nor any other brain structures examined. In fact, Albert et al. (1999) found that the 5-HT_1A receptor-stimulated production of cAMP in HEK 293 cells requires the coexpression of AC type II, which constitutive activation involves subunits probably originating from G_i2 proteins. Taken together, these data suggest that a positive coupling between 5-HT_1A receptors and G_s might occur only under specific conditions that require both specific cellular milieu and particular AC subtypes that are not found in rat brain extracts.

In agreement with previous data in recombinant systems (Bertin et al., 1992; Raymond et al., 1993; Garnovskaya et al., 1997), clear-cut interaction of native 5-HT_1A receptors with G_i3 subunit was demonstrated in the cortex, the hypothalamus, the anterior raphe region, and the hippocampus of the rat brain. However, in the hippocampus, immunoaffinity chromatography experiments indicated that 5-HT_1A receptors coupled mainly with G_o and, to a lower extent, with G_i3. On the other hand, immunoprecipitation experiments evidenced that 5-CT similarly increased [³⁵S]GTPS binding onto both G-protein subtypes, suggesting that 5-HT_1A receptors could activate G_o and G_i3 with the same efficacy. Such discrepancies might be related to immunoaffinity chromatography conditions, in which only one active state of 5-HT_1A receptor could be present and interact essentially with the G_o subunit. In contrast, in immunoprecipitation experiments, the high-efficacy agonist 5-CT might stimulate two different active states of the receptor coupled to either G_o or G_i3 subunit (Kenakin, 1995). On the other hand, G_o and G_i3 subunits may display a difference in the rate of GDP dissociation, as already shown in the case of G_o and G_i1 (Remmers et al., 1999). Therefore, the G_i3-GTP complex would be less stable and G_i3 intrinsic GTPase activity higher than that of G_o subunit. Such characteristics would explain the lower signal obtained with G_i3 in immunoaffinity chromatography experiments. Finally, this coupling could also involve specific regulators of G-protein signaling (RGSs). Indeed, differences have been reported between RGS regulating G_o-versus G_i-GTPase activity (Lan et al., 2000).

The coupling of native 5-HT_1A receptors to G_o in the hippocampus corroborates previous results from electrophysiological experiments. Relevant studies demonstrated that 5-HT_1A receptor stimulation opens a GIRK conductance through the activation of G_o1-protein subtype in hippocampal granule cells (Oleskevich, 1995). It is interesting to note that more recent data from knockout mice evidenced that, in the hippocampus, G_o is the predominant G protein used for coupling both GABA_B and adenosine receptors to K⁺ channels (Greif et al., 2000). This conclusion can probably be extended to 5-HT_1A receptors, because the latter have been shown to share the same pool of G proteins with GABA_B and adenosine receptors in the hippocampus (Mannoury la Cour et al., 2004). In contrast, no interaction with G_o has been detected in the anterior raphe area in which we found that 5-HT_1A receptors physically coupled to G_i3 only.

This disparity between the hippocampus and the anterior raphe area is particularly relevant regarding the differential regulation of 5-HT_1A receptors. Long-term inactivation of 5-HT reuptake by SSRI treatment in rats and 5-HTT gene disruption in 5-HTT-/- mice induce a functional desensitization of 5-HT_1A autoreceptors within the DRN but no adaptive changes of 5-HT_1A heteroreceptors in the hippocampus (Le Poul et al., 2000; Mannoury la Cour et al., 2001). Transductional modifications are probably at the origin of such regional differences in adaptive regulation of 5-HT_1A receptors. Indeed the intronless structure of the 5-HT_1A receptor gene is incompatible with the possible existence of several forms of the receptor protein. This desensitization seemed to be associated with a decrease in 5-CT-stimulated [³⁵S]GTPS binding only in the DRN, suggesting an alteration of receptor/G-protein coupling in this region (Fabre et al., 2000; Hensler, 2002). Our results suggest that desensitization could implicate an alteration of the coupling of 5-HT_1A receptors with G_i3 but not G_o subunits. It is interesting that a recent in vivo study indicated that overexpression of RGS4 within the DRN attenuated specific G_i-mediated 5-HT_1A receptor signaling, leading to a decrease in 5-HT_1A autoreceptor functional response (Beyer et al., 2004). In contrast, such a mechanism would not exist in the hippocampus in which 5-HT_1A receptors mediate K⁺ channel opening essentially through G_o subunits (Oleskevich, 1995).

Therefore, in regions in which 5-HT_1A receptors are coupled with several G proteins, adaptive compensatory changes might occur to preserve the functional characteristics of the receptors. This might take place in the hippocampus (Greif et al., 2000) and in the cerebral cortex, in which the 5-HT_1A receptor/G-protein coupling is unaffected by long-term treatment with fluoxetine (Hensler, 2002). In the hypothalamus, 5-HT_1A receptors seemed to be coupled to four different G subunits, G_o, G_i1, G_i3, and G_z. In line with our data, a recent study demonstrated the existence of 5-HT_1A receptor-G_z interaction in the hypothalamic paraventricular nucleus using G_z antisense oligodeoxynucleotides (Serres et al., 2000). It is interesting that hypothalamic 5-HT_1A receptors have also been shown to be functionally desensitized after long-term SSRI administration. This adaptive change was reported to be associated with a reduction in the levels of G_o, G_i1, and G_i3 proteins (Li et al., 1997) and a decrease in membrane-bound G_z protein (Raap et al., 1999). Such a down-regulation of all G proteins coupled to hypothalamic 5-HT_1A receptors (i.e., the absence of any opposite compensatory changes among these proteins) probably accounts for 5-HT_1A receptor desensitization in this particular region.

In conclusion, our data demonstrate that, in the rat brain, regional differences exist regarding the G protein subtypes that interact with native 5-HT_1A receptors. These differences are particularly striking in the anterior raphe area versus the hippocampus, in which differential adaptive changes in 5-HT_1A receptors have been reported repeatedly after long-term blockade of 5-HT reuptake. Determinations of 1) G_i3 and G_o mRNA and protein levels, 2) associated G subunits, and 3) the stoichiometry between G_i3/G_o and G subunits, specifically in cells expressing 5-HT_1A receptors, will ultimately provide key data concerning the regional differences in 5-HT_1A receptor signaling and regulation. In addition, deciphering the mechanisms through which differential coupling occurs is definitively the further goal to be achieved. In particular it will be necessary to identify the different partner proteins that interact with these particular G proteins in the transduction cascade downstream of 5-HT_1A receptor stimulation. The probable presence of several RGS proteins in 5-HT_1A receptor-expressing cells, the heterogeneity of G protein-coupled receptor kinases, and possible regional differences in 5-HT_1A receptor-dependent effectors (such as GIRK channels) has also to be considered in studies aimed at explaining the functional and regulatory heterogeneity of brain 5-HT_1A receptors.

	Acknowledgements

 
We are grateful to Wyeth-Ayerst for the generous gift of WAY 100635.

	Footnotes

 
This research was supported by grants from the Institut National de la Santé et de la Recherche Médicale (INSERM), Université Pierre et Marie Curie, Bristol-Myers Squibb Foundation (Unrestricted Biomedical Research Grant Program), and the European Community NewMood Program (LSHM-CT-2003-503474). C.M.L.C. was the recipient of fellowships from the Fondation pour la Recherche Médicale and INSERM (poste d'accueil) during performance of this work.

This work was previously presented in abstract form ( Mannoury La Cour C, El Mestikawy S, Rumajogee P, Bernard R, Miquel MC, Hamon M, and Lanfumy L (2001) Regional differences in G proteins coupled to 5-HT_1A receptors in the rat brain (Abstract). Soc Neurosci Abstr 27: 380.7 ).

ABBREVIATIONS: 5-HT_1A, 5-hydroxytryptamine-1A; 5-CT, 5-carboxamidotryptamine; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate; AC, adenylyl cyclase; 5-HTT, 5-hydroxytryptamine transporter; [³⁵S]GTPS, 5'-O-(3-[³⁵S]thio)triphosphate; DRN, dorsal raphe nucleus; PBS, phosphate-buffered saline; RGS, regulator of G-protein signaling; SSRI, selective serotonin reuptake inhibitor; GIRK, G-protein-gated inwardly rectifying K⁺; SPA, scintillation proximity assay; 8-OH-DPAT, 8-hydroxy-2-dipropylaminotetralin; WAY 100635, N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexane carboxamide.

¹ Current affiliation: Institut de Recherches Servier, Croissy sur Seine, France.

Address correspondence to: Dr. L. Lanfumey, UMR 677 INSERM/UPMC, Neuropsychopharmacologie, Facultéde Médecine Pierre et Marie Curie, Site Pitié-Salpétrière, 91, Boulevard de l'Hôpital, 75634 Paris Cedex 13, France. E-mail: lanfumey{at}ext.jussieu.fr

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