J Biol Chem, Vol. 274, Issue 50, 35469-35474, December 10, 1999

Constitutive G_i2-dependent Activation of Adenylyl Cyclase Type II by the 5-HT1A Receptor
INHIBITION BY ANXIOLYTIC PARTIAL AGONISTS^*

Paul R. Albert, Naghmeh Sajedi, Sylvie Lemonde^§, and Mohammad H. Ghahremani^¶

From the Neuroscience Research Institute, Departments of Medicine and Cellular and Molecular Medicine, University of Ottawa, Ottawa, Canada K1H 8M5 and the ^¶ Department of Pharmacology and Therapeutics, McGill University, Montreal, Canada H3G 1Y6

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

The 5-HT1A receptor is implicated in depression and anxiety. This receptor couples to G_i proteins to inhibit adenylyl cyclase (AC) activity but can stimulate AC in tissues (e.g. hippocampus) that express ACII. The role of ACII in receptor-mediated stimulation of cAMP formation was examined in HEK-293 cells transfected with the 5-HT1A receptor, which mediated inhibition of basal and G_s-induced cAMP formation in the absence of ACII. In cells cotransfected with 5-HT1A receptor and ACII plasmids, 5-HT1A agonists induced a 1.5-fold increase in cAMP level. Cotransfection of 5-HT1A receptor, ACII, and G_i2, but not G_i1, G_i3, or G_o, resulted in an agonist-independent 6-fold increase in the basal cAMP level, suggesting that G_i2 preferentially coupled the receptor to ACII. The 5-HT1B receptor also constitutively activated ACII. Constitutive activity of the 5-HT1A receptor was blocked by pertussis toxin and the G antagonist, CT, suggesting an important role for G-mediated activation of ACII. The Thr-149  Ala mutation in the second intracellular domain of the 5-HT1A receptor disrupted G-selective activation of ACII. Spontaneous 5-HT1A receptor activity was partially attenuated by 5-HT1A receptor partial agonists with anxiolytic activity (e.g. buspirone and flesinoxan) but was not altered by full agonists or antagonists. Thus, anxiolytic activity may involve inhibition of spontaneous 5-HT1A receptor activity.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

The 5-HT1A receptor functions as an inhibitory somatodendritic autoreceptor on serotonergic neurons of the raphe nuclei and as a post-synaptic receptor in a variety of serotonergic targets (1-4). A number of partial agonists of the 5-HT1A receptor have been shown to synergize with serotonin reuptake blockers in treatment of depression (1, 4-7). The antidepressant action of these compounds appears to involve both agonist-mediated down-regulation of presynaptic 5-HT1A receptors and activation of the post-synaptic receptors (that are resistant to desensitization) to result in enhanced serotonergic neurotransmission. By contrast, the anxiolytic actions of 5-HT1A partial agonists may involve inhibition of serotonergic activity by enhancing autoreceptor activation and reducing post-synaptic receptor action. Homozygous null 5-HT1A receptor mutant mice display enhanced serotonin release and anxiety-associated behaviors (8-10), consistent with the idea that hyperactivity of the serotonin system by reduction in 5-HT1A autoreceptor activity results in anxiety disorders.

The 5-HT1A receptor mediates inhibitory signaling by coupling to pertussis toxin-sensitive (PTX-sensitive)¹ G proteins (G_i and G_o) to mediate a variety of intracellular changes including inhibition of cAMP accumulation, activation of potassium channels, and inactivation of calcium channels (2, 3). However, in hippocampal membrane preparations, 5-HT1A receptor activation stimulates adenylyl cyclase activity (11-13), an action that can be blocked by the specific antagonist WAY100,135 (11). Activation of the G_i-coupled 5-HT1A or GABA-B receptors potentiated the electrophysiological actions of the G_s-coupled -adrenergic receptor in hippocampal CA1 neurons (14). The ACII protein has been localized to the dendrites and cell bodies of neurons of the hippocampus (15). This subtype of adenylyl cyclase is known to be conditionally stimulated by G subunits derived from activation of G_i/G_o proteins (16, 17). These data suggest that the 5-HT1A receptor may couple to, or potentiate G_s-mediated actions in the central nervous system by activation of G-regulated adenylyl cyclases like ACII.

Recently, we have shown that modulation of G protein subunits by expression of antisense G_i1 subunit cDNA can switch inhibitory coupling of the 5-HT1A receptor to mediate a PTX-sensitive stimulation of cAMP in GH4 pituitary cells, which express ACII endogenously (18). We hypothesized that in G_i1-depleted GH4 cells, the 5-HT1A receptor may activate ACII via release of G subunits derived from other G_i/G_o proteins. Using a cotransfection approach, we have identified a specific requirement for ACII in coupling of the 5-HT1A receptor to enhance AC activity. Furthermore, cotransfection of 5-HT1A receptor, ACII, and G_i2 specifically, resulted in an agonist-independent increase in cAMP that was not inhibited by receptor antagonists. Interestingly, 5-HT1A partial agonists but not full agonists or antagonists inhibited spontaneous coupling of the receptor to stimulate cAMP accumulation. Thus, part of the anti-anxiety activity of 5-HT1A ligands may be to silence the spontaneous activity of the 5-HT1A receptor.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Materials-- All chemicals were reagent grade. PTX was from Sigma, St. Louis. HEK-293 cells were obtained from Dr. R. Dunn, Montreal. Rat G_i1, G_i2, G_i3, G_oA, and ACII cDNAs were generously provided by Dr. Randall Reed, Johns Hopkins University, Baltimore. Human 5-HT1B receptor cDNA (19) was kindly provided by Dr. Brian O'Dowd, University of Toronto and the human dopamine-D1 cDNA (20) was from Dr. Qun-Yong Zhou, University of California, Davis. The pCMV-GAL plasmid was obtained from ATCC, Bethesda, MD. [³H]DPAT (228 Ci/mmol) was obtained from Amersham Pharmacia Biotech. Flesinoxan was a gift of Dr. Pierre Blier, McGill University, Montreal, Canada; (+)WAY100,135 (the active stereoisomer) was a generous gift from A. Fletcher, Wyeth-Ayerst; BMY-7378 was from Bristol-Myers Squibb; other 5-HT1A ligands were from Research Biochemicals Inc., Natick, MA. Rabbit anti-rat G_o and anti-rat G_icommon polyclonal antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA); anti-G_i2 polyclonal antibody was from Upstate Biotechnology.

Cell Culture and Transfection-- HEK-293 cells were maintained in DMEM medium (4.5 mg/l d-glucose) + 8% fetal calf serum at 37 °C, 5% CO₂. The 1.9-kilobase BamHI/XbaI fragment of the rat 5-HT1A receptor or receptor mutants was subcloned into BamHI/XbaI-cut pcDNA3 (Invitrogen) to generate p3-D_BX or mutants and the rat G_i1, G_i2, G_i3, G_oA, and ACII cDNAs were ligated into the EcoRI site of pcDNA3. The opossum CT construct was cloned as described previously (21, 22). For transfection, HEK-293 cells were plated at 5 × 10⁶ cells/10-cm dish and incubated overnight. Calcium phosphate precipitate (2 ml/dish) containing p3-ACII, 12 µg; p3-D_BX, 12 µg; pCMV-GAL, 6 µg; or p3-G_i/G_o, 15 µg or as indicated, was overlaid with 8 ml/dish DMEM + 8% fetal calf serum, 20 mM Hepes (pH 7.0), and incubated at 37 °C for 4-6 h in 5% CO₂. The calcium phosphate was aspirated and replaced with 8 ml/dish DMEM + 8% fetal calf serum, 20 mM Hepes (pH 7.0) for incubation overnight at 37 °C and 5% CO₂. The cells were then plated onto 6-well plates for cAMP and -galactosidase assays.

cAMP Assay-- 24 h after plating cells in 6-well dishes, the cells were rinsed with DMEM and incubated at 37 °C in 1 ml/well DMEM, 0.01% bovine serum albumin, 20 mM Hepes (pH 7.0), 100 µM isobutylmethylxanthine for 15-20 min. Experimental compounds were added to triplicate wells as indicated. Media were recovered and centrifuged at 13,000 × g (30 s) to remove floating cells, and the supernatant was recovered and stored at 20 °C. Attached cells were extracted using reporter lysis buffer (Promega), centrifuged for 10 min at 4 °C, and stored at 20 °C. Media were assayed for cAMP by specific RIA, as described previously. Cell extracts were assayed for -galactosidase activity to monitor transfection efficiency. Statistical analyses of the data were done using the GraphPad Prism software, and groups were compared using unpaired t test with Welsh's correction to determine significance (p < 0.05).

-Galactosidase Assay-- The transfected cells were rinsed with PBS, resuspended in 200 µl of reporter lysis buffer, incubated for 15 min at room temperature, scraped, frozen, and thawed to complete cell lysis. The lysates were centrifuged (14,000 rpm, 20 s), and the supernatant was recovered for measurement of -galactosidase activity. Equal volumes (30 µl each) of cell extract and 0.3 mM 4-methylumbelliferyl--D-galactoside substrate in 15 mM Tris (pH 8.8) were mixed gently, incubated in the dark at 37 °C for 30 min, and the reaction was terminated upon addition of 50 µl of Stop solution (300 mM glycine, 15 mM EDTA (pH 11.2)). The sample was transferred to 2 ml of Z buffer (60 mM Na₂HPO₄, 40 mM NaH₂PO₄, 10 mM KCl, 1 mM MgSO₄), and fluorescence was measured at _EX = 350 nm, _EM = 450 nm on a Perkin-Elmer LS-50 spectrofluorometer (Buckinghamshire, United Kingdom). The variation of -galactosidase activity between wells observed within a transfection was <6%, and between transfections was <7%.

X-Galactosidase Staining-- Cells were fixed in 2% formaldehyde, 0.2% glutaraldehyde in PBS for 10 min and rinsed 2-3 times with PBS and stained overnight using the X-galactosidase solution (5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 2 mM MgCl₂, 1 mg/ml Xgal in PBS) in a humidified container at 37 °C. The proportion of stained cells was quantitated from representative photographic frames (2 photos/well). The transfection efficiency attained was 36.5 ± 5.4% (mean ± S.E.), and the variation in transfection efficiency between different transfections was 7.5% by this method.

Western Blot Analysis-- Cells (10⁷/10-cm plate) were harvested and resuspended in 200 µl of RIPA-L buffer (10 mM Tris (pH 8), 1.5 mM MgCl₂, 5 mM KCl, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 1% Nonidet P-40, 0.1% sodium lauryl sulfate, 0.5% sodium deoxycholate, 5 µg/ml leupeptin) on ice. The cell lysate was sheared through a 25-gauge needle, incubated on ice for 30 min, and centrifuged (10,000 × g, 10 min, 4 °C). The supernatant was recovered, stored at 80 °C, and assayed for protein content by the bicinchoninic acid protein assay kit (Pierce). Lysates (20-50 µg/lane) were electrophoresed on sodium lauryl sulfate-containing polyacrylamide gels at 100 V, 40 mA for 1 h, blotted on polyvinylidene difluoride membranes for 1 h at 250 mA. Blots were incubated overnight in 5% casein in TBS-T (10 mM Tris, 150 mM NaCl (pH 8.0), 0.1% Tween 20) at 4 °C. The blots were then incubated overnight with primary antibody, followed by 30-min incubation with horseradish peroxidase-conjugated secondary antibody at room temperature in Tris-buffered saline-Tween, and the peroxidase product was developed using the enhanced chemiluminescence for Western blot protocol.

Ligand Binding Assay-- Membranes were prepared from cotransfected HEK-293 cells as described (23) and stored as pellets at 80 °C. Membrane pellets were resuspended in TME (75 mM Tris, pH 7.4, 12.5 mM MgCl₂, 1 mM EDTA), aliquotted (100 µg in 50 µl) in tubes containing 200 µl of TME and [³H]DPAT (20 nM) or [³H]5-HT (50 nM) and incubated at room temperature for 40 min. The binding reaction was applied to Whatman GF/C filters under vacuum and rinsed three times with 3 ml of ice-cold 50 mM Tris (pH 7.4). The filters were placed in 3 ml of scintillation fluid (Ultima-Gold, Packard) and counted for 2 min using the direct-disintegrations/min program of the Tricarb TR2100 counter (Packard). 5-HT (10 µM) was used to define nonspecific binding.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Inhibitory Coupling of the 5-HT1A Receptor-- The 5-HT1A receptor couples to inhibition of cAMP accumulation in most cell types. Inhibition of cAMP formation by 5-HT1A receptor activation was examined in HEK-293 cells transiently cotransfected with the G_s-coupled dopamine-D1 receptor (20) and the 5-HT1A receptor (Fig. 1). In three experiments, dopamine induced a 7.6 ± 0.9-fold increase in cAMP level that was inhibited by 49 ± 3% upon activation of 5-HT1A receptors with DPAT. DPAT-mediated inhibition of dopamine-induced cAMP accumulation was reversed by the 5-HT1A receptor antagonist spiperone. Thus, the 5-HT1A receptor is coupled to inhibition of G_s-mediated cAMP accumulation in HEK-293 cells.

View larger version (49K): [in this window] [in a new window]   Fig. 1.   Inhibition of dopamine-stimulated cAMP formation by 5-HT1A receptor activation in HEK-293 cells. HEK-293 cells were transfected transiently by calcium phosphate coprecipitation with 12 µg/transfection each of the human dopamine-D1 and rat 5-HT1A receptor expression plasmids and cAMP production was measured in triplicate dishes (see "Experimental Procedures"). Data are presented as mean ± S.E. The dishes were treated with DPAT (2 µM), dopamine (DA, 2 µM) or both (DA/DPAT) in the absence or presence of the indicated concentration of spiperone, and similar results were obtained in three independent experiments. The 5-HT1A receptor antagonist spiperone blocked DPAT-induced inhibition of dopamine-D1 induced cAMP formation.

5-HT1A Receptor Coupling to ACII-- We addressed whether the 5-HT1A receptor was coupled to enhancement of cAMP levels upon cotransfection with an expression plasmid encoding ACII (Fig. 2). In HEK-293 cells transfected transiently with the rat 5-HT1A receptor cDNA alone (Fig. 2A, first lane), addition of the selective 5-HT1A agonist DPAT (1 µM) inhibited basal cAMP accumulation by 40%, as observed in other cell lines (24). By contrast, in cells cotransfected with 5-HT1A receptor and ACII cDNAs, DPAT induced a 1.5-fold increase in cAMP accumulation (Fig. 2A, second lane; Table I), which was blocked by pretreatment with 50 ng/ml PTX (data not shown). DPAT had no effect in non-transfected cells or cells transfected with ACII alone. Thus, the 5-HT1A receptor couples to endogenous G proteins to stimulate adenylyl cyclase activity upon cotransfection with ACII, as observed for other G_i-coupled receptor subtypes (16, 25-27). The 5-HT1A receptor may also weakly activate G_s, because activation of ACII requires both G release from G_i and minimal G_s activity (17). However in the absence of transfected ACII, DPAT-induced inhibition of basal cAMP is observed (Table I) indicating that the 5-HT1A receptor couples via G_i to inhibit the activity of endogenous AC subtypes in HEK-293 cells.

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Constitutive 5-HT1A receptor activity in the presence of Gi2 and ACII. HEK-293 cells were transfected transiently by calcium phosphate coprecipitation with 12 µg each of the indicated plasmids, and cAMP production was measured. Transfection efficiency was monitored by cotransfection of -galactosidase plasmid and quantitated as described (see "Experimental Procedures"). DPAT (1 µM) was added to 5-HT1A-transfected cells acutely during the assay. Data are presented as mean ± S.E. of triplicate samples for experiments that were repeated at least three times (see Table I). A, 5-HT1A receptor-mediated stimulation of ACII. Cotransfection of plasmids encoding the 5-HT1A receptor (5-HT1A), Gi2 (i2), or ACII led to enhancement of cAMP levels. B, PTX induced uncoupling of the 5-HT1A receptor from Gi2/ACII. Requirement for 5-HT1A, Gi2, and ACII. Various plasmids, including Gi2 (i2) and GoA (o) were transfected in equal amounts (12 µg each). PTX (50 ng/ml) was present overnight in the indicated samples.

View this table: [in this window] [in a new window]   Table I G protein selectivity for 5-HT1A receptor constitutive activity The table presents a summary of data from multiple experiments as in Fig. 2 in which HEK-293 cells were cotransfected with expression plasmids for 5-HT1A receptor, ACII, -galactosidase, and either no G protein (Control), or Go, Gi1, Gi2, or Gi3 as indicated. The level of basal cAMP (normalized to the level of cAMP in Gi2-transfected cells) or DPAT (1 µM)-induced change in cAMP (expressed as percent basal) is presented as the mean ± S.E. of the number of independent experiments indicated in parentheses. Data were compared by unpaired t-test with ***p < 0.001 compared to control for basal cAMP; and *p < 0.05; **p < 0.01 compared to untreated for DPAT-induced cAMP.

G_i2-dependent Constitutive 5-HT1A Receptor Activation-- Upon cotransfection of 5-HT1A receptor, G_i2, and ACII cDNAs, a pronounced agonist-independent increase in cAMP level was observed that was not altered by addition of the agonist, DPAT (Fig. 2A, Table I). Although constitutive activity of the 5-HT1A receptor/G protein complex has been indirectly suggested by the spontaneous binding of GTPS in membranes from cells transfected with 5-HT1A receptors (28-30), the present results provide the first direct evidence of agonist-independent coupling of the 5-HT1A receptor in the absence of GTPS. Constitutive enhancement of basal cAMP levels required cotransfection of 5-HT1A receptor, G_i2, as well as of ACII cDNA (Fig. 2) and was blocked by >80% following pretreatment with 50 ng/ml PTX (Fig. 2B), further indicating the participation of G_i/G_o proteins. By contrast, little increase in cAMP was observed upon cotransfection with G_o.

The G protein specificity of agonist-independent stimulation of cAMP levels by the 5-HT1A receptor was examined further by co-transfecting equal amounts of 5-HT1A receptor, ACII, and either G_i1, G_i2, G_i3, or G_o cDNAs in HEK-293 cells (Table I). Cotransfection of receptor and ACII with G_i2, but not G_i3, G_i1, or G_o, resulted in a 6-fold increase in basal cAMP levels compared with cells transfected with receptor and ACII (16% versus 100%). In multiple experiments, the rank order of G protein preference (G_i2 > G_i3, G_o, G_i1) is similar to that identified for G protein interaction with the bacterially expressed 5-HT1A receptor protein (31). Thus, the extent of constitutive activation of the 5-HT1A receptor appears to reflect the extent of coupling between receptor and G protein and is most dependent on the G protein subtype G_i2.

Addition of the full agonist DPAT resulted in a small increase in cAMP level in cells not transfected with exogenous G proteins (Control, Table I), as observed above. DPAT induced no change or a small decrease in cAMP levels in cells cotransfected with G_i1 or G_i2. In contrast, DPAT inhibited cAMP levels by 30% in cells transfected with G_o (Table I), suggesting that this G protein mediates inhibitory receptor coupling in cells transfected with ACII. A slight DPAT-induced increase in cAMP level was observed with G_i3 but did not achieve significance. Thus, the spontaneous activity of the 5-HT1A receptor was specific for the presence of G_i2 and was insensitive to the full agonist DPAT.

Expression of 5-HT1A Receptor, ACII, and G Proteins-- The relative inactivity of G_o (or G_i1, G_i3) to mediate constitutive coupling of the 5-HT1A receptor could be because of inefficient expression of the receptor, ACII, or the G protein upon cotransfection in HEK-293 cells. The level of 5-HT1A receptors expressed in G_o- and G_i2-transfected cells was not significantly different as detected by specific binding of [³H]DPAT (Table II). Because the ACII subtype is regulated by activation of PKC, functional expression of ACII was detected indirectly by measuring the change in cAMP level induced by the PKC activator, TPA. In non-transfected HEK-293 cells, no response to TPA was observed, consistent with a lack of ACII immunoreactivity observed in these cells (15). In cells cotransfected with plasmids for 5-HT1A receptor, ACII and either G_o or G_i2, a strong response to TPA (5- to 6-fold) was detected that did not differ between G_o or G_i2 (Table II). This result suggests that robust and equivalent expression of ACII was obtained in both transfections.

View this table: [in this window] [in a new window]   Table II Content of 5-HT1A receptor and ACII in cotransfected HEK-293 cells Cells were cotransfected with 5-HT1A receptor, ACII, -galactosidase and either Go or Gi2 expression plasmids as indicated. -Galactosidase activity was measured as an index of overall transfection efficiency and was equivalent between the transfections. For measurement of 5-HT1A receptor levels, membranes were prepared and subjected to ligand binding with [3H]DPAT (20 nM), with 10 µM 5-HT added to determine non-specific binding. Data are presented as mean ± S.E. of three independent experiments. In separate transfections, the -fold induction over basal cAMP following addition of TPA (100 nM) was measured as an index of ACII content. In non-transfected cells, TPA did not alter significantly the basal cAMP level (0.82 ± 0.20 -fold basal). Data represent the mean ± S.E. of three independent experiments.

Although transfection efficiencies were equivalent for each G protein tested (35%, see "Experimental Procedures"), it remained possible that the inactivity of G_o to mediate constitutive activation of the 5-HT1A receptor was because of a lack of expression of G_o protein. The level of G protein expression was assessed directly by Western blot analysis of membranes from G_o- or G_i2-transfected cells (Fig. 3). In two independent transfections, protein extracts from non-transfected HEK-293 cells displayed an undetectable level of G_o immunostaining, whereas samples from cells transfected with G_o cDNA expression plasmid displayed robust expression of G_o that was proportional to the amount of protein/well (Fig. 3). By contrast, endogenous G_i proteins were detected in both non-transfected and transfected cells. In cells transfected with p3-G_i2, a 4.8 ± 0.75-fold (n = 4) increase in G_i2 protein level relative to control was observed. A smaller 2.0 ± 0.2-fold control increase was detected in G_i2-transfected cells using the less selective anti-G_i-common antibody which also detected endogenous G_i proteins in non-transfected cells. In summary, there was an equivalent induction of G_o and G_i2 proteins upon separate cotransfection with 5-HT1A receptor and ACII plasmids. Thus, the inactivity of G_o to mediate constitutive coupling of the 5-HT1A receptor to ACII was not because of inefficient expression of the G_o protein, 5-HT1A receptor, or ACII.

View larger version (11K): [in this window] [in a new window]   Fig. 3.   G protein expression in Go- and Gi2-transfected HEK-293 cells. Independent transient transfections with Go or Gi2 (as indicated above), 5-HT1A receptor, ACII, and -galactosidase expression plasmids were performed in HEK-293 cells as in Fig. 2. Controls represent samples from non-transfected cells. Membranes were prepared and processed for Western blot analysis (see "Experimental Procedures"). Either 20, 25, or 50 µg of protein from nontransfected or G-transfected cells from independent transfections were examined. The Go blot was probed using anti-Go antibody (upper) or stripped and reprobed with anti-Gi-common antibody (lower); the Gi2 blot was probed using anti-Gi2 (upper) or anti-Gi-common antibody (lower).

Concentration-dependence for G_i2 Plasmid-- The concentration dependence of p3-G_i2 cDNA for 5-HT1A receptor-mediated activation of ACII and inhibition of PGE1-stimulated cAMP levels was examined in parallel (Fig. 4). Upon cotransfection with 5-HT1A receptor and ACII plasmids, the p3-G_i2 plasmid induced a concentration-dependent enhancement of cAMP levels, with an EC₅₀ value of 1.7 µg of G_i2 cDNA (Fig. 4A). The response appeared to saturate at a DNA concentration that was approximately equimolar for 5-HT1A receptor, G protein, and ACII plasmid (i.e. approximately 15 µg). For comparison, the concentration dependence of G_i2 cDNA for 5-HT1A receptor-mediated inhibition cAMP levels was examined. PGE1 induced a 3-fold increase in cAMP above the constitutive level of cAMP in HEK-293 cells transfected with 5-HT1A, ACII, and G_i2 (Fig. 4B). Increasing amounts of G_i2 inhibited PGE1-mediated enhancement of cAMP levels only at the highest plasmid concentration (50 µg). Thus, the requirement for G_i2 cDNA was ten-fold greater for agonist-independent inhibition of PGE1-stimulated cAMP than for activation of ACII by the 5-HT1A receptor. The concentration dependence on G_i2 cDNA suggests that the extent of constitutive activation of the 5-HT1A receptor depends on the content of G_i2 and on the signaling pathway (i.e., stimulation versus inhibition of adenylyl cyclase) examined.

View larger version (12K): [in this window] [in a new window]   Fig. 4.   Plasmid concentration-dependence of Gi2 for 5-HT1A receptor-mediated stimulatory and inhibitory actions. All transfections were in HEK-293 cells and contained equal amounts of 5-HT1A receptor and ACII plasmids and increasing amounts of Gi2 plasmid as indicated (p3-Gi2). The level of cAMP was assayed in the absence (A) and presence (B) of PGE1 (1 µM). Results obtained in the presence of PGE1 (B) represent the PGE1-induced increase in cAMP normalized to the basal cAMP level at each concentration of p3-Gi2, i.e. -fold basal cAMP. Data are presented as mean ± S.E. of triplicate samples.

Role of the i2 Domain in 5-HT1A Receptor Coupling to ACII-- To identify the receptor domain implicated in constitutive activation of ACII by the 5-HT1A receptor, we utilized a point mutant (5-HT1A-i2, Fig. 5) in the second intracellular loop (T149A) of the 5-HT1A receptor that couples to G_i-mediated inhibition of cAMP, but not to various G-mediated pathways such as activation of PLC2 or inactivation of calcium channels (23). The 5-HT1A-i3 is a receptor mutant with three point mutations that retains coupling to PLC-mediated calcium mobilization (32). When cotransfected with G_i2 and ACII, the 5-HT1A-i2 mutant failed to increase cAMP over the level obtained in the presence of PTX. By contrast, the 5-HT1A-i3 mutant displayed agonist-independent enhancement of cAMP levels similar to the wild-type 5-HT1A receptor (Fig. 5). The levels of receptor expressed were similar for each clone although the value for the 5-HT1A-i2 clone was slightly lower; this may reflect the lower affinity of this mutant for [³H]DPAT (23). Thus, the second intracellular domain of the 5-HT1A receptor appears to dictate coupling to G subunits resulting in activation of ACII, as well as other G-regulated effectors (33).

View larger version (34K): [in this window] [in a new window]   Fig. 5.   Receptor and domain specificity of 5-HT1A constitutive activity. Equal amounts of the indicated receptor or 5-HT1A receptor mutant were cotransfected with Gi2 and ACII in HEK-293 cells. The 5-HT1A mutants were in the second intracellular loop T149A (5-HT1A-i2 (23)) or in three putative PKC phosphorylation sites in the third intracellular loop (5-HT1A-i3 (32)). In parallel experiments (n = 3-5), the specific binding for each receptor was (pmol/transfection): 5-HT1A, 2.07 ± 0.23; 5-HT1A-i2, 1.27 ± 0.06; 5-HT1A-i3, 1.72 ± 0.29; 5-HT1B, 2.2 ± 0.3.

Because the 5-HT1B receptor is structurally homologous to the 5-HT1A receptors, we tested whether this receptor subtype mediated agonist-independent activity (Fig. 5). Upon cotransfection with ACII and G_i2, the 5-HT1B receptor mediated a 5-fold increase in cAMP levels that was inhibited by PTX. The smaller effect of the 5-HT1B receptor may reflect a lower constitutive activity compared with the 5-HT1A receptor because the levels of receptor expressed were similar. Other receptor subtypes like the dopamine-D1 (G_s coupled) or the dopamine-D2S (G_i/G_o-coupled) did not increase cAMP in the absence of agonist (data not shown). Previous studies have indicated that the dopamine-D2S receptor does not couple to G_s in the presence of ACII (16), suggesting that the activation of ACII by 5-HT1A receptors does not result from basal activation of G_s by other receptors endogenous to HEK-293 cells. This supports the hypothesis that the 5-HT1A receptor does couple to Gs (weakly) in the HEK-293 cells, despite the inability to detect biochemically an interaction between the receptor and G_s (28, 31, 34-36). Although 5-HT1A and 5-HT1B receptors inhibit cAMP synthesis in most tissues and cell lines, these results indicate that, in cells where ACII is predominant, these receptors spontaneously couple to stimulate cAMP accumulation.

Inhibition of Constitutive Activity of the 5-HT1A Receptor-- The activity of a variety of ligands and protein inhibitors to modulate 5-HT1A receptor coupling to G_i2 and ACII was examined and compared with the action of PTX, which entirely uncouples the receptor (Fig. 6). Because ACII is conditionally activated by G subunits from G_i/G_o proteins (17), the importance of G subunits was examined by cotransfection of an equal amount of expression plasmid containing the carboxyl-terminal domain of GRK2 (CT), a G antagonist (37, 38). CT inhibited spontaneous coupling of the 5-HT1A receptor with maximal inhibition equivalent to that achieved with PTX. This suggests that the 5-HT1A receptor couples spontaneously to the G_i2 heterotrimer to release G subunits, resulting in a CT-sensitive activation of ACII. Transfection of G_i2 apparently augments the activity of endogenous G subunits to couple to ACII in the presence of the 5-HT1A receptor, presumably by increasing the level of G_i2 heterotrimer. Co-regulation of G and G subunit protein levels has been observed in G_i2 nullizygous mice that are depleted of G_i2 and specific G subunits (39).

View larger version (71K): [in this window] [in a new window]   Fig. 6.   Inhibition of 5-HT1A constitutive activity by uncoupling agents and partial agonists. All cells were cotransfected with equal amounts of 5-HT1A receptor, Gi2, and ACII plasmids and subjected to cAMP assay in the presence of the indicated compounds except for control (no additions), +PTX (overnight pretreatment with 50 ng/ml PTX), and CT (cotransfected with addition of an equal amount of CT plasmid (21)). The following compounds were used: (+)WAY100,135 (WAY); Flesinoxan (Fles.); BMY-7378 (BMY); buspirone (Busp.); spiperone (Spip.); NAN-190 (NAN); pindolol, (Pind.). The data are expressed as percent control, with addition of 0.1% Me2SO as the vehicle control for spiperone, NAN-190, and pindolol. The data represent mean ± S.E. of 3-5 independent assays with statistical significance compared with control as indicated: *p < 0.05; **p < 0.01.

The activity of 5-HT1A agonists and antagonists was tested in cells transfected with 5-HT1A receptor, G_i2, and ACII (Fig. 6). Ligands for the 5-HT1A receptor have been previously characterized as full or partial agonists or neutral antagonists (40-43). Several partial agonists (e.g. buspirone, BMY-7378) of the 5-HT1A receptor possess anti-anxiety activity (5, 44-46), and 5-HT1A agonists and antagonists potentiate the anti-depressant response to serotonin reuptake blockers (1, 4, 6, 7). Full agonists (e.g., DPAT, 5-HT) and antagonists (spiperone, (+)WAY100,135, NAN-190, pindolol) lacked or had minimal inhibitory activity compared with control. Lack of inhibition by antagonists suggests that G_i2-dependent constitutive activation of 5-HT1A receptor is indeed agonist-independent and not mediated by endogenous 5-HT. Spiperone has been characterized as an inverse agonist that induces a small (30%) decrease in 5-HT1A receptor-induced GTPS binding to Chinese hamster ovary cell membranes (29). The lack of effect of spiperone or (+)WAY100,135 to inhibit spontaneous coupling of the 5-HT1A receptor was not due to their inactivity in the HEK cells because both spiperone (Fig. 1) and (+)WAY100,135 (data not shown) blocked 5-HT1A-mediated inhibition of cAMP formation. Determination of inverse agonism in membrane preparations in the presence of the high affinity G protein ligand GTPS may not reflect modulation of receptor-G protein coupling to specific effectors in intact cells which contain GTP/GDP. The lack of inverse agonist activity of spiperone in coupling to G_i2/ACII suggests that as observed for agonists, inverse agonists may show greater efficacy for some effectors but not others (47-49). Depending on the effector studied, agonists of the 5-HT1A receptor can have different efficacies of coupling. A "tight" coupling of the 5-HT1A receptor to inhibition of cAMP is observed, and all agonists mediate some response; however, for calcium mobilization or pH change, a "loose" coupling is apparent in which some agonists are ineffective (40, 50). Based on our results, spiperone induced a conformation of the 5-HT1A receptor that permits coupling to G_i2/ACII but inhibits G_i-mediated inhibition of G_s-stimulated adenylyl cyclase activity.

The partial agonists buspirone, BMY-7378, and especially flesinoxan, all demonstrated inhibitory activity, which was maximal at 1 or 10 µM. By comparison, pretreatment with PTX or cotransfection with CT induced a greater reduction in cAMP levels, reflecting a more complete uncoupling of the receptor by these agents. The partial agonism of these ligands to incompletely stimulate the inactive 5-HT1A receptor, and to inhibit partially the constitutively active receptor, suggests that the partial agonists promote an incompletely active conformation of the 5-HT1A receptor upon binding to the receptor. Furthermore, there appears to be a correlation between the clinical effectiveness of these compounds as anti-anxiety drugs and their ability to reduce coupling of the 5-HT1A receptor to ACII.

It has been proposed that anxiety results in part from a hyperactivity of the serotonin system and that anxiolytic 5-HT1A partial agonists inhibit serotonergic neurotransmission by activating the presynaptic autoreceptor (51); hence the knockout mice that lack 5-HT1A receptors display both serotonergic hyperactivity and anxiety-related behavior (8-10). Our results suggest that in addition to partial activation of the presynaptic 5-HT1A autoreceptor to inhibit raphe firing, anti-anxiety agents may also inhibit constitutive, as well as 5-HT-induced activation of postsynaptic 5-HT1A receptors. It is tempting to speculate that 5-HT1A receptor ligands that inhibit constitutive receptor activity may have potentially greater therapeutic efficacy as anti-anxiety or antidepressant agents than ligands that lack this activity. The cotransfection paradigm presented here for detecting inhibitory activity at the 5-HT1A receptor may provide an effective screening method for identifying anti-anxiety agents.

In summary, we have identified stimulatory coupling of the 5-HT1A receptor to adenylyl cyclase in the presence of ACII that becomes agonist-independent upon co-transfection with G_i2. Coupling to ACII required mobilization of G subunits and was mediated by a putative G coupling domain in the i2 loop of the 5-HT1A receptor. Partial 5-HT1A receptor agonists that have anti-anxiety properties inhibited spontaneous receptor coupling to ACII, whereas full agonists or antagonists lacked this activity; thus, inhibition of constitutive 5-HT1A receptor activity may contribute to the therapeutic actions of these compounds.

	ACKNOWLEDGEMENTS

We thank Christine Forget for excellent technical assistance. We also thank Drs. Randall Reed, Brian K. O'Dowd, and Qun-Yong Zhou for providing plasmid constructs.

	FOOTNOTES

^* This work was supported by grants from the Medical Research Council, Canada and the National Cancer Institute, Canada.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Novartis/MRC Michael Smith Chair in Neurosciences. To whom correspondence should be addressed: Neuroscience Research Institute, University of Ottawa, 451 Smyth Rd., Ottawa, Canada K1H 8M5. Tel.: 613-562-5800, ext. 8307; Fax: 613-562-5403; E-mail: palbert@ uottawa.ca.

^§ MRC Scholar.

Supported by the Ministry of Iran and a Schizophrenia Society of Canada scholarship.

	ABBREVIATIONS

The abbreviations used are: PTX, pertussis toxin; AC, adenylyl cyclase; G, G protein alpha subunit; CT, carboxyl-terminal fragment of -adrenergic receptor kinase; DPAT, 8-hydroxy-(2-(N,N-di-propylamino)-1,2,3,4-tetrahydronaphthalene; HEK-293, human embryonic kidney cells; PGE1, prostaglandin E1; PKC, protein kinase C; 5-HT, serotonin; TPA, 12-O-tetradecanoylphorbol-13-acetate; i2, i3, second (third) intracellular; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; GTPS, guanosine 5'-3-O-(thio) triphosphate.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES