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Activation of Adenylyl Cyclase by Endogenous G_s-Coupled Receptors in Human Embryonic Kidney 293 Cells Is Attenuated by 5-HT₇ Receptor Expression

Department of Pharmacology, University of Oslo, Oslo, Norway

Received for publication May 31, 2005.

Accepted for publication September 27, 2005.

	Abstract

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Human 5-hydroxytryptamine₇ (5-HT₇) receptors display characteristics shared with receptors believed to form a tight physical coupling with G protein in the absence of ligand. Some receptors apparently preassociated with G_i/o and G_q/11 are reported to inhibit the signaling of other similarly coupled G protein-coupled receptors by limiting their access to activate a common G protein pool. Therefore, we determined whether 5-HT₇ receptor expression was sufficient to limit signaling of endogenously expressed G_s-coupled receptors in human embryonic kidney (HEK) 293 cells. Using the ecdysone-inducible expression system, which allows for the titration of increasing receptor density in the same clonal cell line, we compared the effects of 5-HT_4(b) and 5-HT_7(a,b,d) receptor expression on adenylyl cyclase (AC) stimulation by the endogenous G_s-coupled -adrenergic (AR) and prostanoid EP (EPR) receptors. AR- and EPR-stimulated AC activity was attenuated by 5-HT₇ receptor expression in both membrane preparations and intact HEK293 cells. AR- and EPR-stimulated AC activity was unaffected by expression of the G_s-coupled 5-HT₄ receptor. The mechanism of this heterologous desensitization seems independent of protein kinase A activation, nor does it occur at the level of G protein activation because 1) AR- and EPR-stimulated AC activity was not restored to control values when G_s was overexpressed; and 2) ₁AR and ₂AR activation of G_s was unaffected by the expression of 5-HT₇ receptors. In addition, overexpression of AC isoforms was unable to rescue AR- and EPR-stimulated AC activity. Therefore, 5-HT₇ receptors probably limit access and/or impede activation of AC by AR and EP receptors. Although the 5-HT₇ receptor may preassociate with G protein and/or AC, the mechanism of this heterologous desensitization remains elusive.

The cubic ternary complex model incorporates the existence of such an inactive receptor coupled to a G protein (Weiss et al., 1996). Experimental support for the existence of receptors tightly associated (preassociated) to their respective G protein, in the absence of ligand, has been reported for the CB₁-cannabinoid receptor (Vasquez and Lewis, 1999; Mukhopadhyay et al., 2000), the Mel_1a melatonin receptor (Roka et al., 1999) and the vasoactive intestinal peptide VPAC₁ receptor (Shreeve, 2002). Although the CB₁ receptor-G_i/o association is sensitive to the destabilizing effect of guanine nucleotides (Mukhopadhyay et al., 2000), high-affinity agonist binding at the Mel_1a receptor is resistant to both the destabilizing effect of guanine nucleotides and pertussis toxin (Roka et al., 1999). At the 5-HT₇ receptor, two groups have reported that a high proportion of recombinant human 5-HT_7(a) receptors exist in the high-affinity (presumably G protein-coupled) state (Adham et al., 1998; Alberts et al., 2001). The insensitivity of the high-affinity agonist binding of the human 5-HT₇ receptor to the destabilizing effect of guanine nucleotides (Alberts et al., 2001; Krobert et al., 2001) is another indication the 5-HT₇ receptor and G_s protein form a tight complex.

Expression of the G_i/o-coupled CB₁ receptor in superior cervical ganglia attenuated the ability of ₂AR and somatostatin receptors to activate G_i/o, and it was proposed that the CB₁ receptor, because of its preassociation with G_i/o, sequesters a proportion of the available G protein pool (Vasquez and Lewis, 1999). As a result, the available G protein pool is reduced, limiting activation by other G_i/o-coupled receptors and subsequently their respective signaling ability.

The cubic ternary complex model also proposes the existence of a ligand-occupied inactive receptor coupled to G protein. Stabilization of an inactive CB₁ receptor-G_i/o complex by the inverse agonist SR141716A inhibited insulin- and insulin-like growth factor 1-mediated activation of mitogen-activated protein kinase through G_i/o (Bouaboula et al., 1997). Likewise, purinergic receptor-stimulated G_q/11 activation and subsequent Ca²⁺ mobilization is attenuated in the presence of the guinea pig histamine H₁ receptor inverse agonist mepyramine, presumably by stabilization of an H₁ receptor-G_q/11 complex (Fitzsimons et al., 2004). Taken together, these findings provide experimental evidence for a ligand-occupied inactive G protein-coupled state of the receptor. Furthermore, they indicate that an inactive receptor-G protein preassociation can limit the access of other G protein-coupled receptors to activate a common G protein pool.

The primary objective of the current study was to determine whether 5-HT₇ receptors represented an example of a preassociated G_s-coupled receptor. Therefore, we tested whether 5-HT₇ receptor expression alone was sufficient to limit the signaling of endogenously expressed G_s-coupled receptors in HEK293 cells. To test this hypothesis, we used the ecdysone-inducible expression system, which permitted reproducible expression of increasing receptor density in the same clonal cell line. Using this expression system, we compared the effects of 5-HT_4(b) and 5-HT_{7(a, b, and d)} receptor expression on AC stimulation by the endogenous AR and prostanoid EP receptors.

	Materials and Methods

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Materials. Serotonin, (-)isoproterenol, timolol, alprenolol, GDP, guanosine 5'-[-thio]triphosphate (GTPS), and H89 were from Sigma-Aldrich (St. Louis, MO). Methiothepin (metitepine, 1-[10,11-dihydro-8-(methylthio)dibenzo[b,f]thiepin-10-yl]-4-methylpiperazine) maleate and 8-hydroxy-2-dipropylaminotetralin (8-OH-DPAT) hydrobromide were from Tocris Cookson Inc. (Bristol, UK). Prostaglandin E₁ was from Cayman Chemical (Ann Arbor, MI). Renzapride (BRL24924) hydrochloride, Zeocin, penicillin-streptomycin, G-418, ponasterone A, LipofectAMINE, and LipofectAMINE 2000 were from Invitrogen (Carlsbad, CA). Forskolin was from Calbiochem (San Diego, CA). Supersignal Dura West was from Pierce Chemical (Rockford, IL). Anti-pRas-GRF1 was from Cell Signaling Technology (Beverly, MA). Anti-HA-probe and Anti-G_s/olf were from Santa Cruz Biotechnology (Santa Cruz, CA). Sheep anti-rabbit IgG-HRP was from GE Healthcare (Little Chalfont, Buckinghamshire, UK).

Radiochemicals. [³H]5-Carboxamidotryptamine (5-CT; 60-102 Ci/mmol), [³H]GR113808 (84 Ci/mmol), (-)-3-[¹²⁵I]iodocyanopindolol (ICYP) (2000 Ci/mmol), [-³²P]ATP (400 Ci/mmol), [2,8-³H]cAMP (30-42 Ci/mmol), [³H]CGP12177 (37 Ci/mmol), [N⁶-methyl-³H]mesulergine (87 Ci/mmol), and [-³⁵S]GTPS (1033 Ci/mmol) were from GE Healthcare.

Construction of Expression Vectors, Establishing Inducible EcR293 Cell Lines, and Transfection Construction of Expression Vectors. The human 5-HT_4(b) and 5-HT_7(a) receptors were cloned and stably transfected into the inducible cell line EcR293 (Invitrogen) as described previously (Bruheim et al., 2003). For expression of the human 5-HT_7(b) and 5-HT_7(d) receptors, previously cloned receptor cDNA (Krobert et al., 2001) was excised from the plasmid pcDNA3.1 (Invitrogen) with NheI and BamHI and transferred to the expression vector pInd (Invitrogen). EcR293 cells were transfected with plasmid DNA [pInd containing human 5-HT_7(b) or 5-HT_7(d)] using LipofectAMINE (Invitrogen) according to the manufacturer's protocol.

Human ₁ and ₂ adrenoceptors were excised from the plasmid pAGA-2 (Levy et al., 1993) with EcoRI and XbaI and transferred to pcDNA3.1.

Selection of EcR293 Cell Lines Stably Expressing 5-HT_7(b) or 5-HT_7(d) Receptors. EcR293 cells were cultured in 5-HT-free medium (UltraCULTURE general purpose serum-free medium; Cambrex Bio Science Walkersville, Inc., Walkersville, MD), supplemented with L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 µg/ml), and Zeocin (0.2 mg/ml). Forty-eight hours after transfection, serial dilutions of transfected cells were plated in 96-well plates and the neomycin analog G-418 (0.2 mg/ml; geneticin) was added. Limiting dilutions of isolated single colonies of cells transformed to the neomycin-resistant phenotype were performed to achieve single clonal cell lines. Single colonies were expanded and tested for ponasterone A-induced (10 µM for 24 h) expression of serotonin receptors by radioligand binding assay. For titration of receptor density, ponasterone A (0.1-10 µM) was added to the growth medium 24 h before conducting experiments.

Transfection of HEK293 or EcR293 Cell Lines. HEK293 cells (American Type Culture Collection, Manassas, VA) were grown in Dulbecco's modified Eagle's medium (Cambrex Bio Science Walkersville) with 10% fetal calf serum (EuroClone, Milano, Italy), penicillin (100 U/ml), and streptomycin (100 µg/ml). HEK293 or EcR293 cells inducibly expressing the 5HT_7(a) receptor were transiently transfected with LipofectAMINE 2000 (Invitrogen) according to the manufacturer's protocol. Cells were transfected with the following plasmids: human 5-HT_7(a) (Krobert et al., 2001), human ₁AR or ₂AR, AC5, AC6, or AC7 (all in pcDNA3.1; all AC clones were generous gifts from Dr. Dermot M. F. Cooper, Department of Pharmacology, University of Cambridge, Cambridge, UK), human G_s(S) or G_s(L) (both in pcDNA3.1 obtained from the University of Missouri-Rolla cDNA Resource Center, http://www.cdna.org), control vector (pcDNA3.1), or full-length murine HA-Ras-GRF1 wild type (in pKH3 mammalian expression plasmid; Mattingly et al., 1994; Mattingly and Macara, 1996), where indicated. After transfection, HEK293 cells were cultured in UltraCULTURE supplemented with L-glutamine (2 mM), penicillin (100 U/ml), and streptomycin (100 µg/ml) for 48 h. Twenty-four hours after transfection, EcR293 cells were induced with ponasterone A (10 µM) (where indicated) for an additional 24 h.

Membrane Preparation, Radioligand Binding, and Adenylyl Cyclase Assay. Membranes were prepared as described previously (Krobert et al., 2001). Radioligand binding assays for 5-HT₇, 5-HT_4(b), and AR were performed with 1.3 to 1.7 nM [³H]5-CT, 0.2 to 0.5 nM [³H]GR113808, or 20 to 100 pM (-)-3-[¹²⁵I]ICYP, respectively, as described previously (Krobert et al., 2001). B_max was estimated as described previously (Krobert et al., 2001) on the basis of a K_d value of 0.31 nM, 21 pM, and 6.8 pM for [³H]5-CT, [³H]GR113808, and (-)-3-[¹²⁵I]CYP, respectively. Adenylyl cyclase activity was measured and analyzed by determining conversion of [-³²P]ATP to [³²P]cAMP in membranes, as described previously (Krobert et al., 2001). Isoproterenol- and prostaglandin E₁ (PgE₁)-stimulated AC activities (performed in triplicates) are reported as the percentage activity relative to cells not expressing or not induced to express 5-HT₇ (or 5-HT_4(b)) receptors (control).

Cell-Surface Receptor Binding. Cell-surface AR density was determined as described previously (Clark and Knoll, 2002) with the following modifications: cells were trypsinized, pelleted, and resuspended in UltraCULTURE. Approximately 400,000 cells were plated in each well of a 96-well, round-bottomed microtiter plate and incubated with the hydrophilic compound [³H]CGP12177 (10 nM) for 1 h at 4°C. Nonspecific binding was determined by the inclusion of 1 µM alprenolol in parallel wells. Greater than 95% of cells remained intact after detachment as assessed by trypan blue staining. At the end of the incubation period, the plates were harvested in the same manner as for radioligand binding. B_max was estimated as described above using a K_d of 0.76 nM for [³H]CGP12177. Determination of cell-surface serotonin receptor density was performed similar to AR density. Approximately 60,000 cells per well were incubated with the hydrophobic compounds [³H]GR113808 (1 nM) or [³H]mesulergine (90 nM) with or without the hydrophobic antagonists SB207266 (10 µM) or methiothepin (10 µM) (for the 5-HT_4(b) and 5-HT_7(a) receptors, respectively) or hydrophilic 5-HT (100 µM). The incubation was carried out for 3 h at 13°C, which allows for equilibrium of ligand binding and inhibits sequestration or the return of sequestered receptors to the cell surface (Hausdorff et al., 1989). Plates were harvested as described above. The percentage of receptors on the cell surface was calculated as specific radioligand binding displaced by the hydrophilic ligand. Specific cell-surface receptor density was calculated as the difference between total radioligand binding and that resistant to displacement by the hydrophilic ligand and B_max was estimated as described above using a K_d of 21 pM and 9.2 nM for [³H]GR113808 and [³H]mesulergine, respectively.

cAMP Accumulation. Cells were plated and subsequently induced (where indicated) 24 h before the experiment in 12-well plates (Falcon; BD Biosciences Discovery Labware, Bedford, MA). Cells were incubated with 0.5 mM 3-isobutyl-1-methylxantine (Sigma-Aldrich) for 10 min and stimulated with isoproterenol, PgE₁, or increasing concentrations of 5-HT for 5 min. The reaction was stopped by the addition of trichloroacetic acid (Sigma-Aldrich) to a final concentration of 5%. cAMP content was determined by a radioimmunoassay as described previously (Skomedal et al., 1980). Isoproterenol- and PgE₁-stimulated cAMP accumulation were performed in quadruplicate and are reported as stimulated cAMP content relative to cells not induced to express either 5-HT_7(a) or 5-HT_4(b) receptors (control groups). Increasing concentrations of 5-HT were performed in duplicate, and data were fit to the equation Y = a + (b - a)x/(c + x) where a is basal cAMP accumulated, b is maximal cAMP accumulated stimulated by the agonist, c is EC₅₀, and x is the concentration of agonist.

GTPS Binding Assay Specific for G_s. Agonist-stimulated G_s-protein activation was determined in membrane preparations by measuring the stimulation of [³⁵S]GTPS binding coupled to an antibody capture-based scintillation proximity assay, as described previously (Cussac et al., 2002). Membranes were preincubated for 30 min with indicated agonists in a buffer containing 20 mM HEPES, pH 7.4, 50 mM MgCl₂, 100 mM NaCl, and 1 µM GDP. The reaction was started with the addition of [³⁵S]GTPS (0.3 nM in a final volume of 200 µl in 96-well optiplates; PerkinElmer Life and Analytical Sciences, Boston, MA). After 60-min incubation at room temperature, 20 µl of Nonidet P-40 (Sigma-Aldrich) was added (0.27% final concentration), and plates were incubated for 30 min under gentle agitation. Anti-G_s/olf (10 µl; 1.74 µg/ml final dilution) was then added to each well before an additional 30-min incubation period. Scintillation proximity assay beads coated with anti-rabbit antibodies (GE Healthcare) were added in a volume of 50 µl at a dilution indicated by the manufacturer, and the plates were incubated for 3 h with gentle agitation. The plates were then centrifuged (10 min, 1300g) immediately followed by radioactivity detection in a Topcount microplate scintillation counter (PerkinElmer). Nonspecific binding was measured by parallel wells incubated with GTPS (100 µM). Agonist-stimulated G_s activation is reported as the fold increase in specific binding compared with basal G_s activation.

Western Blotting. EcR293 cells inducibly expressing 5-HT_7(a) receptors were cultured in 35-mm dishes and transfected with the indicated plasmids. Cells were stimulated as indicated, then washed and lysed in ice-cold cell lysis buffer (1% SDS, 1 mM Na₃VO₄, and 50 mM Tris-HCl, pH 7.4, at room temperature), scraped with a Teflon cell scraper, sheared through a 25-gauge syringe, and immediately frozen in liquid N₂. The thawed cell lysates were cleared at 13,000g at 4°C, and the protein concentrations in the supernatants were quantified using the BC assay protein quantitation kit (Uptima, Monticon, France) using bovine serum albumin as a standard. Equal amounts of cell lysate proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and electroblotted onto polyvinylidene difluoride (PVDF) membranes. The membranes were incubated with primary antibodies (anti-phospho-Ras-GRF1, 1:1000; anti-HA-probe, 1:2000; anti-G_s/olf, 1:1000) in 5% nonfat dry milk in phosphate-buffered saline with 0.05% Tween and thereafter incubated with sheep anti-rabbit IgG HRP-conjugated secondary antibody. The immobilized HRP-conjugated secondary antibody was visualized with Supersignal Dura West extended-duration chemiluminescent substrate and analyzed with a BioChemie system (UVP Inc., Upland, CA).

Protein Measurements. Protein concentration was measured with the Micro BC Assay Reagent Kit (Uptima) using bovine serum albumin as a standard.

Statistics. Paired Student's t test was performed using GraphPad Prism 4.00 for Windows (GraphPad Software, Inc., San Diego, CA).

	Results

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Increasing 5-HT_7(a) Receptor Density Does Not Increase 5-HT Potency. We have reported previously that the potency of 5-HT to stimulate AC increased with increasing receptor density in membrane preparations from EcR293 cell lines induced to express the 5-HT_4(b) receptor but not the 5-HT_7(a) receptor (Bruheim et al., 2003). Our first objective was to determine whether intact cells displayed the same phenomenon to eliminate the possibility that this finding was an artifact of membrane preparations. The potency of 5-HT (pEC₅₀) to stimulate AC was examined in intact EcR293 cells expressing either low or high densities of 5-HT_4(b) or 5-HT_7(a) receptors. As shown in Table 1, the potency of 5-HT was increased (leftward shift of 0.8 in pEC₅₀ value) only in EcR293 cells expressing high densities of the 5-HT_4(b) receptor. In contrast, the potency of 5-HT was unchanged in EcR293 cells expressing high densities of the 5-HT_7(a) receptor, even though 5-HT_7(a) receptor density exceeded that of 5-HT_4(b) by more than 4-fold (Table 1). These data confirm the absence of a classic spare receptor effect in intact cells and indicate that this property is an inherent characteristic of 5-HT_7(a) receptor function in vivo.

Partial Agonists Become Full Agonists with Increasing Receptor Density at 5-HT_4(b) but Not 5-HT_7(a) Receptors. We have proposed that the potency of 5-HT for stimulation of AC through the 5-HT_7(a) receptor is independent of receptor-G_s stoichiometry, consistent with a model in which the 5-HT_7(a) receptors are tightly associated with G protein independent of agonist binding (Bruheim et al., 2003). In such a system, partial agonists are not expected to become full agonists in the presence of spare receptors. Therefore, to confirm and extend support for the existence of a stable complex between inactive 5-HT_7(a) receptors and G proteins, we determined the efficacy of 8-OH-DPAT and renzapride, agonists at the 5-HT₇ and 5-HT₄ receptors, respectively, at low and high receptor density in EcR293 cells. At the 5-HT_7(a) receptor, 8-OH-DPAT remained a partial agonist at both the lower (3.6 ± 1.0 pmol/mg of protein) and higher (7.9 ± 0.1 pmol/mg of protein) receptor densities tested, eliciting a maximal response of 81 ± 1% and 75 ± 1%, respectively, of that obtained with the full agonist 5-HT (Fig. 1, top graph). At low 5-HT_4(b) receptor density (0.72 ± 0.48 pmol/mg of protein), renzapride displayed partial agonist activity, eliciting a maximal response 87 ± 3% of that obtained with the full agonist 5-HT. However, and in contrast to the 5-HT_7(a) receptor, the efficacy of renzapride was equal (100 ± 1%) to the full agonist 5-HT at high receptor densities (3.8 ± 0.1 pmol/mg of protein; Fig. 1, bottom graph).

View larger version (28K): [in this window] [in a new window]   Fig. 1. Partial agonists become full agonists at high receptor densities at 5-HT4 but not 5-HT7 receptors. The top graph shows AC activity in response to increasing concentrations of 8-OH-DPAT and 5-HT in membranes from EcR293 cell lines expressing the 5-HT7(a) receptor at low (2.7 pmol/mg of protein; open symbols) and high (7.8 pmol/mg of protein; closed symbols) receptor density. The bottom graph shows AC activity in response to increasing concentrations of renzapride and 5-HT in membranes from EcR293 cell lines expressing the 5-HT4(b) receptor at low (0.24 pmol/mg of protein; open symbols) and high (3.7 pmol/mg of protein; closed symbols) receptor density. AC activity was measured as described under Materials and Methods, and the data shown are representative of those obtained from three independent experiments.

View larger version (17K): [in this window] [in a new window]   Fig. 2. Increasing 5-HT7 receptor density increases its contribution to basal AC activity and inhibits constitutive activity of the 2AR. A, effect of methiothepin (1 µM) on basal AC activity in membrane preparations of EcR293 cells expressing increasing 5-HT7(a) receptor densities. AC activity was measured as described under Materials and Methods, and data are presented as the percentage reduction of basal AC activity. Data shown are mean ± S.E.M. of six experiments from two independent EcR293 clones expressing 5-HT7(a) receptors. Similar data were obtained with clones expressing the 5-HT7(b) and the 5-HT7(d) receptors (data not shown). B, effect of timolol (10 µM) on basal AC activity in membrane preparations of EcR293 cells transiently expressing 2AR in the presence or absence of 5-HT7(a) receptors. Data presented are mean ± S.E.M. of nine experiments. 2AR receptor density was 2.2 ± 1.0 pmol/mg of protein in noninduced and 2.4 ± 1.1 pmol/mg of protein in EcR293 cell membranes induced to express 5-HT7(a) receptors. 5-HT7(a) receptor density was 0.011 ± 0.004 and 11.1 ± 1.4 pmol/mg of protein in noninduced and induced cells, respectively. *, timolol-mediated reduction of basal AC activity was significantly attenuated by 5-HT7(a) receptor expression (p < 0.05).

View larger version (18K): [in this window] [in a new window]   Fig. 3. Expression of 5-HT7 receptors attenuates endogenous Gs-coupled receptor signaling. The figure shows isoproterenol- (, Iso) and PgE1-() (both 10 µM) stimulated AC activity in membranes of EcR293 cells (A and C) or cAMP accumulation in intact EcR293 cells (B and D) induced to express increasing densities of either 5-HT7 (A and B) or 5-HT4(b) (C and D) receptors. All data are expressed as isoproterenol- and PgE1-stimulated AC activity or cAMP accumulation above basal as a percentage of control (uninduced cells). Total cAMP accumulated was measured after 5-min stimulation with isoproterenol or PgE1 in intact cell experiments. Data shown for 5-HT7 receptor membranes are collapsed across the three splice variants because there was no difference between the splice variants. Data are mean ± S.E.M. of 10 experiments obtained from two 5-HT7 clonal cell lines for each splice variant (A), seven cAMP accumulation experiments from two 5-HT7(a) clonal cell lines (B), eight experiments collapsed from two 5-HT4(b) clonal cell lines (C), or six cAMP accumulation experiments from one 5-HT4(b) clonal cell line (D).

-Adrenoceptor Cell Surface Receptor Density Is Not Modified by 5-HT₄ or 5-HT₇ Receptor Expression. The 5-HT₇ receptor splice variants display varying degrees of constitutive internalization in the absence of ligand (Guthrie et al., 2005). This property may promote endocytosis or limit cell-surface expression of endogenous AR and EPR. To determine whether a reduction of endogenous cell-surface receptors accompanied the reduced activation of AC, we measured the effect of 5-HT_4(b) and 5-HT_7(a) receptor expression upon cell-surface AR density. A high percentage of 5-HT_4(b) and 5-HT_7(a) receptors (64 ± 4 and 70 ± 6, respectively) are on the cell surface of EcR293 cells induced to express high 5-HT receptor densities (Table 2). These values are similar to that reported by Guthrie et al. (2005) in HEK293 cells. High-density expression of 5-HT₄ or 5-HT₇ receptors did not modify the density of endogenous AR on the cell surface of EcR293 cells (Table 2) or in EcR293 cells coexpressing transiently transfected ₂ARs (data not shown). These data indicate that a reduction of cell-surface AR and EPR is not mediating the attenuated AR and EPR activation of AC.

View this table: [in this window] [in a new window]   TABLE 2 Effect of 5-HT4(b) and 5-HT7(a) receptor expression on -adrenoceptor cell-surface receptor density The density of -adrenoceptors and serotonin receptors at the cell surface was determined in EcR293 cells induced to express either 5-HT4(b) or 5-HT7(a) receptors. Cell-surface receptor density was determined as described under Materials and Methods. Data shown are the mean ± S.E.M. from four experiments.

Mechanism of Attenuated Endogenous G_s-Coupled Receptor AC Activation Is Protein Kinase A-Independent. To determine whether high constitutive activity of the 5-HT₇ receptor, through sustained activation of AC and subsequent activation of protein kinase A (PKA), mediated the heterologous desensitization of the endogenous AR and EPR, we inhibited PKA activity with H89. As shown in Fig. 4A, isoproterenol- and PgE₁-stimulated AC activities in EcR293 cells expressing 5-HT_7(a) receptors remained similarly attenuated both in the presence and absence of H89. However, 5-HT-induced PKA-dependent phosphorylation of Ser916 on Ras-GRF1 (Norum et al., 2005) was inhibited by H89 in EcR293 cells expressing 5-HT_7(a) receptors (Fig. 4B), indicating that H89 inhibits PKA activity under these experimental conditions. Therefore, it is unlikely that a PKA-dependent mechanism of heterologous desensitization is mediating the attenuation of signaling of the endogenous AR and EPR.

View larger version (33K): [in this window] [in a new window]   Fig. 4. PKA activation is not the mechanism of attenuation of Gs-coupled receptor responses. A, isoproterenol- or PgE1-stimulated (both at 10 µM) AC activity in membranes of EcR293 cells induced to express 5-HT7(a) receptors in the presence or absence of the PKA inhibitor H89. EcR293 cells were incubated with either ponasterone A (10 µM, induced group) or vehicle (ethanol, noninduced group; control). Sister plates of induced and noninduced cells were incubated with H89 (20 µM) or vehicle (50% ethanol) for 3 x 8 h (24 h total incubation). Data shown are AC activity in membrane preparations as a percentage of the noninduced group and are mean ± S.E.M. from four experiments. 5-HT7(a) receptor density was 4.4 ± 0.8 pmol/mg of protein in vehicle and 4.2 ± 0.7 pmol/mg of protein in H89-treated groups. B, EcR293 cells were transfected with HA-Ras-GRF1 24 h before inducing 5-HT7(a) receptor expression. During incubation with ponasterone A (10 µM), the cells were coincubated 3 x 8 h with 20 µM H89 or vehicle. After the 24-h induction period, cells were stimulated with 10 µM 5-HT or vehicle for 5 min, lysed, and proteins were separated on 6% SDS-PAGE, electroblotted to PVDF membrane, and probed with anti-pRas-GRF1 (top). Total HA-Ras-GRF1 was detected with anti-HA (bottom). The blot shown is representative of three experiments.

5-HT₇ Receptors Limit the Ability of Endogenous AR and Prostanoid EP Receptors to Activate AC but Not G_s. We have proposed that a strong physical (pre)association of the 5-HT₇ receptor with G_s in the absence of ligand accounts for the atypical properties of 5-HT₇ receptor function. If the endogenous AR and EPR use the same pool of G_s as 5-HT₇ receptors, and 5-HT₇ receptors preassociate with G_s, access of AR and EPR to G_s may be impeded as 5-HT₇ receptor density increases. To determine whether the amount of G_s was limiting, we overexpressed G_{s(S or L)} protein together with 5-HT_7(a) receptors. Overexpression of G_s had no effect on isoproterenol- and PgE₁-stimulated AC activities, whether tested by G_{s(S or L)} overexpression in EcR293 cells induced to express 5-HT_7(a) receptors (Fig. 5A) or tested by cotransfection of HEK293 cells by 5-HT_7(a) and G_{s(S or L)} (Fig. 5B). In both systems, isoproterenol- and PgE₁-stimulated AC activity remained attenuated by 5-HT₇ receptor expression. It is interesting that isoproterenol-stimulated ARs were able to activate G_s equally well in the presence or absence of 5-HT₇ receptors, as revealed by GTPS binding (Fig. 6A). The fact that ARs are able to activate G_s, whereas AR activation of AC remained attenuated in the presence of 5-HT_7(a) receptors (Fig. 6B), suggests that access and/or availability of AC to activated G_s is limiting. To determine whether the amount of AC was limiting, we overexpressed AC5, a subtype of AC shown to be activated by the 5-HT_7(a) receptor in HEK293 cells (Baker et al., 1998). Isoproterenol- and PgE₁-stimulated AC activities in EcR293 cells induced to express 5-HT₇ receptors were not altered by overexpressing AC5 (Fig. 7A), even though forskolin-stimulated AC activity increased 2-fold (Fig. 7B), indicating that AC5 was properly expressed in the membrane preparations tested. Likewise, overexpression of AC6 (shown previously to be activated by ARs in HEK293 cells; Krupinski et al., 1992) or AC7 did not rescue isoproterenol- and PgE₁-stimulated AC activities (data not shown).

View larger version (18K): [in this window] [in a new window]   Fig. 5. Overexpression of Gs does not rescue AR- or EPR-stimulated AC activity. The figure shows isoproterenol- (Iso) and PgE1-stimulated AC activity (both at 10 µM) in membranes from EcR293 cells induced to express 5-HT7(a) receptors (A) or HEK293 cells transiently expressing 5-HT7(a) receptors (B) in the presence or absence of transient Gs(S or L) overexpression. Data shown are collapsed from experiments with Gs(S) and Gs(L), because no significant differences were noted. A, 24 h before 5-HT7(a) receptor induction by ponasterone A or vehicle, EcR293 cells were transfected with Gs(S or L) or control vector (pcDNA3.1). AC activity was assayed 24 h after induction of 5-HT7(a) receptor expression, 48 h after transfection of Gs. Data are mean ± S.E.M. of four experiments and are reported as a percentage of control (noninduced EcR293 cells). B, HEK293 cells were transiently cotransfected with Gs(S or L) or control vector (pcDNA3.1) and 5-HT7(a) [or control vector (pcDNA3.1)] and were grown for 48 h before AC assay. Data are mean ± S.E.M. of four experiments and are reported as a percentage of control [HEK293 cells not transfected with 5-HT7(a)]. 5-HT7(a) receptor density was 18 ± 3 and 5.9 ± 2.4 pmol/mg of protein in A and B, respectively. C, EcR293 cells transiently transfected with Gs(S or L) (where indicated) and induced to express 5-HT7(a) receptors (where indicated) were lysed and proteins separated on 10% SDS-PAGE, electroblotted to PVDF membranes, and probed with anti-Gs/olf.

View larger version (18K): [in this window] [in a new window]   Fig. 6. AR activation of Gs is unaffected by 5-HT7 receptor expression. A, isoproterenol-stimulated (10 µM) [35S]GTPS binding at Gs (Gs was isolated by an antibody capture technique as described under Materials and Methods) was assayed in membrane preparations from EcR293 cells induced to express 5-HT7(a) receptors (where indicated) and coexpressing transiently transfected 1AR, 2AR or control (pcDNA3.1). [35S]GTPS binding is presented as fold increase of basal. B, isoproterenol (10 µM)-stimulated AC activity (picomoles per milligram of protein per minute above basal) in the same EcR293 cell membrane preparations as above. Receptor density was 0.37 ± 0.09 and 0.80 ± 0.37 pmol/mg of protein for the 1AR and 2AR, respectively, and the presence of 5-HT7(a) receptors (9.1 ± 1.8 pmol/mg of protein) did not alter AR densities. Data shown are mean ± S.E.M. of four experiments performed in triplicate.

View larger version (19K): [in this window] [in a new window]   Fig. 7. Overexpression of adenylyl cyclase does not rescue AR- or EPR-stimulated AC activity. A, isoproterenol- (Iso) and PgE1-stimulated AC activity (both at 10 µM) in membranes from EcR293 cells induced to express 5-HT7(a or b) receptors in the presence or absence of transient AC5 overexpression. Data are reported as a percentage of control (noninduced EcR293 cells). B, forskolin (100 µM)-stimulated AC activity in membranes from EcR293 cells induced to express 5-HT7(a or b) receptors (where indicated) in the presence or absence (control vector, pcDNA3.1) of transient AC5 overexpression. Data are mean ± S.E.M. of five experiments. 5-HT7 receptor density was 6.7 ± 2.3 and 6.1 ± 2.1 pmol/mg of protein in induced cells without or with AC5 overexpression, respectively.

	Discussion

Top Abstract Materials and Methods Results Discussion References

Several findings in this study indicate that constitutive activation of AC and subsequent activation of PKA by 5-HT₇ receptors do not mediate the heterologous desensitization of AR and EPR. First and foremost, the 5-HT_4(b) receptor also constitutively activates AC, because the 5-HT_4(b) inverse agonist SB207266 reduced basal cAMP accumulation (51 ± 2% reduction by 10 µM SB207266) in intact EcR293 cells induced to express 5-HT_4(b) receptors (1.54 ± 0.07 pmol/mg of protein, n = 3). Expression of 5-HT₄ in numerous cell lines has revealed that 5-HT₄ receptors have a high constitutive activity even at low and physiological levels (Bockaert et al., 2004). In addition, mouse 5-HT_4(b) receptors constitutively activate AC in intact COS-7 cells to levels equivalent to the human constitutively active mutant ₂AR (Claeysen et al., 1999). Therefore, attenuation of isoproterenol- and PgE₁-stimulated AC activity would also be expected in the presence of 5-HT_4(b) receptors if constitutive activation of AC was the key determinant mediating PKA-dependent heterologous desensitization. However, as shown in Fig. 3, C and D, expression of the 5-HT_4(b) receptor did not inhibit isoproterenol- or PgE₁-stimulated AC activity. Second, attenuation of the isoproterenol- and PgE₁-stimulated AC activity was not affected when 5-HT₇ constitutive AC activity was blocked by the presence of 5-HT₇ inverse agonists (seven inverse agonists tested: methiothepin, clozapine, metergoline, spiperone, SB269970, methysergide, and mesulergine; data not shown). This indicates that the pool of G_s contributing to 5-HT₇ constitutive activity is not involved in attenuation of AR- or EPR-stimulated AC activity. Third, isoproterenol- and PgE₁-stimulated AC activity was not restored to control values when PKA activity was inhibited by H89 (Fig. 4). Fourth, AR activation of G_s was not attenuated (Fig. 6) as would be expected if AR were desensitized through the classic mechanisms.

On the other hand, the ability of the endogenous receptors to access G_s may be impeded by 5-HT₇ receptor expression. Vasquez and Lewis (1999) propose that the CB₁ receptor, because of its preassociation with G_i/o in the absence of ligand, sequesters a proportion of the available G protein pool. As a result, the available G protein pool is reduced, limiting activation by other G_i/o-coupled receptors. In support, Vasquez and Lewis (1999) have demonstrated that expression of the G_i/o-coupled CB₁ receptor in superior cervical ganglia attenuated the ability of ₂AR and somatostatin receptors to activate G_i/o. To presume that 5-HT₇ receptors are sequestering and limiting access to G protein, it is a prerequisite to demonstrate that the 5-HT₇ receptor is similarly preassociated with G_s.

In fact, such a basis exists, because 5-HT₇ receptors (as opposed to the 5-HT_4(b) receptor) exhibit multiple properties similar to other G protein-coupled receptors known to form a tight complex with G protein in the absence of ligand (Roka et al., 1999; Vasquez and Lewis, 1999; Mukhopadhyay et al., 2000; Shreeve, 2002). For example, a very high fraction of 5-HT₇ receptors exist in a high-affinity agonist binding state, which is insensitive to the destabilizing effect of guanine nucleotides (Alberts et al., 2001; Krobert et al., 2001). In addition, the mode of G protein coupling of the 5-HT₇ receptor is incongruent with the predictions of the operational model of agonism (Black and Leff, 1983), because the 5-HT_7(a) receptor does not display a classic spare receptor phenomenon (Bruheim et al., 2003). In the present studies, using intact EcR293 cells, we confirm that the potency of 5-HT remains unchanged at high 5-HT_7(a) receptor density, whereas the potency of 5-HT increases at high 5-HT_4(b) receptor density, in accordance with our observations in cell membranes (Bruheim et al., 2003). Furthermore, we show that the efficacy of the 5-HT₇ partial agonist 8-OH-DPAT remains unchanged relative to 5-HT at high 5-HT₇ receptor densities, extending support for the absence of a spare receptor phenomenon. In contrast, the 5-HT₄ partial agonist renzapride becomes a full agonist relative to 5-HT at high receptor densities (Fig. 1) as expected in the presence of spare receptors, in accordance with the operational model of agonism. From these data, we have proposed that the potency of 5-HT for stimulation of AC through the 5-HT_7(a) receptor is independent of receptor-G_s stoichiometry. This is consistent with a model in which the inactive conformational state of 5-HT_7(a) receptors is tightly associated with G protein (possibly complexed in a fixed stoichiometry), independent of agonist binding (Bruheim et al., 2003). This is in contrast to the 5-HT_4(b) receptor, which may associate with G protein independent of agonist binding, only or primarily when in the active conformational state. We propose that these characteristics distinguish the 5-HT₇ from the 5-HT_4(b) and may be related to the ability of 5-HT₇ receptors to attenuate signaling through other G_s-coupled receptors. It is well established that the 5-HT₇ receptor has a high constitutive activity, measured as AC activity (Krobert and Levy, 2002), a property that by definition gives rise to the active conformational state of the 5-HT₇ receptor coupled with G_s. In fact, we show that 5-HT₇ accounts for an increasing percentage of basal AC activation with increasing receptor density (accounting for up to 65% of total basal AC activity), at the expense of other G_s-coupled receptors (Fig. 2). Taken together, these findings support the concept of a tight association between both the inactive and active conformational state of the 5-HT₇ receptor and G_s protein.

Although the property of preassociation between the 5-HT₇ receptor and G_s protein may contribute to the attenuation of isoproterenol- and PgE₁-stimulated AC activity, it is unlikely to do so by sequestering or limiting access to a common G_s pool for the following reasons: 1) overexpression of G_s in the presence of high 5-HT₇ receptor density did not restore isoproterenol- and PgE₁-stimulated AC activity to control levels (Fig. 5); 2) ₁- and ₂AR activation of G_s is unaffected by high expression of 5-HT₇ receptors (Fig. 6), indicating that AR can access, couple to, and activate a pool of G_s. The primary implication of these findings is that the mechanism of heterologous desensitization is not occurring at the level of G protein activation. Therefore, it is unlikely that traditional desensitization mechanisms (i.e., G protein-coupled receptor kinase and PKA-dependent phosphorylation) are underlying the 5-HT₇-mediated effect, because these mechanisms result in the uncoupling of receptor from G protein. Furthermore, the mechanism used by the 5-HT₇ receptor differs from that of the CB₁ receptor, because overexpression of G_i/o rescued the ability of the endogenous ₂AR and somatostatin receptors to activate G_i/o (Vasquez and Lewis, 1999). The fact that ₁- and ₂AR activation of AC remains attenuated (Fig. 6B), even though they can activate G_s (Fig. 6A), indicates that the 5-HT₇ receptor in some way limits access to or impedes activation of AC directly. As we have proposed previously, 5-HT₇ receptor activation of AC conforms to a model assuming a preassociated signaling complex that includes G protein and AC (Bruheim et al., 2003). Therefore, the amount of AC available for activation by G_s may become the limiting component, because the approximate molar ratio of receptor/G protein/AC has been estimated as 1:200:3 (Alousi et al., 1991; Post et al., 1995). However, our data indicate that the amount of AC is also not the limiting component, because overexpression of AC5, AC6, or AC7 (AC isoforms known to interact with AR and 5-HT₇ receptors; Krupinski et al., 1992; Baker et al., 1998) did not rescue isoproterenol- or PgE₁-stimulated AC activity (data shown only for AC5 in Fig. 7). In certain cell types, AR but not EPR activation of AC was elevated when overexpressing AC6, presumably because AC6 colocalized only in the microdomain containing the AR (Ostrom et al., 2000). This suggests that the newly synthesized AC may not be accessible by AR and EPR in EcR293 cells, possibly because of compartmentalization of AC and the receptors into different microdomains. On the other hand, AR and EPR have access to AC; however, the presence of 5-HT₇ receptors somehow impedes the ability of these receptors to activate AC. Further study is needed to determine this elusive and potentially novel mechanism of heterologous desensitization mediated by 5-HT₇ receptor expression.

	Acknowledgements

 
We are grateful to Dr. Dermot M. F. Cooper (University of Cambridge, Cambridge, UK) for providing the plasmids encoding AC5, AC6, and AC7 and Dr. Raymond R. Mattingly (Wayne State University, Detroit, MI) for providing the plasmids encoding Ras-GRF1.

	Footnotes

 
This work was supported by The Norwegian Council on Cardiovascular Diseases, The Research Council of Norway, The Norwegian Cancer Society, Anders Jahre's Foundation for the Promotion of Science, The Novo Nordisk Foundation, The Family Blix Foundation, and grants from the University of Oslo.

Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

doi:10.1124/mol.105.015396.

ABBREVIATIONS: 5-HT, 5-hydroxytryptamine; AC, adenylyl cyclase; AR, -adrenergic receptor; EPR, prostanoid EP receptor; PKA, protein kinase A; H89, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrobromide; GR113808, {1-[2-(methylsulphonylamino)ethyl]-4-piperidinyl}methyl-1-methyl-1H-indole-3-carboxylate; CGP12177, (-)-4-(3-t-butylamino-2-hydroxypropoxy)-benzimidazol-2-one; HEK, human embryonic kidney; HA, hemagglutinin; BRL24924, renzapride; 5-CT, 5-carboxamidotryptamine; ICYP, iodocyanopindolol; PgE₁, prostaglandin E₁; PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride; HRP, horseradish peroxidase; SB269970, (2R)-1-[(3-hydroxyphenyl)-sulfonyl]-2-[2-(4-methyl-1-piperidinyl)ethyl]pyrrolidine; SB207266, N-((1-butyl-4-piperidinyl)methyl)-3,4-dihydro-2H-(1,3)oxazino(3,2-a)indole-10-carboxamide; 8-OH-DPAT, 8-hydroxy-2-dipropylaminotetralin; GTPS, guanosine 5'-[-thio]triphosphate; SR141716A, N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride.

Address correspondence to: Dr. Finn Olav Levy, Department of Pharmacology, University of Oslo, P.O. Box 1057 Blindern, N-0316 Oslo, Norway. E-mail: f.o.levy{at}medisin.uio.no

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