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Originally published In Press as doi:10.1074/jbc.M309200200 on November 10, 2003

J. Biol. Chem., Vol. 279, Issue 7, 5152-5161, February 13, 2004
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Constitutively active Gq/11-coupled Receptors Enable Signaling by Co-expressed Gi/o-coupled Receptors*

Remko A. Bakker, Paola Casarosa{ddagger}, Henk Timmerman, Martine J. Smit§, and Rob Leurs

From the Leiden/Amsterdam Center for Drug Research, Division of Medicinal Chemistry, Vrije Universiteit Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands

Received for publication, August 19, 2003 , and in revised form, October 7, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Co-expression of guanine nucleotide-binding regulatory (G) protein-coupled receptors (GPCRs), such as the Gi/o-coupled human 5-hydroxytryptamine receptor 1B (5-HT1BR), with the Gq/11-coupled human histamine 1 receptor (H1R) results in an overall increase in agonist-independent signaling, which can be augmented by 5-HT1BR agonists and inhibited by a selective inverse 5-HT1BR agonist. Interestingly, inverse H1R agonists inhibit constitutively H1R-mediated as well as 5-HT1BR agonist-induced signaling in cells co-expressing both receptors. This phenomenon is not solely characteristic of 5-HT1BR; it is also evident with muscarinic M2 and adenosine A1 receptors and is mimicked by mastoparan-7, an activator of Gi/o proteins, or by over-expression of G{beta}{gamma} subunits. Likewise, expression of the Gq/11-coupled human cytomegalovirus (HCMV)-encoded chemokine receptor US28 unmasks a functional coupling of Gi/o-coupled CCR1 receptors that is mediated via the constitutive activity of receptor US28. Consequently, constitutively active Gq/11-coupled receptors, such as the H1R and HCMV-encoded chemokine receptor US28, constitute a regulatory switch for signal transduction by Gi/o-coupled receptors, which may have profound implications in understanding the role of both constitutive GPCR activity and GPCR cross-talk in physiology as well as in the observed pathophysiology upon HCMV infection.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
GPCRs,1 which can be activated by a diverse array of stimuli, represent the largest group of integral membrane proteins involved in signal transduction. As such, GPCRs are the primary therapeutic target for many of today's drugs (1). The heterotrimeric G proteins mediate signaling from a large number of diverse GPCRs to a variety of intracellular effectors (see Refs. 2 and 3). A large body of work investigating the mechanisms underlying receptor-G protein interactions supports a network of interactions between signaling pathways that converge and diverge at multiple levels, enabling cells to coordinate responses to diverse environmental stimuli (4).

The hitherto existing knowledge of GPCR signal transduction pathways are founded largely upon experimental data obtained by the individual stimulation of the receptor of interest by specific ligands, either in heterologous expression systems or native tissues. Yet, under physiological conditions cells are permanently co-stimulated by various agonists. Investigations using receptor co-stimulation with agonists have recently shown cooperative effects of Gi/o and Gs (5), Gi/o and Gq/11 (6), and Gs and Gq/11 (7), the ability of Gi/o-coupled receptors to activate Gq-coupled receptors to transduce their signals by G{beta}{gamma} exchange (8), as well as G{beta}{gamma}-mediated glucocorticoid receptor transactivation (9). Such synergistic signaling mechanisms have been suggested to play an important role in signal adaptation (8) and may have a significant role in both physiological and pathophysiological processes (5).

Investigations of GPCRs expressed in surrogate cell systems have greatly modified our understanding of the pharmacological properties of GPCRs and consequently of the mechanistic drug-receptor models used to simulate drug action (10, 11). One of the prominent recent additions to our understanding of GPCR action is the occurrence of constitutive, agonist-independent GPCR activity (12–15). Currently, all models consider agonist-independent activity secondary to spontaneous isomerization of the receptors between the inactive and active receptor state(s), which couple(s) to the G protein; agonists are considered to preferentially bind to the active receptor state, whereas inverse agonists preferentially bind to the inactive receptor state (11). Despite initial concerns, the physiological relevance of constitutive GPCR activity is now accepted, and it appears not only to be a common property of GPCRs but, in various instances, also to be involved in the initiation or progression of disease (15). Various polymorphic GPCR variants have been shown to be highly constitutively active (15, 16), but also certain wild-type GPCRs, such as the histamine H3 receptor, exhibit high constitutive activity in vivo (17). Moreover, viral infection of cells may also result in the expression of virally encoded GPCRs that exhibit high levels of constitutive activity (18, 19).

To date, the impact of constitutive GPCR activity on signaling properties on co-expressed GPCRs has not been studied in full detail, despite the potential (patho)physiological relevance. In the present study, we investigated the potential cross-talk between a variety of Gi/o- and constitutively active Gq/11-coupled receptors using a heterologous expression system. Our data indicate that both activated histamine H1R and HCMV-encoded receptor US28 can result in the propagation of Gi/o-coupled receptor dependent signaling. Therefore, active Gq/11-coupled receptors constitute a regulatory role in the regulation of Gi/o-coupled receptor signaling events. As a consequence, the synergistic activation of signaling cascades that is observed upon co-expression of constitutively active receptors may be modulated by ligands acting at either Gi/o- or Gq/11-coupled receptors.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—pNF-{kappa}B-Luc was obtained from Stratagene (La Jolla, CA). ATP disodium salt, bovine serum albumin, CGS-12066A maleate, chloroquine diphosphate, cholera toxin, DEAE-dextran (chloride form), histamine dihydrochloride, mastoparan-7, mepyramine (pyrilamine maleate), pargyline hydrochloride, pertussis toxin, polyethyleneimine, tripelennamine hydrochloride, 3,3',5,5'-tetramethylbenzidine liquid substrate system for ELISA, triprolidine hydrochloride, and Tween 20 were purchased from Sigma. The human chemokine RANTES (CCL5) was obtained from Peprotech (Rocky Hill, NJ). D-Luciferin was obtained from Duchefa Biochemie BV (Haarlem, The Netherlands), glycerol from Riedel-de-Haën (Germany), Triton X-100 from Fluka (Switzerland), and goat anti-mouse horseradish peroxidase conjugate from Bio-Rad. Cell culture media, penicillin, and streptomycin were obtained from Invitrogen. Fetal calf serum was obtained from Integro B.V. (Dieren, The Netherlands) and dialyzed fetal calf serum from HyClone® Laboratories Inc. [3H]Mepyramine (30 Ci/mmol), [3H]5-HT (20 Ci/mmol), and myo-[2-3H]inositol (17 Ci/mmol) was obtained from Amersham Biosciences.

The gifts of acrivastine (The Wellcome Foundation Ltd.), GR-127935 (Dr. P. R. Saxena), mianserin hydrochloride (Organon N.V., Oss, The Netherlands), (R)- and (S)-cetirizine hydrochloride (UCB Pharma), SB-224289 (SmithKline Beecham), and pcDEF3 (Dr. J. Langer) and of the cDNAs encoding bovine G{gamma}2 (Dr. M. Lohse), G{beta}2 (Dr. I. Iyengar), Clostridium botulinum c3 exoenzyme (Dr. S. Narumiya), US28 (encoded by VHL/E HCMV strain, GenBankTM accession number L20501 [GenBank] , bases 219000–220263) in pcDNA3 (Dr R. Doms), the human CCR1 receptor in pcDNA3 (Dr. C. Tensen), the human serotonin 5-HT1B receptor in pKCREH (Dr. N. Stam), the human muscarinic M2 receptor in pcD (Dr. R. Maggio), the human adenosine A1 receptor in pcDNA3 (Dr. S. A. Rivkees), the porcine {alpha}2A adrenergic receptor fused to the pertussis toxin (PTX)-insensitive mutant rat G{alpha}oC351I ({alpha}2A-G{alpha}oC351I) (20) (Dr. G. Milligan), and the human histamine H1 receptor (Dr. H. Fukui) are gratefully acknowledged.

DNA Constructs—{Delta}(2–22)-US28 and the HA-tagged versions of both wild-type and {Delta}(2–22)-US28 were generated by PCR as described previously (18). The single amino acid mutation for US28-R129A was introduced using the Altered Sites® II in vitro mutagenesis system (Promega, Madison, WI) as described earlier (22). All constructs were verified by dideoxy sequencing.

Cell Culture and Transfection—COS-7 African green monkey kidney cells were maintained at 37 °C in a humidified 5% CO2, 95% air atmosphere in Dulbecco's modified Eagle's medium containing 2 mM L-glutamine, 50 IU/ml penicillin, 50 µg/ml streptomycin, and 5% (v/v) fetal calf serum. COS-7 cells were transiently transfected using the DEAE-dextran method as described previously (23, 24). The total amount of DNA transfected was maintained constant by the addition of either pcDEF3 or pcDNA3.

[3H]Inositol Phosphate Formation—Cells were seeded in 24-well plates and labeled overnight in inositol-free culture medium supplemented with 1 µCi/ml myo-[2-3H]inositol 24 h after transfection. Subsequently, the medium was aspirated, and cells were incubated with drugs for 1 h at 37 °C in Dulbecco's modified Eagle's medium containing 25 mM Hepes (pH 7.4) and 20 mM LiCl. Incubations were stopped by aspiration of the culture medium and the addition of cold 10 mM formic acid. After 90 min of incubation at 4 °C, [3H]inositol phosphates were isolated by anion exchange chromatography as we described previously (23, 24) and counted by liquid scintillation.

Reporter Gene Assay—Cells transiently co-transfected with pNF-{kappa}B-Luc (125 µg/1·107 cells, Stratagene) and plasmid DNA encoding the various receptors (25 µg/1·107 cells) were seeded in 96-well blackplates (Costar) in serum-free culture medium and incubated with drugs. After 48 h, cells were assayed for luminescence by aspiration of the medium and the addition of 25 µl/well luciferase assay reagent (0.83 mM ATP, 0.83 mM D-luciferin, 18.7 mM MgCl2, 0.78 µM Na2H2P2O7, 38.9 mM Tris (pH 7.8), 0.39% (v/v) glycerol, 0.03% (v/v) Triton X-100, and 2.6 µM dithiothreitol). After 30 min luminescence was measured for 3 s/well in a Victor2 Multilabel Counter (PerkinElmer Life Sciences).

H1R Binding Studies—COS-7 cells used for radioligand binding studies were harvested 48 h after transfection and homogenized in ice-cold H1-binding buffer. The COS-7 cell homogenates were incubated for 30 min at 25 °C in 50 mM Na2/K-phosphate buffer (pH = 7.4) in 400 µl with 1 nM [3H]mepyramine. The nonspecific binding was determined in the presence of 1 µM mianserin. The incubations were stopped by rapid dilution with 3 ml of ice-cold 50 mM Na2/K-phosphate buffer (pH = 7.4). The bound radioactivity was separated by filtration through Whatman GF/C filters that had been treated with 0.3% polyethyleneimine. Filters were washed twice with 3 ml of buffer, and radioactivity retained on the filters was measured by liquid scintillation counting.

5-HT1BR Binding Studies—COS-7 cells used for 5-HT1BR binding studies were harvested 48h after transfection and homogenized in ice-cold 5-HT1BR-binding buffer (50 mM Tris-HCl (pH 7.4), containing 4 mM CaCl2, 100 µM ascorbic acid, and 10 µM pargyline). 5-HT1BR binding studies were performed using ~7 nM [3H]5-HT. The COS-7 cell homogenates were incubated for 30 min at 30 °C in 5-HT1BR-binding buffer in 400 µl with 7 nM [3H]5-HT. The nonspecific binding was determined in the presence of 1 µM GR-127935. The incubations were stopped by rapid dilution with 3 ml of ice-cold 5-HT1BR-binding buffer. The bound radioactivity was separated by filtration through Whatman GF/C filters that had been treated with 0.3% polyethyleneimine. Filters were washed twice with 3 ml of buffer, and radioactivity retained on the filters was measured by liquid scintillation counting. A KD of 6.8 nM was subsequently used to calculate the expression levels of the 5-HT1BR (25).

US28 Receptor Binding Studies—The transfected COS-7 cells used for radioligand binding studies were seeded in 24-well plates; 48 h after transfection, binding was performed on whole cells for 3 h at 4 °C using [125I]CCL5 (RANTES) in binding buffer (50 mM HEPES, pH 7.4, 1 mM CaCl2, 5 mM MgCl2, and 0.5% bovine serum albumin). After incubation, cells were washed four times at 4 °C with binding buffer supplemented with 0.5 M NaCl. Nonspecific binding was determined in the presence of 0.1 µM unlabeled CCL5.

ELISA—48 h after transfection, receptor expression in COS-7 cells was measured using an ELISA as described previously (18). A mouse anti-HA monoclonal antibody was used as primary antibody and a goat anti-mouse-horseradish peroxidase conjugate as secondary antibody. The 3,3',5,5'-tetramethylbenzidine liquid substrate system for ELISA was used as substrate, and the optical density was measured in a Victor2 at 450 nm.

Analytical Methods—All data shown are expressed as means ± S.E. The data from radioligand-binding and functional assays data were evaluated by a nonlinear, least squares curve-fitting procedure using Graphpad Prism® (GraphPad Software, Inc., San Diego, CA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Constitutive H1R Activity and Inverse Agonistic Properties of Antihistamines—Transient expression of human H1R in COS-7 cells resulted in a high affinity binding site for the H1R radioligand [3H]mepyramine (KD = 1.7 ± 0.2 nM, Bmax = 4.6 ± 0.1 pmol/mg protein, data not shown). H1R activates both PLC- and NF-{kappa}B-mediated enhancement of gene transcription as measured by the accumulation of [3H]inositol phosphates and NF-{kappa}B-driven reporter gene assays, respectively, in an agonist-dependent and -independent manner (Fig. 1). Moreover, the H1R-selective antagonist mepyramine acts as an inverse H1R agonist for the constitutive H1R-mediated activation of these signal transduction pathways. These data are in agreement with previous results indicating the constitutive H1R activity and inverse H1R agonistic properties of various antihistamines (24, 26). The H1R-mediated effects on PLC and NF-{kappa}B activation are PTX-insensitive (see also Fig. 2A), indicating that the Gi/o family of G proteins is not involved in these H1R-mediated responses (24, 26). Moreover, we have shown that constitutive H1R-mediated activation of these pathways can be enhanced selectively via co-expression of G proteins that belong to the Gq/11 family of G proteins (24), confirming that the H1R is coupled to G{alpha}q/11 proteins for modulation of these signaling events in these cells.



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  		FIG. 1.
H1R and 5-HT1BR signal transduction in COS-7 cells. A and B, histamine-stimulated (•) activation and mepyramine-mediated ({circ}) inhibition of constitutive H1R-mediated activation of inositol phosphate formation (A) and NF-{kappa}B (B) in cells expressing human H1R. Representative curves are shown. C, inositol 1,4,5-trisphosphate (InsP3) accumulation in cells co-expressing H1R and 5-HT1BR. Mepyramine (10 µM; Mep) selectively inhibits the constitutive H1 activity as well as the 5-HT1B-mediated PLC activation induced by the selective 5-HT1B agonist CGS (10 µM). D, NF-{kappa}B activation in COS-7 cells expressing 5-HT1BR or co-expressing H1R, as measured in a bioluminescent reporter gene assay. CGS (10 µM)-induced 5-HT1BR-mediated NF-{kappa}B activation is inhibited by PTX treatment of the cells (100 ng/ml for 48 h). Mepyramine (10 µM) inhibits constitutive H1R-mediated as well as 5-HT1BR-mediated NF-{kappa}B activation induced by CGS (10 µM) in cells co-expressing H1R and 5-HT1BR. Also indicated is the effect of histamine (10 µM) on NF-{kappa}B activation in cells co-expressing the 5-HT1B and H1 receptors. Data shown are the average of four experiments, each performed in triplicate, and are presented as a percentage of basal activation.

 



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  		FIG. 2.
Effects of co-expression of Gi/o-coupled receptors, together with human H1R, on NF-{kappa}B activation. A, effects of PTX treatment (100 ng/ml) on NF-{kappa}B activation in cells expressing either human H1R or H1R together with either 5-HT1BR or the adenosine A1 receptor. B, effects of co-expression of H1R together with the muscarinic M2 receptor on NF-{kappa}B activation. Also the effects of the muscarinic agonist carbachol (10 µM; Car) and the effects of PTX (100 ng/ml) and mepyramine (10 µM; Mep) on this agonist treatment are shown. Shown are representative experiments, each performed in triplicate. RLU, relative light unit.

 
Constitutive Activation of NF-{kappa}B by the h5-HT1BR—We evaluated the human 5-HT1BR for constitutive activity upon transient expression of this receptor in COS-7 cells. Expression of the receptor was verified by [3H]5-HT radioligand binding studies, indicating 5-HT1BR expression levels of ~310 fmol/mg protein (data not shown). We also assessed the capacity of 5-HT1BRs to mediate activation of NF-{kappa}B. In 5-HT1BR-expressing cells the selective 5-HT1B agonist CGS-12066A (CGS) (27) and 5-HT stimulate NF-{kappa}B activation only ~0.6-fold (Fig. 1D). Despite the functional 5-HT1BR expression in these cells, we did not detect constitutive 5-HT1BR activation in our reporter gene assay, as the selective inverse 5-HT1BR agonist SB-224289 (28) was without effect on basal NF-{kappa}B activation in 5-HT1BR-expressing cells (data not shown). Consistent with the notion of a Gi/o-coupled 5-HT1BR receptor, 5-HT1BR-mediated NF-{kappa}B activation was PTX-sensitive (Fig. 1D), indicating the involvement of Gi/o proteins in 5-HT1BR-mediated NF-{kappa}B activation.

5-HT1BR Signaling in Cells Co-expressing H1R—In agreement with previous reports on agonist-mediated signaling (25, 29), stimulation of COS-7 cells co-expressing H1R and 5-HT1BR with 10 µM CGS, a selective 5-HT1BR-agonist (27), results in a marked elevation of the inositol phosphate accumulation (Fig. 1C). Surprisingly, however, treatment of cells co-expressing both H1R and 5-HT1BR with 10 µM CGS resulted in a robust PLC and NF-{kappa}B activation, yielding almost the same efficacy as histamine (His), which was completely sensitive to the inverse H1R agonist mepyramine (10 µM, Fig. 1, C and D), indicating the importance of constitutive H1R activity for the observed response to CGS. CGS is without effect on H1R-mediated NF-{kappa}B activation in cells transfected with only H1R-cDNA (Fig. 3A), whereas mepyramine (10 µM) does not influence CGS-induced 5-HT1BR-mediated NF-{kappa}B activation in cells transfected with only the 5-HT1BR-cDNA (data not shown), indicating that these ligands are selective for their respective receptors. Moreover, also in [3H]5-HT and [3H]mepyramine binding studies both ligands turned out to behave as selective ligands (data not shown). H1R expression levels were unaffected by co-expression of 5-HT1BR as determined by [3H]mepyramine radioligand binding studies (Bmax = 4.6 ± 1.3 pmol/mg protein, data not shown).



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  		FIG. 3.
Dose-dependent inhibition of constitutive and agonist-induced 5-HT1BR receptor-mediated NF-{kappa}B activation in cells co-expressing H1 and 5-HT1B receptors in COS-7 cells. A, dose-dependent inhibition of constitutive H1R-mediated NF-{kappa}B activation by mepyramine in the presence ({circ}) or absence (*) of 1 µM CGS in cells expressing human H1R (hH1), and the inhibition of constitutive H1R-mediated NF-{kappa}B activation by mepyramine in the presence (•) or absence ({circ}) of 1 µM CGS in cells co-expressing H1R and 5-HT1BR. RLU, relative light units. B, effects of 1 µM CGS stimulation (filled symbols) on the stereospecific inhibition of NF-{kappa}B activation by R-cetirizine (squares) and S-cetirizine (circles) in cells co-expressing H1R and 5-HT1BR upon stimulation of the cells with 1 µM CGS. C, dose-dependent inhibition of 5-HT1B-mediated NF-{kappa}B activation by SB-224289 in cells co-expressing H1R and 5-HT1BR (•) relative to the inhibition of the constitutive H1R-mediated NF-{kappa}B activation by mepyramine in cells transiently expressing H1R ({circ}). Shown are representative experiments, each performed in triplicate. D, effects of SB-224289 (SB) and PTX (100 ng/ml) on constitutive NF-{kappa}B activation in cells coexpressing both human H1 and 5-HT1B receptors. Data are expressed relative to the basal NF-{kappa}B activation observed in cells co-expressing H1R and 5-HT1BR.

 
As seen in Figs. 2A and 3, A and C, co-expression of both GPCRs not only results in CGS-induced 5-HT1BR-mediated NF-{kappa}B activation but also in a 2–3-fold increase in basal NF-{kappa}B activation. The fold over basal activation of NF-{kappa}B by histamine is reduced from 4.8 ± 0.4-fold (n = 76), when H1R is expressed alone, to 3.3 ± 0.5-fold (n = 9)-fold, upon co-expression of the 5-HT1BR. These data do not reflect a suppression of the response to histamine when H1R is co-expressed with 5-HT1BR; however, because of the increase in the level of basal signaling, it does result in a reduced fold of stimulation. The constitutive NF-{kappa}B activation observed upon co-expression of both GPCRs is partially PTX-sensitive but was completely inhibited by mepyramine (Fig. 2A). PTX treatment of the cells had no effect on mepyramine-treated cells (Fig. 2A). H1R is known to exhibit stereospecificity toward the enantiomers of the partial inverse H1R agonist cetirizine (24, 26). Indeed, the inhibition of CGS-induced NF-{kappa}B activation by the enantiomers of cetirizine is found to be stereospecific in cells coexpressing H1R and 5-HT1BR (Fig. 3B).

The PTX-sensitive increase in basal H1R-mediated NF-{kappa}B activation that is observed upon 5-HT1BR co-expression, but not the constitutive H1R activity itself, is potently inhibited by the inverse 5-HT1BR agonist SB-224289 (28) (Fig. 3, C and D). SB-224289 inhibited constitutive NF-{kappa}B activation in cells coexpressing H1 and 5-HT1B receptors to the level of basal NF-{kappa}B activation as observed in H1R-expressing cells not expressing the 5-HT1BR, indicating the presence of constitutive 5-HT1BR-activity upon co-expression with H1R (Fig. 3C). The observed pIC50 value of 8.0 ± 0.2 is in good agreement with literature data for SB-224289 acting on 5-HT1BR (28). SB-224289 is without effect on H1R-mediated NF-{kappa}B activation in cells only expressing the H1R, indicating that SB-224289 does not exert its effects of inhibition of constitutive NF-{kappa}B activation in cells co-expressing H1 and 5-HT1B receptors by acting at H1Rs.2

Effects of Co-expression of Other Gi/o-coupled Receptors on Constitutive H1R Activity—To test whether these observations are exclusive for 5-HT1BR, we co-expressed H1R with other Gi/o-coupled receptors: the adenosine A1 and the muscarinic M2 receptors. Co-expression of H1R with the A1 or M2 receptor indeed resulted in a similar increase in basal NF-{kappa}B activation, which was largely PTX-sensitive (Fig. 2).

As observed after co-expression with the 5-HT1BR, PTX-insensitive NF-{kappa}B activation is fully inhibited by mepyramine, indicating that the increased NF-{kappa}B activation depends on constitutive H1R signaling. Stimulation of the M2 receptor with the muscarinic agonist carbachol results in a further increased mepyramine- and PTX-sensitive NF-{kappa}B activation (Fig. 2B). We also tested the ability of a Gi/o-coupled {alpha}2A adrenergic receptor, in which the C terminus is fused to a G{alpha}o subunit that harbors a mutation that renders the G{alpha}o insensitive to PTX ({alpha}2A-GoC351I) (20), to enhance NF-{kappa}B activation of co-expressed H1Rs. As expected, stimulation of the {alpha}2A receptor with the specific agonist clonidine resulted in a PTX-insensitive increase in NF-{kappa}B activation that could be fully inhibited by mepyramine, indicating that the PTX-insensitive G{alpha}oC351I subunit fused to the {alpha}2A receptor mediates NF-{kappa}B activation via H1R (data not shown).

Effects of Co-expression of Gi/o- and Gq/11-coupled Chemokine Receptors—To investigate whether the observed responses are H1R-specific or more generally relevant, we tested the effect of the co-expression of two chemokine receptors on their signaling properties. We co-expressed the Gq/11-coupled virally encoded chemokine receptor US28, a chemokine receptor homolog encoded by HCMV, which binds with high affinity the CC-chemokine CCL5 (RANTES) Fig. 4, together with the human Gi/o-coupled chemokine receptor CCR1. We previously reported the high constitutive activity of US28 in activation of both PLC- and NF-{kappa}B-mediated enhancement of gene transcription as measured by the accumulation of [3H]inositolphosphates and NF-{kappa}B-driven reporter gene assays, respectively (23) (Fig. 4, A, B, and E). CCL5 binds with high affinity to receptor US28 but does not increase the level of activation of receptor US28, and therefore CCL5 acts as a neutral antagonist for this receptor (Fig. 4A) (23). In contrast, CCL5 binds to CCR1 with high affinity and acts as a CCR1 agonist, inducing calcium mobilization in a PTX-sensitive manner (see Ref. 30). When tested for activation of NF-{kappa}B, CCR1 does not exhibit basal NF-{kappa}B activation, and stimulation of CCR1 with CCL5 results in a small, PTX-sensitive NF-{kappa}B activation. Upon co-expression of receptor US28 and CCR1, however, CCL5 elicits a robust agonist response that is completely PTX-sensitive, indicating the involvement of Gi/o proteins in CCL5-mediated NF-{kappa}B activation (Fig. 4A). The binding of CCL5 to receptor US28 is not involved in the observed phenomenon as shown by co-expression of CCR1 with an N-terminal truncation mutant of receptor US28 ({Delta}(2–22)-US28), in which the first 22 residues of receptor US28 are deleted. The N-terminal truncation mutant {Delta}(2–22)-US28 receptor does not exhibit [125I]CCL5 binding as observed by [125I]CCL5 saturation binding analysis (Fig. 4D), in line with reports that chemokine binding to chemokine receptors is critically dependent on the N terminus of the receptor (31). We used an ELISA to confirm cell surface expression of {Delta}(2–22)-US28 (45% of wild-type receptor US28, Fig. 4C). {Delta}(2–22)-US28 still activates both NF-{kappa}B (Fig. 4B) and PLC constitutively (Fig. 4E), and co-expression of ({Delta}(2–22)-US28 with CCR1 resulted in CCL5-induced CCR1-mediated activation of NF-{kappa}B (Fig. 4B). In contrast, a mutant US28 receptor (US28-R129A) (22), which is expressed at the cell surface (Fig. 4, C and D), and which binds CCL5 with unchanged affinity in comparison with wild-type receptor US28 (Fig. 4D) but is devoid of constitutive activity (Fig. 4, B and E), does not transduce CCR1-mediated NF-{kappa}B activation upon co-expression with CCR1 (Fig. 4B); this indicates the importance of the constitutive activity of receptor US28 for the observed response to CCL5.



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  		FIG. 4.
Effects of CCL5 on NF-{kappa}B activation in cells expressing either CCR1 or US28 or co-expressing both CCR1 and US28 receptors compared with control cells (Mock). A, effects of co-expression of wild-type CCR1 and US28 receptors and PTX treatment (100 ng/ml) on CCL5-induced (100 nM) NF-{kappa}B activation. RLU, relative light unit. B, effects of co-expression of CCR1 and wild-type US28, {Delta}(2–22)-US28, or US28-R129A on CCL5-induced (100 nM) NF-{kappa}B activation. Similar results have been obtained using N-terminal HA-tagged US28 receptors.2 C, cell surface expression of N-terminal HA-tagged US28 receptors as monitored by ELISA using HA-specific antibodies. WT, wild type. D, cell surface expression of US28, US28-R129A, and {Delta}(2–22)-US28 receptors was monitored by whole cell [125I]CCL5 binding. E, effects of wild-type US28, {Delta}(2–22)-US28, and US28-R129A on constitutive inositol phosphate accumulation.

 
The effects of CCL5 that are observed upon co-expression of CCR1 and receptor US28 are similar to the effects observed for 5-HT1BR agonists in cells co-expressing H1R and 5-HT1BR. The co-expression of the Gq/11-coupled receptor US28 with the Gi/o-coupled CCR1 results in the alteration of CCR1 signaling capabilities. By co-expressing receptor US28 and CCR1, CCR1 acquires the capability to signal through NF-{kappa}B upon agonist (CCL5) stimulation. However, it does not appear to result in increased constitutive receptor activity, as we did not observe modulation of basal NF-{kappa}B activation in cells expressing receptor US28 upon co-expression of CCR1 (Fig. 4A).

Mechanism of Synergistic NF-{kappa}B Activation by Co-expressed Gi/o- and Gq/11-coupled Receptors—Various GPCRs may activate signal transduction pathways leading to NF-{kappa}B activation (see Fig. 7). Both the H1R and receptor US28 are reported to activate G proteins belonging to both the Gi/o and Gq/11 families of G proteins (23, 24, 26, 32–35). Yet, G{alpha}i subunits are not implicated in the H1R or receptor US28-mediated activation of phospholipase C or NF-{kappa}B, as PTX treatment of the cells did not alter H1R or receptor US28-mediated [3H]inositol phosphate production or NF-{kappa}B activation (23, 24) (see also Figs. 2A and 4A).



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  		FIG. 7.
Schematic representation of NF-{kappa}B activation by Gq/11- and Gi/o-coupled GPCRs. Gq/11-coupled GPCRs may activate NF-{kappa}B via both G{alpha}q- and G{beta}{gamma}-activated pathways (23, 24, 59, 60), which may involve phosphatidylinositol 3-kinase (PI3K) (59)-, RhoA (44, 45, 61–64)-, Akt (60)-, and protein kinase C (PKC)-dependent pathways (65, 66). Gi/o-coupled GPCRs may activate NF-{kappa}B via an Akt-dependent pathway (67), a G{beta}{gamma}-, c-Src-, and {beta}-arrestin 1-dependent pathway, or a PLC- and protein kinase C-independent (21) pathway. DAG, diacylglycerol; InsP3, inositol 1,4,5-trisphosphate.

 
Co-expression of H1R or receptor US28 with G{alpha}q or G{alpha}11 resulted in a G{alpha} expression level-dependent increase in (constitutive) H1R- or receptor US28-mediated NF-{kappa}B activation and inositol 1,4,5-trisphosphate production (23, 24) (Fig. 5A). Scavenging of G{beta}{gamma} subunits by co-expression with either G{alpha}t (Fig. 5) or GRK2 (data not shown) effectively reduced constitutive H1R and receptor US28-mediated NF-{kappa}B activation, whereas co-expression with G{beta}{gamma} subunits resulted in the elevation of the constitutive receptor activity of both H1R and receptor US28 (23, 24) (Fig. 5A). In line with published data on GPCR-mediated activation of NF-{kappa}B (see also Fig. 7) these data implicate both the G{alpha}q/11 and G{beta}{gamma} subunits, which are released upon H1R or receptor US28-mediated activation of Gq/11 proteins, in the activation of signal transduction pathways resulting in activation of NF-{kappa}B (23, 24).



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  		FIG. 5.
Assessment of G protein involvement in NF-{kappa}B activation in COS-7 cells. A, effects of co-expression of G{alpha}11 and G{alpha}t proteins and G{beta}2{gamma}1 subunits on constitutive H1R and receptor US28-mediated NF-{kappa}B activation. Shown are representative experiments, each performed in triplicate. Dashed lines indicate the basal levels of NF-{kappa}B activation observed in control cells (Mock) and in cells transiently expressing human H1R (H1) or the virally encoded chemokine receptor US28. Also shown are the effects of the H1R agonist histamine (10 µM) and inverse H1R agonist mepyramine (10 µM, Mep) on H1R-mediated NF-{kappa}B activation upon co-expression of G{alpha}11 and G{alpha}t proteins and G{beta}2{gamma}1 subunits. B, effects of co-expression of the Rho inhibitor C. botulinum c3 exoenzyme (C3) on constitutive and agonist-induced (10 µM His) H1R-mediated NF-{kappa}B activation in COS-7 cells. Co-expressions were performed by co-transfection of equal amounts of the cDNA encoding H1R and the respective cDNAs coding for G{alpha}11, G{alpha}t, G{beta}2, and G{gamma}1 and the c3 exoenzyme, respectively, whereas the total amount of transfected cDNA was maintained constant using pcDEF3.

 
Activation of H1R (36–38) and G{alpha}q signaling (39, 40), as well as NF-{kappa}B activation, have been linked to the activation of small G proteins and especially the Rho-like G proteins (41–45). We therefore investigated the role of Rho signaling in the H1 receptor-mediated activation NF-{kappa}B. C. botulinum c3 exoenzyme, which prevents the coupling of RhoA, -B, and -C to its downstream effectors by irreversible ADP-ribosylation, has often been used to demonstrate the involvement of Rho in GPCR signaling, including NF-{kappa}B activation (42). Co-expression of the c3 exoenzyme abrogates all, both constitutive as well as agonist-induced, human H1R-mediated NF-{kappa}B activation (Fig. 5B).

We have previously reported that constitutively activated (Q205L) G{alpha}i/o proteins do not enhance H1R-mediated NF-{kappa}B activation (24), which indicates that activation of Gi/o-coupled receptors most likely results in the release of G{beta}{gamma} subunits from activated G{alpha}i/o proteins, which then may serve to potentiate signaling mediated by Gq/11-couled receptors. To mimic activation of Gi/o-coupled receptors we used mastoparan-7 (M7), a relatively stable analogue of mastoparan, to directly activate G{alpha}i/o proteins and to stimulate both G{alpha}i/o- and G{beta}{gamma}-mediated signal transduction pathways (46). M7 induced a PTX-sensitive NF-{kappa}B activation in cells expressing 5-HT1BR to a similar extent as the 5-HT1BR agonist CGS (Fig. 6A). M7 also induces NF-{kappa}B activation in cells expressing H1R; this effect is effectively blocked by the inverse H1R agonist mepyramine (Fig. 6B), demonstrating that activated G{alpha}i/o proteins may enhance constitutive H1R-mediated NF-{kappa}B activation. Mepyramine did not affect M7-induced NF-{kappa}B activation in cells that do not express H1R (Fig. 6A).



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  		FIG. 6.
Activation of Gi/o proteins by mastoparan-7 on NF-{kappa}B activation in COS-7 cells. A, effects of M7 (10 µM) on NF-{kappa}B activation in cells expressing 5-HT1BR compared with NF-{kappa}B activation induced by the 5-HT1BR agonist CGS (10 µM) and effects of mepyramine (10 µM; Mep) and PTX (100 ng/ml) on M7-induced NF-{kappa}B activation. B, effects of M7 (10 µM) on NF-{kappa}B activation in cells expressing H1R and effects of mepyramine (10 µM) on M7-induced NF-{kappa}B activation.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We co-transfected COS-7 cells with cDNAs encoding various GPCRs and examined agonist-induced as well as constitutive GPCR-mediated stimulation of PLC- and NF-{kappa}B-regulated gene expression. Expression of either the human histamine H1R or the HCMV-encoded receptor US28 in COS-7 cells results in the activation of both PLC- and NF-{kappa}B-regulated gene expression in an agonist-dependent (H1R) and -independent manner (H1R and receptor US28) (23, 24, 26). H1R- or receptor US28-mediated activation of PLC and NF-{kappa}B is PTX-insensitive (23, 24) (Figs. 1, 2, and 4), indicating that Gi/o proteins are not involved in these H1R- and receptor US28-mediated responses in COS-7 cells. By co-expression of a variety of G protein subunits, we have shown earlier that G{alpha}q/11 as well as G{beta}{gamma} subunits are involved in NF-{kappa}B activation via H1R or receptor US28 (23, 24) (Fig. 7), whereas for both receptors, the activation of PLC is mediated via the classical G{alpha}q/11 pathway (23, 24). In the present study we have investigated the effects of co-expression of various Gi/o-coupled receptors on agonist-induced and constitutive activity of the Gq/11-coupled H1R and receptor US28 using a heterologous expression system.

In agreement with reports on synergistic agonist-induced PLC activation in cells co-expressing H1R and Gi/o-coupled 5-HT1BR (25, 29), stimulation of cells co-expressing both receptors with the selective 5-HT1B agonist CGS-12066A (27) results in a marked synergistic elevation of inositol phosphate accumulation (Fig. 1). Moreover, upon co-expression of 5-HT1BR with H1R, CGS elicited a robust PTX-sensitive activation of NF-{kappa}B, whereas CGS had no such effects on NF-{kappa}B activation in cells lacking 5-HT1BRs (Fig. 1).

Unexpectedly, we found that H1R-mediated effects of H1R-selective inverse H1R agonists such as mepyramine and cetirizine include the inhibition of CGS-induced 5-HT1BR-mediated activation of both PLC and NF-{kappa}B in cells co-expressing both 5-HT1BR and H1R (Figs. 1, 2, and 4). In contrast, inverse H1R agonists had no such effects on 5-HT1BR-mediated signaling in cells lacking H1Rs. Radioligand binding studies confirmed the H1R selectivity of the inverse H1R agonist, indicating that inverse H1R agonists do not modulate 5-HT1BR signaling via a direct interaction with either Gi/o-coupled 5-HT1BR or with the activated Gi/o proteins. We concluded from these results that the interaction of inverse H1R agonists with H1Rs induces biological responses that negatively interfere with 5-HT1BR signaling. Because these experiments were performed in the absence of histamine, it seems that the constitutive activity of the H1R (24, 26) is a critical determinant for the observed functional 5-HT1BR-mediated responses to the 5-HT1BR agonist.

The co-expression of Gq/11- and Gi/o-coupled receptors may not only give rise to agonist induced signaling of Gi/o-coupled receptors but may also affect constitutive signaling. In fact, co-expression of H1R and 5-HT1BR allows the detection of otherwise undetectable constitutive 5-HT1BR activity. Co-expression of 5-HT1BR together with H1R resulted in a significantly increased constitutive activation of NF-{kappa}B (Figs. 2 and 3), which was completely inhibited by the inverse H1R agonist mepyramine but also, partially, by the inverse 5-HT1BR agonist SB-224289 (28). This inverse 5-HT1BR agonist inhibited only the increased constitutive NF-{kappa}B activation that is observed upon co-expression of the 5-HT1BR but not the constitutive H1R-mediated NF-{kappa}B activation (Fig. 3). Based on these findings we conclude that the otherwise minimal 5-HT1BR agonist-induced or undetectable constitutive 5-HT1BR responses are enhanced through constitutive H1Rs.

We subsequently investigated whether the observed phenomena are receptor-specific by testing the effects of co-expression of other Gq/11- and Gi/o-coupled receptors on NF-{kappa}B activation. To test whether these phenomena were specific for 5-HT1BR, we co-expressed several other Gi/o-coupled receptors with H1R. Stimulation of cells co-expressing the muscarinic M2 receptor (M2R) with H1R with the M2R agonist carbachol resulted in a PTX-sensitive activation of NF-{kappa}B. Similar to our findings upon H1R/5-HT1BR co-expression, the inverse H1R agonist mepyramine potently inhibited carbachol-induced M2R signaling in cells co-expressing H1Rs (Fig. 2). As observed for co-expression of 5-HT1BR, we saw an increased constitutive activation of NF-{kappa}B upon co-expression of M2R with H1R. Because similar observations were made with the Gi/o-coupled adenosine A1 (Fig. 2) and the adrenergic {alpha}2 receptors, constitutively active Gq/11-coupled H1R seems to act as a regulatory switch for important cellular functions of Gi/o-coupled GPCRs, including the activation of PLC- and NF-{kappa}B-mediated gene transcription.

To assess whether the observed phenomenon is limited to H1R, we tested an unrelated GPCR, the virally encoded chemokine receptor US28, which is highly constitutively active (23), for its capacity to transduce signaling events mediated through activation of Gi/o-coupled receptors. As observed in the experiments with cells co-expressing the 5-HT1B, M2, or {alpha}2 receptors together with H1R, activation of the human Gi/o-coupled chemokine receptor CCR1 with the CCR1 agonist CCL5 induces a robust PTX-sensitive NF-{kappa}B activation in cells co-expressing the constitutively active Gq/11-coupled receptor US28 (Fig. 4). In contrast, CCL5 does not affect NF-{kappa}B-mediated gene transcription in cells that express CCR1 but not receptor US28. Moreover, CCL5 has no effect on cells that express receptor US28, for which CCL5 is a neutral antagonist (3). However, the binding of CCL5 to US28 is not involved in the observed phenomenon, as CCL5 also induces a robust PTX-sensitive NF-{kappa}B activation in cells co-expressing CCR1 together with the constitutively active {Delta}22-US28 receptor, which is an N-terminal deletion mutant receptor US28 that does not bind CCL5. Co-expression of CCR1 with a mutant receptor US28 (US28-R129A) (22) that is devoid of constitutive receptor activity confirmed that the constitutive activity of receptor US28 is crucial for CCL5-mediated activation of NF-{kappa}B by co-expressed CCR1 receptors (Fig. 4). These data show that in COS-7 cells the active state of the Gq/11-coupled receptors, such as H1R and receptor US28, is crucial for signaling by various Gi/o-coupled receptors. Apparently, constitutively active Gq/11-coupled receptors act as regulators of Gi/o-coupled receptor-mediated signal transduction cascades.

GPCRs activate NF-{kappa}B through a wide variety of signaling mechanisms (Fig. 7). The signal transduction pathways of Gq/11- and Gi/o-coupled receptors provide ample opportunities for cross-talk, ultimately converging at the level of NF-{kappa}B activation. Our previous data implement both G{alpha}q/11 and the released G{beta}{gamma} subunits in the activation of NF-{kappa}B by H1R and receptor US28 (23, 24). We have shown by co-expression or scavenging of G{beta}{gamma} subunits that G{beta}{gamma} subunits are essential but not exclusive signaling moieties for NF-{kappa}B activation. Recent evidence indicates that active G{alpha}q/11 proteins activate RhoA (40), which can lead to activation of NF-{kappa}B via PLD (47, 48). Previous studies have also reported RhoA as a signaling partner for H1R (49, 50). Rho-mediated signaling has been implicated in, for example, H1R-mediated airway hyper-responsiveness (38) and activation of PLD (43). Co-expression of C. botulinum c3 exoenzyme, which is known to inactivate the small G protein Rho (42), completely abolishes H1R-mediated NF-{kappa}B activation (Fig. 5), implying that Rho is required in the H1R-mediated activation of NF-{kappa}B.

Although the activation of Gi/o-coupled receptors may result in a limited degree of NF-{kappa}B activation, the expression of activated G{alpha}i proteins in COS-7 cells does not result in NF-{kappa}B (23, 24) or Rho activation (51). It has been suggested that Gi/o proteins are not sufficient or necessary for GPCR-mediated activation of Rho (52). To obtain evidence that the G{beta}{gamma} subunits of Gi/o proteins have a role in Gi/o-coupled receptor-mediated NF-{kappa}B activation, we stimulated COS-7 cells with mastoparan-7. Activation of heterotrimeric G{alpha}i/o proteins using M7 results in the release of both the activated G{alpha}i/o and G{beta}{gamma} subunits (46). M7 induced PTX-sensitive NF-{kappa}B activation in COS-7 cells expressing 5-HT1BR to a similar extent as the 5-HT1BR agonist CGS (Fig. 6). Moreover, M7 stimulated NF-{kappa}B activation in cells expressing the H1R to a similar extent as achieved by co-expression of G{beta}{gamma} subunits (Figs. 5 and 6); this response was completely sensitive to the inverse H1R agonist mepyramine. Activation of Gi/o-coupled receptors may therefore provide additional free G{beta}{gamma} subunits that synergize with constitutive H1R-mediated NF-{kappa}B activation.

While this manuscript was in preparation, constitutively active Gi/o-coupled cannabinoid CB1 receptors (CB1R) were shown to sensitize MAPK activation by the Gq/11-coupled orexin 1 receptor (OX1R), upon co-expression in Chinese hamster ovary cells (53). In this study orexin-mediated MAPK activation is shown to be sensitive to an inverse CB1R agonist (53). The authors explain the observed synergism by heterodimerization of the two GPCRs (53). In view of our findings with M7 and the apparent lack of specificity for GPCR combinations, we do not consider heterodimerization a major determinant for our observations. However, our study does underscore the importance of constitutive GPCR activity in the modulation of cell signaling.

Our findings are consistent with the reported necessity for preactivation of various Gq/11-coupled receptors for Gi/o-coupled receptor-mediated signaling both in vitro and ex vivo (54). Pretreatment of blood vessels with histamine, for example, is known to yield an enhanced contractile response to 5-HT, which is mediated by previously "silent" 5-HT receptors (55). The conditional synergy might have important (patho)physiological significance, because constitutive activity of Gq/11-coupled receptors might be prominent in (patho)physiology (33). Multiple mechanisms have previously been implicated in the NF-{kappa}B activation that is observed upon cytomegalovirus infection (56). HCMV infection of smooth muscle cells may result not only in the expression of constitutively active US28 receptors (18) but also in Gq/11-mediated (31) as well as PTX-sensitive NF-{kappa}B activation (57). Our present data suggest that expression of receptor US28 upon cytomegalovirus infection may be implicated in both Gq/11-mediated and PTX-sensitive NF-{kappa}B activation through the unmasking of signaling responses of Gi/o-coupled receptors. Therefore, these results provide a new mechanism by which the expression of constitutively active GPCRs, which may be constitutively active under physiological conditions (17) because of receptor mutation (15, 16) or viral infection (18, 19), may regulate signaling events through sensitization of cellular communication that may ultimately result in the initiation and/or progression of disease. These findings may also attribute potentially new roles to naturally occurring inverse agonists (see Ref. 58). Future studies will be required to address these issues in more detail.

In conclusion, our findings reveal a heretofore unrecognized role for constitutively active Gq/11-coupled receptors in the signaling events initiated by Gi/o-coupled receptors. Constitutively active Gq/11-coupled receptors constitute a regulatory switch for signal transduction by Gi/o-coupled receptors to unmask signaling events. The observed conditional synergy may be a key to the identification of the constitutive activity of native Gq/11-coupled GPCRs in vivo and could be useful as a sensitive screening strategy in drug discovery (e.g. for "de-orphanizing" receptors). Our findings suggest that endogenous levels of constitutive GPCR tone can be considerably higher than anticipated on cellular receptor number and expression levels of G proteins or effector molecules. Also, our data indicate that selective inverse agonists may affect signaling events that are induced upon activation of unrelated GPCRs. Although the implications of the present observations remain to be fully ascertained, these data clearly demonstrate the importance of cellular environment for GPCR function.


   FOOTNOTES
 
* This research was supported in part by UCB Pharma (Belgium) and the European Union BIOMED 2 program, "Inverse Agonism, Implications for Drug Research." The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Supported by Altana Nederland. Back

§ Supported by the Royal Dutch Academy of Arts and Sciences. Back

To whom correspondence should be addressed. Tel.: 31-20-444-7579; Fax: 31-20-444-7610; E-mail: leurs{at}few.vu.nl.

1 The abbreviations used are: GPCR, guanine nucleotide-binding regulatory (G) protein-coupled receptor; 5-HT1BR, 5-hydroxytryptamine receptor 1B receptor; RANTES (CCL5), regulated on activation, normal T cell expressed and secreted; CGS, CGS-12066A; COS-7, African green monkey cells; CRE, cyclic-AMP responsive element; G protein, guanine nucleotide-binding regulatory protein; H1R, histamine 1 receptor; M2R, muscarinic M2 receptor; MAPK, mitogen-activated protein kinase; M7, mastoparan-7; PTX, pertussis toxin; HA, hemagglutinin; ELISA, enzyme-linked immunosorbent assay; HCMV, human cytomegalovirus; PL, phospholipase. Back

2 R. A. Bakker, P. Casarosa, H. Timmerman, M. J. Smit, and R. Leurs, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Edwin P. S. van de Ketterij for technical assistance.



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 EXPERIMENTAL PROCEDURES
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 DISCUSSION
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