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Originally published In Press as doi:10.1074/jbc.M204753200 on May 29, 2002

J. Biol. Chem., Vol. 277, Issue 32, 28631-28640, August 9, 2002
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Selective Inhibition of Heterotrimeric Gs Signaling

TARGETING THE RECEPTOR-G PROTEIN INTERFACE USING A PEPTIDE MINIGENE ENCODING THE Galpha s CARBOXYL TERMINUS*

David S. FeldmanDagger §, A. Musa Zamah, Kristen L. Pierce, William E. Miller, Francine KellyDagger , Antonio RapacciuoloDagger , Howard A. RockmanDagger , Walter J. Koch||, and Louis M. LuttrellDagger **DaggerDagger

From the Departments of Dagger  Medicine,  Biochemistry, and || Surgery, Duke University Medical Center, Durham, North Carolina 27710 and the ** Geriatrics Research, Education, and Clinical Center, Durham Veterans Affairs Medical Center, Durham, North Carolina 27705

Received for publication, May 14, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The blockade of heptahelical receptor coupling to heterotrimeric G proteins by the expression of peptides derived from G protein Galpha subunits represents a novel means of simultaneously inhibiting signals arising from multiple receptors that share a common G protein pool. Here we examined the mechanism of action and functional consequences of expression of an 83-amino acid polypeptide derived from the carboxyl terminus of Galpha s (GsCT). In membranes prepared from GsCT-expressing cells, the peptide blocked high affinity agonist binding to beta 2 adrenergic receptors (AR) and inhibited beta 2AR-induced [35S]GTPgamma S loading of Galpha s. GsCT expression inhibited beta 2AR- and dopamine D1A receptor-mediated cAMP production, without affecting the cellular response to cholera toxin or forskolin, indicating that the peptide inhibited receptor-Gs coupling without impairing G protein or adenylyl cyclase function. [35S]GTPgamma S loading of Galpha q/11 by alpha 1BARs and Galpha i by alpha 2AARs and Gq/11- or Gi-mediated phosphatidylinositol hydrolysis was unaffected, indicating that the inhibitory effects of GsCT were selective for Gs. We next employed the GsCT construct to examine the complex role of Gs in regulation of the ERK mitogen-activated protein kinase cascade, where activation of the cAMP-dependent protein kinase (PKA) pathway reportedly produces both stimulatory and inhibitory effects on heptahelical receptor-mediated ERK activation. For the beta 2AR in HEK-293 cells, where PKA activity is required for ERK activation, expression of GsCT caused a net inhibition of ERK activation. In contrast, alpha 2AAR-mediated ERK activation in COS-7 cells was enhanced by GsCT expression, consistent with the relief of a downstream inhibitory effect of PKA. ERK activation by the Gq/11-coupled alpha 1BAR was unaffected by GsCT. These findings suggest that peptide G protein inhibitors can provide insights into the complex interplay between G protein pools in cellular regulation.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Heptahelical, or G protein-coupled, receptors represent the single most diverse class of cell surface receptors, both evolutionarily and within the human genome. The basic unit of G protein-coupled receptor signaling is composed of three parts as follows: a heptahelical receptor, a heterotrimeric G protein,1 and an effector, such as a G protein-regulated enzyme or ion channel. The binding of an extracellular agonist ligand to the receptor changes its conformation so as to permit productive coupling with the G protein, thereby catalyzing the exchange of GTP for GDP on the Galpha subunit, and dissociation of Galpha -GTP from Gbeta gamma subunits. Regulation of effectors is achieved through their interaction with free GTP-bound Galpha or Gbeta gamma subunits. Based upon data from crystallographic, biochemical, and mutagenesis studies, physical coupling of receptor and G protein is thought to involve primarily the second and third intracellular domains of the receptor, which make physical contact with the carboxyl terminus of the Galpha subunit (1-6). In particular, the last ~50 amino acids of the Galpha subunit are important for discriminating between different receptor subtypes and between different functional states of the receptor (3, 4, 6-9).

Pharmacologic agents that act as agonists or antagonists of heptahelical receptors represent the most common type of drug in clinical use today. Irrespective of chemical composition, these agents share a common mechanism of action in that they act extracellularly either to mimic, or to preclude, agonist binding at its receptor. By interacting with the molecular determinants of ligand binding in the extracellular or transmembrane domains of the receptor, often remarkable receptor subtype-specific agonist or antagonist effects can be obtained.

An alternative approach to antagonism of heptahelical receptor signaling is to target the receptor-G protein interface with agents that block coupling between the receptor and G protein intracellularly. Such an approach differs fundamentally from classical heptahelical receptor pharmacology in that the blockade of receptor-G protein coupling might be expected to produce G protein-specific, rather than receptor-specific, antagonism. Several successful applications of this strategy, using polypeptides derived from the putative contact surfaces on the receptor, or the G protein Galpha subunit, have been reported. For example, cellular expression of peptides derived from the third intracellular domains of the Gq/11-coupled alpha 1B adrenergic receptor (AR) and M1 muscarinic acetylcholine receptor, the Gi-coupled alpha 2AAR and M2 acetylcholine receptor, and the Gs-coupled D1A dopamine receptor have been shown to inhibit Gq/11-, Gi-, and Gs-coupled receptor signaling, respectively (10, 11).

Analogous strategies have been applied using modified Galpha subunits or Galpha subunit-derived peptides. Cellular expression of a mutant Galpha s containing three point mutations that impair its function strongly inhibits Gs-dependent stimulation of adenylyl cyclase in cultured cells (12). Modified xanthine nucleotide-binding mutants of Galpha o (13-14) and Galpha 16 (15) inhibit signaling by Gi-coupled receptors when expressed in COS-7 cells, whereas xanthine nucleotide-binding mutants of Galpha 11 and Galpha 16 (15) inhibit Gq-coupled receptor signaling. Smaller peptides, derived from the carboxyl terminus of Galpha subunits, have been shown to produce similar inhibitory effects in membrane preparations and in intact cells (16-20). Cellular expression of a minigene encoding the last 55 amino acids of Galpha q inhibits Gq/11-coupled receptor signaling (18). Minigene plasmids encoding oligopeptides representing the carboxyl termini of Galpha i, Galpha q, Galpha 12, and Galpha 13 have recently been employed to determine the contribution of different G protein pools to signaling by M2 muscarinic and thrombin receptors (19, 20).

To determine whether an expressible peptide could be identified that interrupts signaling at the receptor-Gs interface, we have prepared a series of minigene constructs encoding varying length polypeptides derived from the carboxyl terminus of Galpha s. In this paper, we characterize the mechanism of action and consequences of expression of an 83-amino acid Galpha s carboxyl-terminal polypeptide (GsCT). We find that the GsCT peptide selectively inhibits receptor-Gs coupling in isolated plasma membranes and second messenger production in intact cells, without affecting Gq/11 or Gi signaling. When employed to examine the role of Gs in regulation of the ERK MAP kinase cascade, we find that GsCT expression reveals both stimulatory and inhibitory effects of Gs in response to activation of Gs-, Gi-, and Gq/11-coupled adrenergic receptors. These data indicate that expression of peptides derived from the carboxyl terminus of Galpha s can induce G protein-specific blockade of Gs-coupled receptor signaling. By selectively blocking a single G protein pool, this approach can potentially provide insights into the contribution of different G protein pools to complex signaling processes.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- HEK-293 and COS-7 cells were from the American Type Culture Collection. Tissue culture media, fetal bovine serum (FBS), geneticin (G418), and penicillin/streptomycin were from Invitrogen. FuGENE 6 was from Roche Molecular Biochemicals. 3',5'-[3H]cAMP was from Amersham Biosciences. myo-[3H]Inositol and [35S]GTPgamma S were from PerkinElmer Life Sciences. Monoclonal anti-Galpha s, anti-Galpha i1/2, and anti-Galpha q/11 IgG were from Calbiochem and PerkinElmer Life Sciences. Polyclonal anti-FLAG and anti-HA were from Santa Cruz Biotechnology. Anti-FLAG M2 affinity-agarose was from Sigma. Polyclonal anti-ERK1/2 and anti-phospho-ERK1/2 IgG were from Cell Signaling Technology. Horseradish peroxidase-conjugated donkey anti-mouse IgG was from Amersham Biosciences. Cholera toxin, H89, forskolin, isoproterenol, and 6-chloro-PB hydrobromide were from Sigma. Bordetella pertussis toxin was from List Biological. The cDNAs encoding the hamster alpha 1BAR and the human alpha 2AAR were provided by R. J. Lefkowitz. The cDNA encoding the human D1A dopamine receptor was from M. G. Caron. The cDNA encoding the bovine Galpha s subunit was provided by A. G. Gilman.

Construction of Minigenes Encoding the Galpha s Carboxyl Terminus-- The construction of the Galpha s peptide minigenes is depicted schematically in Fig. 1A. The PCR was employed to amplify cDNA encoding amino acids 286-395, 313-395, or 337-395 of bovine Galpha s and the translation stop codon. Restriction sites at the 5' and 3' ends of the Galpha s-derived sequence were incorporated into the oligonucleotide primers used for DNA amplification. For the construct encoding a Gs-derived peptide capable of post-translational prenylation, the DNA sequence TGCGTCCTCTCTT, encoding the peptide sequence CVLS, was incorporated into the 3' end of the cDNA sequence prior to the stop codon. For the hemagglutinin (HA) epitope-tagged constructs, the Galpha s carboxyl-terminal sequences were subcloned as EcoRI to SalI fragments into a modified pcDNA3.1 that contained a Kozak sequence and an amino-terminal HA epitope upstream of the cloning sites. For the glutathione S-transferase (GST)-tagged constructs, Galpha s carboxyl-terminal sequences were subcloned into the pEBG vector, which encodes an amino-terminal GST epitope. The minigene plasmid encoding the third intracellular domain of the human D1A dopamine receptor (D1A3i) in pRK5 was prepared as described previously (10).

Cell Culture and Transfection-- COS-7 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% FBS and 50 µg/ml penicillin/streptomycin. HEK-293 cells were maintained in minimum essential medium supplemented with 10% fetal bovine serum and 50 µg/ml penicillin/streptomycin. Transient transfection of 40-50% confluent cultures of COS-7 or HEK-293 cells in 100-mm dishes was performed using a ratio of 3 µl of FuGENE 6 per µg of plasmid DNA, according to the manufacturer's directions. Empty pcDNA3.1 vector DNA was added to each transfection as needed to keep the mass of DNA constant. A stable HEK-293 cell line expressing the GsCT minigene, Galpha s-(313-395), was prepared by calcium phosphate transfection using 5 µg/ml G418 for selection, as described previously (21). Minigene expression following transient or stable transfection was detected by protein immunoblotting using antisera directed against the Galpha s carboxyl terminus. All assays on transiently transfected cells were performed after 48-72 h. Prior to assay, transfected cells were split into multiwell plates, as appropriate, and incubated overnight in growth medium supplemented with 0.5% FBS and 10 mM HEPES, pH 7.4.

Competition and Saturation Binding Assays-- Plasma membrane preparations for use in binding assays, [35S]GTPgamma S loading of Galpha subunits, and immunoblotting were prepared by differential centrifugation. Monolayers of appropriately transfected COS-7 or HEK-293 cells were scraped into 4 °C lysis buffer (10 mM Tris-HCl, pH 7.4, 5 mM EDTA) and subjected to Dounce homogenization. Membranes were isolated by sequential centrifugation at 300 × g for 3 min to remove cell nuclei and unbroken cells, and 40,000 × g for 30 min to collect plasma membranes. The supernatant from the second centrifugation represented the cytosolic fraction. For beta 2AR competition binding analyses, membranes were resuspended in binding buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA) at a concentration of 0.5 mg of protein/ml. Membrane aliquots (20 µg of protein) were incubated with [125I]-cyanopindolol for 30 min at 37 °C in the presence of varying concentrations of isoproterenol (0-10-5 M) and then filtered over Whatman GF-C filters and washed to separate unbound ligand. Nonspecific binding was determined in the presence of 25 µM alprenolol. To confirm that assays of GsCT effects were performed under conditions of equal receptor expression, the level of alpha 1BAR, alpha 2AAR, beta 2ARonco, and D1A dopamine receptor expression in HEK-293 and COS-7 cells was determined by saturation binding analysis, as described previously (11).

[35S]GTPgamma S Loading of Galpha Subunits-- Assays of [35S]GTPgamma S loading of endogenous Galpha s, Galpha q/11, and Galpha i1/2 subunits were performed on cell membranes prepared from HEK-293 cells. Membrane pellets were resuspended in TME buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.4) containing 2 mM EDTA and 4.8 mM MgCl2 for Gs loading assays or 100 µM EDTA and 120 µM MgCl2 for Gq/11 and Gi loading assays. [35S]GTPgamma S loading was performed by incubating 25 µg of membrane protein in TME buffer, 1 µM GDP, and 30 nM [35S]GTPgamma S, plus agonist or vehicle, for 5 min at 30 °C in a total volume of 100 µl. Reactions were terminated by solubilizing the membranes for 30 min at 4 °C in IP/Stop buffer (150 mM NaCl, 0.5% Nonidet P-40, 20 mM MgCl2, 100 µM GDP, 100 µM GTP, 1% aprotinin, 50 mM Tris-HCl, pH 7.5). Specific G protein subunits were isolated by immunoprecipitation for 1 h at 4 °C using monoclonal antisera specific for Galpha s, Galpha q/11, or Galpha i1/2, collected on protein A-Sepharose. Immune complexes on Sepharose were washed three times with IP/Stop buffer, and [35S]GTPgamma S bound to the immunoprecipitated Galpha subunits was determined by liquid scintillation counting. [35S]GTPgamma S binding in the presence of 25 mM MgCl2 was used as a positive control. Immunoprecipitations performed in the absence of primary antibody were used to determine nonspecific background.

cAMP Production-- Appropriately transfected HEK-293 or COS-7 cells were split into 6-well plates and serum-starved overnight. Monolayers were preincubated with 1 µM 3-isobutyl-1-methylxanthine for 15 min at 37 °C, prior to stimulation with agonist for 6-10 min as described in the figure legends. Reactions were terminated by aspirating medium and adding 250 µl/well of cAMP buffer (4 mM EDTA, 50 mM Tris-HCl, pH 7.5) on ice. Monolayers were collected by scraping into Eppendorf tubes, boiled for 10 min, and clarified by microcentrifugation at 14,000 rpm for 15 min. The cAMP content of the supernatants was determined according to the manufacturer's instructions using the Biotrack [3H]cAMP Assay System from Amersham Biosciences (22). Data were normalized to protein content as determined by Bradford assay of the cell lysates and expressed as pmol of cAMP/mg cell protein.

Phosphatidylinositol Hydrolysis-- Appropriately transfected HEK-293 or COS-7 cells were split into 6-well plates and incubated for 18-24 h with myo-[3H]inositol at 4 µCi/ml in low serum growth medium. After labeling, cells were washed once with phosphate-buffered saline (PBS) and preincubated for 1 h in PBS at 37 °C followed by fresh PBS containing 20 mM LiCl for 20 min. Cells were then stimulated for 1 h with agonist. Reactions were terminated by the addition of 1.0 ml of 0.4 M perchloric acid and neutralized with 0.4 ml of 0.72 M KOH and 0.6 M KHCO3. Total inositol phosphates were isolated by anion exchange chromatography on Dowex AG1-X8 columns and quantified by liquid scintillation spectroscopy, as described (11).

Phosphorylation of ERK1/2-- Appropriately transfected COS-7 cells were split to 6-well plates and incubated for 18-24 h in low serum growth medium in the presence or absence of inhibitors, as indicated. Agonist stimulation was carried out for 5 min, after which monolayers were washed once in 4 °C PBS and lysed in 200 µl of Laemmli sample buffer. For the determination of total cellular ERK1/2 and phospho-ERK1/2, aliquots containing ~20 µg of cell protein were resolved by SDS-PAGE. ERK1/2 and phospho-ERK1/2 were detected by protein immunoblotting using polyclonal anti-ERK1/2 and anti-phospho-ERK1/2 antisera, respectively, with horseradish peroxidase-conjugated polyclonal donkey anti-rabbit IgG used as secondary antibody. Immune complexes were visualized by enzyme-linked chemiluminescence and quantified using a Fluor-S MultiImager. In each experiment, equal loading of ERK1/2 protein was confirmed by probing parallel immunoblots using anti-ERK1/2 antisera.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cellular Expression of a Polypeptide Derived from the Carboxyl Terminus of Galpha s Inhibits Gs-coupled Receptor Signaling by Blocking Receptor-G Protein Coupling-- To create a peptide inhibitor of receptor-Gs coupling, we initially prepared a series of minigene constructs encoding 59, 83, and 110 amino acid polypeptides derived from the carboxyl terminus of bovine Galpha s. These polypeptides contain the major region of Galpha s thought to mediate contact with the intracellular domains of GPCRs but lack the sequences that contact adenylyl cyclases. As shown schematically in Fig. 1A, each minigene was composed of a minimal Kozak sequence, followed by the Galpha s-derived cDNA and a 3'-untranslated region. To facilitate detection of the expressed polypeptides, HA or GST epitopes were incorporated into the amino termini of each construct. Transient expression studies in COS-7 cells revealed robust expression of the 83- and 110-amino acid Galpha s-derived polypeptides. We were unable to detect expression of the 59-amino acid construct, suggesting that the polypeptide product was subject to rapid intracellular degradation. Fig. 1B shows an immunoblot of whole cell lysates from COS-7 cells transiently transfected with three versions of the 83-amino acid peptide as follows: GST-Galpha s-(313-395) (lane 2), HA-Galpha s-(313-395) (lane 3), and a modified HA-Galpha s-(313-395)-CVLS bearing the protein prenylation sequence CVLS at the carboxyl terminus (lane 4). As shown in Fig. 1C, transient transfection of COS-7 cells with increasing amounts of the HA- Galpha s-(313-395) plasmid produced a progressive increase in peptide expression that reached levels significantly in excess of the expression of endogenous Galpha s isoforms. As shown, minigene expression had no significant effect on the level of endogenous Galpha s expression.


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  		Fig. 1.   Construction of minigenes for expression of polypeptides derived from the Galpha s carboxyl terminus. A, schematic representation of minigene constructs encoding the carboxyl-terminal 59, 83, and 110 amino acids of bovine Galpha s. B, protein immunoblot (IB) of total cell lysates from COS-7 cells transiently transfected with empty vector, pEBG-GST-Galpha s-(313-395), pcDNA3.1-HA-Galpha s-(313-395), and pcDNA3.1-HA-Galpha s-(313-395)-CVLS (5 µg/100-mm dish), performed using antisera directed against the carboxyl terminus of Galpha s. C, representative immunoblot of COS-7 cell lysates transiently transfected with increasing amounts of the pcDNA3.1-HA-Galpha s-(313-395) plasmid (0-10 µg/100-mm dish). B and C, the position of the endogenous p45 and p52 isoforms of Galpha s, as well as the GsCT minigene products, are as indicated. D, effect of GST-Galpha s-(313-395), HA-Galpha s-(313-395), and HA-Galpha s-(313-395)-CVLS expression on D1A dopamine receptor-mediated cAMP production. COS-7 cells were transiently transfected with the pRK5-D1AR (2 µg/100-mm dish), plus either empty vector, pEBG-GST- Galpha s-(313-395), pcDNA3.1-HA-Galpha s-(313-395) or pcDNA3.1-HA-Galpha s- (313-395)-CVLS (8 µg/100-mm dish), and basal and 6-chloro-PB hydrobromide-stimulated cAMP production was determined as described under "Experimental Procedures." Data were normalized to the basal cAMP level measured cells transfected with D1AR plus empty vector (1.25 pmol/mg protein). Data shown represent the mean ± S.D. for triplicate determinations in one of five separate experiments. UTR, untranslated region.

To determine whether expression of Galpha s-derived peptides affected signaling by a Gs-coupled GPCR, we measured basal and agonist-stimulated cAMP production in COS-7 cells transiently expressing the D1A dopamine receptor and either the GST-Galpha s-(313-395), HA-Galpha s-(313-395), or HA-Galpha s-(313-395) -CVLS minigenes. As shown in Fig. 1D, the GST-Galpha s-(313-395) peptide had no significant effect on D1A receptor-mediated cAMP production, despite robust levels of expression. In contrast, expression of either HA epitope-tagged version of the construct led to a marked reduction in the cAMP response. Interestingly, addition of the prenylation sequence CVLS to the carboxyl terminus of the HA-Galpha s-(313-395) peptide, which might be expected to enhance membrane localization of the peptide, did not significantly increase its effectiveness. Based upon these data, we selected the unmodified HA epitope-tagged version of the 83-amino acid polypeptide Galpha s-(313-395) (GsCT) for further characterization.

The basic unit of heptahelical receptor signaling consists of receptor, heterotrimeric G protein, and effector. As depicted schematically for the beta 2AR-Gs-adenylyl cyclase module in Fig. 2A, it is possible to stimulate cAMP production in cells either by applying agonist, by activating Gs directly using cholera toxin, or by activating adenylyl cyclase directly using forskolin. To determine the effect of the GsCT polypeptide on adenylyl cyclase activation by beta 2ARs, we employed a stable GsCT-expressing HEK-293 cell line. Fig. 2B compares the cAMP response of parental and GsCT-expressing HEK-293 cells to stimulation with the beta 2AR agonist, isoproterenol, cholera toxin, or forskolin. In the presence of GsCT, isoproterenol-stimulated cAMP production stimulation was attenuated by ~68% compared with parental HEK-293 cells. In contrast, cAMP production occurring in response to receptor-independent activation of Gs with cholera toxin, or of adenylyl cyclase with forskolin, was indistinguishable between the two cell lines. These data suggest that the GsCT peptide inhibits receptor-G protein coupling without directly impairing G protein or adenylyl cyclase function.


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  		Fig. 2.   Inhibition of beta 2AR coupling to Gs by stable expression of the GsCT minigene in HEK-293 cells. A, diagram of the heptahelical receptor-G protein-adenylyl cyclase unit, showing the site of action of agonist hormone (H), cholera toxin, and forskolin, all of which stimulate cAMP production. The target of antagonist drugs, which block hormone binding to the receptor, and the putative locus of GsCT action, at the receptor-G protein interface, are shown. B, effect of isoproterenol (Iso), cholera toxin (CTX), and forskolin on cAMP production in parental HEK-293 cells and HEK-293 cells stably expressing the GsCT minigene. Cells were treated with vehicle, isoproterenol (10 µM) for 6 min, cholera toxin (100 ng/ml) for 12 h, or forskolin (5 × 10-4 M) for 6 min, prior to the determination of cAMP content as described. Data shown represent the mean ± S.E. values from four separate experiments. *, less than control, p < 0.01. NS, not stimulated.

To elucidate further the mechanism of the inhibition produced by GsCT expression, we assayed the effect of the peptide on the affinity of beta 2AR for agonist binding. As shown in Fig. 3A, the GsCT peptide, like the endogenous Galpha s protein, was present almost exclusively in the plasma membrane fraction following cell fractionation. Neither agonist exposure nor coexpression of FLAG epitope-tagged beta 2AR increased the amount of GsCT in the membrane fraction, suggesting that the peptide inherently partitions into the membrane. As shown in Fig. 3B, the GsCT peptide specifically immunoprecipitates with the FLAG-beta 2AR from cotransfected cells, suggesting that once associated with the membrane, the peptide is capable of binding to the receptor.


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  		Fig. 3.   Effect of GsCT on high affinity agonist binding to the beta 2AR. A, partitioning of GsCT between cytosol and membrane fractions. COS-7 cells were transfected with the HA-GsCT minigene (8 µg/100-mm dish) in the presence or absence of FLAG-beta 2AR (2 µg/dish) and treated for 5 min with isoproterenol (10 µM) or vehicle prior to the preparation plasma membrane and cytosolic fractions. Immunoblots of 2% of the protein from each fraction were performed using antisera directed against the carboxyl terminus of Galpha s. The position of the endogenous p45 and p52 isoforms of Galpha s, as well as the GsCT minigene product, are as indicated. B, coprecipitation of HA-GsCT with FLAG-beta 2AR. COS-7 cells were transfected with the HA-GsCT minigene (8 µg/100-mm dish) and FLAG-beta 2AR (2 µg/dish), alone or in combination, as indicated. FLAG immunoprecipitates (IP) were subjected to immunoblotting (IB) using polyclonal anti-FLAG (left panel) and polyclonal anti-HA antisera (right panel) to detect the FLAG-beta 2AR and HA-GsCT peptide, respectively. The positions of FLAG-beta 2AR and coprecipitated HA-GsCT peptide are as indicated. C, agonist displacement curves for endogenous beta 2AR in COS-7 cell membranes in the presence or absence of GTPgamma S or HA-GsCT. Isoproterenol (Iso) displacement of [125I]cyanopindolol (125I-CYP) was performed using plasma membranes prepared from untransfected COS-7 cells or cells transfected with GsCT (20 µg/150-mm dish). Agonist affinities and abundance were calculated by nonlinear regression analysis with one- and two-site models. Calculated values were as follows: control membranes, high affinity site 5.4 × 10-10 M (22%) and low affinity site 1.25 × 10-7 M (78%); control membranes plus GTPgamma S, low affinity site 1.08 × 10-7 M (100%); GsCT membranes, high affinity site 3.1 × 10-10 M (7%) and low affinity site 2.1 × 10-7 M (93%). Data shown represent the mean ± S.E. for duplicate determinations in two to three separate experiments.

In the absence of exogenous guanine nucleotide, many GPCRs exhibit characteristic high and low affinity states for agonist binding. The high affinity state is thought to represent pre-coupling of the GPCR to GDP-bound heterotrimeric G protein, whereas the low affinity state represents free GPCR. In the presence of a nonhydrolyzable GTP analogue, such as GTPgamma S, which causes irreversible dissociation of G protein subunits, only the low affinity state of the receptor is present. Fig. 3C compares competition binding curves generated for the displacement of the beta 2AR antagonist [125I]-cyanopindolol by isoproterenol in COS-7 cells membranes in the presence of either GTPgamma S or GsCT. In control membranes, the competition binding curve fits a two-site model with the high affinity site composing 22% of the total specific [125I]-cyanopindolol-binding sites. In the presence of GTPgamma S, the curve was shifted to the right, with only a single low affinity site present. In membranes from cells expressing the GsCT, the curve was similarly right-shifted, such that the high affinity site composed only 7% of the total. No significant differences were detected in the EC50 values for the high and low affinity sites between control and GsCT-containing membranes. These data strongly support the hypothesis that GsCT binding to the beta 2AR precludes receptor-G protein coupling.

Because GsCT expression appeared to target the receptor-G protein interface, we sought to determine whether the effect of GsCT expression was specific for Gs by assaying receptor-stimulated [35S]GTPgamma S loading of endogenous G proteins in membranes isolated from parental and GsCT-expressing HEK-293 cells. For these assays, endogenous beta 2ARs or transiently expressed alpha 1BARs and alpha 2AARs were employed to stimulate the endogenous pools of Gs, Gq/11, and Gi, respectively. As shown in Fig. 4A, membranes from GsCT-expressing cells showed a 72% decrement in the isoproterenol-induced increase in [35S]GTPgamma S loading of Galpha s compared with membranes from parental cells, with no significant effect on basal Galpha s loading. As shown in Fig. 4, B and C, no significant differences in basal or agonist-stimulated G protein loading were observed when alpha 1BAR-mediated Galpha q/11 and alpha 2AAR-mediated Galpha i loading in GsCT expressing and parental cells were compared. Thus, GsCT expression led to Gs-specific inhibition of receptor-G protein coupling.


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  		Fig. 4.   Effect of GsCT expression on GTPgamma S loading of endogenous Galpha s, Galpha q/11, and Galpha i pools. A, comparison of basal and isoproterenol-stimulated binding of [35S]GTPgamma S to endogenous Galpha s in membranes isolated from parental (HEK-293) and HA-GsCT-expressing HEK-293 (GsCT-293) cells. Membrane fractions were incubated with [35S]GTPgamma S in the presence and absence of isoproterenol (10 µM) for 10 min, prior to detergent solubilization and immunoprecipitation of Galpha s for the determination of [35S]GTPgamma S binding as described. B, comparison of basal and phenylephrine (1 µM) stimulated binding of [35S]GTPgamma S to endogenous Galpha q/11 in membranes isolated from parental and GsCT-expressing HEK-293 cells transiently expressing hamster alpha 1BAR. C, comparison of basal and UK14304 (10 µM)-stimulated binding of [35S]GTPgamma S to endogenous Galpha i1/2 in membranes isolated from parental and GsCT-expressing HEK-293 cells transiently expressing human alpha 2AAR. In each panel, data shown represent the mean ± S.E. for four separate experiments. * less than control, p < 0.01.

GsCT Expression Results in G Protein-specific Inhibition of Heptahelical Receptor Signaling-- If expression of the GsCT polypeptide selectively uncouples heptahelical receptors from Gs, one would expect it to inhibit the generation of Gs-dependent, but not Gq/11- or Gi-dependent second messengers after stimulation of receptors coupled to these G protein pools. To test this hypothesis, we employed a transfected COS-7 cell system in which various heptahelical receptors were transiently expressed in the presence or absence of GsCT. Fig. 5A depicts the effects of increasing GsCT expression on cAMP production in response to stimulation of coexpressed Gs-coupled D1A dopamine receptors in COS-7 cells. At the highest levels of expression, GsCT inhibited D1A receptor-mediated cAMP production to an extent comparable with that obtained by expression of a 59-amino acid polypeptide derived from the third intracellular domain of the D1A receptor (D1AR3i). We have shown previously that the D1AR3i peptide, which represents the receptor side of the putative receptor-G protein interface, inhibits D1A receptor-mediated cAMP production when expressed in HEK-293 and COS-7 cells (10, 11).


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  		Fig. 5.   Effect of GsCT expression on agonist-stimulated cAMP production by the Gs-coupled D1A dopamine and beta 2 adrenergic receptors. A, effect of increasing GsCT expression on agonist-stimulated cAMP production in COS-7 cells transiently expressing D1A dopamine receptors (D1AR). Cells in 100-mm dishes were cotransfected the pRK5-D1AR plasmid, along with the indicated amounts of either pcDNA3.1-HA-Galpha s-(313-395) or pRK5-D1AR3i. The production of cAMP in response to 6 min of exposure to the dopamine receptor agonist 6-chloro-PB hydrobromide (10 µM) was determined as described. B, dose-response curves for 6-chloro-PB hydrobromide-stimulated cAMP production in COS-7 cells in the presence and absence of coexpressed HA-GsCT or D1A3i peptides. Cells were cotransfected the pRK5-D1A receptor plasmid along with either the pcDNA3.1-HA-Galpha s-(313-395) or pRK5-D1A3i plasmid (10 µg/100-mm dish). The production of cAMP in response to 6 min of exposure to the indicated concentration of 6-chloro-PB hydrobromide was determined as described. C, dose-response curves for isoproterenol-stimulated cAMP production in parental HEK-293 cells and HEK-293 cells stably expressing HA-GsCT (GsCT-293). The production of cAMP in response to 6 min of exposure to the indicated concentration of isoproterenol was determined as described. A-C, data shown represent the mean ± S.E. for four separate experiments. *, less than control, p < 0.05. D, effect of HA-GsCT expression on cAMP production in COS-7 cells transiently expressing a constitutively active mutant of the beta 2AR (beta 2ARonco). COS-7 cells were transfected with plasmid encoding the beta 2ARonco (2 µg/100-mm dish) plus either empty vector (control) or pcDNA3.1-HA-Galpha s-(313-395). Determinations of cAMP production were made under basal conditions and following treatment for 10 min with isoproterenol (1 µM) or forskolin (1 µM) or for 30 min with the inverse agonist ICI118551 (10 µM). Data shown represent the mean ± S.D. values of triplicate determinations in one of three identical experiments.

Fig. 5B depicts the dose-response relationship for D1A receptor-stimulated cAMP production in COS-7 cells expressing a comparable level of receptor (0.9-1.15 pmol/mg membrane protein) in the presence or absence of coexpressed GsCT or D1A3i. In the presence of either polypeptide, 6-chloro-PB hydrobromide-stimulated cAMP production was inhibited by at least 70% at each agonist concentration tested. The observed inhibition was not surmountable by even supersaturating concentrations of agonist. As shown in Fig. 5C, similar, apparently noncompetitive inhibition of beta 2AR-mediated cAMP production was observed in the stable GsCT-expressing HEK-293 cell line. In the GsCT-expressing cells, isoproterenol-stimulated cAMP production was attenuated by at least 66% at each agonist concentration.

For the overexpressed D1AR in COS-7 cells, and to a lesser extent the endogenous beta 2AR in HEK-293 cells, GsCT expression reduced basal as well as agonist-stimulated cAMP levels. To determine whether this effect was due to inhibition of basal receptor-Gs coupling or to an additional receptor-independent effect of GsCT, we compared the effect of GsCT expression with that of the beta 2AR inverse agonist ICI118551 (23, 24). In these assays we employed a constitutively activated point mutant of the beta 2AR, beta 2ARonco (23), because the higher basal levels of cAMP generated by the mutated receptor facilitated measurement of the effects of GsCT and ICI118551 on basal cAMP. As shown in Fig. 5D, basal cAMP production in COS-7 cells expressing the beta 2ARonco was increased 4-fold in the presence of agonist and inhibited by 66% in the presence of maximally effective concentrations of ICI118551. As with the wild type beta 2AR, both basal and agonist-stimulated cAMP production were attenuated in cells expressing GsCT. Treatment with ICI118551 had little additional effect on cAMP levels in the GsCT-expressing cells. The cAMP response to a submaximal dose of forskolin was equivalent between the two cell populations. The lack of additivity of the effects of GsCT and ICI118551 on beta 2ARonco signaling suggests that the predominant effect of the GsCT is mediated through its effects on receptor-G protein coupling.

The apparently noncompetitive pattern of inhibition we observed is consistent with expression of an inhibitor that competes with the endogenous G protein pool for access to ligand-bound receptor. Increasing agonist concentration would have no effect on the ratio of GsCT to functional Gs heterotrimer and would thus not be expected to surmount the inhibitory effect of the polypeptide. Consistent with this, we found that the maximal extent of GsCT-induced inhibition of D1A receptor-mediated cAMP production did vary with the level of receptor expression. At a D1A receptor density of <0.75 pmol/mg, the cAMP response to saturating concentrations of agonist was almost completely blocked by high levels of GsCT expression. Increasing D1A receptor expression to levels >1.5 pmol/mg partially overcame the inhibition (data not shown). In the presence of a saturating concentration of agonist, increasing receptor expression might increase the likelihood of an activated receptor encountering a functional Gs heterotrimer, leading to less inhibition of second messenger generation.

The alpha 1BAR stimulates phosphatidylinositol (PI) hydrolysis primarily by Gq/11-dependent activation of the phospholipase C (PL-C) beta 1 isoform (25). As shown in Fig. 6A, alpha 1BAR-mediated PI hydrolysis is unaffected by coexpression of increasing amounts of the GsCT polypeptide. The alpha 2AAR weakly stimulates PI hydrolysis by Gi-dependent activation of the PL-C beta 2 and beta 3 isoforms (26). As shown in Fig. 6B, alpha 2AAR-mediated PI hydrolysis was likewise unaffected by GsCT expression. Collectively, these data suggest that expression of the GsCT polypeptide produces G protein-specific inhibition of heptahelical receptor-G protein coupling. Its effects are generalizable to multiple Gs-coupled receptors, in that beta 2AR and D1A dopamine receptor cAMP production are similarly affected, but are specific for signals mediated by Gs, in that Gq/11- and Gi-dependent PI hydrolysis is unimpaired.


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  		Fig. 6.   Effect of GsCT expression on agonist-stimulated PI hydrolysis by the Gq/11-coupled alpha 1B and Gi-coupled alpha 2A adrenergic receptors. A, effect of increasing GsCT expression on agonist-stimulated PI hydrolysis in COS-7 cells transiently expressing alpha 1BAR. Cells in 100-mm dishes were cotransfected the pRK5-alpha 1BAR plasmid, along with the indicated amounts of pcDNA3.1-HA-Galpha s-(313-395). PI hydrolysis in response to 1 h of exposure to phenylephrine (1 µM) was determined as described. B, effect of increasing GsCT expression on agonist-stimulated PI hydrolysis in COS-7 cells transiently expressing alpha 2AAR. Cells in 100-mm dishes were cotransfected the pRK5-alpha 2AAR plasmid, along with the indicated amounts of pcDNA3.1-HA-Galpha s- (313-395). PI hydrolysis in response to 1 h of exposure to the alpha 2AAR agonist, UK14304 (10 µM), was determined as described. Data are presented in arbitrary units, such that the basal amount of [3H]inositol phosphate detected in cells not expressing GsCT was assigned a value of 1. In each panel, the data shown represent the mean ± S.E. of triplicate determinations in four separate experiments.

Use of GsCT to Examine the Contribution of Gs to ERK Activation by beta 2 Adrenergic, alpha 1B Adrenergic, and alpha 2A Adrenergic Receptors-- The role of Gs proteins in GPCR-mediated ERK activation is complex. As depicted schematically in Fig. 7, previous studies have indicated that activation of protein kinase A (PKA) by Gs-coupled receptors can produce both stimulation and inhibition of ERK activity. In HEK-293 cells (27), cardiac myocytes (28), and pancreatic acinar cells (29), beta 2AR-mediated ERK activation involves both PKA and activation of pertussis toxin-sensitive G proteins. It has been proposed that phosphorylation of the beta 2AR by PKA switches receptor coupling from Gs to Gi, allowing the receptor to mediate pertussis toxin-sensitive ERK1/2 activation through a Gbeta gamma subunit-dependent pathway (27). On the other hand, PKA-mediated phosphorylation of Raf-1 has been shown to attenuate growth factor-stimulated ERK activation in several cell types (30-33). Thus, the net effect of Gs stimulation on GPCR-mediated ERK activation likely reflects a balance between two opposing mechanisms of regulation.


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  		Fig. 7.   Putative stimulatory and inhibitory effects of PKA phosphorylation on activation of the ERK1/2 MAP kinase cascade by beta 2ARs. Activation of the Gs-adenylyl cyclase (AC)-PKA pathway results in PKA-mediated phosphorylation of the beta 2AR. PKA phosphorylation increases receptor coupling to pertussis toxin-sensitive Gi proteins, resulting in Gbeta gamma subunit and Ras-dependent activation of the ERK1/2 pathway. At the same time, PKA activation exerts an inhibitory effect on ERK1/2 activation by phosphorylating the MAP kinase kinase kinase, Raf1.

Having determined that expression of the GsCT polypeptide leads to selective inhibition of Gs-mediated signaling, we employed the construct to examine the contribution of Galpha s to ERK activation by beta 2, alpha 2A, and alpha 1B adrenergic receptors. For the beta 2AR, which is endogenously expressed, we compared isoproterenol-stimulated ERK1/2 phosphorylation in parental HEK-293 cells with that in stable GsCT-expressing HEK-293 cells. As shown in Fig. 8A, beta 2AR-mediated ERK phosphorylation in HEK-293 cells was inhibited by pretreatment with either the PKA inhibitor, H89, or with pertussis toxin, consistent with the previously described roles of PKA and Gi in the pathway (27). When isoproterenol-stimulated ERK1/2 phosphorylation was compared in parental and GsCT-expressing HEK-293 cells, a significant reduction was observed in the cells expressing the GsCT peptide. These data, shown in Fig. 8B, are consistent with the proposed requirement for Gs activation in beta 2AR signaling to ERK.


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  		Fig. 8.   Effect of GsCT expression on beta 2AR-mediated ERK1/2 phosphorylation. A, effect of the PKA inhibitor H89 and pertussis toxin on beta 2AR-stimulated ERK1/2 activation in HEK-293 cells. Cells in 6-well plates were preincubated with H89 (10 µM) for 30 min or with pertussis toxin (100 ng/nl, PTX) for 16 h, prior to 5 min of stimulation with isoproterenol (Iso) (10 µM). Phospho-ERK1/2 levels in whole cell lysates were determined by immunoblotting (IB) as described. The immunoblot shown is representative of at least three separate experiments. B, effect of GsCT on beta 2AR-stimulated ERK1/2 activation in parental HEK-293 and GsCT-expressing HEK-293 cells (GsCT-293). Serum-starved cells in 6-well plates were stimulated for 5 min of stimulation with isoproterenol (10 µM) prior to determination of phospho-ERK1/2 levels as described. The upper panel depicts a representative immunoblot. Data shown in the lower panel represent the mean ± S.E. for three separate experiments. *, less than control, p < 0.05.

The alpha 2AAR couples to Gi/o family G proteins and in COS-7 cells mediates ERK activation through a pertussis toxin-sensitive pathway that is blocked by expression of a Gbeta gamma subunit sequestrant polypeptide derived from the carboxyl terminus of G protein-coupled receptor kinase 2 (34). However, the alpha 2AAR also couples to Gs, particularly at high levels of receptor expression (35). In contrast to the beta 2AR, alpha 2AAR coupling to both Gi and Gs is a constitutive property of the receptor, not one that is modulated by PKA phosphorylation. Furthermore, Gs activation apparently antagonizes GPCR-stimulated ERK activation in COS-7 cells, because expression of an activated mutant of Galpha s, or treatment with the cell-permeant cAMP analog, 8-bromo-cAMP, attenuates ERK activation in response to either isoproterenol or epidermal growth factor in this system (33). As shown in Fig. 9A, alpha 2AARs transiently expressed in COS-7 cells, like beta 2ARs in HEK-293 cells, activate ERK1/2 via pertussis toxin-sensitive G proteins. However, in contrast to the beta 2AR system, treatment with H89 enhances, rather than inhibits, alpha 2AAR-mediated ERK activation. This presumably reflects relief of the inhibitory effect of PKA on ERK activation that results from phosphorylation of c-Raf1. As shown in Fig. 9B, transfection of COS-7 cells with increasing amounts of the GsCT plasmid, like H89 treatment, caused a progressive enhancement of alpha 2AAR-mediated ERK phosphorylation.


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  		Fig. 9.   Effect of GsCT expression on agonist-stimulated PI hydrolysis by the Gq/11-coupled alpha 1BAR and Gi-coupled alpha 2AAR. A, effect of the PKA inhibitor H89 and pertussis toxin on alpha 2AAR-stimulated ERK1/2 phosphorylation in COS-7 cells. Cells in 6-well plates were preincubated with H89 (10 µM) for 30 min or with pertussis toxin (100 ng/nl, PTX) for 16 h, prior to 5 min of stimulation with UK14304 (10 µM). Phospho-ERK1/2 levels in whole cell lysates were determined by immunoblotting (IB) as described. The immunoblot shown is representative of at least three separate experiments. B, effect of increasing GsCT expression on alpha 2AAR-mediated ERK1/2 activation in COS-7 cells. Cells in 100-mm dishes were cotransfected the pRK5-alpha 2AAR plasmid, along with the indicated amounts of pcDNA3.1-HA-Galpha s-(313-395), prior to passage into 6-well plates. Phospho-ERK1/2 levels after 5 min of stimulation with UK14304 (10 µM) were determined as described. C, effect of increasing GsCT expression on alpha 1BAR-stimulated ERK1/2 activation in COS-7 cells. Cells in 100-mm dishes were cotransfected the pRK5-alpha 1BAR plasmid, along with the indicated amounts of pcDNA3.1-HA-Galpha s-(313-395), prior to passage into 6-well plates. Phospho-ERK1/2 levels after 5 min of stimulation with phenylephrine (1 µM) were determined as described. B and C, the radiograph depicts a representative immunoblot. Data shown represent the mean ± S.E. for three separate experiments. *, greater than control, p < 0.05.

In COS-7 cells, stimulation of transiently expressed alpha 1BARs leads to ERK activation that is pertussis toxin-insensitive (36) but blocked by expression of a polypeptide derived from the carboxyl-terminal 55 amino acids of Galpha q (18). As shown in Fig. 9C, transfection of COS-7 cells with increasing amounts of the GsCT plasmid had no effect on alpha 1BAR-mediated ERK phosphorylation. Thus, ERK1/2 activation by a receptor that does not activate Gs was unaffected by GsCT expression.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The precise structural determinants underlying activation of heterotrimeric G proteins by heptahelical receptors are incompletely understood. Crystallographic analysis of the structure of Galpha s has indicated that receptor coupling specificity is likely determined by a surface formed by the continuous carboxyl-terminal alpha -helix between Asp-368 and Leu-394, and the loop between the alpha 5-helix and beta 6-strand (6). Contact between the Galpha subunit and the second and third intracellular domains of heptahelical receptors determines the efficiency and specificity of the receptor-G protein interaction (3, 37, 38). The Galpha subunit carboxyl-terminal helix may insert into a cavity between the third and sixth receptor transmembrane domains of the heptahelical receptor bundle that forms as a consequence of agonist-induced conformational changes (39). NMR studies have demonstrated that short polypeptides derived from the Galpha s carboxyl terminus form stable alpha -helices in solution. In isolated plasma membranes, 11-amino acid peptides representing the carboxyl termini of Galpha i1/2 or Galpha o modulate ligand binding to the adenosine A1 receptor by disrupting the high affinity receptor-G protein complex (16). Similarly, modified 16-21-amino acid peptides derived from the carboxyl terminus of Galpha s inhibit high affinity agonist binding to the adenosine A2A receptors, and impair A2A receptor-mediated adenylyl cyclase activation (17). These data suggest that the isolated carboxyl-terminal alpha -helix can interact with a receptor in a manner that precludes productive receptor-G protein coupling.

We have examined the mechanism of action and functional consequences of expression of an 83-amino acid polypeptide derived from the carboxyl terminus of Galpha s in intact cells. Expression of the GsCT peptide impaired adenylyl cyclase activation by Gs-coupled beta 2 adrenergic and D1A dopamine receptors, without affecting the response to cholera toxin or forskolin, suggesting that the peptide specifically impairs receptor-G protein coupling. At a constant level of receptor expression, the inhibition was not surmountable by increasing agonist concentration. The magnitude of the effect was partially reversed by increasing receptor density, consistent with the hypothesis that the peptide competes with the endogenous Gs pool for access to ligand-occupied receptors. Furthermore, the inhibition was apparently specific for Gs, because PI hydrolysis induced by stimulation of the Gq/11-coupled alpha 1BAR and Gi-coupled alpha 2AAR was unaffected by GsCT expression.

A significant aspect of the approach of using receptor- or G protein-derived peptides to inhibit heptahelical receptor signaling is that the resulting antagonism affects a class of G protein, rather than a specific receptor. Such reagents differ from pharmacologic antagonists of ligand binding in that expression of a single polypeptide should be able to uncouple multiple receptors from a single G protein pool. In this regard, their function is more like B. pertussis toxin, which uncouples all Gi/o family proteins from their cognate receptors by catalyzing the ADP-ribosylation of a carboxyl-terminal cysteine residue on the Galpha subunit.

Because of their ability to selectively uncouple specific G proteins from multiple receptors, peptide inhibitors of receptor-G protein coupling may be useful for determining the contribution of a given G protein pool to signaling by a receptor that couples to multiple G proteins. Minigene constructs encoding the carboxyl termini of Galpha i, Galpha q, Galpha 12, and Galpha 13 have recently been employed to examine the contribution of different G protein pools to second messenger generation by the thrombin receptor in endothelial cells (20). We have employed GsCT expression to examine the role of Galpha s in a complex process, activation of the ERK MAP kinase cascade in fibroblasts, where Gs activation has been reported previously to produce both stimulation and inhibition of ERK activity. Consistent with previous reports, we found that beta 2AR-mediated ERK activation, which is blocked by PKA inhibition, is inhibited by GsCT, whereas alpha 2AAR-mediated ERK activation, which is accentuated by PKA inhibition, is enhanced in cells expressing GsCT (27-33). These data support a dual role for PKA in ERK activation by the beta 2AR, where PKA phosphorylation of the receptor promotes receptor-Gi coupling and pertussis toxin-sensitive ERK activation (27), but where PKA phosphorylation of Raf1 attenuates ERK activation downstream of Ras (30-33). In the case of the constitutively Gi/Gs-coupled alpha 2AAR, only the downstream inhibitory effect of Gs activation, which is relieved by GsCT expression, is discernible.

Tissue-specific expression of peptide G protein inhibitors has already provided valuable information about the roles of individual G proteins in complex physiologic responses in vivo. Cardiomyocyte-specific expression of a 55-amino acid peptide derived from the carboxyl terminus of Galpha q reduces cardiac hypertrophy (18) and inhibits activation of the ERK and c-Jun amino-terminal kinase MAP kinase cascades (40), in response to surgically induced pressure overload in a transgenic murine model, underscoring the important role of Gq/11 proteins in this process. Recombinant adenovirus-mediated expression of a Gbeta gamma subunit sequestrant polypeptide derived from the carboxyl terminus of G protein-coupled receptor kinase 2 (41), which results in generic inhibition of Gbeta gamma subunit-mediated signaling events (34, 42), blocks ERK activation and vascular smooth muscle hypertrophy in a rat carotid artery model of vascular restenosis (43). The availability of specific polypeptide inhibitors of Galpha s signaling, such as the GsCT minigene, may provide the opportunity to obtain similar insights into the complex roles of Gs in control of cellular hypertrophy and proliferation in a variety of tissues.

    ACKNOWLEDGEMENTS

We thank Grace Irons, Sabrina Exum, and Randy Durren for excellent technical assistance.

    FOOTNOTES

* This work was supported by a Howard Hughes Postdoctoral Research Fellowship for Physicians (to D. S. F.) and National Institutes of Health Grant DK55524 (to L. M. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Present address: Division of Cardiology, P. O. Box 250623, Dept. of Medicine, Medical University of South Carolina, Charleston, SC 29425. E-mail: feldmds@musc.edu.

Dagger Dagger To whom correspondence should be addressed: N3019 GRECC, Durham Veterans Affairs Medical Center, 508 Fulton St., Durham, NC 27705. Tel.: 919-286-0411, Ext. 7196; Fax: 919-416-5823; E-mail: luttrell@receptor-biol.duke.edu.

Published, JBC Papers in Press, May 29, 2002, DOI 10.1074/jbc.M204753200

    ABBREVIATIONS

The abbreviations used are: G protein, heterotrimeric GTP-binding protein; AR, adrenergic receptor; D1AR3i, D1A dopamine receptor third intracellular domain; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; Galpha i, alpha subunit of the heterotrimeric Gi protein; Galpha q/11,