Different Effects of Gsalpha Splice Variants on beta 2-Adrenoreceptor-mediated Signaling
THE beta 2-ADRENORECEPTOR COUPLED TO THE LONG SPLICE VARIANT OF Gsalpha HAS PROPERTIES OF A CONSTITUTIVELY ACTIVE RECEPTOR*

Roland SeifertDagger §, Katharina Wenzel-SeifertDagger §, Tae Weon LeeDagger , Ulrik GetherDagger , Elaine Sanders-BushDagger parallel , and Brian K. KobilkaDagger **Dagger Dagger

From the Dagger  Howard Hughes Medical Institute, ** Division of Cardiovascular Medicine, Stanford University Medical School, Stanford, California 94305-5428

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

The beta 2-adrenoreceptor (beta 2AR) couples to the G-protein Gs to mediate adenylyl cyclase activation. The splice variants of Gsalpha differ by a 15-amino acid insert between the Ras-like domain and the alpha -helical domain. The long splice variant of Gsalpha (Gsalpha L) binds GDP with lower affinity than the short splice variant (Gsalpha S), but the impact of this difference on the interaction of Gsalpha with the beta 2AR is not known. We studied the beta 2AR/Gsalpha interaction using receptor/G-protein fusion proteins (beta 2ARGsalpha S and beta 2ARGsalpha L) expressed in Sf9 cells. Fusion of the beta 2AR to Gsalpha promotes efficient coupling as shown by high-affinity agonist binding and GTPase and adenylyl cyclase activation and ensures fixed stoichiometry between receptor and G-protein. Importantly, fusion does not change the fundamental properties of the beta 2AR or Gsalpha . The beta 2AR in beta 2ARGsalpha L showed hallmarks of constitutive activity (increased potency and intrinsic activity of partial agonists, increased efficacy of inverse agonists, and increased basal GTPase activity) compared with the beta 2AR in beta 2ARGsalpha S. The apparent constitutive activity of the beta 2AR in beta 2ARGsalpha L may be due to the lower GDP affinity of Gsalpha L compared with Gsalpha S, i.e. Gsalpha L is more often nucleotide-free than Gsalpha S and, therefore, more frequently available to stabilize the beta 2AR in the active (R*) state. This study demonstrates that subtle structural differences between closely related G-protein alpha -subunits can have important consequences for the functional properties of a G-protein-coupled receptor.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Numerous hormones and neurotransmitters exert their effects through G-protein-coupled receptors (GPCRs)1 (1-4). The beta 2-adrenoreceptor (beta 2AR), a prototypical GPCR, interacts with the G-protein Gs, causes GDP/GTP exchange at its alpha -subunit (Gsalpha ) and, thereby, leads to activation of adenylyl cyclase (AC). Recently, the ternary complex model of GPCR activation has been extended to explain the finding that GPCRs can activate G-proteins, even in the absence of agonist, and that certain receptor ligands, namely inverse agonists, can suppress the G-protein activation mediated by agonist-free GPCRs (5-14). The agonist-independent activity of a GPCR is referred to as constitutive activity. The extended ternary complex model (two-state model) assumes that agonists stabilize GPCRs in the active (R*) state, while inverse agonists stabilize the inactive (R) state. Although constitutive GPCR activity can be most easily observed when receptors are overexpressed (10-12) or mutated (7, 8, 13), it also occurs at physiological receptor expression levels (5, 6, 9, 14). Hallmarks of constitutive GPCR activity are increased potency and efficacy of partial agonists, increased efficacy of inverse agonists, and elevated basal G-protein activity (5-14). These properties of constitutive activity are generally associated with GPCR function, and little is known about the ability of different G-proteins to influence the efficacy and potency of ligands.

Gsalpha exists as a short (Gsalpha S) and a long (Gsalpha L) splice variant. Compared with Gsalpha S, Gsalpha L contains additional 15 amino acids inserted at position 72 of the polypeptide chain, and there is an exchange of glutamate for aspartate at position 71 (15, 16) (Fig. 1A). Based on the alpha -carbon model of the alpha -subunit of the retinal G-protein tansducin (17), the sequence within which the 15-amino acid insert is localized in Gsalpha L serves as a linker between the Ras-like domain and the alpha -helical domain (Fig. 1B). The guanine nucleotide-binding site is embedded between these two domains. Thus, a change in this linker sequence might be expected to influence the binding kinetics of guanine nucleotides. In fact, purified Gsalpha L releases GDP more than twice as fast as Gsalpha S (18).

The results of a previous study indicate that Gsalpha S may be more effective than Gsalpha L in activating AC (19), but with regard to beta 2AR coupling, studies have not revealed significant differences between Gsalpha S and Gsalpha L (18, 20, 21). Studying differences in the interaction of structurally very similar G-proteins with a given GPCR is technically difficult, because functional interactions between receptors and G-proteins are strongly influenced by their relative expression levels (22). Specifically, defined receptor/G-protein stoichiometries have to be achieved to be able to detect subtle differences in GPCR/G-protein coupling.

To facilitate the examination of receptor/G-protein interactions we constructed fusion protein DNAs in which the C terminus of the beta 2AR was linked to the N terminus of Gsalpha S (beta 2ARGsalpha S) or Gsalpha L (beta 2ARGsalpha L) (Fig. 1A) and expressed the fusion proteins in Sf9 cells. Fusion proteins have a fixed ratio of receptor to alpha -subunit (23, 24). Thus, ambiguities in data analysis because of varying stoichiometry of the signaling partners can be eliminated. Using the fusion protein approach, we observed that the beta 2AR coupled to Gsalpha L has properties of constitutively active GPCR.

    EXPERIMENTAL PROCEDURES
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Procedures
Results
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References

Materials-- Rat Gsalpha L DNA was kindly provided by Dr. R. R. Reed (Johns Hopkins University, Baltimore, MD) (25). For generation of recombinant baculoviruses encoding for rat Gsalpha L, its DNA sequence was transferred into the baculovirus transfer vector pVL 1392 (11). [gamma -32P]GTP (6000 Ci/mmol) and [alpha -32P]ATP (3000 Ci/mmol) were from NEN Life Science Products. [3H]Dihydroalprenolol ([3H]DHA) (85-90 Ci/mmol) was from Amersham Corp. Anti-Gsalpha antibody was from Calbiochem. Guanosine 5'-phosphorothioate (GMPS) was from U. S. Biochemical Corp. All other nucleotides were from Boehringer Mannheim (Mannheim, Germany). Sources of other materials have been described elsewhere (11, 13, 26).

Construction of beta 2ARGsalpha L and beta 2ARGsalpha S DNAs-- beta 2ARGsalpha L DNA was generated by a two-step PCR protocol using Pfu polymerase. A set of fusion primers (sense and antisense), encoding 18 base pairs from the C terminus of the beta 2AR, 18 base pairs encoding a hexahistidine tag, and 21 base pairs from the N terminus of Gsalpha L, were synthesized. In PCR 1A, the sequence between a primer 5' of the EcoRV site of the human beta 2AR and the antisense fusion primer was amplified using beta 2AR DNA in pGEM-3Z as template. In this vector, referred to as pGEM-3Z-SF-beta 2AR-6His, the beta 2AR is tagged at the N terminus with the cleavable influenza-hemagglutinin signal sequence followed by the Flag epitope (IBI, New Haven, CT), and the C terminus of the beta 2AR is tagged with a hexahistidine tail (Fig. 1A) (26). In PCR 1B, the sequence between the sense fusion primer and the antisense primer with an extra SalI site 3' of the stop codon of Gsalpha L was amplified using rat Gsalpha L DNA in pGEM-3Z as template. In PCR 2, the products of PCRs 1A and 1B were annealed and the sense primer 5' of the EcoRV site in the beta 2AR sequence and the antisense primer 3' of the stop codon of Gsalpha L were used. In this way, a fragment encoding the C terminus of the beta 2AR, a hexahistidine tag, and Gsalpha L was obtained. This fragment was digested with EcoRV and SalI and cloned into pGEM-3Z-SF-beta 2AR-6His digested with EcoRV and SalI to obtain the full-length fusion protein DNA sequence (pGEM-3Z-SF-beta 2AR-6His-Gsalpha L). For generation of beta 2ARGsalpha S DNA, a set of deletion primers (sense and antisense) and an antisense primer 3' of the EcoRI site of Gsalpha L were synthesized. In PCR 3A, the sequence between a primer 5' of the EcoRV site in the beta 2AR and the antisense deletion primer was amplified using pGEM-3Z-SF-beta 2AR-6His-Gsalpha L as template. In PCR 3B, the sequence between the sense deletion primer and the antisense primer 3' of the EcoRI site of Gsalpha L was amplified using the same template as in PCR 3A. In PCR 4, the products of PCRs 3A and 3B were annealed, and the sense primer 5' of the EcoRV site in the beta 2AR and the antisense primer 3' of the EcoRI site of Gsalpha L were used. In this way, a DNA fragment encoding the C terminus of the beta 2AR, a hexahistidine tail and the N-terminal portion of Gsalpha S, missing the sequence for amino acids 72-86 in Gsalpha L and encoding the Glu-71 right-arrow Asp substitution (15), was created (Fig. 1A). This fragment was digested with EcoRV and EcoRI and cloned into pGEM-3Z-SF-beta 2AR-6His-Gsalpha L digested with EcoRV and EcoRI. PCR-generated DNA sequences were confirmed by enzymatic sequencing. Fusion protein DNAs were cloned into the baculovirus transfer vector pVL 1392 (11). Recombination of viruses was confirmed by reverse transcriptase PCR.

Cell Culture-- Recombinant baculoviruses were generated and amplified as described (11). Sf9 cells were seeded at 3.0 × 106 cells/ml and infected with 1:50 or 1:500 dilutions of high titer virus stocks. Cells were cultured for 24-48 h to obtain various expression levels of fusion proteins and beta 2AR. For co-expression studies, Sf9 cells were infected with a 1:10,000 dilution of a high titer beta 2AR baculovirus stock and a 1:50 dilution of a high titer Gsalpha L baculovirus stock to achieve a receptor to G-protein stoichiometry of ~1:100. Cells were cultured for 48 h. Membranes were prepared according to Gether et al. (11).

[3H]DHA Binding-- For determination of Kd and Bmax values, Sf9 membranes (5 µg of protein) were suspended in 500 µl of buffer containing 75 mM Tris/HCl, pH 7.4, 12.5 mM MgCl2, and 1 mM EDTA, supplemented with 0.1-10 nM [3H]DHA and 0.2% (w/v) bovine serum albumin. Nonspecific binding was assessed in the presence of 10 µM (-)-alprenolol (ALP). Incubations were performed for 90 min at 25 °C and shaking at 200 rpm. Competition binding experiments were carried out with 15-30 µg of membrane protein with 1 nM [3H]DHA in the presence of unlabeled ligands at various concentrations without or with guanosine 5'-O-(3-thiotriphosphate) (GTPgamma S) (10 µM). In some experiments, tubes contained 1 nM [3H]DHA, 1 µM salbutamol (SAL), and various nucleotides at increasing concentrations.

GTPase Activity-- Assay tubes (100 µl) contained 10 µg of membrane protein, 0.1 µM [gamma -32P]GTP (0.1-0.5 µCi/tube), 1.0 mM MgCl2, 0.1 mM EDTA, 0.1 mM ATP, 1 mM adenylyl imidodiphosphate, 5 mM creatine phosphate, 40 µg of creatine kinase, 0.2% (w/v) bovine serum albumin in 50 mM Tris/HCl, pH 7.4, and ligands at various concentrations. Reactions were conducted for 20 min at 25 °C and were terminated by the addition of 900 µl of a slurry consisting of 5% (w/v) activated charcoal and 50 mM NaH2PO4, pH 2.0. Reaction mixtures were centrifuged for 15 min at room temperature and 15,000 × g. Seven-hundred µl of the supernatant fluid of reaction mixtures were removed and [32P]Pi was determined by liquid scintillation counting.

AC Activity-- Assay tubes (50 µl) contained 15 µg of membrane protein, 1 µM GTP, 40 µM [alpha -32P]ATP (2.5 µCi/tube), 2.7 mM mono(cyclohexyl)ammonium phosphoenolpyruvate, 0.125 IU of pyruvate kinase, 1 IU of myokinase, 0.1 mM cAMP, 5 mM MgCl2, 0.4 mM EDTA, 30 mM Tris/HCl, pH 7.4, and ligands at various concentrations. Reactions were conducted for 20 min at 37 °C. Separation of [32P]cAMP from [alpha -32P]ATP was performed as described (27).

Western Blot Analysis-- Solubilized Sf9 membrane proteins (5-10 µg of protein/lane) were separated by SDS-PAGE (8% (w/v) acrylamide). Proteins were visualized using either M1 antibody or anti-Gsalpha antibody and the ECL Western blotting system (Amersham). Gsalpha L expression in Sf9 membranes was quantitated by immunoblotting with anti-Gsalpha antibody using defined amounts of beta 2ARGsalpha fusion protein as standard.

Miscellaneous-- Protein was determined using the Bio-Rad DC protein assay kit (Bio-Rad). Data were analyzed by nonlinear regression, using the program Prism (GraphPad, Prism, San Diego, CA). Statistical comparisons between beta 2ARGsalpha S and beta 2ARGsalpha L were done with the Wilcoxon test. Data are given as means ± S.D. of three to seven independent experiments performed in duplicate or triplicate.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Expression of beta 2ARGsalpha S and beta 2ARGsalpha L in Sf9 Membranes-- Expression of fusion proteins in Sf9 membranes was confirmed by SDS-PAGE using the M1 monoclonal antibody to detect the N-terminal Flag epitope of the beta 2AR (Fig. 1A). The nonfused beta 2AR expressed in Sf9 cells runs as a broad glycosylated 52-kDa protein in SDS-PAGE (11, 26). The apparent molecular masses of Gsalpha S and Gsalpha L are 45 and 52 kDa, respectively (16). Accordingly, the apparent molecular masses of beta 2ARGsalpha S and beta 2ARGsalpha L were expected to be 97 and 104 kDa, respectively. The data obtained are in agreement with this expectation (Fig. 1C). Immunoblots with an anti-Gsalpha antibody confirmed the presence of Gsalpha in the fusion proteins and the difference in apparent molecular mass between beta 2ARGsalpha S and beta 2ARGsalpha L. In membranes from uninfected cells, no immunoreactive bands in the 97-104-kDa region were detected with the M1 and anti-Gsalpha antibody (data not shown).


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Fig. 1.   Structure of beta 2ARGsalpha fusion proteins and three-dimensional model of transducin-alpha . A, schematic structure of fusion proteins. The DNA of the human beta 2AR, tagged with the Flag epitope at the N terminus and a hexahistidine tag at its C terminus, was fused to the DNA of Gsalpha S or Gsalpha L. The differences in amino acid sequence between Gsalpha S and Gsalpha L are given in the single letter code. B, three-dimensional alpha -carbon model of the alpha -subunit of transducin. Blue, alpha -helical domain; gray, Ras-like domain; red, linker 1 (where insert is located in Gsalpha L); orange, linker 2; green, alpha 5-helix; yellow, GDP (van der Waals representation). The membrane would be in a horizontal plane below the molecule. The N terminus of transducin-alpha is at the lower right. C, immunological characterization of fusion proteins. Sf9 membranes were separated by SDS-PAGE, transferred to nitrocellulose, and probed with M1 antibody or anti-Gsalpha antibody as described under "Experimental Procedures." Numbers on the left indicate molecular masses of marker proteins. Shown are autoluminograms of gels containing 8% (w/v) acrylamide.

Ligand Binding Properties of beta 2ARGsalpha S and beta 2ARGsalpha L, Comparison with the Nonfused beta 2AR-- The Kd values of [3H]DHA for beta 2ARGsalpha S and beta 2ARGsalpha L were very similar (Table I). In competition experiments, we studied the effects of (-)-isoproterenol ((-)-ISO), (+)-isoproterenol ((+)-ISO), SAL, dobutamine (DOB), (-)-ephedrine (EPH), dichloroisoproterenol (DCI) and ICI 118,551 (ICI) on [3H]DHA binding. (-)-ISO binds to beta ARs with higher affinity than (+)-ISO, but both stereoisomers are full agonists (28-30). SAL, DOB, EPH, and DCI are partial beta 2AR agonists (7, 10, 11), and ICI is an inverse agonist (8, 11-13). At both fusion proteins, full and strong partial agonists ((-)-ISO, (+)-ISO, SAL, and DOB) showed a high- and low-affinity binding component (Table I). The high-affinity agonist binding was abolished by GTPgamma S. For agonists with lower intrinsic activity (EPH and DCI), high- and low-affinity binding sites were not discriminated by curve fitting analysis, but GTPgamma S still reduced the affinity of these ligands to beta 2ARGsalpha fusion proteins. There were no significant differences in the low- and high-affinity Ki values of the agonists studied between beta 2ARGsalpha S and beta 2ARGsalpha L. There was a trend toward higher fractions of high-affinity agonist-binding sites for full agonists and strong partial agonists at beta 2ARGsalpha S compared with beta 2ARGsalpha L, but this was significant only for (+)-ISO.

                              
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Table I
Binding properties of beta 2AR ligands at beta 2ARGsalpha S and beta 2ARGsalpha L
[3H]DHA binding was determined as described under "Experimental Procedures" in membranes expressing beta 2ARGsalpha S or beta 2ARGsalpha L at 3.5-6.5 pmol/mg. Competition binding isotherms were analyzed by nonlinear regression for best fit to single-site or two-site binding. Kh and Kl designate the dissociation constants for the high- and low-affinity state of the beta 2AR, respectively. %Rh indicates the percentage of high-affinity binding sites. When competition isotherms were best fit to a single-site model, the respective Ki values are listed under Kl. Ki (+GTPgamma S) indicates the Ki values obtained in the presence of 10 µM GTPgamma S. Data shown represent the means ± S.D. of five to seven independent experiments performed in duplicate or triplicate. Ki values are expressed in nanomolar.

In Sf9 membranes expressing the nonfused Flag epitope- and hexahistidine-tagged beta 2AR (11) at similar levels (4.0-6.5 pmol/mg) as beta 2ARGsalpha fusion proteins (3.5-6.5 pmol/mg), the Kd value for [3H]DHA was 0.36 ± 0.03 nM. ICI inhibited [3H]DHA binding with a Ki value of 1.2 ± 0.3 nM. (-)-ISO inhibited [3H]DHA binding to the nonfused beta 2AR according to a steep monophasic function (Ki, 200 ± 13 nM). The (-)-ISO competition curve was not affected by GTPgamma S (10 µM) (Ki, 201 ± 37 nM). The lack of high-affinity agonist binding was also observed of the avian beta AR expressed in Sf9 cells (31). In membranes from uninfected Sf9 cells, no specific [3H]DHA binding was detected (data not shown), indicative for the absence of endogenous beta 2ARs. Collectively, these data show that in the beta 2ARGsalpha fusion proteins, the receptor productively interacts with the attached G-protein to induce high-affinity agonist binding, while the interaction of the beta 2AR with endogenous G-proteins of Sf9 cells is not efficient enough to result in measurable high-affinity agonist binding. In addition, the antagonist and agonist binding properties of the beta 2AR in beta 2ARGsalpha fusion proteins compare favorably with the ligand binding properties of nonfused beta 2AR (Table I) (7, 8, 12, 28-30).

Regulation of GTPase Activity in beta 2ARsalpha Fusion Proteins, Comparison with a Co-expression System Consisting of beta 2AR and Gsalpha -- Activation of the GTPase of Gs by agonist-occupied beta ARs can be studied with great sensitivity in reconstituted systems (32, 33), but in most plasma membrane systems, the GTPase stimulation induced by beta ARs is small relative to the high background GTPase activity of other cellular G-proteins with higher GTP turnover than Gs and the presence of low-affinity nucleotidases (34, 35). In S49 lymphoma cell membranes, a prototypical system for studying beta 2AR/Gs interaction (23, 36), the beta 2AR and Gsalpha are expressed at levels of ~0.2 and ~20 pmol/mg, respectively, i.e. there is an ~100-fold molar excess of G-protein compared with receptor (37). We co-expressed the beta 2AR at a level of 1.4 pmol/mg with Gsalpha L at a level of ~100 pmol/mg in Sf9 membranes, achieving a similar receptor/G-protein ratio as in S49 lymphoma cells, and studied the regulation of GTPase activity by (-)-ISO and ICI. However, despite the relatively high expression of beta 2AR and Gsalpha L at a stoichiometry similar to that in the mammalian cell line, we detected only marginal activation of GTPase by agonist in Sf9 membranes and failed to see inhibition by inverse agonist (Fig. 2A). Similar results were obtained when the expression level of beta 2AR was increased to 11.8 pmol/mg (data not shown). In marked contrast, (-)-ISO increased GTP hydrolysis in membranes expressing beta 2ARGsalpha (5.0 pmol/mg) by up to 245% above basal, and ICI reduced GTP hydrolysis by up to 50% (Fig. 2B). These findings demonstrate that fusion of the beta 2AR to Gsalpha greatly facilitates detection of ligand-regulated GTP hydrolysis in Sf9 membranes.


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Fig. 2.   Comparison of the ligand regulation of GTPase activity in Sf9 membranes expressing the beta 2AR and Gsalpha L as separate proteins and in membranes expressing the beta 2ARGsalpha L fusion protein. GTP hydrolysis was determined with 100 nM [gamma -32P]GTP as substrate as described under "Experimental Procedures." Reaction mixtures contained (-)-ISO and ICI at the indicated concentrations and membranes expressing the beta 2AR (1.4 pmol/mg) plus Gsalpha L (~100 pmol/mg) (A) or membranes expressing beta 2ARGsalpha L (5.0 pmol/mg) (B). Data shown are the means ± S.D. of three to five independent experiments performed in duplicate.

The regulation of GTPase activity in Sf9 membranes expressing beta 2ARGsalpha S and beta 2ARGsalpha L at similar levels (4.5-5.0 pmol/mg) was compared. (-)-ISO increased GTP hydrolysis in membranes expressing beta 2ARGsalpha S and beta 2ARGsalpha L by up to 315 and 245%, respectively (Fig. 3, A and B). To the best of our knowledge, these are the highest reported agonist stimulations of GTPase by beta ARs in a membrane system (28, 34, 35). It should also be noted that Fig. 3, A and B, show total GTP hydrolysis rates and not only the high-affinity GTPase activity corrected for low-affinity GTPases (34). This fact further underlines the high sensitivity of the GTPase assay with the beta 2ARGsalpha fusion proteins in Sf9 membranes.


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Fig. 3.   Effects of (-)-ISO and ICI on GTPase and AC activity in Sf9 membranes expressing beta 2ARGsalpha S and beta 2ARGsalpha L. GTPase activity (A and B) and AC activity (C and D) in beta 2ARGsalpha S membranes (A and C) and beta 2ARGsalpha L membranes (B and D) were determined as described under "Experimental Procedures" in the presence of (-)-ISO or ICI at various concentrations. For GTPase studies, the expression level of fusion proteins was 4.5-5.0 pmol/mg, and for AC studies, 2.3-2.6 pmol/mg. GTP hydrolysis was determined with 100 nM [gamma -32P]GTP as substrate. AC activity was determined in the presence of 1 µM GTP. Data shown are the means ± S.D. of three to five independent experiments performed in duplicate. The dotted lines are extrapolations of basal GTPase and AC activities to illustrate the relative contributions of (-)-ISO and ICI at the ligand-regulated enzyme activities.

The Effects of Gsalpha S and Gsalpha L on the Efficacy of Agonists and Inverse Agonists at the beta 2ARGsalpha Fusion Proteins-- The precise determination of the intrinsic activities of partial agonists constitutes a major problem in the functional characterization of GPCRs, because the intrinsic activity of a given ligand may depend on numerous variables, i.e. receptor and G-protein expression level and the availability of effector molecules such as AC (11, 22, 38-40). In most studies, the intrinsic activities of partial beta 2AR agonists were characterized by measuring AC activity (7, 10, 11, 41). The AC assay takes advantage of the signal amplification at the Gs level, but it is difficult to control for the impact of Gs and AC availability on intrinsic activities of ligands. We reasoned that with the GTPase activity of beta 2ARGsalpha fusion proteins as parameter, determination of the intrinsic activities of partial agonists should be less ambiguous because of the fixed stoichiometry of the signaling components. Moreover, signal amplification by AC is not required, thereby reducing the number of variables that can influence the determination of intrinsic activity. To validate this assumption, we studied the potencies and intrinsic activities of a series of partial beta 2AR agonists at the GTPase of beta 2ARGsalpha L with expression levels ranging from 0.6 to 7.6 pmol/mg. Within this broad range of expression, we did not observe significant differences in the potency and intrinsic activity of partial beta 2AR agonists (data not shown).

Based on the above results, we determined the effects of a series of agonists with different intrinsic activities and of inverse agonists on GTP hydrolysis in membranes expressing beta 2ARGsalpha S and beta 2ARGsalpha L. The affinity of the beta 2AR for (+)-ISO in beta 2ARGsalpha S and beta 2ARGsalpha L is substantially lower than the affinity for (-)-ISO (Table I). In agreement with the difference in binding affinity, (+)-ISO activated the GTPase of both fusion proteins more than 10-fold less potently than (-)-ISO (Table II). Notably, the potencies of all agonists studied were higher at beta 2ARGsalpha L than at beta 2ARGsalpha S. This difference between the two fusion proteins was significant for all ligands studied except for (-)-ISO (Table II). The difference in potency of partial agonists between beta 2ARGsalpha S and beta 2ARGsalpha L was most prominent for EPH (24-fold). For most ligands ((+)-ISO, SAL, DOB, and DCI), the difference in potency between beta 2ARGsalpha S and beta 2ARGsalpha L was about 3-4-fold.

                              
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Table II
Potencies of full and partial beta 2AR agonists at the GTPase of beta 2ARGsalpha S and beta 2ARGsalpha L
For determination of the potency of ligands, GTP hydrolysis was measured as described under "Experimental Procedures" in membranes expressing beta 2ARGsalpha S and beta 2ARGsalpha L at 4.5-5.0 pmol/mg. Reaction mixtures contained ligands at 0.1 nM to 1 mM as appropriate to obtain saturated concentration-response curves. EC50 values were calculated by nonlinear regression. Data shown represent the means ± S.D. of five to seven independent experiments performed in duplicate or triplicate. Potencies are expressed in nanomolar.

The intrinsic activities of (-)-ISO and (+)-ISO to activate the GTPase of beta 2ARGsalpha S and beta 2ARGsalpha L were similar (Fig. 4A). Analogous data concerning the intrinsic activities of (-)-ISO and (+)-ISO were obtained for nonfused beta ARs (28-30). For both beta 2ARGsalpha S and beta 2ARGsalpha L, ligands activated GTPase in the rank order of intrinsic activity (-)-ISO ~ (+)-ISO >=  SAL > DOB > EPH > DCI > ALP > (-)-propranolol (no intrinsic activity). Of interest, the intrinsic activities of SAL, DOB, EPH, DCI, and ALP at beta 2ARGsalpha L were significantly higher than at beta 2ARGsalpha S. When the intrinsic activities of ligands at the GTPase of beta 2ARGsalpha L are plotted versus the intrinsic activities of ligands at the GTPase of beta 2ARGsalpha S, data are best fitted by a hyperbolic and not a linear function (Fig. 4B). A similar hyperbolic relationship in the intrinsic activities of beta 2AR ligands was found for a (nonfused) constitutively active mutant of the beta 2AR (beta 2ARCAM) in comparison with the (nonfused) wild-type beta 2AR (7). Thus, the beta 2AR in beta 2ARGsalpha L appears to possess some properties of a constitutively active receptor.


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Fig. 4.   Intrinsic activities of full and partial beta 2AR agonists at the GTPase of beta 2ARGsalpha S and beta 2ARGsalpha L. For determination of the intrinsic activities of ligands, GTP hydrolysis was measured as described under "Experimental Procedures" in membranes expressing beta 2ARGsalpha S and beta 2ARGsalpha L at 4.5-5.0 pmol/mg. Reaction mixtures contained ligands at 0.1 nM to 1 mM as appropriate to obtain saturated concentration-response curves. The intrinsic activities were derived from plateau values of concentration-response curves generated by nonlinear regression analysis. A, comparison of the intrinsic activities of various ligands at beta 2ARGsalpha S and beta 2ARGsalpha L. *, p < 0.05. Data shown are the means ± S.D. of five to seven independent experiments performed in duplicate. B, replot of the data shown in A. The intrinsic activities of ligands at beta 2ARGsalpha L were plotted against their intrinsic activities at beta 2ARGsalpha S. Data points were best fitted by a hyperbolic function as assessed by nonlinear regression. The straight line represents the theoretical curve that would have fitted data best if the intrinsic activities of ligands at beta 2ARGsalpha S and beta 2ARGsalpha L had been the same. For comparison, the data for the neutral antagonist (propranolol) (PRO) are included in the panel.

To obtain further evidence for constitutive activation of the beta 2AR in beta 2ARGsalpha L, we studied the effects of the inverse agonist ICI on basal GTPase activity. The basal steady-state GTPase activity with 100 nM [gamma -32P]GTP as substrate was about 3-fold higher for beta 2ARGsalpha L than for beta 2ARGsalpha S (Fig. 3, A and B). In membranes expressing beta 2ARGsalpha S, ICI had a smaller inhibitory effect (15% reduction) on GTP hydrolysis than in membranes expressing beta 2ARGsalpha L (50% reduction). Similar results were obtained with timolol, another inverse agonist at the beta 2AR (10) (data not shown). These inverse agonist studies show that the higher basal GTPase activity in membranes expressing beta 2ARGsalpha L compared with membranes expressing beta 2ARGsalpha S is largely attributable to the activity of the agonist-free beta 2AR.

Regulation of High-affinity Agonist Binding at beta 2ARGsalpha S and beta 2ARGsalpha L by Guanine Nucleotides-- High-affinity agonist binding to GPCRs depends on their interaction with G-protein alpha -subunits, presumably in the nucleotide-free state (1, 42). Occupation of the guanine nucleotide-binding site of alpha -subunits disrupts high-affinity agonist binding (1, 7). To determine the guanine nucleotide binding affinities of Gsalpha S and Gsalpha L in beta 2ARGsalpha fusion proteins, we examined binding of a fixed concentration of the antagonist [3H]DHA in the presence of a subsaturating concentration of the strong partial agonist SAL in membranes expressing beta 2ARGsalpha S and beta 2ARGsalpha . Various nucleotides at increasing concentrations were added to the binding assays. Guanine nucleotide binding to Gsalpha reduces the affinity of the beta 2AR for agonist and, thereby, increases [3H]DHA binding (Fig. 5). In this way, the affinity of G-proteins for nucleotides can be measured. It should be noted that our binding experiments were performed in the absence of a nucleotide-regenerating system, excluding the possibility that effects caused by nucleoside 5'-monophosphates and -diphosphates are due to transphosphorylation. GTP was similarly potent at inhibiting high-affinity agonist binding at beta 2ARGsalpha S and beta 2ARGsalpha L (EC50, 59 ± 15 and 49 ± 20 nM, respectively). In contrast, GDP was far more potent at beta 2ARGsalpha S (EC50, 83 ± 23 nM) than at beta 2ARGsalpha L (EC50, 1.8 ± 0.2 µM). Like GDP, its nucleotidase-resistant phosphorothioate analog, guanosine 5'-O-(2-thiodiphosphate) (GDPbeta S), inhibited agonist binding at beta 2ARGsalpha S more potently than at beta 2ARGsalpha L (EC50, 490 ± 150 nM and 2.2 ± 0.3 µM, respectively). These data show that in the beta 2ARGsalpha fusion proteins, Gsalpha S has a higher affinity for guanosine 5'-diphosphates than Gsalpha L and are in agreement with data obtained with purified Gsalpha L and Gsalpha S (18).


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Fig. 5.   Effects of guanine nucleotides on high-affinity agonist binding in Sf9 membranes expressing beta 2ARGsalpha S and beta 2ARGsalpha L. Binding experiments were carried out as described under "Experimental Procedures" with beta 2ARGsalpha S membranes (A) or beta 2ARGsalpha L membranes (B). Reactions mixtures additionally contained 1 nM [3H]DHA, 1 µM SAL, and guanine nucleotides at increasing concentrations. Data shown are the means ± S.D. of two independent experiments performed in triplicate.

Of interest, even GMP inhibited high-affinity agonist binding at beta 2ARGsalpha S and beta 2ARGsalpha L to some extent, although less potently than GDP (EC50, 55 ± 12 and 15 ± 7 µM, respectively). Substitution of the phosphate group in GMP by a phosphorothioate group, yielding the nucleotidase-resistant GMPS, substantially enhanced the potency and efficacy of the nucleotide to disrupt high-affinity agonist binding at beta 2ARGsalpha S and beta 2ARGsalpha L (EC50, 4.5 ± 1.2 µM and 6.3 ± 3.3 µM, respectively). The data obtained with GMP and GMPS provide strong support for the suggestion that it is the nucleotide-free form of Gsalpha , which confers high agonist-affinity to the beta 2AR (1, 42).

Regulation of AC Activity in Sf9 Membranes Expressing beta 2ARGsalpha S and beta 2ARGsalpha L by Agonist and Inverse Agonist-- The analysis of AC activity in Sf9 membranes expressing beta 2ARGsalpha S and beta 2ARGsalpha L must take into consideration the fact that Sf9 cells express endogenous Gsalpha -like G-proteins (10, 11, 30, 31). This is of particular relevance because for AC studies, we expressed fusion proteins at relatively low levels to avoid AC availability becoming the limiting factor (38). However, the AC activity in membranes from uninfected Sf9 cells in the presence of 10 µM GTPgamma S was ~5.5-fold lower than in Sf9 membranes expressing beta 2ARGsalpha L at 2.6 pmol/mg (0.089 ± 0.015 nmol/mg/20 min versus 0.491 ± 0.054 nmol/mg/20 min). These data show that even under maximal stimulation of AC, the contribution of endogenous Gsalpha -like G-proteins in Sf9 cells to total AC activity is small.

When the basal AC activity in the presence of 1 µM GTP in membranes expressing beta 2ARGsalpha S and beta 2ARGsalpha L at a similar level (2.3-2.6 pmol/mg) was compared, substantial differences between the two fusion proteins became apparent. Specifically, the AC activity in membranes expressing beta 2ARGsalpha S was almost twice as high as in membranes expressing beta 2ARGsalpha L (Fig. 3, C and D). (-)-ISO increased AC activity in beta 2ARGsalpha S membranes by up to 80%, while in membranes expressing beta 2ARGsalpha L, (-)-ISO increased AC activity only by 45%. The EC50 values of (-)-ISO were 51 ± 17 nM for beta 2ARGsalpha S and 17 ± 18 nM for beta 2ARGsalpha L. Despite the fact that the basal AC activity in membranes expressing beta 2ARGsalpha L was considerably lower than in membranes expressing beta 2ARGsalpha S, the inhibitory effect of ICI in membranes expressing beta 2ARGsalpha L (50% reduction) was substantially greater than in membranes expressing beta 2ARGsalpha S (10% reduction). Similar results were obtained with the inverse agonist timolol (data not shown). Thus, the AC data corroborate the GTPase data, pointing to constitutive activity of the beta 2AR in beta 2ARGsalpha L.

AC Regulation in Sf9 Membranes Expressing beta 2ARGsalpha S and beta 2ARGsalpha L in the Absence of Exogenous Guanine Nucleotides-- In the presence of GTP, agonists at Gs-coupled GPCRs cause AC activation (1, 2). However, in the absence of added guanine nucleotides, agonists at Gs-coupled receptors can reduce AC activity (43, 44). The most likely explanation for these observations is that agonists induce release of prebound guanine nucleotide from Gsalpha , generating guanine nucleotide-free Gsalpha and, thereby, reducing AC activity. Indeed, (-)-ISO reduced the basal AC activity in membranes expressing beta 2ARGsalpha S by about 30% and with an IC50 of 40 ± 12 nM (Fig. 6). In contrast, (-)-ISO had no significant deactivating effect on AC activity in membranes expressing beta 2ARGsalpha L. These results suggest that the nucleotide-binding pocket of Gsalpha L in beta 2ARGsalpha L is already nucleotide-free.


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Fig. 6.   Deactivation of AC by (-)-ISO in the absence of exogenous nucleotides. AC activity in beta 2ARGsalpha S and beta 2ARGsalpha L membranes was determined as described under "Experimental Procedures" in the absence of exogenous guanine nucleotides and in the presence of (-)-ISO at different concentrations. The expression level of fusion proteins was 2.3-2.6 pmol/mg. Data shown are the means ± S.D. of three to four independent experiments performed in duplicate.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The beta 2AR Fused to Gsalpha L Has Properties of a Constitutively Active Receptor-- Previous studies have shown that there are subtle differences in the GDP affinities of purified Gsalpha S and Gsalpha L (18) and that Gsalpha S may activate AC more efficiently than Gsalpha L (19). However, studies aiming to reveal differences between Gsalpha S and Gsalpha L in their coupling to the beta 2AR have remained inconclusive because of the difficulties to ensure exactly defined receptor/G-protein stoichiometry (18, 20, 21). This is important because functional interactions between GPCRs and G-proteins are strongly influenced by their relative expression levels (11, 22, 40). To circumvent this problem, we constructed fusion proteins in which the C terminus of the beta 2AR was linked to the N terminus of Gsalpha S or Gsalpha L (Fig. 1A), thereby guaranteeing a defined stoichiometry of receptor to G-protein and increasing the efficiency of receptor/G-protein coupling. Using this approach we observed that the efficacy and potency of partial agonists acting on the beta 2AR were significantly higher when the receptor was fused to Gsalpha L than when the receptor was fused to Gsalpha S (Fig. 4 and Table II). Moreover, the basal GTPase and AC activities in membranes expressing beta 2ARGsalpha L were more sensitive to inverse agonists than the corresponding activities in membranes expressing beta 2ARGsalpha S (Fig. 3). These functional properties of the beta 2AR fused to Gsalpha L are similar to those of the beta 2ARCAM (7, 8, 13).

According to the two-state model of receptor activation, GPCRs exist either in an inactive state R or an active state R*. These two states are in equilibrium, and the R* state can be stabilized by agonists, partial agonists, and nucleotide-free Gsalpha , while the R state is stabilized by inverse agonists (7-14). The results of our agonist binding studies are in agreement with the two-state model (Table I) and strongly support the suggestion that guanine nucleotide-free Gsalpha is necessary to form a high-affinity complex with the beta 2AR (Fig. 5) (1, 42). It has been proposed that in constitutively active receptor mutants, the equilibrium between R and R* is shifted toward R* (7, 8, 13). Experimentally, this results in an increased potency and efficacy of partial agonists, increased efficacy of inverse agonists, and increased basal G-protein activity (7, 8, 11, 13).

The apparent stabilization of the beta 2AR in the R* state in beta 2ARGsalpha L relative to the beta 2AR in beta 2ARGsalpha S can be revealed in experiments in which the outcome of multiple G-protein activation/deactivation cycles is monitored, i.e. the steady-state GTPase assay and the AC assay. As shown in Fig. 4 and Table II, the potency and intrinsic activity of a series of partial beta 2AR agonists to activate GTPase was significantly higher for beta 2ARGsalpha L than for beta 2ARGsalpha S. Moreover, the basal GTPase activity in membranes expressing beta 2ARGsalpha L was approximately 3-fold higher than in membranes expressing beta 2ARGsalpha S at a comparable level (Fig. 3, A and B). This elevated basal GTPase activity in membranes expressing beta 2ARGsalpha L can be reduced by ICI to a level near the basal level of membranes expressing beta 2ARGsalpha S. In contrast to membranes expressing beta 2ARGsalpha L, ICI has little effect on the basal GTPase activity in membranes expressing beta 2ARGsalpha S. These properties of constitutive activity of the beta 2AR in beta 2ARGsalpha L may be due to differences in the way Gsalpha L and Gsalpha S interact with the beta 2AR. In particular, Gsalpha L has a lower affinity for GDP than Gsalpha S (Fig. 5) (18). Therefore, Gsalpha L may be more often guanine nucleotide-free and more often available for stabilizing R* than Gsalpha S.

In contrast to basal GTPase activity, membranes expressing beta 2ARGsalpha S had a higher basal and (-)-ISO-stimulated AC activity than membranes expressing beta 2ARGsalpha L (Fig. 3, C and D). However, ICI inhibited the elevated basal AC activity in membranes expressing beta 2ARGsalpha S by only 10%, while ICI inhibited the lower basal AC activity in membranes expressing beta 2ARGsalpha L by 50%. Therefore, the elevated basal AC activity in membranes expressing beta 2ARGsalpha S is likely due to the intrinsic properties of Gsalpha S rather than to the beta 2AR in the fusion protein. A previous study had already shown that Gsalpha S is more effective in activating AC than Gsalpha L (19). Since GTP hydrolysis is the major mechanism by which G-proteins are deactivated (1, 2, 28, 34), the higher basal and (-)-ISO-stimulated GTPase activity in membranes expressing beta 2ARGsalpha L could indicate that Gsalpha L spends less time in the active GTP-bound state than Gsalpha S and, therefore, is less effective in stimulating AC.

The data shown in Fig. 6 suggest that Gsalpha in its GDP-liganded form may be able to stimulate AC and, thereby, to contribute to the higher basal AC activity in membranes expressing beta 2ARGsalpha S. Specifically, in the absence of added guanine nucleotides, (-)-ISO reduces basal AC activity in membranes expressing beta 2ARGsalpha S. Under these conditions, (-)-ISO can promote dissociation of previously bound GDP, but binding of GTP cannot occur. In contrast, in the absence of added guanine nucleotides, AC activity in membranes expressing beta 2ARGsalpha L is lower, and there is no significant reduction in basal activity following the addition of (-)-ISO. This observation is consistent with the lower affinity of Gsalpha L for GDP compared with Gsalpha S (Fig. 5) (18) and indicates that most of the Gsalpha L in beta 2ARGsalpha L has already released its GDP.

Of interest, there is no major differences in the apparent ability of Gsalpha L and Gsalpha S to stabilize the beta 2AR in the R* state in binding experiments (Table I). Similar results were previously obtained by Freissmuth et al. (20) in a reconstituted system. These data can be explained by the fact that agonist competition studies were performed at equilibrium and in the absence of exogenous guanine nucleotides. Under these conditions, Gsalpha L in beta 2ARGsalpha L membranes is already largely GDP-free so that the R* state accumulates rapidly, while in beta 2ARGsalpha S membranes, agonists induce GDP release from Gsalpha S and, thereby, facilitate accumulation of the R* state (Fig. 6). In contrast, in GTPase studies and AC experiments with added GTP, there is continuous cycling of the beta 2AR between R and R* so that differences in the apparent proportions of the two receptor states can be more readily detected.

Our studies regarding differential effects of Gsalpha splice variants on beta 2AR signaling were facilitated by using receptor/G-protein fusion proteins. However, our data indicate that fusing receptor to G-protein does not alter the fundamental properties of either component. In particular, the binding properties of beta 2AR agonists and antagonists were not altered by fusion to Gsalpha (Table I) (7, 8, 12, 28-30, 45). In addition, GTPgamma S efficiently activated AC in membranes expressing beta 2ARGsalpha L, indicating that fusion of Gsalpha to the beta 2AR does not impair the interaction of the G-protein with AC. Moreover, the relative potencies of GTPgamma S and guanylyl imidodiphosphate to activate AC are preserved in fusion proteins (data not shown). Finally, the Km values of the (-)-ISO-stimulated GTPases of beta 2ARGsalpha L and beta 2ARGsalpha S (279 ± 10 and 144 ± 23 nM, respectively) are in agreement with values reported for reconstituted systems (32).

Physiological Considerations-- Although constitutive activation of GPCRs is easily observed with high receptor expression levels (10-12), this is not a prerequisite. There are several examples in the literature documenting constitutive GPCR activity at physiological or near-physiological expression levels (5, 6, 9, 14). These data raise the possibility that constitutive activity of GPCRs is of relevance in vivo and that the R* state can be more readily stabilized or detected by specific G-protein alpha -subunits. In agreement with such a concept is the finding that increases in expression of specific G-proteins can increase high-affinity agonist binding and can promote constitutive receptor activation (40, 46).

Gsalpha S and Gsalpha L are differentially expressed in various tissues (47). In addition, the expression of Gsalpha S and Gsalpha L changes during erythroid differentiation (48), during multiple passages of HIT insulinoma cells (19), and in uterine smooth muscle during pregnancy (49). These findings could point to different roles of Gsalpha S and Gsalpha L in cell functions. The expression of Gsalpha splice variants also changes in pathological situations. Specifically, in preterm labor, only Gsalpha S is expressed in the uterus, whereas in the normal pregnant uterus, both Gsalpha isoforms are present (49). It remains to be determined of whether the lack of Gsalpha L expression in preterm labor is the basis for the poor therapeutic efficiency of partial beta 2AR agonists as tocolytic drugs (49).

Conclusion-- The 15-amino acid insert by which Gsalpha L differs from Gsalpha S (Fig. 1B) lowers the GDP affinity of the G-protein. Using fusion proteins of the beta 2AR with Gsalpha splice variants, which ensure precise receptor/G-protein stoichiometry, we could show that the subtle differences in GDP affinity between Gsalpha S and Gsalpha L have important consequences for the interaction with the beta 2AR, i.e. Gsalpha L confers to the beta 2AR some properties of a constitutively active receptor. Future studies will have to examine the effects of partial and inverse agonists of the beta 2AR in tissues and cells expressing Gsalpha S and Gsalpha L at different levels and to further define the physiological and pharmacological implications of the differences that we have discovered for the interaction of the beta 2AR with the two splice variants of Gsalpha . Because the overall properties of the beta 2AR and Gsalpha and their interaction were not changed as a result of fusion, this approach may be applied to a broad variety of receptors and G-proteins to uncover subtle differences in the interaction of closely related G-protein alpha -subunits with GPCRs.

    ACKNOWLEDGEMENTS

We are indebted to Dr. Henry R. Bourne for providing the three-dimensional model of transducin-alpha and most helpful discussion. We thank Dr. Hans Schambye for his help with the preparation of the manuscript.

    FOOTNOTES

* This work was supported by the Howard Hughes Medical Institute and National Institutes of Health Research Grant R01-MH34007 (to E. S.-B.).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.

§ Recipients of a research fellowship of the Deutsche Forschungsgemeinschaft.

Present address: Dept. of Cellular Physiology, Institute of Medical Physiology 12.5, The Panum Institute, University of Copenhagen, Blegdamsvej 3, DK-2100 Copenhagen N, Denmark.

parallel Permanent address: Dept. of Pharmacology, Vanderbilt School of Medicine, Nashville, TN 37232-6600.

Dagger Dagger To whom correspondence should be addressed: Howard Hughes Medical Institute, B-157, Beckman Center, Stanford University Medical School, Stanford, CA 94305-5428. Tel.: 650-723-7069; Fax: 650-498-5092; E-mail: kobilka{at}cmgm.stanford.edu.

1 The abbreviations used are: GPCR(s), G-protein-coupled receptor(s); beta 2AR, beta 2-adrenoreceptor; beta 2ARCAM, constitutively active mutant of the beta 2AR; Gsalpha , alpha -subunit of the G-protein Gs; Gsalpha L, long splice variant of the alpha -subunit of Gs; Gsalpha S, short splice variant of the alpha -subunit of Gs; beta 2ARGsalpha L, fusion protein consisting of the beta 2-adrenoreceptor and the long splice variant of Gsalpha ; beta 2ARGsalpha S, fusion protein of the beta 2-adrenoreceptor and the short splice variant of Gsalpha ; DCI, dichloroisoproterenol; [3H]DHA, [3H]dihydroalprenolol; EPH, (-)-ephedrine; GDPbeta S, guanosine 5'-O-(2-thiodiphosphate); GMPS, guanosine 5'-phosphorothioate; GTPgamma S, guanosine 5'-O-(3-thiotriphosphate); (-)-ISO; (-)-isoproterenol; (+)-ISO, (+)-isoproterenol; ICI, ICI 118,551; SAL, salbutamol; DOB, dobutamine; ALP, (-)-alprenolol; AC, adenylyl cyclase; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction.

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Abstract
Introduction
Procedures
Results
Discussion
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