The G Protein beta  Subunit Is a Determinant in the Coupling of Gs to the beta 1-Adrenergic and A2a Adenosine Receptors*

William E. McIntireDagger, Gavin MacCleery, and James C. Garrison

From the Department of Pharmacology, University of Virginia Health System, Charlottesville, Virginia 22908

Received for publication, December 13, 2000, and in revised form, February 7, 2001


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The signaling specificity of five purified G protein beta gamma dimers, beta 1gamma 2, beta 2gamma 2, beta 3gamma 2, beta 4gamma 2, and beta 5gamma 2, was explored by reconstituting them with Gs alpha  and receptors or effectors in the adenylyl cyclase cascade. The ability of the five beta gamma dimers to support receptor-alpha -beta gamma interactions was examined using membranes expressing the beta 1-adrenergic or A2a adenosine receptors. These receptors discriminated among the defined heterotrimers based solely on the beta  isoform. The beta 4gamma 2 dimer demonstrated the highest coupling efficiency to either receptor. The beta 5gamma 2 dimer coupled poorly to each receptor, with EC50 values 40-200-fold higher than those observed with beta 4gamma 2. Strikingly, whereas the EC50 of the beta 1gamma 2 dimer at the beta 1-adrenergic receptor was similar to beta 4gamma 2, its EC50 was 20-fold higher at the A2a adenosine receptor. Inhibition of adenylyl cyclase type I (AC1) and stimulation of type II (AC2) by the beta gamma dimers were measured. beta gamma dimers containing Gbeta 1-4 were able to stimulate AC2 similarly, and beta 5gamma 2 was much less potent. beta 1gamma 2, beta 2gamma 2, and beta 4gamma 2 inhibited AC1 equally; beta 3gamma 2 was 10-fold less effective, and beta 5gamma 2 had no effect. These data argue that the beta  isoform in the beta gamma dimer can determine the specificity of signaling at both receptors and effectors.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Signal transduction involving heterotrimeric G proteins1 is a universal mechanism for the integration of extracellular stimuli such as hormones, neurotransmitters, odorants, and light (1, 2). The components involved in this signaling cascade are diverse, including a large number of receptors, G protein alpha  and beta gamma subunits and effectors. Even though the diversity of the proteins in this system could potentially account for the known specificity of signaling in differentiated cells, the mechanisms for determining specificity are not completely defined. The beta -adrenergic receptor is one of the most well characterized seven transmembrane spanning receptors, and provides an excellent example of selective coupling to a particular alpha  subunit, Gs. When activated, Gs can stimulate all nine adenylyl cyclase isoforms (3, 4). The G protein beta gamma dimer, when released after receptor activation, is also able to regulate adenylyl cyclase (5). However, the regulation of the various isoforms of adenylyl cyclase by the beta gamma dimer is much more selective; apparently, only AC2, AC4 (6, 7), and AC7 (8) are stimulated by beta gamma , whereas the neuronal-specific AC1 (4) and possibly AC5 and AC6 are inhibited by the dimer (9). Moreover, there is evidence that AC2 does not respond well to dimers composed of certain beta  and gamma  subunits (10) or to dimers containing the phosphorylated gamma 12 subunit (11). Thus, to understand fully the regulation of adenylyl cyclase by a Gs-coupled receptor, one needs to know which beta gamma dimers are most likely to support receptor G protein coupling and the effects of beta gamma dimers on the various isoforms of adenylyl cyclase.

The number of functionally distinct beta gamma dimers is potentially very large, with seven G protein beta  isoforms (including two splice variants) and 12 gamma  isoforms characterized to date (12-14). Most in vitro studies involving coupling of receptors to Gs alpha  or regulation of adenylyl cyclases by distinct beta gamma dimers have used dimers containing the beta 1, beta 2, or beta 5 subunits (15, 16). The ubiquitous cellular and tissue distribution of Gs alpha  provides the potential for interaction with all five beta  isoforms and underscores the importance of understanding the role of the different beta  isoforms on signaling pathways involving Gs alpha . For example, the antisense studies of Kleuss et al. (17-19) suggest that specific isoforms of the heterotrimer couple to different receptors, and a number of in vitro studies imply that defined beta gamma dimers may be released upon receptor activation (16, 20, 21). In addition, isolation of G protein heterotrimers from a variety of tissues using chromatography or immunoprecipitation has shown that certain beta  and gamma  subunits preferentially associate with one another as well as with distinct alpha O isoforms (22, 23). These data suggest that specific combinations of G protein subunits do exist in vivo and may have specialized roles in various signaling cascades.

To examine the roles of the various beta  subunits in receptor-Gs coupling, and in regulating adenylyl cyclase, recombinant Gs alpha  and beta gamma dimers containing beta 1-5 complexed with gamma 2 were expressed in baculovirus-infected Sf9 insect cells and purified. Proteins were then reconstituted into partially purified Sf9 cell membranes overexpressing either the beta 1-adrenergic receptor, the A2a adenosine receptor, AC1 or AC2. The effects of the beta 1-5gamma 2 combinations were measured in four assays as follows: 1) the ability to couple the Gs alpha  subunit to the beta 1-adrenergic receptor; 2) the ability to couple the Gs alpha  subunit to the A2a adenosine receptor; 3) the ability to stimulate AC2; and 4) the ability to inhibit AC1. Clear differences were observed among the five beta gamma dimers in both receptor coupling and effector regulation, suggesting that the diversity of the beta  subunit contributes extensively to signaling specificity.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Construction of Recombinant Baculoviruses-- Construction of baculoviruses encoding the beta 1, beta 2, beta 5, gamma 2, gamma 2FH, the Gs alpha  and, Gi1 alpha  subunits has been described (11, 24-26). The viruses encoding AC1 and AC2 were the kind gift of R. Iyengar (27, 28). Baculoviruses encoding the rat beta 1-adrenergic receptor and the human A2a adenosine receptor were gifts from E. Ross (University of Texas, Southwestern Medical Center) and J. Linden (University of Virginia), respectively (29). The human beta 3 cDNA (30), a gift from S. R. Ikeda (Guthrie Institute), was excised from pCI with EcoRI and NotI; the mouse beta 4 cDNA (31), a gift from W. F. Simonds (National Institutes of Health) was excised from pcDNA3 with BamHI and XbaI. The human beta 3s cDNA, a gift from D. Rosskopf (Institute for Pharmacology, Essen, Germany), is a truncated variant of the full-length beta 3 cDNA, in which the beta 3s protein product has a deletion of amino acids 168-208 (32); excision of the beta 3s cDNA from pGEMT was accomplished with BamHI and PstI. The existing restriction sites were used to ligate digestion products into the multiple cloning site immediately downstream of the polyhedron promoter in the baculovirus transfer vector, pVL1393. All clones were sequenced to confirm the fidelity of the cDNA in pVL1393. Recombinant baculoviruses for beta 3, beta 3s, and beta 4 were prepared by co-transfection of linear wild type BaculoGold® viral DNA (PharMingen) with pVL1393 transfer vectors containing the specific beta  sequences into Sf9 cells as described (26) and purified with one round of plaque purification (33).

Expression and Purification of Recombinant G Protein alpha  and beta gamma Dimers-- Sf9 cells were infected with recombinant baculoviruses encoding the desired alpha  and/or beta gamma dimer combinations at a multiplicity of infection of three and harvested 48-60 h after infection. beta gamma dimer combinations containing beta 1-4 and gamma 2 were purified by Gi1 alpha  affinity chromatography as described (34). The dimer containing beta 5 was expressed with a gamma 2 subunit engineered to have a hexahistidine and FLAG tag (26) at the N terminus (gamma 2HF) and purified from isolated Sf9 cell membranes by FLAG affinity and Ni2+ affinity chromatography, followed by anion exchange chromatography (16).

Mass spectrometry was used to examine post-translational processing of the gamma 2 subunit. Purified beta gamma dimers were analyzed by matrix-assisted laser desorption ionization-mass spectrometry to obtain masses of the gamma  subunits as described in Lindorfer et al. (35). For beta gamma dimers with a protein concentration of less than 150 ng/µl, acetone precipitation was used to concentrate the protein before mass analysis (36). Post-translational processing of the gamma 2 isoform includes cleavage of the N-terminal methionine, acetylation of the resulting N-terminal alanine, geranylgeranylation at the cysteine four residues from the C terminus, cleavage of three C-terminal residues, and carboxymethylation of the resulting C-terminal geranylgeranylated cysteine. These post-translational modifications have been observed in gamma  subunits isolated from bovine brain (37) and in Sf9 cells (35). The predicted mass of the properly processed gamma 2 isoform is 7750 Da; insertion of a His-Flag (HF) tag at the N terminus increases the predicted mass to 10,013 Da. Mass spectra of the purified beta gamma isoforms containing either gamma 2 or gamma 2HF demonstrated that the major mass in each spectrum was compatible with these predicted masses within the accuracy of the instrument (35). For example, in one set of purified beta gamma dimers, the experimental masses of the gamma 2 subunits were as follows: beta 1gamma 2, 7758 Da; beta 2gamma 2, 7764 Da; beta 3gamma 2, 7760 Da; beta 4gamma 2, 7759 Da; and beta 5gamma 2FH, 10,020 Da.

Attempts were made to purify beta 3s combined with various gamma  subunits. A protein with the appropriate molecular weight was expressed in Sf9 cells as judged by immunoblotting with a beta -common antibody (PerkinElmer Life Sciences 808); however, the major barrier to purification was that it was not possible to solubilize the protein from the Sf9 cell pellet. For example, soluble extracts of whole cell pellets prepared using 1% (v/v) Genapol, 1% (w/v) CHAPS, or 1% (w/v) Cholate contained little beta 3s protein in supernatant fractions that could be detected with the beta -common antibody. Expression of various alpha  and or the gamma 2FH, gamma 5, and gamma 7 subunits with beta 3s in Sf9 cells did not affect the solubility of the protein (data not shown), and thus characterization of this protein was not pursued.

Gs alpha  was overexpressed with a beta 1 subunit engineered to have a hexahistidine and FLAG tag (11) at the N terminus (beta 1HF), along with gamma 2HF, and purified using a modification of the method described by Kosaza and Gilman (38). Briefly, harvested cells were resuspended in half the infection volume with cell lysis buffer (20 mM Tris, pH 8.0, 10 µM GDP, 17 µg/ml PMSF, and 2 µg/ml pepstatin, leupeptin, and aprotinin). After resuspension, cells were lysed by nitrogen cavitation (25), and membranes were collected by centrifugation at 28,000 × g for 20 min at 4 °C. A Potter-Elvehjem homogenizer was used to resuspend the pellets in a quarter of the original resuspension volume (~63 ml) of cell lysis buffer containing 10 µg/ml DNase. After a 15-min incubation on ice, membranes were collected again by centrifugation at 28,000 × g for 20 min at 4 °C and resuspended with a Potter-Elvehjem homogenizer in a volume of extraction buffer (20 mM Tris, pH 8.0, 150 mM NaCl, 1 mM MgCl2, 0.5% (v/v) Genapol, 1 mM beta -mercaptoethanol, 50 µM GDP, 17 µg/ml PMSF, and 2 µg/ml of leupeptin, aprotinin, and pepstatin) equivalent to 10 times the weight of the original cell pellet. Membranes containing expressed G protein were resuspended and stirred with extraction buffer for 1 h at 4 °C, followed by centrifugation at 142,000 × g for 1 h at 4 °C; the solubilized Gs alpha /beta 1FHgamma 2FH supernatant extracts (typically about 100 ml) were flash-frozen in liquid nitrogen and stored at -80 °C.

To begin the Gs alpha  purification, the extract was diluted with an equal volume of Ni2+ column buffer (20 mM Tris, pH 8.0, 150 mM NaCl, 1 mM MgCl2, 0.2% (v/v) Genapol, 1 mM beta -mercaptoethanol, 10 µM GDP, 5 mM imidazole, 17 µg/ml PMSF, and 2 µg/ml pepstatin, leupeptin, and aprotinin) and loaded onto a Ni2+-NTA Superflow column at 2 ml/min. Unless otherwise noted, all steps were performed at 4 °C. The volume of the column bed was ~5% of the volume of the Genapol extract. The column was washed with 6 column volumes of Ni2+ column buffer, 6 column volumes of Ni2+ column buffer containing 300 mM NaCl, and 3 more column volumes of Ni2+ column buffer. At this point, the column and buffers were warmed to room temperature for 10-20 min, and Gs alpha  was activated and eluted with 4 column volumes of activation buffer (Ni2+ column buffer containing 50 mM MgCl2, 10 mM NaF, and 30 µM AlCl3) also warmed to room temperature. Although the increased temperature facilitates activation of the alpha  subunit, this step should be completed as quickly as possible, as functional activity of alpha  decreases with prolonged elevation of temperature. Pilot experiments using SDS-PAGE to identify the Gs alpha  subunit indicated that the first 8 ml of eluate after the void volume contained the protein. Thereafter, these fractions were collected on ice and pooled. The fractions containing Gs alpha  were diluted 5-fold with 15Q buffer (20 mM Tris, pH 8.0, 1 mM EDTA, 1 mM MgCl2, 0.1% (w/v) CHAPS, 1 mM DTT, 10 µM GDP) and loaded onto a 200-µl 15Q anion exchange column. This dilution facilitates adsorption of the protein to the column by reducing of the Cl- concentration to ~50 mM. In addition to concentrating the protein, the 15Q step is necessary to remove AlF<UP><SUB>4</SUB><SUP>−</SUP></UP>, imidazole, and Genapol, which would itself be concentrated along with the protein in the next concentration step. After the protein was loaded, the column was washed with 15Q buffer containing 10 mM NaCl (15Q buffer A) for 20 min at 1 ml/min. Protein was then eluted with 15Q buffer containing 600 mM NaCl (15Q buffer B) in a linear gradient of 0-50% 15Q buffer B over 15 min. One-ml fractions were collected, and 12% SDS-PAGE followed by either immunoblotting or silver staining with purified Gs alpha  as a standard was used to determine which fractions contained Gs alpha .

Fractions from the 15Q column containing Gs alpha  were pooled and concentrated with a Centricon 30 that had been passivated with a 1% BSA solution as described (16). The concentrated protein was diluted 10-fold with 15Q buffer containing 100 mM NaCl to reduce the high NaCl concentration that resulted from the elution from the 15Q column, and then concentrated once more to a volume of 100-200 µl, aliquoted, and stored at -80 °C. The yield of purified Gs alpha  from 10 g of Sf9 cell pellet (wet weight) was typically 10-20 µg. All protein estimates were determined using scanning densitometry of silver-stained gels as described previously (26), with standard curves generated from ovalbumin standards.

Gi1 alpha  was purified by a similar method with the following exceptions. The Ni2+ column buffer contained 20 mM Tris, pH 8.0, 150 mM NaCl, 0.2% (w/v) CHAPS, 1 mM beta -mercaptoethanol, 10 µM GDP, 5 mM imidazole, 17 µg/ml PMSF, and 2 µg/ml pepstatin, leupeptin, and aprotinin. Protease inhibitors and imidazole were removed from the elution step, and the Gi1 alpha  was taken directly to a Centricon 30 where it was concentrated and diluted 10-fold with Ni2+ column buffer supplemented with 2 mM MgCl2. This step was repeated, and 100-200 µl of Gi1 alpha  at 100-200 ng/µl were stored in aliquots at -80 °C. As one criterion for the viability of the Gs and Gi1 alpha  subunits, the ability of the proteins to bind GTPgamma S in solution was measured. The stoichiometry of nucleotide binding of two preparations of Gs alpha  averaged 0.9 mol/mol. The Gi1 alpha  subunit bound GTPgamma S at a stoichiometry of ~0.3 mol/mol and also coupled effectively to the A1 adenosine receptor in assays similar to the one shown in Fig. 2A (data not shown).

Preparation of Membranes Containing Recombinant beta 1-Adrenergic Receptors, A2a Adenosine Receptors, or Adenylyl Cyclases-- Sf9 cells were infected with recombinant baculoviruses encoding either the rat beta 1-adrenergic receptor, the human A2a adenosine receptor, and either type I or type II adenylyl cyclase (27, 28). In the case of the beta 1-adrenergic receptor, harvested cells were resuspended in membrane homogenization buffer (20 mM HEPES, pH 7.5, 2 mM MgCl2, 1 mM EDTA, 17 µg/ml PMSF, and 2 µg/ml leupeptin and aprotinin), and cells were lysed by nitrogen cavitation. The cell lysate was centrifuged at 750 × g to pellet unbroken cells and nuclei. Membranes were prepared from the supernatant of the low speed spin by centrifugation at 28,000 × g for 20 min at 4 °C. Endogenous G proteins were then stripped from the membranes or inactivated by incubation with urea. Membranes containing the beta 1-adrenergic receptor were homogenized with resuspension buffer (50 mM HEPES, pH 7.5, 3 mM MgSO4, 1 mM EDTA, 17 µg/ml PMSF, and 2 µg/ml leupeptin and aprotinin) containing 7 M urea and allowed to incubate for 30 min at 4 °C. Resuspension buffer was used to dilute the membranes to 4 M urea prior to centrifugation at 142,000 × g for 30 min at 4 °C. Membranes were washed twice with resuspension buffer, and homogenization buffer was used to resuspend the membranes, which were stored in aliquots at -80 °C. Membranes containing the A2a adenosine receptor were prepared using essentially the same method, except that homogenization buffer consisting of 25 mM HEPES, pH 7.5, 100 mM NaCl, 1% (w/v) glycerol, 17 µg/ml PMSF, and 2 µg/ml leupeptin, and aprotinin was used throughout the preparation. Radioligand binding experiments with [125I]iodocyanopindolol and 125I-ZM-241385 were used to determine receptor number and affinity for the beta 1-adrenergic and A2a adenosine receptors, respectively. Stripping membranes with urea did not greatly affect the pharmacological properties of these two receptors (data not shown). The GTPgamma S binding experiments presented under "Results" were obtained using a single preparation of membranes expressing either the beta 1-adrenergic or the A2a adenosine receptor. Membranes expressing AC1 or AC2 were prepared as described previously (28). Total membrane protein concentration was determined by BCA assay using bovine serum albumin as a standard, and aliquots of membranes were stored at -80 °C.

Measurement of Agonist-stimulated GTPgamma S Binding to Gs alpha  after Reconstitution with beta gamma into Membranes Expressing either the beta 1-Adrenergic Receptor or the A2a Adenosine Receptor-- Kinetic parameters of agonist-stimulated binding of [35S]GTPgamma S to Gs alpha  in the presence of different concentrations of beta 1gamma 2 were established with time course experiments. Aliquots of Sf9 cell membranes containing the beta 1-adrenergic receptor were pelleted by centrifugation and resuspended in 100-400 µl of GTPgamma S binding buffer (25 mM HEPES, pH 7.4, 100 mM NaCl, 1 mM EDTA, 5 mM MgCl2, 1 mM DTT, 0.1% BSA, 0.5 µM GDP, and 1 µM AMP-PNP) with a 28-gauge needle. The membrane suspension was reconstituted with 5 nM Gs alpha  subunit such that the Gs alpha :receptor ratio was 26:1; different concentrations of beta 1gamma 2 were then added and allowed to incubate for 30 min at 4 °C. The incubation temperature was increased to 25 °C for 10 min to equilibrate the reconstituted system to the reaction temperature; additions of [35S]GTPgamma S (final concentration 10 nM) and isoproterenol (final concentration 1 mM) initiated the time course. The binding of [35S]GTPgamma S to receptor-activated Gs alpha  was measured at 1-min intervals by vacuum filtration. Increasing concentrations of beta gamma increased the rate of receptor-catalyzed exchange of GDP for GTPgamma S on Gs alpha . The observed rates were relatively linear (see Fig. 2A), thus the effect of beta gamma was quantified by the amount of [35S]GTPgamma S binding measured at a reaction time of 7 min. Seven minutes were chosen as a compromise that allowed a measurable signal before the reaction rate slowed.

By using this protocol, 10 different beta gamma concentrations, ranging from 0.08 to 20 nM beta xgamma 2, were examined to determine the efficiency of coupling Gs alpha  to receptor. Membranes were reconstituted with 5 nM Gs alpha  and the indicated beta gamma concentrations in a total volume of 30 µl per tube. The concentrations of beta gamma were prepared by serial dilution with GTPgamma S binding buffer containing 0.1% CHAPS; 2 µl of each beta gamma concentration was diluted to the final incubation volume of 40 µl, giving a CHAPS concentration of 0.005% for all but the highest concentrations of beta gamma . After the incubation protocols described above, 8 µl of buffer containing [35S]GTPgamma S (~1,000,000 dpm) and isoproterenol were added to each tube to start the 7-min reaction. The reaction was terminated as described above, and efficiency of coupling was determined by plotting [35S]GTPgamma S binding as a function of beta gamma concentration (Fig. 2B). Receptor specificity for Gs alpha  was demonstrated using the same protocol by reconstitution of Gi1 alpha  with the beta 1-adrenergic receptor (Fig. 2B).

A slight modification of this protocol was used to obtain dose response experiments with beta gamma and the A2a adenosine receptor. The Gs alpha :receptor ratio was 1.3:1, and in order to break down endogenous adenosine that is continuously generated in membrane preparations, adenosine deaminase was added to the membrane suspension before the 30-min incubation at a concentration of 14 units of activity/ml. The A2a adenosine receptor was activated with 100 nM 5'-N-ethylcarboximide adenosine.

Measurement of Adenylyl Cyclase Activity-- In addition to activated Gs alpha , the diterpene forskolin can stimulate all isoforms of adenylyl cyclase, whereas Ca2+/calmodulin can stimulate AC1, AC3, and AC8 (3). Gs alpha  and forskolin were natural choices for this study, as they both activated AC1 and AC2, whereas Ca2+/calmodulin will not activate AC2. Forskolin was used to activate successfully AC1, but inhibition of the activity by beta gamma was not as robust as in the case of the Gs alpha -activated AC1 (data not shown); Gs alpha  was therefore chosen as the activator for both AC1 and AC2. GTPgamma S-activated Gs alpha  was prepared by incubation of protein with gel filtration buffer (50 mM HEPES, pH 8.0, 150 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 1 mM DTT, and 0.1% CHAPS) with the addition of 5 mM MgCl2 (10 mM total) and 10 µM GTPgamma S for 30 min at 30 °C. Unbound GTPgamma S was removed by centrifugation with a 2-ml P6 desalting column equilibrated with gel filtration buffer using the method described in Yasuda et al. (39). Reconstitutions of Gs alpha  and beta gamma were performed as described by Lindorfer et al. (16). Maximal activity of AC1 and AC2 was confirmed by stimulation with increasing concentrations of GTPgamma S-activated Gs alpha . Cyclic AMP production was measured using a radioimmunoassay (40). Enzymatic activity reached a maximal rate of ~5 nmol of cAMP/min/mg protein for both AC1 and AC2, which is consistent with previous preparations from this laboratory (16), and with published data (41).

For experiments involving the effect of different beta gamma isoforms on adenylyl cyclase, activated Gs alpha  is required at concentrations below what is necessary for maximal stimulation of ACII; therefore, 10 nM activated Gs alpha  was chosen as the concentration suitable for co-activation of AC2. Complete activation of AC1 is required to observe inhibition by beta gamma ; for this reason, 50 nM Gs alpha  was used for activation of AC1. Adenylyl cyclase experiments utilized a single preparation of each membrane type.

Calculation and Expression of Results-- In experiments using the beta 1-adrenergic and A2a adenosine receptors, data from at least three experiments and two different sets of beta gamma dimers were normalized as a percentage of maximal GTPgamma S binding as determined by the one-site binding curves generated by GraphPad Prism. After normalization, the data were averaged for each beta gamma isoform, and GraphPad Prism was used to obtain estimates of the EC50 values and statistical analysis of the binding curves. These data are presented in Table I.

GraphPad Prism was used to estimate EC50 and Vmax values for the potentiation of Gs alpha -stimulated AC2 activity by beta gamma . At least three experiments using data from two different sets of beta gamma were analyzed, and average values were reported in Table I. For AC1, GraphPad Prism was used to generate inhibition curves for each of the experiments with the different beta gamma dimers; the data were then normalized as percent inhibition of the estimated rate of cAMP production with 50 nM GTPgamma S-activated Gs alpha  in the absence of beta gamma . Normalized data from at least three experiments and two different sets of beta gamma dimers were averaged and analyzed by GraphPad Prism to obtain IC50 estimates (Table I). Statistical significance for differences among binding curves for both receptors and AC1 and AC2 was determined using the F-statistic; this technique is able to discern small but significant differences between two binding curves (42).

Materials-- Reagents for Sf9 cell culture and purification of beta gamma dimers has been described previously (16, 25, 26, 34). 125I-ZM-241385 was a kind gift from J. Linden, University of Virginia; baculovirus transfer vector was from Invitrogen; the BaculoGold kit was from PharMingen; DNase, GDP, imidazole, isoproterenol, and HEPES were from Sigma; adenosine deaminase, CHAPS, and GTPgamma S were from Roche Molecular Biochemicals; P-6 desalting gel was from Bio-Rad; 10% Genapol C-100 was from Calbiochem; Ni2+-NTA Superflow resin was from Qiagen; [35S]GTPgamma S and [125I]iodocyanopindolol from PerkinElmer Life Sciences; Source 15Q anion exchange resin was from Amersham Pharmacia Biotech; type HA 0.45-µm nitrocellulose filters and Centricon 30 concentrators were from Millipore. All other materials were of the highest available purity.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Preparation of G Protein alpha  and beta gamma Subunits-- Fig. 1A presents a silver-stained gel showing the purity of the five beta gamma dimers used in this study. Fig. 1B presents a similar gel showing the purity of the Gs alpha  used. Significantly, both the beta gamma dimers and the Gs alpha  subunit were purified using biological affinity columns. The dimers containing beta 1-4 were purified with a Gi1 alpha -agarose column ensuring that the proteins bound to alpha  subunits with high affinity and that the C terminus of the gamma  subunit was properly modified (see "Experimental Procedures"). Even so, a beta  doublet was occasionally observed in the SDS-PAGE analysis of beta 3gamma 2. The reasons for this behavior are not understood. The beta 5gamma 2HF dimer was purified from the membrane fraction of Sf9 cells using Ni2+/FLAG chromatography. Mass spectrometry was used to demonstrate that the gamma  subunit in this dimer was modified to the same extent as dimers purified on the Gi1 alpha -agarose column, and the biological activity of beta 5gamma 2HF was vetted by determining its ability to activate PLC-beta (10, 16) to the same extent as beta 1gamma 2 (data not shown). Gs alpha  was purified by AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> elution from a Gs alpha :beta 1HFgamma 2HF heterotrimer bound to a Ni2+-NTA-agarose column. The activity of the Gs alpha  preparation was verified by its ability to activate AC1 and AC2. Fig. 1, C and D, shows that the alpha  subunit activated these cyclases 4-8-fold, with EC50 values of 1.9 nM for AC2 and 8.5 nM for AC1. These results are similar to other values reported in the literature for Gs alpha  expressed in Sf9 cells (43), validating the quality of both the purified Gs alpha  and the membrane preparations of AC1 and AC2.


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Fig. 1.   Purity of G protein alpha  and beta  subunits. A, the five isoforms of the beta  subunit were overexpressed in Sf9 insect cells with the gamma 2 or gamma 2FH subunit and purified by Gi1 alpha  affinity chromatography (beta 1-4gamma 2) or Ni2+-NTA affinity chromatography (beta 5gamma 2FH). B, Gs alpha  was overexpressed in Sf9 insect cells with a beta 1gamma 2 dimer containing a hexahistidine tag. The heterotrimer was adsorbed to a Ni2+-NTA column, and Gs alpha  was eluted specifically with AlF<UP><SUB>4</SUB><SUP>−</SUP></UP>. Purity of beta gamma dimers (250 ng of each isoform) and Gs alpha  (150 ng) was visualized by silver staining after separation by 12% SDS-PAGE; positions of molecular weight markers are indicated at the right. C, Sf9 cell membranes expressing AC2 were incubated with increasing concentrations of Gs alpha  activated with GTPgamma S, and cAMP levels were determined using a radioimmunoassay; the calculated EC50 for the experiment shown is 1.9 nM. D, Sf9 cell membranes expressing AC1 were characterized as in C; the calculated EC50 for the experiment shown is 8.5 nM.

The Ability of Different beta gamma Subunits to Support Coupling of Receptors to Gs alpha -- A major goal of this study was to examine the possibility that different beta  subunits interact selectively with certain G protein-coupled receptors. Since exchange of GDP for GTP is the first step of G protein signaling subsequent to receptor activation, an agonist-dependent GTPgamma S-binding assay was used. Fig. 2A presents an experiment performed with membranes expressing the beta 1-adrenergic receptor reconstituted with Gs alpha  and two concentrations of the beta gamma dimer. Note that the rate of isoproterenol-stimulated GTPgamma S binding is nearly linear and highly dependent on the concentration of beta gamma dimer. The triangles in Fig. 2A represent a basal rate of GTPgamma S binding to membranes reconstituted with Gs alpha ; this rate was observed in the absence of beta gamma (as illustrated in the figure) or with a fully reconstituted system in the absence of isoproterenol. Coupling of receptor to G protein is a composite of many biochemical interactions, the most important being the interactions of alpha -beta gamma and receptor-alpha -beta gamma . Receptor-beta gamma interactions were probed with a variation of the protocol designed to be poised on the concentration of the beta gamma dimer. To define precisely the ability of beta 1gamma 2 to support coupling of Gs alpha  to the beta 1-adrenergic receptor, a reaction time of 7 min was chosen to generate concentration-response curves with the different beta gamma isoforms. A representative concentration-response curve performed with beta 1gamma 2 is presented in Fig. 2B; the apparent EC50 estimated from this curve is 0.7 nM. The fidelity of receptor-alpha interactions was confirmed by the reconstitution of the beta 1-adrenergic receptor with Gi1 alpha , which demonstrates that even high concentrations of beta 1gamma 2 (20 nM) do not support isoproterenol-dependent GTPgamma S binding (Fig. 2B) to the Gi1 alpha  subunit. In contrast, when the same Gi1 alpha  was reconstituted with beta 1gamma 2 into membranes containing the A1 adenosine receptor, robust agonist-dependent GTPgamma S binding was observed (data not shown, but see Ref. 39).


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Fig. 2.   Concentration dependence of beta 1gamma 2 on coupling Gs alpha  to the beta 1-adrenergic receptor. A, time course of [35S]GTPgamma S binding to Sf9 cell membranes containing the beta 1-adrenergic receptor and reconstituted with 5 nM Gs alpha  and 0, 0.5, or 10 nM beta 1gamma 2. Ten nM [35S]GTPgamma S and 1 mM isoproterenol (Iso) were added at time 0, and bound nucleotide was determined at 1-min intervals by vacuum filtration. B, increasing concentrations of beta 1gamma 2 were reconstituted with Gs alpha  or Gi1 alpha  into membranes containing the beta 1-adrenergic receptor, and the reaction was initiated as described in C; the amount of [35S]GTPgamma S bound was determined at the 7-min time point. The EC50 for beta gamma supported coupling of Gs alpha  to receptor was 0.7 nM as determined by fitting the data to a one-site model.

The experiment performed with Gs alpha  in Fig. 2B was repeated using each of the five beta gamma isoforms, and the normalized data are shown in Fig. 3A. Highest in coupling efficiency was beta 4gamma 2, with an EC50 of 0.5 nM; beta 2gamma 2 was considerably less efficient with an EC50 of 2.7 nM. There were slight but statistically significant differences in most of the beta gamma isoforms, with only beta 1gamma 2 and beta 3gamma 2 showing no differences and an EC50 of 1.0 nM. Poorest of all at coupling Gs alpha  to the beta 1-adrenergic receptor was beta 5gamma 2 with an EC50 of 17.1 nM (see Table I).


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Fig. 3.   Comparison of ability of different beta gamma isoforms to couple Gs alpha  to the beta 1-adrenergic and A2a adenosine receptors. A, five beta xgamma 2 isoforms were reconstituted with 5 nM Gs alpha  and membranes containing the beta 1-adrenergic receptor and the efficiency of coupling measured as described under "Experimental Procedures." Data from three experiments were normalized as a percent of maximal binding of [35S]GTPgamma S, and the averaged data plotted; error bars, most of which were within ±10%, were omitted for clarity. B, five beta xgamma 2 isoforms were tested as in A, but with the A2a adenosine receptor. Data from at least three experiments were normalized and plotted as in A. EC50 estimates of the data sets for each beta gamma dimer can be found in Table I. C-G, data from A and B were replotted to highlight differences in each particular beta xgamma 2 isoform between the beta 1-adrenergic receptor (beta ) and the A2a adenosine receptor (A2a). Dotted lines indicate beta gamma concentrations of 1 and 10 nM on the x axis. C, beta 1gamma 2; D, beta 2gamma 2; E, beta 3gamma 2; F, beta 4gamma 2; G, beta 5gamma 2FH.

                              
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Table I
Comparison of the ability of five different beta gamma isoforms to couple Gs alpha  to the beta 1-adrenergic receptor, the A2a adenosine receptor, or to potentiate the activation of ACII by Gs alpha , or inhibit stimulation of ACI by Gs alpha  
EC50 and IC50 values were determined by fitting the averaged data (n >=  3) to single site binding or competition curves as described under "Experimental Procedures"; bold numbers indicate EC50 or IC50 values from the statistical fit, and numbers in parentheses represent the range of values within the 95% confidence interval. Statistical significance (indicated by the superscript) was determined using the F statistic.

To determine if the rank order of affinities determined with the beta 1-adrenergic receptor was the same with another Gs-linked receptor, we examined the ability of the panel of beta gamma dimers to support coupling of Gs alpha  to the A2a adenosine receptor. Normalized data for each of the beta gamma isoforms and the A2a adenosine receptor are shown in Fig. 3B; the coupling efficiencies and statistical analysis are presented in Table I. Highest in affinity was beta 4gamma 2 with an EC50 of 1.3 nM; beta 2gamma 2 and beta 3gamma 2 were lower in affinity, both with EC50 values around 6 nM. Strikingly, beta 1gamma 2 was much less efficient at coupling to the A2a adenosine receptor, with an EC50 of 15.7 nM. Lowest in coupling efficiency was beta 5gamma 2, with an EC50 >100 nM.

Fig. 3, C-G, illustrates that there are striking differences in the ability of the two Gs-linked receptors to couple to the five different beta gamma isoforms. Perhaps the most dramatic differences occurred with the beta 1gamma 2 isoform, which coupled 15-fold more efficiently to the beta 1-adrenergic receptor than to the A2a adenosine receptor (Fig. 3C). Similarly, beta 3gamma 2 demonstrated a 7-fold difference between the two receptors (Fig. 3E). Note that beta 4gamma 2 was the most effective at coupling Gs alpha  to either receptor (Fig. 3F), and that beta 5gamma 2 coupled poorly (Fig. 3G). In contrast, there are minimal differences in the ability of beta 2gamma 2 to couple to either receptor (Fig. 3D). It is important to stress that the only differences in these five sets of experiments are the types of recombinant receptor expressed in the Sf9 membranes. The G protein alpha  and beta gamma subunits reconstituted into the membranes were identical in each case. Thus, the data clearly demonstrate that specific G protein beta  subunits exhibit distinct preferences for different receptors. Importantly, these preferences are a result of interactions of receptor with the type of beta  subunit in the dimer, since the Gs alpha -beta gamma interactions are presumably identical for both receptors.

Activation of AC2 by beta gamma Isoforms-- The dramatic differences in the ability of the panel of beta gamma dimers to couple to Gs-linked receptors imply that different beta gamma dimers might be released by receptor activation to act on downstream effectors. Since beta gamma is a known potentiator of Gs alpha -stimulated AC2 activity, and differences have been observed in the ability of dimers containing the beta 1 or beta 5 subunits to stimulate AC2 (9, 16), the ability of all five beta  subunits to activate AC2 was compared. The role of beta gamma in the activation of AC2 is particularly interesting in that beta gamma can increase the rate of cAMP production approximately 5-fold over the maximal effect of Gs alpha  (Fig. 4), suggesting beta gamma can regulate cAMP levels in vivo. Ten nM GTPgamma S-activated Gs alpha  and increasing concentrations of the five purified beta gamma isoforms were reconstituted with Sf9 membranes expressing AC2, and cAMP production was measured. A representative experiment is presented in Fig. 4. Dimers containing beta 1-4 were similar in their ability to potentiate AC2 activity in the presence of activated Gs alpha , with all dimers increasing cAMP production 4-6-fold (Vmax values ranging from 30 to 40 nmol of cAMP/min/mg of protein). Consistent with previous reports (16), beta 5gamma 2 was significantly less active. Data from at least three similar experiments were normalized and averaged to determine the EC50 values for each beta gamma dimer, and the results are summarized in Table I. The EC50 values for beta 1-4 range from 3.5 to 13 nM, and the value for beta 5gamma 2 is significantly higher at 76 nM. Careful analysis of the data indicates that the beta 2gamma 2 dimer is 3-fold less potent than the beta 1gamma 2 and beta 4gamma 2 dimers. Also of interest is the observation that dimers containing beta 3 and beta 4, which had not been previously tested on adenylyl cyclase, were as effective at stimulation of AC2 as dimers containing beta 1 and beta 2. These results suggest that all four beta  isoforms can effectively participate in Gs alpha  signaling pathways affecting the regulation of cAMP via AC2.


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Fig. 4.   Comparison of the potency of five beta xgamma 2 isoforms in the activation of AC2. Increasing concentrations of the indicated beta xgamma 2 dimers were incubated with 10 nM GTPgamma S activated Gs alpha  and membranes containing AC2. Data shown are representative experiments where each point is the average of duplicates; the calculated EC50 values for the data are as follows: beta 1gamma 2, 8.6 nM; beta 2gamma 2, 14.0 nM; beta 3gamma 2, 7.3 nM; beta 4gamma 2, 2.2 nM; and beta 5gamma 2FH, 76.8 nM. EC50 estimates from the averaged data sets for each beta gamma dimer can be found in Table I.

Inhibition of AC1 by beta gamma Isoforms-- AC1 is in very high concentration in neuronal tissue and is notable among the adenylyl cyclase isoforms in that it can be markedly inhibited by the beta gamma dimer. The activity of AC1 expressed in membranes was demonstrated with purified Gs alpha , which stimulated cAMP production with an EC50 of 8.5 nM (Fig. 1D). This activation of AC1 by Gs alpha  provided the opportunity to compare the inhibitory properties of all five beta gamma dimers. Increasing concentrations of purified beta gamma isoforms were reconstituted with 50 nM GTPgamma S-activated Gs alpha  into Sf9 membranes expressing AC1. A representative experiment illustrating the robust inhibition of AC1 elicited by beta 1gamma 2 is presented in Fig. 5A, where the dimer reduced cAMP production by over 50%. Similar experiments were performed with the full panel of beta gamma dimers. The data were normalized and are presented in Fig. 5B. Dimers containing the beta 1, beta 2, and beta 4 subunits were not different in their ability to inhibit AC1, with IC50 values ranging from 10 to 17 nM. In contrast, the beta 3gamma 2 dimer had an almost 10-fold higher IC50 of 110 nM, and surprisingly, no inhibition was observed with beta 5gamma 2 at the concentrations tested (Fig. 5B and Table I). These data suggest that beta gamma dimers composed of different beta  isoforms, especially those containing beta 3 and beta 5, may differentially regulate the type I and type II adenylyl cyclase isoforms.


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Fig. 5.   Comparison of the potency of five beta xgamma 2 isoforms at inhibiting Gs alpha -stimulated AC1 activity. A, representative experiment where increasing concentrations of beta 1gamma 2 were reconstituted with 50 nM GTPgamma S-activated Gs alpha  and membranes containing AC1; each point is the average of duplicates. The calculated IC50 for the data shown is 8.1 nM. B, increasing concentrations of five beta xgamma 2 isoforms were reconstituted with 50 nM GTPgamma S-activated Gs alpha  and membranes as in A. The y axis is expressed as percent of maximal cAMP production in the absence of beta gamma . Each point is the average of at least three separate experiments, ± S.E. Average values for all of the experiments can be found in Table I.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Although considerable research has focused on the role of the alpha  subunit in receptor signaling via G proteins, it is also clear that the beta gamma dimer is required for coupling the alpha  subunit to receptors (16, 39, 44, 45), and both the beta  and gamma  subunits appear to contribute to the interaction. Current evidence indicates that the C-terminal 10 amino acids of the gamma  subunit and the nature of the prenyl group (farnesyl versus geranylgeranyl) are very important determinants of the coupling of G proteins to receptors (20, 21, 39). Compared with alpha  and gamma , less is known about the important domains in the beta  subunit, but cross-linking experiments indicate that the C terminus of the beta  subunit is able to interact directly with the receptor (46). However, the regions close to the C terminus of the beta  subunit are likely to be quite discrete because mutations in amino acids His311, Arg314, and Trp332 have no effect on the ability of the dimers to support receptor coupling but cause a major disruption in the ability of the beta gamma dimer to activate PLC-beta or AC2 (47).

Whereas the diversity of the beta  and gamma  subunits offers attractive possibilities for determining the specificity of cellular signaling, the functional significance of this heterogeneity has not been completely elucidated. Some insight has come from an elegant set of experiments in which antisense mRNAs to various G protein alpha  and beta gamma subunits were injected into the nuclei of GH3 cells, leading to the observation that specific receptors couple to distinct isoforms of the G protein heterotrimer. These experiments indicated that the M4 muscarinic receptor preferred a heterotrimer composed of alpha O1:beta 3gamma 4, whereas the somatostatin receptor preferred a heterotrimer composed of alpha O2:beta 1gamma 3 (17-19). These results have been extended to other systems using the antisense approach (48), where it has been demonstrated that selectivity of receptor coupling to alpha  subunits of the Gi (49), Gq (50), and G12/13 (51) families depends upon the composition of the beta gamma dimer. However, few experiments have examined the composition of beta gamma dimers that couple receptors to Gs alpha . Moreover, reproduction of this signaling specificity using in vitro systems has proven difficult.

A major contribution of the experiments presented in this report is that a panel of highly active recombinant beta gamma dimers was used in a sensitive assay to compare the ability of five beta  subunits to couple to two different Gs-linked receptors. The ability of a defined heterotrimer to support coupling of an alpha  subunit to a particular receptor depends primarily on the affinity of the receptor for the heterotrimer (48), and presumably on the affinity of the interaction between the alpha  and beta gamma subunits (16, 26). Since the order of coupling efficiencies in the present experiments was quite different between the beta 1-adrenergic and the A2a adenosine receptors, the possibility that the differences observed here were due to differences in the alpha :beta gamma affinity seems unlikely. Thus, the order of EC50 values for each receptor is most likely a reflection of its preference for the particular beta  subunit examined. The beta 5gamma 2 dimer, which couples M1 muscarinic receptors efficiently to the Gq alpha  subunit (16), was particularly ineffective at coupling the Gs alpha  subunit to either receptor. In contrast, the beta 4gamma 2 dimer was consistently highest in coupling efficiency for either receptor. Even more surprising was the finding that the beta 1gamma 2 dimer, which in most assays of beta gamma function is potent and efficacious, was very poor at coupling Gs alpha  to the A2a adenosine receptor. These results lead to the following conclusions: 1) receptors can discriminate among the G protein heterotrimers based on the beta  isoform alone; 2) all beta 1-4 isoforms can function in signaling cascades involving Gs alpha ; and 3) dimers containing beta 5 are not likely to be released from Gs-coupled receptors. Finally, since different beta gamma dimers may be released upon receptor activation, the downstream effects of the distinct beta  isoforms may have different signaling properties.

One of the immediate downstream targets for beta gamma is the family of receptor kinases that phosphorylate G protein-coupled receptors upon recruitment to the membrane by the dimer (52). Experiments have examined the ability of defined beta gamma dimers to interact with the receptor kinases. For example, dimers containing beta 1 and beta 2 interact with GRK2, the kinase responsible for down-regulation of the beta -adrenergic and A2a adenosine receptors (53) far better than dimers containing beta 3 (54). Another study examined the ability of a variety of defined beta gamma dimers to promote the phosphorylation of both the beta 2-adrenergic receptor and rhodopsin by GRK2. These results indicate a significant difference in the ability of the various beta gamma dimers to promote phosphorylation of the beta 2-adrenergic receptor or rhodopsin and suggest that the type of beta  subunit could determine selectivity between the two receptors (55). Thus, even though beta 1-4 are over 80% identical, the accumulating evidence suggests the type of beta  subunit in the dimer may have a larger role in determining signaling specificity than previously appreciated.

Another immediate downstream target for the beta gamma subunit is the effector adenylyl cyclase (3). Results presented here demonstrate that dimers containing beta 1, beta 2, or beta 4 were able to regulate either type of adenylyl cyclase effectively. In contrast, beta 5gamma 2 was not particularly effective at inhibiting AC1 or in stimulating AC2 (16). Intriguingly, beta 3gamma 2 was almost 10-fold weaker at inhibiting AC1 as compared with the beta 1-4 isoforms (Table I), whereas it was equally effective on AC2. These results suggest that upon stimulation of certain Gs-linked receptors, co-activation of AC2 by beta gamma is relatively nonspecific, whereas inhibition of AC1 by beta gamma is more selective and may be receptor-dependent. This specificity of interaction between AC1 and the different beta  isoforms suggests that dimers containing the beta 3 subunit have signaling roles distinct from those containing beta 1, beta 2, or beta 4.

The regions of the beta gamma dimer that are thought to interact with AC1 and AC2 have been examined using competition experiments with synthetic peptides and alanine mutagenesis. Peptides identical to residues 86-105 and 115-135 of the beta 1 subunit were able to inhibit stimulation of AC2 by the beta gamma dimer, implicating these residues of the beta  subunit (56), as well as others (57), as sites on the molecule that interact with AC2. Moreover, the QEHA peptide, which represents a sequence from AC2 thought to interact with the beta gamma dimer, was able to bind directly to the beta  subunit. Molecular modeling of the QHEA peptide-beta gamma interaction also identified the region of beta  defined by residues 75-165 as a potentially important effector-binding domain (58). Mutagenesis experiments have suggested that three residues in the beta  subunit involved in alpha :beta interface, Asp228, Asp246, and Trp332, are important for the activation of AC2 but have no effect on the ability of the dimer to inhibit AC1 (59). Studies of the outer surface of the beta  torus show that Asn132 in blade two of the protein is important for inhibition of AC1, but seems to have little effect on activation of AC2 (60). The observation that ADP-ribosylation of Arg129 of beta 1, a residue also present in beta 2-4, prevents the inhibition of AC1 by the beta gamma dimer supports the argument that this is an important domain in the interaction of the dimer with AC1 (61).

Examination of the regions identified by the experiments discussed above in the beta 1-4 subunits shows minimal differences in the amino acid sequence. This is consistent with the observation that these four beta gamma dimers activated AC2 equally. A similar conclusion applies to the ability of three of the dimers to inhibit AC1; the intriguing exception was beta 3gamma 2, which was far less effective. Unfortunately, there are no obvious differences in the amino acid sequence of the beta 3 subunit in these regions to explain the differences in activity, suggesting that some other region in the molecule is also involved in the interaction with AC1. There are, however, sequence variations that could explain the lack of activity of beta 5gamma 2 on either isoform of cyclase. In contrast to the near identity of the beta 1-4 subunits in the regions outlined above that are thought to interact with AC2 and AC1, the beta 5 subunit has 13 amino acid differences in these regions when compared with the beta 1 subunit. Moreover, there is a two-amino acid insert in the region between residues 130-132, a site identified by ADP-ribosylation experiments as being important for the inhibition of AC1 (61). Although not definitive, these differences provide a reasonable starting point for future experiments.

Despite the homology in amino acid sequence among these beta  isoforms, differences in localization have been observed. For example, in contrast to the other beta  isoforms that were localized to the membrane, beta 3 was observed predominantly in cytoplasmic fractions in both heart (62) and retina (63). This property of beta 3 does not seem likely to be responsible for the observed differences in inhibition of AC1, since beta 3gamma 2 was just as effective as the other beta gamma dimers at activating the membrane-bound protein AC2 and supporting coupling to the beta 1-adrenergic and A2a adenosine receptors. A more reasonable explanation is that certain residues unique to beta 3 impart specificity either through altering the contacts with an effector molecule such as AC1, or those unique residues slightly influence the conformation of the beta  subunit, thereby altering interactions with other proteins. Whatever the reason, one important conclusion is that differences in AC1 inhibition may result from the release of different beta gamma dimers by receptor activation.

This concept appears to apply especially well to dimers containing the beta 5 subunit. Even though the current data suggest that the beta 5gamma 2 dimer is unlikely to be released from Gs-linked receptors, it clearly can be released by activation of Gq-linked receptors (16). However, accumulating evidence shows that the beta 5gamma 2 dimer does not regulate a variety of effectors, including AC1, AC2, phosphatidylinositol 3-kinase, PLC-beta 3 and the mitogen-activated protein kinase pathway (this report and Refs. 64 and 65). Even though the beta 5gamma 2 dimer did not regulate adenylyl cyclase in our experiments, transfection of the dimer into COS-7 cells caused an inhibition of both AC1 and AC2 (66). These conflicting data may be explained by other potential partners for beta 5, such as RGS 6, 7, 9, and 11 (67, 68), which may impinge upon the adenylyl cyclase cascade in vivo. This evidence of multiple partners for the beta 5 subunit suggests the beta 5 protein may have functions not normally associated with beta  subunits and makes the physiological role of beta 5 on effectors unclear. Especially interesting is the possibility that the beta 5 subunit may exist as a monomer and exchange between gamma  subunits and RGS proteins (69). Although the role of beta 5 in signaling is clearly complex, one conclusion from this information is that receptors that couple to and release dimers containing the beta 5 subunit are less likely to generate cross-talk between signaling systems because of the limited effect of beta 5 containing dimers on downstream effectors.

Brain is one tissue where all of the signaling components studied in this report are expressed at high levels (70-73). Thus, the differential effects of the beta  isoforms demonstrated in this report could have major effects on signaling cascades in the brain. Some experimental support for this concept comes from experiments showing that small amounts of beta gamma derived from Gs activation can inhibit the neuronal-specific AC1 in vivo (74). Further information needed to corroborate these proposed differences in beta gamma signaling includes cellular and subcellular localization of these molecular components. Once the subcellular architecture in these tissues is better understood with respect to G proteins, signaling models based on specific G protein beta  subunits can be refined with precision.

    ACKNOWLEDGEMENTS

We thank Dr. Joel Linden for the baculovirus encoding the A2a adenosine receptor; Dr. Elliott Ross for the baculovirus encoding the beta 1-adrenergic receptor; and Dr. Ravi Iyengar for the baculoviruses encoding type I and type II adenylyl cyclase. We also thank Qi Wang for expert technical assistance; the University of Virginia Biomolecular Research Facility for DNA sequencing and mass spectrometric analysis; and the University of Virginia Diabetes Core Facility for the cAMP assays.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grant RO1-DK-19952.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.

Dagger Supported by a National Institutes of Health fellowship in Diabetes and Hormone Action. To whom correspondence should be addressed: Dept. of Pharmacology, University of Virginia Health System, P. O. Box 800735, 1300 Jefferson Park Ave., Charlottesville, VA 22908. Tel.: 804-924-9976; Fax: 804-924-5207; E-mail: wem2p@virginia.edu.

Published, JBC Papers in Press, February 14, 2001, DOI 10.1074/jbc.M011233200

    ABBREVIATIONS

The abbreviations used are: G proteins, guanine nucleotide-binding regulatory proteins; Sf9 cells, Spondoptera frugiperda cells; DTT, dithiothreitol; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; AMP-PNP, 5'-adenylylimidodiphosphate; PMSF, phenylmethylsulfonyl fluoride; BSA, bovine serum albumin; Genapol C-100, polyoxyethylene (10) dodecyl ether; GRK, G protein receptor kinase; RGS, regulator of G protein signaling; AC1 or AC2, adenylyl cyclase type I or type II; PLC-beta , phosphatidylinositol-specific phospholipase C-beta isoform; GTPgamma S, guanosine 5'-3-O-(thio)triphosphate; Ni2+-NTA, Ni2+-nitrilotriacetic acid; PAGE, polyacrylamide gel electrophoresis.

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ABSTRACT
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RESULTS
DISCUSSION
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