G Protein beta  Subunit Types Differentially Interact with a Muscarinic Receptor but Not Adenylyl Cyclase Type II or Phospholipase C-beta 2/3*

Yongmin HouDagger §, Vanessa ChangDagger §, Austin B. Capper, Ronald Taussig, and N. GautamDagger ||**

From the Departments of Dagger  Anesthesiology and || Genetics, Washington University School of Medicine, St. Louis, Missouri 63110 and the  Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109-0636

Received for publication, November 16, 2000, and in revised form, March 15, 2001

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In comparison with the alpha  subunit of G proteins, the role of the beta  subunit in signaling is less well understood. During the regulation of effectors by the beta gamma complex, it is known that the beta  subunit contacts effectors directly, whereas the role of the beta  subunit is undefined in receptor-G protein interaction. Among the five G protein beta  subunits known, the beta 4 subunit type is the least studied. We compared the ability of beta gamma complexes containing beta 4 and the well characterized beta 1 to stimulate three different effectors: phospholipase C-beta 2, phospholipase C-beta 3, and adenylyl cyclase type II. beta 4gamma 2 and beta 1gamma 2 activated all three of these effectors with equal efficacy. However, nucleotide exchange in a G protein constituting alpha obeta 4gamma 2 was stimulated significantly more by the M2 muscarinic receptor compared with alpha obeta 1gamma 2. Because alpha o forms heterotrimers with beta 4gamma 2 and beta 1gamma 2 equally well, these results show that the beta  subunit type plays a direct role in the receptor activation of a G protein.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The G protein beta gamma complex regulates the activity of a diverse set of effectors, including phospholipases, adenylyl cyclases, and ion channels (1). There is evidence that the beta  subunit in the complex interacts directly with effectors (2-5). There are five beta  subtypes (beta 1-beta 5) as well as an alternatively spliced version of beta 5 (known as beta 5-long) (6-11). beta 1-beta 4 share over 80% identity with one another, whereas beta 5 shares only ~50% identity with the other beta  subunits (12). The divergence between beta 5 and the other beta  subunits is consistent with the functional differences between beta 5 and beta 1 observed in effector regulation in a variety of systems (4, 13, 14). The high sequence similarity of beta 1-beta 4 suggests that their functions are conserved. Although some experiments indicated little difference in effector modulating capability among these beta  subunit types, other experiments suggest otherwise. The G protein-coupled receptor kinase GRK3 binds beta gamma complexes consisting of beta 1, beta 2, and beta 3, but only beta 1 and beta 2 bind to the related kinase GRK2 (15). Other results indicate the selective mediation of cross-talk between G proteins and protein kinase C modulation of N-type channels by the beta 1 subunit type (16).

Experiments focusing on the specific role of individual G protein subunit types have provided evidence for a certain level of selectivity in the interaction of alpha  subunit types with receptors (17). Evidence for similar selectivity of interaction between gamma  subunit types and receptors also exists (18-20). In contrast there is limited evidence for beta  subunit type selectivity in receptor interaction. Whole-cell experiments using antisense oligonucleotides directed against specific beta  subunit cDNAs selectively disrupted signaling from particular receptors (21). Although the selective interaction of beta  subunit types with receptors could give rise to this result, such selectivity has not been shown so far.

Among the five beta  subunits, beta 4 is the least studied. Its role in effector regulation and receptor interaction remains unclear. To examine its effect on the beta gamma regulation of effectors and G protein interaction with a receptor, we expressed purified recombinant beta 4gamma 2 and beta 1gamma 2 complexes and compared their abilities to regulate the activity of three different G protein effectors: PLC-beta 2,1 PLC-beta 3, and adenylyl cyclase type II (AC-II). Next, we examined the abilities of heterotrimers made up of alpha obeta 1gamma 2 and alpha obeta 4gamma 2 to couple to the M2 muscarinic receptor in a reconstituted system containing purified M2 and G protein subunits. The results indicate that in comparison to beta 1, the beta 4 subunit does not differentially modulate effector function but does differentially affect the receptor activation of a G protein. These results indicate that the particular beta  subunit type present in a heterotrimer influences the effectiveness of the receptor activation of that G protein. Because these experiments were performed with purified receptor and G protein subunits, these results also indicate that the beta  subunit plays a direct role in the receptor activation of a G protein.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- PIP2 and phosphatidylethanolamine were obtained from Avanti Polar Lipids. [3H]PIP2 was from PerkinElmer Life Sciences. Where a source is not stated, reagent was obtained from Sigma.

Construction of Recombinant Baculoviruses-- beta 4-Expressing baculovirus was constructed using the Bac-to-Bac baculovirus expression system from Life Technologies, Inc. Because sequencing revealed a very long cytosine-rich region at the 5'-end of the original beta 4 cDNA (kind gift from Dr. M. I. Simon, California Institute of Technology), a synthetic oligonucleotide cassette made up of DX19103 (5'-GGCCGCATGAGCGAGCTGGAGCAG) and DX19104 (5'-CTGCTCCAGCTCGCTCATGC) was constructed to replace this region. The cassette encodes the first six amino acids of beta 4. It was introduced into the beta 4-pFastBac construct using NotI/PvuII sites. The methods used for the construction of baculoviruses expressing His-PLC-beta 2, His-PLC-beta 3, adenylyl cyclase type II, the G protein beta 1 subunit, and the G protein His-gamma 2 subunit have been published (22-26).

Expression and Purification of beta gamma Complexes from Sf9 Insect Cells-- Sf9 cells were maintained in suspension in IPL-41 medium (Life Technologies, Inc.) containing 1% Pluronic F68, 10% heat-inactivated fetal bovine serum (Atlanta Biologicals), and 50 µg/ml gentamicin. Cells were grown at 27 °C with constant shaking at 125 rpm. For initial expression studies, Sf9 cells were infected with the beta 4 virus for varying lengths of times. Cell lysates were examined by immunoblotting with B4-specific B4-2 antibody (27) used at 1:600 dilution. The purification of beta gamma subunits was performed essentially as described before (26). Sf9 cells were simultaneously infected with His-gamma 2 baculovirus and either beta 4 or beta 1 baculovirus. Approximately 60 h after infection, cells were lysed by nitrogen cavitation, and the membranes were extracted with 1% cholate. The detergent extract was applied to a column of nickel resin (nickel-nitrilotriacetic acid column from Qiagen) and washed with Buffer A (20 mM Hepes, pH 8.0, 1 mM MgCl2, and 10 mM beta -mercaptoethanol) containing 300 mM NaCl, 0.5% C12E10, and 10 mM imidazole. beta gamma complex was eluted with Buffer A containing 50 mM NaCl, 1% cholate, and 250 mM imidazole. Peak fractions were concentrated to a final concentration of 1-2 mg/ml using centrifugal filtering devices (Centricon YM10 from Millipore). For use in receptor assays, G protein beta gamma complexes were exchanged into 20 mM Hepes, pH 8.0, 1 mM EDTA, 3 mM MgCl2, 3 mM dithiothreitol, 0.1 M NaCl, and 0.7% CHAPS by dialysis. The purity and concentration of beta gamma complexes were assessed by the separation of proteins on SDS-polyacrylamide gel electrophoresis followed by staining with Coomassie Blue and the quantitation of protein bands with laser densitometry analysis. PLC-beta activation assays were used to ensure that the functional proportion of beta 4gamma 2 and beta 1gamma 2 complexes containing different detergents was the same in all samples. The final preparations of all proteins were over 95% pure.

Purification of G Protein alpha  Subunit-- Recombinant Galpha was synthesized in Escherichia coli and purified as described previously (28). Protein purity and quantity were estimated by separation on SDS gels and densitometry. The proportion of the functional alpha  subunit was estimated by GTPgamma S binding assays.

PLC-beta Assays-- PLC-beta 2 and PLC-beta 3 were purified as described previously (22, 23). The beta gamma stimulation of phospholipase C-beta was performed as described (22). Briefly, lipid substrate (50 µM PIP2, 200 µM PE, and [3H]PIP2 (~8000 cpm/assay) was mixed, dried, and ultrasonicated in a buffer containing 50 mM Na-Hepes (pH 7.2), 3 mM EGTA, 80 mM KCl, and 1 mM dithiothreitol. 1 ng of PLC-beta 2 per reaction and 5 ng of PLC-beta 3 per reaction were used. PLC-beta and beta gamma complex were added to the substrate in ice. Reactions were started with the addition of CaCl2 (final concentration 2.8 mM), and reactions were incubated at 30 °C for 15 min. Reactions were terminated by the addition of 10% trichloroacetic acid and bovine serum albumin. After centrifugation, the supernatant containing [3H]phosphatidylinositol 1,4,5-trisphosphate was analyzed by scintillation counting as described before (22).

Adenylyl Cyclase Assays-- Sf9 cells were infected with baculovirus expressing adenylyl cyclase type II, and Sf9 cell membranes were then prepared as described previously (29). Membranes expressing adenylyl cyclase type II were used in adenylyl cyclase assays performed according to the procedure of Smigel (30). alpha s subunit was activated by incubation with 50 mM Na-Hepes (pH 8.0), 5 mM MgSO4, 1 mM EDTA, 1 mM dithiothreitol, and 400 mM GTPgamma S at 30 °C for 30 min; free GTPgamma S was removed by gel filtration. All assays were performed for 10 min at 30 °C in a final volume of 100 µl containing 10 mM MgCl2 and 100 nM GTPgamma S-bound alpha s.

Preparation of Purified and Reconstituted Recombinant M2-- A detailed description of M2 purification, reconstitution, and measurement of G protein stimulation has been published elsewhere (31). His6-tagged M2 was expressed in Sf9 cells by using the recombinant baculovirus (kind gift from Dr. E. Ross). The receptor was purified using the CoCl2 affinity column according to previously published procedures with some modifications (32). The purified His-M2 was reconstituted into brain lipids and characterized by binding to an antagonist, [3H]N-methylscopolamine, as described previously with some modifications (33). The Kd for reconstituted M2 binding to [3H]N-methylscopolamine was determined to be 0.25 nM (34).

GTP Hydrolysis-- Heterotrimeric G protein subunits were formed by incubating 10 nM concentrations of alpha  and beta gamma subunits on ice for 30 min in a buffer containing 25 mM Hepes, 100 mM NaCl, 2 mM MgCl2, 0.1 mM EDTA, 10 µM GDP, and 1 mM dithiothreitol. The reconstituted M2 was incubated with heterotrimeric Go on ice for 30 min with or without RGS4 protein (kind gift from Dr. M. Linder, Washington University). The receptor-G protein complex was then mixed with carbachol or water before gamma -[32P]GTP was added to initiate the reaction at 25 °C. The reaction mixture contained different concentrations of G protein, 1 nM receptor, 0.2 µM GTP, 5 µM GDP, and 1 mM carbachol or 200 µM GTP in a buffer of 20 mM Hepes (pH 8.0), 100 mM NaCl, 2 mM MgCl2, 0.1 mM EDTA, 1 mM dithiothreitol, and 0.5 mg/ml bovine serum albumin. RGS4 concentration was 0.1 µM. Excess 200 µM GTP (1000× more than gamma -[32P]GTP) instead of carbachol serves as a nonspecific control of GTP hydrolysis. Aliquots taken at the indicated time points were diluted with 0.5 ml of ice-cold buffer containing 5% activated charcoal and 50 mM K3PO4. Samples were centrifuged, and radioactivity in supernatants was quantified by scintillation counting.

Measurement of G Protein Heterotrimer Formation-- To measure the formation of heterotrimer, a fixed concentration of beta gamma complex was mixed with various concentrations of alpha o subunit. Initially, 360 nM beta gamma complex was incubated with increasing concentrations of the alpha o subunit (11.25, 22.5, 45, 90, 180, and 360 nM) in ice for 30 min in a 10-µl buffer containing 20 mM Hepes (pH 8.0), 100 mM NaCl, 2 mM MgCl2, 0.1 mM EDTA, 1 mM dithiothreitol, and 0.5 mg/ml bovine serum albumin. Five µl of this mixture were then diluted 10 times to a total of 50 µl of buffer containing 50 mM Na-Hepes (pH 7.2), 3 mM EGTA, 1 mM EDTA, 5 mM MgCl2, 100 mM NaCl, and 1 mM dithiothreitol. 10 µl of this diluted sample containing beta gamma and various concentrations of the alpha  subunit were then added to a total of 60 µl of PLC reaction buffer containing [3H]PIP2 substrate and enzyme for determining the PLC-beta 3 activity as described above (6 nM final concentration of beta gamma complex).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

beta 4gamma 2 and beta 1gamma 2 Protein Expression and Purification-- To study the function and properties of the G protein beta 4 subunit, we constructed a baculovirus expressing beta 4. To confirm the viral expression of beta 4, Sf9 cells were infected with this virus for varying lengths of time; cells were then harvested, lysed, and checked for protein expression by immunoblotting with the B4-2 antibody against the beta 4 protein (Fig. 1A). To produce beta 4gamma 2 dimers, we simultaneously co-infected Sf9 cells with the beta 4 virus and a virus expressing a His-tagged gamma 2 subunit. beta 4gamma 2 was purified by nickel-nitrilotriacetic acid chromatography. beta 1gamma 2 was expressed and purified using a similar approach. beta 4 consistently runs with slightly faster mobility than does beta 1 (Fig. 1B). This is consistent with a report from Asano et al. (35), who studied the native beta 4 protein expressed in bovine tissues. We confirmed that the purified beta 4gamma 2 and beta 1gamma 2 proteins contain the same concentration of detergents by using thin layer chromatography with the appropriate detergent standards (data not shown).


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Fig. 1.   Expression and purification of beta gamma complexes. A, immunoblot analysis of beta 4 protein expression in Sf9 cells with the beta 4-specific antibody (B4-2). Cells were infected with the beta 4-baculovirus for varying amounts of time and then lysed as described under "Experimental Procedures." 10 µg of proteins from each sample were loaded. Lane 1, uninfected cells; lane 2, cells infected for 40 h; lane 3, 48 h; lane 4, 64 h; and lane 5, 72 h. B, Coomassie Blue staining of purified G protein recombinant beta 1gamma 2 and beta 4gamma 2 subunits separated by SDS-polyacrylamide gel electrophoresis on a 12% gel.

Stimulation of Effectors by beta 4gamma 2 and beta 1gamma 2-- To search for potential differences between beta 4 and beta 1 in effector regulation, we focused on three major effectors regulated by G protein beta gamma complexes: PLC-beta 2, PLC-beta 3, and AC-II. The effect of beta gamma complexes on these enzymes was examined. Fig. 2A shows the activation of purified PLC-beta 2 by beta 4gamma 2 and beta 1gamma 2 complexes. Consistent with previous reports where brain beta gamma was tested (36), both beta gamma complexes stimulate PLC-beta 2 more than 4-fold above basal activity with similar effectiveness. The PLC-beta 2 stimulatory properties of both beta gamma complexes are thus essentially identical. Although PLC-beta 2 and PLC-beta 3 are isozymes that are both stimulated by the G protein beta gamma complex, there is evidence that the residues in the beta  subunit that contact these two enzymes are distinct (37). This raised the possibility that although the two beta gamma complexes showed little difference in the activation of PLC-beta 2, they might interact differentially with PLC-beta 3. We therefore examined the stimulation of PLC-beta 3 by beta 4gamma 2 and beta 1gamma 2. Both beta gamma complexes activate PLC-beta 3 20-fold over basal activity (Fig. 2B). The higher stimulation of PLC-beta 3 compared with PLC-beta 2 is consistent with previous reports (36). As in the case of PLC-beta 2, the effectiveness with which beta 1gamma 2 and beta 4gamma 2 activate PLC-beta 3 is similar. Because detergents in the beta gamma preparations may themselves activate PLC-beta (38), we also tested boiled preparations of beta 4gamma 2 and beta 1gamma 2. Stimulation from boiled samples was minimal. Moreover, boiled samples of beta 4gamma 2 and beta 1gamma 2 also showed essentially the same profile of activity, indicating that detergent concentrations and any other nonprotein stimulators of PLC activity are present at equivalent levels (data not shown).


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Fig. 2.   beta 4gamma 2 and beta 1gamma 2 stimulation of PLC-beta 2 and PLC-beta 3 activities. 1 ng of purified PLC-beta 2 (A) or 5 ng of purified PLC-beta 3 (B) were assayed for stimulation with the indicated concentrations of beta 1gamma 2 or beta 4gamma 2 in a lipid mixture containing [3H]PIP2 as described under "Experimental Procedures." Basal activity is defined as activity in the absence of the beta gamma complex. The fold stimulation over basal activity is shown. [3H]phosphatidylinositol 1,4,5-trisphosphate production was measured by scintillation counting. Data in A are the means of three independent experiments performed in duplicate (± S.E.). When examined with the unpaired t test, differences in the activities between beta 4gamma 2 and beta 1gamma 2 at all concentrations were not statistically significant. B is representative of two independent experiments; each experiment was done in duplicate.

AC-II is stimulated by the G protein beta gamma complex in the presence of the activated alpha s subunit. To test whether beta 4gamma 2 and beta 1gamma 2 stimulate this enzyme differentially, we examined AC-II stimulation in the presence of GTPgamma S-bound alpha s. Sf9 cell membranes expressing AC-II were used as a source of the enzyme (29). In the presence of GTPgamma S-bound purified alpha s, beta 4gamma 2 and beta 1gamma 2 activate AC-II significantly over the basal level. Fig. 3 shows that, as in the case of PLC-beta , the extent of maximal stimulation (8-fold) by beta 4gamma 2 or beta 1gamma 2 and the effectiveness of both beta gamma complexes in stimulating AC-II are similar.


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Fig. 3.   beta 4gamma 2 and beta 1gamma 2 stimulation of adenylyl cyclase type II. An adenylyl cyclase assay was performed as described under "Experimental Procedures." Various concentrations of beta gamma complex were used as indicated. Activities are expressed as fold stimulation over control, which was measured in the presence of 100 nM GTPgamma S-bound Galpha s without the beta gamma complex. Data shown are the means of two independent experiments performed in duplicate (± S.E.). Differences in the activities elicited by beta 4gamma 2 and beta 1gamma 2 were not statistically significant. The plot shown is representative of at least three other independent experiments performed in duplicate.

M2 Receptor Activation of Go Containing the beta 1 and beta 4 Subunits-- The receptor stimulation of a G protein can be measured as GTPgamma S binding to the alpha  subunit or as GTPase activity of the alpha  subunit in the presence of an agonist. We have recently determined that in a reconstituted system containing the purified M2 muscarinic receptor and G protein subunits, the GTPase assay is much more sensitive and allows us to measure G protein activation with a relatively low concentration of receptor (1 nM) at ratios of receptor:G protein that are close to 1:1 (34). These conditions are potentially closer to the dissociation constant for M2 interaction with Go (which has not been determined) than to the ratio of receptor to G protein used in the less sensitive GTPgamma S assays. Subtle differences in the receptor interaction of G protein heterotrimers are more likely to be revealed under the conditions used in the GTPase assay. This notion is borne out in the analysis of the G protein gamma  subunit interaction with M2 where differences in coupling were detected using these conditions (34).

The M2 stimulation of alpha obeta 1gamma 2 and alpha obeta 4gamma 2 was examined by assaying GTPase activity in the presence of RGS4. The RGS4 protein, a GTPase-activating protein for the Go/i family, increases the pool of G protein heterotrimers available to the receptor and considerably increases the sensitivity of the reaction (by ~10-fold). As expected, the addition of carbachol increases the GTP hydrolysis rate significantly (Fig. 4). More importantly, the M2-activated GTP hydrolysis of alpha obeta 4gamma 2 was ~200% higher than that of alpha obeta 1gamma 2. This difference was consistently observed at all G protein concentrations tested (Table I). Statistical analysis with the unpaired t test indicated that these differences are significant (p < 0.05). To further verify these data, two independent preparations of each purified beta gamma complex containing beta 1 or beta 4 were examined again. These preparations provided similar results. As mentioned before, the concentration of detergent (CHAPS) in the purified beta 1gamma 2 and beta 4gamma 2 stocks was determined using thin layer chromatography (data not shown). This analysis indicated that the detergent concentrations in the samples were similar and were not the cause for the differential receptor activation. Furthermore, when the ability of the beta 1gamma 2 and beta 4gamma 2 complexes were examined in PLC-beta 3 activation assays, the results indicated that the functional proportions in the stocks of both subunit complexes were the same. Finally, the possibility that these differences arose from the differential interaction of the beta  subunit types with the RGS4 protein was tested by assaying the M2-stimulated GTPase activity in the absence of the RGS protein. Again alpha obeta 4gamma 2 was consistently 2-3-fold more active than alpha obeta 1gamma 2 (Table I), indicating that the differential stimulation of GTPase activity resulted from receptor rather than RGS protein interaction.


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Fig. 4.   M2-stimulated GTPase activity of alpha obeta 1gamma 2 and alpha obeta 4gamma 2. The G protein concentration was 2 nM. Other details of the GTP hydrolysis assay are described under "Experimental Procedures." The GTPase activity was expressed as Pi produced (fmol) in a 5-µl reaction. Experiments have been replicated at least three times, and representative data are shown.

                              
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Table I
M2-promoted GTPase activity of Go protein containing beta 1 and beta 4

Efficiency of Heterotrimerization of beta 1gamma 2 and beta 4gamma 2 Subunits with alpha o-- Because receptors interact effectively only with the heterotrimer and not with the individual subunits, the difference observed in the M2 receptor-stimulated activity between alpha obeta 1gamma 2 and alpha obeta 4gamma 2 could be attributable to the differential heterotrimer formation between alpha o and these two beta gamma complexes. The residues in beta 1 and beta 4 that contact the alpha  subunit are conserved, indicating that the beta 1 and beta 4 affinity for alpha o is likely to be the same (39). However, it was possible that heterotrimerization was differentially affected by divergent residues in beta 1 and beta 4 that were located at a distance from residues that contacted the alpha  subunit. To examine this possibility, we used a recently developed assay for measuring G protein heterotrimer formation (34). This assay is based on evidence that the beta gamma complex has overlapping sites for binding the alpha  subunit and the PLC-beta 3 enzyme (40). Thus heterotrimerization prevents beta gamma complex interaction with PLC-beta 3, leading to the inhibition of the beta gamma complex-stimulated PLC-beta 3 activity. Because the assay is sensitive, it can be used to examine the alpha o-beta gamma interaction at the same subunit concentrations (1-10 nM) used in the M2-stimulated GTPase assays above. In contrast, the ADP-ribosylation assay that has been used extensively in the past is less sensitive (requiring more than ~1 µM subunits), and in addition, it is complicated by the lack of knowledge regarding the mechanistic basis of the beta gamma enhancement of the alpha  subunit ADP-ribosylation by pertussis toxin. As shown in Fig. 5, the beta gamma complex-stimulated PLC-beta activity is inhibited in a dose-dependent manner by the alpha o subunit. The alpha o concentration dependence of this inhibition of beta 1gamma 2- and beta 4gamma 2-stimulated PLC beta  activity is similar, indicating that both complexes form a heterotrimer with alpha o with equal effectiveness.


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Fig. 5.   The beta 1gamma 2 and beta 4gamma 2 subunits form heterotrimer equally well with alpha o. The beta gamma subunit (6 nM) was incubated with the indicated concentration of alpha  subunit on ice for 30 min to allow heterotrimer formation. PLC-beta 3 activity catalyzed by free beta gamma but not alpha -beta gamma was determined to monitor the progress of complex beta gamma with the alpha  subunit. The activity stimulated by free beta gamma serves as a positive control representing the maximum PLC activity (100%), whereas the enzyme activity by alpha o alone was used as a negative control. The PLC activity was assayed as described under "Experimental Procedures." Values shown are the means (± S.E.) from three independent experiments. Differences in inhibition between beta 1gamma 2 and beta 4gamma 2 at various concentrations of alpha o were not statistically significant in an unpaired t test.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Because the G protein beta gamma complex is known to interact directly with and modulate various effectors and the beta  subunit is known to contact effectors, we first compared the relative abilities of the beta 4gamma 2 and beta 1gamma 2 complexes to stimulate three common effectors regulated by the beta gamma complex: PLC-beta 2, PLC-beta 3, and AC-II. Both beta gamma complexes activated each of the effectors with similar potency. This observation is consistent with the conservation between beta 1 and beta 4 of 93% of similar amino acids (Fig. 6A). It is unclear whether this result indicates that residues not conserved between beta 1 and beta 4 play no significant role in the regulation of effectors examined here. Comparing these results with previous mutational studies of beta 1, which implicate particular residues in PLC-beta regulation, does not resolve this question. beta  subunit mutants analyzed in three different studies (40-42) did not involve residues that are divergent between beta 1 and beta 4. In another study (43), several residues were mutated simultaneously. It is therefore difficult to interpret these results in terms of the divergence in the beta 1/beta 4 primary structures. Among the few single residues that were mutated in this study, Asp-303 is the only one that is not conserved between beta 1 and beta 4 (Fig. 6A). This mutant beta 1gamma complex stimulated PLC-beta 2 normally. Although very limited, this result is consistent with results presented here.


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Fig. 6.   Differences in the amino acid sequences of the beta 1 and beta 4 subunits mapped on the beta gamma complex three-dimensional structure. A, primary structures of beta 1 and beta 4 subunits with the differences highlighted. Residues located in the putative receptor-interacting surface of the beta gamma complex are numbered. Residues in the other beta  subunit types at these loci are shown. (beta  subunit types from top to bottom are beta 2, beta 3, and beta 5.) Arrows indicate beta  strands in the folded beta  subunit (panel B). Four beta  strands make up a sheet. Sheets are denoted as lines labeled S1-S7. B, structure of the G protein subunit complex (alpha tbeta 1gamma 1 from Lambright et al. (50)). Dark gray, alpha  subunit; light gray, beta  subunit; and black, gamma  subunit. Open circles denote residues that are nonconservative changes between the beta 1 and beta 4 subunit amino acid sequences. The positions of these residues in the primary structure of beta 1/beta 4 are: residue 1, -31; residue 2, -35; residue 3, -37; residue 4, -39; residue 5, -302; residue 6, -303; and residue 7, -305. In the phosducin-beta gamma complex the prenyl group, farnesyl (C-15), is buried in the pocket between beta  sheets S6 and S7 (49). Prepared with Ras Top 1.3 by P. Valadon.

It has been known for many years that the beta gamma complex is essential for heterotrimeric G protein interaction with a receptor (44). The reasons for this requirement have been less clear. There is increasing evidence to support the interaction of the C-terminal domain of the gamma  subunit with a receptor. This evidence comes from studies of rhodopsin-Gt coupling using peptides and mutant gamma 1 subunits (45, 46). It is also supported by the ability of a peptide specific to the gamma 5 subunit type but not the gamma 7 or gamma 12 subunit type to inhibit muscarinic receptor-mediated signaling in superior cervical ganglion neurons (20). More recently, the gamma  subunit type in a heterotrimer has been shown to influence the M2 receptor-stimulated nucleotide exchange (34). Here we used the same assay to detect a consistent 2-3-fold difference in M2-stimulated GTP hydrolysis between alpha obeta 4gamma 2 and alpha obeta 1gamma 2. Because these experiments were performed in the presence of an RGS protein, the difference in activity reflects a difference in receptor-stimulated nucleotide exchange. The measurement of heterotrimer formation between alpha o and beta 1gamma 2 or beta 4gamma 2 showed that both heterotrimers form with equal effectiveness and ruled out the possibility that the difference in receptor-stimulated nucleotide exchange arose from differences in heterotrimer formation. Receptor-stimulated GTPase assays performed in the absence of the RGS protein showed that the differences arose at the site of receptor interaction and not from differential interaction with the RGS protein.

The differences in receptor-stimulated activity between alpha obeta 4gamma 2 and alpha obeta 1gamma 2 therefore indicate that the distinct primary structures of beta 1 and beta 4 influence the interaction of the heterotrimer with the receptor. Evidence that the gamma  subunit interacts with receptors and previous evidence that the alpha  subunit C and N termini interact with receptors (47) help identify the surface of the G protein that contacts the receptor (Fig. 6B). Inspection of the amino acid sequences of the beta 1 and beta 4 subunits indicates differences that are distributed over the sequence (Fig. 6A). For these differences to play a role in receptor interaction, they most likely need to be accessible to the receptor and therefore located on the outer surface of the molecule. The location of the different amino acids between the beta 1 and beta 4 subunits on the three-dimensional structure of the beta  subunit indicates that two clusters of residues (31-39 and 302-305) are located on parallel strands of the beta  subunit, as shown in Fig. 6B, although the two clusters are far apart in the primary structure, located toward the N and C termini of the beta  subunit (Fig. 6A). These residues are on the outer surface of the molecule. Most strikingly, these residues are on the surface that has been inferred to contact the receptor; note the location of the C termini of the alpha  subunit and the gamma  subunit (Fig. 6B). Finally, several of these residues show divergence between the various beta  subunit types (Fig. 6A).

Antisense oligonucleotides specific to beta 1 and beta 3 have previously been shown to selectively inhibit somatostatin and muscarinic M4 receptor-mediated Ca2+ channel activity (21). The precise point in the signaling pathway that was perturbed by the introduction of the oligonucleotides has not been elucidated so far. Inferences about the relationship between those results and the differential stimulation of alpha o in the presence of beta 1/beta 4 cannot be drawn both for this reason and because the physiological effect of the differential M2 stimulation of alpha obeta 4gamma 2 versus alpha obeta 1gamma 2 is not known.

The recent crystal structure determination of the inactive form of rhodopsin indicates that the intracellular portion of the receptor spans a little over 40 Å. It is unclear whether the activated forms of other receptors will expose intracellular surfaces that are considerably larger in surface area than 40 Å in surface area. The distances between the C terminus of the gamma  subunit and several of the residues in the beta  subunit clusters are much less than 40 Å, so the beta  subunit domains and the C terminus of the gamma  subunit can interact with a receptor simultaneously. In contrast, the distances between these domains in the beta  subunit and the C terminus of the alpha  subunit are over 50 Å, and it seems less likely that these regions can interact with a receptor at the same time. However, even domains that are far apart in the G protein can interact with the receptor in a temporally sequential fashion. Direct contact between the beta  subunit region containing residues 302-305 and the receptor is consistent with a previous study that showed the cross-linking of a beta  subunit peptide covering residues 281-340 with an alpha 2-adrenergic receptor-derived peptide (48). Divergences in the amino acid sequence between beta 1 and beta 4 may thus contribute directly to differences in interaction through differential contact with corresponding residues in the receptor. This would result in the G proteins containing these subunit types being activated selectively, as seen here. Alternatively, the residues in the two clusters, 31-39 and 302-305, may influence the three-dimensional structure of the receptor-interacting surface without contacting the receptor directly. The elucidation of the crystal structure of a beta gamma complex that retains the prenyl modification bound to phosducin indicates that this is a possibility. It has been inferred from the elucidation of this structure that the prenyl moiety is buried in a cavity between beta  sheets 6 and 7 of the folded beta  subunit structure (49) (Fig. 6B). This region of the beta  subunit is susceptible to conformational changes; the conformation of this region has been inferred to be different in the free beta gamma complex compared with beta gamma bound to phosducin (49). It is thus possible that the differences between beta 1 and beta 4 in the two clusters of residues highlighted in Fig. 6B can have an effect on the conformational state of this prenyl binding cavity between beta  sheets 6 and 7. Because there is evidence that the prenyl moiety and the last several C-terminal residues of the gamma  subunit contact the receptors, such a rearrangement would indirectly affect the receptor interaction of the heterotrimer by altering the accessibility of the gamma  subunit tail. Recent results seem to indicate that this is the less likely mechanism. In a study by Myung and Garrison (42), mutations were introduced in the prenyl binding cavity and in the region of the beta  subunit that undergoes conformational changes on binding to phosducin. These mutant forms of the beta  subunit did not affect the G protein interaction with the A1 adenosine receptor as measured by the ability of the G proteins to stabilize the high affinity binding state of A1 receptors. It is unclear whether the GTPase assays used here will detect differences between the beta  subunit mutants and wild type used in that study.

Overall, the importance of these results is that the differential receptor interaction of beta  subunit types indicates a direct role for the beta  subunit in receptor activation of a G protein.

    ACKNOWLEDGEMENTS

We thank Dr. I. Azpiazu of our laboratory for purified M2 and alpha o proteins as well as valuable discussions. We thank Dr. A. Smrcka for PLC-B.

    FOOTNOTES

* This work was supported by Grants GM53645 (to R. T.) and GM46963 (to N. G.) from the National Institutes of Health.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.

§ These authors contributed equally to this work.

** To whom correspondence should be addressed: Box 8054, Washington University School of Medicine, St. Louis, MO 63110. Tel.: 314-362-8568; E-mail: gautam@morpheus.wustl.edu.

Published, JBC Papers in Press, March 21, 2001, DOI 10.1074/jbc.M010424200

    ABBREVIATIONS

The abbreviations used are: PLC, phospholipase C; Sf9 cells, Spodoptera frugiperda cells; AC-II, adenylyl cyclase type II; PIP2, phosphatidylinositol 4,5-bisphosphate; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; GTPgamma S, guanosine 5'-O-(3-thiotriphosphate); RGS, regulator of G protein signaling.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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