G Protein β Subunit Types Differentially Interact with a Muscarinic Receptor but Not Adenylyl Cyclase Type II or Phospholipase C-β2/3*

In comparison with the α subunit of G proteins, the role of the β subunit in signaling is less well understood. During the regulation of effectors by the βγ complex, it is known that the β subunit contacts effectors directly, whereas the role of the β subunit is undefined in receptor-G protein interaction. Among the five G protein β subunits known, the β4subunit type is the least studied. We compared the ability of βγ complexes containing β4 and the well characterized β1 to stimulate three different effectors: phospholipase C-β2, phospholipase C-β3, and adenylyl cyclase type II. β4γ2 and β1γ2 activated all three of these effectors with equal efficacy. However, nucleotide exchange in a G protein constituting αoβ4γ2 was stimulated significantly more by the M2 muscarinic receptor compared with αoβ1γ2. Because αo forms heterotrimers with β4γ2 and β1γ2equally well, these results show that the β subunit type plays a direct role in the receptor activation of a G protein.

The G protein ␤␥ complex regulates the activity of a diverse set of effectors, including phospholipases, adenylyl cyclases, and ion channels (1). There is evidence that the ␤ subunit in the complex interacts directly with effectors (2)(3)(4)(5). There are five ␤ subtypes (␤ 1 -␤ 5 ) as well as an alternatively spliced version of ␤ 5 (known as ␤ 5 -long) (6 -11). ␤ 1 -␤ 4 share over 80% identity with one another, whereas ␤ 5 shares only ϳ50% identity with the other ␤ subunits (12). The divergence between ␤ 5 and the other ␤ subunits is consistent with the functional differences between ␤ 5 and ␤ 1 observed in effector regulation in a variety of systems (4,13,14). The high sequence similarity of ␤ 1 -␤ 4 suggests that their functions are conserved. Although some experiments indicated little difference in effector modulating capability among these ␤ subunit types, other experiments suggest otherwise. The G protein-coupled receptor kinase GRK3 binds ␤␥ complexes consisting of ␤ 1 , ␤ 2 , and ␤ 3 , but only ␤ 1 and ␤ 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 ␤ 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 ␣ subunit types with recep-tors (17). Evidence for similar selectivity of interaction between ␥ subunit types and receptors also exists (18 -20). In contrast there is limited evidence for ␤ subunit type selectivity in receptor interaction. Whole-cell experiments using antisense oligonucleotides directed against specific ␤ subunit cDNAs selectively disrupted signaling from particular receptors (21). Although the selective interaction of ␤ subunit types with receptors could give rise to this result, such selectivity has not been shown so far.
Among the five ␤ subunits, ␤ 4 is the least studied. Its role in effector regulation and receptor interaction remains unclear.
To examine its effect on the ␤␥ regulation of effectors and G protein interaction with a receptor, we expressed purified recombinant ␤ 4 ␥ 2 and ␤ 1 ␥ 2 complexes and compared their abilities to regulate the activity of three different G protein effectors: PLC-␤2, 1 PLC-␤3, and adenylyl cyclase type II (AC-II). Next, we examined the abilities of heterotrimers made up of ␣ o ␤ 1 ␥ 2 and ␣ o ␤ 4 ␥ 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 ␤ 1 , the ␤ 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 ␤ 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 ␤ subunit plays a direct role in the receptor activation of a G protein.
nologies, Inc.) containing 1% Pluronic F68, 10% heat-inactivated fetal bovine serum (Atlanta Biologicals), and 50 g/ml gentamicin. Cells were grown at 27°C with constant shaking at 125 rpm. For initial expression studies, Sf9 cells were infected with the ␤ 4 virus for varying lengths of times. Cell lysates were examined by immunoblotting with B 4 -specific B4-2 antibody (27) used at 1:600 dilution. The purification of ␤␥ subunits was performed essentially as described before (26). Sf9 cells were simultaneously infected with His-␥ 2 baculovirus and either ␤ 4 or ␤ 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 MgCl 2 , and 10 mM ␤-mercaptoethanol) containing 300 mM NaCl, 0.5% C 12 E 10 , and 10 mM imidazole. ␤␥ 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 ␤␥ complexes were exchanged into 20 mM Hepes, pH 8.0, 1 mM EDTA, 3 mM MgCl 2 , 3 mM dithiothreitol, 0.1 M NaCl, and 0.7% CHAPS by dialysis. The purity and concentration of ␤␥ 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-␤ activation assays were used to ensure that the functional proportion of ␤ 4 ␥ 2 and ␤ 1 ␥ 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 ␣ Subunit-Recombinant G␣ 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 ␣ subunit was estimated by GTP␥S binding assays.
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). ␣ s subunit was activated by incubation with 50 mM Na-Hepes (pH 8.0), 5 mM MgSO 4 , 1 mM EDTA, 1 mM dithiothreitol, and 400 mM GTP␥S at 30°C for 30 min; free GTP␥S was removed by gel filtration. All assays were performed for 10 min at 30°C in a final volume of 100 l containing 10 mM MgCl 2 and 100 nM GTP␥S-bound ␣ 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). His 6 -tagged M2 was expressed in Sf9 cells by using the recombinant baculovirus (kind gift from Dr. E. Ross). The receptor was purified using the CoCl 2 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, [ 3 H]N-methylscopolamine, as described previously with some modifications (33). The K d for reconstituted M2 binding to [ 3 H]N-methylscopolamine was determined to be 0.25 nM (34). [ 3 H]phosphatidylinositol 1,4,5-trisphosphate production was measured by scintillation counting. Data in A are the means of three independent experiments performed in duplicate (Ϯ S.E.). When examined with the unpaired t test, differences in the activities between ␤ 4 ␥ 2 and ␤ 1 ␥ 2 at all concentrations were not statistically significant. B is representative of two independent experiments; each experiment was done in duplicate. mM K 3 PO 4 . 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 ␤␥ complex was mixed with various concentrations of ␣ o subunit. Initially, 360 nM ␤␥ complex was incubated with increasing concentrations of the ␣ o subunit (11.25, 22.5, 45, 90, 180, and 360 nM) in ice for 30 min in a 10-l buffer containing 20 mM Hepes (pH 8.0), 100 mM NaCl, 2 mM MgCl 2 , 0.1 mM EDTA, 1 mM dithiothreitol, and 0.5 mg/ml bovine serum albumin. Five l of this mixture were then diluted 10 times to a total of 50 l of buffer containing 50 mM Na-Hepes (pH 7.2), 3 mM EGTA, 1 mM EDTA, 5 mM MgCl 2 , 100 mM NaCl, and 1 mM dithiothreitol. 10 l of this diluted sample containing ␤␥ and various concentrations of the ␣ subunit were then added to a total of 60 l of PLC reaction buffer containing [ 3 H]PIP 2 substrate and enzyme for determining the PLC-␤3 activity as described above (6 nM final concentration of ␤␥ complex).

RESULTS
␤ 4 ␥ 2 and ␤ 1 ␥ 2 Protein Expression and Purification-To study the function and properties of the G protein ␤ 4 subunit, we constructed a baculovirus expressing ␤ 4 . To confirm the viral expression of ␤ 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 ␤ 4 protein (Fig. 1A). To produce ␤ 4 ␥ 2 dimers, we simultaneously co-infected Sf9 cells with the ␤ 4 virus and a virus expressing a His-tagged ␥ 2 subunit. ␤ 4 ␥ 2 was purified by nickel-nitrilotriacetic acid chromatography. ␤ 1 ␥ 2 was expressed and purified using a similar approach. ␤ 4 consistently runs with slightly faster mobility than does ␤ 1 (Fig.  1B). This is consistent with a report from Asano et al. (35), who studied the native ␤ 4 protein expressed in bovine tissues. We confirmed that the purified ␤ 4 ␥ 2 and ␤ 1 ␥ 2 proteins contain the same concentration of detergents by using thin layer chromatography with the appropriate detergent standards (data not shown).
Stimulation of Effectors by ␤ 4 ␥ 2 and ␤ 1 ␥ 2 -To search for potential differences between ␤ 4 and ␤ 1 in effector regulation, we focused on three major effectors regulated by G protein ␤␥ complexes: PLC-␤2, PLC-␤3, and AC-II. The effect of ␤␥ complexes on these enzymes was examined. Fig. 2A shows the activation of purified PLC-␤2 by ␤ 4 ␥ 2 and ␤ 1 ␥ 2 complexes. Consistent with previous reports where brain ␤␥ was tested (36), both ␤␥ complexes stimulate PLC-␤2 more than 4-fold above basal activity with similar effectiveness. The PLC-␤2 stimulatory properties of both ␤␥ complexes are thus essentially identical. Although PLC-␤2 and PLC-␤3 are isozymes that are both stimulated by the G protein ␤␥ complex, there is evidence that the residues in the ␤ subunit that contact these two enzymes are distinct (37). This raised the possibility that although the two ␤␥ complexes showed little difference in the activation of PLC-␤2, they might interact differentially with PLC-␤3. We therefore examined the stimulation of PLC-␤3 by ␤ 4 ␥ 2 and ␤ 1 ␥ 2 . Both ␤␥ complexes activate PLC-␤3 20-fold over basal activity (Fig. 2B). The higher stimulation of PLC-␤3 compared with PLC-␤2 is consistent with previous reports (36). As in the case of PLC-␤2, the effectiveness with which ␤ 1 ␥ 2 and ␤ 4 ␥ 2 activate PLC-␤3 is similar. Because detergents in the ␤␥ preparations may themselves activate PLC-␤ (38), we also tested boiled preparations of ␤ 4 ␥ 2 and ␤ 1 ␥ 2 . Stimulation from boiled samples was minimal. Moreover, boiled samples of ␤ 4 ␥ 2 and ␤ 1 ␥ 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).
AC-II is stimulated by the G protein ␤␥ complex in the presence of the activated ␣ s subunit. To test whether ␤ 4 ␥ 2 and ␤ 1 ␥ 2 stimulate this enzyme differentially, we examined AC-II stimulation in the presence of GTP␥S-bound ␣ s . Sf9 cell membranes expressing AC-II were used as a source of the enzyme (29). In the presence of GTP␥S-bound purified ␣ s , ␤ 4 ␥ 2 and ␤ 1 ␥ 2 activate AC-II significantly over the basal level. Fig. 3 shows that, as in the case of PLC-␤, the extent of maximal stimulation (8-fold) by ␤ 4 ␥ 2 or ␤ 1 ␥ 2 and the effectiveness of both ␤␥ complexes in stimulating AC-II are similar.
M2 Receptor Activation of G o Containing the ␤ 1 and ␤ 4 Subunits-The receptor stimulation of a G protein can be measured as GTP␥S binding to the ␣ subunit or as GTPase activity of the ␣ 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 G o (which has not been determined) than to the ratio of receptor to G protein used in the less sensitive GTP␥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 ␥ subunit interaction with M2 where differences in coupling were detected using these conditions (34).
The M2 stimulation of ␣ o ␤ 1 ␥ 2 and ␣ o ␤ 4 ␥ 2 was examined by assaying GTPase activity in the presence of RGS4. The RGS4 protein, a GTPase-activating protein for the G o / i family, in- 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 P i produced (fmol) in a 5-l reaction. Experiments have been replicated at least three times, and representative data are shown.
creases the pool of G protein heterotrimers available to the receptor and considerably increases the sensitivity of the reaction (by ϳ10-fold). As expected, the addition of carbachol increases the GTP hydrolysis rate significantly (Fig. 4). More importantly, the M2-activated GTP hydrolysis of ␣ o ␤ 4 ␥ 2 was ϳ200% higher than that of ␣ o ␤ 1 ␥ 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 Ͻ 0.05). To further verify these data, two independent preparations of each purified ␤␥ complex containing ␤ 1 or ␤ 4 were examined again. These preparations provided similar results. As mentioned before, the concentration of detergent (CHAPS) in the purified ␤ 1 ␥ 2 and ␤ 4 ␥ 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 ␤ 1 ␥ 2 and ␤ 4 ␥ 2 complexes were examined in PLC-␤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 ␤ subunit types with the RGS4 protein was tested by assaying the M2-stimulated GTPase activity in the absence of the RGS protein. Again ␣ o ␤ 4 ␥ 2 was consistently 2-3-fold more active than ␣ o ␤ 1 ␥ 2 (Table I), indicating that the differential stimulation of GTPase activity resulted from receptor rather than RGS protein interaction.
Efficiency of Heterotrimerization of ␤ 1 ␥ 2 and ␤ 4 ␥ 2 Subunits with ␣ 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 ␣ o ␤ 1 ␥ 2 and ␣ o ␤ 4 ␥ 2 could be attributable to the differential heterotrimer formation between ␣ o and these two ␤␥ complexes. The residues in ␤ 1 and ␤ 4 that contact the ␣ subunit are conserved, indicating that the ␤ 1 and ␤ 4 affinity for ␣ o is likely to be the same (39). However, it was possible that heterotrimerization was differentially affected by divergent residues in ␤ 1 and ␤ 4 that were located at a distance from residues that contacted the ␣ 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 ␤␥ complex has overlapping sites for binding the ␣ subunit and the PLC-␤3 enzyme (40). Thus heterotrimerization prevents ␤␥ complex interaction with PLC-␤3, leading to the inhibition of the ␤␥ complex-stimulated PLC-␤3 activity. Because the assay is sensitive, it can be used to examine the ␣ o -␤␥ interaction at the same subunit concentrations (1-10 nM) used in the M2stimulated GTPase assays above. In contrast, the ADP-ribosylation assay that has been used extensively in the past is less sensitive (requiring more than ϳ1 M subunits), and in addition, it is complicated by the lack of knowledge regarding the mechanistic basis of the ␤␥ enhancement of the ␣ subunit ADP-ribosylation by pertussis toxin. As shown in Fig. 5, the ␤␥ complex-stimulated PLC-␤ activity is inhibited in a dose-dependent manner by the ␣ o subunit. The ␣ o concentration dependence of this inhibition of ␤ 1 ␥ 2 -and ␤ 4 ␥ 2 -stimulated PLC ␤ activity is similar, indicating that both complexes form a heterotrimer with ␣ o with equal effectiveness. DISCUSSION Because the G protein ␤␥ complex is known to interact directly with and modulate various effectors and the ␤ subunit is known to contact effectors, we first compared the relative abilities of the ␤ 4 ␥ 2 and ␤ 1 ␥ 2 complexes to stimulate three common effectors regulated by the ␤␥ complex: PLC-␤2, PLC-␤3, and AC-II. Both ␤␥ complexes activated each of the effectors with similar potency. This observation is consistent with the conservation between ␤ 1 and ␤ 4 of 93% of similar amino acids (Fig.  6A). It is unclear whether this result indicates that residues not conserved between ␤ 1 and ␤ 4 play no significant role in the regulation of effectors examined here. Comparing these results with previous mutational studies of ␤ 1 , which implicate particular residues in PLC-␤ regulation, does not resolve this question. ␤ subunit mutants analyzed in three different studies (40 -42) did not involve residues that are divergent between ␤ 1 and ␤ 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 ␤ 1 /␤ 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 ␤ 1 and ␤ 4 (Fig. 6A). This mutant ␤1␥ complex stimulated PLC-␤2 nor-FIG. 5. The ␤ 1 ␥ 2 and ␤ 4 ␥ 2 subunits form heterotrimer equally well with ␣ o . The ␤␥ subunit (6 nM) was incubated with the indicated concentration of ␣ subunit on ice for 30 min to allow heterotrimer formation. PLC-␤3 activity catalyzed by free ␤␥ but not ␣-␤␥ was determined to monitor the progress of complex ␤␥ with the ␣ subunit. The activity stimulated by free ␤␥ serves as a positive control representing the maximum PLC activity (100%), whereas the enzyme activity by ␣ alone was used as a negative control. The PLC activity was assayed as described under "Experimental Procedures." Values shown are the means (Ϯ S.E.) from three independent experiments. Differences in inhibition between ␤ 1 ␥ 2 and ␤ 4 ␥ 2 at various concentrations of ␣ o were not statistically significant in an unpaired t test. (1 nM M2) tested, the P i production was linear (as indicated in this table and in Fig. 4). c The differences in GTPase activity between ␣ o ␤ 1 ␥ 2 and ␣ o ␤ 4 ␥ 2 at all concentrations are statistically significant (p Ͻ 0.05). d GTPase activity was determined as described before and expressed as nM P i /min Ϯ S.E. During the time course of the experiment (Ͻ10 min), the GTPase activity increases in a linear fashion. The data are therefore the mean from at least four (G o ) points (similar to Fig. 4). e ND, not determined.
mally. Although very limited, this result is consistent with results presented here. It has been known for many years that the ␤␥ 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 ␥ subunit with a receptor. This evidence comes from studies of rhodopsin-G t coupling using peptides and mutant ␥ 1 subunits (45,46). It is also supported by the ability of a peptide specific to the ␥ 5 subunit type but not the ␥ 7 or ␥ 12 subunit type to inhibit muscarinic receptor-mediated signaling in superior cervical ganglion neurons (20). More recently, the ␥ 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 2-3fold difference in M2-stimulated GTP hydrolysis between ␣ o ␤ 4 ␥ 2 and ␣ o ␤ 1 ␥ 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 ␣ o and ␤ 1 ␥ 2 or ␤ 4 ␥ 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 ␣ o ␤ 4 ␥ 2 and ␣ o ␤ 1 ␥ 2 therefore indicate that the distinct primary structures of ␤ 1 and ␤ 4 influence the interaction of the heterotrimer with the receptor. Evidence that the ␥ subunit interacts with receptors and previous evidence that the ␣ subunit C and N termini interact with receptors (47) help identify the surface FIG. 6. Differences in the amino acid sequences of the ␤ 1 and ␤ 4 subunits mapped on the ␤␥ complex three-dimensional structure. A, primary structures of ␤ 1 and ␤ 4 subunits with the differences highlighted. Residues located in the putative receptor-interacting surface of the ␤␥ complex are numbered. Residues in the other ␤ subunit types at these loci are shown. (␤ subunit types from top to bottom are ␤ 2 , ␤ 3 , and ␤ 5 .) Arrows indicate ␤ strands in the folded ␤ subunit (panel B). Four ␤ strands make up a sheet. Sheets are denoted as lines labeled S1-S7. B, structure of the G protein subunit complex (␣ t ␤ 1 ␥ 1 from Lambright et al. (50)). Dark gray, ␣ subunit; light gray, ␤ subunit; and black, ␥ subunit. Open circles denote residues that are nonconservative changes between the ␤ 1 and ␤ 4 subunit amino acid sequences. The positions of these residues in the primary structure of ␤ 1 /␤ 4 are: residue 1, Ϫ31; residue 2, Ϫ35; residue 3, Ϫ37; residue 4, Ϫ39; residue 5, Ϫ302; residue 6, Ϫ303; and residue 7, Ϫ305. In the phosducin-␤␥ complex the prenyl group, farnesyl (C-15), is buried in the pocket between ␤ sheets S6 and S7 (49). Prepared with Ras Top 1.3 by P. Valadon. of the G protein that contacts the receptor (Fig. 6B). Inspection of the amino acid sequences of the ␤ 1 and ␤ 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 ␤ 1 and ␤ 4 subunits on the three-dimensional structure of the ␤ subunit indicates that two clusters of residues (31-39 and 302-305) are located on parallel strands of the ␤ 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 ␤ 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 ␣ subunit and the ␥ subunit (Fig. 6B). Finally, several of these residues show divergence between the various ␤ subunit types (Fig. 6A).
Antisense oligonucleotides specific to ␤ 1 and ␤ 3 have previously been shown to selectively inhibit somatostatin and muscarinic M4 receptor-mediated Ca 2ϩ 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 ␣ o in the presence of ␤ 1 /␤ 4 cannot be drawn both for this reason and because the physiological effect of the differential M2 stimulation of ␣ o ␤ 4 ␥ 2 versus ␣ o ␤ 1 ␥ 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 ␥ subunit and several of the residues in the ␤ subunit clusters are much less than 40 Å, so the ␤ subunit domains and the C terminus of the ␥ subunit can interact with a receptor simultaneously. In contrast, the distances between these domains in the ␤ subunit and the C terminus of the ␣ 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 ␤ subunit region containing residues 302-305 and the receptor is consistent with a previous study that showed the cross-linking of a ␤ subunit peptide covering residues 281-340 with an ␣ 2 -adrenergic receptor-derived peptide (48). Divergences in the amino acid sequence between ␤ 1 and ␤ 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 ␤␥ 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 ␤ sheets 6 and 7 of the folded ␤ subunit structure (49) (Fig. 6B). This region of the ␤ subunit is susceptible to conformational changes; the conformation of this region has been inferred to be different in the free ␤␥ complex compared with ␤␥ bound to phosducin (49). It is thus possible that the differences between ␤ 1 and ␤ 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 ␤ sheets 6 and 7. Because there is evidence that the prenyl moiety and the last several C-terminal residues of the ␥ subunit contact the receptors, such a rearrangement would indirectly affect the receptor interaction of the heterotrimer by altering the accessibility of the ␥ 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 ␤ subunit that undergoes conformational changes on binding to phosducin. These mutant forms of the ␤ 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 ␤ subunit mutants and wild type used in that study.
Overall, the importance of these results is that the differential receptor interaction of ␤ subunit types indicates a direct role for the ␤ subunit in receptor activation of a G protein.