Selective Role of G Protein γ Subunits in Receptor Interaction*

Receptor stimulation of nucleotide exchange in a heterotrimeric G protein (αβγ) is the primary event-modulating signaling by G proteins. The molecular mechanisms at the basis of this event and the role of the G protein subunits, especially the βγ complex, in receptor activation are unclear. In a reconstituted system, a purified muscarinic receptor, M2, activates G protein heterotrimers αi2β1γ5 and αi2β1γ7 with equal efficacy. However, when the α subunit type is substituted with αo, αoβ1γ7 shows a 100% increase in M2-stimulated GTP hydrolysis compared with αoβ1γ5. Using a sensitive assay based on βγ complex stimulation of phospholipase C activity, we show that both β1γ5 and β1γ7 form heterotrimers equally well with αo and αi. These results indicate that the γ subunit interaction with a receptor is critical for modulating nucleotide exchange and is influenced by the subunit-type composition of the heterotrimer.

The G protein cycle is primarily regulated by the interaction of heterotrimeric G protein and cell surface receptors. Both ␣ and ␤␥ subunits are required for interaction with receptor (1)(2)(3). The G protein ␣ subunit has been demonstrated to interact with and selectively couple to receptors, especially muscarinic receptors (4). The role of the ␤␥ complex in interaction with the receptor is, however, less well understood. There is evidence for interaction of the G protein ␥ subunit with receptors (5). There are also indications for specificity in this interaction. Results from experiments in pituitary GH3 cells using antisense oligonucleotides specific to different ␤ or ␥ subunit types indicated that signaling stimulated by different receptors can be specifically inhibited (6). The ␥1 subunit type allowed effective coupling of Gt with rhodopsin in contrast to ␥2 and ␥3 (7). In superior cervical ganglion (SCG) 1 neurons peptides spe-cific to the ␥5 subunit type disrupted signaling from the M2/M4 muscarinic receptors, whereas peptides specific to ␥7 and ␥12 had no effect (8). Because this implied that the M2 muscarinic receptor selectively interacts with a G protein containing ␥5 but not ␥7, we tested the ability of the M2 receptor to activate G proteins containing these two subunits. Earlier studies addressing the question of G protein specificity for receptors used whole cells or crude membranes from cells. Experiments with intact cells do not definitively allow identification of the site at which the disruption in signaling occurs. Crude membranes contain endogenous G proteins, receptors, and other components that may affect analysis of specificity in receptor-G protein interactions. To more rigorously and directly examine the effect of G protein subunit constitution on receptor-G protein coupling, we reconstituted a purified muscarinic receptor, M2, in lipids and measured its ability to activate G proteins containing different ␥ subunits. The M2 receptor is known to couple to members of the Gi/o family but not Gq (9). To examine whether the subunit-type constitution of a heterotrimer influenced receptor interaction we tested different combinations of the ␣ and ␥ subunit types ␣o␤␥5, ␣o␤1␥7, ␣i2␤1␥5, and ␣i2␤1␥7. Recombinant G protein subunits were purified from insect cells, and heterotrimers constituting different combinations of ␣ and ␥ subunits were assembled. We measured M2stimulated GTP␥S binding and GTPase activity using defined G protein heterotrimers. GTP␥S binding assays were performed at a ratio of the G protein ␣ subunit to receptor of 100:1 (1 nM receptor). Because subtle differences in receptor activation could be missed under these conditions, we developed GTPase assays to measure receptor activation of a G protein at a ratio of G protein to receptor approaching 1:1 (1 nM receptor). These assays detected consistent and significant differences in the ability of the M2 receptor to activate ␣o␤1␥5 compared with ␣o␤1␥7. In contrast, when the ␣ subunit type was substituted with ␣i2 there was no difference in receptor-stimulated GTPase activity between ␣i2␤1␥5 and ␣i2␤1␥7. The difference in receptor-stimulated activity between ␣o␤1␥5 and ␣o␤1␥7 could be due to differential heterotrimer formation between ␣o and these ␤␥ complexes. To test this possibility we developed a novel phospholipase C (PLC)-based assay to measure heterotrimer formation. This assay indicated that the differences in GTPase activity between ␣o␤␥5 and ␣o␤␥7 arose as a result of differential receptor coupling rather than differential heterotrimer formation.

Expression and Purification of Recombinant G Protein Subunits-G
protein ␤␥ subunits were expressed in the baculovirus Sf9 cell system. The purification was essentially performed according to the procedures described before (10). The purity and quantity of these ␤␥ proteins were assessed by separating by SDS gel electrophoresis, staining with Coomassie Blue, scanning with a laser densitometer, and comparing with protein standards. G proteins ␣o and ␣i2 were purified from Escherichia coli using published methods (11). RGS4 was a gift from Dr. M. Linder, Washington University.
Measuring PLC Activity-The ␤␥-stimulated PLC assay was performed using a procedure as stated previously (12).
Purification, Reconstitution, and Functional Characterization of Recombinant M2-His-tagged M2 was expressed in Sf9 cells and purified using a CoCl 2 affinity column (13). Purified M2 was reconstituted into brain lipids (Folch type VII; Sigma) and characterized by binding to antagonist, [ 3 H]N-[ 3 H]methylscopolamine (NMS). The receptor (50 pM) was incubated with various concentrations of [ 3 H]NMS at room temperature for 60 min in a binding buffer containing 20 mM sodium phosphate (pH 7.4) and 10 mM MgCl 2 . The reactions were terminated by filtration through Whatman GF/B membranes, and the filters were then washed with ice-cold binding buffer before counting the radioactivity.
GTP␥S Binding and GTP Hydrolysis-The formation of heterotrimeric Go protein, the M2-G protein complex, GTP␥S binding, and GTP hydrolysis assays are described in the legends for Figs. 2 and 3.
Measuring Heterotrimer Formation-To form the G protein heterotrimer, 360 nM ␤␥ complex was initially incubated with increasing concentrations of ␣ subunit in ice for 30 min to obtain a wide range of ␤␥:␣ ratios in a buffer containing 20 mM Hepes (pH 8.0), 100 mM NaCl, 2 mM MgCl 2 , 0.1 mM EDTA, 1 mM DTT, and 0.5 mg/ml BSA. This mixture was then diluted 10-fold in a buffer containing 50 mM Hepes (pH 7.2), 3 mM EGTA, 1 mM EDTA, 5 mM MgCl 2 , 100 mM NaCl, and 1 mM DTT. 10 l of these diluted samples containing ␤␥ and various concentrations of ␣ subunit was then added to 50 l of PLC reaction buffer containing [ 3 H]PIP 2 substrate and PLC ␤3 for determining enzyme activity as described above.

RESULTS AND DISCUSSION
Purification and Functional Characterization of Recombinant G Protein Subunits and M2-␤␥ complexes purified as described above were over 95% pure (Fig. 1A). When PLC ␤3 isozyme stimulation by ␤1␥5 or ␤1␥7 complexes was measured, the similar levels of activation of PLC by both ␤␥ complexes indicated that the functional proportion of each ␤␥ complex was the same (Fig. 1B). In addition, we ensured that the concentration of detergent in the purified ␤1␥5 and ␤1␥7 samples were identical, using thin layer chromatography and detergents at various concentrations as standards (data not shown). His-M2 has been shown to possess similar properties to native M2 after reconstitution into lipids (13). His-M2 receptor was purified as described earlier. The purified M2 was ϳ90% pure as assessed by the methods used to quantify the G protein subunits. Reconstituted M2 had a K d for an antagonist, NMS, similar to native M2 (14) (Fig. 1C).
M2-stimulated GTP␥S Binding to Go Containing ␥5 or ␥7-To allow for accurate titration of various concentrations of G protein subunits with purified receptor, we developed a system in which receptor alone was first reconstituted into lipids, quantified, and then assayed for its ability to activate varying concentrations of added G protein (Fig. 2, see legend). ␣o was assayed in the presence of two different ␤␥ complexes, ␤1␥5 and ␤1␥7. The reconstituted M2 efficiently activates ␣o in a ␤␥-dependent manner ( Fig. 2A). At an ␣:␤␥:M2 ratio of 100: 10:1 with 1 nM M2, no differences were noted in the ability of M2 to activate ␣o␤1␥5 and ␣o␤1␥7 ( Fig. 2A). It was possible that at lower concentrations of ␣o and an ␣:M2 ratio closer to 1:1, subtle differences would be detected. However, GTP␥S binding at lower concentrations of ␣o (e.g. 10 nM) was not sufficiently above background for use in these assays. Given this constraint, it was possible only to examine the effect of  varying concentrations of ␤␥ complex, as well as agonist, on M2-stimulated GTP␥S binding to ␣o␤1␥5 and ␣o␤1␥7. These assays did not reveal any differences between the heterotrimers (Fig. 2, B and C). ␤␥ complex concentrations below 10 nM could not be examined because of the low activity detected above background.
Go but Not Gi Containing Different ␥ Subunits Shows Differences in M2-stimulated GTPase Rates-The M2-promoted GTP␥S binding assay could mask differential activation of heterotrimers containing different ␥ subunits for the following two related reasons: (i) the excess of ␣ subunit relative to receptor (100:1) present in the GTP␥S assay and (ii) the lack of amplification in the signal, because an ␣ subunit bound to the nonhydrolyzable GTP␥S analog undergoes only one cycle of activation unlike GTP-bound ␣ subunit. We therefore examined M2-activated GTP hydrolysis by defined Go/i protein heterotrimers. M2 stimulation with carbachol increased GTPase activity 5-10-fold over the activity in the absence of agonist. The addition of RGS4, a GTPase-activating protein for the Go/i family (15), promoted an additional 10-fold increase in GTPase activity (data not shown). This increase in sensitivity allowed us to measure G protein activation at ratios of ␣ to receptor close to 1:1 with a constant concentration of 1 nM M2. When defined heterotrimers were assayed under these conditions, the receptor-stimulated GTPase activity of ␣o␤1␥7 was 100% higher than ␣o␤1␥5 (see Fig. 3A and Table I). This difference was seen at all G protein concentrations tested (Table I). The difference was also seen with ␤1␥5 and ␤1␥7 preparations purified independently indicating that the difference was not because of contaminants in one particular preparation of the ␤␥ complexes. Statistical analysis using the Student's t test showed that the differences are significant (p Ͻ 0.05). To test whether the ␣ subunit type in the heterotrimer influenced this differential activity, we compared the receptor-stimulated GTPase activity of ␣i2␤1␥5 and ␣i2␤1␥7. Surprisingly, there was no difference in GTPase activity between the two heterotrimers at various ratios of Gi2:M2 (see Fig. 3B and Table I).
The ␤1␥5 and ␤1␥7 Subunits Form Heterotrimers Equally Well with ␣o or ␣i2-Formation of the heterotrimer is essential for G protein interaction with a receptor (3) (Fig. 2). One explanation for the difference in the receptor-stimulated GTPase activity between ␣o␤1␥5 and ␣o␤1␥7 could be differential efficacy of heterotrimer formation between ␤1␥5 and ␤1␥7 with ␣o. To test this possibility, we developed an assay for measuring G protein heterotrimer formation. Thus far, efficacy of heterotrimer formation has been measured using pertussis toxin-mediated ADP ribosylation of the ␣ subunit, which is enhanced by the ␤␥ complex. However, the mechanistic basis for ␤␥ complex requirement in this assay is unclear. Also, the enhancement by the ␤␥ complex is catalytic and requires relatively high concentrations of the subunits (Ͼ1 M) inappropriate for measuring heterotrimer formation under conditions identical to the GTPase assays used here (Ͻ10 nM subunits). To overcome these problems we developed an alternative approach. This approach was based on the idea that the ␤␥ complex contains overlapping binding sites for both the ␣ subunit and PLC ␤3 (16). Thus, binding of ␣i/o to ␤␥ complex would prevent ␤␥ interaction with PLC ␤3. Heterotrimer formation should thus lead to inhibition of ␤␥-stimulated PLC activity. As shown in Fig. 4, this assay is highly sensitive. ␤␥-stimulated PLC activity is inhibited by the addition of ␣o or ␣i subunit. Inhibition is dependent on the concentration of ␣ subunit added. The assay is sensitive in the nM range of ␣ and ␤␥ subunits. Both ␤1␥5 and ␤1␥7 form a heterotrimer with ␣o and ␣i2 equally well (Fig. 4, A and B).
G Protein ␣ and ␥ Subunit Interaction with Receptor-Indi-cations that the ␥ subunits may impart specificity to receptor-G protein interaction have come from the analysis of different receptors (7,8,17,18). In studies with the muscarinic receptor, a peptide specific to the C terminus of the ␥5 subunit was shown to inhibit coupling of G protein to the M2 receptor and also disrupt a muscarinic receptor (M2/M4)-mediated signaling pathway in SCG neurons (8). In comparison, a homologous peptide from ␥7 was ineffective indicating that the ␥5 subunit interacted with the M2/M4 class of receptors, whereas ␥7 did not. To test whether the results of the peptide activity in intact cells could be reproduced in a reconstituted system, we expressed, purified, and assayed the ability of G proteins containing the same ␥ subunit types to functionally couple to the purified M2 muscarinic receptor. By using a sensitive functional assay we sought to elucidate the underlying mechanistic bases for any differences in coupling. The results show a clear difference in the ability of ␣o heterotrimers containing ␥5 or ␥7 to be activated by M2 receptors. ␣o␤1␥7 has a distinctly higher GTP hydrolysis rate compared with ␣o␤1␥5. A simple interpretation of this would be that ␣o␤1␥7 couples more efficiently with M2 than ␣o␤1␥5. However, we favor an alternative interpretation in the context of earlier experiments, which indicate that the C terminus of the ␥5 subunit more effectively interacts with M2 than ␥7 (8). Because ␤1␥5 and ␤1␥7 interact equally 1000 times more than radiolabeled GTP) served as a control for nonspecific activity. Aliquots were taken at indicated time points and quenched in 0.5 ml of ice-cold buffer containing 5% activated charcoal and 50 mM K 3 PO 4 . Samples were centrifuged, and supernatants were quantified by scintillation counting. Experiments have been repeated at least five times, and representative data are shown. well with ␣o, the difference in GTPase activity has to arise from a difference in the rate of receptor-stimulated nucleotide exchange between the two heterotrimers. It is likely that the ␥5 subunit interacts appropriately with the M2 receptor, whereas ␥7 does not, and the increased GTPase activity is a consequence of more rapid "leaky" nucleotide exchange from the resulting inappropriate configuration of the ␣o subunit with reference to the ␤1␥7 complex. This interpretation is consistent with a previous conclusion that the ␤␥ complex plays a direct or indirect role at the receptor surface in controlling nucleotide exchange in the ␣ subunit (19). Strikingly, the difference seen between ␣o␤1␥7 and ␣o␤1␥5 disappears when ␣o is substituted with ␣i2. One possibility is that ␣i2 interacts with a different site on the receptor compared with ␣o thus changing the overall conformation of the G protein heterotrimer at the receptor surface. There are evidences from the analyses of different receptors for such differential interaction with G protein subtypes (20). The distinctly differential rate of M2-induced Go and Gi GTPase rates (Fig. 3), as well as GTP␥S binding (21), are also consistent with this scenario. Regardless of the mechanism, this result indicates that the heterotrimer composition influences receptor-stimulated nucleotide exchange.
Because the G protein subunits are families of proteins it has been thought that particular combinations of ␣ and ␤␥ subtypes may differentially regulate signaling (22). The results here indicate that different ␣ subunit and ␥ subunit types, through specific interactions with a receptor, can coordinately modulate receptor-stimulated nucleotide exchange resulting in differential signaling kinetics.