©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Functional Analysis of a Dominant Negative Mutant of G(*)

(Received for publication, September 16, 1994; and in revised form, December 5, 1994)

Vladlen Z. Slepak Arieh Katz Melvin I. Simon (§)

From the Biology Division, 147-75, California Institute of Technology, Pasadena, California 91125

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The key event in receptor-catalyzed activation of heterotrimeric G proteins is binding of GTP, which leads to subunit dissociation generating GTP-bound alpha subunits and free beta complexes. We have previously identified a mutation that abolished GTP binding in Galpha(o) (S47C) and demonstrated that the mutant retained the ability to bind beta and could act in a dominant negative fashion when expressed in Xenopus oocytes (Slepak, V. Z., Quick, M. W., Aragay, A. M., Davidson, N., Lester, H. A., and Simon, M. I.(1993) J. Biol. Chem. 268, 21889-21894). In the current work, we investigated the effects of the homologous mutant of Galpha (S48C) upon signaling pathways reconstituted in transiently transfected COS-7 cells. We found that expression of the Galpha S48C mutant prevented stimulation of phospholipase C (PLC) beta2 by free beta subunit complexes. This effect of Galpha(i) S48C was not readily reversible in contrast to the inhibitory effect of wild-type Galpha, which could be reversed upon activation of the cotransfected muscarinic M2 receptor, presumably by release of beta from the G protein heterotrimer. Coexpression of Galpha(i) S48C or the wild-type Galpha also dramatically decreased G-mediated stimulation of PLC by C5a in the cells transfected with cDNAs encoding C5a receptor and Galpha. Activation of PLC via endogenous G(q) or G in the presence of alpha1C adrenergic receptors was similarly attenuated by coexpression of Galpha(i) or Galpha(i) S48C. Pertussis toxin treatment of the transfected cells enhanced the inhibition of the receptor-stimulated PLC by wild-type Galpha(i) subunits but did not influence the effects of the dominant negative mutant. The enhancement of the wild-type Galpha(i) inhibitory effect by pertussis toxin can be explained by stabilization of Galpha(i) binding to beta as a result of ADP-ribosylation, while Galpha(i) S48C mutant binds beta irreversibly even without pertussis toxin treatment. Therefore, a feasible mechanism to rationalize the attenuation of the Galpha and G-mediated activation of PLC by cotransfected Galpha(i) is the competition between Galpha(i) and Galpha or G for the beta complexes, which are necessary for the G protein coupling with receptors. These experiments provide new evidence for the role of beta in the integration of signals controlling phosphoinositide release through different Galpha families.


INTRODUCTION

Many receptors of hormones, neuromediators, and growth factors transmit signals via heterotrimeric G proteins. Receptor-induced activation leads to dissociation of G protein subunits, generating alpha subunits charged with GTP and free beta complexes. In recent years, G protein-mediated signaling has proven to be far more complex than just a combination of ``linear'' pathways from specific receptors to their effectors. The number of cloned genes encoding G protein subunits, effectors, and receptors includes hundreds of members(1, 2, 3, 4) . Furthermore, in many signaling pathways, activity of effectors is modulated not solely by Galpha subunits as was traditionally thought but also by Gbeta complexes(5, 6, 7, 8, 9, 10, 11) . One of the ways to define the specific links within the network of G proteins, multiple receptors, and effectors is by blocking the signaling circuits by expressing dominant negative mutants of different Galpha subunits in vivo. Mutations of glycine residues in the conservative sequence DVGGQR of the Galpha subunits were found to reduce GTP binding and activation of the G proteins. Expression of these mutants inhibited pathways controlled by Galpha(s) and Galpha(i)(12, 13, 14, 15) . We identified another interesting mutation that abolished GTP binding in Galpha(o)-Galpha(o) S47C and demonstrated that the mutant retained the ability to bind beta and could suppress G protein-mediated signal transduction in Xenopus oocytes (16) .

In the current work, we studied the effects of expression of the Galpha, Galpha, and their dominant negative mutants Galpha(i) S48C and Galpha(o) S47C on reconstituted signaling pathways in transiently transfected COS-7 cells. We found that Galpha(i) can attenuate activation of PLC (^1)by hormone receptors coupled to the enzyme via members of the G(q) family and that the most likely molecular mechanism for this inhibition is competition for beta subunits. Our data provide further evidence for the interdependence of G protein-mediated signaling pathways and the important role of beta subunits in controlling these interactions.


EXPERIMENTAL PROCEDURES

Site-directed Mutagenesis and Construction of COS Cell Expression Vectors

The Galpha(i) S48C, Galpha(q) S47C, and Galpha S56C mutations were introduced by using polymerase chain reaction(17) . The cDNAs were inserted into pCMV vector as previously described(18) . The Galpha(o) S47C mutant was subcloned into pCMV from pG(o)alpha bacterial expression vector(16) . Preparation of pCMV vectors containing the cDNAs for alpha1C adrenergic and C5a receptors, PLC beta2, and G protein alpha, beta, and subunits was previously described(18, 19) .

Cell Culture and Transfection

COS-7 cells were maintained in DMEM containing 10% FCS. 1 times 10^5 cells/well were seeded in 12-well plates 1 day before transfection. The total amount of DNA in all transfections was 1.0 µg/well. The amount of each type of DNA in each set of experiments was equal, and pCIS encoding beta-galactosidase was used to maintain a constant amount of DNA. To each well, 1.0 µg of DNA mixed with 10 µl of lipofectamine (Life Technologies, Inc.) in 0.5 ml of Opti-MEM (Life Technologies, Inc.) was added, and 5 h later, 0.5 ml of 20% FCS in DMEM was added to the cells. Cells were assayed for inositol phosphate levels or harvested for protein expression analysis 48 h after transfection.

Analysis of Phosphoinositide Release

1 day post-transfection, the medium was removed, and the cells were washed with phosphate-buffered saline and incubated in 0.4 ml of medium of inositol-free DMEM with 10% dialyzed FCS containing 10 µCi/ml myo-[2-^3H]inositol (DuPont NEN). 24 h later, the cells were washed with phosphate-buffered saline, 200 µl of inositol-free medium containing 10 mM LiCl was added, and the cells were incubated for 25 min at 37 °C. Each reaction was stopped by adding 200 µl of ice-cold mix of 10% perchloric and 0.2% phytic acids and incubating the cells on ice for 10 min. Then, 200 µl of the supernatant was transferred to a microcentrifuge tube and neutralized with 2 M KOH. After centrifugation, the supernatant was loaded on 0.5 ml of AG1-X8 anion exchange column (200-400 mesh, formate form, Bio-Rad) separated and counted as described earlier(18, 19) . All the data represent duplicate determinations in a single experiment. The error bar expresses the range of the duplicates. Three additional experiments gave similar results.

Pertussis Toxin Labeling

The membranes were prepared from the transfected COS cells as described(19) . Membranes (30 µl, 1-2 mg/ml protein) were preincubated with guanine nucleotides for 15 min at 22 °C, and then [P]NAD (5 µl, 0.1 µM, 500,000 cpm) and activated pertussis toxin (5 µl, 10 ng) was added. After 30 min of incubation, the samples were mixed with 10 µl of a 5 times SDS-polyacrylamide gel electrophoresis sample buffer, heated for 3-5 min at 95 °C and analyzed on 10% polyacrylamide gel as previously described(20) .


RESULTS AND DISCUSSION

Substitution of serine 47 for cysteine in Galpha(o) abolished GTP binding, but the mutant retained its interaction with beta. Due to the apparent inability to release beta upon the hormonal activation, this mutant behaved as dominant negative in the G(o)-mediated signaling pathway reconstituted in Xenopus oocytes(16) . To obtain a similar mutant of Galpha(i), we replaced the homologous Ser-48 with Cys by modifying the cDNA of Galpha. We tested this mutant to determine if it would inhibit pertussis toxin-sensitive pathways of PLC stimulation in mammalian cells. In these pathways, PLC is apparently activated by free beta complexes (8, 9, 10) that are released from G protein heterotrimers upon hormonal stimulation. Fig. 1demonstrates that in the COS-7 cells cotransfected with cDNAs for beta, , and PLC beta2, there is a 4-6-fold stimulation of phosphoinositide hydrolysis compared with cells expressing PLC beta2 alone. Cotransfection of both wild-type Galpha(i) cDNA and the Galpha(i) S48C mutant abolished the beta-induced PLC activity, apparently due to sequestration of free beta. However, the behavior of mutant and the wild-type Galpha subunits was different with respect to receptor-mediated activation of PLC. In the cells cotransfected with muscarinic M2 receptors (Fig. 1B), carbachol stimulation resulted in an increase of inositol phosphates released in the presence of beta and wild-type Galpha(i). In contrast, no ligand-induced activity was observed in cells cotransfected with the S48C mutant of Galpha(i), presumably because it bound beta irreversibly.


Figure 1: Influence of Galpha(i) and its S47C mutant on stimulation of PLC beta2 by free beta. A, cells were cotransfected with cDNAs encoding phospholipase beta2 (0.2 µg), Gbeta subunit (beta(1), 0.2 µg), G (0.2 µg) (2 or 5 were used in different experiments), and wild-type (WT) or mutant Galpha (0.3 µg). Total amount of DNA was adjusted to 1.0 µg by CMV-LZ cDNA. Cells were then labeled with [^3H]inositol, and levels of inositol phosphates were determined. Transfection, labeling, and analysis of inositol phosphate release were performed as previously described(15) . B, cells were cotransfected also with cDNA for muscarinic M2 receptor, and levels of released inositol phosphates were determined after incubation of the cells with indicated concentrations of carbachol.



PLC beta2 can be stimulated by two pathways: pertussis toxin-sensitive (by beta subunits released upon the activation of G proteins (members of the G(i) family)) and pertussis toxin-insensitive (by Galpha subunits of the G(q) family). If both pathways share the same pool of beta subunits, the dominant negative mutants of G(i) family alpha subunits would not only block the stimulation of PLC by beta but also prevent its activation through G(q) family alpha subunits. To test this idea, we cotransfected COS-7 cells with cDNAs encoding the C5a receptor and Galpha, reconstituting a pathway for G-mediated stimulation of endogenous PLC by C5a (Fig. 2). Coexpression of either wild-type Galpha subunit or its mutant S48C in the system did not have a significant effect on the basal level of inositol phosphate release but markedly reduced the C5a stimulation. Similar inhibitory effects were observed upon cotransfection with other pertussis toxin-sensitive Galpha subunits such as Galpha, Galpha(o), or the Galpha(o) S47C mutant (data not shown). Western analysis demonstrated that introduction of these proteins did not change the level of Galpha expression (Fig. 2B). This suggested, that the inhibitory effect of Galpha(i) proteins is due to specific interaction with components of the reconstituted signaling pathway and not because of interference with transcription/translation machinery of the transfected cells. The maximal level of inhibition of the C5a receptor-PLC pathway by Galpha(i) was around 70%. It is possible that the inhibitory effect could have been larger if all of the transfected cells could take up and express all three transfected cDNAs. The effect of Galpha(i) was proportional to the amount of protein expressed in the cells, i.e. higher concentrations of Galpha(i) caused stronger inhibition of the C5a-induced PLC activity (Fig. 3). This observation suggests that inhibition occurred due to competition between G(i) and G alpha subunits for the interaction with other protein components involved in the C5a induction of PLC activity.


Figure 2: Inhibition of C5a-stimulated PLC activity by Galpha(i) and its S48C mutant in transiently transfected COS-7 cells. Cells were transfected with C5a receptor (0.2 µg) and Galpha (0.2 µg) cDNAs together with cDNAs (0.6 µg) corresponding to wild-type (WT) Galpha, its S48C mutant, or pCMV-LZ (control). A, levels of inositol phosphates released were determined with no ligand or in the presence of 0.25 µM C5a. B and C, Western blot analysis of the transfected COS-7 cells. Cells were treated the same way as those for the PLC assay with exception that [^3H]inositol was omitted from the media. On the day of the PLC assay, they were harvested and subjected to Western analysis with antibodies raised against Galpha(i) (B) or Galpha (C).




Figure 3: Inhibition of C5a-stimulated PLC activity by different amounts of Galpha(i) S48C mutant. Cells were transfected with C5a receptor (0.2 µg) and Galpha (0.2 µg) cDNAs and indicated amounts of Galpha S48C mutant cDNA. The total amount of DNA per transfection was adjusted to 1.0 µg with pCMV-LZ. Levels of inositol phosphates were determined with no ligand (openbar) or in the presence of 0.25 µM of C5a (graybars). Inset, Western analysis of identically treated cells with antibodies raised against Galpha(i) (GalphaS48C) and Galpha.



Both Galpha(i) and Galpha have been shown to couple to the C5a receptor(21, 22, 23) . It is possible that Galpha(i) sequesters beta, which may be necessary for coupling of C5a receptor with Galpha. Alternatively, G(i) and G may compete for interaction with the receptor. Coupling of G(i) with its cognate receptors can be abolished after ADP-ribosylation with pertussis toxin. We found, however, that ADP-ribosylation of wild-type Galpha(i) promoted even further inhibition of the C5a-induced PLC activation, while cells expressing only C5a and Galpha were insensitive to the toxin (Fig. 4). This observation implies that Galpha(i) attenuates the C5a-induced activation of PLC by competing with the Galpha not on the receptor but rather by competing for beta. It is known that ADP-ribosylation stabilizes association of the alpha and beta subunits(24) . In the pertussis toxin-treated cells, the wild-type Galpha(i) binds beta stronger and therefore competes with Galpha for beta more efficiently. Further support for this notion comes from cotransfecting Galpha and C5a receptor together with the S48C mutant of Galpha(i). Treatment of these cells with pertussis toxin did not lead to any further inhibition because the mutant has been shown to bind beta irreversibly even without pertussis toxin treatment(13) . To ensure that both wild-type and the mutant G(i)alpha were ADP-ribosylated equally, we treated the membrane preparations of the COS-7 cells with pertussis toxin in the presence of [P]NAD. Fig. 4(inset) demonstrates that both proteins were labeled to a similar extent, and, as in the case with Galpha(o)(13) , GTPS did not influence labeling of the S48C mutant but drastically reduced the modification of the wild-type Galpha(i). These experiments suggest that the mechanism for the attenuation of hormone-activated PLC by Galpha(i) is based upon sequestering of beta that can be ``shared'' with Galpha. It is unlikely that competition occurs at the effector level because previous evidence argues against the interaction of G(i) family proteins with PLC(10, 18, 19) . The possibility that Galpha(i) causes the inhibition of PLC indirectly by activation of a different effector is also ruled out because the Galpha(i) dominant negative mutant, which cannot bind GTP and be activated, is more potent in attenuation of hormone-stimulated PLC than wild-type Galpha(i).


Figure 4: Influence of pertussis toxin (PTX) on inhibition of C5a-stimulated PLC by Galpha(i) or its S48C mutant in transfected COS-7 cells. Cells were transfected with C5a receptor (0.2 µg), Galpha (0.2 µg) cDNAs and cDNAs (0.6 µg) corresponding to wild-type Galpha, its S48C mutant, or pCMV-LZ (control). Levels of inositol phosphates were determined after treatment with (blackbars) or without (graybars) 200 µg/ml pertussis toxin for 4 h at 37 °C prior to addition of 0.25 µM C5a. Inset, influence of 100 µM GTPS on pertussis toxin-catalyzed ADP-ribosylation of wild-type Galpha(i) and the S48C mutant. Cell membranes were obtained and treated with pertussis toxin in the presence of [P]NAD, 10 µM GDP, and with or without 0.1 mM GTPS as described under ``Experimental Procedures.'' Proteins were then resolved by SDS electrophoresis. Gels were stained with Coomassie Blue, dried, and exposed to x-ray film.



If it is binding of beta that is responsible for the interference of Galpha(i) with the Galpha (Galpha)-mediated signaling, we would expect that Galpha(i) would inhibit ligand induction through receptors that do not interact with Galpha(i), such as alpha1C adrenergic receptors(25) . This was indeed the case; in the cotransfected COS-7 cells, alpha1C adrenergic receptor-PLC coupling, which is apparently mediated by endogenous G(19) , was inhibited by Galpha(i) and Galpha(o) (Fig. 5). As found with the C5a receptor, this inhibition was significantly enhanced by pertussis toxin treatment. Therefore, it is quite likely that endogenous beta is a limiting factor for the coupling of Galpha or Galpha with their cognate receptors.


Figure 5: Effect of Galpha(i) and Galpha(o) on stimulation of PLC by alpha1C adrenergic receptor in transiently transfected COS-7 cells. Cells were transfected with alpha1C-adrenergic receptor (0.4 µg) cDNA and cDNAs (0.6 µg) corresponding to wild-type Galpha, Galpha, or pCMV-LZ. Stimulation of inositol phosphate release by 10 µM norepinephrine (NE) was determined after (blackbars) or with no (graybars) treatment with pertussis toxin (PTX).



Recently published crystal structures of transducin alpha subunit complexes with GTPS (26) and GDP (27) show that the hydroxyl group of serine 43, which is homologous to serine 48 in Galpha(i), coordinates with Mg found in the GTP binding pocket. The conversion of the -OH to -SH in a Ser Cys mutant apparently does not cause a major disruption of the overall structure of Galpha protein, since Galpha(i) S48C can still bind beta. Because the serine residue is conserved in the Galpha family, we introduced the Ser Cys mutation into different Galpha subunits to use them for inhibition of specific signaling pathways. However, the mutations introduced in Galpha(q) (S47C), Galpha (S56C) resulted in a null phenotype (data not shown). The mutants were expressed in cells at the same level as wild-type proteins according to Western analysis but failed to reveal any functional activity. They did not stimulate PLC and did not inhibit hormone-stimulated PLC or the stimulation of the enzyme by free beta complexes. It is noteworthy that introduction of other putative dominant negative mutations, G203T and G204A, into Galpha(q) and Galpha also resulted in a null phenotype, (^2)while the homologous mutations G203T and G204A in G(i)(12, 13, 14, 15) or G(o)(16) proved to be functional. Interestingly, mutants G203T and G204A of recombinant Galpha(o) had very similar biochemical properties, yet only G203T Galpha(i) behaved as a dominant negative in vivo while G204A resulted in null phenotype(14) . At this point, we do not understand exactly why the mutants of G(q) family alpha subunits were inactive. However, these results suggest that there are local differences in structure and activity even in highly conserved regions of Galpha subunits. Detailed comparison of the crystal structures of G(i) and G(q) alpha subunits will shed light on the differences between these highly homologous proteins.

Cross-talk between the different G protein-mediated signaling pathways has been previously demonstrated(28, 29, 30) ; for instance, adenylate cyclase type IV was found to integrate signals coming through G(s), G(i), and G(q)(31) . Here, we demonstrate that Galpha(i)-like proteins can alter the pathways regulating PLC via G(q) family G proteins. Can this occur in vivo? Some indirect evidence supports the existence of such mechanisms. For example, in vivo most of the signaling through C5a receptor is pertussis toxin sensitive, whereas in vitro C5a receptor couples to pertussis toxin-insensitive G. In neutrophils where Galpha is the most abundant Galpha protein, these contradictory observations can be reconciled if a mechanism similar to the one shown on Fig. 4took place, i.e. ADP-ribosylation increased affinity of Galpha(i) for beta, thus preventing coupling of receptor to G. Therefore, the apparent pertussis toxin sensitivity of G-mediated signaling can be explained as a result of depleting the pathway of beta. In light of such a possibility, the interpretation of the experiments on pertussis toxin treatment of cells must be done with care; ADP-ribosylation not only can uncouple G(i) from its cognate receptor, but it can shift the equilibrium alpha(i) + beta = alphaibeta toward the heterotrimer, thus reducing the available pool of beta subunits and affecting other pathways. This mechanism implies that beta is a limiting factor for G (G(q))-mediated signaling. Recent data show that beta can be bound by other proteins such as effectors PLC beta(8, 9, 10) , PLA2(6) , inositol kinase(32) , and K channel(33, 34) , ras-related proteins(35) , phosducin(36) , receptor kinase, and other proteins containing pleckstrin homology domains(37, 38) , calmodulin(39) , etc. Therefore, the GDP-bound Galpha subunits compete for the binding to beta not only with different Galpha but also with these ``other'' proteins. It is clear that relative affinities of the different Galpha subunits for beta complexes are critical for specific channeling of signals. Another important notion is that regulation of the expression level of the G protein subunits could provide additional diversity to signal transduction pathways in various cells.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed.

(^1)
The abbreviations used are: PLC, phosholipase C; GTPS, guanosine 5`-3-O-(thio)triphosphate; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum.

(^2)
A. Katz, T. Wilkie, and D. Wu, unpublished data.


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