J Biol Chem, Vol. 273, Issue 46, 30183-30188, November 13, 1998

Interaction of the Xanthine Nucleotide Binding Go Mutant with G Protein-coupled Receptors^*

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

	ABSTRACT

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We constructed a double mutant version of the  subunit of Go that was regulated by xanthine nucleotides instead of guanine nucleotides (GoX). We investigated the interaction between GoX and G protein-coupled receptors in vitro. First, we found that the activated m2 muscarinic cholinergic receptor (MAChR) could facilitate the exchange of XTPS for XDP in the GoX heterotrimer. Second, the GoX complex was able to induce the high affinity ligand-binding state in the N-formyl peptide receptor (NFPR). These experiments demonstrated that GoX was able to interact effectively with G protein-coupled receptors. Third, we found that the empty form of GoX, lacking a bound nucleotide and , formed a stable complex with the m2 muscarinic cholingeric receptor associated with the plasma membrane. Finally, we investigated the interaction of GoX with receptor in COS-7 cells. The empty form of GoX bound tightly to the receptor and was not activated because XTP was not available intracellularly. We tested the ability of GoX to inhibit the activities of several different G protein-coupled receptors in transfected COS-7 cells and found that GoX specifically inhibited Go-coupled receptors. Thus the modified G proteins may act as dominant-negative mutants to trap and inactivate specific subsets of receptors.

	INTRODUCTION

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Hundreds of seven-transmembrane receptors activate heterotrimeric G proteins and transduce signals across cell membranes in eukaryotic cells. The stimulated receptors catalyze the exchange of GTP for GDP bound to G protein  subunits. Activated GTP-bound  subunits and free subunits regulate a variety of cellular effectors including enzymes and ion-channels (1-3). Signaling is normally initiated by the binding of agonist to receptor, which stabilizes the receptor in an active conformation. Receptors function to stimulate the dissociation of GDP bound to the G protein  subunits. The subsequent binding of GTP to the empty  subunit promotes the conformational change of G and dissociation of the subunits. The G protein  subunit in the nucleotide-free state appears to be an important intermediate in the activation. From studies of rhodopsin and transducin, it has been postulated that the empty G protein (nucleotide-free) forms a stable complex with the receptor (4). Both empty forms of Gi and Go  subunits have been purified under harsh conditions (1 M (NH₄)₂SO₄ and 20% glycerol), and they were unstable (5).

We recently reported that a mutant version of Go, GoX (GoD273N/Q205L), was regulated by xanthine nucleotides, not by guanine nucleotides (6). GoX bound XDP¹ and XTP instead of GDP or GTP. GoX bound G protein subunits only in the presence of XDP, and XTP stimulated dissociation of the GoX heterotrimer. XTP-bound GoX underwent a conformational change similar to the activated wild-type Go. In the present study, we investigated the interaction between GoX and G protein-coupled receptors. We found that GoX mutant proteins retained the receptor binding specificity of the wild-type Go and were able to interact with Go-coupled receptors, such as the m2 muscarinic cholinergic receptor (MAChR), N-formyl peptide receptor (NFPR), and thrombin receptor, but not with m1 MAChR or thyrotropin-releasing hormone (TRH) receptor which do not couple to wild-type Go. Because the concentrations of XDP and XTP are relatively low in vivo (7), GoX mutant proteins are essentially nucleotide-free unless exogenous xanthine nucleotides are provided. Thus, GoX provides an excellent model to study the receptor interaction of empty G protein  subunits. Consistent with the previously reported studies on the empty form of transducin (4), our data are most readily interpreted as showing that "empty" GoX can form a stable complex with appropriate receptors on the membrane.

	EXPERIMENTAL PROCEDURES

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Materials-- Purified bovine retinal transducin were generous gifts from Dr. O. Nakanishi (Division of Biology, Caltech). Xanthine and guanine nucleotides were from Sigma. All the radioactive ligands including [³⁵S]ATPS, [³⁵S]GTPS, [³H]QNB, and fML[³H]P were from NEN Life Science Products.

Expression and Purification of His₆-tagged Go-- Both wild-type Go and mutant GoX were subcloned into the Escherichia coli expression vector pET-15b (Novagen) with His₆ tag at the N terminus. These clones were used to transform the E. coli strain BL21(DE3), and proteins were expressed. Expression and purification of these proteins was described previously (6, 8). After harvesting the culture, cell extracts were resuspended in the binding buffer (5 mM imidazole, 0.5 M NaCl, 160 mM Tris-HCl, pH 7.9, and 1 mM Me). The His₆-tagged proteins were purified from Ni²⁺-NTA column according to the protocol provided by Novagen. Purified proteins were stored in TED buffer (20 mM Tris-HCl, pH 8.0, 1 mM EDTA, and 1 mM dithiothreitol) with 0.1 mM MgCl₂ and 0.1 mM nucleotide diphosphate (GDP or XDP as appropriate).

Membrane Preparation from Baculovirus-infected Sf9 Cells-- Sf9 cells were grown and infected with recombinant baculoviruses encoding either m2 MAChR or NFPR (9-11). Infected cells were centrifuged and resuspended at <10⁷ cells/ml in HME/PI buffer (20 mM NaHepes, pH 8.0, 2 mM MgCl₂, 1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, and 10 µg/ml leupeptin). The cell suspension was homogenized by 10 strokes in a glass homogenizer followed by passing through a 27 gauge hypodermic needle several times. The homogenate was briefly centrifuged at 3,000 × g for 10 min, and then the supernatant was collected and centrifuged at 30,000 × g for 30 min. The pellet was washed once with HME/PI, and the final pellet was resuspended in HME/PI at a protein concentration of 5 mg/ml.

Synthesis of XTPS-- XTPS was synthesized from XDP and ATPS with nucleotide diphosphate kinase (NDK) as described previously (12). To produce ³⁵S-labeled XTPS, the reaction contained 10 µM XDP, 1 µM [³⁵S]ATPS, and 10 units of NDK (Sigma) in 100 µl of NDK buffer (1 mM MgCl₂, 5 mM dithiothreitol, and 20 mM Tris-HCl, pH 8.0). The mixture was incubated at room temperature for 2 h. The resulting concentration of [³⁵S]XTPS was about 1 µM (1 µCi/pmol). The radiochemical purity of XTPS was monitored by TLC on Avicel/PEAE plates (Analtech) in 0.07 N HCl.

Nucleotide Binding of Purified Go-- Binding of [³⁵S]GTPS and [³⁵S]XTPS to the recombinant Go or the mutant proteins was performed as described (6). The binding reaction contained 0.5 µg of purified protein in TED buffer, with 0.1 mM MgCl₂, 1 µM ATP, and 0.1 µM GTPS or XTPS (20,000 cpm/pmol). For the time course experiments, 20-µl aliquots were withdrawn from a 200-µl reaction, diluted 10-fold with ice-cold TED buffer containing 0.1 mM MgCl₂, filtered through 45-µm nitrocellulose, washed, and dried. The amount of bound radioactivity was determined by scintillation counting.

Radioligand Binding of Receptors-- The ligand binding assays of membrane-bound receptors were performed as described (9-11). The total concentration of m2 MAChR and the affinities of NFPR were determined by incubating membranes with 2 nM [³H]QNB or various concentrations of fML[³H]P for 1 h in 20 mM Tris-HCl, pH 7.4, 12.5 mM MgCl₂, and 1 mM EDTA at 30 °C in a final volume of 0.5 ml. Nonspecific binding was defined as binding that was not displaced by 10 µM atropine for m2 MAChR or 10 µM fMLP for NFPR. Unbound ligands were removed by filtration through Whatman GF/C filters and washing four times using ice-cold binding buffer. The amount of bound radioactivity was determined by scintillation counting.

Binding of Go on Sf9 Cell Membranes-- 0.2 µg of purified Go or Go mutant proteins were incubated with 100 µg of Sf9 cell membranes in TED buffer of a final volume of 100 µl at room temperature for 1 h. The membranes were centrifuged and subjected to Western blot using antibodies against Go.

Cell Culture and Transfection-- COS-7 cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. 1 × 10⁵ cells/well were seeded in 12-well plates 1 day before transfection. All transfection assays contained a total amount of 1 µg of DNA, and pCIS encoding -galactosidase was used to maintain a constant amount of DNA. To each well, 1 µg of DNA was mixed with 5 µl of LipofectAMINE (Life Technologies, Inc.) in 0.5 ml of Opti-MEM (Life Technologies, Inc.), and 5 h later, 0.5 ml of 20% fetal calf serum in Dulbecco's modified Eagle's medium was added to the medium. After 48 h, cells were assayed for inositol phosphate levels as described previously (13, 14).

	RESULTS

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Stimulation of XTPS Binding of GoX by Activated M2 Muscarinic Receptor-- To test if GoX could interact with G protein-coupled receptors, we investigated the receptor-stimulated nucleotide binding of GoX. Activated G protein-coupled receptors are known to facilitate the binding of GTPS to G protein  subunits. It has been reported that recombinant m2 MAChR from Sf9 cells stimulated the binding of GTPS to wild-type Go 2-3-fold in response to muscarinic agonists (9, 10). We infected Sf9 cells with recombinant baculoviruses encoding m2 MAChR and prepared membranes. The concentration of receptor was about 20 pmol/mg of membrane protein, determined from [³H]QNB binding. We have previously shown that GoX mutant proteins bind only xanthine nucleotides, but not guanine nucleotides. In this experiment, we reconstituted purified GoX with Sf9 cell membranes containing m2 MAChR in the presence of XDP and G protein subunits purified from bovine retina, and followed agonist-dependent stimulation of XTPS binding to GoX. We found that carbachol accelerated the XTPS binding of GoX, in a fashion similar to the acceleration of GTPS binding observed with wild-type Go (Fig. 1a). In control experiments using wild-type Sf9 cell membranes, both atropine and carbachol had no effect on the XTPS binding of GoX (Fig. 1a). subunits were required for the carbachol-dependent stimulation of nucleotide binding (Fig. 1b), suggesting that only the trimeric complex of GoX with can be activated to exchange XDP for XTPS by the ligand-bound receptors.

View larger version (21K): [in this window] [in a new window]   Fig. 1.   M2 MAChR stimulated XTPS binding of GoX. a, 0.5 µg of purified GoX was incubated with 1 µg of , 100 µg of m2 MAChR membranes, or control Sf9 cell membranes and with 10 µM XDP in TEDM buffer (20 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1 mM dithiothreitol, and 1 mM MgCl2) for 30 min at room temperature, and then the mixture was diluted 10-fold with TEDM buffer containing 0.1 µM [35S]XTPS (20,000 cpm/pmol) and 100 µM carbachol or 2 µM atropine at time 0. 20-µl aliquots were withdrawn and assayed for the bound nucleotides at the indicated times. b, 0.5 µg of purified wild-type Go or GoX were subjected to the similar nucleotide binding assay as in panel a with GTPS or XTPS under indicated conditions. Only data at 20 min were shown as the percentage of maximum binding.

High Affinity Ligand Binding of N-formyl Peptide Receptor Induced by GoX-- Another well documented indication of receptor-G protein interaction is that GTP or GTPS inhibits the high affinity binding of G protein-coupled receptors to their agonists. NFPR receptors expressed in Sf9 cells are known to be in the low ligand affinity state (~60 nM fMLP) (11), presumably because of the lack of mammalian Gi-like G proteins in Sf9 cells, whereas NFPR in neutrophils and NFPR expressed in mouse L cells exhibited high affinity ligand binding (0.5-3 nM fMLP) (15, 16). Therefore, we reconstituted GoX with NFPR from Sf9 cells and investigated whether GoX could induce the high affinity ligand binding state in NFPR receptors. Sf9 cell membranes containing NFPR receptors were prepared as described under "Experimental Procedures." The expression level of NFPR was about 20 pmol/mg of membrane protein, determined by fML[³H]P binding. We incubated the NFPR with wild-type Go or mutant GoX in the presence of and varieties of nucleotides and determined their affinities for the agonist fMLP. As expected, NFPR expressed in Sf9 cells showed low ligand affinity binding of fMLP (~100 nM), and GoX alone did not affect ligand binding (Fig. 2a). More interestingly, NFPR exhibited high affinity ligand binding (~10 nM) when GoX, , and XDP were present (Fig. 2a). Both and XDP were required to induce the high ligand affinity state of NFPR, and XTP inhibited the fMLP binding of the receptors (Fig. 2b). In the control experiments, wild-type Go heterotrimer was also found to convert the NFPR to the high affinity ligand binding state, which was inhibited by GTPS (Fig. 2b). These experiments demonstrated that the heterotrimeric complex of GoX can interact efficiently with NFPR.

View larger version (22K): [in this window] [in a new window]   Fig. 2.   High affinity ligand binding of NFPR induced by GoX. a, 10 µg of NFPR membranes or wild-type Sf9 cell membranes were incubated with various concentrations of fML[3H]P for 1 h in 20 mM Tris-HCl, pH 7.4, 12.5 mM MgCl2, and 1 mM EDTA at 30 °C in a final volume of 0.5 ml, in the presence of 0.1 µg of GoX, 0.2 µg of , and 100 µM XDP, or GoX alone. The amount of bound radioligand was then determined. Nonspecific binding was defined as binding in the presence of 10 µM cold fMLP, which was less than 10% of total ligand binding, and was subtracted before analyzing. b, NFPR was incubated with 50 nM fML[3H]P and various reagents under the same conditions as panel a.

Binding of GoX with M2 Muscarinic Receptor on Sf9 Cell Membranes-- The previous two experiments showed that GoX heterotrimer could interact with the G protein-coupled receptors efficiently and that the interaction was similar to the interaction between wild-type Go and receptors. To investigate receptor interaction of GoX more directly, we studied binding of GoX to receptor containing Sf9 cell membranes. Purified wild-type Go or GoX were incubated with Sf9 cell membranes containing m2 MAChR in the presence of different reagents. The membranes were then pelleted and subjected to Western blotting using antibodies against Go to see if Go remained bound to the membrane. In the control experiments using wild-type Sf9 cell membranes without m2 MAChR, both wild-type Go and GoX did not remain associated with the membrane. However, wild-type Go was bound to membrane when it was coincubated with . Similarly, GoX stayed on the membrane when in complex with in the presence of XDP (data not shown). These experiments using wild-type Sf9 cell membranes showed that Go bound to the membranes only in the complex form, presumably because facilitates membrane association. In the experiments using membranes containing m2 MAChR, we found somewhat surprisingly that GoX bound to receptor-containing membranes even in the absence of carbachol and without (Fig. 3, lane 12), whereas wild-type Go did not (Fig. 3, lane 1), suggesting GoX alone was able to bind to receptor. Interestingly, both XDP and XTP abolished the interaction between GoX and m2 MAChR-containing membranes and released GoX from the membrane fraction (Fig. 3, lanes 7 and 8), whereas GDP or GTP had no effect (data not shown), suggesting that the nucleotide-free form of GoX can recognize and bind to Go-mediated receptor. As expected, GoX stayed on the membrane when both XDP and were present (Fig. 3, lane 10), and XTP promoted dissociation of the GoX complex (Fig. 3, lane 9). In the case of wild-type Go, the binding pattern was the same between membranes with or without the receptors, and XDP or XTP had no effect on binding (Fig. 3, lanes 1-5). In a titration experiment, quantitation of GoX revealed that the amount of GoX bound to the membrane increased linearly until it reached saturation, and the level of saturation was proportional to the amount of receptor incubated in the reaction (Fig. 4, a-c). Furthermore, similar experiments using Sf9 cell membranes containing NFPR were also performed, and the results were similar (data not shown). These experiments indicated that the empty form of GoX, without a bound nucleotide and , could form a stable complex with receptor. In summary, these data suggest that GoX with XDP bound and bind to membranes, whereas the XTP form is found to be cytoplasmic. The nucleotide-free form is able to bind to Go-mediated receptors.

View larger version (26K): [in this window] [in a new window]   Fig. 3.   Binding of GoX to m2 MAChR on Sf9 cell membranes. 0.2 µg of wild-type Go (with 100 µM GDP) or GoX were incubated with 100 µg of m2 MAChR membranes in TED buffer of a final volume of 100 µl at room temperature for 1 h with indicated reagents. The membrane then was centrifuged and subjected to Western blot using antibodies against Go. All nucleotide concentrations were 100 µM, and the amount of was 0.5 µg. Lane 13 shows the total amount of Go used in each assay.

View larger version (32K): [in this window] [in a new window]   Fig. 4.   Titration of GoX bound to m2 MAChR on Sf9 cell membranes. The binding assays were done under the same conditions described in Fig. 3 legend. a, indicated amount of GoX was incubated with 100 µg of m2 MAChR membranes in TED buffer. b, the relative intensities of bands in panel a were quantitated. c, quantitated binding of 0.2 µg of GoX with indicated amount of m2 MAChR membranes. Wild-type Sf9 cell membranes were used to maintain a constant amount of membranes of 200 µg in each binding reaction.

Dominant-negative Effect of GoX on Receptor Activation in COS-7 Cells-- Because our experiments suggested that empty GoX was able to bind to the receptor in vitro, we went on to test for this interaction in intact cells. Indeed, we found that GoX was able to interact with receptors and inhibit their activities in COS-7 cells consistent with the observation that GoX did not dissociate from the receptors without xanthine nucleotides. Thrombin receptors are known to couple with G proteins from both the Gi and Gq families (17). In COS-7 cells transfected with the thrombin receptor, endogenous Gq is activated by the addition of thrombin and stimulates PLC isoforms to elevate cellular IP₃ concentration. Inhibition of receptor activation in transfected cells by wild-type G proteins was observed before (18). Thus if cells are cotransfected with both the thrombin receptor and wild-type Go subunit, the activation of Gq is inhibited because of the competition of Go for the receptor or endogenous G protein subunits (Fig. 5a). We cotransfected thrombin receptor and GoX to determine whether GoX could compete with endogenous Gq for the receptors. Indeed, we found that GoX inhibited Gq activity stimulated by thrombin, and the inhibition was proportional to the amount of GoX cDNA used in the transfection (Fig. 5a). Because GoX in the absence of XDP does not interact with and does not affect the -stimulated PLC activity in COS-7 cells (6), the inhibition by GoX of Gq activation stimulated by the thrombin receptor must come from the competitive binding of GoX to the receptor. Similar experiments were performed with m1 MAChR and TRH receptor which were known to couple only to the Gq family of G proteins and not to the Go family (10, 19, 20). GoX had no effect on the activation of m1 MAChR or TRH receptor (Fig. 5, b and c). On the other hand, wild-type Go, which can compete for endogenous , inhibited both m1 MAChR and TRH receptor stimulation as expected. In COS-7 cells, the activation of thrombin receptor, m1 MAChR, and TRH receptor share the same Gq pathway downstream of the receptor. Because GoX inhibited only the thrombin receptor activity, but had no effect on m1 MAChR or TRH receptor, we concluded that GoX inhibited thrombin receptor stimulation by competitive binding to the receptor.

View larger version (45K): [in this window] [in a new window]   Fig. 5.   The negative inhibitory effects of GoX on receptor-stimulated PLC activation in COS-7 cells. 1 × 105 cells/well were seeded in a 12-well plate and then were transfected with cDNAs encoding the indicated G proteins (GoDN designates GoD273N) and thrombin receptor (a), m1 MAChR (b), TRH receptor (c), or m2 MAChR (d). In panels a-c, the amount of receptor cDNA used in each well was 0.25 µg, and the amount of Go cDNA was 0.75 µg/well unless otherwise indicated. In panel d, the amount of both m2 MAChR and G15 cDNA was 0.2 µg/well and that of Go was 0.6 µg/well. The total amount of cDNA for each well was adjusted to 1.0 µg by the addition of CMV-LacZ cDNA. After cells were labeled with [3H]inositol overnight, they were incubated in the medium containing 0.1 unit/ml thrombin (a), 1 µM carbachol (b and d), or 1 µM TRH (c) before levels of inositol phosphates were determined.

To test if GoX could bind to other Go-coupled receptors in cells, we looked into the interaction between GoX and m2 MAChR. Because m2 MAChR couples only to the Gi family of G proteins and not to the Gq family (9, 21), we could not assay their interaction in the same way as the thrombin receptor by monitoring PLC activities in COS-7 cells transfected with the receptor and GoX. Therefore, we constructed an artificial pathway by cotransfecting both m2 MAChR and G15 into COS-7 cells. G15 is known as a promiscuous G protein that can be activated by all kinds of G protein-coupled receptors, and G15 also activates PLC isoforms (21). In cells cotransfected with both m2 MAChR and G15, we were able to activate endogenous PLC isoforms by the addition of the muscarinic agonist carbachol. We found that this m2 MAChR stimulation pathway could also be inhibited by GoX (Fig. 5d). All these experiments suggested that GoX was able to interact with G protein-coupled receptors in cells and retained the receptor specificity of wild-type Go; it coupled with thrombin receptor and m2 MAChR, but not with m1 MAChR or TRH receptor. Furthermore, GoX exhibited dominant-negative inhibitory effects against these receptors in cells.

	DISCUSSION

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GoX (GoD273N/Q205L) was the first reported mutant of heterotrimeric G protein  subunits that bound xanthine nucleotides, not guanine nucleotides (6). It bound only in the presence of XDP and could be activated by XTP. We continued to study the interaction of GoX with G protein-coupled receptors in this report. The interaction of G proteins and their receptors is best demonstrated in two experiments: agonist-stimulated GTPS binding of G protein  subunits and inhibition of high affinity ligand binding of the receptors by GTPS. To test if GoX can interact with G protein-coupled receptors and be activated by their agonists, we reconstituted purified GoX with Sf9 cell membranes containing m2 MAChR or NFPR. First, we found that binding of XTPS to GoX was stimulated by the muscarinic agonist carbachol in the presence of m2 MAChR. In similar experiments using wild-type Go, GTPS binding was also stimulated by carbachol. In both cases, was required for the carbachol-dependent nucleotide binding, suggesting that only GoX heterotrimer could interact with the receptors effectively. Second, we tested GoX to determine whether it could induce the high affinity state in NFPR receptors expressed in Sf9 cells. The NFPR expressed in these cells is known to be in the low affinity state probably because of lack of mammalian Gi-like proteins in Sf9 cells (11). In our experiments, we found that GoX could convert NFPR into the high affinity state in the presence of and XDP, and this effect was inhibited by XTP. These two experiments demonstrated that GoX, when in complex with and XDP, could interact with G protein-coupled receptors effectively and be activated by the agonists.

Because cells lack xanthine nucleotides, GoX provides an excellent model to study empty G protein  subunits. The empty form of G is an important intermediate in receptor activation and has long been proposed to form a stable complex with activated receptors. However stable interaction between empty G proteins and their receptors was only reported in the transducin-rhodopsin system. Empty transducin apparently formed a stable complex with light-activated rhodopsin and stayed on the rod outer segment membrane. Interestingly, deactivation of the rhodopsin did not lead to the dissociation of transducin from the complex (4). In this report, we showed that empty GoX was able to bind to the receptor on the membrane in the absence of subunits and without agonists, and the interaction could be abolished by either XDP or XTP. The amount of GoX associated on the membranes with m2 MAChR was proportional to the amount of receptor at saturation. Interestingly, binding of GoX alone did not convert the receptor to the high ligand affinity conformation, which required the complex. Therefore, the binding of GoX alone to the receptor is not functional in contrast with the binding in the presence of and XDP.

Because GoX appears to form a stable complex with the receptor, we tested whether GoX could inhibit receptor activation in cells. In transfected COS-7 cells, we showed that GoX was able to inhibit thrombin receptor or m2 MAChR stimulated PLC activities via the Gq or G15 pathway, but had no effect on m1 MAChR or TRH receptor stimulation. Because both thrombin receptor and m2 MAChR are known to couple with wild-type Go, and m1 MAChR and TRH receptor only couple with Gq, we interpret the data to mean that GoX retained the receptor specificity of wild-type Go and was able to interact with Go-coupled receptors in cells. The inhibitory binding of GoX enables us to specifically block Go-coupled receptors in certain systems. This could be a useful means to analyze different receptor-stimulated signal transduction pathways, and could perhaps be useful in drug screening associated with G protein-coupled receptors.

In the previous report (6), we showed that the single Go mutant, GoD273N, lost the ability to bind either guanine nucleotides or xanthine nucleotides and could not bind under any conditions. Surprisingly, GoD273N can still bind to receptors. In transfected COS-7 cells, we found that GoD273N inhibited thrombin receptor and m2 MAChR activation, in a fashion similar to GoX (Fig. 5, a and d). GoD273N also retained the same receptor specificity as wild-type Go; i.e. it had no effect on m1 MAChR or TRH receptor stimulated pathways (Fig. 5, b and c). In the Sf9 cell membrane binding assay, it only bound to the m2 MAChR membranes, not to the control wild-type Sf9 cell membranes. However in contrast to GoX, GoD273N was not released from the m2 MAChR membranes by XDP or XTP, consistent with its inability to bind nucleotides (data not shown). The reason that GoD273N mutant proteins do not bind xanthine nucleotides is not clear. Apparently it must have a structure similar to that of the empty Go which enables it to bind receptors, but the structure is probably not stable locally around the nucleotide binding pocket. Nevertheless, GoD273N may also be useful as a dominant-negative inhibitor of receptor functions.

	ACKNOWLEDGEMENTS

The recombinant baculovirus encoding m2 MAChR was a generous gift from Dr. E. Ross. We thank members of Dr. Simon's lab for helpful discussions, and Dr. Tau-Mu Yi for comments on the manuscript.

	FOOTNOTES

^* 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.

The abbreviations used are: XDP, xanthine diphosphate; XTP, xanthine triphosphate; MAChR, muscarinic cholinergic receptor; NFPR, N-formyl peptide receptor; TRH, thyrotropin-releasing hormone; NDK, nucleotide diphosphate kinase; IP₃, inositol 1,4,5-trisphosphate; PLC, phospholipase C; GTPS, guanosine 5'-O-(3-thiotriphosphate); XTPS, xanthine 5'-O-(3-thiotriphosphate); [³H]QNB, quinuclidinylbenzilate; fML[³H]P, formyl-methionyl-leucyl-phenylalanine.

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