©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Characterization of G Family G Proteins G (G), G (G), and G Expressed in the Baculovirus-Insect Cell System (*)

(Received for publication, October 31, 1994; and in revised form, December 29, 1994)

Fumio Nakamura (§) Mariko Kato Kimihiko Kameyama Toshihide Nukada (¶) Tatsuya Haga (**) Hiroyuki Kato (1) Tadaomi Takenawa (1) Ushio Kikkawa (2)

From the  (1)Department of Biochemistry, Institute for Brain Research, Faculty of Medicine, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113, theDepartment of Molecular Oncology, Institute of Medical Science, University of Tokyo, Shiroganedai 4-6-1, Minato-ku, Tokyo 108, and the (2)Biosignal Research Center, Kobe University, Rokkodai-cho 1-1, Nada-ku, Kobe 657, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The alpha subunits of G(q) family G proteins, Galpha(G(14)alpha), Galpha(Galpha), and G(q)alpha were expressed with G protein beta(1) and (2) subunits in insect cells using a baculovirus system. The trimeric forms of G proteins, G (Galphabeta), G (Galphabeta), and G(q) (G(q)alphabeta), were solubilized by 1% sodium cholate and purified by sequential chromatography on three kinds of columns. G, G, and G(q) activated phospholipase C-beta purified from bovine brain in the presence of aluminum fluoride to the same extent. Muscarinic acetylcholine receptor m1 subtype stimulated the guanosine 5`-O-(3-thiotriphosphate) (GTPS) binding to G, G, and G(q) in the presence of similar concentrations of carbamylcholine. When m1 receptor, G protein, and phospholipase C-beta were reconstituted in lipid vesicles, each subtype of G(q) family G proteins mediated the activation of phospholipase C-beta by carbamylcholine in the presence of either 1 µM GTPS or 1 mM GTP. Phospholipase C-beta stimulated the GTPase activity of G, G, and G(q) in the presence of m1 receptor and carbamylcholine but did not stimulate the GTPase activity of G(o). Protein kinase C phosphorylated m1 receptor and phospholipase C-beta, but the phosphorylation did not significantly affect the ability of the m1 receptor to stimulate phospholipase C-beta in the reconstitution system of purified proteins.


INTRODUCTION

The G(q)(^1)family of GTP-binding regulatory proteins (G proteins) (^2)consists of four kinds of alpha subunits(1) . The cDNA clones of G(q)alpha and Galpha were isolated from mouse brain library by Strathmann and Simon(2) . We have identified bovine cDNA clones encoding G(q) family alpha subunits designated Galpha and Galpha (3) that correspond to murine G(14)alpha and Galpha, respectively(2, 4) . Human Galpha and its mouse version Galpha expressed in hematopoietic tissues have also been identified as members of the G(q) family(4, 5) .

G(q) family alpha subunits are thought to activate phospholipase C-beta (PLC-beta) in a pertussis toxin-insensitive manner. A mixture of G(q) and G (G(q)/G) purified from bovine liver or a mixture of G(q)alpha and Galpha from bovine brain was reported to activate PLC-beta in the presence of GTPS (6) or aluminum fluoride(7) . In contrast to Galpha and G(q)alpha, the function of Galpha is as yet unknown. Whereas the G(14)alpha (Galpha) transiently expressed in COS-7 cells was reported to activate purified PLC-beta(1)(8) , Galpha coexpressed with metabotropic glutamate receptors in Xenopus oocytes was found to inhibit the PLC activity in oocytes(9) . Here, we have examined whether purified Galpha stimulates or inhibits the activity of purified PLC-beta.

Muscarinic acetylcholine receptors (mAChRs) consist of five subtypes that are coupled to their effectors via G proteins. Among the five subtypes of mAChRs, m1, m3, and m5 subtypes activate PLC in a pertussis toxin-insensitive manner, whereas m2 and m4 subtypes inhibit adenylate cyclase via pertussis toxin-sensitive G proteins(10, 11) . G(q) family G proteins are thought to mediate the former pathway. The mixture of G(q) and G reconstituted in lipid vesicles with m1 mAChR was reported to bind GTPS and subsequently activate PLC-beta in an agonist-dependent manner(12) . It has been shown that PLC-beta stimulates the GTPase activity of G(q)/G in the presence of m1 mAChR and carbamylcholine(13) . It has not been determined, however, if there are any quantitative differences between G(q) and G regarding their interactions with m1 mAChR and PLC-beta. Furthermore, it is not known if Galpha can also be activated by m1 mAChR or activate PLC-beta(1) or whether its GTPase is activated by PLC-beta.

It is difficult to isolate and characterize individual members of G(q) family G proteins because of their limited expression in native tissues. Recently, the recombinant forms of G(q)alpha, Galpha, and Galpha have been purified from Sf9 insect cells that coexpressed beta(2)(2) subunits (14, 15) . We have independently found that the coexpression of beta subunits with G(q) family alpha subunits in Sf9 insect cells facilitated the solubilization of the alpha-subunits and enabled us to purify the trimeric G proteins.

Activation of PLC leads to the breakdown of phosphatidylinositol 4,5-bisphosphate (PIP(2)) and the generation of the intracellular messengers, inositol 1,4,5-trisphosphate (IP(3)) and diacylglycerol(16) . Diacylglycerol activates certain isotypes of protein kinase C(17, 18) . Experiments in vivo have shown that the activation of protein kinase C leads to the inhibition of mAChR-mediated PLC activation(19, 20, 21, 22) . It would provide the most straightforward explanation if protein kinase C phosphorylates the components in the pathway of PLC activation and attenuates its signal transduction. In fact, protein kinase C is reported to phosphorylate m1 mAChR (23) and PLC-beta(1)(24) in vitro. The effect of phosphorylation by protein kinase C on m1 mAChR-mediated stimulation of PLC-beta can now be examined in a reconstitution system using purified proteins.

In the present studies, we have expressed and purified each subtype of the G(q) family G proteins, Galpha, Galpha, and G(q)alpha, and reconstituted them in lipid vesicles with m1 mAChR, PLC-beta, or both. Each of the G(q) family G proteins was found to be activated by m1 mAChR and to activate PLC-beta with essentially the same efficiency. Protein kinase C phosphorylated m1 mAChR and PLC-beta, but the activation of PLC-beta by carbamylcholine was not affected by the phosphorylation.


EXPERIMENTAL PROCEDURES

Materials

[S]GTPS and [^3H]QNB were purchased from DuPont NEN, [^3H]PIP(2) was from American Radiolabeled Chemicals, Inc., [P]ATP was from Amersham International plc, and [P]GTP was from the Hungarian Academy of Science. The cDNA encoding mouse G(q)alpha subunit was kindly donated by Dr. M. I. Simon from the California Institute of Technology at Pasadena; Sf9 cells and recombinant baculoviruses containing cDNA encoding the human m1 and m2 mAChR were donated by Dr. E. M. Ross at the University of Texas Southwestern Medical Center at Dallas.

Construction of Galpha Transfer Vectors

The baculovirus transfer vectors containing sequences for G protein alpha subunits (pVLGalpha, pVLGalpha, pVLG(q), and pVLG(o)) were constructed as follows. The NcoI-SmaI fragment containing the entire Galpha coding region was excised from pGL1(3) , where the NcoI site was filled in with T4 DNA polymerase to yield a blunt end and attached by an EcoRI linker prior to SmaI digestion. The fragment was inserted between the EcoRI and SmaI sites of the baculovirus transfer vector pVL1392. The NaeI-EcoRI fragment, including the Galpha coding sequence, was excised from pGL7 (3) and ligated to the SmaI-EcoRI sites of pVL1393. The NotI-SspI fragment (1.2 kilobase pairs) was excised from the P3 plasmids containing the G(q)alpha cDNA (2) and was inserted between the NotI and SmaI sites of pVL1392. The NcoI-HincII fragment containing the entire coding sequence of bovine G(o)alpha1 that was excised from the clone pG(o)alpha3 was filled in with T4 DNA polymerase, attached to an EcoRI linker, and inserted into the EcoRI site of pVL1392.

We have constructed a transfer vector in which both coding regions of beta and subunits were placed downstream of the polyhedrin promoters. A polymerase chain reaction-amplified coding sequence of bovine (2) subunit (9, 25) was ligated to the SmaI-EcoRI sites of pVL1393 (pVL13932). The 1.9 kilobase pair NaeI-VspI fragment containing a polyhedrin promoter, a coding sequence of the (2) subunit, and a poly(A) signal sequence was excised from pVL13932. The fragment was filled in with T4 DNA polymerase and ligated to the EcoRV site of pVL1393. Among obtained vectors, the vector in which two promoters were arranged back-to back, named pVL3R2, was selected for further construction. The coding sequence of bovine beta(1) subunit excised from pGbeta4 (26) was inserted between the SmaI and XbaI sites of pVL3R2.

Isolation and Inoculation of Recombinant Baculoviruses

Recombinant viruses were generated and isolated according to the standard procedures(27, 28) . Spodoptera frugiperda (Sf9) cells were cultured in 75 cm^2 flasks containing 12 ml of IPL-41 medium supplemented with 4% fetal bovine serum at 28 °C. Cells were grown in 150 ml of the same medium supplemented with 0.1% pluronic F-68 with constant stirring at 60 rpm. Cells were usually seeded at a density of 0.4 times 10^6 cells/ml in 150 ml of medium and allowed to multiply for 3-4 days up to 2.5-3 times 10^6 cells/ml. Grown cells were spun down and suspended in 5-7 ml of the same medium. To this cell suspension, 12-14 ml of alpha subunit recombinant virus (6 times 10^9 plaque-forming units/ml) and 3-4 ml of beta(1)(2) recombinant virus (6 times 10^9 plaque-forming units/ml) were added. After incubation for 1 h, the cell suspension was transferred into a spinner flask with 130 ml of the medium and cultured in an atmosphere of 50% O(2)/50% N(2) for 65-72 h. Harvested cells were stored at -80 °C until use.

Preparation of G Proteins

All purification steps were carried out at 4 °C. The cells (2-3 times 10^9) from the six spinner flasks were resuspended in 280 ml of ice cold homogenization buffer containing 20 mM Tris-HCl buffer (pH 8.0), 1 mM EDTA, 1 mM DTT, and a mixture of protease inhibitors (2.5 µg/ml pepstatin, 0.002 mg/ml phenylmethylsulfonyl fluoride, 0.02 mg/ml leupeptin, and 0.5 mM benzamidine). Cells were homogenized with 10-15 strokes in a Potter-type homogenizer. The homogenates were centrifuged at 100,000 times g for 30 min. Pellets were washed once in 280 ml of the homogenization buffer, resuspended in 200 ml of the homogenization buffer supplemented with 1% sodium cholate, and gently stirred for 2 h. After centrifugation at 100,000 times g for 45 min, the supernatant fraction (210 ml) was applied to a DEAE-Sephacel column (50 ml) pre-equilibrated with the homogenization buffer containing 1% sodium cholate (Buffer A). The column was washed with 50 ml of Buffer A and eluted with a gradient consisting of 100 ml each of Buffer A and Buffer A supplemented by 500 mM NaCl. The alpha subunits, together with beta subunits, were eluted between 120 and 200 mM NaCl. Aliquots of each column fraction were subjected to SDS-PAGE and immunostaining. The G protein-rich fractions (30 ml) were diluted 5-fold with Buffer B (20 mM Hepes-NaOH buffer (pH 7.0), 1 mM EGTA, 1 mM DTT, and protease inhibitors) and then applied to a heptylamine column (20 ml) that had been washed with 40 ml of buffer B supplemented with 0.2% sodium cholate. The column was eluted with a gradient consisting of 70 ml each of Buffer B supplemented with 0.2% sodium cholate and 200 mM NaCl and Buffer B containing 1.75% sodium cholate and 50 mM NaCl. The G protein trimers were eluted between 0.7 and 1.0% sodium cholate. The G protein-rich fractions were supplemented with 5 mM potassium phosphate and applied to a hydroxylapatite column (5 ml), which had been pre-equilibrated with Buffer C (20 mM Hepes-NaOH buffer (pH 7.0), 1 mM EGTA, 1 mM DTT, and 0.8% sodium cholate) supplemented with protease inhibitors and 5 mM potassium phosphate. The column was washed with Buffer C containing 5 mM potassium phosphate and eluted with a linear gradient of Buffer C and the Buffer C containing 150 mM potassium phosphate. beta subunits were eluted at 5-25 mM potassium phosphate, and then the alphabeta trimers were eluted between 50 and 75 mM potassium phosphate.

Phospholipase C Assays

Phospholipase C-beta (PLC-beta) was purified from bovine brain by a method modified from those of Ryu et al.(29) and Rhee et al.(30) . PLC assay was performed according to Smrcka et al.(7) . Briefly, substrate was provided as vesicles containing 2 µg of phosphatidylinositol 4,5-bisphosphate (PIP(2)), 20 µg of phosphatidylethanolamine, and 10 nCi of [^3H]PIP(2) in a Hepes buffer solution (43 mM Hepes-NaOH buffer (pH 7.0), 2 mM EGTA, 1 mM DTT, 20 mM NaCl, 30 mM KCl, 5 µM GDP, 2 mM MgCl(2), and 0.06% sodium cholate in 50 µl, final volume). CaCl(2) was supplemented to yield approximately 1 µM free Ca. Where necessary, 20 µM AlCl(3) and 6 mM NaF were added as activators of G protein. The reaction was initiated by addition of 10-20 fmol of PLC-beta and proceeded for 10 min at 37 °C. The reaction was terminated by addition of 600 µl of methanol/CHCl(3) mixture (2:1, v/v), 200 µl of CHCl(3), and 200 µl of 0.1 M HCl, followed by separation of the aqueous and lipid phases as described(7) . [^3H]IP(3) in the aqueous phase was quantitated by a liquid scintillation counter.

Reconstitution of mAChRs and G Proteins

Recombinant mAChRs, m1 and m2 subtypes, were purified by a single-step affinity chromatography from Sf9 cells expressing human m1 or m2 subtypes(31) . Reconstitution of mAChRs and G proteins was performed as described previously(32, 33) .

For PLC assay, m1 mAChR and G proteins (20-80 pmol) were reconstituted in a modified HEN buffer (20 mM Hepes-NaOH (pH 7.0), 1 mM EGTA, and 160 mM NaCl). Reconstituted vesicles (10-20 µl) were mixed with phospholipid vesicles (10 µl) and an assay buffer (final concentrations: 20 mM Hepes-NaOH buffer (pH 7.0), 1 mM EGTA, 10 mM MgCl(2), 30 mM NaCl, 1 µM GDP, and 10 µM free Ca in 20 µl, final volume). As mAChR ligands, carbamylcholine and atropine were also supplemented to be 1 mM or 10 µM, respectively. The assay was initiated by the addition of a mixture of GTPS and PLC-beta (final concentrations: 100 nM and 10 fmol/tube, respectively, in 10 µl, final volume). The reaction was performed for 0-15 min at 30 °C.

[^3H]QNB and [S]GTPS Binding Assays

For [^3H]QNB binding, the reconstituted vesicle containing mAChRs and G proteins (10 µl) was incubated with 1 nM QNB ([^3H]QNB, 50-60 cpm/fmol) in the presence or absence of 10 µM atropine for 60 min at 30 °C in HEN buffer (20 mM Hepes-KOH (pH 8.0), 1 mM EGTA, and 160 mM NaCl in 500 µl, final volume). For the [S]GTPS binding assay, the same reconstituted vesicle (10-20 µl) was mixed with HEN buffer (80-90 µl) supplemented with 1 mM DTT, 20 mM MgCl(2), and 50 nM GTPS ([S]GTPS, 30-40 cpm/fmol), and either 1 mM carbamylcholine or 10 µM atropine. The mixtures were incubated for 0-15 min at 30 °C and then diluted with chilled solution (20 mM Tris-HCl (pH 8.0), 100 mM NaCl, and 25 mM MgCl(2) in 0.5 ml, final volume) containing 0.1 mM GTP. Bound forms of [^3H]QNB and [S]GTPS were assayed as described previously(32, 33) . Specific activities of purified m1 and m2 receptors were estimated to be approximately 2 nmol/mg protein. Recoveries of m1 and m2 receptors in the reconstitution experiments were 5-16 and 10-20%, respectively. The specific activities of G proteins reconstituted with m1 receptors were estimated to be 1-2 nmol/mg protein, as assessed by [S]GTPS binding activity.

GTPase Assay

The GTPase assay was performed according to Berstein et al.(13) . Briefly, reconstituted vesicles (10 µl) containing mAChR and G proteins were mixed with an assay mixture (final concentrations: 20 mM Hepes-NaOH buffer (pH 7.0), 1 mM EGTA, 100 mM NaCl, 5 mM MgCl(2), 0.5 µM GDP, 1 µM [-P]GTP (30-50 cpm/fmol), and either 1 mM carbamylcholine or 100 µM atropine in 30 µl, final volume). The reaction was initiated by addition of PLC-beta (final concentration: 0-10 nM in 10 µl, final volume) and was proceeded at 30 °C for 15 min. GTP hydrolysis was measured by charcoal precipitation(34) .

Phosphorylation by Protein Kinase C

Phosphorylation was performed as described previously(23) . Briefly, reconstituted vesicles (180 µl) containing m1 mAChRs were supplemented with G (20.8 pmol), PLC-beta (1.5 pmol), and protein kinase C (a mixture of alpha, beta, and subtypes in 2.6 pmol, final volume). Protein kinase C was purified as described previously(35) . The vesicles were incubated with or without 100 µM ATP for 1 h at 30 °C in Buffer D (20 mM Hepes-NaOH buffer (pH 7.0), 1 mM EGTA, 100 mM NaCl, 1 mM GTP, 1 µM GDP, 1 mM carbamylcholine, and 10 µM free Ca). The reaction mixture was passed through a small column of Sephadex G-50 (2 ml) to remove ATP. The PLC assay was initiated by addition of the void volume fraction from the Sephadex column (20 µl) to the mixture of substrate and ligands (final concentrations: 20 mM Hepes-NaOH buffer (pH 7.0), 1 mM EGTA, 10 mM MgCl(2), 1 mM DTT, 1 µM GDP, 1 mM GTP, 1 µM free Ca, and either 1 mM carbamylcholine or 10 µM atropine in 30 µl, final volume). The reaction was performed for 0-20 min at 30 °C. To confirm incorporation of [P]phosphates, the reconstituted vesicles were incubated with 12.5 µM [P]ATP in Buffer D for 1 h at 30 °C. An aliquot (20 µl) was directly subjected to SDS-polyacrylamide gel (12%) electrophoresis followed by autoradiography and then Cerenkov counting of the receptor band. Phosphorylation of PLC-beta and G protein alpha subunit was estimated from the density of the autoradiography.

Miscellaneous Procedures

To detect the subunits of G proteins, Tricine-buffered SDS-polyacrylamide gel electrophoresis was employed(36) . G protein concentrations were determined by the protein-dye binding method(37) . The amount of active G proteins may be less than that estimated by protein assay, because the maximum binding of [S]GTPS in the presence of carbamylcholine and m1 mAChR was only 10-20% of the amount of G proteins estimated from protein assay. Antiserum against the carboxyl terminus of Galpha was prepared as described previously(3) . A Konica Immunostaining Kit or 3,3`-diaminobenzidine tetrahydrochloride was used as a substrate for peroxidase. Free Ca concentration was estimated by a chelation calculation program(38) .


RESULTS

Expression and Purification of Recombinant G Proteins

We have expressed Galpha in Sf9 cells alone or simultaneously with the G protein beta and subunits and have solubilized these proteins with 1% sodium cholate. Immunoblot analysis showed that the yield of solubilized Galpha was approximately 10 times higher when beta subunits were coexpressed as compared with when alpha subunits were expressed alone (compare lanes1 and 2 in Fig. 1). The coexpression of beta subunits also facilitated the solubilization of Galpha and G(q)alpha (not shown). Endogenous proteins that react with anti-G antibody, probably intrinsic G(q)alpha or related proteins, were also found to be solubilized by the same procedure (lane 3 in Fig. 1). Some degradation of recombinant Galpha, particularly in the preparation expressed without beta subunits, was detected.


Figure 1: Expression in Sf9 cells of Galpha and beta(1)(2) subunits and their solubilization. Sf9 cells (2-3 times 10^7) were infected with Galpha virus (lane 1), beta(1)(2) virus (lane 3), or both (lane 2). P2 fractions were prepared from these cells 3 days after infection and solubilized with 1% sodium cholate. An aliquot from solubilized preparations (40 µg as protein) was subjected to SDS-PAGE (acrylamide, 12%). Arrowheads indicate the position of Galpha. A, Coomassie Blue staining; B, immunoblot staining with an antibody against a carboxyl-terminal peptide of Galpha.



We have purified the trimeric form of G proteins from cells expressing Galpha, Galpha, or G(q)alpha simultaneously with the beta subunits. For comparison, recombinant G(o)alpha was also expressed in Sf9 cells and purified with beta subunits using the same procedure. Fig. 2shows Coomassie staining patterns following SDS-PAGE of the fractions in each purification step of recombinant G. The Galpha and beta(1) subunits were observed in the particulate fractions as bands of 42 and 36 kDa, respectively. Both Galpha and beta subunits could be partially solubilized with a buffer solution containing 1% sodium cholate. The yield of solubilization was higher for beta subunits compared with alpha subunits.


Figure 2: SDS-PAGE of fractions generated during the purification of recombinant G. Aliquots (10 µg) from each step of the purification procedure of recombinant G were subjected to SDS-PAGE (acrylamide, 12%) followed by Coomassie Blue staining.



The recombinant proteins were partially purified from the cholate extract using DEAE-Sephacel and heptylamine-Sepharose column chromatography. A hydroxylapatite column was used to separate free beta subunits and alphabeta trimers, which were eluted with 5-25 and 50-75 mM potassium phosphate buffer, respectively (not shown). Table 1summarizes the results of purification of a recombinant G. Similar results were obtained for purification of G and G(q). Starting with 900 ml of cell culture suspension, final yields of trimeric G proteins ranged from 0.2 to 0.5 mg.



Fig. 3A shows the SDS-PAGE patterns of purified G, G, G(q), and G(o). Molecular masses of alpha subunits were estimated to be 43, 42, 42, and 39 kDa for G, G, G(q), and G(o), respectively. Molecular masses of beta(1) and (2) subunit were estimated to be 36 and 8 kDa, respectively (Fig. 3, A and B). The intensity of the stained band for each of the subunits Galpha, Galpha, G(q)alpha, and G(o)alpha was essentially the same as that of the stained band for each of the copurified beta subunits. These results indicate that each of these alpha subunits is capable of forming a stoichiometric complex with beta(1)(2) subunit and was purified as an alphabeta trimer.


Figure 3: SDS-PAGE of purified recombinant G proteins. A, purified G, G, G(q), and G(o) (1 µg of protein each) were subjected to SDS-PAGE (acrylamide, 12%). B, beta subunits purified from bovine brain (lane 1, 51.5 µg) or recombinant beta(1)(2) subunits purified from Sf9 cells (lane 2, 34.8 µg) were electrophoresed in a 16.5% SDS-polyacrylamide gel (16% T, 3% C gel) as described(35) . The gels were stained with Coomassie Blue. The arrowhead indicates the position of the subunit.



Two forms of G(q)alpha, a major (42 kDa) and a minor (44 kDa) form, were observed. The minor form may represent a polypeptide translated from the polyhedrin initiator derived from the pVL1392 vector.

Activation of Phospholipase C-beta by G Proteins

To determine whether each subtype of G(q) family G proteins activates PLC-beta or not, each of the subunits G, G, and G(q) was reconstituted with PLC-beta in lipid vesicles. In the presence of aluminum fluoride, G, G, or G(q) activated PLC-beta 13-19-fold (Fig. 4, A-C). In the absence of aluminum fluoride, these G proteins only slightly enhanced (1.5-3-fold) the activity of PLC-beta in a dose-dependent manner. Under the assay conditions employed, the concentrations of G, G, and G(q) giving a half-maximal effect were estimated to be 7.37 ± 4.87, 9.47 ± 6.89, and 11.5 ± 6.3 nM, respectively. The PLC activity in the presence of excess G proteins was also not different among G, G, and G(q). These results indicate that the AlF(4)-bound forms of G, G and G(q) have essentially the same affinity for PLC-beta and the same ability to activate PLC-beta. In contrast, G(o) only slightly activated PLC-beta (1.5-3-fold), and its activation was not enhanced by the addition of aluminum fluoride (Fig. 4D).


Figure 4: Activation of brain PLC-beta by recombinant G, G, and G(q). Phospholipid vesicles containing purified G proteins (0-7.7 pmol) were incubated with PLC-beta purified from bovine brain (10 fmol) for 10 min at 37 °C in the absence (circle) or presence (bullet) of AlF(4). The assay was carried out in duplicate. Data were fitted to a Michaelis-Menten equation.



Reconstitution of mAChRs and G Proteins

We have reconstituted m1 or m2 mAChRs with each subtype of G(q) family G proteins in lipid vesicles in order to test the specificity of coupling between receptors and G proteins. The m1 mAChRs markedly stimulated [S]GTPS binding to the G(q) family G proteins in the presence of 1 mM carbamylcholine, but they only slightly stimulated the [S]GTPS binding in the presence of 10 µM atropine (Fig. 5). In contrast with m1 mAChRs, m2 mAChRs did not stimulate the [S]GTPS bindings to these G proteins. Fig. 6shows the effect of concentrations of carbamylcholine on the [S]GTPS binding to G, G, and G(q) reconstituted with m1 mAChRs. The concentrations of carbamylcholine giving a half-maximal effect on the [S]GTPS binding were estimated to be 6.42 ± 3.15, 5.41 ± 2.00, and 3.47 ± 1.97 µM for G, G, and G(q), respectively, and these values were not significantly different from each other. These results indicate that the agonist-bound form of m1 mAChR may activate G, G, or G(q) with similar potency.


Figure 5: Stimulation by carbamylcholine of [S]GTPS binding to G proteins reconstituted with m1 mAChRs. The m1 (bullet and circle) or m2 ( and box) mAChRs were reconstituted with G proteins in lipid vesicles, and [S]GTPS binding activity in the vesicles was assayed in the presence of 1 mM carbamylcholine (bullet and ) or 10 µM atropine (circle and box) as described under ``Experimental Procedures.'' The amounts of m1 and m2 mAChRs in one tube were 73.2 and 63.9 fmol, respectively. The vesicles in one tube contained 600-650 fmol of G proteins.




Figure 6: Effect of carbamylcholine concentrations on the [S]GTPS binding. Each tube contained 14.0 fmol of m1 mAChRs and 300-400 fmol of G proteins. Each sample was incubated for 8 min at 30 °C.



GDP is known to have a lower affinity for G(i) or G(o) reconstituted with m2 mAChR in the presence of agonist than in the presence of antagonist(33, 39, 40) . We have examined whether GDP has different affinities for G reconstituted with m1 mAChR in the presence or absence of agonists. As shown in Fig. 7, GDP inhibited the GTPS binding in the presence or absence of agonist in a dose-dependent manner, and the higher concentration of GDP was required to inhibit the GTPS binding in the presence of agonist. The concentrations of GDP giving 50% inhibition of the [S]GTPS binding were estimated to be 0.2 and 4 µM in the absence and presence of carbamylcholine, respectively. In contrast, the concentrations of cold GTPS giving 50% inhibition of the [S]GTPS binding were not different in the presence of carbamylcholine and atropine (not shown).


Figure 7: Effect of GDP concentrations on [S]GTPS binding to G. Experiments were performed as described in the legend to Fig. 5, except that different concentrations of GDP were included and incubation time was 15 min. The amounts of reconstituted m1 mAChR and G in each tube were 35.0 and 215 fmol, respectively.



Reconstitution of m1 Receptor, G Protein, and PLC-beta

Fig. 8shows that the activity of PLC-beta reconstituted with m1 mAChR and each member of G(q) family G proteins was enhanced by carbamylcholine in the presence of GTPS. A similar stimulation was observed when 1 mM GTP was used instead of GTPS, as shown later (see Fig. 10, B and C). The activity of PLC-beta in the presence of carbamylcholine and G, G, or G(q) was estimated to be 16, 17, or 30 fmolbulletmin/mg of G protein, respectively.


Figure 8: Stimulation by carbamylcholine of PLC-beta reconstituted with m1 mAChRs and G proteins. The m1 mAChRs and G proteins were reconstituted in lipid vesicles as described under ``Experimental Procedures.'' The reaction was initiated by addition of a mixture of PLC-beta and GTPS (final concentrations of 0.2 and 100 nM, respectively) and proceeded for 0-10 min at 30 °C. The vesicles in one tube contained 13.4 fmol of m1 mAChRs and 300 fmol of G, 700 fmol of G, or 800 fmol of G(q).




Figure 10: Effect of protein kinase C on the carbamylcholine-stimulated PLC activity. A, lipid vesicles containing m1 mAChR, G(q) family G proteins, and PLC-beta were incubated with protein kinase C (16 nM) and [P]ATP (12.5 µM) for 1 h at 30 °C, and then an aliquot of the reaction mixture was subjected to SDS-PAGE and autoradiography. The bands with apparent molecular masses of 150, 80, and 59 kDa correspond to PLC-beta, protein kinase C, and m1 mAChR, respectively. A weakly phosphorylated band of 42 kDa corresponds to the alpha subunit of G proteins. The band with an apparent molecular mass of 29 kDa was not identified. B and C, lipid vesicles containing m1 mAChR, G(q) family G proteins, and PLC-beta were incubated with protein kinase C (125 nM) in the presence or absence of ATP (100 µM) for 1 h at 30 °C, and then the vesicles were separated from free ATP through a Sephadex G-50 column (2 ml). PLC activity of the void volume fraction was examined in the presence of 1 mM GTP and 1 mM carbamylcholine (bullet and ) or 10 µM atropine (circle and box), where samples treated with protein kinase C in the presence or absence of ATP were represented by dashed lines ( and box) or solid lines (bullet and circle), respectively. The amounts of m1 mAChR were 5.9-9.0 fmol/tube. Data in C are the means of three independent experiments.



Stimulation of the GTPase Activity of G(q) Family G Proteins by PLC-beta

PLC-beta(1) is known to accelerate the GTPase activity of a mixture of G(q) and G(13) . We have extended these experiments and examined the effect of PLC-beta on the GTPase activity of G, G, and G(q) reconstituted with m1 mAChR (Fig. 9). The GTPase activity of G, G, and G(q) but not of G(o) was greatly enhanced by PLC-beta in the presence of carbamylcholine. The GTPase activity of G(q) family G proteins was 60-90 fmol/min in the presence of 10 nM PLC-beta and 1 mM carbamylcholine, whereas the GTPase activity was barely detectable in the absence of either carbamylcholine or PLC-beta (1-5 fmol/min). The concentrations of PLC-beta giving a half-maximal effect on the GTPase of G, G, and G(q) were estimated to be 1.36 ± 0.50, 2.71 ± 1.17, and 3.07 ± 1.43 nM, respectively (averages and standard deviations for three independent experiments). These results indicate that PLC-beta acts not only as a G protein effector but also as a GTPase-activating protein for all three G proteins. In sharp contrast with G(q) family G proteins, the GTPase activity of G(o) reconstituted with m2 mAChR was not affected by addition of PLC-beta under exactly the same conditions, although the GTPase activity was stimulated by carbamylcholine (Fig. 9D). It should also be noted that G(o) exhibits a much higher GTPase activity than G(q) family G proteins in the absence of PLC-beta and agonists.


Figure 9: Effect of PLC-beta concentrations on the GTPase activity of G proteins. The mAChR and G proteins were reconstituted in lipid vesicles as described under ``Experimental Procedures.'' The reaction was started by addition of various concentrations of PLC-beta and then incubated for 15 min at 30 °C in the presence of 1 mM carbamylcholine (bullet) or 10 µM atropine (circle). The vesicles in each tube contained 70 fmol of m1 mAChR and 300-350 fmol of G(q) family G proteins (A, B, and C) or 30 fmol of m2 mAChR and 550 fmol of G(o) (D). A half-maximal stimulation of was observed in the presence of 0.78 nM PLC-beta for G, 1.87 nM PLC-beta for G, and 1.52 nM G(q) in this assay.



Effect of Phosphorylation by Protein Kinase C

The experiments described above demonstrate the establishment of a reconstitution system for the signal transduction from m1 mAChRs to PLC-beta. We next examined if this signal transduction is affected by protein kinase C, as was expected from in vivo experiments. When the lipid vesicles containing m1 mAChR, G, and PLC-beta were incubated with protein kinase C (a mixture of alpha, beta, and subtypes) and [P]ATP, m1 mAChR was predominantly phosphorylated. The amount of [P]phosphate recovered in the band corresponding to m1 mAChRs (59 kDa) was estimated to be 4 mol/mol of [^3H]QNB binding site. PLC-beta was also phosphorylated (0.58 mol/mol), but Galpha, Galpha, G(q)alpha, and beta subunits were barely phosphorylated (<0.01 mol/mol) (Fig. 10A).

Reconstituted vesicles containing m1 mAChR, G, and PLC-beta were subjected to phosphorylation by protein kinase C in the presence of 0.1 mM ATP, followed by the assay of PLC activity in the presence of GTP. Control samples were treated in the same way except for the omission of ATP. The PLC-beta activity either in the absence or presence of 1 mM carbamylcholine was not affected by whether the vesicles had been treated with protein kinase C in the presence or absence of ATP (Fig. 10B). Fig. 10C shows the dose-response curves for stimulation by carbamylcholine of the PLC activity of samples treated with protein kinase C in the presence or absence of ATP. The PLC activity of samples treated in the presence of ATP tends to require higher concentrations of carbamylcholine. The concentration of carbamylcholine giving a half-maximal effect was estimated to be 21.0 ± 5.1 and 32.4 ± 11.5 µM for samples treated with protein kinase C in the absence or presence of ATP.


DISCUSSION

We have expressed three kinds of G(q) family alpha subunits, Galpha, Galpha, and G(q)alpha, together with beta(1) and (2) subunits in Sf9 cells and purified each of them as an alphabeta trimer. Consistent with previous results(14, 15) , we also found that the coexpression of beta subunits was necessary for functionally active G proteins to be solubilized. In addition, we noticed that the yield of active G proteins was increased by replacing air with 50% O(2)/50% N(2) during the cell culture (41) and by infecting the cell with alpha and beta recombinant virus with a ratio of 3-4:1 instead of 1:1. Relatively lower amounts of beta recombinant virus were used because the expression level of beta subunits was found to be much higher than that of alpha subunits, when a recombinant virus encoding Galpha, beta, and subunits in tandem was used (not shown). We could purify alphabeta trimers of G, G, and G(q) to apparent homogeneity by three-step column chromatographies, and these purified trimers were active with respect to interaction with m1 mAChRs and PLC-beta. These results indicate that Galpha, Galpha, or G(q)alpha may make a functionally active complex with beta(1)(2) subunits.

The present reconstitution studies provide direct evidence that G, as well as G and G(q), is capable of activating PLC-beta. We did not find any significant differences among G, G, and G(q) as far as their interaction with PLC-beta. This finding is consistent with and extends the report by Hepler et al.(14) but is not consistent with the previous report that the potency of G(14)alpha (Galpha) in activating PLC-beta was half as much as that of G(q)alpha or Galpha(8) . This discrepancy may reflect the differences in the expression level between G(14)alpha and Galpha or G(q)alpha. We noticed that the expression level in COS-7 cells was lower for Galpha compared with Galpha (not shown).

Berstein et al.(12) have presented evidence for the functional interaction between the m1 mAChR and a mixture of G(q) and G and indicated that both G(q) and G are responsive to the m1 mAChR. We have confirmed and extended their results and have shown that G, G, or G(q) may mediate the signal transduction from the m1 mAChR to PLC-beta with similar potency and efficacy. These findings, however, are not in accord with the previous results indicating that the formation of IP(3) induced by the activation of metabotropic glutamate receptors mGluR1 (9) or thyrotropin-releasing hormone receptor (42) in Xenopus oocytes was accelerated by the coexpression of Galpha but was inhibited by the coexpression of Galpha or G(q)alpha. The reason for this discrepancy is not known. A possible explanation is that the PLC-beta expressed in Xenopus oocytes has different properties from the PLC-beta in bovine brain. The amphibian PLC-beta is known to be relatively distant from any known mammalian PLC-betas, with the closest identity of 64% to mammalian PLC-beta(3)(43) . This explanation remains to be examined by reconstituting purified frog PLC-beta with G(q) family G proteins.

The stimulation by carbamylcholine of IP(3) formation in the reconstituted vesicles containing m1 mAChR, G, and PLC-beta was observed in the presence of 1 mM GTP as well as in the presence of 1 µM GTPS ( Fig. 8and Fig. 10). This result indicates that the presence of three protein components is sufficient for the signal transduction from carbamylcholine to IP(3) in the presence of endogenous guanine nucleotide. Berstein et al.(12) have reported that the stimulation by carbamylcholine of IP(3) formation is observed in the presence of GTPS but not in the presence of GTP. The reason for the discrepancy is not known, but is not due to the lack of stimulation of GTPase activity by PLC-beta. We have confirmed and extended the result by Berstein et al.(13) and shown that the GTPase activities of G, G, and G(q) are stimulated by PLC-beta approximately to the same extent. Concentrations of PLC-beta giving a half-maximal effect were similar among the three G proteins, indicating that PLC-beta interacts with these G proteins with similar affinity.

The m1 mAChR may interact with all of G, G, and G(q), but the m2 mAChR apparently did not interact with any of them. A slight activation by the m2 mAChR of [S]GTPS binding to a mixture of G(q) and G in the previous report (12) may represent a contamination by G(o) and G(i) in the G(q)/G preparation purified from brain or liver. We have found that it is very difficult to prepare G(q) preparations free from G(i)/G(o) starting from intact tissue, although it is possible to avoid the problem by using recombinant G proteins. The PLC-beta may interact with all of G, G, and G(q) but does not interact with G(o), as was evident from the lack of stimulation by G(o) of PLC activity and of stimulation by PLC of GTPase activity of G(o). These results demonstrate the strict specificity of the interactions of G(q) family G proteins with their receptor and effector, in contrast with the apparent lack of specificity among the three G(q) family G proteins.

The stimulation of protein kinase C by phorbol esters is known to lead to the desensitization of the activation of PLC mediated by mAChRs ((20, 21, 22) ; for review see (19) ). In the present studies, we have shown that the phosphorylation of m1 mAChR and PLC-beta does not affect the stimulation of PLC-beta by carbamylcholine in the reconstitution system. We cannot, however, exclude the possibility that the phosphorylation by protein kinase C reduces the affinity of the interaction of G proteins with m1 mAChR or PLC-beta, but the effect has not been detected by the presence of excessive amounts of these proteins in the reconstitution system. It is also possible that other types of protein kinase C may be involved in the phosphorylation and the desensitization. Alternatively, it is likely that the presence of three components of the m1 mAChR, G protein, and PLC-beta is sufficient for the signal transduction from acetylcholine to IP(3) but is not sufficient for the regulation of the signal transduction.

In summary, we have expressed and purified three kinds of G(q) family G proteins, G, G, and G(q), and reconstituted them with the m1 mAChR and PLC-beta in lipid vesicles. In the reconstitution system, we have shown that 1) these G proteins mediated the activation of PLC-beta by m1 mAChR in the presence of agonist and GTP, 2) the GTPase activity of these G proteins is enhanced by PLC-beta, and 3) the signal transduction from the m1 mAChR to PLC is not affected by phosphorylation by protein kinase C of the m1 mAChR and PLC-beta.


FOOTNOTES

*
This work was supported in part by grants from the Ministry of Education, Science, and Culture of Japan, the Japan Research Foundation for Clinical Pharmacology, and the Yamada Science Foundation. 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.

§
Recipient of fellowships from the Japan Society for the Promotion of Science for Japanese Junior Scientists from April, 1992 to March, 1994. Present address: Dept. of Neurology, School of Medicine, Yale University, 333 Cedar St., New Haven, CT 06510.

Present address: Dept. of Neurochemistry, Psychiatric Research Inst. of Tokyo, Kamikitazawa 2-1-8, Setagaya-ku, Tokyo 156, Japan.

**
To whom correspondence should be addressed. Tel.: 81-3-5689-7331; Fax: 81-3-3814-8154.

(^1)
G(q), G(q)alphabeta; G, Galphabeta; G, Galphabeta; G(o), G(o)alpha(1)beta. Galpha and Galpha are the bovine homologues of mouse G(14)alpha and Galpha, respectively.

(^2)
The abbreviations used are: G protein, GTP-binding regulatory protein; mAChRs, muscarinic acetylcholine receptors; PLC, phosphoinositide-specific phospholipase C; DTT, dithiothreitol; GTPS, guanosine 5`-O-(3-thiotriphosphate); QNB, L-quinuclidinyl benzilate; PIP(2), phosphatidylinositol 4,5-bisphosphate; IP(3), inositol 1,4,5-trisphosphate; PAGE, polyacrylamide gel electrophoresis; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.


ACKNOWLEDGEMENTS

We thank Dr. M. I. Simon of the California Institute of Technology for the donation of cDNA of G(q)alpha, Dr. M. D. Summers for permission to use the baculovirus vectors, Dr. E. M. Ross for the m1 and m2 mAChR baculoviruses, Dr. K. Haga for purification of m1 and m2 mAChRs, and Dr. D. W. Saffen for comments and for the editing of the manuscript.


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