Role of Isoprenoid Lipids on the Heterotrimeric G Protein gamma  Subunit in Determining Effector Activation*

Chang-Seon MyungDagger , Hiroshi YasudaDagger §, Wendy W. LiuDagger , T. Kendall Hardenparallel , and James C. GarrisonDagger **

From the Dagger  Department of Pharmacology, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908 and the parallel  Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599-7365

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Post-translational prenylation of heterotrimeric G protein gamma  subunits is essential for high affinity alpha -beta gamma and alpha -beta gamma -receptor interactions, suggesting that the prenyl group is an important domain in the beta gamma dimer. To determine the role of the prenyl modification in the interaction of beta gamma dimers with effectors, the CAAX (where A indicates alipathic amino acid) motifs in the gamma 1, gamma 2, and gamma 11 subunits were altered to direct modification with different prenyl groups. Six recombinant beta gamma dimers were overexpressed in baculovirus-infected Sf9 insect cells, purified, and examined for their ability to stimulate three phospholipase C-beta isozymes and type II adenylyl cyclase. The native beta 1gamma 2 dimer (gamma  subunit modified with geranylgeranyl) is more potent and effective in activating phospholipase C-beta than either the beta 1gamma 1 (farnesyl) or the beta 1gamma 11 (farnesyl) dimers. However, farnesyl modification of the gamma  subunit in the beta 1gamma 2 dimer (beta 1gamma 2-L71S) caused a decrement in its ability to activate phospholipase C-beta . In contrast, both the beta 1gamma 1-S74L (geranylgeranyl) and the beta 1gamma 11-S73L (geranylgeranyl) dimers were more active than the native forms. The beta 1gamma 2 dimer activates type II adenylyl cyclase about 12-fold; however, neither the beta 1gamma 1 nor the beta 1gamma 11 dimers activate the enzyme. As was the case with phospholipase C-beta , the beta 1gamma 2-L71S dimer was less able to activate adenylyl cyclase than the native beta 1gamma 2 dimer. Interestingly, neither the beta 1gamma 1-S74L nor the beta 1gamma 11-S73L dimers stimulated adenylyl cyclase. The results suggest that both the amino acid sequence of the gamma  subunit and its prenyl group play a role in determining the activity of the beta gamma -effector complex.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Heterotrimeric G proteins1 are transducers of numerous extracellular signals from seven transmembrane receptors to intracellular effectors (1-4). G proteins are composed of alpha , beta , and gamma  subunits and associate with the inner side of the plasma membrane. Receptor activation catalyzes the exchange of GDP for GTP on the alpha  subunit, resulting in dissociation of the GTP-liganded alpha  subunit from the beta gamma dimer (1). Both the GTP-bound form of the alpha  subunit and the released beta gamma dimer regulate a variety of effectors, including PLC-beta (5, 6) and adenylyl cyclases (7, 8). To date, beta  subunits and 11 gamma  subunits have been identified in mammalian systems (9-15). Selective assembly of beta gamma heterodimers from these proteins may produce a large number of unique complexes that differ in their interactions with alpha  subunits, receptors, and effectors. While the first four beta  subunits identified, beta 1-beta 4, are 85-90% identical in primary amino acid sequence (9), the gamma  subunits are much more divergent. For example, the gamma 1 and gamma 5 subunits are only 25% identical. Thus, the gamma  subunits may impart some specificity to the beta gamma signal.

The G protein gamma  subunits are subject to post-translational modification by the addition of isoprenoid lipids to an invariant cysteine residue in the CAAX motif at their C terminus (15-17). The last amino acid (X) of the CAAX motif is an important determinant for modification by one of two distinct isoprenoids, the 15-carbon farnesyl group or the 20-carbon geranylgeranyl group. Among the 11 known gamma  subunits, the gamma 1, gamma 8, and gamma 11 subunits are thought to be modified with the addition of a 15-carbon farnesyl group, whereas the other gamma  subunits contain a 20-carbon geranylgeranyl group (10). The gamma 11 subunit was recently identified as a widely expressed gamma  subunit that is 76% identical to the gamma 1 subunit (10, 18). However, the function of beta gamma dimers containing the gamma 11 subunit has not been studied. Moreover, the significance of farnesyl versus geranylgeranyl modification of the gamma  subunit in beta gamma subunit-mediated activation of effectors has not been established.

Lipid modification is important for anchoring the beta gamma subunit to the membrane (17, 19, 20), but the prenyl group may also play a major role in determining functional interaction with other proteins (15, 17, 21). For instance, prenylation of the gamma  subunit is necessary for formation of an active transducin alpha -beta gamma complex (22, 23), for translocation of the beta -adrenergic receptor kinase to the plasma membrane (24), for high affinity interactions with either alpha  subunits or adenylyl cyclases (25), and for stimulation of PLC-beta by the beta 1gamma 1 dimer (26). Since the gamma  subunits may have different primary amino acid sequences and different prenyl groups at their C terminus, investigators have attempted to determine whether the type of prenyl group is an important determinant of the activity of the beta gamma dimer. Some studies suggest that beta gamma dimers containing the farnesylated gamma 1 subunit couple rhodopsin to the Gt alpha  subunit more effectively (27, 28), while others show a small increase in the activity of beta gamma dimers containing a geranylgeranylated gamma 1 subunit in receptor-coupling assays (29). We have determined that beta gamma dimers containing a geranylgeranyl group on the gamma  subunit couple the Gi alpha  subunit to the A1 adenosine receptor more effectively than those containing a farnesyl group (30). Overall, these results suggest that the type of prenyl group on the gamma  subunit does play an important role in determining the activity of beta gamma dimers.

Most investigators have considered that the prenyl group is involved in tethering the beta gamma subunit to the membrane (17, 19, 20). However, the recent x-ray crystal structure of a phosducin-beta gamma complex, which was crystallized with an intact gamma  subunit containing a farnesyl group, shows that the isoprenoid group folds into a hydrophobic pocket formed by blades 6 and 7 of the beta  propeller (31). Interestingly, the conformation of the beta  subunit in this complex is different from the conformation of the free beta gamma dimer (32). Thus, it is possible that there is an active conformation of the beta gamma dimer, which occurs upon binding to effectors, and that the prenyl group participates in establishing this conformation. These observations provide a compelling reason to examine the ability of beta gamma dimers in which the gamma  subunit contains different isoprenoid lipids in regulating effectors.

We have reported previously that the prenyl group on the gamma 1 and gamma 2 subunits can be altered by mutation of the last amino acid (X) in the CAAX motif at their C terminus (30, 33). Expression of the mutant gamma  subunits in Sf9 cells with a beta  subunit allows formation of functional beta gamma dimers in which the lipid on the gamma  subunit has been changed from farnesyl to geranylgeranyl or vice versa (30, 33). Using this approach, we have purified six recombinant beta gamma dimers containing gamma  subunits with native or altered prenyl groups and verified that the altered CAAX sequences result in the expected post-translational modifications using mass spectrometry. We examined the effects of the beta 1gamma 2, beta 1gamma 1, and beta 1gamma 11 dimers and their prenyl mutants on the activity of PLC-beta and type II adenylyl cyclase. The results indicate that both the primary amino acid sequence of the gamma  subunits and the type of prenyl group on the gamma  subunit are important determinants of the activation of PLC-beta and type II adenylyl cyclase.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Construction of Recombinant Baculoviruses-- The recombinant baculoviruses encoding the alpha S, beta 1, gamma 1, gamma 2 subunits (34, 35), and the gamma 1 and gamma 2 subunits with altered prenylation sequences in the CAAX motifs, gamma 1-S74L and gamma 2-L71S, were prepared as described (33). A full-length cDNA encoding the human gamma 11 protein was identified in the expressed sequence tag data base and obtained from Research Genetics, Inc. (gamma 11, GenBankTM accession number H00850). To minimize the length of the construct 5' from the ATG start codon, the polymerase chain reaction was used to add a SmaI restriction site to the 5' end of gamma 11. For the gamma 11 cDNA, the primers used were: sense primer, 5'-TCCCGGGCGAAAATGCCTGCC-3'; antisense primer, 5'-CCACTTAGGATGCAGTTTCTCCC-3'. The polymerase chain reaction products were subcloned into the pCNTR shuttle vector, and the gamma 11 coding sequence excised from pCNTR with SmaI and XbaI was ligated into these sites in the baculovirus transfer vector, pVL1393. The cDNA encoding a gamma 11 with the altered prenylation sequence in the CAAX motif, gamma 11-S73L, was constructed with the same strategy. Modification of the C-terminal CAAX motif of gamma 11 was performed using polymerase chain reaction to introduce a CVIS right-arrow CVIL into the CAAX sequence. To produce the gamma 11-S73L cDNA, the primers used were: sense primer, 5'-TCCCGGGCGAAAATGCCTGCC-3'; antisense primer, 5'-TTTATAAAATAACACAGCTGCC-3'. The polymerase chain reaction products were subcloned into the pCNTR shuttle vector, digested with SmaI and XbaI, and ligated into these sites in the pVL1393 transfer vector. The completed constructs of both gamma 11 and gamma 11-S73L in the pVL1393 transfer vector were sequenced to confirm the fidelity of the gamma  sequences. Recombinant baculoviruses were produced by co-transfecting each recombinant plasmid DNA with linear wild type BaculoGold® viral DNA (PharMingen) into Sf9 cells as described (36). The recombinant baculoviruses were purified by one round of plaque purification (37).

Expression and Purification of Recombinant G Protein beta gamma Subunits-- G protein alpha  and beta gamma subunits were overexpressed by infecting Sf9 insect cells with recombinant baculoviruses as described (34, 35). Sf9 cells were co-infected at a multiplicity of infection of 3 with the appropriate beta  and gamma  recombinant baculoviruses and harvested 48 h after infection. The beta gamma subunits were extracted from frozen cell pellets with 0.1% Genapol C-100 and purified on a DEAE column followed by affinity chromatography on a Gi1-alpha -agarose column as described (35). The Gs alpha  subunit used in the adenylyl cyclase assays was prepared from a 0.1% (w/v) CHAPS extract of crude cell lysates as described (36).

Purification of Phospholipase C-beta -- Recombinant turkey PLC-beta and human PLC-beta 1 and PLC-beta 2 were purified to homogeneity following overexpression in baculovirus-infected Sf9 insect cells using chromatography on Q-Sepharose FF, heparin-Sepharose CL-6B, HPHT hydroxylapatite, Sephacryl S-300, and FPLC Mono Q HR 5/5 columns as described previously (38, 39).

Analysis of the Post-translational Processing of gamma  Subunits by Mass Spectrometry-- Full processing of the gamma  subunit requires the addition of either a farnesyl or a geranylgeranyl group to the C-terminal cysteine in the CAAX motif, the removal of the three C-terminal amino acids (AAX), and the addition of a carboxymethyl group to the C terminus (16, 33). To determine the extent of the post-translational lipid modification of the gamma  subunits, the six purified beta gamma dimers used in this study were analyzed by electrospray ionization mass spectrometry as described previously (33). The result of this analysis showed that 96% of the native gamma 1 subunit contained the expected farnesyl group and had a completely processed C terminus. Similarly, 87% of the prenyl mutant, gamma 1-S74L, contained the geranylgeranyl group and was fully processed at its C terminus. Ninety-one percent of the gamma 2 subunit and 70% of the gamma 11 subunit were found to have the expected geranylgeranyl and farnesyl groups, respectively, and a completely processed C terminus. The prenyl mutants of these two gamma  subunits, gamma 2-L71S and gamma 11-S73L, were fully processed with expected isoprenoid lipids to the extent of 95 and 87%, respectively. Therefore, all gamma  subunits used in this study contain the expected isoprenoid lipid at the C terminus and are capable of high-affinity interactions with alpha  subunits, receptors and effectors.

Preparation of Large Unilamellar Vesicles-- Phospholipid vesicles were prepared as described (40). Briefly, phospholipids were mixed at a molar ratio of 4:1 of phosphatidylethanolamine to PIP2 with [inositol-2-3H]PIP2 in a buffer containing 50 mM HEPES, pH 8.0, 3 mM EGTA, 80 mM KCl, 1 mM dithiothreitol and the mixture dried under argon. The dried lipid was suspended with 3.0 ml of the above buffer to give final concentrations of 1 mM phosphatidylethanolamine, 250 µM PIP2, and 800 cpm/µl [3H]PIP2 and hydrated in the dark at room temperature. Extruded large unilamellar vesicles were formed from multilamellar vesicles by vortexing the lipid solution followed by 10 extrusion cycles through a stack of two polycarbonate filters using a mini-extruder (Avanti Polar Lipids, Inc.) (41). After extrusion, the final lipid concentration (phosphatidylethanolamine and PIP2) was about 1100 µM. Extruded large unilamellar vesicles were stored under argon at 4 °C until use.

Reconstitution of beta gamma Dimers to Large Unilamellar Vesicles by Gel Filtration-- CHAPS-solubilized beta gamma subunits (ranging from 10 ng to 10 µg) were mixed with vesicles in a volume of 200 µl. The final CHAPS concentration in the mixture was held to 0.01%. After reconstituting the mixture on ice for 30 min, the vesicles were applied to a 2-ml gel filtration column prepared with Ultrogel AcA34 as described previously (40, 42). The column was pre-equilibrated at 4 °C with buffer containing 20 mM HEPES, pH 8.0, 2 mM MgCl2, 100 mM NaCl, and 1 mM EDTA (450 µl/min). The vesicles reconstituted with beta gamma dimers were eluted at the void volume of the column in a total volume of about 900 µl. This protocol has been demonstrated to remove more than 98% of the detergent from the vesicles (42, 43) and to resolve the vesicles from the free beta gamma dimers (40). The concentration of lipid in each fraction was monitored by counting the incorporated [3H]PIP2, and the amount of beta gamma protein inserted into the vesicles was quantified by silver staining as described below. The second fraction from the AcA34 column contained vesicles with the least amount of free beta gamma dimer and were used in all experiments (40).

Measurement of Phospholipase C-beta Activity-- Reaction buffer containing 50 mM HEPES, pH 8.0, 17 mM NaCl, 67 mM KCl, 0.83 mM MgCl2, 0.17 mM EDTA, 3 mM EGTA, 1 mM dithiothreitol, and 1 mg/ml BSA was added to 70 µl of gel-filtered vesicles (fraction 2) in a total volume of 80 µl. Controls were performed using column-reconstituted vesicles without beta gamma dimers. The reactions were initiated by adding 10 ng of PLC-beta and 3 µM free Ca2+ and placing the mixture in a 30 °C water bath. After a 15-min incubation, the reactions were terminated by transferring each assay tube to a 4 °C ice bath and adding 200 µl of ice-cold 10% trichloroacetic acid followed by the addition of 100 µl of 10 mg/ml BSA (40). The assay mixtures were centrifuged to remove precipitated protein and intact PIP2 and the [3H]inositol 1,4,5-trisphosphate released into the supernatant quantitated by liquid scintillation spectrometry (44, 45).

Measurement of Adenylyl Cyclase Activity-- Adenylyl cyclase activity was measured as described previously (36, 46). Briefly, Sf9 insect cells were infected with recombinant baculovirus encoding rat type II adenylyl cyclase (47) and harvested 48 h after infection. Sf9 membranes containing type II adenylyl cyclase (5 µg of protein/assay tube) were reconstituted with GTPgamma S-activated Gs alpha  subunit (48) and varying concentrations of beta gamma dimers on ice for 30 min. The reaction was initiated by addition of the reconstituted membranes to the reaction buffer containing 25 mM HEPES, pH 8.0, 10 mM phosphocreatine, 10 units/ml of creatine phosphokinase, 0.4 mM 3-isobutyl-1-methylxanthine, 10 mM MgCl2, 0.5 mM ATP, and 0.1 mg/ml BSA and incubated for 7 min at 30 °C. The assay was stopped by addition of 0.1 N HCl and cyclic AMP measured using an automated radioimmunoassay (49).

Electrophoresis-- For quantitation of the beta gamma concentration in vesicles, the beta gamma dimers reconstituted into phospholipid vesicles were electrophoresed on a 12% acrylamide, sodium dodecyl sulfate-polyacrylamide gel and stained with silver according to the method of Bloom et al. (50). The beta gamma concentrations in the gel were estimated using the amount of stained beta  subunit protein as compared with ovalbumin standards run in the same gel. The stained proteins were quantitated using a Bio-Image scanning densitometer and the Whole Band® software (Bio-Image, Ann Arbor, MI) as described (36, 40). The accuracy of this procedure was verified by comparison with the BCA (Pierce) protein assay (40). To better display the mobility of the gamma  subunits, Tricine/SDS-polyacrylamide gels were run according to the procedure of Schagger and von Jagow (51). The separating gel contained 16.5% total acrylamide, 0.4% bisacrylamide, and 10% (v/v) glycerol. The stacking gel contained 4% total acrylamide and 0.1% bisacrylamide. Gels were run at constant voltage (~100 volts) at 10 °C for 4-5 h. Resolved proteins were stained with silver according to the method of Morrissey (52) with the modification that the dithiothreitol incubation was reduced to 15 min.

Calculation and Expression of Results-- Experiments presented under "Results" are representative of three or more similar experiments. Data expressed as concentration-response curves were fit to sigmoid curves using the fitting routines in the GraphPad PrismTM software. Statistical differences between the curves were determined using all the individual data points from multiple experiments to calculate the F statistic as described (53).

Materials-- All reagents used in the culture of Sf9 cells and for the expression and purification of G protein beta gamma subunits have been described previously in detail (34, 35). The baculovirus transfer vector, pVL1393, was purchased from Invitrogen; the BaculoGold® kit from PharMingen; 10% Genapol C-100 and phosphatidylinositol 4,5-bisphosphate from Calbiochem®; phosphatidylethanolamine (bovine heart) from Avanti Polar Lipids, Inc.; [inositol-2-3H]phosphatidylinositol 4,5-bisphosphate from NEN Life Science Products; CHAPS from Roche Molecular Biochemicals; BSA (fatty acid-free) from Sigma; the pCNTR shuttle vector from 5 Prime right-arrow 3 Prime, Inc. (Boulder, CO); Q-Sepharose FF, heparin-Sepharose CL-6B, Sephacryl S-300, and FPLC Mono Q HR 5/5 columns from Amersham Pharmacia Biotech; and HPHT hydroxylapatite from Bio-Rad. All other reagents were of the highest purity available.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

To determine the role of the prenyl modification of the gamma  subunit in the interaction of beta gamma dimers with effectors, the CAAX motifs in the gamma 1, gamma 2, and gamma 11 subunits were altered to direct the addition of different prenyl isoprenoids. Each gamma  subunit was co-expressed with the beta 1 subunit in Sf9 insect cells, and the six beta gamma dimers, beta 1gamma 1, beta 1gamma 1-S74L, beta 1gamma 2, beta 1gamma 2-L71S, beta 1gamma 11, and beta 1gamma 11-S73L, were purified using alpha i1-agarose affinity chromatography and tested for their ability to activate two effectors, PLC-beta and type II adenylyl cyclase. All six gamma  subunits were analyzed by mass spectrometry to ensure that their CAAX motifs were appropriately processed. The results of the analysis showed that each gamma  subunit had the expected isoprenoid lipids at its C terminus with the extent of processing ranging from 70 to 95% (see "Experimental Procedures"). Therefore, all beta gamma dimers used in this study are capable of high-affinity interactions with alpha  subunits, receptors, and effectors. The purity of these dimers is shown in Fig. 1A. The purity of the three recombinant PLC-beta isoforms used in this study is shown in Fig. 1B.


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Fig. 1.   Sodium dodecyl sulfate-polyacrylamide electrophoresis of purified G protein beta gamma subunits and three PLC-beta isoforms. A, purified, recombinant beta gamma subunits of defined subtypes were electrophoresed on a 16% acrylamide, 10% (v/v) glycerol, sodium dodecyl sulfate-Tricine gel and stained with silver. The migration of the beta  and gamma  subunits are indicated on the left. B, three isoforms of purified, recombinant PLC-beta were electrophoresed on a 8% acrylamide SDS-PAGE and stained with silver. The mobility of the molecular weight standards is indicated on the left.

Differential Effect of Three Different beta gamma Dimers on PLC-beta and Adenylyl Cyclase Activity-- We compared the ability of beta gamma dimers with gamma  subunits containing either a farnesyl or a geranylgeranyl moiety to stimulate PLC-beta . An assay system was used in which purified beta gamma dimers were reconstituted into extruded phospholipid vesicles and the vesicles separated from mono-dispersed beta gamma dimers and the detergent used to solubilize G proteins on an Ultrogel AcA34 column (40). The data in Fig. 2A illustrate the ability of three beta gamma dimers to activate recombinant turkey PLC-beta . Avian PLC-beta was used initially, because it has a lower basal activity and higher beta gamma subunit-stimulated activity than either the mammalian PLC-beta 1 or PLC-beta 2 isozymes (39) (see also Fig. 3). The beta 1gamma 2 dimer activated turkey PLC-beta about 10-fold with an estimated EC50 value of 0.7 nM (refer to Table II). The beta 1gamma 11 and beta 1gamma 1 subunits activated turkey PLC-beta with estimated EC50 values of 4.1 and 1.9 nM, respectively. The Vmax values were 50-60% of those observed with the beta 1gamma 2 dimer (refer to Table II). Therefore, the beta 1gamma 2 dimer is more potent and effective in activating PLC-beta than either beta 1gamma 1 or beta 1gamma 11, and the potency of beta 1gamma 11 is less than that of beta 1gamma 1. The data in Fig. 2B compare the ability of the three beta gamma dimers to activate type II adenylyl cyclase. The beta 1gamma 2 dimer activated type II adenylyl cyclase about 12-fold with an estimated EC50 value of 14 nM. However, neither beta 1gamma 1 (54) nor beta 1gamma 11 effectively activated type II adenylyl cyclase. Since the gamma  subunits were combined with the same beta 1 subunit, these results indicate that differences in the activation of either PLC-beta or type II adenylyl cyclase are due to differences in the gamma  subunit. The major differences between the known gamma  subunits are their divergent amino acid sequences and their lipid modifications. Interestingly, the data in Fig. 2A show that beta gamma dimers containing the gamma 2 subunit modified with a geranylgeranyl group are more potent and effective in activating both effectors than those containing either the gamma 1 or the gamma 11 subunit modified with a farnesyl group. Therefore, to address the possibility that the identity of the prenyl group on the gamma  subunit is an important determinant of the dimer's activity, the CAAX motif in the gamma 2 subunit was altered to direct the addition of the farnesyl group and the ability of the beta 1gamma 2-L71S dimer to activate three PLC-beta isoforms was examined.


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Fig. 2.   Differential abilities of three beta gamma dimers to regulate PLC-beta and adenylyl cyclase. A, the ability of the beta 1gamma 1 and beta 1gamma 11 dimers to activate phospholipase C-beta as compared with that of the beta 1gamma 2 dimer. Extruded phospholipid vesicles containing phosphatidylinositol 4,5-bisphosphate were reconstituted with 0.01, 0.03, 0.1, 0.3, 1.0, 3.0, or 10.0 µg of each beta gamma dimer on ice for 30 min. The mixture was applied to a 2-ml Ultrogel AcA34 column, fraction 2 was collected, and the ability of beta 1gamma 1 (open circles), beta 1gamma 2 (closed squares), and beta 1gamma 11 (closed triangles) in fraction 2 to stimulate turkey PLC-beta activity was measured as described under "Experimental Procedures." The difference between the effect of beta 1gamma 2 and either beta 1gamma 1 or beta 1gamma 11 was statistically significant (p < 0.0001). B, comparison of the ability of three beta gamma dimers to stimulate type II adenylyl cyclase. Sf9 cells were infected with a recombinant baculovirus encoding type II adenylyl cyclase, membranes prepared, and the cyclase reaction performed with the indicated concentrations of three recombinant beta gamma dimers as described under "Experimental Procedures." The results are representative of six similar experiments, each performed in duplicate. Sigmoid curves were fit to the data and statistical differences between the curves determined as described under "Experimental Procedures." The difference between the effect of beta 1gamma 2 and either beta 1gamma 1 or beta 1gamma 11 was statistically significant (p < 0.0001).


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Fig. 3.   Comparison of the activity of the beta 1gamma 2 and beta 1gamma 2-L71S dimers to activate three different isoforms of purified PLC-beta . A, the ability of beta 1gamma 2 (modified with geranylgeranyl; closed squares) and beta 1gamma 2-L71S (modified with farnesyl; open squares) to stimulate recombinant turkey PLC-beta (tPLC-beta ) was measured as described under "Experimental Procedures." The difference between the effect of beta 1gamma 2 and beta 1gamma 2-L71S was statistically significant (p < 0.001). B, analogous experiments were performed with recombinant human PLC-beta 2 (hPLC-beta 2). The difference between the effect of beta 1gamma 2 and beta 1gamma 2-L71S was statistically significant (p < 0.001). C, analogous experiments were performed with human PLC-beta 1 (hPLC-beta 1).

Effect of beta 1gamma 2 and beta 1gamma 2-L71S on PLC-beta Activity-- The data in Fig. 3 show that the beta 1gamma 2 dimer stimulated PLC-beta with a subnanomolar potency and that exchanging the prenyl group on the gamma 2 subunit from geranylgeranyl to farnesyl (beta 1gamma 2-L71S) caused a diminution in the ability of the beta 1gamma 2 dimer to activate all three isoforms of PLC-beta . The farnesyl-modified beta 1gamma 2-L71S dimer was less potent and effective in activating turkey PLC-beta than the native beta 1gamma 2 dimer (Fig. 3A). Fitting the data to sigmoid curves estimated that the maximal stimulation with the beta 1gamma 2-L71S dimer decreased by 35% and that the EC50 value increased from 0.7 to 1.2 nM (refer to Table II). Similarly, the beta 1gamma 2-L71S dimer was less potent and effective than the native beta 1gamma 2 dimer in the activation of human PLC-beta 2 (Fig. 3B). The data in Fig. 3C indicate that the beta gamma subunits produced little effect on the activity of PLC-beta 1 as demonstrated previously (5, 44, 45, 55, 56). Thus, beta gamma subunits stimulated activity in the order of tPLC-beta  >=  PLC-beta 2 >>> PLC-beta 1. Overall, these results indicate that the prenyl group on the gamma  subunit is an important determinant of the interaction between PLC-beta and beta gamma dimers.

Partition of Different Recombinant beta gamma Dimers into Phospholipid Vesicles-- Since most studies of G protein activation of PLC-beta have used the reconstitution of pre-aggregated lipid with alpha  or beta gamma subunits to activate PLC-beta , a potential problem might arise if beta gamma dimers containing different isoprenoid lipids did not partition equally into pre-aggregated lipid layers. This is especially important considering the different chain lengths of the lipids associated with the native gamma 1, gamma 2, or gamma 11 subunits. To determine the partitioning into vesicles of the six different beta gamma dimers used in this study, fractions 2 and 3 from the AcA34 column were collected and the amount of protein inserted into the vesicles was measured as described (40). Table I shows that both native beta 1gamma 1 and beta 1gamma 11 dimers containing farnesyl groups partitioned equally with the native beta 1gamma 2 dimer containing a geranylgeranyl group. About 27% of the beta gamma dimers added to the reaction mixture were incorporated into the phospholipid vesicles isolated in fractions 2 and 3. Moreover, the beta gamma dimers with altered CAAX sequences partitioned equally with native beta gamma dimers. Therefore, the length of the prenyl chain does not significantly affect the concentration of beta gamma dimers reconstituted into phospholipid vesicles containing phosphatidylinositol 4,5-bisphosphate.

                              
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Table I
Partition of different recombinant beta gamma dimers into phospholipid vesicles
Purified beta gamma subunits were mixed with phospholipid vesicles, reconstituted on ice for 30 min, applied to a 2-ml Ultrogel AcA34 column at 4 °C, and fractions 2 and 3 eluting at the void volume of the column used for measurement of the amount of dimer inserted into the vesicles. The amount of phospholipid eluted and beta gamma dimers bound to the vesicles were quantitated as described under "Experimental Procedures." Data are representative of three separate experiments.

Effect of beta 1gamma 1, beta 1gamma 1-S74L, beta 1gamma 11, and beta 1gamma 11-S73L on PLC-beta Activity-- It was important to determine whether switching the farnesyl group to geranylgeranyl improves the ability of either the beta 1gamma 1 or the beta 1gamma 11 dimer to activate PLC-beta . Thus, the CAAX motifs in the gamma 1 and gamma 11 subunits were altered to direct the addition of a geranylgeranyl group, and the ability of both the beta 1gamma 1-S74L and beta 1gamma 11-S73L dimers to activate either turkey PLC-beta or human PLC-beta 2 was examined. The data in Fig. 4A show that the geranylgeranyl-modified beta 1gamma 1-S74L dimer exhibited an increased potency and maximal activity compared with the native beta 1gamma 1 dimer. Similarly, the geranylgeranyl-modified beta 1gamma 11-S73L dimer was more potent and effective in activating turkey PLC-beta than the native beta 1gamma 11 dimer. The beta 1gamma 1-S74L and beta 1gamma 11-S73L dimers also exhibited increased capacity to stimulate human PLC-beta 2 activity (Fig. 5). These four beta gamma dimers (beta 1gamma 1, beta 1gamma 1-S74L, beta 1gamma 11, and beta 1gamma 11-S73L) showed little effect on the activity of PLC-beta 1 (data not shown, but see Fig. 3C). The data in Table II summarize the potency and efficacy of the six beta gamma dimers used in this study on the activation of these two PLC-beta isoforms. The beta 1gamma 1-S74L dimer exhibited an about 1.6-fold increase in maximal activity as compared with the native beta 1gamma 1 dimer and the EC50 value decreased from 1.9 to 1.1 nM. The estimated maximal activity of the beta 1gamma 11-S73L dimer increased about 1.3-fold and the EC50 value decreased from 4.1 to 1.6 nM. Similar changes were observed in the ability of the dimers to activate human PLC-beta 2. Taken together, the data indicate that beta gamma dimers with a gamma  subunit containing a geranylgeranyl group interact with higher affinity with PLC-beta than beta gamma dimers with a gamma  subunit containing a farnesyl group.


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Fig. 4.   Comparison of the activity of native and altered beta gamma dimers to stimulate recombinant turkey PLC-beta . A, the ability of beta 1gamma 1 (modified with farnesyl; open circles) and beta 1gamma 1-S74L (modified with geranylgeranyl; closed circles) dimers to stimulate recombinant turkey PLC-beta (tPLC-beta ) was measured as described under "Experimental Procedures." The difference between the effect of beta 1gamma 1 and beta 1gamma 1-S74L was statistically significant (p < 0.001). B, analogous experiments were performed with beta 1gamma 11 (modified with farnesyl; closed triangles) and beta 1gamma 11-S73L (modified with geranylgeranyl; open triangles) dimers. The difference between the effect of beta 1gamma 11 and beta 1gamma 11-S73L was statistically significant (p < 0.001).


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Fig. 5.   Comparison of the activity of native and altered beta gamma dimers to stimulate recombinant human PLC-beta 2. A, phospholipid vesicles reconstituted with beta 1gamma 1 (modified with farnesyl; open circles) or beta 1gamma 1-S74L (modified with geranylgeranyl; closed circles) dimer were incubated with recombinant human PLC-beta 2 (hPLC-beta 2) and PLC-beta activity measured as described under "Experimental Procedures." The data were fit to sigmoid curves. The difference between the effect of beta 1gamma 1 and beta 1gamma 1-S74L was statistically significant (p < 0.001). B, analogous experiments were performed with beta 1gamma 11 (modified with farnesyl; closed triangles) and beta 1gamma 11-S73L (modified with geranylgeranyl; open triangles) dimers. The difference between the effect of beta 1gamma 11 and beta 1gamma 11-S73L was statistically significant (p < 0.001).

                              
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Table II
Comparison of EC50 and Vmax of six beta gamma dimers on the activation of two PLC-beta isoforms
Phospholipid vesicles containing phosphatidylinositol 4,5-bisphosphate were reconstituted with different recombinant beta gamma dimers on ice for 30 min. After the incubation, the mixture was applied to a 2-ml Ultrogel AcA34 column at 4 °C and fraction 2 eluting at the void volume used for the measurement of PLC-beta activity. Fraction 2 was incubated for 15 min at 30 °C in the presence of purified recombinant turkey PLC-beta (tPLC-beta ) or human PLC-beta 2 (hPLC-beta 2) as described under "Experimental Procedures." The data are expressed as mean ± S.E. and the average of three determinations, each performed in duplicate. The EC50 (nM) and Vmax (µmol/mg of PLC/min) values were estimated by fitting of the averaged data to sigmoid curves.

Effect of the Prenyl Group on the Activity of Type II Adenylyl Cyclase-- To determine the role of the isoprenoid group in the interaction with an effector which is membrane-associated, the capacity of the six beta gamma dimers to activate type II adenylyl cyclase was also examined. The data in Fig. 6A indicate that switching the prenyl group on the gamma 2 subunit from geranylgeranyl to farnesyl caused a significant decrement in the ability of the beta 1gamma 2 dimer to activate type II adenylyl cyclase. Our previous experiments showed that about the same amount of the beta 1gamma 2 and beta 1gamma 2-L71S (about 25% of the beta gamma subunits added to a reconstitution mixture) are incorporated into Sf9 cell membrane, indicating regardless of the prenyl group on the gamma  subunit, the dimers partition equally into the Sf9 cell membrane (30). Thus, the result from Fig. 6A suggests that the type of prenyl group is important for the interaction of the beta gamma dimer with adenylyl cyclase. As expected, the beta 1gamma 1 dimer did not activate type II adenylyl cyclase (54). Interestingly, the beta 1gamma 11 dimer was completely ineffective on type II adenylyl cyclase. Surprisingly, neither the beta 1gamma 1-S74L nor the beta 1gamma 11-S73L dimer containing a geranylgeranyl group on the gamma  subunit were able to significantly stimulate type II adenylyl cyclase (Fig. 6, B and C). The data in Table III summarize the potency and efficacy of the six beta gamma dimers on the activation of type II adenylyl cyclase. The farnesyl-modified beta 1gamma 2-L71S dimer was about 60-65% less active, and its EC50 value was increased from 14 to 76 nM. This change in potency and maximal activity is larger than that observed with PLC-beta (see Fig. 3). Thus, beta gamma dimers with a gamma  subunit containing a geranylgeranyl group interact with higher affinity with type II adenylyl cyclase than those with a gamma  subunit containing a farnesyl group. While these results are not surprising, the observation that neither the gamma 1 or the gamma 11 subunit modified with a geranylgeranyl group have any activity on type II cyclase was unexpected. These observations suggest that the differences in amino acid sequence between gamma 1 and gamma 2 greatly affect the ability of the dimers to activate adenylyl cyclase.


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Fig. 6.   Comparison of the activity of native and altered beta gamma dimers to stimulate type II adenylyl cyclase. A, Sf9 cells were infected with a recombinant baculovirus encoding the type II adenylyl cyclase, membranes prepared, and the cyclase reaction performed with the indicated concentrations of beta 1gamma 2 (modified with geranylgeranyl; closed squares) and beta 1gamma 2-L71S (modified with farnesyl; open squares) as described under "Experimental Procedures." The difference between the effect of beta 1gamma 2 and beta 1gamma 2-L71S was statistically significant (p < 0.0001). B, the cyclase reaction was performed with the indicated concentrations of beta 1gamma 1 (modified with farnesyl; open circles) and beta 1gamma 1-S74L (modified with geranylgeranyl; closed circles) and compared with the effect of beta 1gamma 2 (dotted lines). The difference between the effect of beta 1gamma 2 and beta 1gamma 1 was statistically significant (p < 0.0001), but the difference between beta 1gamma 1 and beta 1gamma 1-S74L was not statistically significant. C, analogous experiments were performed with beta 1gamma 11 (modified with farnesyl; closed triangles) and beta 1gamma 11-S73L (modified with geranylgeranyl; open triangles) dimers. The difference between the effect of beta 1gamma 2 and beta 1gamma 11 was statistically significant (p < 0.0001), but the difference between beta 1gamma 11 and beta 1gamma 11-S73L was not significant.

                              
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Table III
Comparison of EC50 and Vmax of six beta gamma dimers on the activation of type II adenylyl cyclase
Sf9 cells were infected with a recombinant baculovirus encoding the type II adenylyl cyclase, membranes prepared, and the cyclase reaction performed with the range of beta gamma dimer concentrations described under "Experimental Procedures." The data are expressed as mean ± S.E. and the average of three determinations, each performed in duplicate. The EC50 (nM) and Vmax (nmol/mg of protein/min) values were estimated by fitting of the averaged data to sigmoid curves.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

This study demonstrates that beta gamma dimers with gamma  subunits containing different isoprenoids have distinct abilities to activate PLC-beta and type II adenylyl cyclase. The two major findings of this study are (a) the isoprenoid lipid added to the cysteine residue in the CAAX motif of the gamma  subunit plays a role in determining the activity of the beta gamma dimer at both effectors and (b) beta gamma dimers with a gamma  subunit containing a geranylgeranyl group are more potent and effective in activating either PLC-beta or type II adenylyl cyclase than those with a gamma  subunit containing a farnesyl group. This study also presents the first demonstration of the functional activity of a beta gamma dimer containing the gamma 11 subunit. The beta 1gamma 11 dimer is about six times less potent and about 50% less effective in activating PLC-beta than the beta 1gamma 2 dimer and has little or no effect on the type II adenylyl cyclase. Overall, the data presented here indicate that in addition to the primary amino acid sequence of the gamma  subunits, the prenyl group on the gamma  subunit is an important determinant of the activation of PLC-beta and type II adenylyl cyclase.

The recent x-ray crystal structure of a complex of bovine retinal phosducin with the transducin beta gamma subunit in which the gamma  subunit contained a farnesyl group provides a conceptual background for interpreting the results obtained with PLC-beta and adenylyl cyclase (31). Remarkably, the interaction between phosducin and the beta gamma dimer appears to result in a significant conformational change in the beta  subunit as compared with the conformation seen in the free beta gamma dimer (32). This event also appears to cause the insertion of the prenyl group into a hydrophobic pocket in the beta  subunit formed between blades 6 and 7 (31). Interestingly, this conformational change occurs on the outer strands of blades 6 and 7 of the beta  propeller and recent mutagenesis studies show that these regions are critical for the activation of PLC-beta (57). If the hydrophobic pocket within the beta  subunit that binds the prenyl group is able to discriminate between the different length of the farnesyl and geranylgeranyl lipids, different conformational changes might be induced by the gamma 1 and gamma 2 subunits (or their prenyl mutants). This possibility might explain differences in the ability of the various beta gamma dimers to stimulate the activity of PLC-beta and adenylyl cyclase. Overall, these observations suggest that insertion of a prenyl group into the hydrophobic pocket formed in the beta  subunit may be an important part of the mechanism by which the beta gamma dimers activate effectors when released from the alpha  subunit.

The interaction of protein prenyltransferases with their isoprenoid substrates provides another example of a hydrophobic binding pocket determining isoprenoid specificity. Protein prenyltransferases are responsible for the addition of isoprenoid lipid to proteins and are classified by their lipid substrate: protein farnesyltransferase (FTase), protein geranylgeranyltransferase (GGTase-I), and the Rab geranylgeranyltransferase (GGTase-II) (58). G protein gamma  subunits are targets for either FTase or GGTase-I. These two prenyltransferases are heterodimers composed of the common 48-kDa alpha  subunit and distinct beta  subunits: 46-kDa beta  subunit for FTase (beta F) and 43-kDa beta  subunit for GGTase-I (beta GGI) (59-62). Thus, the binding sites for different lipid substrates on these two prenyltransferases are thought to reside in their beta  subunits (58). The x-ray crystal structure determined by Park et al. (63) illustrates that farnesyl diphosphate occupies a hydrophobic pocket in the center of the beta  subunit barrel. Recently, it has been demonstrated that the isoprenoid moiety of farnesyl diphosphate binds in an extended conformation within the hydrophobic cavity of the beta  subunit of the FTase, and the diphosphate moiety binds to a positively charged cleft at the top of this cavity near the subunit interface (64). These findings suggest that the isoprenoid substrate specificity of the hydrophobic binding cavity is determined by the depth of the cavity and that the pocket acts as a ruler discriminating between isoprenoids of different length (64).

The three classes of PLC isozymes, PLC-beta , PLC-gamma , and PLC-delta , contain two highly conserved catalytic domains, X and Y, but only the PLC-beta isoforms are regulated by G proteins (65). These isoforms are distinguished from the other families by an extended C terminus. Deletion of the C-terminal amino acids of PLC-beta 1 and PLC-beta 2 eliminated Gq alpha  subunit-mediated PLC-beta activation (66-68). Peptides corresponding to a portion of the C-terminal region of PLC-beta 1 inhibited activation by Gq alpha  subunit in vitro (67), suggesting that the C-terminal region might be important for the alpha  subunit-induced PLC-beta activation. Since removal of the C-terminal region of PLC-beta 1 and PLC-beta 2 did not impair beta gamma subunit-induced enzyme activation, the remaining domains of the enzyme must provide the beta gamma subunit interaction sites. A PLC-beta 2 fragment corresponding to a region between the X and Y domains (Glu435 to Val641) inhibited beta gamma subunit-mediated activation of PLC-beta 2 in transfected COS-7 cells (69). Peptides from this sequence expressed as a series of fusion proteins bound to purified beta gamma subunits in vitro (69). Overexpression of this region also blocked beta gamma subunit-induced activation of co-transfected PLC-beta 2 in COS cells (69). It has been reported recently that two overlapping PLC-beta 2 fragments corresponding to Asn564-Lys583 and Glu574-Lys593 of PLC-beta 2 inhibited activation of PLC-beta 2 by beta gamma subunits, inhibited beta gamma subunit-dependent ADP-ribosylation of Gi1 alpha  subunit by pertussis toxin, and inhibited beta gamma subunit-dependent activation of phosphoinositide 3-kinase (70). These observations suggest that the region between Glu574 and Lys583 in PLC-beta 2 may be the site that interacts with beta gamma subunits (70).

A number of studies have attempted to determine the sites at which the beta gamma dimer interacts with adenylyl cyclase. Type I adenylyl cyclase is inhibited by Gi alpha  subunits and beta gamma subunits, whereas type II cyclase is activated by the beta gamma subunits in the presence of Gs alpha  subunits (7, 71). In an attempt to identify the sites in adenylyl cyclase that interact with the beta gamma subunits, a chimeric soluble adenylyl cyclase was engineered by linking the type I adenylyl cyclase C1a domain (the N-terminal part of the first cytoplasmic domain) with the type II adenylyl cyclase C2 domain (the second cytoplasmic domain) (72, 73). The chimeric soluble enzyme was unresponsive to the Gi alpha  subunit but was inhibited almost completely by the beta 1gamma 2 dimer, suggesting that the C1a domain is critical for beta gamma subunit-induced inhibition of type I adenylyl cyclase (72, 73). In contrast, a 27-amino acid peptide (956-982) derived from the C2 domain of type II adenylyl cyclase blocked the interaction of beta gamma dimers with several effectors, including PLC-beta , type II adenylyl cyclase, and K+ channels, but did not block beta gamma interaction with alpha  subunits (74). This result suggests that beta gamma dimers can also interact with a region on the C2 domain of type II adenylyl cyclase (74).

Whereas the G protein beta 1-beta 4 subunits are highly conserved in primary amino acid sequence, the gamma  subunits are much more divergent, suggesting that the functional specificity of different beta gamma dimer combinations may be due to the differences in gamma  subunits. The results presented here support this possibility. Combination of different gamma  subunits with the same beta  subunit resulted in beta gamma dimers exhibiting very different ability to activate PLC-beta and adenylyl cyclase. The differences are due both to the primary amino acid sequences of the gamma  subunits and to their lipid modifications. For example, all beta gamma dimers containing a geranylgeranyl group on the gamma  subunit were more potent and effective in activating the two effectors than those containing a farnesyl group. Thus, the type of lipid at the C terminus of the gamma  subunit is important to the ability of beta gamma dimers to activate effectors. Other groups have also obtained data suggesting that the isoprenoid lipid is important in the interaction of beta gamma dimers with effectors. Matsuda et al. (29) have shown that the beta 1gamma 1-S74L dimer containing a mixture of farnesyl and geranylgeranyl groups had a greater ability to inhibit Ca2+/calmodulin-stimulated adenylyl cyclase and stimulate PLC-beta than did the native farnesylated beta 1gamma 1 dimer. However, our data suggest that the amino acid sequence of the gamma  subunit is also an important determinant of the activity of the beta gamma dimer. For example, neither the beta 1gamma 1 nor the beta 1gamma 11 dimer are able to activate type II adenylyl cyclase. In contrast to the results obtained with PLC-beta , switching the prenyl group in the beta 1gamma 1 and beta 1gamma 11 dimers to geranylgeranyl did not increase their ability to activate type II adenylyl cyclase. Overall, these results indicate that both the primary amino acid sequence and the prenyl group of the gamma  subunits are important determinants for the activation of effectors. For the activation of PLC-beta , the type of prenyl group appears to be a major determinant of the activity of beta gamma dimer. In the case of type II adenylyl cyclase, the prenyl group also appears to be important, but the amino acid sequences of certain gamma  subunits, such as gamma 1 and gamma 11, appear to be incapable of activating the enzyme regardless of prenyl group at their C terminus.

Our previous data supports the possibility that charge differences in the gamma  subunit can have a large effect on the ability of beta gamma dimers to activate type II adenylyl cyclase. Phosphorylation of the gamma 12 subunit in the beta 1gamma 12 dimer significantly inhibits the ability of the dimer to stimulate type II adenylyl cyclase (46), and the phosphorylation site has been determined to be at Ser1 in the N terminus of the molecule (11). This result suggests that introduction of negative charges at the N-terminal region of the gamma  subunit inhibits the interaction of the beta gamma dimer with the type II adenylyl cyclase. Interestingly, comparison of the N-terminal 20 amino acids of the gamma 2 with the gamma 1 or gamma 11 subunits shows that gamma 1 and gamma 11 have six negatively charged amino acids whereas gamma 2 has only one. Thus, the negatively charged N terminus of the gamma 1 and gamma 11 subunits may explain the inability of dimers containing these gamma  subunits to activate type II adenylyl cyclase. Preliminary experiments with chimeric gamma  subunits in which the N-terminal 20 amino acids of gamma 2 were replaced with the corresponding amino acids from gamma 1 support this explanation. When expressed with the beta 1 subunit, this dimer has a greatly reduced ability to stimulate type II adenylyl cyclase.2 Thus, multiple domains of the gamma  subunit may be involved in the interaction with different effectors.

    ACKNOWLEDGEMENTS

We thank Dr. Ravi Iyengar for the baculovirus encoding type II adenylyl cyclase, Dr. Anne Hinderliter for advice about lipid vesicle preparations, the University of Virginia Biomolecular Research Facility for DNA sequencing and mass spectrometric analysis, and the University of Virginia Diabetes Core Facility for the cyclic AMP assays.

    FOOTNOTES

* This work was supported by the National Institutes of Health Grants PO1-CA-40042 and RO1-DK-19952 and by United States Public Health Service Grant GM-29536.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.

§ Present address: Dept. of Medicine, University of Tokyo, Tokyo 112-8688, Japan.

Present address: IDEXX Laboratories, Inc., One IDEXX Drive, Westbrook, ME 04092.

** To whom correspondence should be addressed: Dept. of Pharmacology, Box 448 Health Sciences Center, University of Virginia, Charlottesville, VA 22908. Tel.: 804-924-5618; Fax: 804-982-3878; E-mail: jcg8w{at}virginia.edu.

2 C.-S. Myung and J. C. Garrison, unpublished observation.

    ABBREVIATIONS

The abbreviations used are: G proteins, guanine nucleotide-binding regulatory proteins; Sf9 cells, Spodoptera frugiperda cells (ATCC number CRL 1711); PLC-beta , phosphatidylinositol-specific phospholipase C-beta isoform; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; BSA, bovine serum albumin; Genapol C-100, polyoxyethylene (10) dodecyl ether; PIP2, phosphatidylinositol 4,5-bisphosphate; GTPgamma S, guanosine 5'-3-O-(thio)triphosphate; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; FTase, protein farnesyltransferase; GGTase-I, protein geranylgeranyltransferase; GGTase-II, Rab geranylgeranyltransferase.

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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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