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* This work was supported by National Institutes of Health Grants PO1-CA-40042 and RO1-DK-19952. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ‡ Current address: Cell Migration Consortium, P. O. Box 800902, University of Virginia, 1224 W. Main St., Charlottesville, VA 22908-0902. § Current address: Dept. of Pharmacology, College of Pharmacy, Chungnam National University 220 Geung-dong Yuseong, Daejeon 305-764, Republic of Korea.
The ability of G protein α and βγ subunits to activate the p110γ isoform of phosphatidylinositol 3-kinase (PtdIns 3-kinase) was examined using pure, recombinant G proteins and the p101/p110γ form of PtdIns 3-kinase reconstituted into synthetic lipid vesicles. GTP-activated Gs, Gi, Gq, or Go α subunits were unable to activate PtdIns 3-kinase. Dimers containing Gβ1–4 complexed with γ2-stimulated PtdIns 3-kinase activity about 26-fold with EC50 values ranging from 4 to 7 nm. Gβ5γ2 was not able to stimulate PtdIns 3-kinase despite producing a 10-fold activation of avian phospholipase Cβ. A series of dimers with β subunits containing point mutations in the amino acids that undergo a conformational change upon interaction of βγ with phosducin (β1H311Aγ2, β1R314Aγ2, and β1W332Aγ2) was tested, and only β1W332Aγ2 inhibited the ability of the dimer to stimulate PtdIns 3-kinase. Dimers containing the β1 subunit complexed with a panel of different Gγ subunits displayed variation in their ability to stimulate PtdIns 3-kinase. The β1γ2, β1γ10, β1γ12, and β1γ13 dimers all activated PtdIns 3-kinase about 26-fold with 4–25 nm EC50 values. The β1γ11 dimer, which contains the farnesyl isoprenoid group and is highly expressed in tissues containing the p101/p110γ form of PtdIns 3-kinase, was ineffective. The role of the prenyl group on the γ subunit in determining the activation of PtdIns 3-kinase was examined using γ subunits with altered CAAX boxes directing the addition of farnesyl to the γ2 subunit and geranylgeranyl to the γ1 and γ11 subunits. Replacement of the geranylgeranyl group of the γ2 subunit with farnesyl inhibited the activity of β1γ2 on PtdIns 3-kinase. Conversely, replacement of the farnesyl group on the γ1 and γ11 subunit with geranylgeranyl restored almost full activity. These findings suggest that all β subunits, with the exception of β5, interact equally well with PtdIns 3-kinase. In contrast, the composition of the γ subunit and its prenyl group markedly affects the ability of the βγ dimer to stimulate PtdIns 3-kinase.
The generation of phosphatidylinositol 3,4,5-trisphosphate (PIP3)
). The phosphorylated inositol head group provides a docking site for proteins containing pleckstrin homology domains (PH domains) and leads to activation of many enzymes including the phosphoinositol-dependent protein kinase and protein kinase B (Akt). Activation of protein kinase B regulates multiple cellular functions including differentiation, regulation of metabolic events, cell survival, and motility (
A large family of phosphatidylinositol 4,5-bisphosphate 3-kinases (PtdIns 3-kinases) is responsible for generating PIP3 by phosphorylating the D3 position in the inositol ring of phosphatidylinositol 4,5-bisphosphate. These enzymes are divided into 3 classes, Class 1A and 1B, Class II, and Class III, based on their structure and substrate specificity (
). Class 1A and 1B enzymes represent the major receptor-regulated kinases and are heterodimers composed of a regulatory subunit and a catalytic subunit. The three major isoforms of the Class 1A catalytic subunits have molecular masses of about 110 kDa and are termed α, β, and δ. Although there are multiple spliced isoforms of the p85 regulatory subunit for these p110 isoforms, the full-length molecule has two SH2 domains and a SH3 domain that interact with tyrosine-phosphorylated proteins (
The Class 1B catalytic subunit also has a molecular mass of 110 kDa; its catalytic subunit is termed p110γ. The regulatory subunit for this isoform has a molecular mass of 101 kDa, has no recognizable protein-protein interaction domains, and dimerizes with the p110γ catalytic moiety (
). Thus, ligands working through G protein-coupled receptors regulate the p110γ form of PtdIns 3-kinase and, hence, affect cell survival, reorganization of the cytoskeleton, cell shape changes, and cell migration, which are of central importance to the biology of hematopoietic cells (
). Based on experiments performed with mice in which the p110γ isoform of PtdIns 3-kinase has been genetically ablated, this effector has multiple roles in the function of platelets, neutrophils, macrophages, mast cells, and monocytes (
). For example, neutrophils from p110γ –/–mice show no increase in PIP3 levels upon stimulation of the fMet-Leu-Phe receptor, no activation of protein kinase B, and clear defects in the respiratory burst, cell shape changes, and migration (
). The isoforms of the G protein α and βγ subunits contribute extensively to the specificity of this signaling system. Receptors couple selectively to certain α subunits, and most effectors interact selectively with the different isoforms of the α and βγ subunits (
), we investigated the ability of a complete spectrum of G protein α subunits and βγ dimers to activate the p110γ isoform of PtdIns 3-kinase. Using pure, recombinant proteins reconstituted into synthetic lipid vesicles, we find that the p110γ isoform responds selectively to the different βγ dimers and does not respond to activated α subunits. Moreover, the specific form of the γ subunit in the βγ dimer and the prenyl group on its C terminus play major roles in the activation of PtdIns 3-kinase.
Materials—Reagents used for Sf9 cell culture and purification of α and βγ dimers have been described (
). GDP, imidazole, and HEPES were from Sigma. Chaps and GTPγS were from Roche Applied Science. Genapol C-100 was from Calbiochem. Ni2+-NTA Superflow resin was from Qiagen. [3H]PIP2 was from PerkinElmer Life Sciences. [γ-33P]ATP was from ICN. Source™ 15Q anion exchange resin was from Amersham Biosciences. Centricon 30 concentrators were from Millipore, and Ultra 100 concentrators were from Amicon. Phosphatidylethanolamine (PE), phosphatidylinositol, PIP2, and PIP3 were purchased from Avanti Polar Lipids; TLC plates were from Whatman (60 Å silica gel, 250 μm), Restriction enzymes were from New England Biolabs. Synthetic oligonucleotides were from Qiagen. All other materials were of the highest available purity.
Construction of Recombinant Baculoviruses—The methods for constructing the G protein baculoviruses used in this study have been published as follows: Gsα, Giα, Goα (
). The baculoviruses encoding the “conformational change” mutants (H311A, R314A, W332A) and the “prenyl pocket” mutants (T329K, S331A, W339A, V315A, F335A, K337A) in the β1 subunit were prepared as described (
), and used to construct new recombinant baculoviruses. The cDNA for the p101 subunit was used to prepare a p101 baculovirus with a FLAG epitope on the N terminus using complimentary synthetic oligonucleotides to create the FLAG sequence. The new cDNA was subcloned into the XhoI and EcoRI sites in the Sf9 transfer vector pAcSG2, sequenced, and the recombinant virus was prepared via our standard techniques (
). The cDNA for p110γ was modified to contain a hexahistidine tag on the N terminus using complimentary synthetic oligonucleotides, subcloned into the EcoRI and NotI sites in the Sf9 transfer vector pAcSG2, and sequenced, and the recombinant virus prepared as described (
). Cell culture, viral amplification, and baculovirus infections for protein production were performed in the presence of 1.0% antibiotic/antimycotic (10,000 units/ml penicillin G, 10,000 mg/ml streptomycin, and 25 mg/ml amphotericin B). Sf9 insect cells were infected at a multiplicity of infection of 3 with recombinant baculoviruses expressing G protein α and/or βγ subunits or with viruses for the p101 and the p110γ subunits of PtdIns 3-kinase prepared as described above. The incubations were allowed to proceed for ∼60–80 h or until cell viability fell to 80%, and the cells were harvested by centrifugation at 100 × g. For the purification of G protein α and βγ subunits, the cell pellet was washed three times in insect cell phosphate-buffered saline (7.3 mm NaH2PO4, pH 6.2, 58 mm KCl, 47 mm NaCl, 5.0 mm CaCl2) and resuspended in an ice-cold buffer composed of 25 mm HEPES, pH 7.5, 1 mm MgCl2, 120 mm NaCl, 1 mm 2-mercaptoethanol supplemented with a mixture of freshly prepared protease inhibitors (aprotinin, leupeptin, and pepstatin (at 2 mg/ml), benzamidine at 20 mg/ml, and Pefabloc SC Plus at 100 mg/ml) and frozen. Cell pellets were stored in this buffer at –80 °C before G protein purification (see below). Pellets that were to be used for purification of PtdIns 3-kinase were processed immediately as freezing and thawing cell pellets released proteases active on the p101 subunit (see below).
Purification of G Protein βγ Subunits—The βγ dimers were purified using a Gi1α affinity chromatography procedure that uses a His6-tagged α subunit to select properly modified G protein βγ subunits (
). Sf9 insect cells were co-infected with the His6-Gi1α baculovirus along with the specific β and γ baculoviruses of interest, and the cell membranes were prepared as described and extracted with a buffer containing 10 mm Tris, pH 8.0, 25 mm NaCl, 5 μm GDP, the above protease inhibitors, and 0.1% Genapol (
). Extracts were passed over a Ni2+-NTA column and washed with Ni2+-NTA base buffer (20 mm Tris, pH 8.0, 150 mm NaCl, 10 μm GDP, 1 mm β-mercaptoethanol) containing the above mixture of protease inhibitors, 10 mm imidazole, 0.5% Genapol, and 500 mm NaCl. A subsequent wash with base Ni2+-NTA buffer containing 0.1% Chaps was performed before elution of the βγ dimers with Ni2+-NTA base buffer containing 50 mm MgCl2,10mm NaF, and 30 μm AlCl3. Protein was concentrated using an Amicon Ultra 30 concentrator and exchanged twice using Ni2+-NTA base buffer containing 0.1% Chaps. The β5γ2HF dimer was purified as described (
), the γ subunits in the dimers used in this study were fully and properly modified with the geranylgeranyl (γ2, γ10, γ12, γ13) or farnesyl (γ11, γ1) isoprenoid groups. Moreover, the γ subunits engineered to have altered CAAX sequences (e.g. γ2-L71S) were shown to have the expected change in the prenyl group at the C terminus of the γ subunit (i.e. farnesyl on γ2-L71S) (
Purification of G Protein α Subunits—The method for purification of G protein α subunits is based on the affinity procedure used for the βγ purification. The procedures used for purification of the α subunits of Gi, Gq, Gs, and Go were very similar; our methods for purification of Gs and Gq have been described in detail (
). Sf9 cell membranes containing the Gαβ1HF·γ2HF were prepared, the heterotrimer was extracted from the membranes with 0.2% Genapol, the extracts were applied to a Ni2+-NTA column and washed, and the α subunits were purified by elution from the column with a buffer containing 50 mm MgCl2, 10 mm NaF, and 30 μm AlCl3. These procedures are modifications of those described by Kozasa and Gilman (
). The purified α subunits were concentrated to a volume of 100–200 μl, separated into aliquots, and stored at –80 °C. Each preparation of G protein α subunit used in this study was functional based on its ability to couple to recombinant receptors and βγ subunits when reconstituted into Sf9 cell membranes (
Purification of Phosphatidylinositol 3-Kinase—After infection with the recombinant baculoviruses for p101 and p110γ as described above, Sf9 cells were harvested by centrifugation at 100 × g. The cell pellet was not frozen; rather, it was washed three times in insect cell phosphate-buffered saline, then lysed by nitrogen cavitation at 0 °C in a buffer containing 25 mm Hepes, pH 7.5, 120 mm NaCl, and the standard mixture of protease inhibitors. The extract was centrifuged at 1000 × g for 10 min at 4 °C to remove cell debris, then immediately centrifuged at 100,000 × g for 45 min, and 100 ml of supernatant was loaded onto a 1.75-ml Ni2+-NTA column. The Ni2+-NTA column was washed with 17.5 ml (10 bed volumes) of a wash buffer containing 25 mm Hepes, pH 7.5, 120 mm NaCl with the above protease inhibitors and 1% (v/v) Triton X-100. To remove the Triton from the column it was washed with 17.5 ml of the wash buffer in which the Triton was replaced with 280 mm NaCl. Finally, the column was washed with 17.5 ml of the wash buffer containing 10 mm imidazole. PtdIns 3-kinase was eluted from the column with 10 ml of wash buffer containing 70 mm imidazole, 0.1% (v/v) Tween 20, and 1.0% (w/v) betaine. The protein eluted from the Ni2+-NTA column was immediately loaded onto a 3-ml anti-FLAG-agarose affinity gel column, and the column was washed with 30 ml (10 bed volumes) of 25 mm HEPES, pH 7.5, 3 mm MgCl2, 120 mm NaCl, 1 mm EGTA, 0.1% (v/v) Tween 20, 1.0% (v/v) betaine plus the above protease inhibitors. The column was washed with 30 ml of the above buffer containing 1% (v/v) Triton X-100 then 30 ml of the buffer with 280 mm NaCl. The column was eluted with 10 ml of the above buffer containing 0.5 mg/ml FLAG peptide, and 1-ml fractions were collected. To determine the yield and purity of each preparation, fractions collected from the FLAG column were resolved on an 8% SDS gel and stained with Coomassie Blue (Invitrogen, “Simply Blue”). Fractions containing PtdIns 3-kinase were pooled and concentrated to ∼300 ng/μl using an Amicon Ultra 100 concentrator. A typical preparation yielded about 100 μg of protein. The pure protein was stored at –80 °C in the elution buffer.
Assay of Phosphatidylinositol 4,5-Bisphosphate 3-Kinase—The activity of PtdIns 3-kinase was assayed using G protein α subunits or βγ dimers reconstituted into synthetic phospholipid vesicles. Phospholipid vesicles were prepared at a molar ratio of 4:1 PE to PIP2 (1 mm PE, 250 μm PIP2) in a buffer containing 50 mm Hepes, pH 8.0, 80 mm KCl, 3 mm EGTA, and 1 mm dithiothreitol. The phospholipid mixture was dried under argon to evaporate the chloroform and methanol vehicle, resuspended in 0.5 ml of the above HEPES buffer, and vortexed gently. The suspension was then subjected to 10 freeze/thaw cycles to form large multilamellar vesicles. The large multilamellar vesicles were extruded through a polycarbonate filter (0.1-μm-diameter pore size) to form a uniform preparation of large unilamellar vesicles (
). The extruded large unilamellar vesicles were stored under argon at 4 °C until use.
The activity of PtdIns 3-kinase was measured by the ability to produce PIP3 from PIP2 using [γ-33P]ATP to monitor the extent of the reaction. The purified G protein α or βγ subunits were diluted in 20 mm Hepes, pH 7.5, and reconstituted at final concentrations of 0.1–300 nm into the unilamellar vesicles on ice for 30 min in a buffer consisting of 20 mm Hepes, pH 7.5, 1 mm MgCl2, 150 mm NaCl, and 0.04% Chaps. The final Chaps concentration in the assay was kept below 0.01% because the activity of PtdIns 3-kinase is inhibited by concentrations of Chaps above 0.04%. The assay was started by the addition of 10 ng of PtdIns 3-kinase to a 50-μl reaction mixture containing 20 mm Hepes, pH 7.5, 1 mm dithiothreitol, 50 μm ATP, 3 mm MgCl2, 1 mg/ml bovine serum albumin, and 2.0 μCi of [γ-33P]ATP and incubated for 15 min at 30 °C. During the development of this assay it was found that bovine serum albumin concentrations ranging from 0 to 1.0 mg/ml did not affect the basal or Gβγ-stimulated activity of PtdIns 3-kinase. The rate of PIP3 production was linear for 30 min. The reaction was stopped by the addition of ice-cold 1 n HCl, and the lipids were extracted with CHCl3: MeOH (1:1 v/v). The samples were vortexed and centrifuged at 4 °C at 1500 × g for 2 min, the aqueous phase was aspirated off, and the organic phase was retained. The organic phase was washed with ice-cold 1 n HCl followed by a vortex/centrifugation cycle. Samples of the phospholipids retained in the organic phase were resolved by thin layer chromatography on a potassium oxalate-pretreated silica TLC plate (
). The mobile phase was 65 ml of 1-propanol, 4 ml of glacial acetic acid, and 31 ml of deionized water. After development for 2–3 h, the plate was dried and subjected to autoradiography with Amersham Biosciences Hyperfilm MP. The silica plates were scraped using the autoradiograph as a guide to identify the [33P]PIP3, and the radioactivity was quantified by liquid scintillation counting.
Assay of Phospholipase Cβ—The activity of PLC was measured via the ability to produce inositol trisphosphate from PIP2 incorporated into synthetic lipid vesicles in the presence of pure G protein α or βγ subunits (
), but [inositol-2-3H]PIP2 was included in the vesicles to provide a radioactive substrate for PLCβ. The G protein α or βγ subunits were reconstituted into the vesicles on ice for 30 min in 20 mm Hepes, pH 7.5, 1 mm MgCl2, 150 mm NaCl, and 0.04% Chaps. Before reconstitution, they were diluted in the reconstitution buffer so that their final concentration in the vesicles was 0.1–300 nm. As with the PtdIns 3-kinase assay, the final Chaps concentration was kept below 0.01% in the assay. The assay of PLCβ activity was carried out using 5 ng of pure, recombinant turkey PLCβ as described (
). The [3H]inositol trisphosphate released into the aqueous solution was quantified by liquid scintillation spectrometry.
Activation of G Protein α Subunits—Activation of the Gα subunits before reconstitution into the phospholipid vesicles used in the PLCβ and PtdIns 3-kinase assays was achieved by diluting the various α subunits to a final concentration of 1000 nm in a buffer containing 50 mm Hepes, pH 8.0, 1 mm EGTA, 3 mm EDTA, 5 mm MgCl2, 100 mm NaCl, 1% sodium cholate, 1 mm dithiothreitol, and 1 mm GTPγS. The α subunits were incubated in this solution at 30 °C for 30 min before the addition to the unilamellar phospholipid vesicles. The final concentration of the α subunits reconstituted into the vesicles was 0.1–300 nm. The protocols for PtdIns 3-kinase and PLCβ assays as described above were followed with the exception that a final concentration of 10 mm NaF and 20 μm AlCl3 was added to each incubation at the time the PtdIns 3-kinase or avian PLCβ was added to the vesicles.
Gel Electrophoresis and Immunoblotting—The identity and purity of protein samples were confirmed by gel electrophoresis followed by silver staining and/or by Western blot analysis. Preparations were resolved on 8 or 12% SDS gels, and the gels were stained with Coomassie Blue or silver or transferred to nitrocellulose membranes. Visualization of proteins on Western blots with specific antisera was performed using the ECL chemiluminescence system (Amersham Biosciences).
Calculations, Statistics, and Expression of Results—The experiments presented under “Results” are representative of four or more experiments. The EC50 values presented in Table I were obtained by fitting the points to sigmoidal curves using the routines provided in the Graph Pad Prism software. The statistical significance between the fits of different data sets was determined on normalized data using the F statistic (
Pure, recombinant G proteins, and PtdIns 3-kinase were reconstituted into unilamellar synthetic lipid vesicles to investigate the ability of various G protein α subunits and βγ isoforms to stimulate PtdIns 3-kinase activity. All proteins were expressed in Sf9 insect cells infected with recombinant baculoviruses and purified as described under “Experimental Procedures.” The data in Fig. 1 demonstrate the purity of these proteins and the ability of the βγ dimer to markedly activate PtdIns 3-kinase. Fig. 1A presents a silver-stained polyacrylamide gel resolving typical preparations of Gβ1γ2 and Gβ1γ11, and Fig. 1B demonstrates the purity of the p101/p110γ form of PtdIns 3-kinase. Fig. 1C presents a section of an autoradiograph demonstrating the resolution of the 33P-labeled PIP3 from other components in the vesicle assay used to monitor PtdIns 3-kinase activity. Fig. 1D shows the low background activity in the assay and shows that β1γ2 can activate PtdIns 3-kinase more than 20-fold with an EC50 of ∼5 nm. This result agrees with the literature (
); however, there are differing reports about the effect of G protein α subunits on the activity of this enzyme. The original report describing the cloning of the p110γ isoform reported that the Giα and Gtα subunits purified from bovine brain and retina, respectively, caused a stimulation of PtdIns 3-kinase (
). To examine this issue with pure, recombinant proteins, we prepared four different α subunits from the Gs, Gi, and Gq families by purifying them from a βγ affinity column and examined their ability to activate the enzyme. The α subunits were activated using a 30-min incubation with GTPγS and reconstituted into synthetic lipid vesicles, and the activity of PtdIns 3-kinase activity was measured in the presence of AlF–. Fig. 2A compares the ability of pure, recombinant Gq, Gi1,Go, and Gs α subunits to activate PtdIns 3-kinase with that of β1γ2. None of the α subunits tested activated PtdIns 3-kinase, whereas β1γ2 stimulated the enzyme more than 20-fold. To ensure that the protocols used to activate the α subunits were effective, we examined the ability of an aliquot of the activated Gq to stimulate its effector, PLCβ, in a lipid vesicle assay using avian PLCβ (
). This assay was performed at the same time as the PtdIns 3-kinase assay. Fig. 2B indicates that the activated Gq α subunit can stimulate avian PLCβ activity about 10-fold and equally with the β1γ2 dimer. Activation of Gqα provides a stringent control, as the purified form of this α subunit slowly exchanges GTP and is difficult to activate (
To explore the question of whether the many isoforms of the Gβγ dimer differentially activate PtdIns 3-kinase, we prepared a panel of 10 recombinant βγ dimers. The 7 known β subunits fall into two subfamilies, one comprised of β1–4 and one containing β5 and β5L. The γ subunits separate into 5 subfamilies, one with γ1,8,11, one containing γ2,3,4,9, one comprised of γ7,12, and another containing γ5,10 (
). To minimize the complexity of exploring all 84 possible combinations of the existing β and γ subunits, we chose subunits known to be effective activators of PtdIns 3-kinase such as β1 and γ2 and selected β or γ isoforms from each of the subfamilies to compare with these benchmarks. Thus, dimers were prepared containing the β1–5 subunits, each complexed with γ2 as the partner, and a panel containing different γ subunits was prepared with the β1 subunit.
The effects of these 10 different βγ dimers on PtdIns 3-kinase activity are presented beginning in Fig. 3. In agreement with previous experiments (
), Fig. 3 illustrates that dimers containing the β1–3 subunits complexed with the γ2 subunit all activate PtdIns 3-kinase with EC50 values ranging from 4 to 7 nm. Interestingly, the β4γ2 dimer activates PtdIns 3-kinase activity to a greater extent than the β3γ2 dimer; this difference has also been seen with type II adenyl cyclase (
), the β5γ2HF dimer did not activate PtdIns 3-kinase at concentrations up to 100 nm. In experiments not shown, 300–400 nm β5γ2HF did not activate the enzyme. Importantly, this particular preparation of β5γ2HF was an active molecule, as indicated by its ability to activate PLCβ ∼10-fold (not shown), an activation equivalent to that seen with β1γ2 in Fig. 2B. The EC50 values of the five βγ dimers for activation of PtdIns 3-kinase are presented in Table I. The values range from 4 to 7 nm and are not statistically different from each other.
To investigate the regions of the β subunit that might be important for activating PtdIns 3-kinase, we made two types of mutations in the β1 subunit. These mutations have been termed the conformational change and prenyl pocket mutations (
). Based on the two x-ray structures of β1γ1 complexed with phosducin, three amino acids (His-311, Arg-314, and Trp-332) in the β subunit undergo significant conformational changes when the dimer binds to phosducin (
). Two Gβ1 subunits containing point mutations in which these amino acids were changed to alanine (H311A and W332A) bind phosducin and phosducin-like-protein less well than the native β1γ2 or β1γ1 dimers (
). This result and the fact that the dimers are purified from a Gα subunit column suggest that the β subunits with these point mutations are properly folded.
Dimers containing β1 subunits harboring these three mutations were purified in a complex with the γ2 subunit and examined for their ability to activate PtdIns 3-kinase. Fig. 4 illustrates that β subunits containing the H311A and R314A mutations have similar activity as native β1γ2 and that β subunits containing the Trp-332 mutation are less potent. The EC50 value for the Trp-332 mutation is shifted to the right about 4-fold, to 12 nm (Table I). Interestingly, dimers with β subunits harboring the Trp-332 mutation are uniformly less active in assays of multiple effectors including PLCβ, type II adenyl cyclase, K+ channels, Ca2+ channels, and binding to phosducin (
Representative βγ dimers containing members of the 5 families of γ subunits had much larger differences in their activity on PtdIns 3-kinase. The data in Fig. 5 indicate that dimers containing the γ2, γ12, and γ13 subunits were about equally effective in activating PtdIns 3-kinase with EC50 values ranging from 4 to 7 nm. Dimers containing the γ10 subunit were statistically less effective, and dimers containing γ11 did not effectively activate the enzyme. The EC50 values for all combinations tested are shown in Table I. Note that the weak activation of PtdIns 3-kinase caused by β1γ11 had an EC50 value of about 12 nm, suggesting that it bound to PtdIns 3-kinase with reasonable affinity but could not cause the conformational change needed for increased activity.
In addition to differences in amino acid sequence, a major difference between γ11 and the other γ subunits used in these experiments is that γ11 contains a C15 farnesyl group on its C terminus (
). For this reason we tested the role of the prenyl group in the activation of PtdIns 3-kinase by using γ subunits that contained altered CAAX boxes to direct the addition of a C15 farnesyl group to γ2 (γ2-L71S) or a C20 geranylgeranyl group to γ11 (γ11-S74L) or γ1 (γ1-S74L). Thus, the amino acid sequence of the γ subunit is not altered. Dimers containing γ subunits with these mutations have been tested for their activity on PLCβ and type II adenyl cyclase with the result that dimers containing the C15 farnesyl group are less active than those containing the geranylgeranyl group (
The data presented in Fig. 6A indicate that switching the prenyl group on γ2 from geranylgeranyl to farnesyl shifts the activation of PtdIns 3-kinase about 1 log to the right (EC50 from 5.6 to 35 nm, see Table I). As expected (
), Fig. 6B indicates that the β1γ1 dimer, like the similar β1γ11 dimer, poorly activates PtdIns 3-kinase. More importantly, switching the farnesyl group on γ11 (or γ1) to the geranylgeranyl group produces dimers that are nearly as active as the native β1γ2. Note from Fig. 6B that β1γ11-S74L and β1γ1-S74L can activate PtdIns 3-kinase to about the same extent as β1γ2. However, they are still less potent at activating the enzyme (EC50 values of about 53 nm). The requirement of the geranylgeranyl group for proper activation of PtdIns 3-kinase is very similar to the near absolute requirement for the geranylgeranyl group in the stimulation of type II adenyl cyclase by βγ dimers (
). Mutations of the residues in this area designed to destabilize the binding of the prenyl group in its pocket (F335A, K337A, V315A, S331A, W339A, and T329K in Gβ1) reduce the ability of dimers to activate type II adenyl cyclase and PLCβ (
). Accordingly, β1γ2 dimers containing mutations in this area of the β subunit were tested for their ability to activate PtdIns 3-kinase. The data in Fig. 7 indicate that the effects of these prenyl pocket mutations fall into two groups. Dimers containing β1T329Kγ2 and β1S331Aγ2 and β1W339Aγ2 activate PtdIns 3-kinase with EC50 values equal to that observed with the β1γ2 dimer. Dimers containing the F335A, K337A, and V315A point mutations shift the EC50 for activation of PtdIns 3-kinase about 4–5-fold to the right (see Table I). A slightly larger rightward shift in EC50 was observed in the ability of dimers harboring these mutations to activate type II adenyl cyclase and PLCβ (
). The observation that they cause a right shift in the ability of the dimer to activate PtdIns 3-kinase suggests that the prenyl group also plays an important role in the interaction of the γ subunit with this enzyme.
The family of heterotrimeric G proteins is composed of α, β, and γ subunits with many isoforms of each subunit expressed in most cells. The Gα subunits are expressed from 17 different genes and, through the splice variants that exist in cells, form 22 different α subunits. These subunits separate into four major families (
). The β and γ subunits form a dimer that is an integral and important part of the G protein-signaling mechanism. The βγ dimer is required for the receptor to form a high affinity complex with agonists (
). The β subunits are expressed from 5 genes (with 2 splice variants, β3S and β5L), and the γ subunits are expressed from 12 genes. The β subunits separate into 2 families, one containing β1–4, which are about 90% identical, and one containing β5 and β5L, which are only 50% similar to β1 (
). The γ subunits can be divided into 5 subfamilies, one subfamily consists of γ subunits that are modified with farnesyl (C15) at their C terminus (γ subunits 1,8, and 11) and three other subfamilies containing geranylgeranyl (C20) at their C terminus. The second subfamily is comprised of γ subunits 2,3,4,9, whereas the third and fourth subfamilies only consist of two members each, γ subunits 7 and 12, and γ subunits 5 and 10, respectively. Gγ13 is the most divergent γ and forms its own subfamily (
). The large number of targets for the βγ dimer, and the possibility that the many different isoforms of the dimer represent selective signals make it important to determine the signaling specificity inherent in these molecules. The generation of PIP3 in the membrane of hematopoietic cells such as neutrophils, mast cells, and macrophages is markedly increased by Gi-linked receptors such as the fMet-Leu-Phe or C5a receptors. Activation of these receptors releases the βγ dimer (
). Thus, the primary goal of this study was to understand the activation of the p101/p110γ form of PtdIns 3-kinase by the multiple different isoforms of the βγ dimer. The experiments were performed with pure, recombinant proteins to ensure that direct effects of the βγ dimer were measured.
The results demonstrate four major points. First, GTP-activated Gs,Gi,Gq,orGo α subunits were not able to stimulate the p101/p110γ form of PtdIns 3-kinase. These experiments clarify earlier conflicting results on this point in the literature (
). As each α subunit was purified from a column of immobilized βγ dimers after activation with AlCl3 and NaF, which induces a conformational change, the proteins are properly folded, active molecules. Moreover, each preparation of α subunit coupled to the appropriate recombinant receptors (
). These experiments offer clear evidence that the p101/p110γ form of PtdIns 3-kinase falls into the group of G protein effectors that are regulated exclusively by the βγ dimers. Ion channels and the Rac guanine nucleotide exchanger, P-Rex1, are other examples (
). This finding is also consistent with observations that the βγ dimer is a major regulatory molecule released by activation of Gi-coupled receptors such as the fMet-Leu-Phe or C5a receptors in cells expressing the p101/p110γ form of PtdIns 3-kinase such as neutrophils, platelets, mast cells, and macrophages (
), make it difficult to speculate about the meaning of this finding. Third, the isoform of the γ subunit in the dimer had a major effect on its ability to activate PtdIns 3-kinase. Clearly, dimers containing representatives of the γ2, γ10, γ12, and γ13 families all activate PtdIns 3-kinase well. However, dimers containing γ11 are not effective. Finally, the prenyl group on the γ subunit was found to be very important for the activation of PtdIns 3-kinase. Gγ subunits modified with the farnesyl group do not activate the enzyme well. Previous studies have shown that dimers containing γ subunits modified with the farnesyl group are ineffective in activating type II adenyl cyclase (
). However, the present results demonstrate that it is not an effective activator of a major βγ target in these cells, PtdIns 3-kinase. Moreover, the Giα subunit is not able to activate the enzyme (Fig. 2B), yet Gi-linked receptors such as the fMet-Leu-Phe and C5a receptors markedly activate the p101/p110γ form of PtdIns 3-kinase in mast cells, neutrophils, and macrophages (
). Thus, it seems highly unlikely that dimers containing the γ11 subunit are released after activation of fMet-Leu-Phe or C5a receptors. Activation of these receptors must release a dimer such as β1γ2, which is able to activate PtdIns 3-kinase 20–40-fold. Interestingly, dimers containing γ11 are not very effective at activating type II adenyl cyclase (
), these observations suggest that cells containing γ11 could use dimers containing γ11 to couple α subunits to receptors, allowing receptors to activate α subunits without inducing effects on downstream βγ targets. Taken further, this argues that receptor activation may release specific βγ dimers, a concept that gains experimental support from biochemical experiments (
). Thus, in hematopoietic cells, receptors coupled to Gi provide stimulatory inputs, and receptors coupled to Gs provide negative inputs into a major biological process. However, activation of Gs must also release some isoform of the βγ dimer, posing a potential question, Why would an inhibitory pathway release a signaling molecule (the βγ dimer) that can markedly activate the very pathway to be inhibited? Thus, it seems logical that the βγ dimer released by activation of Gs-coupled receptors would not be able to stimulate PtdIns 3-kinase.
There are many possible reasons that βγ dimers released from Gs may not be able to stimulate PtdIns 3-kinase. The dimer might be compartmentalized (
). Another interesting possibility is that the dimer which couples to Gsα in these cells may contain β or γ subunits that do not interact well with the effectors. A significant amount of data suggests that distinct βγ isoforms can selectively regulate effectors (
Another body of literature indicates that specific βγ dimers may be released after activation of specific receptors. Examples include the original antisense experiments performed in GH3 cells, suggesting that specific βγ isoforms couple to muscarinic and somatostatin receptors (
), and results using small interfering RNA to knock down the β2 subunit in the J774.A1 macrophage line, showing loss of this subunit blunts the ability of the C5a receptor to stimulate a rise in intracellular Ca2+ (
). Taken together these results suggest the possibility that activation of specific receptors may release specific βγ dimers. Based on our results, if the dimer released by receptor activation is β1γ11, it is not likely to activate effectors. This hypothesis suggests that receptor activation may not always release a βγ dimer that acts on downstream signaling pathways. This possibility would restrict receptor activation of effectors to those able to interact with that α subunit coupled to the receptor and provides interesting avenues for future experiments in intact cells.
We thank Dr. L. Stephens for the gift of the cDNAs encoding the p101 and p110γ subunits of PtdIns 3-kinase, Dr. T. K. Harden for the gift of purified avian PLCβ, and Dr. D. Siderovski for the gift of the γ13 baculovirus. We are very grateful to Melinda Ring and Qi Wang for excellent assistance in culturing Sf9 insect cells and to Dr. P. Ongusaha for initial experiments showing the feasibility of activating PtdIns 3-kinase with Gβγ.