Heterodimeric Phosphoinositide 3-Kinase Consisting of p85 and p110β Is Synergistically Activated by the βγ Subunits of G Proteins and Phosphotyrosyl Peptide*

Phosphoinositide 3-kinase (PI 3-kinase) is a key signaling enzyme implicated in variety of receptor-stimulated cell responses. Receptors with intrinsic or associated tyrosine kinase activity recruit heterodimeric PI 3-kinases consisting of a 110-kDa catalytic subunit (p110) and an 85-kDa regulatory subunit (p85). We separated a PI 3-kinase that could be stimulated by the βγ subunits of G protein (Gβγ) from rat liver. The Gβγ-sensitive PI 3-kinase appeared to be a heterodimer consisting of p110β and p85 (or their related subunits). The stimulation by Gβγ was inhibited by the GDP-bound α subunit of the inhibitory GTP-binding protein. Moreover, the stimulatory action of Gβγ was markedly enhanced by the simultaneous addition of a phosphotyrosyl peptide synthesized according to the amino acid sequence of the insulin receptor substrate-1. Such enzymic properties could be observed with a recombinant p110β/p85α expressed in COS-7 cells with their cDNAs. In contrast, another heterodimeric PI 3-kinase consisting of p110α and p85 in the same rat liver, together with a recombinant p110α/p85α, was not activated by Gβγ, although their activities were stimulated by the phosphotyrosyl peptide. These results indicate that p110β/p85 PI 3-kinase may be regulated in a cooperative manner by two different types of membrane receptors, one possessing tyrosine kinase activity and the other activating GTP-binding proteins.


Phosphoinositide 3-kinase (PI 3-kinase) is a key signaling enzyme implicated in variety of receptor-stimulated cell responses.
Receptors with intrinsic or associated tyrosine kinase activity recruit heterodimeric PI 3-kinases consisting of a 110-kDa catalytic subunit (p110) and an 85-kDa regulatory subunit (p85). We separated a PI 3-kinase that could be stimulated by the ␤␥ subunits of G protein (G␤␥) from rat liver. The G␤␥sensitive PI 3-kinase appeared to be a heterodimer consisting of p110␤ and p85 (or their related subunits). The stimulation by G␤␥ was inhibited by the GDP-bound ␣ subunit of the inhibitory GTP-binding protein. Moreover, the stimulatory action of G␤␥ was markedly enhanced by the simultaneous addition of a phosphotyrosyl peptide synthesized according to the amino acid sequence of the insulin receptor substrate-1. Such enzymic properties could be observed with a recombinant p110␤/p85␣ expressed in COS-7 cells with their cDNAs. In contrast, another heterodimeric PI 3-kinase consisting of p110␣ and p85 in the same rat liver, together with a recombinant p110␣/p85␣, was not activated by G␤␥, although their activities were stimulated by the phosphotyrosyl peptide. These results indicate that p110␤/ p85 PI 3-kinase may be regulated in a cooperative manner by two different types of membrane receptors, one possessing tyrosine kinase activity and the other activating GTP-binding proteins.
Phosphoinositide 3-kinase (PI 3-kinase) 1 is a key signaling enzyme implicated in the regulation of a broad array of biological responses including receptor-stimulated mitogenesis, oxi-dative burst, membrane ruffling, and glucose uptake (1,2). The activation of PI 3-kinase results in an increase in cellular levels of D-3 phosphorylated phosphoinositides, such as PtdIns(3)P, PtdIns(3,4)P 2 , and PtdIns(3,4,5)P 3 . These products, however, do not serve as the substrates of phospholipase C (3) and thus have been proposed to act as second messengers. In this regard, recent studies have indicated that PtdIns(3,4)P 2 can directly activate certain protein kinase C and Akt (4,5) and PtdIns(3,4,5)P 3 is capable of binding to the Pleckstrin homology domain of guanine nucleotide exchange factor of the small GTP-binding protein ARF1 (6 -8).
At least two types of PI 3-kinase, in terms of mode of the activation, have been described in mammalian cells (2). One is stimulated by membrane-bound receptors activating tyrosine kinase, whereas the other is under the direct control of the heterotrimeric GTP-binding proteins. The well known former type has been structurally characterized as a heterodimer consisting of a 110-kDa catalytic subunit (p110) and an 85-kDa regulatory subunit (p85); the regulatory subunit contains one SH3 and two SH2 domains. Stimulation of tyrosine kinase receptors by extracellular signals phosphorylates specific tyrosine residues located in the YMXM motifs of their own receptors or adaptor molecules, such as insulin receptor substrate-1. These phosphorylated proteins bind to the SH2 domains of p85 and stimulate the lipid kinase activity. The stimulatory effect of these proteins can thus be mimicked in vitro by a synthetic tyrosine-phosphorylated peptide possessing the YMXM motif (9,10). Although several subtypes of p85 and p110 (␣, ␤, and ␦) have been identified (11)(12)(13), differences in their functions have not been well described.
In addition to the tyrosine phosphorylation-dependent activation of PI 3-kinase, it has been reported that the ␤␥ subunits of G proteins also stimulate the lipid kinase activity (14 -18). One report showed that a partially purified PI 3-kinase, which was immunochemically distinct from p110␣ and not associated with p85, is activated by G␤␥ (19). Thus, a novel catalytic subunit of PI 3-kinase, designated as p110␥, has been cloned and shown to be activated in vitro by both the ␣ and ␤␥ subunits of G proteins (14). Receptor-induced translocation of p110␥ to a cytoskeletal fraction has also been reported (20). This isozyme lacks the binding site to p85 and thus does not interact with the regulatory subunit. Quite recently, Stephens et al. (15) reported a heterodimeric PI 3-kinase consisting of a p110␥-related 120-kDa (or 117-kDa) catalytic subunit and a 101-kDa adaptor protein, which is distinct from p85. PI 3-kinase activity of the novel heterodimer was markedly stimulated by G␤␥ but not by a tyrosine-phosphorylated peptide.
In contrast to these reports, two groups including us showed * This work was supported in part by research grants from the Scientific Research Fund of the Ministry of Education, Science, Sports, and Culture of Japan and by Grant JSPS-RFTF 96L00505 from the "Research for the Future" Program of the Japan Society for the Promotion of Science. 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.

EXPERIMENTAL PROCEDURES
Materials-[␥-32 P]ATP, 125 I-labeled protein A, and 125 I-labeled protein G were purchased from NEN Life Science Products. Protein A-Sepharose and anti-mouse IgG-agarose resin were form Pharmacia Biotech Inc. and American Qualex, respectively. Tyrosine-phosphorylated and nonphosphorylated peptides synthesized according to the sequence of insulin receptor substrate-1, NGDYMPMSPKS, were obtained from Kurabou Co. (Osaka, Japan). A monoclonal antibody against the N-terminal SH2 domain of p85␣ ([N]SH2) and a polyclonal antibody against the whole p85 (p85 PAN ) were purchased from Seikagaku Corp. (Tokyo, Japan). Polyclonal antibodies against p110 (␣ and ␤) were purchased from Santa Cruz Biotechnology, Inc. All other reagents were from commercial sources and of analytical grade.
DNA Constructs-The bovine p85␣ cDNA was subcloned into pc-DLSR␣296 eukaryotic expression vectors for transient transfection (21). The bovine p110␣ cDNA was subcloned between the HindIII and SalI sites of the pCMV5. The pCMV5 plasmid was a gift from D. W. Russell, University of Texas Southwestern Medical Center. The human p110␤ cDNA was obtained by reverse transcriptase polymerase chain reaction with mRNA of Jurkat cells that had been treated with dibutyryl cAMP and ligated between the HindIII and SalI sites of the pCMV5. To modify the C-terminal end of p110 (␣ and ␤), the fragments in which the p110 (␣ and ␤) coding region were extended by sequences encoding amino acids ELG as a hinge region, which precedes the 11amino acid Myc epitope (EQKLISEEDLN), followed by a stop codon. The sequences were confirmed by DNA sequence analysis.
Cell Culture and Transfection-COS-7 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 IU/ml penicillin, 100 g/ml streptomycin, and 0.6 mg/ml glutamine at 37°C in a humidified 5% CO 2 atmosphere. Transfections were performed on 40 -50% confluent monolayers in 100-mm dishes for immunoprecipitation. For transient transfection, the cells were incubated at 37°C in 5 ml of serum-free Dulbecco's modified Eagle's medium containing 40 l of Lipofectin reagent (Life Technologies, Inc.) per 100-mm dish plus 1 g of pCMV5 containing the cDNA encoding Myc tag-conjugated p110 (␣ or ␤) with 5 g of pcDLSR␣296 containing the cDNA encoding p85␣ per 100-mm dish. After 12 h of exposure to the transfection medium, monolayers were replaced with growth medium and incubated for 48 h.
Immunoprecipitation-The transfected COS-7 cells were washed twice with ice-cold phosphate-buffered saline, lysed in 400 l of a lysis buffer consisting of 20 mM Na-HEPES (pH 7.4), 75 mM NaCl, 15 mM NaF, 1 mM Na 3 VO 4 , 10 mM Na 4 P 2 O 7 , 2 mM EDTA, 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, and 2 g/ml aprotinin. The cell extracts, after being precleared with Sepharose 4B resin, were incubated with 9E10 (1 g) and anti-mouse IgG-agarose resin. After the incubation, the beads were washed three times with the lysis buffer and then three more times with 20 mM Tris-HCl (pH 8.0) and 150 mM NaCl. PI 3-kinase activity in the immunocomplex was determined as described below. In some cases, proteins in the immune complex were separated by SDS-PAGE and probed with the antibody against p85, p110␤, or epitope tag.
Assay of PI 3-Kinase Activity-PI 3-kinase activity was assayed with 0.1 mM PtdIns(4,5)P 2 as described previously (17). When the effects of GTP-binding proteins were examined, GDP-bound G i ␣ and/or G␤␥ subunits were mixed and incubated on ice for 5 min before addition of the enzyme preparation.
Purification of PI 3-Kinases from Rat Liver-G␤␥-sensitive PI 3-kinase was purified from rat liver according to a purification protocol of Carpenter et al. (22) with modifications. Rat livers were homogenized in 2.5 volumes of a buffer consisting of 20 mM Tris-HCl (pH 7.4), 2 mM MgCl 2 , 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 10 g/ml leupeptin, 10 g/ml aprotinin, and 2.5 mM phenylmethylsulfonyl fluoride. The homogenate was centrifuged at 10,000 ϫ g for 10 min, and the supernatant was further centrifuged at 150,000 ϫ g for 1 h. The resultant supernatant was diluted with an equal volume of 20 mM Tris-HCl (pH 7.4), 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 g/ml leupeptin, 0.5 g/ml aprotinin, 0.5 mM phenylmethylsulfonyl fluoride, and 10% glycerol (buffer A) and loaded to a 250-ml column of DEAE-Sepharose FF, which had been equilibrated with buffer A. The column was washed with 1,250 ml of buffer A and eluted with a linear gradient of 0 -400 mM KCl in buffer A over 1,000 ml. Fractions containing G␤␥-sensitive PI 3-kinase, which had been eluted with 150 mM KCl, were pooled, concentrated to 20 ml using a Minimodule (Asahikasei Co.), and diluted with 4 volumes of 50 mM MES-NaOH (pH 6.7), 0.5 mM EDTA, 0.5 mM EGTA, 1 mM DTT, 1 g/ml leupeptin, 0.5 g/ml aprotinin, 0.5 mM phenylmethylsulfonyl fluoride, and 10% glycerol (buffer B). The mixture was then loaded on a 50-ml column of CM-Sepharose FF equilibrated with buffer B containing 50 mM KCl. The column was washed with 150 ml of buffer B containing 50 mM KCl and eluted with a linear gradient of 50 -400 mM KCl in buffer B over 200 ml. Fractions containing the activity, which had been eluted with 190 mM KCl, were pooled and concentrated to 7.5 ml using the Minimodule. The concentrate was loaded to a 300-ml column of Sephacryl S-300HR, which had been equilibrated with 20 mM Tris-HCl (pH 7.4), 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 g/ml leupeptin, 0.5 g/ml aprotinin, 0.5 mM phenylmethylsulfonyl fluoride, and 150 mM KCl (buffer C). The PI 3-kinase was eluted at a flow rate of 1.25 ml/min, and the fractions containing the activity were concentrated to 2 ml using a Centriprep-30 (Amicon). The concentrate was diluted with 10 volumes of buffer B and loaded on a 5-ml column of blue Sepharose CL-6B equilibrated with buffer B containing 25 mM NaCl. The column was washed with 50 ml of buffer B containing 25 mM NaCl and eluted with a linear gradient of 25-1000 mM NaCl in buffer B over 25 ml. The PI 3-kinase eluted with 400 mM NaCl was concentrated to 1 ml using a Centricon-30 (amicon) and diluted with 10 volumes of 20 mM Tris-HCl (pH 8.0), 0.5 mM EDTA, 0.5 mM EGTA, 1 mM DTT, and 10% glycerol (buffer D). The mixture was loaded on a 1-ml column of Mono Q HR5/5, which had been equilibrated with buffer D containing 50 mM KCl. The column was washed with 10 ml of buffer D containing 50 mM KCl and eluted with a linear gradient of 50 -300 mM KCl in buffer D over 25 ml. The PI 3-kinase eluted with 170 mM KCl was pooled and concentrated to 0.2 ml using the Centricon-30. The sample was supplemented with 0.1% (w/v) bovine serum albumin before being aliquoted, frozen in liquid N 2 , and stored at Ϫ80°C.
A heterodimeric PI 3-kinase consisting of p110␣/p85 was also separated from rat liver. The p110␣/p85 PI 3-kinase was eluted with 160 mM KCl from the first DEAE-Sepharose column and further separated by the column chromatography of CM-Sepharose FF and Sephacryl S-300HR under the same conditions as the G␤␥-sensitive PI 3-kinase.
Preparation of GTP-binding Protein Subunits-The ␤␥ subunits of GTP-binding proteins were purified from rat brain and stored at a concentration of 20 M in 20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.1 mM EDTA, and 0.5% CHAPS (23). Rat recombinant G␣ i-2 was purified from Escherichia coli and stored at a concentration of 20 M in 50 mM Na-HEPES (pH 7.4), 1 mM EDTA, and 1 mM dithiothreitol (24).

RESULTS
When rat liver cytoplasmic proteins were applied to a DEAE-Sepharose column and eluted with a linear gradient of KCl, there were several peaks of fractions containing PI 3-kinase activity (17). In the previous paper (17), we have identified that a 100-kDa/46-kDa heterodimeric form of PI-3 kinase, which was not tightly retained on the column, was activated by G␤␥. In addition to this isoform, we found that PI 3-kinase, which had been eluted from the column with approximately 150 mM KCl, was also stimulated by G␤␥ upon assay with PtdIns(4,5)P 2 as a substrate. The G␤␥-sensitive PI 3-kinase was further purified by means of sequential chromatography with CM-Sepharose, Sephacryl S-300/HR, blue Sepharose, and Mono Q columns. Fig. 1 shows the PI 3-kinase activity eluted form the final Mono Q column. The kinase activity eluted with about 170 mM KCl (fractions 15-18) was markedly stimulated by G␤␥ (closed circles), although the basal activity assayed FIG. 3. Comparison between the G␤␥-sensitive (p110␤/p85) and p110␣/p85 PI 3-kinases of rat liver. The G␤␥-sensitive PI 3-kinase (p110␤/p85) in the Mono Q fraction and p110␣/p85-rich fraction separated from the same rat liver were subjected to immunoprecipitation with polyclonal antibody against p110␤ and p110␣, respectively. A, after resolution of the immunoprecipitated proteins by SDS-PAGE (8% acrylamide), the proteins were transferred and immunoblotted (IB) with anti-p110␤ (left), p110␣ (center), or p85 PAN (right) antibody. IP, immunoprecipitate. B, the samples immunoprecipitated with the anti-p110␤ (left) or p110␣ (right) antibody were assayed for the PI 3-kinase activity as the substrate of PtdIns(4,5)P 2 . The assay was performed in the presence or absence of the phosphotyrosyl peptide (pY-pep., 100 M), G␤␥ (0.5 M), and/or wortmannin (0.1 M). Bars represent differences between duplicate determinations in a typical study.
FIG. 4. Effects of G␤␥ and the phosphotyrosyl peptide on recombinant p110␤/p85␣ and p110␣/p85␣ PI 3-kinases. The epitopetagged p110 (␤ or ␣) and p85␣ were co-expressed in COS-7 cells and subjected to immunoprecipitation with a monoclonal antibody (9E10) against Myc tag. The immunoprecipitated samples were assayed for the PI 3-kinase activity in the absence or presence of the phosphotyrosyl peptide (pY-pep., 100 M) and/or G␤␥ (0.5 M). Bars represent differences between duplicate determinations in a typical study. PIP 3 , phosphatidylinositol 3,4,5-triphosphate; MT, Myc epitope-tagged. without G␤␥ was quite low (open circles). The G␤␥-sensitive PI 3-kinase fractions contained 110-and 85-kDa proteins, which were recognized with anti-p110␤ and p85 antibodies, respectively (Fig. 1, middle and bottom panels of the inset), and the degree of the G␤␥-stimulated activity was correlated with both protein amounts. There was, however, no protein band immunoreacted with an anti-p110␣ specific antibody in the Mono Q fractions (Fig. 1, top panel of the inset).
We next investigated whether a signal associated with protein tyrosine phosphorylation may also regulate the kinase activity. A tyrosine-phosphorylated peptide, NGDY* MPM-SPKS (Y* indicates phosphotyrosine), derived from the sequence of insulin receptor substrate-1 was used for the present kinase assay. In the chromatography on Mono Q column, the phosphotyrosyl peptide produced only a slight increase in the PI 3-kinase (data not shown, but see Figs. 2C and 3B below). However, the combination of G␤␥ with the phosphotyrosyl peptide caused marked activation of the PI 3-kinase (Fig. 1, closed  triangles). Fig. 2 shows properties of the Mono Q PI 3-kinase, which was assayed with the various concentrations of G␤␥ and the phosphotyrosyl peptide. G␤␥ stimulated the kinase activity in a concentration-dependent manner, regardless of whether the phosphotyrosyl peptide was included in the assay mixture; the half-maximal activation was observed with about 0.5 M G␤␥ ( Fig. 2A). To confirm that the free G␤␥ subunits were responsible for the kinase activation, we examined the effect of GDPbound G␣ i-2 on the G␤␥-sensitive PI 3-kinase (Fig. 2B). As expected, the increasing concentrations of the ␣ subunit induced the progressive inhibition of PI 3-kinase activities stimulated by G␤␥ alone and G␤␥ plus phosphotyrosyl peptide. Fig.  2C shows the effect of the various concentrations of the phosphotyrosyl peptide, together with a control tyrosine-nonphosphorylated peptide, NGDYMPMSPKS. The phosphotyrosyl peptide further enhanced the G␤␥-sensitive PI 3-kinase activity, although the control peptide had no stimulatory effect on the kinase activities assayed in the presence and absence of G␤␥.
The active fractions of the Mono Q column (Fig. 1) were next treated with the specific antibody against the p110␤. The peptides in the immunoprecipitate were separated by SDS-PAGE and then analyzed by immunoblotting with the anti-p85 antibody. As expected, an 85-kDa peptide can be detected in the p110␤ immunoprecipitate. The PI 3-kinase activity in the immunoprecipitate was again found to be stimulated by G␤␥ (Fig.  3B, left panel). The further addition of the phosphopeptide to the assay mixture increased the G␤␥-stimulated activity in the immunocomplex (Fig. 3B, left panel), as is the case in the Mono Q fraction (Fig. 1). The activity in the immunocomplex was completely inhibited by the presence of PI 3-kinase inhibitors, wortmannin (Fig. 3B) (25) or LY294002 (data not shown) (26). Thus, the Mono Q PI 3-kinase activated in a cooperative manner by G␤␥ and the phosphotyrosyl peptide appeared to be a heterodimer consisting of p110␤ and p85 (or their related subunits). This notion is in agreement with the findings that the antibody against the N-terminal SH2 domain of p85 ([N]SH2) recognized the 85-kDa polypeptide of the Mono Q fraction and that the G␤␥-sensitive PI 3-kinase migrated as an apparent molecular mass of 220,000 on a gel filtration column (data not shown).
For a comparison, another heterodimeric PI 3-kinase consisting of p110␣ and p85 was also separated from the same rat liver. The p110␣-subtype specificity in the separated fraction was confirmed by immunoblot analysis (Fig. 3A, middle and right panels). As shown in the left panel of Fig. 3A, there was no protein band cross-reacted with anti-p110␤ antibody in the p110␣/p85 PI 3-kinase fraction. In contrast to the Mono Q fraction containing p110␤/p85, G␤␥ had no stimulatory effect on the PI 3-kinase activity of the p110␣/p85 PI 3-kinase fraction, although its activity was certainly stimulated by the phosphotyrosyl peptide (Fig. 3B, right panel). As expected, the p110␣/p85 PI 3-kinase activity was also inhibited by wortmannin. The phosphotyrosyl peptide alone increased the activity of both p110␤/p85 and p110␣/p85 only slightly, but this effect can be observed reproducibly and significantly.
To confirm that p110␤/p85 PI 3-kinase certainly functions as the target isoform of the synergistic activation by G␤␥ and the phosphotyrosyl peptide, we expressed Myc-tagged p110␤/p85␣ and p110␣/p85␣ in COS-7 cells with their cDNAs. As shown in the left panel of Fig. 4, PI 3-kinase activity of the recombinant p110␤/p85␣ was stimulated by both G␤␥ and the phosphotyrosyl peptide and synergistically by the presence of the two activators. The activity of p110␣/p85␣ was increased by the phosphotyrosyl peptide but was unaffected by G␤␥ regardless of the presence or absence of the phosphotyrosyl peptide (Fig. 4,  right panel). These results suggest that the G␤␥ sensitivity of the heterodimer was carried by p110␤. This notion is supported by a finding that a PI 3-kinase consisting of an undefined 46-kDa peptide and p110␤ is also stimulated by G␤␥ (Ref. 17). 2

DISCUSSION
In the present study, we have separated the target PI 3-kinase of G␤␥-induced stimulation from rat liver cytosol. Immunological analysis (Figs. 1 and 3) suggested that the rat liver G␤␥-sensitive PI 3-kinase is a heterodimer consisting of p110␤ and p85 (or their related subunits). In fact, G␤␥ could stimulate an epitope-tagged p110␤/p85␣ PI 3-kinase, which was expressed in COS-7 cells with their cDNAs (Fig. 4, left panel). Both the purified and the recombinant p110␤/p85 PI 3-kinases were stimulated not only by G␤␥ but also by a synthetic phosphotyrosyl peptide (containing the YMXM motif) that binds to the SH2 domain of p85. A surprising feature of the p110␤/p85 type is its synergistic activation in the presence of the two effectors. The actions of the two effectors are considered to be specific, since (i) G␤␥-induced activation could be inhibited by the GDP-bound form of G␣ i-2 (Fig. 2B) and (ii) a nonphosphorylated peptide with the same sequence had no stimulatory effect (Fig. 2C).
Stephens et al. (19) showed that the PI 3-kinase activity distinct from the p110/p85 heterodimer was activated by G␤␥ but was unaffected by a phosphotyrosyl peptide. In agreement with their results, we have observed the presence in THP-1 cells of the PI 3-kinase activity that is regulated solely by G␤␥ (18). Because the fractions having these activities are reported not to contain p85, the G␤␥-sensitive activities are considered to be different from the p110␤/p85 described in this study. Thus the present paper indicates that there are multiple species of G␤␥-sensitive PI 3-kinase. The possibility that the previously reported enzyme is the p110␤ subunit freed from p85 is unlikely because the enzyme migrates as an apparent molecular mass of 200,000 on a gel filtration column (19). Furthermore, a very recent report by the same authors showed that the enzyme consisted of the p101 regulatory subunit and the ␥-subtype of p110 (15).
Recent studies have revealed that a tyrosine kinase-associated PI 3-kinase plays an important role in cellular signaling mediated by pertussis toxin-sensitive G proteins (27)(28)(29)(30). For examples, an increase in PI 3-kinase activity was observed in immunoprecipitated fractions with anti-phosphotyrosine and anti-src-type protein tyrosine kinase antibodies upon stimulation of the G protein-coupled receptors (27,28), and mitogen-activated protein kinase activation induced by G␤␥ was attenuated by the introduction of a dominant negative p85 mutant (29). Furthermore, we previously reported in monocytic THP-1 cells that stimulation of N-formyl-Met-Leu-Phe receptors, which activate pertussis toxin-sensitive G proteins, potentiated the insulin-induced and thus tyrosine kinase-mediated accumulation of PtdIns(3,4,5)P 3 (18). Such synergistic activation has been observed also in Chinese hamster ovary cells expressing both insulin and formyl-Met-Leu-Phe receptors. 3 These results suggested that p110␤/p85 PI 3-kinase is regulated in the downstream of a pertussis toxin-sensitive GTP-binding protein-coupled receptor. Thus, the synergistic activation of PI 3-kinase indicated in the present in vitro study may be expected to function in the intact cell systems.