Direct Evidence for Involvement of a Guanine Nucleotide-binding ...

THEJOURNAL OF BIOLOGICAL CHEMISTRY (2, 1986 by The American Society of Biological Chemists, Inc.

Vol. 261, No. 25, Issue of September 5, pp. 11558-11562,1986 Printed in U.S.A.

Direct Evidence forInvolvement of a Guanine Nucleotide-binding Protein inChemotactic Peptide-stimulated Formation of Inositol Bisphosphate and Trisphosphate in Differentiated Human Leukemic (HL-60) Cells RECONSTITUTIONWITH TOXIN*

Gi OR Go OF T H E PLASMAMEMBRANESADP-RIBOSYLATED

BY PERTUSSIS

(Received for publication, April 11, 1986)

Akira Kikuchi, Osamu Kozawa, Kozo Kaibuchi, Toshiaki Katadaz, Michio Vis, and Yoshimi Takais From the Department of Biochemistry, Kobe University School of Medicine, Kobe 650 and the $Departmentof Physiological Chemistry, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo 060, Japan

fMet-Leu-Phe (fMLP) stimulated the formation of inositol bis- and trisphosphate in the [3H]inositol-labeled plasmamembranesfrom the human leukemic (HL-60) cellsdifferentiated to neutrophil-like cells by dibutyryl cyclic AMP. The stimulatory effect of fMLP was completely dependent on the simultaneous presence of GTP and Ca2+. The fMLP-stimulatedformation of the phosphorylated inositols was markedly reduced by the prior ADP-ribosylation of the membranes with pertussis toxin. This toxin ADP-ribosylated a M , 40,000 protein, presumably the CY subunit of Gi and/or Go, in the membranes. Reconstitution of the membranes ADP-ribosylated by pertussis toxin with Gi or Go purified from rat brain restored the fMLP-stimulated formation of the phosphorylated inositols. The efficiency of the rat brain Gi and Go in this capacity was roughly equal. The rat brain Gi or Go ADP-ribosylated beforehand by pertussis toxin was inactive in this reconstitution. These results indicate that both rat brain Gi and Go have the potency to couple functionally the fMLP receptor to the phospholipase C-mediated polyphosphoinositidehydrolysis and suggest that Gi or Go may be involved in the mechanism of signal transduction fromthe fMLP receptor to this reaction in the differentiated HL-60 cells.

Among these products, diacylglycerol and IP, serve as messengers for protein kinase C activation and intracellular Ca2+ mobilization, respectively (1, 2). Although the mechanism of signal transduction from receptors to this PI hydrolysis has long been unclear, accumulating evidence strongly suggests that a guanine nucleotide-binding protein(s) may be involved in the regulation of this reaction in several receptor systems. Ithas beendescribed that the PI hydrolysisinduced by compound 48/80 in rat mast cells (3) and by the chemotactic peptide fMLP in human and guinea pig neutrophils (4, 5 ) is inhibited by the action of pertussis toxin which is known to ADP-ribosylate Gi, the inhibitory guaninenucleotide-binding component of adenylate cyclase (6, 7). Based on these observations, it has been suggested that Gi may also be the regulatory component of the PI hydrolysis in these receptor systems. However, it has not yet beenclarified whetherthe purified Gi has indeed the potency to couple the receptor to this reaction. Recently, another typeof a guanine nucleotidebinding protein serving as a substrate for pertussis toxin has been identified in several tissues (8-11). The functionof this novel type of transducer, designated Go, has not yet been clarified. The present studieswere undertaken toclarify whether the purified Gi or Go may have the potency tocouple the receptor to the phospholipase C-mediated P I hydrolysis in a cell-free membrane system. For this purpose, we used the reconstitution system with rat brain Gi or Go of the plasma membranes ADP-ribosylated by pertussis toxin. The plasma membranes In response to various extracellular signals, PI’ including were isolated from the human leukemic (HL-60) cells differphosphatidylinositol, phosphatidylinositol 4-monophosphate, entiatedtoneutrophil-like cells by dibutyryl cyclic AMP, and phosphatidylinositol 4,5-bisphosphate arehydrolyzed by since it has been described that fMLP induces the PI hydrolthe action of phospholipase C to produce diacylglycerol and ysis andthatthisfMLP-induced reaction is inhibited by the respective phosphorylated inositols, IP1, IP2, and IP, (1). pertussis toxin in an intact cell system (12). In this paper,we * This investigation was supported in partby research grants from demonstrate the directevidence that both rat brainGi and Go couple functionally the fMLPreceptor to the Scientific Research Fund of the Ministry of Education, Science have the potency to and Culture, Japan (1985) and the Investigation Committee on Ab- the phospholipase C-mediated PI hydrolysis and to stimulate normalities in HormoneReceptor Mechanism, the Ministry of Health this reaction ina fMLP-dependent manner.

-

~

and Welfare, Japan (1985). The costs of publication of this article were defrayed in part by the payment of page charges. This article EXPERIMENTALPROCEDURES must thereforebe hereby marked “aduertisement” in accordance with Materials and Chemicals-Dibutyryl cyclic AMP and fMLP were 18 U.S.C. Section 1734 solely to indicate thisfact. obtained from Yamasa Chemicals and Sigma, respectively. [3H]InoTo whom all correspondence should be addressed. were from Amersham Corp. and New England The abbreviations used are: PI, phosphoinositide; IPI, inositol1- sitol and [W~’P]NAD monophosphate; IP,, inositol1,4-bisphosphate; IP3, inositol 1,4,5- Nuclear, respectively. All media were from GIBCO. Ultroser G was from LKB. Pertussis toxin was a generous gift from Kakenseiyaku. trisphosphate; fMLP, Net-Leu-Phe; Pipes, piperazine-NJV-bis(2ethanesulfonic acid); Hepes, 4-(2-hydroxyethyl)-l-piperazine- Preactivated pertussis toxin was obtained by incubating the toxin ethanesulfonic acid; EGTA, [ethylene-bis(oxyethylenenitri1o)l with 10 mM dithiothreitol, 1 mM ATP, and 50 mM Tris/HCl at pH 7.5 for 15 min a t 30 “C under the conditions described previously tetraacetic acid; GTP+, guanosine 5”O-thiotriphosphate.

11558

11559

Involvement of G, or Go in IP2 and IP3 Formation (11). Gi and Go werepurified from rat brain membranes by extraction with sodium cholate followed by successive chromatographies on DEAE-Sephacel, ACA-34 Ultrogel, hydroxyapatite, DEAE-Toyopearl 650(S), and hydroxyapatite columns under the conditions specified earlier (11). Isolation of [3HlInositol-labekd Plasma Membranes from the HL60 Cells Differentiated to Neutrophil-like Cells-HL-BO cells were a generous gift from Dr. S. Murao (Kobe University School of Medicine, Kobe, Japan) who originally obtained them from Dr. R. C.Gallo (National Institutes of Health, Bethesda, MD). The cells (1.5 X lo6 cells/ml) were differentiated to neutrophil-like cells by treatment with 500 p~ dibutyryl cyclic AMP for 36 h at 37 "C. The cells were labeled with 3 pCi/ml of [3H]inositol during this differentiation. The conditions for the differentiation and labeling of the cells were the same as those described previously (13). The differentiated cells were washed twice with Ham's F-10 medium containing 0.025% bovine serum albumin and finally suspended at a concentration of 1-1.5 X lo7 cells/ml in Buffer A (10 mM Pipes at pH 7.3 containing 100 mM KC1, 3 mM NaC1, 1 mM ATP, and 3.5 mM MgC1,). The [3H]inositollabeled plasma membranes were isolated by disrupting the cells with N, cavitation followed by Percoll density gradient centrifugation as specified by Borregaard et al. (14). The plasma membranes were resuspended at a concentration of 0.6-1 mg of protein/ml in Buffer B (Buffer A containing 1.25 mM EGTA). This preparation of plasma membranes consisted of mostly plasma membrane vesicles and partly ribosomes and endoplasmic reticula, but did not contain nuclei, mitochondria, and lysosomes, as judged by electron microscopy. In the isolated plasma membranes, the average radioactivities of [3H] inositol incorporated into phosphatidylinositol, phosphatidylinositol 4-monophosphate, and phosphatidylinositol 4,5-bisphosphate were 1, 200, 94, and 47 nCi/mg of protein, respectively. Assay for the Formation of Phosphorylated Inositols in the Plasma Membranes-The [3H]inositol-labeledplasma membranes (about 2.5 X lo4 cpm in PI) were incubated for 1 min at 37 "C in the standard reaction mixture (0.3 ml) containing 50 mM Hepes at pH 7.4, 10 mM LiCl, 5 mM MgC12, 3 mM EGTA, 2.04 mM CaCl,, 5 p~ GTP, and 1 p M fMLP. In this reaction mixture, the free CaZf concentration was about 0.5 p~ as calculated by the equation of Bartfai(15). The reaction was stopped by the addition of chloroform/methanol/HCl (100:200:2).IP,, IP2, andIP, were isolated by DowexAG 1-X8column chromatography under the conditions specified previously (13). Other Procedures-The radioactivity of ,H- and 32P-sampleswas determined with a Packard Instruments Co. Tri-Carb liquid scintillation spectrometer, Model3330. Protein was determined by the method of Lowry et al. (16) with bovine serum albumin as a standard protein.

TABLE I fMLP-stimulated, GTP- and Ca2+-dependentformation of IP, and IP3in the plasma membranes of the differentiated HL-60 cells The [3H]inositol-labeledplasma membranes were incubated for 1 min at 37 "C under the standard assay conditions except that 1 pM fMLP, 5 p~ GTP, or 0.5 p~ Ca2+was added as indicated. Other details are described under "Experimental Procedures." Results are the mean f S.E. of five independent experiments. Additions

IP,

IP*

formation formation ~.~~~~~ ~~

cPm

*

CPm

IP3 formation

cpm

47f 5 5+ 2 467 52 None 124 f 13" 58 9" 518 5 34 Ca2+ 8 f 3 52+ 6 487 f 42 GTP 50f 7 6 f 1 432 f 36 fMLP 242 f 30b 102 1 4 b 497 + 45 Ca2++ G T P 54+ 8 136 f 19 439 + 50 Ca2++ fMLP 8 f 2 58 f 10 GTP 421 + 29 - -~ + fMLP Ca'+ + GTP + fMLP 454 61 386 + 35'179 f 21' "The increase in IP, and IP, formation due to Caz+was significantly greater than no addition with p < 0.01. The increase in IP, and IP, formation due to Ca2+plus GTP was significantly greater than due to Ca2+alone with p < 0.01. The increase in IP, and IP, formation due to Ca2+plus GTP plus fMLP was significantly greater than due to Ca*+plus GTP with p < 0.01.

* *

~~~~

*

20 p~ fully stimulated the formation of IP, a n d IP:+ fMLP reduced the doses of GTP necessary for these reactions withand thereby outaffectingthemaximalreactionvelocities stimulated the reactions at the submaximal doses of GTP. The GTP-stimulated formation of IP, a n d IP, was observed only in thepresence of Ca2+. Ca2+ byitself a t 0.1-2 p M stimulated the formation of IPSand IP, in a dose-dependent manner in the absenceof GTP. Ca2+over 10 pM fully stimulated these reactions. GTP decreased the concentrations of Ca2+for these reactions without affecting the maximal reaction velocities and thereby stimulated the reactions at t h e submaximal concentrationsof this divalent cation. The mode of action of Ca2+in the formation of IP, and IP, is not known, but the similar results have also been described in the membranes from blowfly salivary glands (19), human neutrophils (4) andrathepatocytes (18). Typicalactual data forthe RESULTS of fMLP-stimulated, GTP- and Ca"-dependentformation fMLP-stimulated, GTP- and Ca2+-dependent Formation of IP2and IPSi n the plasma membranes are shown in TableI. IP, and IPS inthe Plasma Membranes-Incubationwith Reduction of the fMLP-stimulated Formation of IPSa n d IP, various doses of fMLP of the differentiated HL-60 cells lainthePlasmaMembranesADP-ribosylated by Pertussis beled with [3H]in~~itol resulted in the formation of IP1, IP,, Toxin-Consistent with the earlier observation in the differand IP3 i n a dose-dependent manner. The doses of fMLP entiatedHL-60cells(12),pretreatment of t h e cellswith necessary for the formation of these three phosphorylated a markedreduction of t h e fMLPpertussistoxincaused inositols were nearly the same,and the doses of fMLP giving stimulated formationof IP,, IP,, a n d IP, when these reactions the maximal and half-maximal reaction velocities were about were measured in the intact cells. Similarly, the fMLP-stim1 and 0.1 p ~ respectively. , Theseresultsagreedwiththe ulated formation of IP, a n d IP, was markedly reduced in the earlier observations (12, 13). When the [3H]inositol-labeled plasma membranes from the cells pretreated with pertussis plasmamembraneswereincubatedwithvariousdoses of toxin as shown in Table 11.' In this experiment, the basal f M L P , IP, a n d IP, were produced in a does-dependent man- reactions induced by GTP a n d Ca2+in the absence of fMLP ner. The doses of fMLP necessary for these reactions in the were not inhibited by the action of pertussis toxin. plasma membranes were nearly identical with those necessary Pertussis toxin ADP-ribosylated a protein in the memIP, production was branes, and the molecular weightof this protein was estimated in intactcells. Under the same conditions, fMLP i n the plasmamembranes.The notstimulatedby to be about40,000 on sodium dodecyl sulfate-polyacrylamide reason for the failure of fMLP to stimulate the formation of gel electrophoresiswithratbrain Gi and Go as reference IP, in the membranes is not known, but the similar results GH, have also been described in the membranes from ratcells *When the formation of IP, and IPS was measured in the intact cells, the pretreatment of the cells with pertussis toxin always caused (17) and rat hepatocytes (18). The fMLP-stimulated formation of IP, and IPS was abso- the reduction of these fMLP-induced reactions in five independent lutely dependent on the simultaneous presence of GTP a n d experiments. However,when the formation of IP,and IP3 were measured in the plasma membranes, the pretreatment of the cells Ca2+.G T P by itself at 1-10 p~ stimulated these reactions in with the toxin sometimes (three times in 10 independent experiments) a dose-dependent manner in the presence of a low concentra- did not reduce these fMLP-induced reactions. The cause for this rare tion (0.5 p ~ of)Ca2+even in the absence of fMLP. GTP over inconsistent phenomenon is not known.

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Involvement of Gi or Go in IPz and IP3 Formation TABLEI1 Reduction of the fMLP-stimulated formation of IP, and IPS in the cells pretreated with pertussis toxinor in the p h m a membranes from thecells pretreated with the toxin The [3H]inositol-labeled cellswere washed twice with Ham's F-10 medium containing 0.025% bovine serum albumin and resuspended at a concentration of 7.5 x lo5 cells/ml in the same medium. The cells werethen treated with or without400 ng/ml of pertussis toxin for 3 h at 30 "C and washed with the same medium. Inthe experiments with the intact cells, the cells were preincubated for 10 min at 37 "C in the presence of 10 mM LiCl and then incubated with or without 1 PM WLP for 1 min at 37 "C under the conditions described previously (13). In the experiments with the plasma membranes, the plasma membranes isolated from the control and ADP-ribosylated cells were incubated for 1 min at 37 "C with or without 1 PM fh4LP in the presence of 5 PM GTP and 0.5 PM Ca2+ under the standard assay conditions. Other details are described under "Experimental Procedures." Results are the mean f S.E. of five cell-level andseven membrane-level exDeriments uerformed indenendentlv. ~~

Experiment

With intact cells

IP, formation

IP, formation

Treatment +WLP -WLP +WLP -WLP

None

Pertussis toxin

+

1752 208 1720 2 189

+WLP

"LP cpm

2 9 5 8 f 313 1985+ 218"

With plasma membranes None Pertussis toxin

CPm

9 2 + 18 104+ 21

+

242 30 238+ 23

2214+11306 443 + 68" 386+ 102 35 262 f 31"

~

IPSformation

+

cpm

2 12+ 5

5 3 6 f 51 82 10"

f 14 98+ 8

1 7 9 f 21 11Of 16"

+

The effect of pertussis toxin was significant withp < 0.01. proteins. Since Gi and Go except transducin are the proteins of IP2 and IP3 ina GTP-dependent manner, and this fMLPthat are known to be ADP-ribosylated by the action of per- stimulated poly-PI hydrolysis is inhibited by the ADP-ribotussis toxin (6-ll), theM , 40,000 protein(s) ADP-ribosylated sylation of Gi or Go by pertussis toxin. The formation of IP2 by this toxin in the plasma membranes of the differentiated and IPS may be derived from the phospholipase C-mediated HL-60 cells might be the cy subunit of Gi and/or Go. poly-PI hydrolysis as well described in other cell systems (1). Reconstitution of the ADP-ribosylated Plasma Membranes These results strongly suggest that either Gi or Go may be with Rat Brain Gior G,-In the ADP-ribosylated plasma involved in the fMLP-stimulated poly-PI hydrolysis also in membranes prepared from the cells pretreated with pertussis thedifferentiatedHL-60 cells. This suggestion is further toxin, the fMLP-stimulated formation of IP2 and IP3 was confirmed by the result that reconstitution with rat brain Gi markedly reduced as compared to that of the control mem- or Go of the membranes ADP-ribosylated by pertussis toxin branes as described above and also shown in Fig. 1. However, restores the fMLP-stimulated poly-PI hydrolysis. The effiaddition of various amountsof either Gi or Go purified highly ciency of these two GTP-binding proteins in this capacity is from rat brain to the ADP-ribosylated membranes restored roughly equal. This result clearly indicates that both Gi and the fMLP-stimulated formation of IP2 and IPS in adoseGo have the equal potency to couple functionally the fMLP dependent manner. Theefficiency of the rat brainGi and Go receptor to the poly-PI hydrolysis. However, it cannot be in this capacity was roughly the same. The maximal stimu- concluded which transducer, Gi or Go, may be involved in vivo lation of these reactions was elicited by the addition of the in thiscoupling in the differentiated HL-60cells. ratbrain Gi or Go inanamountsimilartothat of the Ithas beensuggested thatGi may be involvedin the endogenous proteins servingas substrates for pertussis toxin, phospholipase C-mediated P I hydrolysisinduced by compresumably Gi and/or Go, in the differentiated HL-60 cell pound 48/80 in rat mastcells (3) or by fMLP in human and membranes. The amount of these membrane proteins was guinea pig neutrophils (4,5). However, our result that Go as quantitated with rat brain Gi as a reference protein by mea- well as Gi has the potency toregulate the poly-PI hydrolysis suring the transfer of [32P]ADP-ribose into the membrane has raised the possibility that this transducer instead of Gi proteins by the actionof pertussis toxin under the conditions may be involved in this reactionalso in these pertussis toxinspecified (11). Rat brain Gi or Go added to the ADP-ribosylated membranes did not affect the basal reactions induced sensitivereceptor systems. Incontrasttothesepertussis recentlybeendeby GTP andCa2+in the absenceof fMLP. When these GTP- toxin-sensitive receptor systems,ithas scribed that the phospholipase C-mediated PI hydrolysis inbinding proteinswere beforehand ADP-ribosylated by pertusduced by thyrotropin-releasing hormone in rat GH3 cell memsis toxin and then added to the ADP-ribosylated membranes, branes (17) and by vasopressin in rat hepatocyte membranes they were inactive to restore the fMLP-stimulated formation (18) is dependent on GTP and its stable analogs but is not of IP, and IP3 in the reconstitution system. The Gi and Go employed in these experimentswere purified sensitive t o pertussis toxin. It has also been shown that the from rat brain membranesby extraction with sodium cholate PI hydrolysis induced by acetylcholine in chick heart cells followed by severalcolumn chromatographiesas specified (20) and thrombin in mouse fibroblasts (21) is not inhibited previously (11).These preparations of the GTP-binding pro- by this toxin. Based on these observations, it has been sugteins were more than 95% pure as judged by sodium dodecyl gested that in these pertussis toxin-insensitive receptor syssulfate-polyacrylamidegel electrophoresis andwere not cross- tems, a guanine nucleotide-binding protein(s) other than Gi contaminated by each other as demonstrated inFig. 2. These and Go may serve as a regulatory component of this reaction. preparations were not also contaminated by G,, the stimula- Evidence is not available whether this novel transducer is tory guanine nucleotide-binding componentof adenylate cy- present also in the pertussis toxin-sensitive cells described above or whether this transducer,if present, may be involved clase (6). in the regulation of the PIhydrolysis in these cell types. DISCUSSION It has been described that the ADP-ribosylation of the a In the plasma membranes from the HL-60 cells differen- subunit of Gi by pertussis toxin causes theuncoupling of the signal tiated to neutrophil-likecells, fMLP stimulates the formation receptor to the transducer and thereby inhibits the

Involvement of Gi or Goin IPz and IP3Formation

ID

T

11561

0 m

N

u

-Y

”

0

C

rT

”

FIG.1. Reconstitution of the ADP-ribosylated plasma memb r a n e s w i t h rat brain Gi or Go.The [3H]inositol-labeled plasma membranes were isolated from the cells treated with or without pertussis toxin. Treatment of the cells with this toxin was carried out as described in the legend to Table 11. The isolated membranes were suspended at a concentration of 1 mgof protein/ml in Buffer B. Various amounts of rat brain Gi or Go were added to the ADPribosylated membranes (30 pg of protein containing about 3ng of Gi plus Go). Each mixture was preincubated for 10 min a t 30 “C in the reaction mixture (0.1 ml) containing 25 mM Hepes at pH 8.0 and 5 mM MgCI2 and then incubated for 1 min a t 37 “C with or without 1 p~ fMLP in the presence of 5 p~ GTP and 0.5 p~ Ca2+under the standard assay conditions. In anotherexperiment, rat brain Gi or Go, ADP-ribosylated beforehand by pertussis toxin, was added to the ADP-ribosylated membranes. The ADP-ribosylation of the GTPbinding proteins was carried out by incubating rat brain Gi or G, (each 0.3 pg of protein) with 10 pg/ml of the preactivated pertussis toxin for 20 min a t 30 “C under the conditions specified (11)except Other that 100 p~ NAD was employed instead of 2.5 p~ [LU-~~PINAD. details are described under “ExperimentalProcedures.” A and C, with the control membranes; B and D, with the ADP-ribosylated memwith fMLP; - - -, without fMLP; 0, with intact Gi; 0, branes. -, with intact Go;A,with fMLP plus ADP-ribosylated Gi; A, with fMLP plus ADP-ribosylated Go. A and B, IPz formation; C and D, IP3 formation. Results are themean +. S.E. of three independent experiments. The effect of the ratbrain Gi or Gowas significant (* indicates p < 0.01).

FIG.2. P u r i t y of rat brain Gi a n d Go.Gi and Go werepurified from rat brain membranes as described under “Experimental Procedures.” The purified preparation of G , Go, or their mixture was subjected separately to chromatographyon aDEAE-Toyopearl65O(S) column (1 X 10 cm).Elution was performed with a 25-ml linear concentration gradient of NaCl(25 to200 mM),and fractions of 1ml each were collected. The Gi and Go were quantitated by measuring the maximal incorporation of [32P]ADP-riboseinto the proteins in each fraction by the action of pertussis toxin and also by measuring the [35S]GTPyS-bindingactivity of each fraction. In another set of experiments, the ratbrain Gi or Go waslabeled with [32P]ADP-ribose by the action of pertussis toxinand thensubjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The gel was stained with Coomassie Brilliant Blue. After destaining, the gel was dried and exposed to an x-ray film to prepare the autoradiograph. All the procedures and assay conditions used in theseexperiments were described earlier (11).The left and right panels indicate the result of column chromatographies and gel electrophoresis, respectively. A, a mixture of Gi and Go;B, Gi; C, Go.0, [32P]ADP-ribosylation;A, [35S] GTPyS bound; -, absorbance a t 280 nm. D, protein staining; E, autoradiograph. Lane a, a mixture of GI and Go; lane b, Gi; lane c, Go. a, p, and y indicate the subunits ofGi and Go. ai and or, are the a transduction from the receptor to adenylate cyclase (7). It has subunit of Gi and Go,respectively.

not been clarified whether the ADP-ribosylation of the CY subunit of Go bythe toxin also causes the similar uncoupling of the receptor to this transducer, but this mechanism is highly possible. Therefore, our resultsfrom the reconstitution experiments suggest that exogenous Gi or Go may interact with the fMLP receptor in place of the endogenous Gi or Go which is uncoupled with the receptor due to the ADP-ribosylation of their LY subunits by pertussis toxin and thereby may restore the fMLP-stimulated poly-PI hydrolysis. The basal formation of IP2 and IP3 obtained in the absence of fMLP is induced by GTP andCa”, and these basal reactions are not inhibited by the ADP-ribosylation of the membranes by pertussis toxin, indicating that the ADP-ribosylated Gi or Go may be still as active as the intact proteins to stimulate the poly-PI hydrolysis. The basal formation of IP2and IP3 in

the control and ADP-ribosylated membranes is not affected by the addition of exogenous Gi or Go, but theexact interpretation for this result is not known. The precise mode of action of Gi or Go in theregulation of the phospholipase C-mediated poly-PI hydrolysis in the differentiated HL-60 cells is now under investigation. Acknowledgments-We are grateful to Dr. A. Mizoguchi of the Kobe University School of Medicine for producing the electron micrographs and to J. Yamaguchi for skillful secretarial assistance. REFERENCES 1. Berridge, M. J., and Irvine, R.F. (1984) Nature 312,315-321 2. Takai, Y., Kikkawa, U., Kaibuchi, K., and Nishizuka, Y. (1984)

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Involvement of Gi or Go in IPz and IP3 Formation

Adu. Cyclic Nucleotide Protein Phosphorylation Res. 18,119158 3. Nakamura, T., and Ui, M. (1985) J. Biol. Chem. 260,3584-3593 4. Smith, C. D., Lane, B.C., Kusaka, I., Verghese,M. W., and Snyderman, R. (1985) J. Biol. Chem. 260,5875-5878 5. Ohta, H., Okajima, F., and Ui, M. (1985) J. Biol. Chern. 260, 15771-15780 6. Gilman, A. G. (1984) Cell 36,577-579 7. Ui, M. (1984) Trends Phurmacol. Sci. 5 , 277-279 8. Sternweis, P.C.,and Robishaw, J. D.(1984) J. Biol. Chem. 259, 13806-13813 9. Malbon, C. C., Rapiejko, P. J., and Garcii-Sainz. J. A. (1984) FEBS Lett. 176,301-306 10. Halvorsen, S. W., and Nathanson, N.M. (1984) Biochemistry 23,5813-5821 11. Katada, T., Oinuma, M., and Ui, M. (1986) J. Biol. Chem. 261, 8182-8191

12. Brandt, S. J., Dougherty, R. W., Lapetina, E. G., and Niedel. J. E. (1985) Proc. Natl. Acad. Sci. U. S. A . 82,3277-3280 13. Kikuchi, A., Kozawa, O., Hamamori, Y., Kaibuchi, K., and Takai, Y.(1986) Cancer Res., in press 14. Borregaard, N., Heiple, J. M., Simons, E. R., and Clark, R. A. (1983) J. Cell Biol. 97,52-61 15. Bartfai, T. (1979) Adu. Cyclic Nucleotide Res. 10, 219-242 16. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 1 9 3 , 265-275 17. Lucas, D. O., Bajjalieh, S. M., Kowalchyk, J. A., and Martin, T. F.J. (1985) Biochem. Biophys. Res. Commun. 132,721-728 18. Uhing, R. J., Prpic, V., Jiang, H., and Exton, J. H. (1986) J. Biol. Chem. 261,2140-2146 19. Litosch, I., Wallis, C., and Fain, J. N. (1985) J.Biol. Chem. 260, 5464-5471 20. Masters, S. B., Martin, M. W., Harden, T. K., and Brown, J. H. (1985) Biochem. J. 227,933-937 21. Murayama, T., and Ui, M. (1985) J. Biol. Chem. 260,7226-7233