Epidermal growth factor-induced phosphoinositide guanosine ...

CELL REGULATION, VOI. 1, 615-620, August 1990

Epidermal growth factor-induced phosphoinositide hydrolysis in permeabilized 3T3 cells: lack of guanosine triphosphate dependence and inhibition by tyrosine-containing peptides

Gijs F. Verheijden, Ingrid Verlaan, Joseph Schlessinger*, and Wouter H. Moolenaar Division of Cellular Biochemistry The Netherlands Cancer Institute 1066 CX Amsterdam, The Netherlands *Rorer Biotechnology, Inc. King of Prussia, Pennsylvania 19406 The possible involvement of a stimulatory guanosine triphosphate (GTP)-binding (G) protein in epidermal growth factor (EGF)-induced phosphoinositide hydrolysis has been investigated in permeabilized NIH-3T3 cells expressing the human EGF receptor. The mitogenic phospholipid lysophosphatidate (LPA), a potent inducer of phosphoinositide hydrolysis, was used as a control stimulus. In intact cells, pertussis toxin partially inhibits the LPA-induced formation of inositol phosphates, but has no effect on the response to EGF. In cells permeabilized with streptolysin-O, guanosine 5'-O-(3thiotriphosphate) (GTPyS) dramatically increases the initial rate of inositol phosphate formation induced by LPA. In contrast, activation of phospholipase C (PLC) by EGF occurs in a GTP-independent manner. Guanine 5'-O-(2-thiodiphosphate) (GDP,8S) which keeps G proteins in their inactive state, blocks the stimulation by LPA and GTPyS, but fails to affect the EGF-induced response. Tyrosine-containing substrate peptides, when added to permeabilized cells, inhibit EGF-induced phosphoinositide hydrolysis without interfering with the response to LPA and GTPyS. These data suggest that the EGF receptor does not utilize an intermediary G protein to activate PLC and that receptor-mediated activation of effector systems can be inhibited by exogenous substrate peptides.

Introduction

Many growth factors, hormones, and neurotransmitters are known to stimulate the breakdown of phosphoinositides. This receptor-mediated response gives rise to at least two second messengers, inositol 1,4,5-trisphos1990 by The American Society for Cell Biology

phate, which mediates Ca2" release from internal stores, and diacylglycerol, which activates protein kinase C (Nishizuka, 1986; Berridge, 1987). The hydrolysis of phosphoinositides can be catalyzed by any of the phospholipase C (PLC)1 isozymes that, in many cases, are thought to be stimulated via an as-yet-unidentified guanosine triphosphate (GTP)-binding (G) protein (putative Gp; Cockcroft, 1987). In some cells, receptor-mediated phosphoinositide hydrolysis is inhibited by pertussis toxin, which inactivates certain G proteins of the afl^y heterotrimeric form, whereas in other cells this toxin has no effect, suggesting that at least two different forms of Gp are involved (Boyer et al., 1989). It appears that G proteins mediate PLC activation by a wide variety of agonists, including many neurohormones (Uhing et al., 1986; Smith et al., 1987; Hasegawa-Sasaki et al., 1988; Tilly et aL, 1990) and the mitogenic phospholipid lysophosphatidate (LPA) (van Corven et al., 1989), as assessed by several criteria, in particular the ability of stable guanine nucleotide analogues to modulate PLC activity in cell-free preparations. A characteristic feature of receptors that utilize heterotrimeric G proteins for signal transduction is that they are integral membrane proteins with seven membranespanning segments (reviewed in Hanley and Jackson, 1987). Whether growth factor receptors of the protein tyrosine kinase family with a single transmembrane domain, such as the epidermal growth factor (EGF) receptor, may similarly couple to G proteins to stimulate PLC is uncertain. In case of the platelet-derived growth factor (PDGF) receptor, contrasting results have been ' The abbreviations used are DMEM, DUlbecco's modified Eagle's medium; EGF, epidermal growth factor; GDP, guanosine diphosphate; GDPJ3S, guanine 5'-O-(2-thiodiphosphate); Gp, putative G protein for PLC; G protein, GTP binding protein; GTP, guanosine triphosphate; GTP-yS, guanine 5'-0-(3-thiotriphosphate); LPA, lysophosphatidic acid (1oleoylglycerol-3-phosphate); PDGF, platelet-derived growth factor; PLC, phosphoinositide-specific phospholipase C.

615

G.F. Verheijden et al.

reported with respect to the mechanism of activation of PLC. Hasegawa-Sasaki et al. (1 988) suggested that the PDGF receptor does not couple to a G protein to activate PLC, whereas two other groups reported that PDGF-induced phosphoinositide hydrolysis in membranes has an absolute requirement for guanosine 5'-O-(3thiotriphosphate) (GTPyS) (Kamata and Kung, 1988; Huang and Ives, 1989). Phospholipid turnover induced by the insulin receptor tyrosine kinase has also been claimed to proceed via a specific pertussis toxin-sensitive G protein (Luttrell et aL., 1988). EGF stimulates phosphoinositide turnover in various cell types, including A431 cells (Pike and Eakes, 1987; Tilly et aL., 1988; Wahl and Carpenter, 1988) and 3T3 transfectants (Moolenaar et aL., 1988). Receptor tyrosine kinase activity is essential, although not necessarily sufficient, for this response (Moolenaar et aL., 1988), while recent evidence indicates that the EGF receptor phosphorylates the a-form of PLC at tyrosine residues in vivo (Margolis et aL, 1989; Meisenhelder et aL., 1989; Wahl et aL., 1990). However, whether tyrosine phosphorylation of PLC affects its enzymatic activity is currently impossible to determine. On the other hand, froom studies on hepatocytes and renal epithelial cells, there is

6 LPTX coto Fgure

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Figure 1. Effect of pertussis toxin (PTX) on EGF- and LPAinduced accumulation of inositol phosphates in transfected NIH-3T3 cells. [3H]inositol-labeled cells were preincubated for 2 h in DMEM in the presence or absence of pertussis toxin (100 ng/ml) as indicated. Thereafter, cells were treated for 30 min with 100 ng/ml EGF or 10 ,ug/ml LPA and inositol phosphate formation was determined as described in Methods. A similar experiment was performed with LPA (10 Ag/ml) instead of EGF. Values were normalized to 1 and expressed as means ± SE (n = 3). Control represents 7.5 x 103 cpm/well. 616

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EGF(ng/ml) Figure 2. EGF-induced inositol phosphate formation in intact and streptolysin-0 permeabilized cells. EGF was added to intact (0) or streptolysin-0 permeabilized (0) cells prelabeled with [3H]inositol. After 10 min, incubations were terminated and inositol phosphate formation was determined as described in Methods. Data are expressed as means ± SE (n = 3).

evidence that EGF utilizes a pertussis toxinsensitive G protein to stimulate phosphoinositide metabolism (Johnson and Garrison, 1987; Teitelbaum et al., 1990). Furthermore, recent reports suggest that EGF stimulates adenylate cyclase in cardiac membranes and phospholipase A2 in kidney cells through stimulatory G proteins (Nair et al., 1989; Teitelbaum, 1990). Thus, the emerging picture is by no means complete, and the role of G proteins in EGFstimulated second messenger formation needs to be clarified. In this study we have examined PLC activation in permeabilized NIH-3T3 cells expressing the human EGF receptor, using LPA as a control stimulus known to activate PLC in a GTP-dependent manner (van Corven et al., 1989). Our results demonstrate that EGF, unlike LPA, activates PLC without participation of an intermediary G protein. In addition, we report that small tyrosine-containing substrate peptides specifically inhibit EGF receptor-mediated activation of phospholipase C, probably by competitive interference.

Results and discussion In NIH-3T3 cells overexpressing the wild-type human EGF receptor, both EGF (100 ng/ml) and CELL REGULATION

EGF-induced phosphoinositide hydrolysis

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Figure 3. Time course of inositol phosphate formation in streptolysin-O permeabilized cells. (Left panel) Cultures were stimulated with GTP'yS (1 riM; G), EGF (100 ng/ml; E), or EGF + GTPyS (100 ng/ml and 1 ,uM, respectively; E + G) for the indicated periods of time. Control (C) represents [3H]inositol phosphate formation in the absence of any stimulus. Each data point represents mean ± SE (n = 3). Inset represents [3H]inositol phosphate accumulation during 15 min as a function of GTPyS concentration. (Right panel) Cultures were stimulated with LPA (10 gg/ml; L), GTPyS (1 uM; G), or LPA + GTPyS (10 gg/ml and 1 gM, respectively; L + G) for the indicated periods of time. Each data point represents mean ± SE (n = 3).

the mitogenic phospholipid LPA (10 ag/ml) activate PLC, as revealed by a 2.5- to 5.0-fold increase in the level of total inositol phosphates (Figure 1). As is shown in Figure 1, EGF-induced inositol phosphate formation is not affected by preincubating the NIH-3T3 cells with pertussis toxin for 2 h. In contrast, LPA-induced inositol phosphate formation is significantly reduced by pertussis toxin in these cells (Figure 1). It is noteworthy that in other cells, such as human foreskin fibroblasts, pertussis toxin has no effect on LPA-induced inositol phosphate formation (van Corven et al., 1989), suggesting a degree of cell-specific heterogeneity in the G proteins involved.

meabilization method as revealed by immunoblotting of cell lysates with anti-phosphotyrosine antibody (results not shown). Figure 2 shows that, although permeabilization reduces the absolute amount of total radiolabeled inositol phosphates at each EGF concentration tested, the cells clearly remain responsive to EGF. The concentration dependency is similar to that in intact cells, with half-maximal stimulation of inositol phosphate formation observed at -10 ng/ml EGF. Figure 3A (inset) shows that the nonhydrolyzable GTP-analogue GTPyS activates PLC within 10 min in a dose-dependent manner. Halfmaximal stimulation is observed at a concentration of 1 ,uM. It was confirmed that GTPyS does not affect inositol phosphate metabolism when added to intact cells (not shown). To investigate the kinetics of PLC activation by EGF and LPA in the presence or absence of GTPyS, we terminated incubations at various time points after agonist addition. As shown in Figure 3, A and B, the inositol phosphate response to a half-maximal effective concentration of GTPyS (1 AM) develops rather slowly with time, significant stimulation being observed only after 5-10 min of addition of the nucleotide. Addition of EGF (100 ng/ml) results in increased inositol phosphate formation within a few minutes, without requirement of exogenously added -

PLC activation in permeabilized cells To explore the possible involvement of a GTPbinding protein in the activation of PLC by EGF, we made use of the streptolysin-O permeabilization protocol. This method allows direct access to intracellular compartments while leaving unaffected the G protein-mediated breakdown of inositol lipids (Howell and Gomperts, 1987; Tilly et al., 1990). After treatment with streptolysin-O for 5 min, -950/o of the NIH-3T3 cells were leaky and remained permeable for at least 30 min, as determined by trypan blue staining. We confirmed that EGF-induced receptor tyrosine kinase activity is not affected by this perVol. 1, August 1990

617


GTP-analogue. When EGF is added together with GTPyS, the initial rate of inositol phosphate formation is not significantly increased. The response to EGF and GTPyS together is simply additive rather than synergistic (Figure 3A), strongly suggesting that EGF does not rely on a G protein to stimulate PLC activity. The situation is completely different with LPA, a mitogenic phospholipid that is a potent activator of phosphoinositide breakdown (van Corven et al., 1989; Jalink et al., 1990). Figure 3B shows the response of permeabilized 3T3 cells to LPA in the presence or absence of GTPyS. It is seen that, when LPA and GTPRyS are added together, the initial rate of inositol phosphate formation is dramatically increased. After 5 min, there is a severalfold increase above basal levels, whereas LPA alone causes a barely detectable stimulation over this time period. This kinetic behavior of the LPA response, unlike that of EGF, resembles that of a G protein-coupled receptor, where receptor activation accelerates the exchange of bound guanosine diphosphate (GDP) for GTP on the G protein a subunit. Further support for the notion that the EGF receptor does not couple to a stimulatory G protein comes from experiments using the GDP analogue guanine 5'-O-(2-thiodiphosphate (GDPfS), which keeps G proteins in their inactive GDP-bound state. Figure 4 shows that GDP3S fails to affect inositol phosphate formation induced by EGF, whereas it completely blocks the response to LPA/GTPyS. Tyrosine-containing peptides inhibit EGF-induced PLC activation Synthetic peptides belonging to the family of angiotensins (angiotensin I, DRVYIHPFHL; angiotensin 11, DRVYIHPF) are known to be readily phosphorylated by receptor tyrosine kinases with a Km -1 mM (Hunter and Cooper, 1985). Such tyrosine-containing peptide substrates have been shown to act as competitive inhibitors of EGF receptor autophosphorylation in vitro (Honegger et al., 1988). Using [y-32P]ATP, we confirmed that both angiotensin I and 11 are rapidly phosphorylated and inhibit EGF-induced receptor autophosphorylation in membrane preparations (results not shown). We then studied whether the peptide substrates are capable of inhibiting EGF receptormediated activation of PLC in permeabilized cells. Table 1 shows that the EGF-induced increase in inositol phosphates is up to 500/% inhibited by the presence of angiotensin (at 500 ,uM), whereas basal inositol accumulation pro618

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Figure 4. Effect of GDPf?S on inositol phosphate formation induced by EGF and LPA/GTP-yS. Streptolysin-O permeabilized cells were treated with either EGF (100 ng/ml) or LPA (10 ,ug/ml) + GTPyS (1 pM) in the presence (s) or absence (D) of GDPfS (1 mM) for 10 min. [3H]inositol phosphate formation was determined as described in Methods and normalized to 1.0 (control).

duction is not significantly affected. In contrast, inositol phosphate formation induced by LPA/ GTP-yS is not significantly altered by the substrate peptide (Table 1). These results suggest that tyrosine-containing substrate peptides can act as competitive inhibitors not only of receptor autophosphorylation but also of receptormediated activation of effector enzymes such as PLC.

Conclusions In the present study, we have taken advantage of a recently developed permeabilized cell system for the study of phosphoinositide breakdown evoked by EGF and other stimuli. The results reported here, together with earlier observations, indicate that activation of PLC can be mediated by at least two different mechanisms (reviewed by Boyer et al., 1989). One mechanism involves an as-yet-unidentified G protein (Gp). In some cell types this way of stimulation is sensitive to pertussis toxin (Martin, 1989), which ADP-ribosylates and thereby inactivates the Gj/G. members of the family of heterotrimeric G proteins. Another means of PLC regulation does not appear to depend on a stimulatory G protein, as shown here for the activated EGF receptor in 3T3 cells. Because the ay-form of PLC is phosphorylated on tyrosine residues after activation of EGF and PDGF reCELL REGULATION

EGF-induced phosphoinositide hydrolysis

Table 1. Effect of angiotensins on mitogen-stimulated accumulation of inositol phosphates in permeabilized NIH-3T3 cells

[3H]inositol phosphates (dpm/well) Addition

Control EGF(100ng/ml) LPA (10 ug/ml) + GTPyS (1

ALM)

Control cells

Angiotensin I

Angiotensin II

379 ± 40 814±50 1918 ± 55

478 ± 82 647±116 2125 ± 185

408 ± 28 457± 26 2400 ± 158

Values are the mean ± SE of at least 3 determinations. Cells were permeabilized with streptolysin-O as described in Methods. Angiotensin I and angiotensin 11 were added at a final concentration of 500 AM. After 15 min incubation at room temperature, EGF or LPA + GTPyS was added at the indicated concentrations and incubations were continued for another 15 min at 37°C. The labeling of the cells and the extraction of total [3H]inositol phosphates were performed as described in the Methods. Inhibition by angiotensin I and 11 of the EGF response is 36% and 54%, respectively, as calculated from: 0

dpm (angiotensin + EGF) x 100Y inhibition dpm (EGF) dpm (EGF) - dpm (control)

The response of intact cells to angiotensin and 11 is negligible (not shown).

ceptors, it seems plausible to assume that these phosphorylations are essential for enhancing the enzymatic activity of PLC--y in a G proteinindependent way. Yet, the precise molecular mechanisms whereby EGF stimulates PLC activation remain to be unequivocally established. Our data obtained in 3T3 cells contrast with the recent observations of Teitelbaum et al. (1990) in renal epithelial cells, in which EGF-induced phosphoinositide hydrolysis is enhanced by GTPyS and is inhibited by GDPjS or pertussis toxin (see also Johnson and Garrison, 1987). This raises the interesting possibility that EGF may trigger phosphoinositide hydrolysis by distinctly different mechanisms in different cell types. Finally, we have employed commonly used peptide substrates to inhibit specifically EGF receptor-mediated activation of PLC in permeabilized cells. The results obtained are consistent with a mechanism in which exogenous peptide substrates and PLC-,y compete for the protein tyrosine kinase active site, but further kinetic studies are required to establish this point firmly. Nevertheless, the observation that small phosphorylatable peptides can inhibit PLC activation in permeabilized cell systems offers promising perspectives for the analysis and pharmacological alteration of EGF receptor signal transduction.

Methods Materials The reagents used in this study were obtained from the following suppliers: [3HJmyoinositol, Amersham (Arlington Vol. 1, August 1990

Heights, IL); EGF, Collaborative Research (Bedford, MA); guanine nucleotides, Boehringer Mannheim (Mannheim, Federal Republic of Germany); pertussis toxin, List Biochemicals (Campbell, CA); streptolysin-O, Wellcome Research (Weesp, The Netherlands); angiotensins, Sigma (St. Louis, MO).

Cell culture NIH-3T3 cells (type 2.2) devoid of endogenous EGF receptors were transfected with human wild-type receptors (Honegger et al., 1987). These cells express -3 x 105 EGF receptors per cell as determined by Scatchard analysis and were grown in Dulbecco's modified Eagle's medium (DMEM) with 100% fetal calf serum. The cells were grown to near confluency before being used in the experiments.

Measurements of inositol phosphates Confluent monolayers of transfected NIH-3T3 cells were labeled with 2 ACi/ml of [3H]myoinositol for 48 h. Two hours before stimulation, the cells were shifted to serum-free DMEM. Experiments were continued with either intact or permeabilized cells. Intact cells were incubated with agonist and/or inhibitors in serum-free DMEM containing 10 mM LiCI. Cells were permeabilized with streptolysin-O essentially as described (Howell and Gomperts, 1987). Briefly, cultures were incubated for 5 min in permeabilization buffer (137 mM KCI, 2 mM ethylene glycol-bis(8-aminoethyl ether)N,N,N',N'-tetraacetic acid [EGTAl, 1 mM CaCI2, 2 mM MgCI2, 1 mM Na-ATP, 5 mM piperazine-N,N'-bis(2-ethanesulfonic acid) [PIPES], pH 6.8) containing 0.4 U/mI streptolysin-O. After washing the permeabilized cells 3 times with incubation buffer (130 mM KCI, 10 mM NaCI, 1 mM CaCI2, 2 mM EGTA, 2 mM MgCI2, 1 mM Na-ATP, 5 mM PIPES, pH 6.8) we added agonists and/or inhibitors. The reactions were stopped by replacing the medium by 10% (w/v) icecold trichloroacetic acid. After 10 min, we collected extracts, and, after washing them with diethylether and neutralizing them with Tris-base, we recovered total [3H]inositol phosphates (IP,-fraction) by anion exchange chromatography on Dowex AG 1 x 8 columns (formate formJ as described by Tilly et al. (1987). 619


Acknowledgments We thank Hidde Ploegh for useful comments, Angelique van Rijswijk for technical assistance, and Paulien Sobels for preparing the manuscript. This work was supported by the Dutch Cancer Society.

Received: May 15, 1990. Revised and accepted: July 5, 1990.

References Berridge, M.J. (1987). Inositol trisphosphate and diacylglycerol: two interacting second messengers. Annu. Rev. Biochem. 56, 159-193. Boyer, J.L., Hepler, J.R., and Harden, T.K. (1989). Hormone and growth factor receptor-mediated regulation of phospholipase C activity. Trends Pharmacol. Sci. 10, 360-364. Cockcroft, S. (1987). Polyphosphoinositide phosphodiesterase: regulation by a novel guanine nucleotide binding protein, Gp. Trends Biochem. Sci. 12, 75-78. Hanley, M.R., and Jackson, T. (1987). Return of the magnificent seven. Nature 329, 766-767. Hasegawa-Sasaki, H., Lutz, F., and Sasaki, T. (1988). Pathway of phospholipase C activation initiated with plateletderived growth factor is different from that initiated with vasopressin and bombesin. J. Biol. Chem. 263, 1297012976. Honegger, A.M., Dull, T.J., Felder, S., Van Obberghen, E., Bellot, F., Szapary, D., Schmidt, A., Ullrich, A., and Schlessinger, J. (1987). Point mutation at the ATP binding site of EGF receptor abolishes protein-tyrosine kinase activity and alters cellular routing. Cell 51, 199-209. Honegger, A.M., Dull, T.J., Felder, S., Szapary, D., Komoriya, A., Kris, R., Ullrich, A., and Schlessinger, J. (1988). Kinetic parameters of the protein tyrosine kinase activity of EGFreceptor mutants with individually altered autophosphorylation sites. EMBO J. 7, 3053-3060. Howell, T.W., and Gomperts, B.D. (1987). Rat mast cells permeabilised with streptolysin 0 secrete histamine in response to Ca2l at concentrations buffered in the micromolar range. Biochim. Biophys. Acta. 927, 177-183. Huang, C., and Ives, H.E. (1989). GTPyS potentiates both thrombin- and PDGF-induced inositol phosphate release in permeabilized vascular smooth muscle cells. J. Biol. Chem. 264, 4391-4397. Hunter, T., and Cooper, J.A. (1985). Protein-tyrosine kinases. Annu. Rev. Biochem. 54, 897-930. Jalink, K., van Corven, E.J., and Moolenaar, W.H. (1990). Lysophosphatidic acid, but not phosphatidic acid, is a potent Ca2l-mobilizing stimulus for fibroblasts. J. Biol. Chem. 265, 12232-12239. Johnson, R.M., and Garrison, J.C. (1987). EGF and angiotensin 11 stimulate formation of inositol 1,4,5- and inositol 1,3,4-trisphosphate in hepatocytes. J. Biol. Chem. 262, 17285-17293. Kamata, T., and Kung, H.F. (1988). Effects of ras-encoded proteins and PDGF on inositol phospholipid turnover in NRK cells. Proc. NatI. Acad. Sci. USA 85, 5799-5803. Luttrell, L.M., Hewlett, E.L., Romero, G., and Rogol, A.D. (1988). Pertussis toxin treatment attenuates some effects of insulin in BC3H-1 murine myocytes. J. Biol. Chem. 263, 6134-6141. Margolis, B., Rhee, S.G., Felder, S., Mervic, M., Lyall, R., Levitzki, A., Ullrich, A., Zilberstein, A., and Schlessinger, J. 620

(1989). EGF induces tyrosine phosphorylation of phospholipase C-Il: a potential mechanism for EGF receptor signaling. Cell 57, 1101-1107. Martin, T.F.J. (1989). Lipid hydrolysis by phosphoinositidase C: enzymology and regulation by receptors and guanine nucleotides. In: Inositol Lipids in Cell Signalling, ed. R.H. Michell, A.H. Drummond, and C.P. Downes, New York: Academic Press, 81-112. Meisenhelder, J., Suh, P-G., Rhee, S.G., and Hunter, T. (1989). Phospholipase C-y is a substrate for the PDGF and EGF receptor protein-tyrosine kinases in vivo and in vitro. Cell 57, 1109-1122. Moolenaar, W.H., Bierman, A.J., Tilly, B.C., Verlaan, I., Honegger, A.M., Ullrich, A., and Schlessinger, J. (1988). A point mutation at the ATP-binding site of the EGF-receptor abolishes signal transduction. EMBO J. 7, 707-710. Nair, B.G., Rashed, H.M., and Patel, T.B. (1989). EGF stimulates rat cardiac adenylate cyclase through a GTP-binding regulatory protein. Biochem. J. 264, 563-571. Nishizuka, Y. (1986). Studies and perspectives of protein kinase C. Science 233, 305-312. Pike, L.J., and Eakes, A.T. (1987). EGF stimulates the production of phosphatidylinositol monophosphate and the breakdown of polyphosphoinositides in A431 cells. J. Biol. Chem. 262, 1644-1651. Smith, C.D., Uhing, R.J., and Snyderman, R. (1987). Nucleotide regulatory protein-mediated activation of phospholipase C in human polymorphonuclear leukocytes is disrupted by phorbol esters. J. Biol. Chem. 262, 61 21-6127. Teitelbaum, I. (1990). The EGF receptor is coupled to a phospholipase A2-specific pertussis toxin-inhibitable G protein in cultured rat inner medullary collecting tubule cells. J. Biol. Chem. 265, 4218-4222. Teitelbaum, I., Strasheim, A., and Berl, T. (1990). EGF-stimulated phosphoinositide hydrolysis in cultured rat inner medullary collecting tubule cells. J. Clin. Invest. 85, 10441050. Tilly, B.C., van Paridon, P., Verlaan, I., Wirtz, K., de Laat, S.W., and Moolenaar, W.H. (1987). Inositol phosphate metabolism in bradykinin-stimulated human A431 carcinoma cells. Biochem J. 244, 129-135. Tilly, B.C., van Paridon, P.A., Verlaan, I., de Laat, S.W., and Moolenaar, W.H. (1988). EGF-induced formation of inositol phosphates in human A431 cells. Biochem. J. 252, 857863. Tilly, B.C., Tertoolen, L.G.J., Lambrechts, A.C., Remorie, R., de Laat, S.W., and Moolenaar, W.H. (1990). Histamine Hl-

receptor-mediated phosphoinositide hydrolysis, Ca2l signalling and membrane-potential oscillations in human Hela carcinoma cells. Biochem J. 266, 235-243. Uhing, R.J., Prpic, V., Jiang, H., and Exton, J.H. (1986). Hormone-stimulated polyphosphoinositide breakdown in rat liver plasma membranes. J. Biol. Chem. 261, 2140-2146. van Corven, E.J., Groenink, A., Jalink, K., Eichholtz, T., and Moolenaar, W.H. (1989). Lysophosphatidate-induced cell proliferation: identification and dissection of signaling pathways mediated by G proteins. Cell 59, 45-54. Wahl, M.l., and Carpenter, G. (1988). Regulation of EGFstimulated formation of inositol phosphates in A431 cells by calcium and protein kinase C. J. Biol. Chem. 263, 75817590.

Wahl, M.l., Nishibe, S., Kim, J.W., Kim, H., Rhee, S.G., and Carpenter, G. (1990). Identification of two EGF-sensitive tyrosine phosphorylation sites of phospholipase C-'y in intact HSC-1 cells. J. Biol. Chem. 265, 3944-3948. CELL REGULATION