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Activation of the phosphatidylinositol cycle in spreading cells
Experimental Cell Research 182 (1989) 659-663
Activation of the Phosphatidylinositol
Cycle in Spreading Cells
DGRGTI-IEE BRBUER and CHRIS’IOPH WAGENER’ Institute for Clinical Chemistry and Pathobiochemistry, Medical Faculty, University of Technology, Aachcn, Federal Republic of Germany
Metabolites of the phosphatidylinositol cycle were analyzed in BHK-21 (C13) cells spreading on fibronectin-coated culture plates in comparison with attached nonspreading ceils 45 min after plating. Among the water-soluble metabolites (glycerophosphoinositol, inositol, inositol monophosphate, inositol bisphosphate, inositol trisphosphate, and inosito1 tetrakisphosphate), signiticant elevations were found for inositol monophosphate, inositol bisphosphate, and inositol tetrakisphosphate. In the lipid fraction, phospbatidylinositol 4-monophosphate and phosphatidylinositol 4,5-bisphosphate were significantly elevated. The activation of the phosphatidylinositol cycle in spreading versus nonspreadii attached BHK-21 (C13) cells may be involved in the permissive effect of the extracellular matrix on cell proliferation. Q 1989Academic RCSS, Inc.
The extracellular matrix (ECM) exhibits profound effects on growth control and differentiation. It has been suggested that matrix effects may be mediated through changes in cell shape [l]. In this context, the spreading of mesenchymal and endothelial cells can be regarded as a distinct mitogenic signal 12, 31. The biochemical signal(s) leading to the proliferation of spreading cells is not known. Because the activation of the phosphatidylinositol (PI) cycle plays an important role in both the control of proliferation [4] and cell motility [5], we addressed the question of if the PI cycle may be activated in spreading cells. Materials and Methods Abbreviations: PtdIns, phosphatidylinositol; PtdInsQP, phosphatidylinositol Cmonophosphate; PtdIns(4,5)Pz, phosphatidylinositol4,5-bisphosphate; GroPIns, glycerophosphoinositol; GroPIns(4)P, glycerophosphoinositol Cmonophosphate; GroPIns(4,5)Pz, glycerophosphoinositol A,S-bisphosphate; InsP, inositol monophosphate; InsP2, inositol bisphosphate; InsPr, inositol trisphosphate; InsP,, inositol tetrakisphosphate. When specific isomers of inositol phosphates are intended, the position of phosphates is given (i.e., Ins(l,4,5)Pr for inositol 1,4,5-trisphosphate as opposed to InsPJ for inositol trisphosphate(s) of unspecified or unknown structure). Cell culture. BHK-21 (C13) cells [6] were obtained from Flow. Cells were grown in GMEM supplemented with 10% tryptose phosphate broth and 10% FCS (GIBCO). Cell spreading assay. The spreading assay followed the protocol of Grinell et al. 171.Polystyrol tissue culture dishes (Falcon) were coated with fibronectin purified from human serum 181 at concentrations of 40 pg/ml followed by heat-denaturated BSA (10 mg/ml) [9]. For controls uncoated dishes were used. Confluent monolayers were incubated for 48 h in serum-free, inositol-freeGMEM containing 55 kBq/ml of [3H]inositol. Prior to trypsinisation, excess [‘Hlinositol was removed by washing three times with PBS. Tkypsinized cells were suspended in adhesion medium 171containing 2.5 mM LiCl and 1.0~106 cells/ml (100,000 cells/cm’) were seeded on llbronectin-coated culture
’ To whom reprint requests should be addressed. 659
Copyright Q 1989 by Academic FTCSS,Inc. AU rights of reproduction in my form nsenwJ 0014-4827!%9w3.00
Fig. 1. Effect of flbronectin on cell spreading. BHK-21 (C13) cells were seeded either on dishes coated with 40 pg/ml human plasma fibronectin (a) or on uncoated dishes (b) as described under Materials and Methods and 100,000cells/cm* were inoculated into the dishes and incubated for 45 min. The number of adherent cells and the percentage of spreaded cells were determined by phase-contrast microscopy. X91.
dishes or on uncoated culture dishes. After 45 min of incubation at 37”C, the adhesion medium was removed and the cell cultures were washed two times with PBS. Four fields with 150-200 cells at diierent positions of the dishes were counted to determine the number of adherent cells and the percentage of spread cells using an inverted phase-contrast microscope. Two independent experiments (three dishes per group) were performed. Extraction and determination of inositolphosphates and phosphoinositides. The extraction of inositolphosphates and phosphoinositides from cultured cells was performed according to Berridge et al. [lo]. Inositolphosphates were determined by anion exchange chromatography over Dowex-1 (X8formate form, Sigma) following the protocols of Ellis et al. [ll] and Berridge et al. [12]. The following buffers were used to elute the compounds indicated: distilled water (inositol), 5 mM disodium tetraborate/60 mM sodium formate (GroPIns), 0.2 M ammonium formate/O.l M formic acid (InsP), 0.4 M ammonium formate/O. 1 M formic acid (InsPz), 0.8 M ammonium formate/O. 1 M formic acid (InsP,) [13]. Subsequently, the columns were washed by the addition of 1.0 M ammonium formate10.1 M formic acid to elute Ins(1,3,4,5)P, [13], followed by 2.2 M ammonium formate/O.l M formic acid, and 3.4 M ammonium formate/O.l M formic acid. InsP, InsP,, and InsP3 were identified by comparison with radiolabeled Ins(l)P, Ins(1,4)Pz, and Ins(l,4,5)P3 standards (Amersham). The elution from GroPIns was investigated by a rerunning technique described by Irvine [14]. Polyphosphoinositides were determined by anion exchange chromatography after deacylation by alkaline hydrolysis [11, 151.The following buffers were used to elute the compounds indicated: 5 mM disodium tetraborate/O. 18 M ammonium formate (GroPIns), 0.3 M ammonium formatem. 1 M formic acid (GroPIns(gP), 0.75 M ammonium formate/O. 1 M formic acid (GroPIns(4,5)P& Subsequently, the columns were washed by the addition of 1.0 M ammonium formate/O.l M formic acid. Deacylation and elution protocols were controlled by radiolabeled lipid standards (NEN).
Results The adhesion and spreading rates of BHK-21 (C13) cells on fibronectin-coated culture dishes vs uncoated dishes were determined 45 min after the addition of cells (Fig. 1). After this time, 52.7 to 56.4% of the cells were spread on the fibronectin-coated culture dish whereas no spreading activity was observed on the control dish. The determination of metabolites of the PI cycle was performed at a time point where approximately half of the cells had spread on the fibronectin matrix (45 min). The amount of water-soluble metabolites in the experimental groups relative to the controls is demonstrated in Fig. 2. In comparison with the control groups, significantly higher cpm were obtained in the InsP and InsPz fractions.
Short note
661
p < 0.001
-
“2% -
-
iE E
-
Fig. 2. Fold change of inositol phosphates in spreading vs nonspreading attached BHK-21 (C13) cells. IP, InsP; IP2, InsP*; IPS, Insq; IP,, InsP4. The elution conditions for InsP, InsS, and InsPSwere confirmed by the use of radiolabeled inositol phosphate standards. The elution conditions for InsP, were adopted from [13]. Values from two independent experiments each performed with three experimental and control cultures.
Similarly, the cpm in the fraction eluted with 1.0 M ammonium formate/O.1 M formic acid were significantly higher. The cpm in the GroPIns fraction were significantly lower (PcO.01, not shown). For Inositol and InsP3, no significant difference between experimental and control group could be shown. The percentage of deacylated PtdIns, PtsIns(4)P, and PtsIns(4,5)P2 relative to the total radioactivity of the lipid phase for experimental and control groups is shown in Table 1. It follows from Table 1 that the amount of PtdIns(4)P is 17.2% higher and that of PtdIns(4,5)P2 is 33.3% higher than the corresponding values of the control groups. Discussion
The results reported here indicate that the PI cycle is activated in spreading BHK-2 1 (C 13) cells in comparison with attached rounded cells. Among the watersoluble metabolites of the PI cycle, those metabolites eluting with Ins(4)P and Ins(1,4)Pz standards as well as metabolites eluted with 1.O M ammonium formate/O.1M formic acid were significantly elevated. The analytical method applied does not allow the exact identification of the different isomers of inositol phosphates described recently [16, 171.The high activity in the 1.0 A4ammonium formate/O.l M formic acid fraction most probably results from Ins(1 ,3,4,5)P4[ 131. The main metabolite in the 0.4 M ammonium formate/O.l M formic acid fraction, Ins(l,4)Pr, can result either from phospholipase C-mediated cleavage of
662 Short note
TABLE 1 Percentage cpm of deacylation products of inositol phospholipids in relation to the cpm of total inositol phospholipids Control GroPIns GroPIns(4)P GroPIns(4,5)P,
96.2k2.7 2.9kO.3 2.1kO.4
Cells
spreadon fibronectin 91.7f1.3* 3.4+0.2* 2.gf0.2*
*P
PtdIns(4)P or from metabolization of Ins(l,4,5)P3. In addition, the fraction may contain Ins(3,4)Pz derived from Ins(l,3,4)P3. The possible metabolites in the 0.2 M formate/O.1 M formic acid fraction are Ins(l)P resulting from cleavage of PtdIns as well as the isomers Ins(4)P and Ins(3)P derived from the respective inositol bisphosphates. The finding that in the fractions containing inositol trisphosphates the radioactivity was not significantly elevated is probably due to the rapid metabolization of Ins(l,4,5)P3. The 33.3% increase of PtdIns(4,5)P2 suggests an increased synthesis as a result of the accelerated turnover of PtdIns(4,5)P2 following phospholipase C cleavage. The increased turnover of PtdIns(4)P may be due to either cleavageor further phosphorylation. There are several possibilities to explain the activation of the PI cycle in spreading vs rounded cells. It is thought that the clustering of membrane receptors at the sites of adhesion of spreading cells is analogous to the clustering of membrane receptors of nonadherent cells [18]. Our findings are in accordance with this hypothesis since the PI cycle is activated in resting lymphocytes by agents known to cluster membrane receptors such as concanavalin A or anti-Ig antibodies [l9, 201. An alternate explanation would be the increase of surface area of spreading cells which would allow an increased uptake of nutrients and growth stimulating compounds from the medium [2l, 221. According to BenZe’ev [3], suspended3T6.fibroblasts resume protein synthesis when attached in a rounded configuration while the recovery of mRNA production and the rRNA and DNA synthesis as well as cell growth require extensive cell spreading. The latter two hypotheses would suggest that the stimulation of the PI cycle in spreading cells reflects a general increase of metabolic activity rather than a specific answer to the clustering of membrane receptors. It should be noted, however, that, in the present study, cells were kept as a monolayer under serumfree conditions prior to the spreading experiments. For this reason, the cellular metabolism is not reduced in a manner similar to that of suspended cells and an increased uptake of serum factors by spreading cells can be excluded. Since the PI cycle plays a central role in the control of cell proliferation [4], the activation of the PI cycle may be, at least in part, responsible for the mitogenic stimulation of cell spreading.
Short note 663 References 1. Watt, F. M. (1986) Trends Biochem. Sci. 11, 482-485. 2. Folkman, J., and Moscona, A. (1978) Nature (London) 273, 345-349. 3. Ben-Ze’ev, A. (1980) Cell 21, 365-372. 4. Berridge, M. J. (1987) Biochim. Biophys. Acta 907, 33-45. 5. Lassing, I., and Lindberg, U. (1988) Exp. Cell Res. 174, 1-15. 6. Macpherson, I. (1%3) J. Nat. Cancer Inst. 30, 795-806. 7. Grinell, F., Hays, D. G., and Minter, D. (1977) Exp. Cell Res. 110, 175-190. 8. Miekka, S. I., Ingham, K. C., and Menache, D. (1982) Thromb. Res. 27, 1-14. 9. Yamada, K. M., and Kennedy, D. W. (1984) J. Cell Biol. 99, 29-36. 10. Berridge, M. J., Heslop, J. P., Irvine, R. F., and Brown, K. D. (1984) Biochem. J. 222, 195-201. 11. Ellis, R. B., Galliard, T., and Hawthorne, J. N. (1%3) Biochem. J. 88, 125-131. 12. Bet-ridge, M. J., Dawson, R. M. C., Downes, C. P., Heslop, J. P., and Irvine, R. F. (1983) Biochem. J. 212, 473-482.
13. Batty, I. R., Nahorski, S. R., and Irvine, R. F. (1985) Biochem. J. 232, 211-215. 14. Irvine, R. F. (1986) in Phosphoinositides and Receptor Mechanisms, pp. 89-107, A. R. Liss, New York. 15. Downes, C. P., and Michell, R. H. (1981) Biochem. J. 198, 133-M. 16. Inhom, R. C., Bansal, V. S., and Majerus, P. W. (1987) Proc. Natl. Acad. Sci. USA 84, 2170-2174. 17. Dean, N. M., and Moyer, J. D. (1988) Biochem. J. 250,493-500. 18. Vasiliev, J. M., and Gelfand, I. M. (1982) Proc. Natl. Acad. Sci. USA 79, 2594-2597. 19. Taylor, M. V., Metcalfe, J. C., Hesketh, T. R., Smith, G. A., and Moore, J. P. (1984) Nature (London)
312, 462-465.
20. Bijesterbosch, M. K., Meade, C. J., Turner, G. A., and Klaus, G. G. B. (1985) Cell 41,999-1006. 21. O’Neill, C. H., Riddle, P. N., and Jordan, P. W. (1979) Cell 16, 909-918. 22. O’Neill, C., Jordan, P., and Ireland, G. (1986) Cell 44, 489-496. Received October 14, 1988
43-890336
F’rinted in Sweden
Activation of the Phosphatidylinositol
Cycle in Spreading Cells
DGRGTI-IEE BRBUER and CHRIS’IOPH WAGENER’ Institute for Clinical Chemistry and Pathobiochemistry, Medical Faculty, University of Technology, Aachcn, Federal Republic of Germany
Metabolites of the phosphatidylinositol cycle were analyzed in BHK-21 (C13) cells spreading on fibronectin-coated culture plates in comparison with attached nonspreading ceils 45 min after plating. Among the water-soluble metabolites (glycerophosphoinositol, inositol, inositol monophosphate, inositol bisphosphate, inositol trisphosphate, and inosito1 tetrakisphosphate), signiticant elevations were found for inositol monophosphate, inositol bisphosphate, and inositol tetrakisphosphate. In the lipid fraction, phospbatidylinositol 4-monophosphate and phosphatidylinositol 4,5-bisphosphate were significantly elevated. The activation of the phosphatidylinositol cycle in spreading versus nonspreadii attached BHK-21 (C13) cells may be involved in the permissive effect of the extracellular matrix on cell proliferation. Q 1989Academic RCSS, Inc.
The extracellular matrix (ECM) exhibits profound effects on growth control and differentiation. It has been suggested that matrix effects may be mediated through changes in cell shape [l]. In this context, the spreading of mesenchymal and endothelial cells can be regarded as a distinct mitogenic signal 12, 31. The biochemical signal(s) leading to the proliferation of spreading cells is not known. Because the activation of the phosphatidylinositol (PI) cycle plays an important role in both the control of proliferation [4] and cell motility [5], we addressed the question of if the PI cycle may be activated in spreading cells. Materials and Methods Abbreviations: PtdIns, phosphatidylinositol; PtdInsQP, phosphatidylinositol Cmonophosphate; PtdIns(4,5)Pz, phosphatidylinositol4,5-bisphosphate; GroPIns, glycerophosphoinositol; GroPIns(4)P, glycerophosphoinositol Cmonophosphate; GroPIns(4,5)Pz, glycerophosphoinositol A,S-bisphosphate; InsP, inositol monophosphate; InsP2, inositol bisphosphate; InsPr, inositol trisphosphate; InsP,, inositol tetrakisphosphate. When specific isomers of inositol phosphates are intended, the position of phosphates is given (i.e., Ins(l,4,5)Pr for inositol 1,4,5-trisphosphate as opposed to InsPJ for inositol trisphosphate(s) of unspecified or unknown structure). Cell culture. BHK-21 (C13) cells [6] were obtained from Flow. Cells were grown in GMEM supplemented with 10% tryptose phosphate broth and 10% FCS (GIBCO). Cell spreading assay. The spreading assay followed the protocol of Grinell et al. 171.Polystyrol tissue culture dishes (Falcon) were coated with fibronectin purified from human serum 181 at concentrations of 40 pg/ml followed by heat-denaturated BSA (10 mg/ml) [9]. For controls uncoated dishes were used. Confluent monolayers were incubated for 48 h in serum-free, inositol-freeGMEM containing 55 kBq/ml of [3H]inositol. Prior to trypsinisation, excess [‘Hlinositol was removed by washing three times with PBS. Tkypsinized cells were suspended in adhesion medium 171containing 2.5 mM LiCl and 1.0~106 cells/ml (100,000 cells/cm’) were seeded on llbronectin-coated culture
’ To whom reprint requests should be addressed. 659
Copyright Q 1989 by Academic FTCSS,Inc. AU rights of reproduction in my form nsenwJ 0014-4827!%9w3.00
Fig. 1. Effect of flbronectin on cell spreading. BHK-21 (C13) cells were seeded either on dishes coated with 40 pg/ml human plasma fibronectin (a) or on uncoated dishes (b) as described under Materials and Methods and 100,000cells/cm* were inoculated into the dishes and incubated for 45 min. The number of adherent cells and the percentage of spreaded cells were determined by phase-contrast microscopy. X91.
dishes or on uncoated culture dishes. After 45 min of incubation at 37”C, the adhesion medium was removed and the cell cultures were washed two times with PBS. Four fields with 150-200 cells at diierent positions of the dishes were counted to determine the number of adherent cells and the percentage of spread cells using an inverted phase-contrast microscope. Two independent experiments (three dishes per group) were performed. Extraction and determination of inositolphosphates and phosphoinositides. The extraction of inositolphosphates and phosphoinositides from cultured cells was performed according to Berridge et al. [lo]. Inositolphosphates were determined by anion exchange chromatography over Dowex-1 (X8formate form, Sigma) following the protocols of Ellis et al. [ll] and Berridge et al. [12]. The following buffers were used to elute the compounds indicated: distilled water (inositol), 5 mM disodium tetraborate/60 mM sodium formate (GroPIns), 0.2 M ammonium formate/O.l M formic acid (InsP), 0.4 M ammonium formate/O. 1 M formic acid (InsPz), 0.8 M ammonium formate/O. 1 M formic acid (InsP,) [13]. Subsequently, the columns were washed by the addition of 1.0 M ammonium formate10.1 M formic acid to elute Ins(1,3,4,5)P, [13], followed by 2.2 M ammonium formate/O.l M formic acid, and 3.4 M ammonium formate/O.l M formic acid. InsP, InsP,, and InsP3 were identified by comparison with radiolabeled Ins(l)P, Ins(1,4)Pz, and Ins(l,4,5)P3 standards (Amersham). The elution from GroPIns was investigated by a rerunning technique described by Irvine [14]. Polyphosphoinositides were determined by anion exchange chromatography after deacylation by alkaline hydrolysis [11, 151.The following buffers were used to elute the compounds indicated: 5 mM disodium tetraborate/O. 18 M ammonium formate (GroPIns), 0.3 M ammonium formatem. 1 M formic acid (GroPIns(gP), 0.75 M ammonium formate/O. 1 M formic acid (GroPIns(4,5)P& Subsequently, the columns were washed by the addition of 1.0 M ammonium formate/O.l M formic acid. Deacylation and elution protocols were controlled by radiolabeled lipid standards (NEN).
Results The adhesion and spreading rates of BHK-21 (C13) cells on fibronectin-coated culture dishes vs uncoated dishes were determined 45 min after the addition of cells (Fig. 1). After this time, 52.7 to 56.4% of the cells were spread on the fibronectin-coated culture dish whereas no spreading activity was observed on the control dish. The determination of metabolites of the PI cycle was performed at a time point where approximately half of the cells had spread on the fibronectin matrix (45 min). The amount of water-soluble metabolites in the experimental groups relative to the controls is demonstrated in Fig. 2. In comparison with the control groups, significantly higher cpm were obtained in the InsP and InsPz fractions.
Short note
661
p < 0.001
-
“2% -
-
iE E
-
Fig. 2. Fold change of inositol phosphates in spreading vs nonspreading attached BHK-21 (C13) cells. IP, InsP; IP2, InsP*; IPS, Insq; IP,, InsP4. The elution conditions for InsP, InsS, and InsPSwere confirmed by the use of radiolabeled inositol phosphate standards. The elution conditions for InsP, were adopted from [13]. Values from two independent experiments each performed with three experimental and control cultures.
Similarly, the cpm in the fraction eluted with 1.0 M ammonium formate/O.1 M formic acid were significantly higher. The cpm in the GroPIns fraction were significantly lower (PcO.01, not shown). For Inositol and InsP3, no significant difference between experimental and control group could be shown. The percentage of deacylated PtdIns, PtsIns(4)P, and PtsIns(4,5)P2 relative to the total radioactivity of the lipid phase for experimental and control groups is shown in Table 1. It follows from Table 1 that the amount of PtdIns(4)P is 17.2% higher and that of PtdIns(4,5)P2 is 33.3% higher than the corresponding values of the control groups. Discussion
The results reported here indicate that the PI cycle is activated in spreading BHK-2 1 (C 13) cells in comparison with attached rounded cells. Among the watersoluble metabolites of the PI cycle, those metabolites eluting with Ins(4)P and Ins(1,4)Pz standards as well as metabolites eluted with 1.O M ammonium formate/O.1M formic acid were significantly elevated. The analytical method applied does not allow the exact identification of the different isomers of inositol phosphates described recently [16, 171.The high activity in the 1.0 A4ammonium formate/O.l M formic acid fraction most probably results from Ins(1 ,3,4,5)P4[ 131. The main metabolite in the 0.4 M ammonium formate/O.l M formic acid fraction, Ins(l,4)Pr, can result either from phospholipase C-mediated cleavage of
662 Short note
TABLE 1 Percentage cpm of deacylation products of inositol phospholipids in relation to the cpm of total inositol phospholipids Control GroPIns GroPIns(4)P GroPIns(4,5)P,
96.2k2.7 2.9kO.3 2.1kO.4
Cells
spreadon fibronectin 91.7f1.3* 3.4+0.2* 2.gf0.2*
*P
PtdIns(4)P or from metabolization of Ins(l,4,5)P3. In addition, the fraction may contain Ins(3,4)Pz derived from Ins(l,3,4)P3. The possible metabolites in the 0.2 M formate/O.1 M formic acid fraction are Ins(l)P resulting from cleavage of PtdIns as well as the isomers Ins(4)P and Ins(3)P derived from the respective inositol bisphosphates. The finding that in the fractions containing inositol trisphosphates the radioactivity was not significantly elevated is probably due to the rapid metabolization of Ins(l,4,5)P3. The 33.3% increase of PtdIns(4,5)P2 suggests an increased synthesis as a result of the accelerated turnover of PtdIns(4,5)P2 following phospholipase C cleavage. The increased turnover of PtdIns(4)P may be due to either cleavageor further phosphorylation. There are several possibilities to explain the activation of the PI cycle in spreading vs rounded cells. It is thought that the clustering of membrane receptors at the sites of adhesion of spreading cells is analogous to the clustering of membrane receptors of nonadherent cells [18]. Our findings are in accordance with this hypothesis since the PI cycle is activated in resting lymphocytes by agents known to cluster membrane receptors such as concanavalin A or anti-Ig antibodies [l9, 201. An alternate explanation would be the increase of surface area of spreading cells which would allow an increased uptake of nutrients and growth stimulating compounds from the medium [2l, 221. According to BenZe’ev [3], suspended3T6.fibroblasts resume protein synthesis when attached in a rounded configuration while the recovery of mRNA production and the rRNA and DNA synthesis as well as cell growth require extensive cell spreading. The latter two hypotheses would suggest that the stimulation of the PI cycle in spreading cells reflects a general increase of metabolic activity rather than a specific answer to the clustering of membrane receptors. It should be noted, however, that, in the present study, cells were kept as a monolayer under serumfree conditions prior to the spreading experiments. For this reason, the cellular metabolism is not reduced in a manner similar to that of suspended cells and an increased uptake of serum factors by spreading cells can be excluded. Since the PI cycle plays a central role in the control of cell proliferation [4], the activation of the PI cycle may be, at least in part, responsible for the mitogenic stimulation of cell spreading.
Short note 663 References 1. Watt, F. M. (1986) Trends Biochem. Sci. 11, 482-485. 2. Folkman, J., and Moscona, A. (1978) Nature (London) 273, 345-349. 3. Ben-Ze’ev, A. (1980) Cell 21, 365-372. 4. Berridge, M. J. (1987) Biochim. Biophys. Acta 907, 33-45. 5. Lassing, I., and Lindberg, U. (1988) Exp. Cell Res. 174, 1-15. 6. Macpherson, I. (1%3) J. Nat. Cancer Inst. 30, 795-806. 7. Grinell, F., Hays, D. G., and Minter, D. (1977) Exp. Cell Res. 110, 175-190. 8. Miekka, S. I., Ingham, K. C., and Menache, D. (1982) Thromb. Res. 27, 1-14. 9. Yamada, K. M., and Kennedy, D. W. (1984) J. Cell Biol. 99, 29-36. 10. Berridge, M. J., Heslop, J. P., Irvine, R. F., and Brown, K. D. (1984) Biochem. J. 222, 195-201. 11. Ellis, R. B., Galliard, T., and Hawthorne, J. N. (1%3) Biochem. J. 88, 125-131. 12. Bet-ridge, M. J., Dawson, R. M. C., Downes, C. P., Heslop, J. P., and Irvine, R. F. (1983) Biochem. J. 212, 473-482.
13. Batty, I. R., Nahorski, S. R., and Irvine, R. F. (1985) Biochem. J. 232, 211-215. 14. Irvine, R. F. (1986) in Phosphoinositides and Receptor Mechanisms, pp. 89-107, A. R. Liss, New York. 15. Downes, C. P., and Michell, R. H. (1981) Biochem. J. 198, 133-M. 16. Inhom, R. C., Bansal, V. S., and Majerus, P. W. (1987) Proc. Natl. Acad. Sci. USA 84, 2170-2174. 17. Dean, N. M., and Moyer, J. D. (1988) Biochem. J. 250,493-500. 18. Vasiliev, J. M., and Gelfand, I. M. (1982) Proc. Natl. Acad. Sci. USA 79, 2594-2597. 19. Taylor, M. V., Metcalfe, J. C., Hesketh, T. R., Smith, G. A., and Moore, J. P. (1984) Nature (London)
312, 462-465.
20. Bijesterbosch, M. K., Meade, C. J., Turner, G. A., and Klaus, G. G. B. (1985) Cell 41,999-1006. 21. O’Neill, C. H., Riddle, P. N., and Jordan, P. W. (1979) Cell 16, 909-918. 22. O’Neill, C., Jordan, P., and Ireland, G. (1986) Cell 44, 489-496. Received October 14, 1988
43-890336
F’rinted in Sweden