Cellular Signalling Vol. 4, No. 6, pp. 747-755, 1992. Printed in Great Britain.
0898-6568/92 $5.00 + 0.00 © 1992 Pergamon Pretts Lid
'CROSS-TALK' BETWEEN PHOSPHOLIPASE C AND ADENYLYL CYCLASE INVOLVES REGULATION OF G-PROTEIN LEVELS IN GH 3 RAT PITUITARY CELLS EYvrm~ J. PAULSSEN,RUTH H. PAULSSEN,* KAARE M. GAUTVIKand JAN O. GORDELADZE Institute of Medical Biochemistry, University of Oslo, Norway (Received 7 July 1992; and accepted 10 August 1992)
Abstract--We have investigated the possibility that adenylyl cyclase (AC) activity and membrane protein levels of the ~-subunits of the stimulatory and inhibitory G-proteins of AC (Gsa and Gi.#) in cultured prolactinproducing rat pituitary adenoma cells (GH 3 cells) are modulated by phospholipase C (PLC)-generated second messengers. Pretreatment of cells (6-48 h) with ionomycin (1 t~M) or l-oleoyl-2-acetylglyceroi (OAG; 1/aM) showed that ionomycin regulated Gsa levels in a time-dependent, biphasic manner; a two-fold increase followed a 40% initial reduction, while OAG lowered G~t levels by more than 50% at all time-points. Gi.2~ levels remained unchanged by both pretreatments. OAG, but not ionomycin, increased basal AC activity without increasing enzyme protein levels. Alterations in AC responsiveness to peptide hormones (e.g. thyroliberin and vasoactive intestinal peptide) correlated to membrane G, protein ct-subunit content. These results demonstrate the involvement of G-protein translation regulation as one mechanism of 'cross-talk' between the PLC- and AC-dependent signalling pathways. Key words: G-protein, adenylyl cyclase, GH cells, ionomycin, l-oleoyl-2-acetylglycerol, phospholipase C, 'cross-talk'.
INTRODUCTION GH3 CELLSare clonal rat pituitary tumour cells that produce and spontaneously secrete prolactin and growth hormone to the culture medium [1]. Hormone synthesis and secretion are subject to regulation by a variety of physiological and pharmacological agents, such as the neuropeptides thyroliberin (TRH), vasoactive intestinal peptide (VIP) and somatostatin (SRIF) [2]. The main mode of action of TRH is activation of phosphoinositide turnover by phospholipase C (PLC) [3-5]. The coupling of TRH to adenylyl cyclase (AC) via G s is of unknown biological importance [6]. VIP activates AC through coupling to Gs, whereas * Author to whom correspondenceshould be addressed. Abbreviations: AC--adenylyl cyclase; DAG----diacylglycerol; Gpp(NH)p-lguanosine 5'-LBy-imido]trisphosphate; IP--inositol phosphate; OAG--l-oleoyl-2.acetylglycerol; PIP2--phosphatidylinositol-4,5-bisphosphate; PKC--protein kinase C; PLC--phospholipase C; SRIF---somatostatin; TRH--thyroliberin;VIP--vasoactive intestinal peptide. 747
SRIF inhibits AC activity through a Gi.2-mediated pathway [2,6,7]. PLC catalyses the hydrolysis of phosphatidyl-inositol-4,5-bisphosphate (PIP2) into inositol 1,4,5-trisphophate (IP3) and 1,2-diacylglycerol (DAG), which both act as second messengers in signal transduction (for review, see [8,9]). Whereas DAG is believed to be the main activator of protein kinase C (PKC), a biphasie increase of intracellular Ca 2+ follows the formation of IP 3. Although independent, these pathways act synergistically in conveying PLC effects [9]. Ionomycin, a calcium ionophore that raises intracellular Ca 2+ concentration and thus mimics the IP 3 pathway, has been used in several studies [10-12]. 1-Oleoyl-2-acetylglycerol (OAG) and phorbol esters have been shown to activate PKC in a manner parallel to DAG, and thus may resemble the second pathway of PLC action [13, 14]. Interplay of G-protein-coupled second messenger systems, 'cross-talk', has been shown
748
E.J. PAU~S~Net al.
to be o f major importance in cellular response to extracellular signals [15]. Both T R H and VIP have been shown to enhance G ~ protein levels in the G H 3 cells [16]. Hence, alterations of steady-state levels of G-protein subunits may thus change the responsiveness to hormone signals regulating hormone synthesis and secretion in G H 3 cells. In this work the aim was to study the influence o f the dual pathways of phosphoinositide turnover by PLC on G H 3 cell membrane levels of G-proteins involved in AC activation (G,~, Gi.2~) and the responsiveness of AC to stimulation by T R H and VIP and inhibition by SRIF. MATERIALS AND METHODS Cell culture GH 3 cells [1] were grown for 5 days subsequent to sub-cultivation in plastic tissue culture flasks containing Ham's F-10 medium [17] supplemented with 6.5% horse and 3% foetal calf sera at 37°C in a humidified atmosphere of 95% air and 5% CO2 [18,19]. Penicillin (50IU/ml) and streptomycin (50 #g/ml; GIBCO), and amphotericin B (2.5/~g/ml; Flow Laboratories) were added to the culture medium. Culture medium was changed every 2-3 days. Experimental design The experiments were carried out such that all cells were harvested simultaneously at the end of the experiment. Ionomycin (1/an; Sigma) or OAG (1 /~M; Sigma) was added 48, 24, 12 and 6 h before cell harvest. Medium was changed at the start of the experiment and 24 h before the cells were harvested. Preparation of subcellular particulate fractions The medium was removed and the cells scraped in ice-cold 150raM NaCI, 10raM Tris--HCl, pH7.5 and pelleted (300g, 10 rain, 4°C). Crude membrane fractions were prepared as previously described [20]. Adenylyl cyclase assay AC activity was measured in 20-#i afiquots of crude subceilular fractions which were diluted in homogenization buffer to obtain 40-55/~g of protein per assay tube according to Pauluen et al. [20]. The total incubation volume was 50#1 and contained ! mM ATP (including 1.2 × 106 c.p.m, of [~-32P]ATP
(Amersham), 10/tM GTP, 2.8mM M g C I 2 , 1.4mM EDTA, I mM cAMP [containing approx. 5 x 10 3 c.p.m, of [8-3H]cAMP (Amersham)], 20 mM creatine phosphate, 0.2mg/ml creatine kinase, 0.02mg/ml myokinase and 25mM Tris-HCl, pH7.4, in the absence or presence of TRH (1/zM), VIP (1 #M), SRIF (I/tM) or guanosine 5'-Lg~,-imido]trisphosphate [Gpp(NH)p] (20 #M). Incubations were carried out at 35°C for 20 min. Reactions were stopped with 0.1ml of a solution comprising 10mM cAMP, 40 mM ATP and 1% sodium dodecyl sulphate. The [32p]cAMP formed and the [3H]cAMP added to monitor recovery (65-80%) were isolated as described previously using combined Dowex and aluminium oxide chromatography [21]. The enzyme activity was linear with time up to 60 min and protein concentration up to 150/zg protein (data not shown). PLC assay This method was adapted from the one previously described [22]. Aliquots (20/d) of diluted crude membrane suspensions (40-55 #g protein) in 10 mM Tris--HCl, pH 7.4, I mM EDTA, were mixed with 10/zl incubation mixture (100 mM Tris--HC1, pH 6.5, 400/zM GTP, 2.2 mM CaCI z, I mM MgC12and 10/~1 of TRH (10/zM) or Gpp(NH)p (400/~M) in 2.4-ml microcentrifuge tubes on ice). Three microlitres (42,000 c.p.m.) of a [3H]PIP2(New England Nuclear) stock solution in 2% sodium cholate was added to each tube, and incubation carried out at 35°C for 5 rain. The reaction was stopped by adding in succession 150/d CHCI3/CH3OH/HCI (1:2:0.02), 50/zl CHCI3 and 50/zl 2M KCI. After vortexing and phase separation at 5000g in a microcentrifuge, 100-/d aliquots of the aqueous layers were counted in a liquid scintillation counter. In the controls, approximately 2% of the radioactivity was retained in the aqueous phase. The enzyme activity was linear with time up to 20 min and protein concentration up to lO0 #g protein (data not shown). Western blotting and immunostaining Membrane proteins were prepared from crude membrane fractions and analysed on sodium dodecyl sulphate-polyacrylamide gel electrophoresis prior to transfer and immunostaining as described previously [20]. The quality and quantity of the electrophoresis and transfer were ascertained by Coomassie Blue staining of parallel filters (data not shown). The antisera against the stimulatory G-protein ~,-subunit G,~ (denominated RM) and the inhibitory G-protein r,-subunits Gi.l~/Gi.2~ (denominated AS) of which the former is not detected in G H 3 cells [20] have previously been described [23] and were used at
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Gsa G~-2a FIG. 1. Western blot analysis and quantification of Gs~t- and Gi.2~t-subunit levels in GH 3 cells treated with ionomycin and OAG. Membrane protein from GH 3 cells treated with 1 pM ionomycin (A) or 1 pM OAG (B) was analysed by immunoblotting and detection with specific antisera against Gs~t protein subunit (RM) and Gi.~/G~.2~t (AS) as described in Materials and Methods. Semiquantitative estimations of the relative intensities of the appropriate signals were determined by densitometric scanning of the exposed autoradiograms. The basal value at each time-point defines the relative value of 1. The experiment was carried out twice with similar results. 749
'Cross-talk' between PLC and AC in GH 3 cells
1:200-1:400 final dilution. The specificity of the antiserum is reported to be as follows [23]: G,ct antiserum (RM) is specific for both known ~-subunits of G~ (52,000 and 45,000 M,). Gi.l~,/Gi.2~t antiserum (AS) detects both Gi. l and Gi. 2 ~t-subunits, of which the former is not found in the GH 3 cells [20]. Semiquantitative data from autoradiograms were obtained with a universal densitometer (Vitatron TLD 100).
RESULTS
Alterations of the G-protein ot-subunit levels by ionomycin and OAG To ascertain the effect of ionomycin and D A G on the levels of the G-protein ~t-subunits of Gs and Gi.2, G H 3 cells were grown in the presence o f 1/~M ionomycin or 1 #M O A G for 6-48 h as described in Materials and Methods. Untreated cells served as controls. Cell membrane protein was analysed by immunoblotting and G-protein levels were quantified by densitometric scanning o f immunoblots (Fig. 1). The following results were obtained. Ionomycin pretreatment resulted in a transient decrease of the protein levels of the ~-subunit of stimulatory G-protein, Gs (Fig. 1A) up to 12h of incubation. G,~ levels decreased to approximately 50% o f untreated controls after 6 h incubation with ionomycin, and an increase to 200% of controls was
751
observed as 48 h. In comparison, levels of Gi.2~ remained unchanged in response to ionomycin pretreatment (Fig. IA). In contrast, O A G reduced Gs0t protein levels to approximately 20% of controls after 6 h incubation, followed by a slight subsequent increase to less than 50% of controls (Fig. 1B). Protein levels of Gi.2~ remained again unchanged. Levels of Go~ protein also remained constant under both pretreatment schemes (data not shown).
Basal and modulated AC responsiveness in GH a cells pretreated with ionomycin and OAG To illustrate the time-dependent effect of continuous ionomycin or D A G exposure on the sensitivity of the membrane signalling systems involving AC, cell membrane preparations were analysed for basal AC activity and AC response to T R H , VIP, SRIF and Gpp(NH)p as indicated in Materials and Methods. The results of hormone stimulation of AC activity are shown in Fig. 2 as net modulation (actual activity minus basal), as percentage of controls (100%). Figure 2 shows the effect of prolonged ionomycin (A) or O A G (B) exposure on AC responsiveness. Basal AC activity was not altered during the time-course of 48h ionomycin pretreatment (Table 1), whereas O A G caused a significant
TABLE 1. BASAL AC ^ND PLC ACTIVITIESIN GH 3 CELLSDURING PROLONGED PRETREATMENT WITH IONOMYCIN OR OAG Treatment group
6h
12h
48h
Ionomycin OAG
5.3±0.1 8.3±0.4*
5.2±0.1 7.4±0.1"
5.2±0.3 6.3±0.4*
AC
Ionomycin OAG
5.1±0.3 8.3±1.1"
5.3±0.1 9.4±0.8*
5.3±0.2 8.8±0.8*
PLC
GH 3 cells were grown in the presence of I/zM ionomycin or 1 /~M OAG for up to 48 h. Enzyme activities were measured in cell membrane fractions as indicated in Materials and Methods. AC activity is shown as pmol cAMP/rag protein/rain. Basal AC activity in untreated cells (controls) was 5.2+0.1. PLC activity is given as 103 c.p.m. IP3/mg protein/min. Basal PLC activity in untreated cells (controls) was 5.0+0.2. * P = ~, < 0.05, Wileoxon rank test, stimulated vs control. CELLS416-d
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Treatment time (hrs) FIG. 2. The effect of ionomycin and OAG on modulated AC activity in GH3 cells. AC activity was measured in membranes from cells grown in the presence of 1/aM ionomycin (A) and 1/aM OAG (B) as described in Materials and Methods. Hormone or guanyl nucleotide effects on AC were measured in the presence of either TRH (1/aM), VIP (1/aM), SRIF (10/aM) or Gpp0NH)p (20/aM) in the enzyme assay. The results are shown as percentage modulatory effect on AC [stimulation by TRH, VIP and Gpp(NH)p, inhibition by SRIF] of control values throughout the pretreatment period (6--48h), as a mean of triplicates. *P = a < 0.05 (significant difference from controls; Wilcoxon rank test).
al.
increase of basal AC activity, most notable after 6 h. Forskolin-stimulated AC activity was not changed during the exposure to ionomycin or O A G (Table 2). As shown in Fig. 2A, TRH-stimulated AC activity was transiently reduced by 60% in G H 3 cells treated with ionomycin. The reduction was followed by an increase in AC responsiveness to approximately 140% of controls. In parallel, VIP-elicited AC activity was reduced by 20% after 6 - 1 2 h and increased up to 150% after 48 h. Gpp(NH)p-induced and SRIF-inhibited AC activity remained unchanged for the whole pretreatment period. Thus, the T R H - and VIP-sensitive AC showed a biphasic pattern after ionomycin pretreatment which is in accordance with the obtained protein results (Fig. 1). In comparison, AC responsiveness to T R H was almost abolished by pretreatment of G H 3 cells with O A G (Fig. 2B). The reduction was close to linear with time, and significant already after 6 h. Responsiveness to inhibition by SRIF displayed a transient reduction (to approximately 40% of controls) at 6 h. Gpp(NH)p-elicited AC activity showed a smaller, but significant reduction of approximately 15-20%, for the whole time-period. AC responsiveness to VIP stimulation changed in an irregular pattern due to O A G pretreatment, but was generally reduced (at 6 and 48 h).
GH3 CELLS DURING PROLONGED PRETREATMENT WITH IONOMYCIN OR O A G
TABLE 2. FORSKOLIN-STIMULATED A C ACTIVITY IN
Treatment group
6h
12 h
48 h
Ionomycin OAG
11.7+0.1 10.7+0.4
12.6+0.1 11.0+0.t
13.6+0.3 13.2+0.4
AC
GH 3 cells were grown in the presence of 1/aM ionomycin or 1 /aM OAG for up to 48 h. Forskolin-stimulated AC activity was measured in cell membrane fractions as indicated in Materials and Methods. AC activity is shown as pmol cAMP/rag protein/rain. Forskolin-stimulatecl AC activity in non-stimulated cells (control) was 12.2+0.2. * P = a < 0.05, Wileoxon rank test, stimulated vs control.
'Cross-talk' between PL C and A C in G H 3 cells
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cant increase of basal enzyme activity. PLC activation by TRH (Fig. 3) transiently decreased about 35% at 6h, followed by an increase of about 55% at 48 h. Gpp(NH)p-enhanced PLC activity increased gradually to approximately 150% of controls after 48 h incubation in the presence of ionomycin. Pretreatment of GH 3 cells with OAG (Fig. 3B) did not result in a significant change in Gpp(NH)p-stimulated PLC activity, whereas TRH-modulated PLC activity decreased dramatically to 25 and 10% of controls after 12 and 48 h, respectively.
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FIG. 3. The effect of ionomycin and OAG on modulated PLC activity in GH3 cells. PLC activity was measured in membranes from cells grown in the presence of 1/aM ionomycin (A) or 1 # OAG (B) as described in Materials and Methods. PLC activity was estimated in the presence of 42,000 c.p.m. [~H]PIP2, 20/aM GTP and 100/aM Ca2+. Enzyme activities are expressed as l03 c.p.rn. IPs mostly IP3 and IP, formed/mg protein/rain and shown as mean + S.D. of triplicate determinations. Stimulations of PLC activity were achieved (for 5 min at 35°C) in the presence of either 1/aM TRH or 20#M Gpp(NH)p. The assay was repeated twice with similar results. *P = ~ < 0.05, Wilcoxon rank test, stimulated vs control.
Basal and modulated PLC activity in GH a cells treated with ionomycin and OAG For comparison, GH a membrane preparations from pretreated cells with ionomycin and OAG were also analysed for basal PLC activity and PLC responsiveness to TRH and Gpp(NH)p as indicated in Materials and Methods. As shown in Table 1, basal PLC activity was not changed by ionomycin pretreatment, whereas OAG caused a signifi-
In the present study we have demonstrated that ionomycin and OAG regulate G-protein steady-state levels as well as PLC and AC responsiveness in GH 3 cells in a specific manner. Ionomycin, which increases intraceUular Ca 2+ mainly by mobilizing Ca 2+ from intracellular stores [12], transiently reduced the levels of the g-subunits of the stimulatory G-protein, Gs, which was paralleled by a transient decrease of AC responsiveness to stimulation by TRH and VIP. Since basal and forskolin-stimulated AC activity was unchanged in response to pretreatment with ionomycin, it appears that the response to hormones is probably not due to changes in membrane content of AC's catalytic unit. A similar transient alteration of PLC responsiveness to TRH is, however, also observed, which does not exclude modulatory actions at the receptor level. The alterations in TRH and VIP stimulation of AC were approximately parallel, but the effect was more pronounced for TRH activation. This discrepancy was also observed through inhibition of G, coupling by antisense RNA expression and antibody blocking [6], and it may be due to differences in the ability of the receptors for TRH and VIP to utilize the available G,. The existence of separate G,~ pools has also been postulated [24,25]. Contrary to ionomycin, OAG alters basal
754
E.J. PAUI.S~N et al.
AC activity. However, forskolin-stimulated AC activity remained unchanged, indicating unaltered cellular quantities of AC holoenzyme. Increased basal AC activity may be explained by enhancement of AC by phosphorylation by PKC. Phorbol esters have been shown to enhance basal AC activity through Gi-inactivation in GH4CI cells [26] and purified AC protein is shown to be a substrate for PKC [27]. These may be important mechanisms of cross-talk between the AC and PLC membrane signal systems. Decreased levels of G,~ protein due to OAG pretreatment were followed by a slight reduction of AC responsiveness to VIP. The degree of reduction was somewhat less than would be expected by the large decrease of G,a levels, possibly due to the increased sensitivity of PKC-stimulated AC enzyme to G s coupling, as previously postulated [15]. The almost total abolition of AC responsiveness to TRH following pretreatment with OAG was parallel to the reduction in TRH-stimulated PLC activity. This phenomenon resembles the homologous densensitization of TRH responsive AC activation and phosphoinositide turnover following TRH pretreatment of GH3 cells alone [28]. The latter is possibly due to a PKC-dependent reduction in TRH receptor expression [29]. Inhibition of AC activity by SRIF has been shown to be linked through a pathway involving coupling to Gi.z [6]. The observation that AC responsiveness to SRIF is unaltered parallel to the lack of changes in steady-state levels of Gi.2g is in agreement with our previous results. The absence of alterations in the ability of the non-hydrolysable GTP-analogne Gpp(NH)p to stimulate AC activity, even under conditions that alter G,a content, is also in agreement with previous observations [6]. Such findings call for a revised model of Gpp(NH) action or point to altered post-translational G-protein modifications. Several reports have discussed the interplay between AC- and PLC-dependent membrane signalling systems (for reviews, see [15,30,31]). The enzyme action of PKC has been shown to
regulate the coupling sensitivity of G-protein-mediated signals, involving their receptors [32], G-proteins [26, 33-36] and effector enzymes [27]. Recent reports have focused on the involvement of regulated G-protein expression in the cross-talk between different signal transduction pathways. Indeed, alteration of steady-state levels of G-proteins may be an important mode of action for both peptide [37-41] and steroid hormones [42,43]. Acknowledgements---We thank DR A. M. SPIEGEL for supplying the antisera against the various G-protein ~-subunits. This work received support from the Norwegian Cancer Society, the Norwegian Research Council for Science and the Humanities, the Anders Jahre Foundation for the Promotion of Sciences, and the Nordic Insulin Foundation, Copenhagen, Denmark. The authors are grateful for technical assistance from Ms A. K. F~LDm~M, Ms H. TmLmE~ and Ms A. Bm.sExa.
REFERENCES I. Tashjian A. H. Jr (1979) Meth. Enzym. 58, 527-535. 2. Gautvik K. M., Bjzro T., Slctholt K., Ostbcrg B, C., Sand O., Torjcsen P., Gordeladzc J. O., Iverscn J.-G. and Haug E. (1988) Molecular Mechanisms in Secretion. Alfred Benzon Symposium 25 (Thorn N. A., Treiman M. and
Pedersen O. H., Eds), pp. 211-227. Munksgaard, Copenhagen. 3. Drust D. S. and Martin T. F. J. (1984) J. biol. Chem. 259, 14,520-14,530. 4. Gershengorn M. C. and Thaw C. (1985) Endocrinology 116, 591-596. 5. Straub (3. E. and Gershengorn M. C. (1986) J. biol. Chem. 261, 2712-2717. 6. Paulssen R. H., Paulssen E. J., Gautvick K. M. and Gordeladze J. O. (1992) Fur. J. Biochem. 204, 413-418.
7. Bjzro T., Sand O., ~stberg B. C., Gordeladze J. O., Torjesen P., Gautvik K. M. and Haug E. (1990) Biosci. Rep. 10, 189-199. 8. Drummond A. H. (1986) J. exp. Biol. 124, 337-358. 9. Bcrridge M. J. (1987) A. Rev. Biochem. 56, 159-193. 10. Liu C. and Hermann T. E. 0978) J. biol. Chem. 253, 5892-5894. 11. Toeplitz B. K., Cohen A. I., Funke P. T.,Parker W. L. and Gougoutas J. Z. (1979) J. Am. Chem. Soc. 101, 3344-3353.
'Cross-talk' between PLC and AC in GH3 cells 12. Fasolato C. and Pozzan T. (1989) J. biol. Chem. 264, 19,630-19,636. 13. Nishizuka Y. (1984) Nature 308, 693-698. 14. Nishizuka Y. (1986) Science 233, 305-312. 15. Bouvier M. (1990) Ann. N. Y. Acad. Sci. 594, 120-129. 16. Paulssen E. J., Paulsscn R. H., Gautvik K. M. and Gordeladze J. O. (1992) Biochem. Pharmac. 44, 471-477. 17. Ham R. G. (1963) Expl Cell Res. 29, 515-526. 18. Gautvik K. M., Gordeladz¢ J. O., Jahnsen T., Haug E., Hansson V. and Lystad E. (1983) J. biol. Chem. 258, 10,304-10,311. 19. Bjoro T., Haug E., Sand O. and Gautvik K. M. (1984) Molec. cell. Endocr. 37, 41-50. 20. Paulsscn E. J., Paulsscn R. H., Haugen T. B., Gautvik K. M. and Gordeladz¢ J. O. (1991) Molec. cell. Endocr. 76, 45-53. 21. Salomon Y., Londos C. and RodbcU M. (1974) ,4nalyt. Biochem. 58, 541-548. 22. Jackowski S., Rettenmier C. W., Sherr C. J. and Rock C. O. (1986) J. biol. Chem. 261, 4978-4985. 23. Simonds W. F., Goldsmith P. K., Codina J., Unson C. G. and Spiegel A. M. (1989) Proc. natn. Acad. Sci. U.S.A. 86, 7809-7813. 24. Ishikawa Y., Bianchi C., Nadal-Ginard B. and Homcy C. J. (1990) J. biol. Chem. 265, 8458-8462. 25. Raymond J. R., Albcrs F. J., Middleton J. P., Lefkowitz R. J., Caron M. G., Obcid L. M. and Dennis V. W. (1990) J. biol. Chem. 266, 372-379. 26. Gordeladze J. O., Bjoro T., Torjesen P. A., Ostbcrg B. C., Haug E. and Gautvik K. (1989) Eur. J. Biochem. 183, 379-406. 27. Yoshimasa T., Sibley D. R., Bouvier M., Lefkowitz R. J. and Caron M. G. (1987) Nature 327, 67-70.
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28. Drummond A. H. (1985) Nature 315, 752-755. 29. Fujimoto J., Straub R. E. and Gcrshengorn M. C. (1991) Molec. Endocr. 5, 1527-1532. 30. Houslay M. D. (1991) Eur. J. Biochem. 195, 9-27. 31. Di Marzo V., Galadari S. H. I., Tippins J. R. and Morris H. R. (1991) Life Sci. 49, 247-259. 32. Bouvier M., Guilbault N. and Bonin H. (1991) FEBS Lett. 279, 243-248. 33. Garcia-S~inz J. A. and Guti6rrez-Venegas G. (1989) FEBS Lett. 257, 427-430. 34. Pyne N. J., Murphy G. J., Milligan G. and Houslay M. D. (1989) FEBS Lett. 243, 77-82. 35. Bushfield M. and Houslay M. D. (1990) Biochem. Soc. Trans. 18, 456. 36. Hern/mdez-Sotomayor S. M. T., Macias-Silva M., Malbon C. C. and Garcia-Sfiinz J. A. (1991) Am. J. Physiol. 260, C259-C265. 37. Lee R. T., Brock T. A., Tolman C., Bloch K. D., Seidman J. G. and Ncer E. J. (1989) FEBS Lett. 249, 139-142. 38. Hadcock J. R., Ros M., Watkins D. C. and Malbon C. C. (1990) J. biol. Chem. 265, 14,784-14,790. 39. Reithmann C., Gierschik P., Werdan K. and Jakobs K. H. (1990) Br. J. clin. Pharmac. 30, 118S-120S. 40. Saunier B., Dib K., Dclemer B., Jacquemin C. and Corr6ze C. (1990) J. biol. Chem. 265, 19,942-19,946. 41. Chan S. D. H., Strewler G. J. and Nissenson R. A. (1990) J. biol. Chem. 265, 20,081-20,084. 42. Chang F. and Bourne H. R. (1987) Endocrinology 121, 1711-1715. 43. Bouvier C., Lagac6 G. and Collu R. (1991) Molec. cell. Endocr. 79, 65-73.
0898-6568/92 $5.00 + 0.00 © 1992 Pergamon Pretts Lid
'CROSS-TALK' BETWEEN PHOSPHOLIPASE C AND ADENYLYL CYCLASE INVOLVES REGULATION OF G-PROTEIN LEVELS IN GH 3 RAT PITUITARY CELLS EYvrm~ J. PAULSSEN,RUTH H. PAULSSEN,* KAARE M. GAUTVIKand JAN O. GORDELADZE Institute of Medical Biochemistry, University of Oslo, Norway (Received 7 July 1992; and accepted 10 August 1992)
Abstract--We have investigated the possibility that adenylyl cyclase (AC) activity and membrane protein levels of the ~-subunits of the stimulatory and inhibitory G-proteins of AC (Gsa and Gi.#) in cultured prolactinproducing rat pituitary adenoma cells (GH 3 cells) are modulated by phospholipase C (PLC)-generated second messengers. Pretreatment of cells (6-48 h) with ionomycin (1 t~M) or l-oleoyl-2-acetylglyceroi (OAG; 1/aM) showed that ionomycin regulated Gsa levels in a time-dependent, biphasic manner; a two-fold increase followed a 40% initial reduction, while OAG lowered G~t levels by more than 50% at all time-points. Gi.2~ levels remained unchanged by both pretreatments. OAG, but not ionomycin, increased basal AC activity without increasing enzyme protein levels. Alterations in AC responsiveness to peptide hormones (e.g. thyroliberin and vasoactive intestinal peptide) correlated to membrane G, protein ct-subunit content. These results demonstrate the involvement of G-protein translation regulation as one mechanism of 'cross-talk' between the PLC- and AC-dependent signalling pathways. Key words: G-protein, adenylyl cyclase, GH cells, ionomycin, l-oleoyl-2-acetylglycerol, phospholipase C, 'cross-talk'.
INTRODUCTION GH3 CELLSare clonal rat pituitary tumour cells that produce and spontaneously secrete prolactin and growth hormone to the culture medium [1]. Hormone synthesis and secretion are subject to regulation by a variety of physiological and pharmacological agents, such as the neuropeptides thyroliberin (TRH), vasoactive intestinal peptide (VIP) and somatostatin (SRIF) [2]. The main mode of action of TRH is activation of phosphoinositide turnover by phospholipase C (PLC) [3-5]. The coupling of TRH to adenylyl cyclase (AC) via G s is of unknown biological importance [6]. VIP activates AC through coupling to Gs, whereas * Author to whom correspondenceshould be addressed. Abbreviations: AC--adenylyl cyclase; DAG----diacylglycerol; Gpp(NH)p-lguanosine 5'-LBy-imido]trisphosphate; IP--inositol phosphate; OAG--l-oleoyl-2.acetylglycerol; PIP2--phosphatidylinositol-4,5-bisphosphate; PKC--protein kinase C; PLC--phospholipase C; SRIF---somatostatin; TRH--thyroliberin;VIP--vasoactive intestinal peptide. 747
SRIF inhibits AC activity through a Gi.2-mediated pathway [2,6,7]. PLC catalyses the hydrolysis of phosphatidyl-inositol-4,5-bisphosphate (PIP2) into inositol 1,4,5-trisphophate (IP3) and 1,2-diacylglycerol (DAG), which both act as second messengers in signal transduction (for review, see [8,9]). Whereas DAG is believed to be the main activator of protein kinase C (PKC), a biphasie increase of intracellular Ca 2+ follows the formation of IP 3. Although independent, these pathways act synergistically in conveying PLC effects [9]. Ionomycin, a calcium ionophore that raises intracellular Ca 2+ concentration and thus mimics the IP 3 pathway, has been used in several studies [10-12]. 1-Oleoyl-2-acetylglycerol (OAG) and phorbol esters have been shown to activate PKC in a manner parallel to DAG, and thus may resemble the second pathway of PLC action [13, 14]. Interplay of G-protein-coupled second messenger systems, 'cross-talk', has been shown
748
E.J. PAU~S~Net al.
to be o f major importance in cellular response to extracellular signals [15]. Both T R H and VIP have been shown to enhance G ~ protein levels in the G H 3 cells [16]. Hence, alterations of steady-state levels of G-protein subunits may thus change the responsiveness to hormone signals regulating hormone synthesis and secretion in G H 3 cells. In this work the aim was to study the influence o f the dual pathways of phosphoinositide turnover by PLC on G H 3 cell membrane levels of G-proteins involved in AC activation (G,~, Gi.2~) and the responsiveness of AC to stimulation by T R H and VIP and inhibition by SRIF. MATERIALS AND METHODS Cell culture GH 3 cells [1] were grown for 5 days subsequent to sub-cultivation in plastic tissue culture flasks containing Ham's F-10 medium [17] supplemented with 6.5% horse and 3% foetal calf sera at 37°C in a humidified atmosphere of 95% air and 5% CO2 [18,19]. Penicillin (50IU/ml) and streptomycin (50 #g/ml; GIBCO), and amphotericin B (2.5/~g/ml; Flow Laboratories) were added to the culture medium. Culture medium was changed every 2-3 days. Experimental design The experiments were carried out such that all cells were harvested simultaneously at the end of the experiment. Ionomycin (1/an; Sigma) or OAG (1 /~M; Sigma) was added 48, 24, 12 and 6 h before cell harvest. Medium was changed at the start of the experiment and 24 h before the cells were harvested. Preparation of subcellular particulate fractions The medium was removed and the cells scraped in ice-cold 150raM NaCI, 10raM Tris--HCl, pH7.5 and pelleted (300g, 10 rain, 4°C). Crude membrane fractions were prepared as previously described [20]. Adenylyl cyclase assay AC activity was measured in 20-#i afiquots of crude subceilular fractions which were diluted in homogenization buffer to obtain 40-55/~g of protein per assay tube according to Pauluen et al. [20]. The total incubation volume was 50#1 and contained ! mM ATP (including 1.2 × 106 c.p.m, of [~-32P]ATP
(Amersham), 10/tM GTP, 2.8mM M g C I 2 , 1.4mM EDTA, I mM cAMP [containing approx. 5 x 10 3 c.p.m, of [8-3H]cAMP (Amersham)], 20 mM creatine phosphate, 0.2mg/ml creatine kinase, 0.02mg/ml myokinase and 25mM Tris-HCl, pH7.4, in the absence or presence of TRH (1/zM), VIP (1 #M), SRIF (I/tM) or guanosine 5'-Lg~,-imido]trisphosphate [Gpp(NH)p] (20 #M). Incubations were carried out at 35°C for 20 min. Reactions were stopped with 0.1ml of a solution comprising 10mM cAMP, 40 mM ATP and 1% sodium dodecyl sulphate. The [32p]cAMP formed and the [3H]cAMP added to monitor recovery (65-80%) were isolated as described previously using combined Dowex and aluminium oxide chromatography [21]. The enzyme activity was linear with time up to 60 min and protein concentration up to 150/zg protein (data not shown). PLC assay This method was adapted from the one previously described [22]. Aliquots (20/d) of diluted crude membrane suspensions (40-55 #g protein) in 10 mM Tris--HCl, pH 7.4, I mM EDTA, were mixed with 10/zl incubation mixture (100 mM Tris--HC1, pH 6.5, 400/zM GTP, 2.2 mM CaCI z, I mM MgC12and 10/~1 of TRH (10/zM) or Gpp(NH)p (400/~M) in 2.4-ml microcentrifuge tubes on ice). Three microlitres (42,000 c.p.m.) of a [3H]PIP2(New England Nuclear) stock solution in 2% sodium cholate was added to each tube, and incubation carried out at 35°C for 5 rain. The reaction was stopped by adding in succession 150/d CHCI3/CH3OH/HCI (1:2:0.02), 50/zl CHCI3 and 50/zl 2M KCI. After vortexing and phase separation at 5000g in a microcentrifuge, 100-/d aliquots of the aqueous layers were counted in a liquid scintillation counter. In the controls, approximately 2% of the radioactivity was retained in the aqueous phase. The enzyme activity was linear with time up to 20 min and protein concentration up to lO0 #g protein (data not shown). Western blotting and immunostaining Membrane proteins were prepared from crude membrane fractions and analysed on sodium dodecyl sulphate-polyacrylamide gel electrophoresis prior to transfer and immunostaining as described previously [20]. The quality and quantity of the electrophoresis and transfer were ascertained by Coomassie Blue staining of parallel filters (data not shown). The antisera against the stimulatory G-protein ~,-subunit G,~ (denominated RM) and the inhibitory G-protein r,-subunits Gi.l~/Gi.2~ (denominated AS) of which the former is not detected in G H 3 cells [20] have previously been described [23] and were used at
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Gsa G~-2a FIG. 1. Western blot analysis and quantification of Gs~t- and Gi.2~t-subunit levels in GH 3 cells treated with ionomycin and OAG. Membrane protein from GH 3 cells treated with 1 pM ionomycin (A) or 1 pM OAG (B) was analysed by immunoblotting and detection with specific antisera against Gs~t protein subunit (RM) and Gi.~/G~.2~t (AS) as described in Materials and Methods. Semiquantitative estimations of the relative intensities of the appropriate signals were determined by densitometric scanning of the exposed autoradiograms. The basal value at each time-point defines the relative value of 1. The experiment was carried out twice with similar results. 749
'Cross-talk' between PLC and AC in GH 3 cells
1:200-1:400 final dilution. The specificity of the antiserum is reported to be as follows [23]: G,ct antiserum (RM) is specific for both known ~-subunits of G~ (52,000 and 45,000 M,). Gi.l~,/Gi.2~t antiserum (AS) detects both Gi. l and Gi. 2 ~t-subunits, of which the former is not found in the GH 3 cells [20]. Semiquantitative data from autoradiograms were obtained with a universal densitometer (Vitatron TLD 100).
RESULTS
Alterations of the G-protein ot-subunit levels by ionomycin and OAG To ascertain the effect of ionomycin and D A G on the levels of the G-protein ~t-subunits of Gs and Gi.2, G H 3 cells were grown in the presence o f 1/~M ionomycin or 1 #M O A G for 6-48 h as described in Materials and Methods. Untreated cells served as controls. Cell membrane protein was analysed by immunoblotting and G-protein levels were quantified by densitometric scanning o f immunoblots (Fig. 1). The following results were obtained. Ionomycin pretreatment resulted in a transient decrease of the protein levels of the ~-subunit of stimulatory G-protein, Gs (Fig. 1A) up to 12h of incubation. G,~ levels decreased to approximately 50% o f untreated controls after 6 h incubation with ionomycin, and an increase to 200% of controls was
751
observed as 48 h. In comparison, levels of Gi.2~ remained unchanged in response to ionomycin pretreatment (Fig. IA). In contrast, O A G reduced Gs0t protein levels to approximately 20% of controls after 6 h incubation, followed by a slight subsequent increase to less than 50% of controls (Fig. 1B). Protein levels of Gi.2~ remained again unchanged. Levels of Go~ protein also remained constant under both pretreatment schemes (data not shown).
Basal and modulated AC responsiveness in GH a cells pretreated with ionomycin and OAG To illustrate the time-dependent effect of continuous ionomycin or D A G exposure on the sensitivity of the membrane signalling systems involving AC, cell membrane preparations were analysed for basal AC activity and AC response to T R H , VIP, SRIF and Gpp(NH)p as indicated in Materials and Methods. The results of hormone stimulation of AC activity are shown in Fig. 2 as net modulation (actual activity minus basal), as percentage of controls (100%). Figure 2 shows the effect of prolonged ionomycin (A) or O A G (B) exposure on AC responsiveness. Basal AC activity was not altered during the time-course of 48h ionomycin pretreatment (Table 1), whereas O A G caused a significant
TABLE 1. BASAL AC ^ND PLC ACTIVITIESIN GH 3 CELLSDURING PROLONGED PRETREATMENT WITH IONOMYCIN OR OAG Treatment group
6h
12h
48h
Ionomycin OAG
5.3±0.1 8.3±0.4*
5.2±0.1 7.4±0.1"
5.2±0.3 6.3±0.4*
AC
Ionomycin OAG
5.1±0.3 8.3±1.1"
5.3±0.1 9.4±0.8*
5.3±0.2 8.8±0.8*
PLC
GH 3 cells were grown in the presence of I/zM ionomycin or 1 /~M OAG for up to 48 h. Enzyme activities were measured in cell membrane fractions as indicated in Materials and Methods. AC activity is shown as pmol cAMP/rag protein/rain. Basal AC activity in untreated cells (controls) was 5.2+0.1. PLC activity is given as 103 c.p.m. IP3/mg protein/min. Basal PLC activity in untreated cells (controls) was 5.0+0.2. * P = ~, < 0.05, Wileoxon rank test, stimulated vs control. CELLS416-d
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Treatment time (hrs) FIG. 2. The effect of ionomycin and OAG on modulated AC activity in GH3 cells. AC activity was measured in membranes from cells grown in the presence of 1/aM ionomycin (A) and 1/aM OAG (B) as described in Materials and Methods. Hormone or guanyl nucleotide effects on AC were measured in the presence of either TRH (1/aM), VIP (1/aM), SRIF (10/aM) or Gpp0NH)p (20/aM) in the enzyme assay. The results are shown as percentage modulatory effect on AC [stimulation by TRH, VIP and Gpp(NH)p, inhibition by SRIF] of control values throughout the pretreatment period (6--48h), as a mean of triplicates. *P = a < 0.05 (significant difference from controls; Wilcoxon rank test).
al.
increase of basal AC activity, most notable after 6 h. Forskolin-stimulated AC activity was not changed during the exposure to ionomycin or O A G (Table 2). As shown in Fig. 2A, TRH-stimulated AC activity was transiently reduced by 60% in G H 3 cells treated with ionomycin. The reduction was followed by an increase in AC responsiveness to approximately 140% of controls. In parallel, VIP-elicited AC activity was reduced by 20% after 6 - 1 2 h and increased up to 150% after 48 h. Gpp(NH)p-induced and SRIF-inhibited AC activity remained unchanged for the whole pretreatment period. Thus, the T R H - and VIP-sensitive AC showed a biphasic pattern after ionomycin pretreatment which is in accordance with the obtained protein results (Fig. 1). In comparison, AC responsiveness to T R H was almost abolished by pretreatment of G H 3 cells with O A G (Fig. 2B). The reduction was close to linear with time, and significant already after 6 h. Responsiveness to inhibition by SRIF displayed a transient reduction (to approximately 40% of controls) at 6 h. Gpp(NH)p-elicited AC activity showed a smaller, but significant reduction of approximately 15-20%, for the whole time-period. AC responsiveness to VIP stimulation changed in an irregular pattern due to O A G pretreatment, but was generally reduced (at 6 and 48 h).
GH3 CELLS DURING PROLONGED PRETREATMENT WITH IONOMYCIN OR O A G
TABLE 2. FORSKOLIN-STIMULATED A C ACTIVITY IN
Treatment group
6h
12 h
48 h
Ionomycin OAG
11.7+0.1 10.7+0.4
12.6+0.1 11.0+0.t
13.6+0.3 13.2+0.4
AC
GH 3 cells were grown in the presence of 1/aM ionomycin or 1 /aM OAG for up to 48 h. Forskolin-stimulated AC activity was measured in cell membrane fractions as indicated in Materials and Methods. AC activity is shown as pmol cAMP/rag protein/rain. Forskolin-stimulatecl AC activity in non-stimulated cells (control) was 12.2+0.2. * P = a < 0.05, Wileoxon rank test, stimulated vs control.
'Cross-talk' between PL C and A C in G H 3 cells
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cant increase of basal enzyme activity. PLC activation by TRH (Fig. 3) transiently decreased about 35% at 6h, followed by an increase of about 55% at 48 h. Gpp(NH)p-enhanced PLC activity increased gradually to approximately 150% of controls after 48 h incubation in the presence of ionomycin. Pretreatment of GH 3 cells with OAG (Fig. 3B) did not result in a significant change in Gpp(NH)p-stimulated PLC activity, whereas TRH-modulated PLC activity decreased dramatically to 25 and 10% of controls after 12 and 48 h, respectively.
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DISCUSSION
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FIG. 3. The effect of ionomycin and OAG on modulated PLC activity in GH3 cells. PLC activity was measured in membranes from cells grown in the presence of 1/aM ionomycin (A) or 1 # OAG (B) as described in Materials and Methods. PLC activity was estimated in the presence of 42,000 c.p.m. [~H]PIP2, 20/aM GTP and 100/aM Ca2+. Enzyme activities are expressed as l03 c.p.rn. IPs mostly IP3 and IP, formed/mg protein/rain and shown as mean + S.D. of triplicate determinations. Stimulations of PLC activity were achieved (for 5 min at 35°C) in the presence of either 1/aM TRH or 20#M Gpp(NH)p. The assay was repeated twice with similar results. *P = ~ < 0.05, Wilcoxon rank test, stimulated vs control.
Basal and modulated PLC activity in GH a cells treated with ionomycin and OAG For comparison, GH a membrane preparations from pretreated cells with ionomycin and OAG were also analysed for basal PLC activity and PLC responsiveness to TRH and Gpp(NH)p as indicated in Materials and Methods. As shown in Table 1, basal PLC activity was not changed by ionomycin pretreatment, whereas OAG caused a signifi-
In the present study we have demonstrated that ionomycin and OAG regulate G-protein steady-state levels as well as PLC and AC responsiveness in GH 3 cells in a specific manner. Ionomycin, which increases intraceUular Ca 2+ mainly by mobilizing Ca 2+ from intracellular stores [12], transiently reduced the levels of the g-subunits of the stimulatory G-protein, Gs, which was paralleled by a transient decrease of AC responsiveness to stimulation by TRH and VIP. Since basal and forskolin-stimulated AC activity was unchanged in response to pretreatment with ionomycin, it appears that the response to hormones is probably not due to changes in membrane content of AC's catalytic unit. A similar transient alteration of PLC responsiveness to TRH is, however, also observed, which does not exclude modulatory actions at the receptor level. The alterations in TRH and VIP stimulation of AC were approximately parallel, but the effect was more pronounced for TRH activation. This discrepancy was also observed through inhibition of G, coupling by antisense RNA expression and antibody blocking [6], and it may be due to differences in the ability of the receptors for TRH and VIP to utilize the available G,. The existence of separate G,~ pools has also been postulated [24,25]. Contrary to ionomycin, OAG alters basal
754
E.J. PAUI.S~N et al.
AC activity. However, forskolin-stimulated AC activity remained unchanged, indicating unaltered cellular quantities of AC holoenzyme. Increased basal AC activity may be explained by enhancement of AC by phosphorylation by PKC. Phorbol esters have been shown to enhance basal AC activity through Gi-inactivation in GH4CI cells [26] and purified AC protein is shown to be a substrate for PKC [27]. These may be important mechanisms of cross-talk between the AC and PLC membrane signal systems. Decreased levels of G,~ protein due to OAG pretreatment were followed by a slight reduction of AC responsiveness to VIP. The degree of reduction was somewhat less than would be expected by the large decrease of G,a levels, possibly due to the increased sensitivity of PKC-stimulated AC enzyme to G s coupling, as previously postulated [15]. The almost total abolition of AC responsiveness to TRH following pretreatment with OAG was parallel to the reduction in TRH-stimulated PLC activity. This phenomenon resembles the homologous densensitization of TRH responsive AC activation and phosphoinositide turnover following TRH pretreatment of GH3 cells alone [28]. The latter is possibly due to a PKC-dependent reduction in TRH receptor expression [29]. Inhibition of AC activity by SRIF has been shown to be linked through a pathway involving coupling to Gi.z [6]. The observation that AC responsiveness to SRIF is unaltered parallel to the lack of changes in steady-state levels of Gi.2g is in agreement with our previous results. The absence of alterations in the ability of the non-hydrolysable GTP-analogne Gpp(NH)p to stimulate AC activity, even under conditions that alter G,a content, is also in agreement with previous observations [6]. Such findings call for a revised model of Gpp(NH) action or point to altered post-translational G-protein modifications. Several reports have discussed the interplay between AC- and PLC-dependent membrane signalling systems (for reviews, see [15,30,31]). The enzyme action of PKC has been shown to
regulate the coupling sensitivity of G-protein-mediated signals, involving their receptors [32], G-proteins [26, 33-36] and effector enzymes [27]. Recent reports have focused on the involvement of regulated G-protein expression in the cross-talk between different signal transduction pathways. Indeed, alteration of steady-state levels of G-proteins may be an important mode of action for both peptide [37-41] and steroid hormones [42,43]. Acknowledgements---We thank DR A. M. SPIEGEL for supplying the antisera against the various G-protein ~-subunits. This work received support from the Norwegian Cancer Society, the Norwegian Research Council for Science and the Humanities, the Anders Jahre Foundation for the Promotion of Sciences, and the Nordic Insulin Foundation, Copenhagen, Denmark. The authors are grateful for technical assistance from Ms A. K. F~LDm~M, Ms H. TmLmE~ and Ms A. Bm.sExa.
REFERENCES I. Tashjian A. H. Jr (1979) Meth. Enzym. 58, 527-535. 2. Gautvik K. M., Bjzro T., Slctholt K., Ostbcrg B, C., Sand O., Torjcsen P., Gordeladzc J. O., Iverscn J.-G. and Haug E. (1988) Molecular Mechanisms in Secretion. Alfred Benzon Symposium 25 (Thorn N. A., Treiman M. and
Pedersen O. H., Eds), pp. 211-227. Munksgaard, Copenhagen. 3. Drust D. S. and Martin T. F. J. (1984) J. biol. Chem. 259, 14,520-14,530. 4. Gershengorn M. C. and Thaw C. (1985) Endocrinology 116, 591-596. 5. Straub (3. E. and Gershengorn M. C. (1986) J. biol. Chem. 261, 2712-2717. 6. Paulssen R. H., Paulssen E. J., Gautvick K. M. and Gordeladze J. O. (1992) Fur. J. Biochem. 204, 413-418.
7. Bjzro T., Sand O., ~stberg B. C., Gordeladze J. O., Torjesen P., Gautvik K. M. and Haug E. (1990) Biosci. Rep. 10, 189-199. 8. Drummond A. H. (1986) J. exp. Biol. 124, 337-358. 9. Bcrridge M. J. (1987) A. Rev. Biochem. 56, 159-193. 10. Liu C. and Hermann T. E. 0978) J. biol. Chem. 253, 5892-5894. 11. Toeplitz B. K., Cohen A. I., Funke P. T.,Parker W. L. and Gougoutas J. Z. (1979) J. Am. Chem. Soc. 101, 3344-3353.
'Cross-talk' between PLC and AC in GH3 cells 12. Fasolato C. and Pozzan T. (1989) J. biol. Chem. 264, 19,630-19,636. 13. Nishizuka Y. (1984) Nature 308, 693-698. 14. Nishizuka Y. (1986) Science 233, 305-312. 15. Bouvier M. (1990) Ann. N. Y. Acad. Sci. 594, 120-129. 16. Paulssen E. J., Paulsscn R. H., Gautvik K. M. and Gordeladze J. O. (1992) Biochem. Pharmac. 44, 471-477. 17. Ham R. G. (1963) Expl Cell Res. 29, 515-526. 18. Gautvik K. M., Gordeladz¢ J. O., Jahnsen T., Haug E., Hansson V. and Lystad E. (1983) J. biol. Chem. 258, 10,304-10,311. 19. Bjoro T., Haug E., Sand O. and Gautvik K. M. (1984) Molec. cell. Endocr. 37, 41-50. 20. Paulsscn E. J., Paulsscn R. H., Haugen T. B., Gautvik K. M. and Gordeladz¢ J. O. (1991) Molec. cell. Endocr. 76, 45-53. 21. Salomon Y., Londos C. and RodbcU M. (1974) ,4nalyt. Biochem. 58, 541-548. 22. Jackowski S., Rettenmier C. W., Sherr C. J. and Rock C. O. (1986) J. biol. Chem. 261, 4978-4985. 23. Simonds W. F., Goldsmith P. K., Codina J., Unson C. G. and Spiegel A. M. (1989) Proc. natn. Acad. Sci. U.S.A. 86, 7809-7813. 24. Ishikawa Y., Bianchi C., Nadal-Ginard B. and Homcy C. J. (1990) J. biol. Chem. 265, 8458-8462. 25. Raymond J. R., Albcrs F. J., Middleton J. P., Lefkowitz R. J., Caron M. G., Obcid L. M. and Dennis V. W. (1990) J. biol. Chem. 266, 372-379. 26. Gordeladze J. O., Bjoro T., Torjesen P. A., Ostbcrg B. C., Haug E. and Gautvik K. (1989) Eur. J. Biochem. 183, 379-406. 27. Yoshimasa T., Sibley D. R., Bouvier M., Lefkowitz R. J. and Caron M. G. (1987) Nature 327, 67-70.
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28. Drummond A. H. (1985) Nature 315, 752-755. 29. Fujimoto J., Straub R. E. and Gcrshengorn M. C. (1991) Molec. Endocr. 5, 1527-1532. 30. Houslay M. D. (1991) Eur. J. Biochem. 195, 9-27. 31. Di Marzo V., Galadari S. H. I., Tippins J. R. and Morris H. R. (1991) Life Sci. 49, 247-259. 32. Bouvier M., Guilbault N. and Bonin H. (1991) FEBS Lett. 279, 243-248. 33. Garcia-S~inz J. A. and Guti6rrez-Venegas G. (1989) FEBS Lett. 257, 427-430. 34. Pyne N. J., Murphy G. J., Milligan G. and Houslay M. D. (1989) FEBS Lett. 243, 77-82. 35. Bushfield M. and Houslay M. D. (1990) Biochem. Soc. Trans. 18, 456. 36. Hern/mdez-Sotomayor S. M. T., Macias-Silva M., Malbon C. C. and Garcia-Sfiinz J. A. (1991) Am. J. Physiol. 260, C259-C265. 37. Lee R. T., Brock T. A., Tolman C., Bloch K. D., Seidman J. G. and Ncer E. J. (1989) FEBS Lett. 249, 139-142. 38. Hadcock J. R., Ros M., Watkins D. C. and Malbon C. C. (1990) J. biol. Chem. 265, 14,784-14,790. 39. Reithmann C., Gierschik P., Werdan K. and Jakobs K. H. (1990) Br. J. clin. Pharmac. 30, 118S-120S. 40. Saunier B., Dib K., Dclemer B., Jacquemin C. and Corr6ze C. (1990) J. biol. Chem. 265, 19,942-19,946. 41. Chan S. D. H., Strewler G. J. and Nissenson R. A. (1990) J. biol. Chem. 265, 20,081-20,084. 42. Chang F. and Bourne H. R. (1987) Endocrinology 121, 1711-1715. 43. Bouvier C., Lagac6 G. and Collu R. (1991) Molec. cell. Endocr. 79, 65-73.
