©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cyclic GMP-dependent Protein Kinase Blocks Pertussis Toxin-sensitive Hormone Receptor Signaling Pathways in Chinese Hamster Ovary Cells (*)

Alexander Pfeifer (§) , Bernd Nürnberg (1), Simone Kamm , Martina Uhde (1), Günter Schultz (1), Peter Ruth , Franz Hofmann

From the (1) Institut für Pharmakologie und Toxikologie, Technische Universität München, Biedersteiner Strae 29, D-80802 München, Federal Republic of Germany and the Institut für Pharmakologie, Universitätsklinikum Rudolf Virchow, Freie Universität Berlin, Thielallee 67-73, D-14195 Berlin, Federal Republic of Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

cGMP-dependent protein kinase (cGMP kinase) has been implicated in the regulation of the cytosolic calcium level ([Ca]). In Chinese hamster ovary (CHO) cells stably transfected with the cGMP kinase I (CHO-cGK cells), cGMP kinase suppressed the thrombin-induced increase in inositol 1,4,5-trisphosphate and [Ca] (Ruth, P., Wang, G.-X., Boekhoff, I., May, B., Pfeifer, A., Penner, R., Korth, M., Breer, H., and Hofmann, F. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 2623-2627). Cholecystokinin activated intracellular calcium release via a pertussis toxin (PTX)-insensitive pathway in CHO-cGK cells. cGMP kinase did not attenuate the CCK-stimulated [Ca]. In contrast, cGMP kinase suppressed calcium influx stimulated by insulin-like growth factors 1 and 2 (IGF-1 and IGF-2) via PTX-sensitive pathways. The effects of PTX and cGMP kinase on [Ca] were not additive. 8-Bromo-cGMP had no effect on [Ca] stimulated by IGF-1 or IGF-2 in wild type CHO cells. These results suggested that cGMP kinase inhibited the different signaling pathways by the phosphorylation of a PTX-sensitive G protein. cGMP kinase phosphorylated the subunits of G, G, and G in vitro. Phosphorylation stoichiometry was 0.4 mol of phosphate/mol of Gafter reconstitution of heterotrimeric Gin phospholipid vesicles. The subunit of Gwas also phosphorylated in vivo. These results show that cGMP kinase blocks transduction of distinct hormone pathways that signal via PTX-sensitive Gproteins.


INTRODUCTION

Nitric oxide and atrial natriuretic factor activate guanylyl cyclases and increase cGMP levels. Cyclic GMP decreases [Ca]() in a number of cells including smooth muscle and platelets by activation of the cGMP kinase (for extensive discussion see Refs. 1-4). It was postulated that cGMP kinase stimulates Caremoval from the cytosol by activation of the calmodulin-stimulated Ca-ATPase, by phosphorylation of phospholamban and stimulation of calcium uptake into the sarcoplasmic reticulum, or by phosphorylation of the IPreceptor (see Refs. 1-4, 5, and 6 for further references). Cyclic GMP has also been reported to inhibit calcium influx by modulation of cAMP-hydrolyzing phosphodiesterases (7, 8) , by phosphorylation of calcium-activated potassium channels leading to hyperpolarization of the membrane (9, 10) , or by phosphorylation-dependent inhibition of L-type calcium channel activity (11, 12) .

Rapoport (13) suggested that cGMP kinase alters hormone signaling by interfering with the production of IP. The latter mechanism was supported later by other groups (14, 15) . Recently we showed that cGMP kinase inhibited thrombin-stimulated IPsynthesis and the rise in [Ca] in CHO-cGK cells (16) . The general validity of the latter mechanism was unknown and was investigated further in this study.

CHO cells contain a number of hormone receptors that increase [Ca] by different mechanisms, i.e. a receptor that is activated by high concentrations of CCK and signals via PTX-insensitive G proteins (17) and high affinity, low density receptors that are activated by IGF-1 and IGF-2 (18) . The IGF-1 receptor, a receptor tyrosine kinase, stimulated calcium influx through a nonspecific cation ion channel in Balb/c 3T3 cells (19) . The pathway leading to channel opening involved a PTX-sensitive Gprotein (20) . Interestingly, IGF-2 receptor activation stimulated Cainflux by a similar pathway (21) . The IGF-2 receptor has no tyrosine kinase activity (22) but contains a single transmembrane helix that has been reported to couple to several Gproteins (23) . This IGF-2 receptor may be identical with the cation-independent mannose 6-phosphate receptor.

CHO cells express a number of PTX-sensitive G proteins including G,G, G, and G(24, 25) , suggesting that pathways may exist in CHO cells that are similar to those found in Balb/c 3T3 cells. This paper shows that cGMP kinase suppressed specific signaling via PTX-sensitive pathways, probably by phosphorylation of the subunit of a Gprotein.


EXPERIMENTAL PROCEDURES

Stable Transfection of CHO Cells

CHO cells were transfected by electroporation with the expression vector p91023I (26) containing the coding sequence of the cGMP kinase I isozyme and, in a second reading frame, the coding sequence of the dihydrofolate reductase (27) . CHO-WT and CHO-cGK cells were grown for 3-4 days on coverslips in Dulbecco's modified Eagle's medium supplemented with 10% non-dialyzed and dialyzed fetal calf serum, respectively.

Measurement of Calcium Transients in CHO Cells

[Ca] was determined by fluorescence measurement of the calcium-sensitive indicator Fura-2 as described previously (16) . In brief, cells were loaded for 1 h with 2.5 µ M Fura-2/AM at 37 °C. PTX-pretreated cells were incubated in Dulbecco's modified Eagle's medium containing 100 ng/ml PTX for 8 h prior to Fura-2/AM loading. Coverslips were then washed with the NaCl/Hepes buffer (140 m M NaCl, 6.6 m M KCl, 1.18 m M MgSO, 2 m M CaCl, 10 m M glucose, 5 m M Hepes, pH 7.4) and superfused with this buffer. cGMP kinase stimulation was achieved by superfusion of the cells for 10 min with 1 m M 8-Br-cGMP included in the NaCl/Hepes buffer. The cells were placed in the light path of an Axiovert-35 microscope (Zeiss) equipped with a xenon light source and a 340/380-nm filter wheel (Luigs & Neumann) alternating at a frequency of 2 Hz. Fura-2 fluorescence of a single cell was monitored by a photomultiplier unit (Hamamatsu 928 SF) attached to the microscope. Changes in the 340/380-nm ratio were analyzed and displayed on an ATARI-ST computer. The cells were superfused by micropipettes loaded with the agents to be tested. Superfusion was started by applying air pressure to the pipette (Lorenz MPCU-3) and lasted, if not otherwise indicated, for 60 s. Apparent [Ca] values were calculated according to Ref. 28. All data are expressed as means ± S.E., with the number of cells shown in parentheses.

Purification of G Proteins

PTX-sensitive G proteins were purified from bovine brain as described (29) . Briefly, membranes were prepared and stored in TEM buffer (20 m M Tris-HCl, pH 8.0, 1 m M EDTA, 20 m M -mercaptoethanol). All procedures were carried out at 4 °C. Thawed membranes were extracted for 1 h with TEM containing 0.9% sodium cholate. After centrifugation at 70,000 g, the clear supernatant was mixed with ethylene glycol to a final concentration of 30% (v/v), followed by an addition of 10 µ M GDP. Purified mixtures of G, G/G, heterotrimeric G/G, and Gwere obtained after sequential chromatography on a 1.5-liter DEAE-Sepharose Fast Flow column (Pharmacia Biotech Inc.), a 1.2-liter AcA 34 gel filtration column (Serva), and a 0.65-liter heptylamine-Sepharose column (30) . Heterotrimeric G proteins containing different subtypes of G/Gwere separated on a Mono Q column (Pharmacia), employing a method described by Codina et al. (31) . Repetitive fast protein liquid chromatography runs resulted in fractions containing G, G, G, or G. Gwas contaminated with small amounts of G, Gwith small amounts of G, and Gwith small amounts of Gas detected by immunoblot analysis. The purified G proteins were stored in 20 m M TEM buffer containing 100-300 m M NaCl, 11 m M CHAPS, and 0.1% Lubrol.

Reconstitution of G Proteins into Phospholipid Vesicles

Purified heterotrimeric G proteins were reconstituted into phospholipid vesicles containing azolectin. About 6 µg of heterotrimeric Gor Gwere mixed with azolectin and sodium cholate and incubated for 1 h. Vesicles containing G proteins were formed by passing the solution over a gel filtration column eluted with 20 m M Hepes, pH 7.4, containing 0.1 m M EDTA, 1 m M dithiothreitol, and 100 m M NaCl. G proteins reconstituted into phospholipid vesicles eluted in the void volume and were immediately used for further experiments.

GTPS Binding to G Proteins

For quantification of purified and reconstituted G proteins, [S]GTPS binding to G proteins was essentially performed as described (29) . Aliquots of 3 µl were incubated with [S]GTPS (100,000 cpm/tube; 500 n M GTPS) in a total volume of 60 µl/tube for 60 min at 32 °C. Nonspecific binding was estimated by the addition of 2 m M GTP.

Phosphorylation of G Proteins

Purified G proteins (9-20 pmol) were phosphorylated for 10 min at 30 °C in 40 µl of 50 m M MES buffer, pH 6.9, containing 0.4 m M EGTA, 1.6 m M CHAPS, 0.015% Lubrol, 1 m M MgCl, 10 m M NaCl, 10 m M dithiothreitol in the absence and presence of 10 µ M 8-Br-cGMP, 0.1 m M [-P]ATP (1500 cpm/pmol), and 780 n M cGMP kinase holoenzyme. Reconstituted G proteins (2.5-5 pmol) were phosphorylated for 10 min at 30 °C in 200 µl of 15 m M Hepes, pH 7.4, 0.075 m M EDTA, 75 m M NaCl, 0.75 m M dithiothreitol, 1 m M magnesium acetate, 10 µ M 8-Br-cGMP, 0.1 m M [-P]ATP (1500 cpm/pmol), and 780 n M cGMP kinase holoenzyme. In both cases, reactions were stopped with Laemmli's buffer (final concentration: 1% SDS, 5% mercaptoethanol, 5% glycerol, 0.01% bromphenol blue) followed by SDS-gel electrophoresis as described below.

Photolabeling and Immunoprecipitation of Membrane Proteins

Photolabeling of G protein subunits by [P]GTP azidoanilide was performed as described (32) . Each tube contained freshly prepared membranes obtained from 10CHO cells. Immunoprecipitations of G proteins were performed according to Nürnberg et al. (29) and Harhammer et al. (33) . Essentially, G proteins reconstituted into phospholipid vesicles and phosphorylated by cGMP kinase were acetone-precipitated and dissolved in 40 µl of a solution containing 4% SDS at room temperature. After 5 min, 280 µl of the immunoprecipitation buffer (10 m M Tris-HCl, pH 7.4, 150 m M NaCl, 1 m M EDTA, 1 m M dithiothreitol, 1% (w/v) sodium deoxycholate, 1% (v/v) Nonidet P-40, 0.3 µ M aprotinin, and 0.2 µ M phenylmethanesulfonyl fluoride) was added, followed by 30 µl of antiserum AS8 or AS6. Peptide antibodies AS6 (anti-) and AS8 (anti-) were characterized previously (34) . The mixture was gently shaken at 4 °C. After 2 h the solution was supplemented with 60 µl of a slurry containing 15% Protein A-Sepharose beads. This mixture was shaken overnight. Protein A-Sepharose beads were pelleted and washed once with immunoprecipitation buffer and twice with immunoprecipitation buffer containing 300 m M NaCl. Bound proteins were eluted by adding Laemmli's buffer and loaded onto discontinuous SDS gels. The separating gels contained 11% (w/v) acrylamide. Dried gels were exposed to x-ray films. Thereafter, the phosphorylated bands were cut out, dissolved in scintillation mixture, and counted.

Metabolic Labeling of Cells and Immunoprecipitation of G Proteins

For detection of cGMP kinase-dependent phosphorylation of G proteins in CHO-WT and CHO-cGK cells, 10cells/tube were incubated in a small volume of buffer A (137 m M NaCl, 1 m M MgCl, 2.7 m M Hepes, pH 7.4, devoid of phosphate). Subsequently, cells were incubated with [P]orthophosphate (0.5 mCi/tube) in buffer A for 2 h at 30 °C. Thereafter, cells were disrupted mechanically in the presence of 20 m M -mercaptoethanol, 25 m M EDTA, 2 m M pyrophosphate, 2 m M vanadate, 0.0005% aprotinin, and 1 m M benzamidine. The whole cell homogenate was centrifuged at 500 g (10 min, 4 °C), and the supernatant was centrifuged a second time at 30,000 g (10 min, 4 °C). The pellet was solubilized by adding 40 µl of lysis buffer (1% SDS, 100 m M phosphate buffer, pH 8, 4 m M EDTA, 2 m M pyrophosphate, 2 m M vanadate, 0.0005% aprotinin, 1 m M benzamidine) for 15 min at room temperature. Subsequently, 280 µl of precipitation buffer (50 m M phosphate buffer, pH 7.2, 150 m M NaCl, 2 m M EDTA, 5 m M sodium fluoride, 1% sodium deoxycholate, 1% Triton X-100, 0.5% SDS, 2 m M pyrophosphate, 2 m M vanadate, 0.0005% aprotinin, 1 m M benzamidine) was added, followed by centrifugation at 30,000 g (10 min, 4 °C). The supernatant was transferred to a second tube and incubated with 20 µl of nonimmune rabbit serum for 30 min at 4 °C. Subsequently, 30 µl of a solution containing 37.5% of Protein A bound to Sepharose beads were added for another 3.5 h. The slurry was pelleted, and the supernatant was transferred to a third tube and incubated with 40 µl of rabbit antiserum AS373 or nonimmune rabbit serum. AS373 was generated against the peptide ((C)DVIIKNNLKDCGLF) specific for the C terminus of Gand G. The mixture was gently shaken overnight at 4 °C. The solution was finally supplemented with 60 µl of a suspension containing 12.5% Protein A bound to Sepharose beads. This mixture was shaken for another 3 h. Protein A-Sepharose beads were pelleted and washed once with precipitation buffer and twice with precipitation buffer containing 300 m M NaCl. Bound proteins were eluted by adding Laemmli's buffer and loaded onto discontinuous SDS gels with an 11% separating gel. Dried gels were exposed to x-ray films.

Materials

The dihydrofolate reductase-deficient CHO cell line DG44 was kindly provided by L. Chasin, Columbia University, New York. Dialyzed calf serum was purchased from Life Technologies, Inc. Dulbecco's modified Eagle's medium was from Biochrom, Berlin. CCK-8 (sulfated) was obtained from Bachem. Thrombin, CCK (pancreozymin) from porcine intestine, PTX, azolectin, and genistein were from Sigma. 8-Br-cGMP was purchased from Boehringer Mannheim. [-P]ATP was from Amersham Buchler. S-Labeled GTPS and [-P]GTP were purchased from DuPont NEN. cGMP kinase was purified as described previously (35) . All other reagents were of highest purity commercially available.


RESULTS

Stimulation of Calcium Transients by CCK

Previously it was shown that thrombin and CCK signal through different G proteins in CHO cells (17) . The stimulation of [Ca] by thrombin was completely suppressed by cGMP kinase in CHO-cGK but not in CHO-WT cells (16) . The general validity of this observation was tested. Individual CHO-cGK cells were superfused with CCK at various concentrations (Fig. 1). Superfusion of the cell with 100 n M CCK elicited a rapid increase in [Ca] (Fig. 1). At this threshold concentration, 82% of cells responded with an average increase in [Ca] of 155 ± 55 n M (11 cells) above basal levels. At concentrations from 100 n M to 300 µ M CCK, the calcium signal typically consisted of an initial transient rise in [Ca] followed by oscillations superimposed on an elevated plateau (Fig. 1). CCK stimulation with concentrations above 300 µ M resulted in a slightly different response; CCK induced a large peak, which rapidly decreased to a much lower but still elevated non-oscillating plateau level. The calcium transients induced by CCK were not affected by chelating the extracellular Cawith EGTA (Fig. 1). Thus, CCK mobilized Cafrom intracellular stores, supporting the previous notion that CCK activated IPsynthesis via a G protein-mediated stimulation of phospholipase C in CHO cells (17) .


Figure 1: CCK stimulates intracellular calcium release in CHO cells. CHO-cGK cells were superfused with NaCl/Hepes buffer containing 2 m M CaCl( upper panel and lower left) or NaCl/Hepes buffer without CaClbut containing 5 m M EGTA ( lower right). CCK addition at the concentrations indicated is shown by an arrow. Representative experiments of at least eight individual cells for each condition are shown.



CCK dose dependently stimulated [Ca] up to 9-fold with a half-maximal effective concentration of 120 µ M in both CHO-WT and CHO-cGK cells (Fig. 2). A similar response was observed when CHO-cGK cells were superfused with the sulfated CCK-8 octapeptide (data not shown). Similar high concentrations of CCK have also been used by others to stimulate IPproduction in CHO cells (17) , suggesting that the properties of this receptor significantly differ from those of the cloned CCK-A and CCK-B receptors (36, 37) , but this receptor may share some properties with the very low affinity CCK receptor postulated for pancreas acinar cells (38) .

Activation of cGMP Kinase Prevented Thrombin- but Not CCK-induced Calcium Transients

The activation of the cGMP kinase in CHO-cGK cells did not attenuate the Catransients induced by 100 µ M CCK (Fig. 3) or that of any other concentration of CCK tested (). In contrast to CCK, the thrombin-induced calcium transients were completely suppressed by activation of cGMP kinase (Fig. 3).


Figure 3: 8-Br-cGMP suppresses thrombin- but not CCK-stimulated Ca transients in CHO-cGK cells. Coverslips were pretreated with NaCl/Hepes buffer ( left) or with NaCl/Hepes buffer containing 1 m M 8-Br-cGMP ( right). At the arrow, individual cells were stimulated with 100 µ M CCK ( upper part) or 200 n M thrombin ( lower part) for 60 s. Recordings are representative for at least eight individual cells.



These results indicated that CCK and thrombin signal via pathways that are differently affected by cGMP kinase. The lack of any effect of cGMP kinase activation on the CCK signaling pathway confirms the previous finding (16) that the IP-triggered calcium release channels, the endoplasmic reticulum Ca-ATPase, the plasma membrane Ca-ATPase, and other calcium removal systems were not the target for cGMP kinase in CHO cells, as implied for other cell types (see Refs. 1-4, 5, and 6). On the other hand, this result did not support the general validity of the previous findings that active cGMP kinase prevents hormone-triggered IPsynthesis, since activation of both the thrombin and the CCK receptor increased IPlevels in CHO cells (16, 17) .

Several possibilities were tested to explain the observed difference. It is known that thrombin induces tyrosine phosphorylation (39, 40, 41) , including phosphorylation and thereby activation of phospholipase C (40) . Phospholipase C is phosphorylated in vitro by cAMP kinase (42) , suggesting that it is also a potential target for cGMP kinase. In addition, inhibition of tyrosine phosphorylation blocked the mitogenic action of thrombin without affecting the Carelease from internal stores (40) . It was reported that CCK exerted some of its effects by tyrosine phosphorylation in pancreas acinar cells (41) . However, CCK-induced amylase release was inhibited by genistein distal of Camobilization (43) . Hence, although unlikely, we tested the possibility whether phospholipase C activation or tyrosine phosphorylation of other proteins contributed to the increase of [Ca] by thrombin and CCK in CHO cells. CHO-cGK cells were preincubated with 200 µ M genistein. The cells were then superfused with 200 n M thrombin or 100 µ M CCK, hormones that stimulated [Ca] to mean peak levels of 618 ± 155 n M (9 cells) or 800 ± 102 n M (5 cells). Both values are similar to the peak Cavalues in the absence of genistein (compare with Tables I and II). These results indicated that tyrosine phosphorylation was not involved in the signal transduction pathways or in the calcium-releasing mechanisms stimulated by thrombin or CCK in CHO cells.

PTX Pretreatment Suppressed Thrombin-induced but Not CCK-induced Calcium Transients

Another possible cause for the distinct effects of cGMP kinase was that thrombin and CCK signal via different G proteins and phospholipase Cs. Preincubation of CHO-cGK cells with 100 ng/ml PTX for 8 h completely prevented the thrombin-stimulated calcium transient ( Fig. 4and ). Thrombin stimulated [Ca] in only 1 out of 18 PTX-pretreated cells. In contrast, PTX preincubation did not suppress the increase in [Ca] stimulated by CCK. Superfusion of a PTX-pretreated CHO cell with 300 µ M CCK stimulated [Ca] to a similar peak value as in the control cell (Fig. 4). Furthermore, PTX pretreatment did not affect [Ca] stimulated by 100 µ M CCK (). Statistical evaluation of control CHO-cGK cells and CHO-cGK cells pretreated with PTX did not result in a significant difference between both cell populations and, therefore, confirmed that PTX pretreatment did not affect the CCK-activated [Ca].


Figure 4: Pretreatment with PTX prevents thrombin- but not CCK-induced Ca transients in CHO-cGK cells. The cells were incubated with buffer ( left) or 100 ng/ml PTX ( right) for 8 h. Thereafter, the cells were superfused with 300 µ M CCK ( upper part) or 200 n M thrombin ( lower part) for 60 s.



It was possible that the CHO-cGK cells were composed of two subpopulations in which the hormone-stimulated [Ca] was differently affected by cGMP kinase and PTX. To exclude this unlikely possibility, individual CHO-cGK cells were superfused consecutively with either agonist. The CHO-cGK cells were first superfused with thrombin and then, after a short wash out, with CCK. The thrombin-induced Catransients were suppressed by both cGMP kinase and PTX pretreatment (Fig. 5). In contrast, neither PTX nor 8-Br-cGMP affected the CCK response in the same cell. [Ca] values in the presence of 300 µ M CCK were increased from 0.17 ± 0.02 to 1.1 ± 0.2 µ M (5 cells) after 8-Br-cGMP and from 0.14 ± 0.02 to 1.0 ± 0.2 µ M (8 cells) after PTX pretreatment. These values are similar to those obtained with CCK in the absence of any pretreatment (compare with ).


Figure 5: Ca transients in CHO-cGK cells consecutively stimulated by thrombin and CCK. Two individual CHO-cGK cells were preincubated with 1 m M 8-Br-cGMP for 15 min ( left) or 100 ng/ml PTX for 8 h ( right). The cells were superfused consecutively with 200 n M thrombin followed by a short washout with NaCl/Hepes buffer without agonist and then with 300 µ M CCK. The arrows indicate the start of the individual superfusions with hormone, which lasted for 60 s each.



The results indicated that in CHO cells thrombin and CCK receptors couple to phospholipase C via a PTX-sensitive and a PTX-insensitive G protein, respectively. cGMP kinase interfered selectively with the signaling of thrombin. The coupling to different G proteins suggested that the involvement of a PTX-sensitive G protein might be required for the suppressive action of cGMP kinase on agonist-induced Catransients.

IGF-2 and IGF-1 Stimulated CaInflux in CHO Cells

In order to test whether the susceptibility to the inhibitory action of cGMP kinase could in fact be attributed to the type of G protein involved or was rather an individual characteristic of the thrombin receptor or the phospholipase C coupled to this receptor, we tested the effect of cGMP kinase on other receptors involving Gproteins. CHO cells endogenously express receptors that are activated by IGF-1 and IGF-2 (18) . IGF-1 and IGF-2 activate a calcium-permeable cation channel via a PTX-sensitive G protein in Balb/c 3T3 cells (20, 21) , thereby increasing [Ca]. Both receptors differ in their structure from each other and are quite different from the thrombin receptor (22, 44) .

IGF-2 superfusion of the CHO cells resulted in a rapid rise in [Ca] followed by a sustained plateau superimposed by oscillations (Fig. 6). The long lasting stimulation of [Ca] was reversed upon removal of the hormone. Chelation of the extracellular calcium prevented the IGF-2-induced calcium transient (Fig. 6). Ten n M IGF-2 elicited calcium transients in 94% of the CHO-cGK cells, with an average peak value of 439 n M above basal values of [Ca] (Fig. 7).


Figure 6: IGF-2 and IGF-1 stimulate calcium influx in CHO-cGK cells. Individual CHO-cGK cells were superfused with 10 n M IGF-2 ( left panel) or 50 n M IGF-1 ( right panel) in the presence of NaCl/Hepes buffer containing 2 m M CaCl( upper part) or in the presence of NaCl/Hepes buffer without CaClbut containing 5 m M EGTA ( lower part). The superfusion with IGF-2 and IGF-1 started at the arrows and lasted, if not otherwise indicated, until the end of the shown recording. wash indicates superfusion with NaCl/Hepes buffer of the cell in the absence of IGF-2. Note the different time scales in the left and right panels. Calcium transients activated by IGF-2 ( left) and IGF-1 ( right) are representative for at least 10 cells.




Figure 7: Inhibition of IGF-2- and IGF-1-induced calcium influx by 8-Br-cGMP and PTX. CHO-cGK cells were incubated in the absence ( con) or presence of 1 m M 8-Br-cGMP ( 8-cG) or 100 ng/ml PTX. The mean peak values (10-27 cells/condition) of the calcium transients ( open bars) are superimposed on the corresponding basal [Ca] levels ( shaded bars). Basal values were recorded directly before stimulation with 10 n M IGF-2 ( left) or 50 n M IGF-1 ( right). Please note that cells not responding to the particular treatment have not been excluded from the calculations.



In contrast to IGF-2, CHO-cGK cells responded to IGF-1 with a slow and long lasting rise in [Ca] (Fig. 6). IGF-1 at 50 n M stimulated [Ca] from 112 ± 13 to 322 ± 25 n M (29 cells) in CHO-cGK cells (Fig. 7). Only 3 out of 29 cells showed no response to IGF-1 superfusion. Removal of extracellular Caby EGTA completely abolished the IGF-1-induced increase in [Ca] (Fig. 6). These results suggested that both IGF-2 and IGF-1 activated a Ca-permeable cation channel in CHO-cGK cells. Similar results were obtained in CHO-WT cells.

Inhibition of IGF-2-induced Calcium Influx by cGMP Kinase and PTX

Stimulation of the cGMP kinase in CHO-cGK cells with 8-Br-cGMP substantially reduced the IGF-2-activated calcium influx. Incubation with 8-Br-cGMP decreased IGF-2-stimulated calcium transients by 54% (Fig. 7). An almost identical reduction of the IGF-2 response, namely by 58%, was observed after pretreatment of CHO-cGK cells with 100 ng/ml PTX (Fig. 7). To evaluate whether the partial suppression by cGMP kinase and PTX was mediated by interference with different targets, we tested whether the effects of cGMP kinase and PTX were additive. Treatment of CHO-cGK cells for 8 h with 100 ng/ml PTX, followed by incubation with 1 m M 8-Br-cGMP in the presence of PTX, did not result in a more pronounced inhibition of the IGF-2 response. The simultaneous treatment of 10 cells with 8-Br-cGMP and PTX resulted in a 62% reduction of IGF-2-induced calcium transients.

In contrast to CHO-cGK cells, the IGF-2 response of CHO-WT cells was not altered by 1 m M 8-Br-cGMP. Ten n M IGF-2 increased [Ca] from 70 ± 29 to 430 ± 94 n M (8 cells) in the absence of 8-Br-cGMP and from 84 ± 22 to 428 ± 69 n M (7 cells) in the presence of 8-Br-cGMP. This indicated that the partial suppression of the IGF-2-stimulated Catransients in CHO-cGK cells with 8-Br-cGMP was mediated by cGMP kinase and not by other cGMP-regulated proteins.

Inhibition of IGF-1-induced Calcium Influx by cGMP Kinase and PTX

The IGF-1-induced rise in [Ca] was also inhibited by activation of cGMP kinase. In the absence of 8-Br-cGMP, 92% of the cells responded to IGF-1. After pretreatment with 8-Br-cGMP, [Ca] did not increase in 79% of the cells after application of 50 n M IGF-1, and only 21% of the cells responded to 50 n M IGF-1 with a reduced rise in [Ca]. Overall, the treatment with 1 m M 8-Br-cGMP caused a reduction by 75% of the IGF-1 response in the CHO-cGK cells (Fig. 7). The IGF-1-induced Catransients were also suppressed by PTX. PTX pretreatment of CHO-cGK cells abolished the response to IGF-1 in 10 of 13 cells. Overall, PTX inhibited the IGF-1 response by 71% (Fig. 7).

The IGF-1 response in CHO-WT cells was not affected by preincubation with 8-Br-cGMP. In the absence of 8-Br-cGMP, IGF-1 increased [Ca] from 109 ± 20 to 280 ± 29 n M (7 cells). In the presence of 8-Br-cGMP, IGF-1 stimulated [Ca] from 135 ± 17 to 293 ± 35 n M (6 cells), demonstrating that inhibition of the Catransients required activation of cGMP kinase.

Effect of Genistein on IGF-2- and IGF-1-induced Calcium Transients

In order to test whether the remaining Cainflux, which was not suppressed by 8-Br-cGMP, resulted from the activation of a pathway involving tyrosine phosphorylation, we studied the effect of genistein on IGF-2- and IGF-1-induced Catransients. Incubation of CHO-cGK cells with 100 µ M genistein for 15 min did not alter the IGF-2-induced calcium influx; in the presence of genistein (100 µ M), IGF-2 elicited a rise in [Ca] from 110 ± 36 n M up to a mean peak value of 504 ± 41 n M (9 cells), a value similar to that obtained in the absence of genistein (Fig. 7).

The IGF-1-induced increase in [Ca] was also not affected by genistein. At concentrations up to 200 µ M, genistein did not inhibit the IGF-1-induced rise of [Ca] in CHO-cGK cells. The basal and peak values of [Ca] after IGF-1 stimulation were 173 ± 19 and 349 ± 34 n M (10 cells), respectively, corresponding to the increase observed in the absence of genistein. These results suggested that either tyrosine phosphorylation did not contribute to the calcium influx, triggered by IGF-1 and IGF-2 in CHO cells, or that genistein did not inhibit the tyrosine phosphorylation step involved in the calcium influx pathway.

Phosphorylation of G Proteins by cGMP Kinase

The results presented so far demonstrate that the signaling pathways affected by cGMP kinase involved PTX-sensitive G proteins. CHO cells are known to express the PTX-sensitive G proteins G, G, G, and G(24, 25) . These observations raised the possibility that cGMP kinase affected the various signal transduction pathways through the modification of a G protein. To examine the potential phosphorylation of G proteins by cGMP kinase, purified heterotrimeric G proteins were tested as in vitro substrates for cGMP kinase.

cGMP kinase added at a high but physiological concentration (1, 16) phosphorylated a protein of about 40 kDa in preparations of G, G, and G(Fig. 8 A). The approximate molecular mass of 40 kDa was identical with that of the subunits of G/Gand indicated that the subunits were phosphorylated. However, the phosphorylation was less than stoichiometric as estimated from counting the radioactive band after dissolving the gel and from the amount of G protein added to the incubation. The substoichiometric phosphorylation was presumably due to the presence of detergents in the reaction mixture carried over with the G protein storage buffer. The detergents may have altered the G protein structurally in such a way that a potential phosphorylation site became more or less accessible for cGMP kinase. To test this possibility, the heterotrimeric Gcomplex was reconstituted in phospholipid vesicles prior to phosphorylation. cGMP kinase phosphorylated the reconstituted Gsubunit at an estimated stoichiometry of about 0.4 mol of phosphate/mol of G(Fig. 8 B). The identity of the phosphorylated band with Gwas ensured by immunoprecipitation of the subunit with the antibody AS8 recognizing all PTX-sensitive G subunits (34) . A phosphorylated immunoprecipitate was not obtained when the antibody AS6 recognizing specifically Gisoforms (34) was used (Fig. 8 B). In contrast to G, a preparation containing Gwas not phosphorylated by cGMP kinase (not shown), suggesting that cGMP kinase phosphorylated preferentially the subunits of the Gsubfamily.


Figure 8: Phosphorylation of G proteins by cGMP kinase. A, G, G, and G( lanes 1-3) were phosphorylated by cGMP kinase and thereafter applied to an SDS gel. The amounts of G, G, and Gin the phosphorylation assays were 12, 9, and 20.4 pmol. The phosphorylated 80-kDa protein corresponds to autophosphorylated cGMP kinase. B, the reconstituted Gprotein (5 pmol) was phosphorylated by cGMP kinase and was then applied directly ( lane 4) or first immunoprecipitated with the ( lane 5) or with the antibody ( lane 6) as described under ``Experimental Procedures.'' Two different autoradiographs of phosphorylated Gproteins are shown.



We tested, therefore, whether or not the subunit of a Gprotein was phosphorylated in vivo in CHO cells. CHO-cGK cells were prelabeled with POand then stimulated with 8-Br-cGMP. Separation of the cell extract on an SDS gel yielded a phosphorylated band at 80 kDa, which was immunoprecipitated with specific cGMP kinase antibodies. Separation of a crude membrane fraction on an SDS gel yielded no pronounced phosphorylated band in the 40-kDa range where the subunit of heterotrimeric G proteins should be localized. A positive result was obtained after solubilization and immunoprecipitation of the membrane fraction with the antibody AS372, which recognizes specifically the and protein (Fig. 9). Precipitation of a membrane preparation prelabeled with azido-GTP with nonimmune serum yielded no labeled band (Fig. 9, lane 1), whereas precipitation of the membrane fraction with the specific antibody resulted in a strong signal at a Mof 41,000 (Fig. 9, lane 2). The same protein band contained radioactive phosphate. Phosphor imaging analysis indicated that the P content of this band was 1.7-fold higher in the protein isolated from CHO-cGK cells than in that from CHO-WT cells. The three characteristics of the phosphorylated band, i.e. the immunoprecipitation of the protein by the G-specific antibody, its labeling by azido-GTP, and its Mof 41,000, strongly supported the notion that the phosphorylated band was the subunit of a Gprotein.


Figure 9: [P]GTP azidoanilide and 8-Br-cGMP induced [P]orthophosphate labeling of G proteins in CHO cells. Lanes 1 and 2, membranes obtained from 10cells were subjected to [P]GTP azidoanilide labeling and immunoprecipitation by nonimmune serum ( lane 1) or AS373 ( lane 2) followed by separation on SDS-polyacrylamide gel electrophoresis. Lanes 3 and 4, 10CHO-WT cells ( lane 3) or 10CHO-cGK cells ( lane 4) were metabolically labeled with induced [P]orthophosphoric acid and subsequently incubated with 8-Br-cGMP. Thereafter, cell membranes were prepared and subjected to immune precipitation employing AS373 followed by separation on SDS-polyacrylamide gel electrophoresis. Radiolabeled proteins were visualized by autoradiography.




DISCUSSION

A previous study showed that cGMP kinase suppressed thrombin-stimulated IPproduction and [Ca] elevation in CHO-cGK cells (16) , suggesting that cGMP kinase may interfere with the generation of second messengers by specific hormones. The general applicability of this hypothesis was tested in the present experiments, which investigated the effects of cGMP kinase on thrombin-, CCK-, IGF-1-, and IGF-2-stimulated Catransients. cGMP kinase suppressed Catransients triggered by thrombin, IGF-1, and IGF-2 via PTX-sensitive G proteins. These effects were specific for cGMP kinase and were not observed in CHO-WT cells. In contrast, Catransients induced by CCK, which was signaling via an unidentified low affinity receptor and a PTX-insensitive G protein, were not affected by cGMP kinase. These different responses toward cGMP kinase activation ruled out the possibility that cGMP kinase phosphorylated and affected the function of plasma membrane or endoplasmic reticulum Ca-ATPases, the IPrelease channel, or other common calcium-lowering mechanisms in CHO-cGK cells.

It was intriguing that the signaling of structurally different hormone receptors, i.e. a seven-membrane-spanning receptor (44) , a receptor with a single transmembrane helix (22) , and a tyrosine kinase-containing receptor (22) , was affected by cGMP kinase. These receptors activated different cellular targets involving IPproduction and opening of a cation channel. The diversity of receptors and targets led to the conclusion that it was unlikely that either of them was phosphorylated by cGMP kinase. In agreement with other groups (40, 43) , the experiments with genistein excluded the possibility that these receptors stimulated [Ca] by a tyrosine phosphorylation step or that cGMP kinase affected such a step.

There was a close correlation between the effects of PTX and cGMP kinase on the different signal transduction processes, suggesting that both enzymes modified the same targets, namely PTX-sensitive heterotrimeric G proteins of the G/Gsubfamily. CHO cells predominantly express Gand Gand at lower concentrations Gand G(24, 25) . The thrombin receptor couples to several G proteins including PTX-sensitive G proteins (17, 32, 45, 46) . In Balb/c 3T3 cells, a PTX-sensitive Gprotein mediated the IGF-1- and IGF-2-stimulated calcium influx (20, 21, 23) . cGMP kinase inhibited the thrombin-, IGF-1-, and IGF-2-stimulated Catransients to the same extent as PTX. This apparent identity suggested that the signal transduction of the thrombin receptor and the IGF-1 and IGF-2 receptors involved a similar or identical G protein in CHO cells, presumably a member of the Gsubfamily. Since these G proteins are the only link involved in both the thrombin and IGF-1/IGF-2 signal transduction pathways (17, 20, 23, 45, 46) , they could be the candidates for cGMP-dependent phosphorylation (see Fig. 10).

G protein preparations containing G, G, and Gcontaminated with small amounts of Gwere phosphorylated by cGMP kinase. Immunoprecipitation of reconstituted Gconfirmed that the subunit was phosphorylated by cGMP kinase. In agreement with the in vitro data cGMP kinase phosphorylated a Gsubunit in vivo. Such a phosphorylation has not been detected previously (47) , presumably since at that time the heterogeneity of the available G protein preparations was not appreciated. Gproteins were not phosphorylated by cGMP kinase, suggesting specificity of the phosphorylation reaction.

The primary sequence of the Gsubunit contains a potential cGMP kinase phosphorylation site (Arg-Lys-Asp-Thr-Lys) in the carboxyl-terminal effector region (48) . The same or similar phosphorylation sites are present in the sequences of the subunits of G, G, G, G, and Gbut not in G, G, G, G, G, G, G, and G. This analysis supports the experimental findings and strengthens further the hypothesis that cGMP kinase attenuated the calcium transients by the phosphorylation of the subunit of a Gprotein.

An interaction of cGMP kinase with PTX-sensitive G proteins was already demonstrated for the renal collecting duct, where cGMP kinase reduced the open probability of an amiloride-sensitive sodium channel, involving a PTX-sensitive G protein (49) . In addition, a few examples exist for the phosphorylation of G proteins by other protein kinases. In platelets, the activation of protein kinase C phosphorylated stoichiometrically G in situ (50) . In S49 lymphoma cells, phosphorylation of a Gprotein by protein kinase C prevented somatostatin-induced inhibition of adenylyl cyclase (51) . In neuroblastoma Glioma hybrid cells Gis phosphorylated by protein kinase C (52) . This reaction attenuates the inhibitory adenylyl cyclase pathway. Therefore, it is not too speculative to assume that cGMP-dependent phosphorylation of a Gprotein interrupted the coupling of the active Gsubunit with its effector in the CHO-cGK cells.

In conclusion, it has been shown that cGMP kinase attenuated signal transduction in PTX-sensitive pathways. The involved receptors and the postulated G protein have growth-promoting effects (19, 22, 38, 39, 45) . It is possible that the mechanism interrupted by cGMP kinase in CHO-cGK cells is responsible for the reported antiproliferative action of cGMP (53) .

  
Table: Activation of cGMP kinase does not affect CCK-induced Catransients

CHO-cGK cells were preincubated without (-) or with (+) 1 m M 8-Br-cGMP for 15 min and then stimulated with 30, 100, or 300 µ M CCK. Basal values were taken directly before addition of CCK. CCK values are the peak values reached after CCK superfusion ( n = number of cells). Note that cells not responding to the particular treatment have not been excluded from the calculations.


  
Table: Effect of PTX on CCK- and thrombin-induced Catransients

CHO-cGK cells were preincubated in the presence (PTX) and absence (Buffer) of PTX and then stimulated with CCK or thrombin as described in the legend to Fig. 4. Basal values were taken directly before addition of the agonists. Thrombin and CCK values are the peak values reached ( n = number of cells). Note that cells not responding to the particular treatment have not been excluded from the calculations.



FOOTNOTES

*
The experimental work was supported by grants from the Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 49-89-3849-3260; Fax: 49-89-3849-3261.

The abbreviations used are: [Ca], cytosolic calcium concentration; cGMP kinase, cGMP-dependent protein kinase; CHO-WT, CHO wild type cells; CHO-cGK, CHO cells expressing cGMP kinase I; IGF, insulin-like growth factor; CCK, cholecystokinin; G protein, guanine nucleotide-binding protein; GTPS, guanosine 5`-(3- O-thio)triphosphate; PTX, pertussis toxin; 8-Br-cGMP, 8-bromo-cGMP; IP, inositol 1,4,5-trisphosphate; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MES, 4-morpholineethanesulfonic acid.


ACKNOWLEDGEMENTS

We are grateful to Dr. K. Spicher for providing the antisera, to Dr. T. Gudermann for synthesis of GTP-azidoanilide, and to E. Roller for the graphical work.


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