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G-proteins and adenylyl cyclase in ovarian granulosa cells of amago salmon (Oncorhynchus rhodurus)
ELSEVIER
Molecular and Cehlar Endocrinology 105 (1994) 83-88
G-proteins and adenylyl cyclase in ovarian granulosa cells of amago salmon (Oncorhynchus rhodurus) Masatoshi Mitaa, Michiyasu Yoshikunib, Yoshitaka Nagahamab-* ‘Teikyo Junior College, Honcho, Shibuya-ku, Tokyo 151, Japan bL.uboratory of Reproductive Biology, National Institute for Basic Biology, Okamki 444, Japan
Received 28 December 1993; accepted 15 July 1994
Abstract The involvement of guanine nucleotide-binding regulatory proteins (G-proteins) and adenylyl cyclase in the gonadotropin stimulation of CAMP was investigated using crude membrane fractions from granulosa cells of amago salmon (Oncorhynchus rhodurus) postvitellogenic ovarian follicles. Although cholera toxin-catalyzed ADP ribosylation occurred in 45 and 50-kDa proteins, only the former was recognized by an antibody against the a-subunit of Gs. With pertussis toxin, only the 41-kDa protein was ADPribosylated. This 41-kDa protein was recognized by an antibody against the a-subunit of Gi. Partially purified chum salmon gonadotropin (SGA) stimulated adenylyl cyclase activity in crude membrane preparations of granulosa cells only in the presence of pertussis toxin in the incubation medium. Adenosine inhibited adenylyl cyclase in the presence of GTP and pertussis toxin reversed it. Unlike SGA, forskolin, which acts upon adenylyl cyclase without G-protein interaction, markedly stimulated adenylyl cyclase activity in the absence of pertussis toxin. These results provide evidence that both stimulator-y (Gs) and inhibitory (Gi) regulation by adenylyl cyclase operates in the granulosa cells of amago salmon postvitellogenic ovarian follicles. It is possible that, although a stimulatory receptor interacts with Gs, its activity is influenced by the functional state of Gi. Keywords:
G-protein; Adenylyl cyclase; Granulosa cell; Amago salmon; Gonadotropin; Steroidogenesis
1. Introduction Guanine nucleotide-binding-proteins (G-proteins) participate in a wide variety of biological processes, patticularly those that require the coupling of an external stimulus to an effector system (Gilman, 1984). G-proteins were first identified as substances that can either stimulate (Gs) or inhibit (Gi) the activity of adenylyl cyclase (Rodbell, 1980; Ui, 1984). The a-subunit of some G proteins is ADP-ribosylated by specific toxins, which results in modulation of the G protein function. For example, Gs is a substrate for cholera toxin-catalyzed ADP-ribosylation, which results in the constitutive activation of adenylyl cyclase (Gill, 1982). Gi is a substrate for the pertussis toxin-catalyzed ADP-ribosylation (Vi, 1984). Cyclic adenosine monophosphate (CAMP) production in several systems is catalyzed by the activity of adenylyl cyclase
xponding
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upon association of the GTP-bound, active a-subunit of Gs with the enzyme. It is generally accepted that in mammals, gonadotropin-induced steroidogenesis is mediated by the Gprotein-adenylyl cyclase-CAMP system. In teleost fishes, gonadotropin stimulates ovarian production of estradiol178 (vitellogenesis-inducing hormone) and progestogens such as 17a,2Q%dihydroxy-4-pregnen-3-one (maturationinducing hormone of salmonid fishes) (Nagahama, 1987a). These actions of gonadotropin are mimicked by drugs such as forskolin, exogenous CAMP or phosphodiesterase inhibitors, that increase the cellular level of CAMP (Chang and Huang, 1982; Bogomolnaya and Yaron, 1984; Tan et al., 1986; Kanamori and Nagahama, 1988b). However, there has been no evidence indicating the involvement of G-proteins and adenylyl cyclase in coupling gonadotropin to CAMP formation. The present experiments were carried out to examine the involvement of G-proteins and adenylyl cyclase in the action of gonadotropin on ovarian granulosa cells of
84
M. Miraet al. I MolecularandCellularEndocrinology 105 (1994) 83-88
amago salmon, Oncorhynchus rhodurus. We first demonstrate the presence of two G-proteins, Gs and Gi, which are responsible for the stimulation or inhibition of adenylyl cyclase. The effect of compounds that modulate the activity of G-proteins on gonadotropin-induced adenylyl cyclase activation was also examined. 2. Materials and methods 2.1. Preparation of crude membrane fractions in granulosa cells Amago salmon at the breeding season were obtained from the Gifu Prefecture Fisheries Experimental Station, Japan. Intact granulosa layers were prepared according to the method described by Kagawa et al. (1982). Granulosa cells were removed from the zona radiata by stirring the granulosa layers in ice-cold Ringer’s solution for 1 h. The mixture was then centrifuged at 1700 X g for 5 min at 0°C. The pelleted granulosa cells were washed three times with Ringer’s solution. The isolated granulosa cells were diluted in 10 mM Tris-HCl (pH 7.6) and homogenized with a Teflon homogenizer in an ice bath. The homogenate was centrifuged at 15 000 X g for 30 min at 4°C. The precipitate was used as the crude membrane fraction. The protein concentration was measured by the method of Lowry et al. (195 1). 2.2. Gel electrophoresis and autoradiography Crude granulosa membrane preparations (0.17 mg protein) were incubated for 30 min at 25°C with activated cholera toxin (5Opglml) or pertussis toxin (lO~g/ml) in the presence of [(r-32P]NAD (29.6 TBq/mmol), 1 mM ATP, 10 mM thymidine, 3 mM MgC12 and 30 mM TrisHCl (pH 7.8) in a total volume of 0.1 ml. 5’-Guanylylimidodiphosphate (GppNHp) (0.1 mM) was added to the cholera toxin sample. At the end of incubation, the reaction mixture was quickly diluted with 1 ml of ice-cold 30 mM Tris-HCl (pH 7.8) containing 3 mM MgC12 and then centrifuged at 10 000 x g for 10 min; the sediment was washed twice by suspension and centrifugation. The precipitate was finally dissolved in 0.06 ml of gel sample buffer and heated for 3 min at 100°C. Twenty-five microliter aliquots were loaded onto each lane of a l-mmthick sodium dodecyl sulfate-polyacrylamide slab (10% gel) (SDS-PAGE) and resolved by electrophoresis as described (Laemmli, 1970). The gels were then stained with Coomassie brilliant blue R-250, destained, dried on filter paper and autoradiographed at -80°C using a Fuji X-ray RX film. 2.3. Immunoblotting Proteins separated by SDS-PAGE were transferred to an Immobilon membrane (Millipore) by electro-blotting (Towbin et al., 1979). The membrane was rinsed in Trisbuffered saline (TBS, 20 mM Tris-HCl, 150 mM NaCl, pH 7.5), blocked with 5% non-fat dry milk in TBS con-
taining 0.1% Tween 20 (TTBS), and incubated with a 1:lOOO dilution of anti Gs or Gi proteins in TBS containing 5% bovine serum albumin overnight at 4°C. After three washes (5 min each) with Tf’BS, the membrane was incubated with a 1: 1000 dilution of alkaline phosphataseconjugated goat anti-rabbit immunoglubolin (Tago). Following three further washes with ‘ITBS, phosphatase activity was visualized by treating the membrane with 0.2 mM 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt and nitroblue tetrazolium in 100 mM diethanolamine buffer (pH 9.5) containing 5 mM MgC12. 2.4. Adenylyl cyclase assay The reaction mixture for the adenylyl cyclase assay contained 1 mM ATP, 6 mM MgC12, 1 mM 3-isobutyl-lmethylxanthine (IBMX), 10 mM creatine phosphate, 0.03 mg of creatine kinase and 40 mM Tris-HCl (pH 7.8). Crude granulosa cell membranes (0.04 mg protein) were incubated in the reaction mixture (0.1 ml) for 10 min at 25°C. The reaction was stopped by adding 0.1 ml of 0.1 M EDTA and boiled for 3 min. Cyclic AMP was measured as previously described (Honma et al., 1977) using a CAMP radioimmunoassay kit (Yamasa Shoyu, Chiba, Japan). The enzyme activity was expressed as nanomoles CAMP formed/l0 min per mg protein. 2.5. Reagents [a-32P]NAD (29.6 TBq/mmol) and anti G-proteins for Gsa (RM/l) and Gia (AS/7) were purchased from DuPont-New England Nuclear (Wilmington, DE). Cholera toxin and pertussis toxin from Seikagaku Kogyo (Tokyo, Japan) were activated by incubation with 40 mM dithiothreitol (DlT) for 30 min at 30°C before use. Guanosine 5’-0-(3-thiotriphosphate) (GTP-yS), GppNHp and forskolin were from Sigma Chemical Co. (St. Louis, MO). Partially purified chum salmon gonadotropin (SGA) was from Syndel Laboratories (Vancouver, B.C., Canada). 3. Results 3.1. G-proteins in amago salmon granulosa cells To identify G-proteins in amago salmon granulosa cells, incorporation of the labelling into crude membrane preparations was determined by SDS-PAGE and autoradiography. When crude membranes were incubated with [cz-~~P]NAD in the presence of cholera toxin, the labelling was incorporated into two proteins with apparent molecular weights of 45 and 50 kDa (Fig. 1). Pertussis toxin labelled only the 41-kDa protein. The level of incorporation of 32P into the 41-kDa protein was much greater than that into the 45- and 50-kDa proteins. In the absence of the toxins, there was no labelling. Immnoblotting using antibodies against the a-subunit of Gs (Gsa) and Gi (Gia) was performed to determine the presence of G-proteins in amago salmon granulosa cells
hf. Mita et al. I Molecular and Cellular Endocrinology 105 (1994) 83-88
85
was observed with cholera toxin at concentrations between 10 and lOOpg/ml. We then examined the effects of cholera toxin (5Opg) and pertussis toxin (1Opg) on intact granulosa cells. Unlike crude membrane preparations, intact granulosa cells did not produce either 17a,20@-DP or CAMP in response to either toxin (data not shown).
66-
4529 -
CT PT NT Fig. 1. Cholera toxin (CT)- and pertussis toxin (PT)-mediated ADPtibosylation of crude membrane preparations of granulosa cells. In the control experiment, no toxin (NT) was added. The experiments were performed as described in Section 2. The gel was autoradiographed for 6 days. Molecular mass standards: myoglobin (17 kDa), carbonic anhydrolase (29 kDa), ovalbumin (45 kDa), bovine serum albumin (66 kDa). Arrows indicate the origin and dye front of the gel.
(Fig. 2). The Gsa and Gia antibodies recognized the 45 and 41 kDa proteins, respectively. Although 32P was incorporated into the 50-kDa protein in the presence of cholera toxin (Fig. l), the anti-m antibody did not recognize this protein (Fig. 2). 3.2. Effect of cholera toxin and pertussis toxin on adenylyl cyclase activity Since cholera toxin and pertussis toxin regulate the hormone-sensitive adenylyl cyclase system, we examined their effects upon adenylyl cyclase activity using crude membrane preparations. As shown in Fig. 3, pertussis toxin stimulated adenylyl cyclase activity, with the maximal stimulation being at a concentration of about lOpg/ml. By contrast, no marked increase in the activity
3.3. Effect of gonadotropin on adenylyl cyclase activity Since our previous studies showed that gonadotropin stimulates accumulation of intracellular CAMP in granulosa cells (Kanamori and Nagahama, 1988b), we examined the effect of gonadotropin on adenylyl cyclase activity in crude membrane preparations. SGA at concentrations between 0.1 and 10 pg/ml did not have any effect on adenylyl cyclase activity irrespective of 0.1 mM GTP (Fig. 4a). In the presence of pertussis toxin (20pgg/ml), SGA stimulated adenylyl cyclase activity only in the presence of GTP (Fig. 4b). We then examined the effects of various doses of GTP on gonadotropin-induced adenylate cyclase activity in the absence or presence of lOpg/ml pertussis toxin. In the presence of pertussis toxin, GTP produced a dosedependent increase in adenylyl cyclase activity. The addition of 0.1 mM GTP markedly enhanced SGAstimulated adenylyl cyclase activity and SGA at 1.O,@ml brought about maximal activation of the enzyme activity. In the presence of l.Opg/ml SGA, GTP induced a concentration-dependent activation of adenylyl cyclase activity, with half maximal stimulation at about 10 PM (Fig. 5b). In contrast, in the absence of pertussis toxin, GTP did not stimulate adenylyl cyclase activity (Fig. 5a). In addition, non-hydrolyzable GTP analogs such as GTP-yS and GppNHp did not affect adenylyl cyclase activity (data not shown).
_(b)
*p 009-(a) :z ‘5 . g 5 0.08 -
. .
f ; 0.07 5 2 0.06 -
45:
0”‘E s 0 0.05 59z
41-
B:
g-,0.04-
-i/i
m
’
0.03 l 0
50
100
[CT] tug/ml)
Gs
Gi
Fig. 2. Immunoblotting after SDS-PAGE of crude membrane preparations of granulosa cells with anti-Gscz (Gs) and -Gia (Gi) antibodies. The positions of the 45 and 41 kDa are indicated. Arrowhead indicates the position of the 50 kDa protein.
0
10
20
[PTI (~g/ml)
Fig. 3. The effect of cholera toxin (CT) (a) and pertussis toxin (PT) (b) on adenylyl cyclase activity in granulosa cells. Crude membranes (0.04 mg protein) of granulosa cells were incubated for 10 min at 25°C in a reaction mixture (0.1 ml) containing 1 mM NAD and 1 mM DTT in the presence of various concentrations of activated CT or PT. The values shown are means of duplicate determinations.
M. Mitu et al. I Molecular and Cellular Endocrinology 105 (1994) 83-88
86
(a)
-I
[Adenosine]
(mM)
Fig. 6. The effect of adenosine on adenylyl cyclase activity in granulosa cells. Crude granulosa cell membranes (0.04 mg protein) were incubated for 10 min at 25°C in a reaction mixture (0.1 ml) in the presence of various concentrations of adenosine with (0) or without (0) 0.1 mM GTP. The values shown are means of duplicate determinations.
0.08 7-
0
0.5
1.0
EGA1
10
(pglmt)
Fig. 4. The effect of chum salmon gonadotropin (SGA) on adenylyl cyclase activity in granulosa cells in the absence (a) and presence (b) of pertussis toxin (PT). Crude membranes (0.04 mg protein) of granuloss cells were incubated for 10 min at 25°C in the reaction mixture (0.1 ml) in the presence of various concentrations of SGA with (e) or without (0) 0.1 mM GTP (a). Additionally, the reaction mixture contained 1 mM NAD, 1 mM DTT and 20,@ml PT (b). The values shown are means of duplicate determinations.
0.08
t 0.08 ;22 ; 0.04 -
.
l
m h 0, 0 0.02 :E cz 0” ‘E (b)
f--
3.4. Effect of adenosine on adenylyl cyclase activity Since adenosine is an agonist coupled with the adenylyl cyclase inhibitory system in a number of tissues (Fain et al., 1972; Londos and Preston, 1977; Londos and Wolff, 1977), we examined whether adenosine inhibits adenylyl cyclase activity in crude membrane preparations of granulosa cells. As shown in Fig. 6, adenosine induced a concentration-dependent inhibition of adenylyl cyclase activity in crude membranes. GTP at a concentration of 0.1 mM enhanced this action, with half-maximal inhibition at 0.1 mM adenosine. Furthermore, pertussis toxin reversed the adenosine-induced inhibition of adenylyl cyclase in a dose-dependent manner (Fig. 7). 3.5. Effect offorskolin on adenylyl cyclase activity Since forskolin mimics the action of gonadotropin on steroidogenesis in amago salmon granulosa cells (Kana-
. :r 0
20
10
[PTI
@g/ml)
Fig. 7. The effect of pertussis toxin (PT) on adenosine-inhibited adenylyl cyclase activity in granulosa cells. Crude granulosa cell membranes (0.04 mg protein) were incubated for 10 min at 25°C in a reaction mixture (0.1 ml) containing 1 mM adenosine, 1 mM NAD and I mM DTT in the presence of various concentrations of activated PT. The values shown are means for duplicate determinations.
M. Mita et al. I Molecular and Cellular Endocrinology 105 (1994) 83-88
[Forskolinl
04)
Fig. 8. The effect of forskolin on adenylate cyclase activity in granulosa cells. Crude granulosa cell membranes (0.04 mg protein) were incubated for 10 mitt at 25°C in the reaction mixture (0.1 ml) in the presence of various concentrations of forskolin. Forskolin was dissolved in dimethyl sulfoxide. The stock solution (100mM) was diluted in distilled water. The values shown are means for duplicate determinations.
mori and Nagahama, 1988b), its effect upon adenylyl cyclase activity was examined using crude membrane preparations of granulosa cells. Forskolin at concentrations of 0.1 PM and 0.1 mM activated adenylyl cyclase by about 2- and 20-fold respectively (Fig. 8).
Discussion Our previous in vitro studies using intact granulosa cells isolated from amago salmon postvitellogenic ovarian follicles demonstrated that gonadotropin promotes the conversion of 17a-hydroxyprogesterone to 17a,2C@ dihydroxy-4-pregnen-3-one (17a,2C@DP) (Nagahama and Adachi, 1985) by stimulating the de novo synthesis of 20/3-hydroxysteroid dehydrogenase (2tJ&HSD) (Nagahama et al., 1985; Young et al., 1986; Nagahama, 1987b). We also showed that this action of gonadotropin occurs through the receptor-mediated formation of CAMP in granulosa cells (Kanamori and Nagahama, 1988a,b). In this study, we provided evidence that G-proteins and adenylyl cyclase couple the gonadotropin receptor to the formation of CAMP in ovarian granulosa cells of amago salmon. In this study, cholera toxin and pertussis toxin, which catalyze the ADP-ribosylation of the a-subunit of Gproteins, were used to investigate the interaction of Gproteins with the control of adenylyl cyclase activity. Exposing crude membrane preparations to cholera toxin resulted in the labelling of several proteins including two major proteins of 45 and 50 kDa. However, immunoblotting revealed that the anti-Gsa antibody recognized only the 45-kDa protein. Since the anti-Gsu antibody used in the present study was raised against a peptide (RMHLRQYELL) highly conserved from human
87
to Drosophila Gsa, it is likely that the 45-kDa protein, but not the 50-kDa protein, is a component of G-proteins in amago salmon granulosa cells. These results are consistent with our recent finding that rainbow trout (Oncorhynchus mykiss) oocyte plasma membranes contain only a single Gs protein (43 kDa) (Yoshikuni and Nagahama, 1994). The ability of pertussis toxin to catalyze the ADPribosylation of the a subunit of Gi has allowed identification of 41-kDa proteins as an a-subunit of Gi in various mammalian cells (Vi, 1984). In this study, pertussis toxin catalyzed the ribosylation of the 41-kDa protein of the amago salmon granulosa cell membranes. This substrate had the electrophoretic mobility of Gi and was the only substrate present in membrane preparations of granulosa cells. Finally, Western blotting revealed that the 41-kDa protein was recognized by the anti-Gia antibody. A 40kDa protein from rainbow trout oocyte plasma membrane preparations was also [32P]ADP-ribosylated in the presence of pertussis toxin (Yoshikuni and Nagahama, 1994). The presence of Gs proteins in amago salmon granulosa cells suggests that Gs is involved in the gonadotropin-induced activation of adenylyl cyclase and formation of CAMP. Under our conditions (a cell-free system using crude membrane preparations), however, SGA was effectively stimulated adenylyl cyclase only when both GTP and pertussis toxin were present in the incubation medium. Furthermore, pertussis toxin by itself stimulated adenylyl cyclase activity. These results suggest that there was a tonic inhibition of adenylyl cyclase response present under our incubation conditions. It is noteworthy that in this study, adenosine inhibited adenylyl cyclase activity. This effect was completely reversed by pertussis toxin, suggesting that adenosine inhibits adenylyl cyclase activity via interaction with Gi. The reaction mixture for assaying adenylyl cyclase activity contained ATP as the substrate. It is possible that ATP was partially hydrolyzed to adenosine by a phosphatase present in our cell-free system of crude membrane preparations of granulosa cells. Thus, one explanation for the stimulatory effect of pertussis toxin is that it reverses the adenosine-mediated activation of Gi protein. Consistent with this hypothesis, forskolin, which activates adenylyl cyclase without Gprotein (Seamon and Daly, 1981; Seamon et al., 1981), markedly stimulated adenylyl cyclase activity in crude membrane preparations of granulosa cells even in the absence of pertussis toxin. In summary, we provided evidence for the presence of both stimulatory (Gs) and inhibitory (Gi) pathways of adenylyl cyclase that operate in the granulosa cells of amago salmon postvitellogenic follicles. It is possible that although a stimulatory receptor interacts exclusively with Gs, its activity is influenced by the functional state of Gi. The physiological role of this inhibitory pathway in the endocrine function of the ovary and the regulation of oogenesis remains to be determined.
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M. Mita et al. I Molecular and Cellular Endocrinology 105 (1994) 83-88
Acknowledgements The study was carried out under the auspices of the NIBB Cooperative Research Program (90-l 13 and 92120) and supported in part by grants (03740369 to M.M. and 02102010 and 04044177 to Y.N.) from the Ministry of Education, Science and Culture of Japan. References Bogomolnaya, A. and Yaron, Z. (1984) Gen. Comp. Endocrinol. 53, 187-196. Chang, Y.S. and Huang, F.L. (1982) Gen. Comp. Endocrinol. 48, 147153. Fain, J.N., Pointer, R.H. and Ward, W.F. (1972) J. Biol. Chem. 247, 6866-6872. Gill, D.M. (1982) in ADP-Ribosylation Reactions Biology and Medicine (Hayaishi, 0. and Ueda, K., eds.), pp. 593-621, Academic Press, New York. Gilman, A.G. (1984) Cell 36,577-579. Honma, M., Satoh, T., Takezawa, J. and Ui, M. (1977) Biochem. Med. 18,257-273. Kagawa, H., Young, G., Adachi, S. and Nagahama, Y. (1982). Gen. Comp. Endocrinol. 47, 44@448.
Kanamori, A. and Nagahama, Y. (1988a) Gen. Comp. Endocrinol. 72, 25-38. Kanamori, A. and Nagahama, Y. (1988b) Gen. Comp. Endocrinol. 72, 39-53. Laemmli, U. K. (1970) Nature 227, 68@685. Londos, C. and Preston, MS. (1977) J. Biol. Chem. 252.5951-5956. Londos, C. and Wolff, J. (1977) Proc. Natl. Acad. Sci. USA 74, 54825486. Lowry, O.H., Rosebrough, N.J., Fan, A.L. and Randall, R.J. (1951) I. Biol. Chem. 193,265-275. Nagahama, Y. (1987a) Zool. Sci. 4, 209-222. Nagahama, Y. (1987b) Dev. Growth Differ. 29, l-12. Nagahama, Y. and Adachi, S. (1985) Dev. Biol. 109.428435. Nagahama, Y., Young, G. and Adachi, S. (1985) Dev. Growth Differ. 27,213-221. Rodbell, M. (1980) Nature 284, 17-22. Seamon, K.B. and Daly, J.M. (1981) J. Biol. Chem. 256,9799-9801. Seamon, K.B., Padgett, W. and Daly, J. (1981) Proc. Natl. Acad. Sci. USA 78.3363-3367. Tan, D. J., Adachi, S. and Nagahama, Y. (1986) Gen. Comp. Endocrinol. 53, 187-196. Towbin, H., Saehelin, T. and Gordon, J. (1979) Proc. Natl. Acad. Sci. USA 76,435&4354. Ui, M. (1984) Trends Pharmacol. Sci. 5.277-279. Yoshikuni, M. and Nagahama, Y. (1994) Dev. Biol., in press. Young, G., Adachi, S. and Nagahama, Y. (1986) Dev. Biol. 118,1-8.
Molecular and Cehlar Endocrinology 105 (1994) 83-88
G-proteins and adenylyl cyclase in ovarian granulosa cells of amago salmon (Oncorhynchus rhodurus) Masatoshi Mitaa, Michiyasu Yoshikunib, Yoshitaka Nagahamab-* ‘Teikyo Junior College, Honcho, Shibuya-ku, Tokyo 151, Japan bL.uboratory of Reproductive Biology, National Institute for Basic Biology, Okamki 444, Japan
Received 28 December 1993; accepted 15 July 1994
Abstract The involvement of guanine nucleotide-binding regulatory proteins (G-proteins) and adenylyl cyclase in the gonadotropin stimulation of CAMP was investigated using crude membrane fractions from granulosa cells of amago salmon (Oncorhynchus rhodurus) postvitellogenic ovarian follicles. Although cholera toxin-catalyzed ADP ribosylation occurred in 45 and 50-kDa proteins, only the former was recognized by an antibody against the a-subunit of Gs. With pertussis toxin, only the 41-kDa protein was ADPribosylated. This 41-kDa protein was recognized by an antibody against the a-subunit of Gi. Partially purified chum salmon gonadotropin (SGA) stimulated adenylyl cyclase activity in crude membrane preparations of granulosa cells only in the presence of pertussis toxin in the incubation medium. Adenosine inhibited adenylyl cyclase in the presence of GTP and pertussis toxin reversed it. Unlike SGA, forskolin, which acts upon adenylyl cyclase without G-protein interaction, markedly stimulated adenylyl cyclase activity in the absence of pertussis toxin. These results provide evidence that both stimulator-y (Gs) and inhibitory (Gi) regulation by adenylyl cyclase operates in the granulosa cells of amago salmon postvitellogenic ovarian follicles. It is possible that, although a stimulatory receptor interacts with Gs, its activity is influenced by the functional state of Gi. Keywords:
G-protein; Adenylyl cyclase; Granulosa cell; Amago salmon; Gonadotropin; Steroidogenesis
1. Introduction Guanine nucleotide-binding-proteins (G-proteins) participate in a wide variety of biological processes, patticularly those that require the coupling of an external stimulus to an effector system (Gilman, 1984). G-proteins were first identified as substances that can either stimulate (Gs) or inhibit (Gi) the activity of adenylyl cyclase (Rodbell, 1980; Ui, 1984). The a-subunit of some G proteins is ADP-ribosylated by specific toxins, which results in modulation of the G protein function. For example, Gs is a substrate for cholera toxin-catalyzed ADP-ribosylation, which results in the constitutive activation of adenylyl cyclase (Gill, 1982). Gi is a substrate for the pertussis toxin-catalyzed ADP-ribosylation (Vi, 1984). Cyclic adenosine monophosphate (CAMP) production in several systems is catalyzed by the activity of adenylyl cyclase
xponding
author. Tel. 81 564 55 7550. Fax 81 564 55 7556.
0167-8140/94/$07.00 0 1994 Elsevier Science Ireland Ltd. All rights reserved SSDI 0167-8140(94)03365-Z
upon association of the GTP-bound, active a-subunit of Gs with the enzyme. It is generally accepted that in mammals, gonadotropin-induced steroidogenesis is mediated by the Gprotein-adenylyl cyclase-CAMP system. In teleost fishes, gonadotropin stimulates ovarian production of estradiol178 (vitellogenesis-inducing hormone) and progestogens such as 17a,2Q%dihydroxy-4-pregnen-3-one (maturationinducing hormone of salmonid fishes) (Nagahama, 1987a). These actions of gonadotropin are mimicked by drugs such as forskolin, exogenous CAMP or phosphodiesterase inhibitors, that increase the cellular level of CAMP (Chang and Huang, 1982; Bogomolnaya and Yaron, 1984; Tan et al., 1986; Kanamori and Nagahama, 1988b). However, there has been no evidence indicating the involvement of G-proteins and adenylyl cyclase in coupling gonadotropin to CAMP formation. The present experiments were carried out to examine the involvement of G-proteins and adenylyl cyclase in the action of gonadotropin on ovarian granulosa cells of
84
M. Miraet al. I MolecularandCellularEndocrinology 105 (1994) 83-88
amago salmon, Oncorhynchus rhodurus. We first demonstrate the presence of two G-proteins, Gs and Gi, which are responsible for the stimulation or inhibition of adenylyl cyclase. The effect of compounds that modulate the activity of G-proteins on gonadotropin-induced adenylyl cyclase activation was also examined. 2. Materials and methods 2.1. Preparation of crude membrane fractions in granulosa cells Amago salmon at the breeding season were obtained from the Gifu Prefecture Fisheries Experimental Station, Japan. Intact granulosa layers were prepared according to the method described by Kagawa et al. (1982). Granulosa cells were removed from the zona radiata by stirring the granulosa layers in ice-cold Ringer’s solution for 1 h. The mixture was then centrifuged at 1700 X g for 5 min at 0°C. The pelleted granulosa cells were washed three times with Ringer’s solution. The isolated granulosa cells were diluted in 10 mM Tris-HCl (pH 7.6) and homogenized with a Teflon homogenizer in an ice bath. The homogenate was centrifuged at 15 000 X g for 30 min at 4°C. The precipitate was used as the crude membrane fraction. The protein concentration was measured by the method of Lowry et al. (195 1). 2.2. Gel electrophoresis and autoradiography Crude granulosa membrane preparations (0.17 mg protein) were incubated for 30 min at 25°C with activated cholera toxin (5Opglml) or pertussis toxin (lO~g/ml) in the presence of [(r-32P]NAD (29.6 TBq/mmol), 1 mM ATP, 10 mM thymidine, 3 mM MgC12 and 30 mM TrisHCl (pH 7.8) in a total volume of 0.1 ml. 5’-Guanylylimidodiphosphate (GppNHp) (0.1 mM) was added to the cholera toxin sample. At the end of incubation, the reaction mixture was quickly diluted with 1 ml of ice-cold 30 mM Tris-HCl (pH 7.8) containing 3 mM MgC12 and then centrifuged at 10 000 x g for 10 min; the sediment was washed twice by suspension and centrifugation. The precipitate was finally dissolved in 0.06 ml of gel sample buffer and heated for 3 min at 100°C. Twenty-five microliter aliquots were loaded onto each lane of a l-mmthick sodium dodecyl sulfate-polyacrylamide slab (10% gel) (SDS-PAGE) and resolved by electrophoresis as described (Laemmli, 1970). The gels were then stained with Coomassie brilliant blue R-250, destained, dried on filter paper and autoradiographed at -80°C using a Fuji X-ray RX film. 2.3. Immunoblotting Proteins separated by SDS-PAGE were transferred to an Immobilon membrane (Millipore) by electro-blotting (Towbin et al., 1979). The membrane was rinsed in Trisbuffered saline (TBS, 20 mM Tris-HCl, 150 mM NaCl, pH 7.5), blocked with 5% non-fat dry milk in TBS con-
taining 0.1% Tween 20 (TTBS), and incubated with a 1:lOOO dilution of anti Gs or Gi proteins in TBS containing 5% bovine serum albumin overnight at 4°C. After three washes (5 min each) with Tf’BS, the membrane was incubated with a 1: 1000 dilution of alkaline phosphataseconjugated goat anti-rabbit immunoglubolin (Tago). Following three further washes with ‘ITBS, phosphatase activity was visualized by treating the membrane with 0.2 mM 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt and nitroblue tetrazolium in 100 mM diethanolamine buffer (pH 9.5) containing 5 mM MgC12. 2.4. Adenylyl cyclase assay The reaction mixture for the adenylyl cyclase assay contained 1 mM ATP, 6 mM MgC12, 1 mM 3-isobutyl-lmethylxanthine (IBMX), 10 mM creatine phosphate, 0.03 mg of creatine kinase and 40 mM Tris-HCl (pH 7.8). Crude granulosa cell membranes (0.04 mg protein) were incubated in the reaction mixture (0.1 ml) for 10 min at 25°C. The reaction was stopped by adding 0.1 ml of 0.1 M EDTA and boiled for 3 min. Cyclic AMP was measured as previously described (Honma et al., 1977) using a CAMP radioimmunoassay kit (Yamasa Shoyu, Chiba, Japan). The enzyme activity was expressed as nanomoles CAMP formed/l0 min per mg protein. 2.5. Reagents [a-32P]NAD (29.6 TBq/mmol) and anti G-proteins for Gsa (RM/l) and Gia (AS/7) were purchased from DuPont-New England Nuclear (Wilmington, DE). Cholera toxin and pertussis toxin from Seikagaku Kogyo (Tokyo, Japan) were activated by incubation with 40 mM dithiothreitol (DlT) for 30 min at 30°C before use. Guanosine 5’-0-(3-thiotriphosphate) (GTP-yS), GppNHp and forskolin were from Sigma Chemical Co. (St. Louis, MO). Partially purified chum salmon gonadotropin (SGA) was from Syndel Laboratories (Vancouver, B.C., Canada). 3. Results 3.1. G-proteins in amago salmon granulosa cells To identify G-proteins in amago salmon granulosa cells, incorporation of the labelling into crude membrane preparations was determined by SDS-PAGE and autoradiography. When crude membranes were incubated with [cz-~~P]NAD in the presence of cholera toxin, the labelling was incorporated into two proteins with apparent molecular weights of 45 and 50 kDa (Fig. 1). Pertussis toxin labelled only the 41-kDa protein. The level of incorporation of 32P into the 41-kDa protein was much greater than that into the 45- and 50-kDa proteins. In the absence of the toxins, there was no labelling. Immnoblotting using antibodies against the a-subunit of Gs (Gsa) and Gi (Gia) was performed to determine the presence of G-proteins in amago salmon granulosa cells
hf. Mita et al. I Molecular and Cellular Endocrinology 105 (1994) 83-88
85
was observed with cholera toxin at concentrations between 10 and lOOpg/ml. We then examined the effects of cholera toxin (5Opg) and pertussis toxin (1Opg) on intact granulosa cells. Unlike crude membrane preparations, intact granulosa cells did not produce either 17a,20@-DP or CAMP in response to either toxin (data not shown).
66-
4529 -
CT PT NT Fig. 1. Cholera toxin (CT)- and pertussis toxin (PT)-mediated ADPtibosylation of crude membrane preparations of granulosa cells. In the control experiment, no toxin (NT) was added. The experiments were performed as described in Section 2. The gel was autoradiographed for 6 days. Molecular mass standards: myoglobin (17 kDa), carbonic anhydrolase (29 kDa), ovalbumin (45 kDa), bovine serum albumin (66 kDa). Arrows indicate the origin and dye front of the gel.
(Fig. 2). The Gsa and Gia antibodies recognized the 45 and 41 kDa proteins, respectively. Although 32P was incorporated into the 50-kDa protein in the presence of cholera toxin (Fig. l), the anti-m antibody did not recognize this protein (Fig. 2). 3.2. Effect of cholera toxin and pertussis toxin on adenylyl cyclase activity Since cholera toxin and pertussis toxin regulate the hormone-sensitive adenylyl cyclase system, we examined their effects upon adenylyl cyclase activity using crude membrane preparations. As shown in Fig. 3, pertussis toxin stimulated adenylyl cyclase activity, with the maximal stimulation being at a concentration of about lOpg/ml. By contrast, no marked increase in the activity
3.3. Effect of gonadotropin on adenylyl cyclase activity Since our previous studies showed that gonadotropin stimulates accumulation of intracellular CAMP in granulosa cells (Kanamori and Nagahama, 1988b), we examined the effect of gonadotropin on adenylyl cyclase activity in crude membrane preparations. SGA at concentrations between 0.1 and 10 pg/ml did not have any effect on adenylyl cyclase activity irrespective of 0.1 mM GTP (Fig. 4a). In the presence of pertussis toxin (20pgg/ml), SGA stimulated adenylyl cyclase activity only in the presence of GTP (Fig. 4b). We then examined the effects of various doses of GTP on gonadotropin-induced adenylate cyclase activity in the absence or presence of lOpg/ml pertussis toxin. In the presence of pertussis toxin, GTP produced a dosedependent increase in adenylyl cyclase activity. The addition of 0.1 mM GTP markedly enhanced SGAstimulated adenylyl cyclase activity and SGA at 1.O,@ml brought about maximal activation of the enzyme activity. In the presence of l.Opg/ml SGA, GTP induced a concentration-dependent activation of adenylyl cyclase activity, with half maximal stimulation at about 10 PM (Fig. 5b). In contrast, in the absence of pertussis toxin, GTP did not stimulate adenylyl cyclase activity (Fig. 5a). In addition, non-hydrolyzable GTP analogs such as GTP-yS and GppNHp did not affect adenylyl cyclase activity (data not shown).
_(b)
*p 009-(a) :z ‘5 . g 5 0.08 -
. .
f ; 0.07 5 2 0.06 -
45:
0”‘E s 0 0.05 59z
41-
B:
g-,0.04-
-i/i
m
’
0.03 l 0
50
100
[CT] tug/ml)
Gs
Gi
Fig. 2. Immunoblotting after SDS-PAGE of crude membrane preparations of granulosa cells with anti-Gscz (Gs) and -Gia (Gi) antibodies. The positions of the 45 and 41 kDa are indicated. Arrowhead indicates the position of the 50 kDa protein.
0
10
20
[PTI (~g/ml)
Fig. 3. The effect of cholera toxin (CT) (a) and pertussis toxin (PT) (b) on adenylyl cyclase activity in granulosa cells. Crude membranes (0.04 mg protein) of granulosa cells were incubated for 10 min at 25°C in a reaction mixture (0.1 ml) containing 1 mM NAD and 1 mM DTT in the presence of various concentrations of activated CT or PT. The values shown are means of duplicate determinations.
M. Mitu et al. I Molecular and Cellular Endocrinology 105 (1994) 83-88
86
(a)
-I
[Adenosine]
(mM)
Fig. 6. The effect of adenosine on adenylyl cyclase activity in granulosa cells. Crude granulosa cell membranes (0.04 mg protein) were incubated for 10 min at 25°C in a reaction mixture (0.1 ml) in the presence of various concentrations of adenosine with (0) or without (0) 0.1 mM GTP. The values shown are means of duplicate determinations.
0.08 7-
0
0.5
1.0
EGA1
10
(pglmt)
Fig. 4. The effect of chum salmon gonadotropin (SGA) on adenylyl cyclase activity in granulosa cells in the absence (a) and presence (b) of pertussis toxin (PT). Crude membranes (0.04 mg protein) of granuloss cells were incubated for 10 min at 25°C in the reaction mixture (0.1 ml) in the presence of various concentrations of SGA with (e) or without (0) 0.1 mM GTP (a). Additionally, the reaction mixture contained 1 mM NAD, 1 mM DTT and 20,@ml PT (b). The values shown are means of duplicate determinations.
0.08
t 0.08 ;22 ; 0.04 -
.
l
m h 0, 0 0.02 :E cz 0” ‘E (b)
f--
3.4. Effect of adenosine on adenylyl cyclase activity Since adenosine is an agonist coupled with the adenylyl cyclase inhibitory system in a number of tissues (Fain et al., 1972; Londos and Preston, 1977; Londos and Wolff, 1977), we examined whether adenosine inhibits adenylyl cyclase activity in crude membrane preparations of granulosa cells. As shown in Fig. 6, adenosine induced a concentration-dependent inhibition of adenylyl cyclase activity in crude membranes. GTP at a concentration of 0.1 mM enhanced this action, with half-maximal inhibition at 0.1 mM adenosine. Furthermore, pertussis toxin reversed the adenosine-induced inhibition of adenylyl cyclase in a dose-dependent manner (Fig. 7). 3.5. Effect offorskolin on adenylyl cyclase activity Since forskolin mimics the action of gonadotropin on steroidogenesis in amago salmon granulosa cells (Kana-
. :r 0
20
10
[PTI
@g/ml)
Fig. 7. The effect of pertussis toxin (PT) on adenosine-inhibited adenylyl cyclase activity in granulosa cells. Crude granulosa cell membranes (0.04 mg protein) were incubated for 10 min at 25°C in a reaction mixture (0.1 ml) containing 1 mM adenosine, 1 mM NAD and I mM DTT in the presence of various concentrations of activated PT. The values shown are means for duplicate determinations.
M. Mita et al. I Molecular and Cellular Endocrinology 105 (1994) 83-88
[Forskolinl
04)
Fig. 8. The effect of forskolin on adenylate cyclase activity in granulosa cells. Crude granulosa cell membranes (0.04 mg protein) were incubated for 10 mitt at 25°C in the reaction mixture (0.1 ml) in the presence of various concentrations of forskolin. Forskolin was dissolved in dimethyl sulfoxide. The stock solution (100mM) was diluted in distilled water. The values shown are means for duplicate determinations.
mori and Nagahama, 1988b), its effect upon adenylyl cyclase activity was examined using crude membrane preparations of granulosa cells. Forskolin at concentrations of 0.1 PM and 0.1 mM activated adenylyl cyclase by about 2- and 20-fold respectively (Fig. 8).
Discussion Our previous in vitro studies using intact granulosa cells isolated from amago salmon postvitellogenic ovarian follicles demonstrated that gonadotropin promotes the conversion of 17a-hydroxyprogesterone to 17a,2C@ dihydroxy-4-pregnen-3-one (17a,2C@DP) (Nagahama and Adachi, 1985) by stimulating the de novo synthesis of 20/3-hydroxysteroid dehydrogenase (2tJ&HSD) (Nagahama et al., 1985; Young et al., 1986; Nagahama, 1987b). We also showed that this action of gonadotropin occurs through the receptor-mediated formation of CAMP in granulosa cells (Kanamori and Nagahama, 1988a,b). In this study, we provided evidence that G-proteins and adenylyl cyclase couple the gonadotropin receptor to the formation of CAMP in ovarian granulosa cells of amago salmon. In this study, cholera toxin and pertussis toxin, which catalyze the ADP-ribosylation of the a-subunit of Gproteins, were used to investigate the interaction of Gproteins with the control of adenylyl cyclase activity. Exposing crude membrane preparations to cholera toxin resulted in the labelling of several proteins including two major proteins of 45 and 50 kDa. However, immunoblotting revealed that the anti-Gsa antibody recognized only the 45-kDa protein. Since the anti-Gsu antibody used in the present study was raised against a peptide (RMHLRQYELL) highly conserved from human
87
to Drosophila Gsa, it is likely that the 45-kDa protein, but not the 50-kDa protein, is a component of G-proteins in amago salmon granulosa cells. These results are consistent with our recent finding that rainbow trout (Oncorhynchus mykiss) oocyte plasma membranes contain only a single Gs protein (43 kDa) (Yoshikuni and Nagahama, 1994). The ability of pertussis toxin to catalyze the ADPribosylation of the a subunit of Gi has allowed identification of 41-kDa proteins as an a-subunit of Gi in various mammalian cells (Vi, 1984). In this study, pertussis toxin catalyzed the ribosylation of the 41-kDa protein of the amago salmon granulosa cell membranes. This substrate had the electrophoretic mobility of Gi and was the only substrate present in membrane preparations of granulosa cells. Finally, Western blotting revealed that the 41-kDa protein was recognized by the anti-Gia antibody. A 40kDa protein from rainbow trout oocyte plasma membrane preparations was also [32P]ADP-ribosylated in the presence of pertussis toxin (Yoshikuni and Nagahama, 1994). The presence of Gs proteins in amago salmon granulosa cells suggests that Gs is involved in the gonadotropin-induced activation of adenylyl cyclase and formation of CAMP. Under our conditions (a cell-free system using crude membrane preparations), however, SGA was effectively stimulated adenylyl cyclase only when both GTP and pertussis toxin were present in the incubation medium. Furthermore, pertussis toxin by itself stimulated adenylyl cyclase activity. These results suggest that there was a tonic inhibition of adenylyl cyclase response present under our incubation conditions. It is noteworthy that in this study, adenosine inhibited adenylyl cyclase activity. This effect was completely reversed by pertussis toxin, suggesting that adenosine inhibits adenylyl cyclase activity via interaction with Gi. The reaction mixture for assaying adenylyl cyclase activity contained ATP as the substrate. It is possible that ATP was partially hydrolyzed to adenosine by a phosphatase present in our cell-free system of crude membrane preparations of granulosa cells. Thus, one explanation for the stimulatory effect of pertussis toxin is that it reverses the adenosine-mediated activation of Gi protein. Consistent with this hypothesis, forskolin, which activates adenylyl cyclase without Gprotein (Seamon and Daly, 1981; Seamon et al., 1981), markedly stimulated adenylyl cyclase activity in crude membrane preparations of granulosa cells even in the absence of pertussis toxin. In summary, we provided evidence for the presence of both stimulatory (Gs) and inhibitory (Gi) pathways of adenylyl cyclase that operate in the granulosa cells of amago salmon postvitellogenic follicles. It is possible that although a stimulatory receptor interacts exclusively with Gs, its activity is influenced by the functional state of Gi. The physiological role of this inhibitory pathway in the endocrine function of the ovary and the regulation of oogenesis remains to be determined.
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Acknowledgements The study was carried out under the auspices of the NIBB Cooperative Research Program (90-l 13 and 92120) and supported in part by grants (03740369 to M.M. and 02102010 and 04044177 to Y.N.) from the Ministry of Education, Science and Culture of Japan. References Bogomolnaya, A. and Yaron, Z. (1984) Gen. Comp. Endocrinol. 53, 187-196. Chang, Y.S. and Huang, F.L. (1982) Gen. Comp. Endocrinol. 48, 147153. Fain, J.N., Pointer, R.H. and Ward, W.F. (1972) J. Biol. Chem. 247, 6866-6872. Gill, D.M. (1982) in ADP-Ribosylation Reactions Biology and Medicine (Hayaishi, 0. and Ueda, K., eds.), pp. 593-621, Academic Press, New York. Gilman, A.G. (1984) Cell 36,577-579. Honma, M., Satoh, T., Takezawa, J. and Ui, M. (1977) Biochem. Med. 18,257-273. Kagawa, H., Young, G., Adachi, S. and Nagahama, Y. (1982). Gen. Comp. Endocrinol. 47, 44@448.
Kanamori, A. and Nagahama, Y. (1988a) Gen. Comp. Endocrinol. 72, 25-38. Kanamori, A. and Nagahama, Y. (1988b) Gen. Comp. Endocrinol. 72, 39-53. Laemmli, U. K. (1970) Nature 227, 68@685. Londos, C. and Preston, MS. (1977) J. Biol. Chem. 252.5951-5956. Londos, C. and Wolff, J. (1977) Proc. Natl. Acad. Sci. USA 74, 54825486. Lowry, O.H., Rosebrough, N.J., Fan, A.L. and Randall, R.J. (1951) I. Biol. Chem. 193,265-275. Nagahama, Y. (1987a) Zool. Sci. 4, 209-222. Nagahama, Y. (1987b) Dev. Growth Differ. 29, l-12. Nagahama, Y. and Adachi, S. (1985) Dev. Biol. 109.428435. Nagahama, Y., Young, G. and Adachi, S. (1985) Dev. Growth Differ. 27,213-221. Rodbell, M. (1980) Nature 284, 17-22. Seamon, K.B. and Daly, J.M. (1981) J. Biol. Chem. 256,9799-9801. Seamon, K.B., Padgett, W. and Daly, J. (1981) Proc. Natl. Acad. Sci. USA 78.3363-3367. Tan, D. J., Adachi, S. and Nagahama, Y. (1986) Gen. Comp. Endocrinol. 53, 187-196. Towbin, H., Saehelin, T. and Gordon, J. (1979) Proc. Natl. Acad. Sci. USA 76,435&4354. Ui, M. (1984) Trends Pharmacol. Sci. 5.277-279. Yoshikuni, M. and Nagahama, Y. (1994) Dev. Biol., in press. Young, G., Adachi, S. and Nagahama, Y. (1986) Dev. Biol. 118,1-8.