Hormonal Regulation of Cyclic AMP-dependent Protein Kinase in

THEJOURNALOF

BIOLOGICAL CHEMISTRY

Vol. 259, No. 23, Issue of December 10. pp. 14778-14782, 1984 Printed in U.S.A.

Hormonal Regulation of Cyclic AMP-dependentProtein Kinase in Cultured Ovarian Granulosa Cells EFFECTSOFFOLLICLE-STIMULATINGHORMONE

AND GONADOTROPIN-RELEASINGHORMONE* (Received for publication, October 31, 1983)

Jean-Marie Darbon,Michael Knecht, TapioRanta, MariaL. Dufau, and Kevin J. Cattg From the Endocrinology and Reproduction Research Branch, National Instituteof Child Health and Human Development, National Institutesof Health, Bethesda, Maryland 20205

The hormonal regulation of CAMP-dependent pro- differentiation is primarily exertedby pituitary FSH,’ which tein kinase was examined in granulosa cells from di- stimulates granulosa cell maturation i n vivo and in vitro (1, ethylstilbestrol-implanted immature rats. Follicle- 2). FSH-induced granulosacell differentiation hasbeen shown stimulating hormone (FSH) increased the number of to be mediated by cAMP (3), presumably via activation of available CAMP-binding sites in a dose- and time-de- CAMP-dependent protein kinase. In most mammalian cells, pendent manner, with a maximum 4-6-fold increase CAMP binds to the R subunits of type I or type I1 protein at 50-100 ng/ml between 6 and 48 h of culture aftera kinase andcauses dissociation of the holoenzyme with release transient decrease in availablesites during the first6 of the C subunits. While the total concentration of CAMPh. The potent gonadotropin-releasing hormone (GnRH) dependent protein kinase appears to be constant in various agonist [D - Ala‘ldes - Gly” - GnRH - N - ethylamide tissues and species (4), the relative proportions of type I and (GnRHa) reduced the FSH-induced increase in CAMP- type I1 vary considerably (5).Differences in the function and binding sites by approximately 50%at 24 and48 h of hormonal regulation of the two classes of protein kinase are culture. Photoaffinity labeling with 8-azid0-[~~P]suggested by changesinthetype I andtype I1 enzymes cAMP revealed the existence of one majorCAMP-bind- throughout thecell cycle (6) and duringadipocyte differentiaing protein (Mr = 55,000 f 400) which appeared to be tion (7). Inthe pigovary, type I1 protein kinase is the the regulatory (R) subunitof type I1 CAMP-dependent predominant form in folliculartissue, while the type I/typeI1 protein kinase. While FSH induced a 5-10-fold in- ratio increases in large follicles and corpora lutea (8). Simicrease in the labelingof R I1 both in vivo and in vitro, larly, estrus rabbitfollicles contain only the type I1 isoenzyme, GnRHa reduced the amountof R I1 induced by FSH in while both typeI and typeI1 kinases are present in the corpus granulosa cells cultured for 48 h. The large increase luteum (9). Also, i n uiuo treatment with estradiol and FSH in R I1 subunit was not accompaniedby a correspond- has been reported to increase the typeI1 CAMP-binding site ing increase in protein kinase activity, which was only (R 11) in rat granulosa cells (10). In the present study, we enhanced by 50% after 48 h of culture with FSH. haveanalyzed thehormonal regulation of protein kinase Fractionation of granulosacell cytosol from FSH- activity and its subunits in cultured rat granulosa cells to treated ovaries on DEAE-cellulose showed a single define the CAMP-dependent events occurring duringthe peak of CAMP-dependent phosphokinase activity with FSH-induceddifferentiation process. We also studiedthe the elution properties of a type I1 protein kinase.How- effect of a potentGnRHagonistonprotein kinasesince ever, the peak of cAMP binding activity(eluted at 0.20 hypothalamic GnRH and itsanalogs exert prominent inhibiM KCl) was not coincidentwith the protein kinase tory actions onCAMP-mediated differentiation in the granuactivity. FSH transiently stimulatedCAMP-dependent losa cell (3, 11, 12). protein kinase activity during the first 10-30 min of culture. GnRHa impaired the FSH-induced early inEXPERIMENTALPROCEDURES crease in protein kinase activity, causing a delay in activation until 60 min. These findings suggestthat a Materials large dose- and time-dependent increase in the content (50-80 Ci/mmol) was purchased from ICN, of CAMP-binding sites may be a major factor in CAMP- a n8-A~ido-[~’PlcAMP d [ T - ~ ~ P ] A T(10-40 P Ci/mmol)and[3H]cAMP (34.5 Ci/mmol) mediated differentiation of granulosa cells. The inhib- were obtained from New England Nuclear. Ovine FSH (NIH-oFSHitory effect of GnRHa on both FSH-induced protein 15) was a gift from the National Pituitary Agency, and GnRHa was kinase activation during the first minutes of culture obtained from Peninsula Laboratories, SanCarlos, CA. and on FSH-induced R I1 synthesis during the subseMethods quent 48 h of culture could be crucial events in the prevention of granulosa cell maturation by GnRH agAnimals and Cell Culture-Twenty-one-day-old diethylstilbestrolonists. implanted female rats were obtained from Hormone Assay Laboratories (Chicago, IL). Six days after diethylstilbestrol treatment, the ovaries were collected and granulosa cells were isolated as previously

Thehormonalcontrol

of ovarian folliculargrowth

and

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate thisfact. T o whom correspondence should beaddressed.

The abbreviations used are: FSH, follicle-stimulating hormone; oFSH, ovine FSH; RI and RII, type I and type I1 CAMP-binding subunits of protein kinase; C, catalytic subunit of protein kinase; GnRH, gonadotropin-releasinghormone; GnRHa, [~-Ala~ldes-Gly’~GnRH-N-ethylamide; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; Mes, 2-(N-rnorpholino)ethanesulfonicacid.

14778

Hormonal Regulation

of CAMP-dependent Protein Kinase

described (13) with the following modifications. Prior toplating, cells were further incubated with 20 pg/ml trypsin for 1 min to lyse dead cells, followed by 7 min with 50 pg/ml egg white trypsin inhibitor and 50 pg/ml DNAse I. Aliquots of 5-10 X lo5 cells, of 85-90% viability by trypan blue exclusion, were added to 35-mm plastic tissue culture dishes in 1.0 ml of McCoy's 5A medium (modified, without serum) supplemented by 10 mM Hepes, pH 7.4,4 mM L-glutamine, 100 units/ ml penicillin, and 100 pg/ml streptomycin sulfate. Various concentrations of oFSH (5-250 ng/ml) and low7M GnRHa (final concentration) were added, and cells were cultured at 37 "C in a humidified 95% air, 5% COS environment for 6-48 h. At the end of the culture period, medium was removed and the cells were washed with phosphatebuffered saline and released from the wells into phosphate-buffered saline with a rubber policeman. After centrifugation, cells were resuspended in 10 mM Tris. HCl, pH 7.4, containing 1 mM EDTA, 50 pg/ml phenylmethylsulfonyl fluoride, and 1 mM dithiothreitol. For some experiments, a similar buffer containing 5 mM instead of 1 mM EDTA and 50 mM benzamidineinstead of phenylmethylsulfonyl fluoride was used. After cell disruption by sonication or by freezingthawing, homogenates were centrifuged a t 30,000 X g for 1h, and the supernatants ("cytosol") were collected. No significant differences in results were observed with either of the buffers or cell disruption procedures. In some experiments, granulosa cells were isolated from ovaries of diethylstilbestrol-implanted rats treated with four injections of 7.5 pg of oFSH every 12 h before death. PHICAMP Binding Assay-Available CAMP-binding sites were quantitatedas previously described (14).Samples of10-20pgof protein were incubated for 1 h at 4 "C in 0.1 ml of 20 mM potassium phosphate, pH 6.5, containing 0.5 mM 1-methyl-3-isobutylxanthine and 0.25 p~ [3H]cAMP(40,000 cpm/pmol). Nonspecific binding was measured by adding M unlabeled CAMP. Freeand bound [3H] cAMP was separated by filtration on 0.45-pm Millipore filters. In all experiments, protein concentrationwas determined by the method of Lowry et al. (15). Histone Kinase Assay-Aliquots of cytosol (10-20 pg of protein) were incubated for 10 min at 30 "C in 0.2 ml of25 mM potassium phosphate, pH 6.5, containing 10 mM magnesium acetate, 1 mg/ml histone type IIa (Sigma), 50 p~ [Y-~'P]ATP(200 cpm/pmol). 32Plabeled histones were precipitated and washed with 10% trichloroacetic acid prior to assay for radioactivity. One unit of kinase activity was defined as the amount of enzyme which transfers 1 pmol of 32P to histone in 1 min a t 30 "C. Photoaffinity Labeling-Covalent binding of 8-a~ido-[~'P]cAMP was performed as described by Liu (7). Samples containing 20-50 pg of protein were incubated in 80 pl of 50 mM Mes, pH 6.2, containing 0.5-1 p~ 8-a~ido-[~'P]cAMP 4at"C for 60 min to reach equilibrium binding and were photolyzed under UV light for 10 min at a distance of 5 cm. The reaction was stopped by addition of 20 p1 of electrophoresis sample buffer containing 0.3 M Tris-HC1, pH 6.7, 10% sodium dodecyl sulfate, 10% @-mercaptoethanol, 40% glycerol, and 0.025% bromphenol blue. Samples were then immediately boiled for 5 min at 90 "C. Autophosphorylation-Samples were incubatedunder the same conditions as in thephotoaffinity labeling experiments in 80 pl of 50 mM Mes, pH 6.2, containing 0.5 mM 1-methyl-3-isobutylxanthine. [y3'P]ATP (0.5 p M ) was added for 10 min at 2 "C, and the reaction was stopped by adding electrophoresis sample buffer and boiling. Polyacrylamide Gel Electrophoresis-Proteins were fractionated by electrophoresis on 4.5% and 10% (w/v) discontinuoussodium dodecyl sulfate-polyacrylamide slab gels as described by Laemmli (16). After protein fixation by trichloroacetic acid and Coomassie Blue staining as previously described (17), the gels were dried and then exposed to Kodak X-Omat AR films for 24-48 h. DEAE-cellulose Chromatography-Granulosa cells from FSHprimed ovarieswere sonicated in 10 mM Tris-HC1, pH 7.4, containing 1 mM EDTA, 1 mM dithiothreitol (buffer A). The 40,000 X g supernatant obtained from homogenates was collected and immediately applied to a DEAE-ce1ulose column(DE52, 1.2 X 6 cm), equilibrated with buffer A. After washing with 15ml of buffer A, a linear gradient of KC1(0-0.4 M ) in buffer A was used to elute proteins from the column. 0.75-ml fractions were collected in tubes containing75 p1 of 30% sucrose, 50 mM EDTA, and 500 mM benzamidine. In Some experiments, 20 mM benzamidine was added to buffer A, and the column was fuxther equilibrated. Salt gradient molarity was determined by conductivity measurements.

14779 RESULTS

When granulosacells were cultured for48 h in thepresence of FSH, there was a dose-dependent increase in the number of available CAMP-binding sites in the cytoplasmic fraction Amaximum 4-6-fold increase in specific cAMP (Fig. U). binding activity was observed with 50-100 ng of FSH, and a significant response was elicited by 10 ng of the gonadotropin. The timecourse of cAMP binding during FSH-induced granulosa cell differentiation (Fig. 1B) revealed a significant decrease in available CAMP-binding sites during the first 6 h of culture.Subsequently,thenumber of CAMP-binding sites continued to fall in control cells, while FSH induced a 3- and 6-fold increase in cAMP bindinga t 24 and 48 h, respectively. In contrast, GnRHa reduced the FSH-induced increase in the number of CAMP-binding sites by approximately 50% after 24 and 48 h of culture. The FSH-inducedincrease in CAMP-binding sites was analyzed by photoaffinity labeling with 8-azid0-[~~P]cAMPin cytosols obtained fromfreshly prepared cells of immature 1500

-A

-

-

T

I

C ._

I

0

T

6

24

I

48

TIME (h.1

FIG. 1. Concentration- and time-dependent increase in CAMP-binding sites in cultured granulosa cells. Approximately 5 X lo5granulosa cells were cultured for 48 h in theabsence or in the presence of increasing concentrations of oFSH(Panel A ) or for various times in the absence (0)or in the presence of 100 ng/ml oFSH (0)orFSH plus M GnRHa (A) (Panel B ) . Available CAMP-binding sites were measured on 30,000 X g supernatants, as indicated under "Experimental Procedures." Data are the mean & S.E. of triplicate measurements from one experiment representative of two or three similar experiments.

14780

Hormonal Regulation of CAMP-dependent Protein Kinase

rats, from cells cultured for 48 h with FSH, andfrom ovarian cells of animals primed with FSH in uiuo. FSH induced a 510-fold increase in thelabeling of a protein band with apparent molecular weight of 55,000 f 400 ( n = 7) (presumably the proteinkinase R I1 subunit) in both in uitro and in vivo differentiated granulosa cells (Fig. 2 4 ) . A slight increase in the labeling of a protein with molecular weight of 50,000 f 500 was observed in cytosols from FSH-treated cells. A third protein band with a molecular weight of 37,500 f 200 was also apparent, despite the use of several protease inhibitors and homogenization procedures. The photoaffinity labeling was specific for CAMP-binding sites, since it was prevented by the addition of excess CAMP. Fig. 2B shows the effect of GnRHa on the FSH-induced increase of R I1 subunit content in cultured granulosa cells. It was clear that GnRHa considerably reduced the amount of R I1 produced with FSH treatment. To confirm that the M , = 55,000 protein was the R I1 subunit, the cytosolic fraction of cells cultured for 48 h with FSH was phosphorylated with a low [y-"PIATP concentration (0.5 p ~ a)t 0 "C to determine if autophosphorylation occurred.Aliquots of thesame cytosol were subjected to photoaffinity labeling with 8 - a ~ i d o - [ ~ ~ P ] c A M P autophosand phorylation. The phosphorylation pattern (Fig. 3A) demonstrates a single-band migrating exactly as the photoaffinity labeled M , = 55,000 protein (lane 3 uersus lane 1). When the cytosol was incubated a t 37 "C for45 min instead of 4 "C prior to the additionof [y-"PIATP, followed by incubation a t 2 "C, a single band of M , = 55,000 was again obtained ( l a n e 2). Since the bandsin lanes 2 and 3 of Fig. 3A were faint due to the short exposure timerequired for the photoaffinity experiment, the autophosphorylation studies were repeated with longer exposure times. Lanes 2 and 3 of Fig. 3B demonstrate a major band of M , = 55,000 with the presenceof other minor bands. The addition of cAMP did not change the phosphorylation pattern (lane 4 ) , suggesting that the R I1 phospho-

A

B 2

1 2 3 CAMP

3

- -

- - -

94

4

-+

iI

=36 630 I FIG. 3. Comparison between photoaffinity labeled and autophosphorylated proteins in granulosa cells cytosols. Cytosols from FSH-treated cells were submittedtophotoaffinity labeling (Panel A, lane I ) and to autophosphorylation (Panel A, lanes 2 and 3, and Panel B, lanes 2-4). Cytosols were incubated for 45 min a t 4 "C (Panel A. lane 3, and Panel B, lanes 3 and 4 ) or a t 37 "C (Panels A and B, lane 2) before autophosphorylation. M cAMP was added during the preincubation period (Panel B, lane 4 ) . Panels A and B are the results of two separate experiments with autoradiographs developed for 1 and 10 days, respectively.

rylation was an intramolecular phenomenon. To determine if the FSH-induced increase in CAMP-binding siteswas associated with a parallel stimulation of protein kinase activity, the phosphorylationof histone by the soluble fraction of granulosa cells was measured after 48 h of culture. FSH increased kinase activity by approximately 50% in the absence of exogenous cAMP (from 40 k 2 to 64 f 1 units of kinase activity), while the maximal enzyme activity in both control and FSH-treated cells in the presence of 5 X lo-' M cAMP was 340 f 11 units. The kinase activity in GnRHatreated cells remained at thecontrol level. Since translocation of the C subunit of CAMP-dependent protein kinase can occur A 0 during the cytosol preparation, homogenization was also perTo F1 F2 formed in the presence of 0.2 M NaCl to suppress binding of CAMP " 1 F 1 To C F F+G the C subunit to particulate fractions (18, 19). However, no changes inCAMP-dependent protein kinaseactivity were observed in FSH-treated cells. T o analyze the apparent discrepancybetween the large FSH-induced increase in CAMP-binding protein content and the absence of a parallel increase in histone kinase activity, Le 43 1 5 5 the granulosa cell protein kinase was analyzed on DEAEcellulose. Only one broad peak of CAMP-dependent protein 50 53 - 37 kinase activity from granulosa cells of FSH-primed ovaries 2 36 was eluted a t about 0.17 M KC1 (Fig. 4). In addition to the 30 difference between this pattern and thatusually observed for the type I and type I1 enzymes, the majorpeak of cAMP 20 binding activity (eluted at 0.2 M KCl) was not coincident with FIG. 2. Characterization of the CAMP-binding sites in gran- the protein kinase activity peak, while a shoulder preceding ulosa cell cytosols by photoaffinity labeling. A, 30,000 X g the major peak of binding corresponded fairly well with the supernatants from freshly prepared granulosa cells from immature enzymatic activity. No enzyme activity was found in the first rats (To), granulosa cells cultured 48 h in the presence of 250 ng/ml column fractions,indicatingtheabsence of free catalytic and granulosa cells obtained from FSH-treated rats (Fz) units. Similar elution patternswere obtained when benzamioFSH (F,), were tested. Photoaffinity labeling was performed in the absence (-) dine was added to the columnbuffer to further minimize or in the presence (+) of 6 X M CAMP. B, cytosols were obtained from freshly prepared granulosa cells (To)and from granulosa cells degradation of protein kinase. Under such conditions, the cultured for 48 h without (C) or with 250 ng/ml oFSH (0or FSH whole elution pattern was slightly shifted toward the left, but plus 10" M GnRHa (F+G).After a 60-min incubationa t 4 "C, cytosols again the peakof cAMP binding activity was eluted after the were photolysed for 10 min (see "Experimental Procedures"). After protein kinase activity peak, and no free CAMP-independent sodium dodecyl sulfate-polyacrylamide gel electrophoresis fractionation, photolabeled proteins were visualized by autoradiography. Mo- activity was detected in thewash and low KC1 concentration fractions.When cytosol was incubated witha saturating lecularweightswere determined by the use of standardprotein amount of ["HICAMP(4 PM) before DEAE-cellulose fractionmarkers.

1

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Hormonal Regulation of CAMP-dependent Protein Kinase

14781

sites cannotbe attributed tooccupancy by the initialsurge in cAMP production stimulatedby FSH (20), since this fall also occurred in control cells which produce negligible levels of 150 CAMP. FSH receptorsshow a similar decline during the first hours of culture in the absence orpresence of hormone (211, 100 and both changes may be simply the consequences of cell isolation and plating. The increasein CAMP-binding sites 50 observed from 6 to 24 h of culture is concomitant witha rise inFSHreceptorsandcAMPsynthesis by 24 h (21, 22). O - M M M M m M e I I I I GnRHa, which has a transient inhibitory effect on cAMP 10 20 30 40 50 60 70 30 min of culture (22), prevents the synthesis during the first FRACTION NUMBER FIG. 4. DEAE-cellulose chromatography of granulosa cell FSH-induced increase in CAMP-binding sites, FSHreceptors, cytosol. 30,000 X g supernatant (6.5 mg of protein) prepared from and adenylatecyclase activity (21,22). Since themajor expresgranulosa cells obtained from the ovaries of FSH-treated rats was sion of luteinizing hormone receptors and steroidogenic enfractionated on a DEAE-cellulose column (1.2 X 6 cm) as indicated zymes by FSH occurs during the second day of culture and is under “Experimental Procedures.”After washing with 15 ml of buffer dependent upon the continued maintenance of cAMP producA (10 mM Tris-HC1, pH 7.4, 1 mM EDTA, 1 mM dithiothreitol), tion (20), the increase in CAMP-binding sites induced byFSH proteins were eluted at a flow rate of 20 ml/h by a linear gradient of procKC1 (0-0.4 M ) in buffer A. Aliquots of 0.75-ml fractions were assayed could be a major regulatory factor in the differentiation for cAMP binding (A) and protein kinase activity in the absence (0) ess. Conversely, GnRHa could exert its inhibitory effect on granulosa cell differentiation by decreasing the content of and in the presence (0)of 5 X M CAMP. CAMP-binding sites. Thephotoaffinity labeling experiments confirmed the prominentstimulation of CAMP-binding sites induced by FSHandtheinhibition of thissynthesis by GnRHa.It appeared that the acuteincrease in CAMP-binding sites was confined tothe R I1 subunit.Thus,the dose- andtimedependent increase in available CAMP-binding sites (Fig. 1) probably reflects this increase in R I1 subunit synthesis. Since the DEAE-cellulose elution pattern did not show the type I protein kinase (usually eluted at or below 0.1 M salt concentration), the minorM , = 50,000 protein band observed after photoaffinity labeling probably represents a degraded formof R 11, rather than the R I subunit. When photoaffinitylabeling 0 30 60 120 210 was performed after preincubation of the cytosol at 37 “ C , INCUBATION TIME Imml labeling of the M , = 50,000 protein was increased (data not FIG. 5. Effect of GnRHa on FSH-induced protein kinase shown), supporting thepossibility of partial proteolysis of R activation. Granulosa cells were incubated, in duplicate, in polypro- 11. However, the autophosphorylation studies only demonpylene tubes duringvarious times in the absence (0)or inthe presence strate the labeling of a single band, even after preincubation M GnRHa (0).Cytosols of 100 ng/ml oFSH (A) or FSH plus were immediately prepared, and histone kinase activity was measured a t 37 “C. Of particular interest was the smallincrease in protein M CAMP. in triplicate, in the absence and in the presence of 5 X kinase activity after48 h of culture, despite thelarge increase Results are expressed as protein kinase activity ratio (-CAMP/ +CAMP). in CAMP-binding sites during FSH-induced granulosa cell differentiation. A similar lack of increase in protein kinase ation, a single peak of [3H]cAMP-Rwas eluted at the same activity in combination with an elevation in CAMP-binding salt molarity as the major cAMP binding activity peak, and sites has been previously reported in ovarian granulosa cells again nofree catalytic activitywas detected (data not shown). obtained from rats treated in vivo with estradiol and FSH The lack of a clear-cut increase in protein kinase activity (10). This discrepancybetween cAMP binding and kinase in FSH-treated cells a t 48 h could question theimplication of activity could be caused by several factors, e.g. the presence proteinkinaseingranulosa cell differentiation. However, of a protein kinase inhibitor in granulosa cell cytosol, a high during the first minutes of culture, FSH stimulated CAMP- level of phosphatase activity, or rapid degradationof the free dependent protein kinase since the activity ratio increased catalytic subunit. It is also possible that compartmentalizawithin 10 min (Fig. 5). This effect of FSH was not sustained, tion between proteinkinase,proteinkinaseinhibitor,and and protein kinase activity decreased after 30-60 min; after phosphatases exists in the cell and is disrupted during ho210 min, the activity ratio was the sameas that found a t 48 mogenization. However, another explanation of our results is h. Of particular interestwas the finding that GnRHa impairedsuggestedby recentreports. WhileR and C subunits are the FSH-induced increase in protein kinase activity, reducing usually supposed to be present in equimolecular amounts in the degree of hormonal activation a t 10 min and causing a the cell (41, R I and/or R I1 have been reported tobe in excess delay in activation until 60 min. over C subunit in neuroblastomaglioma hybrid cells (23) and Friend erythroleukemic cells (24). The DEAE-cellulose eluDISCUSSION tion pattern of granulosa cell cytosol and the shift in the relative to the proteinkinase peak These studies have shown significant hormonal effects on major CAMP binding peak CAMP-dependent proteinkinasesubunitsinculturedrat (eluted asa type I1 kinase) could be explained by an excess of granulosa cells exposed to FSH and GnRHa. FSH caused a the more acidic molecular species R I1 over thetype I1 marked, dose-dependent increase inavailable CAMP-binding holoenzyme. sites subsequent to the early decrease in these sites during We have previously shown an FSH-inducedrise in adenylthe first6 h of culture. The initial reduction CAMP-binding in ate cyclase activity in the first60 min of hormone treatment, 200 r

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14782

Hormonal Regulation of CAMP-dependent Protein

followed by a decline in cAMP formation until 24 h when a secondary rise in cAMP synthesis occurred (22). Whether the latter increase in cAMP is also directly involved in activating protein kinase or is only bound to theCAMP-bindingproteins is not yet clear. Treatment with GnRHa delayed the initial rise incAMP production, apparently due to activation of phosphodiesterase activity (22). We now show that GnRHa also decreased FSH-induced protein kinase activation during the first 30 min of culture, probably due to the transient inhibition of FSH-induced cAMPproduction during this time. Since the presence of GnRHa is required for only a few hours to inhibit granulosa cell development (20), the impairment of protein kinase activity by GnRHa may result in altered phosphorylation of proteins necessary for the maturationprocess. The large dose- and time-dependent increase inCAMPbinding sites during FSH treatment may be a major factor in CAMP-mediated differentiation processes in the granulosa cell. Further investigation will be necessary to define the possible role of the regulatory subunit R I1 on the differentiation process. The inhibitory effect of GnRH on both FSHinduced protein kinase activationin the firstminutes of culture and FSH-inducedR I1 synthesis during the later course of the 48-h culture indicatesthat theearly suppressive action of GnRH on cAMP production has a significant role in the attenuation of granulosa cell maturation by GnRH agonists. REFERENCES 1. Zeleznik, A. J., Midgley, A.R., and Reichert, L. E., Jr. (1974) Endocrinology 95,818-825 2. Richards, J. S.(1980) Physiol. Rev. 60,51-89 3. Knecht, M., Amsterdam, A., and Catt, K. (1981) J. Biol. Chem. 256,10628-10633

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4. Hofmann,F., Bechtel, P. J., and Krebs, E. G. (1977) J. Biol. Chem. 252,1441-1447 5. Nimmo, H. G., and Cohen, P. (1977) Adv. Cyclic Nucleotide Res. 8, 145-266 6. Costa, M., Gerner, E. W., and Russell, D. H. (1976) J. Biol. Chem. 251,3313-3319 7. Liu, A. Y.-C. (1982) J. Biol. Chem. 2 5 7 , 298-306 8. Dimino, M. J., Bieszczad, R. R., and Rowe, M. J. (1981) J. Biol. Chern. 256, 10876-10882 9. Hunzicker-Dunn, M. (1981) J. Biol. Chem. 2 5 6 , 12185-12193 10. Richards, J . S., and Rolfes, A. I. (1980) J . Biol. Chern. 255,54815489 11. Gore-Langton, R. E., LaCroix, M., and Dorrington, J. H. (1981) Endocrinology 108,812-819 12. Hsueh, A. J. W., and Jones, P. B. C. (1981) Endocr. Rev. 2,437461

13. Knecht, M., Katz, M. S., and Catt, K. J . (1981) J. Biol. Chem. 256,34-36 14. Dufau, M.L., Tsuruhara, T., Horner, K.A., Podesta, E., and Catt, K. J. (1977) Proc. Natl. Acad. Sci.U. S. A. 74,3419-3423 15. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 1 9 3 , 265-275 16. Laemmli, U. K. (1970) Nature (Lond.)227, 680-685 17. Darbon, J. M., Ursely, J., and Leymarie, P. (1981) Eur. J. Biochem. 119,237-243 18. Keely, S.L., Corbin, J. B., and Park, C.R. (1973) Proc. Natl. Acad. Sci. U. S. A. 72, 1501-1504 19. Darbon, J. M., Ursely, J., and Leymarie, P. (1976) Febs Lett. 63, 159-163 20. Knecht, M., Amsterdam, A., and Catt, K. J. (1982) Endocrinology 110.865-872 21. Knecht, M., Ranta, T., andCatt, K. J. (1983) Endocrinology 1 1 3 , 949-956 22. Knecht, M., Ranta, T., Katz, M. S., andCatt, K. J. (1983) Endocrinology 112, 1247-1255 23. Walter, U., Costa, M. R. C., Breakefield, X. O., and Greengard, P. (1979) Proc. Natl. Acad. Sci. U. S. A. 7 6 , 3251-3255 24. Schwartz, D. A., and Rubin, C. S. (1983) J. Biol. Chem. 2 5 8 , 777-784