Cyclic AMP and Phorbol Esters Interact Synergistically to Regulate ...

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rionic gonadotropin (hCG) in choriocarcinoma cell lines. Here, we show that treatment of choriocarci- noma cells with activators of protein kinase C, such as.

THEJOURNALOFBIOLOGICAL

CHEMISTRY

Val. 263, No. 30, Issue of October 2 5 , pp. 15578-15583, 1988 Printed in U.S.A.

Cyclic AMP and Phorbol Esters Interact Synergistically to Regulate Expression of the Chorionic GonadotropinGenes* (Received for publication, January 4, 1988)

Bogi AndersenSQlI,Amy Milstedll**,Giulia KennedySV, and JohnH. Nilson$ $$ From the $Departments of Pharmacology and Medicine, School of Medicine, Case Western Reserve University, the §Cleveland Metropolitan General Hospital, and the I(Cleveland Veterans Administration Medical Center, Cleveland, Ohio 44106

Previous studies have shown that activators of the Human chorionic gonadotropin (hCG)’ is a glycoprotein of the hormone composed of an a subunit common to theglycoproprotein kinaseA pathway increase transcription genes encoding the a- and &subunits of human cho- tein hormones and a unique @ subunit that confers receptor rionicgonadotropin (hCG) inchoriocarcinomacell specificity (1).The common a subunit is encoded by a single lines. Here, we show that treatment of choriocarci- gene located on chromosome 6, whereas the hCG @-subunitis noma cellswith activatorsof protein kinaseC, suchas encoded by a family of six genes located on chromosome 19 phorbol myristate acetate (PMA) and dioctanoylglyc- (2-4); of these, at least two are transcriptionally active (5). erol, increases accumulation o f the mRNAs for both The noncovalent association of the hCG a- and @-subunitsis subunits ofhCG by 3-4-fold. In contrast, a phorbol essential for biological activity, and there is accumulating ester which fails to activate protein kinaseC, phorbol evidence suggesting that synthesis of the subunits is regulated 12j3,13a-didecanoate, has no effect onhCG mRNA levels. To test the possibility that these two major intra- by changes in the accumulation of both mRNAs (6, 7). Although physiological regulation of hCG biosynthesis and cellular signaling pathways interact, we treated choriocarcinoma cells with PMA, Eorskolin, or PMA and secretion is poorly understood, there is evidence to suggest forskolin together. Treatment with either agent led toparacrine regulation by polypeptide hormones, including goa 2-3-fold increase in hCGmRNA levels, whereas nadotropin-releasing hormone and epidermal growth factor treatment with both agents resulted in a 9-fold in- (8-13). Polypeptide hormones bind to cell-surface receptors; thus, their signal is most likely transmitted through asecond crease. This synergistic response also occurred when choriocarcinoma cells were treated withPMA and 8- messenger pathway. Cyclic AMP (14) and activators of proBr-CAMP. Furthermore, PMA did not increase intra- tein kinase C, such as phorbol esters (13, 15), are examples of cellular cAMP levels, suggesting that these two path- major intracellular messengers known to stimulate hCG seways interact subsequent to cAMP generation. PMA cretion from choriocarcinoma cell lines. Recent studies indialso increased transcriptionof the hCG a- and &genes cate that cAMP regulates hCG biosynthesis by altering tranby 2-3-fold. Whereas transcription of the a subunit scription of both the hCG a- and @-genes(7, 16). However, geneincreasessynergisticallyaftertreatmentwith the kinetics of their transcriptional responses are different both PMA and forskolin, transcription of the hCG 8- (7), suggesting that cAMP regulates the transcriptional activgene was limited to the increase caused by either agent ity of these genes by different mechanisms. alone. This latter result suggests that regulation of Phorbol esters bind to and activate protein kinase C, a hCGB mRNA accumulation is more complex than that calcium- and phospholipid-dependent protein kinase (17). of a-subunit mRNA and probably involves both tran- These compounds therefore have beenwidelyemployed to scriptional and post-transcriptionalcomponents. study the function of protein kinase C in diverse cells. Whereas phorbol esters stimulate hCG synthesis, it is not known whether this regulation occurs at the level of gene transcription and, if so, whether there is an interaction be* This work was supported in part by the Ohio Edison Biotechnol- tween the CAMP-dependent protein kinase A andprotein ogy Fund (to J. H. N.), National Institutes of Health Grant AM kinase C pathways. Here, we show that activators of protein 28559 (to J. H. N.), the Icelandic Science Foundation (to B. A.), and kinase C increase the transcriptional rate of the hCG a- and the Veterans Administration (to A. M.). The costs of publication of this article were defrayed in part by the payment of page sharges. /?-genes. Furthermore, simultaneous activation of both the This article must therefore be hereby marked “advertisement” In protein kinase A and C pathways leads to an increase in the accumulation of a-subunit and [email protected] which is sigaccordance with 18 U.S.C. Section 1734 solelyto indicate this fact. ll Recipient of a fellowship from the American Heart Association, nificantly greater than theadditive response expected if both Northeastern Ohio Affiliate. ** Present address: Dept. of Brain and Vascular Research, Cleveland Clinic Found., Cleveland, OH 44106. $$Recipient of National Institutes of Health Research Career Developmental Award AM-01316. To whom correspondence should be addressed: Dept. of Pharmacology, School of Medicine, Case Western Reserve University, 2119 Abington Rd., Cleveland, OH 44106.

The abbreviations used are: hCG, human chorionic gonadotropin; PMA, phorbol myristateacetate;8-Br-cAMP, 8-bromoadenosine 3’:5‘-monophosphate; MeZSO, dimethyl sulfoxide; CHO-B cDNA, Chinese hamster ovary cell cDNA; GADPH, glyceraldehyde-3-phosphate dehydrogenase.

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concentrations of 8-Br-CAMPfrom 0.01 to 1.0 mM, or 100 nM PMA and increasing concentrations of 8-Br-CAMP simulta10 neously. Both a-subunit and hCGp mRNA levels increased 9 3-4-fold after treatment with 100 nM PMA. Both mRNAs were increased in a dose-dependent fashion after treatment > with 8-Br-CAMP;the maximum effect (4-6-fold) wasobserved 3 7 with 0.1-1.0 mM 8-Br-CAMP. Combined treatment with 100 a nM PMA and 1.0 mM 8-Br-CAMP resulted in a 12-fold in2 6 a crease in a-subunit mRNA levels and a 16-fold increase in € 5 .-? hCGP mRNA levels(Fig. 5). This is significantly greater than -za, 4 the 7-8-fold increase in a-subunit and hCGB mRNA levels, CT whichwould be expected for astrictly additive effect. A 3 synergistic response was also observed when lower concentra2 tions of 8-Br-CAMP (0.1 and 0.5 mM) were used (Fig. 5). In short, PMA caused an additional increase in both mRNA 1 levels when added with a maximally effective concentration 0 of 8-Br-CAMP. This suggests that PMA cannot be acting Control PMA Forskolin PMA + Forskolin simply by increasing intracellular cAMP levels via enhanceFIG. 4. Synergistic effect of PMA and forskolin on a-sub- ment of adenylate cyclase. To test the possibility that PMA increases intracellular unit and hCG0 mRNA accumulation. JAr cells were treated for 24 h with 100 nM PMA alone, 50 PM forskolin alone, or PMA (100 cAMP levels by inhibiting phosphodiesterase, we measured nM) and forskolin (50 p ~ simultaneously. ) Results are expressed as intracellular cAMP levels directly after treatmentof JAr cells the mean and S.E. of the mean from four experiments. The -fold with 100 nM PMA alone, 50 PM forskolin alone, and PMA induction with the combined treatment was statistically higher for and forskolin for either 1 or 4h(Table 1). As expected, both a-subunit ( p = 0.057) and hCGp ( p = 0.03) mRNAs compared forskolin increased intracellular cAMP levels markedly. In to the expected values if the responses were additive. contrast, PMA decreased both basal and forskolin-stimulated cAMP levels. Collectively, these data suggest that the interpathways acted independently. Surprisingly, however, the action between forskolin and PMA occurs subsequent to the synergistic increase in accumulation of a-subunit mRNA ap- increase in intracellular cAMP levels. The increase in hCG mRNAs after treatment with PMA pears to be mediated solely atthe transcriptional level, whereas the synergistic accumulation of hCGP mRNAis more could be due to increased transcription or to post-transcriptional alterations, such as increased efficiency of RNA proccomplex and probably involves both a transcriptional and a post-transcriptional component. These studies indicate that essing, nuclear/cytoplasmic transport, or mRNA stability. To interaction of the two major intracellular signaling pathways begin to distinguish between these possibilities, we treated JAr cells for 4 h with 100 nM PMA alone, 50 p~ forskolin results in a potentiated effect on hCG gene expression. alone, or PMA and forskolin. Subsequently, nuclei were isoMATERIALS AND METHODS AND RESULTS~ lated, and previously initiated RNA transcripts were allowed to elongate in the presence of 32P-labeled UTP. PMA or The data shown in Figs. 1-3 indicate that PMA anda diacylglycerol analogue, dioctanoylglycerol, increase a-sub- forskolin increased transcription of the a-subunit gene apunit and hCGp mRNAs. Therefore, to determine whether proximately 2-%fold. Treatment with both compounds rethere is an interaction between the PMA- andCAMP-depend- sulted in a 7-fold increase in transcription of the a-subunit ent pathways, a choriocarcinoma cell line (JAr) was treated gene (Fig. 6A), suggesting that the synergistic effect on afor 24 h with 100 nM PMA alone, 50 p M forskolin alone (a plant diterpene that directly activates the catalytic subunitof adenylate cyclase) (27), or PMA (100 nM) and forskolin (50 Alpha Beta T p ~ )Treatment . with either forskolin or PMA led to a 2- and %fold increase in both mRNAs, respectively, whereas joint T treatment resulted in a 9-fold increase in both mRNAs (Fig. 4 z 4). The 9-fold increase is more than 100% higher than the 4- E 10 fold increase expected if the responses were strictly additive. m Similar results were obtained after 4 and 8 h of treatment .-m (data not shown). These results suggest that both the PMA- UT I 5 and CAMP-dependent pathways are active in JAr cells and 0 that their interaction leads to a synergistic response. CTL PMA 0.01 0.1 0.5 1.0 CTL PMA 0.01 0.1 0.5 1.0 Interaction between the CAMP- andPMA-dependent pathrnM 8-Br-CAMP rnM 8-Br-CAMP ways mayoccur prior to cAMP generation. For example, PMA potentiates the effect of forskolin on adenylate cyclase in the FIG. 5. Synergistic effect of PMA and 8-Br-CAMPon hCG 235-1 pituitary cell line (26). Totestthis possibility, we mRNA levels. JAr cells were treated for 24 h with 100 nM PMA treated JAr cells for 24 h with 100 nM PMA alone, increasing alone, different concentrations (0.01, 0.1, 0.5, and 1.0 mM)of 8-Br11

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* Portions of this paper (including “Materials and Methods,” part of “Results,” Figs.1-3, Table 1, and Footnote 3) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal thatis available from Waverly Press.

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cAMP alone, or 100 nM PMA and 8-Br-CAMP at the indicated concentrations simultaneously. Results are expressed as mean and S.E. of the mean of six separate experiments, except for data points representing 0.01, 0.1, and 0.5 mM 8-Br-cAMP, which are the results of a single experiment. The induction of a-subunit ( p = 0.01) and [email protected]( p = 0.005) mRNAs after treatment with 100 nM PMA and 1 mM 8-Br-CAMPcombined was significantly higher than theexpected results if the responses were additive. The expected values of an additive response are indicated by the arrowheads. CTL,control.

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FIG.6. A, nuclear run-off analyses of JAr cells treated with PMA and forskolin. JAr cells were treated for 4 h with 100 nM PMA alone ( P ) ,50 p~ forskolin alone ( F ) , PMA and forskolin jointly (P+F), or media containing vehicle alone (CTL,C). Nuclei were isolated, and aliquots containing approximately 300 pg of DNA were incubated in

subunit mRNA levelsis primarily transcriptional. In contrast, even though transcription of the @-subunitgene increased 2and 5-fold when cells were treated with either PMA or forskolin, respectively, the addition of both compounds failed to change the transcription rate beyond that observed for forskolin alone. This suggests that the synergistic action of cAMP and PMA on the accumulation of @-subunitmRNA is mediated, at least in part, by increased efficiency of one or more of the post-transcriptional events mentioned above. In other experiments (data not shown), we have examined the kinetics of the transcriptional response to PMA. Maximal changes in transcription rate occur within 30 min for either gene and remain unchanged for at least 24 h. By comparison, theextent of a-subunitand [email protected] accumulation reaches a maximum approximately 24-48 h after addition of PMA, suggesting that both mRNAs are relatively stable. Whereas it is tempting to compare maximal changes in transcription and mRNA accumulation (compare Figs. 4 and 6), these experiments were carried out with different isolates of JAr cells. Consequently, it is difficult to determine whether a change in transcription fully accounts for a change in mRNA accumulation. Nevertheless, it is possible to compare a- and @-genetranscription rates and levels of a and @ mRNAs as these measurements were always performed with the same isolate of JAr cells. Thus, the synergistic change in the transcription rate of the a-gene observed after treatment with forskolin and PMA serves as a positive control for the lack of a synergistic change in the transcription rate of the hCG @gene. To determine whether the differential transcriptional response of the hCG a- and @-genesto cAMP and PMA is restricted to JAr choriocarcinoma cells, we measured transcription rates in another choriocarcinoma cell line (BeWo). These cells were treated for 8 h with 100 nM PMA alone, 1 mM 8-Br-CAMPalone, and PMA and 8-Br-CAMP together. PMA alone increased transcription of both genes 2-3-fold, whereas 8-Br-CAMPcaused a 3-4-fold increase in transcription of both genes. Consistent with our data from JAr cells, the combined treatment with 8-Br-CAMP andPMA caused a marked synergistic effect on a-gene transcription, whereas the increase in @-genetranscription was limited to thatcaused by 8-Br-CAMP alone (Fig. 6B).These data further suggest that post-transcriptional mechanisms are responsible for change in accumulation of [email protected] mRNAafter activation by both cAMP and phorbol esters. the presence of 32P-labeled UTP. Subsequently, equal amounts of purified 3ZP-labeledRNA transcripts (2.0 X 10' cpm) were hybridized to individual nitrocellulose filter strips containing 5 pgof purified cloned plasmid cDNAs: Bluescript plasmid (pKS), hCGa cDNA in pKS, hCGP cDNA in pKS, rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH), human a-tubulin cDNA, Chinese hamster ovary cell (CHOB)cDNA, and HeLa @-actincDNA. After autoradiography, we quantitated the hybridization signal by scanning densitometry. Background signal as measured by hybridization to nonspecific pKS DNA was subtracted from each experimental signal. Hybridizations were done in quadruplicate or quintuplicate. Upper, autoradiogram from a representative experiment; lower, mean and S.E.of the mean from densitometric scans of autoradiograms from four or five different filter hybridizations. Similarresults were obtainedin two other experiments. B, nuclear run-off analyses of BeWo cells treated with PMA and 8-Br-CAMP.BeWo cells were treated for 8 h with 100 nM PMA alone (P),1 mM 8-Br-CAMPalone (cA), PMA and 8-Br-CAMP jointly (P+cA), or media containing vehicle alone (CTL,C). Transcription rates were measured as described for A. Upper, autoradiogram from a representative experiment; lower, mean and S.E. of the mean from densitometric scans of two-four different filter hybridizations except for a-gene transcription after PMA treatment., where only one filter hybridization was scannable.

Interaction between

CAMPand PMA in

DISCUSSION

Major signal transduction pathways can operate independently or interact at many levels to regulate cell function (24, 28). Previous investigations from this laboratory and elsewhere have demonstrated a role for cAMP in regulation of gonadotropin gene expression; for example, cyclic AMP analogues induce accumulation of a-subunit and [email protected] levels in threechoriocarcinoma cell lines: BeWo( 7 ) ,JAr (29), and JEG-3 (16, 30). Direct measurements of transcription rates in BeWo ( 7 ) and JEG-3 (16) cells indicate that this increased accumulation of both subunit mRNAs is, at least in part, mediated by increased transcription. Our present data suggest that inaddition to a CAMP-dependent pathway, hCG gene expression is also regulated by activators of protein kinase C. Choriocarcinoma cells therefore provide an excellent model to study the inter-relationship between these two major pathways, especially as it relates to gene expression. The interaction between protein kinase C and A pathways can be classified into two major categories (24). In a bidirectional system, such as that found in platelets, agents like thrombin activate the protein kinase C pathway to stimulate certain cellular functions, whereas agents that activate the protein kinase A pathway antagonize this effect (24). In a monodirectional system, however, these two pathways act in concert. One prediction of monodirectional regulation is that different combinations of two intracellular signaling systems could cooperate positively, thereby producing an intensified biological signal (24). Regulation of gonadotropin gene expression in choriocarcinoma cells appears to satisfy this prediction because simultaneous treatment of JAr and BeWo cells with cAMP and PMA results in a synergistic increase in both asubunitand [email protected] mRNA levels. A synergistic response, however, may not always occur when the two pathways act in concert. For example, in theTT cell line of human medullary thyroid carcinoma, both phorbol esters and cAMP stimulate transcription of the calcitonin gene (31); but in contrast to our results, simultaneous treatmentleads to a strictlyadditive response. Thus, it remains to be seen whether a synergistic response between these two major pathways is limited to a select group of endocrine cells or is a more common property for obtaining full transcriptional activation. Although our data suggest that phorbol esters and cAMP interact synergistically to regulate expression of the gonadotropin genes, the molecular mechanism(s) underlying this process are unknown. Phorbol esters have been shown to potentiate the effect of cholera toxin on induction of cAMP in BeWo cells (15). It is unlikely, however, that thesynergism observed in this study is due to enhancement of adenylate cyclase activity or inhibition of phosphodiesterase by PMA because PMA failed to increase either basal or forskolininduced cAMP levels. Rather, synergism probably results from the interaction of the protein kinase A and C pathways subsequent to generation of CAMP. This could occur at the level of protein phosphorylation and ultimately affect proteinprotein or protein-DNA interaction required for transcription. Although synergism could also occur if cAMP enhances the effect of PMA on protein kinase C, we view this as unlikely for two reasons. First, we are unaware of any precedence for an enhancement of cAMP on the activity of protein kinase C. Second, we used concentrations of PMA that have been shown to activate maximally protein kinase C and are saturating with regard to increases in hCG mRNA levels. Thus, it is unlikely that the marked synergistic effect of PMA and cAMP on both mRNA levels results from potentiation of protein kinase C activity. Another factor that lends additional complexity tothe mechanisms underlying the synergistic response of the gonad-

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otropin genes is our observation that regulation of a-subunit and [email protected] levels appears to occur through different mechanisms. Transcription of the a-subunitgene is enhanced by PMA. Furthermore, changes in the transcription rate of the a-subunitgene appear to account fully for the synergistic effect of cAMP and PMA on accumulation of a-subunit mRNA. In contrast, although PMA alone increased the transcription rate of the hCG @-genesin JAr cells, the synergistic increase in the accumulation of [email protected] with both PMA and forskolin was not associated with an additional increase in the transcription rate. Similarily, cAMP and PMA caused a synergistic increase in transcription of the a-subunit gene in BeWo cells without a concomitant change in [email protected] rate. Together, these data suggest that thesynergistic effect on the accumulation of [email protected] mRNA may result from an alteration in a post-transcriptional process. The notion that a-subunit and [email protected] mRNA concentrations are coordinately regulated by PMA and CAMP, even though the molecular mechanisms underlying this regulation are distinct, is consistent with our recent data indicating that the transcriptional response of the hCG a- and @-genesto cAMP also occurs through a different mechanism ( 7 ) . For example, regulation of the a-subunit gene by cAMP is mediated by an 18-base pair element found in the proximal 160 base pairs of the 5”flanking sequence (32-35). [email protected] has a cis-active element that confers cAMP regulation to a reporter gene, but this element lacks a homolog to the 18-base pair sequence found in the a-subunit 5”flanking sequence and anumber of other CAMP-responsive genes.* Furthermore, cAMP induction of a-subunit gene transcription is independent of ongoing protein synthesis,whereas transcription of the hCG @-generequires ongoing proteinsynthesis(datanot shown). In the future, DNA-mediated gene transfer should allow us to study further the interaction between the cAMP and protein kinase C pathways. In this regard, we have found that 1500 base pairs of the proximal 5“flanking sequence from the human a-subunit gene contain a phorbol ester response element in addition to the previously described cAMP response element.5 Thus, it will beof interest to determine whether the two pathways converge prior to transcriptional activation and act on the same gene sequence, such as has been suggested for the proenkephalin gene (36), or whether the effects are mediated by two separate DNA sequence elements, in which case the synergistic response might result from interaction between proteins binding to these DNA sequences. Acknowledgments-We thank Drs. Richard W. Hanson, Jeffrey B. Virgin, and Joseph A. Bokar for helpful discussions and suggestions. We also thank Dr. George Dubyak for performing the cAMP radioimmunoassays. REFERENCES 1. Pierce, J. G., and Parsons, T.F. (1981) Annu. Reu. Biochem. 50, 465-495 2. Fiddes, J. C., and Goodman, H. M. (1981) J. Mol. Appl. Genet. 1, 3-18 3. Policastro, P. F.,Daniels-McQueen, S., Carle, G., and Boime, I. (1986) J. Biol. Chem. 261,5907-5916 4. Naylor, S. L., Chin, W. W., Goodman, H. M., Lalley, P.A., Grzeschik, K.-H., and Sakaguchi, A. Y. (1983) Sonat. Cell Genet. 9, 757-770 5. Talmadge, K., Boorstein, W. R., Vamvakopoulos, N. C., Gething, M.-J., and Fiddes, J. C. (1984) Nucleic Acids Res. 1 2 , 84158436 J. B. Virgin, and J. H. Nilson, unpublished results. B. Andersen, and J. H. Nilson, unpublished results.

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6. Milsted, A., Silver, B. J., Cox,R. P., and Nilson, J. H. (1985) Endocrinology 117, 2033-2039 7. Milsted, A., Cox, R. P., and Nilson, J. H. (1987) D N A ( N .Y.) 6, 213-219 8. Khodr, G. S., and Siler-Khodr, T.M. (1980) Science 2 0 7 , 315317 9. Iwashita, M., Evans, M. I., and Catt, K. J. (1986) J. Clin. Endocrinol. Metab. 62, 127-133 10. Butzow, R. (1982) Znt. J . Cancer 29.9-11 11. Siler-Khodr, T. M., and Khodr, G. S. (1981) Bwl. Reprod. 25, 353-358 12. Benveniste, R., Speeg, K. V., Carpenter, G., Cohen, S., Lindner, J., and Rabinowitz, D. (1978) J. Clin. Endocrinol. Metab. 46, 169-172 13. Ilekis, J., and Benveniste, R. (1985) Endocrinology 116, 24002409 14. Hussa, R. 0. (1980) Endocr. Reu. 1,268-294 15. Ritvos, O., Jalkanen, J., Huhtaniemi, I., Stenman, U.-H., Alfthan, H., and Ranta, T. (1987) Endocrinology 120, 1521-1526 16. Jameson, J. L., Jaffe, R. C., Gleason, S. L., and Habener, J. F. (1986) Endocrinology 119, 2560-2567 17. Nishizuka, Y. (1984) Nature 308, 693-697 18. Nilson, J. H., Nejedlik, M. T., Virgin, J. B., Crowder, M. E., and Nett, T. M. (1983) J. Biol. Chem. 258, 12087-12090 19. Fort, Ph., Marty, L., Piechaczyk, M., Sabrouty, S. El., Dani, Ch., Jeanteur, Ph., and Blanchard, J. M. (1985) Nucleic Acids Res. 13,1431-1442 20. Cowan, N. J., Dobner, P. R., Fuchs, E. V., and Cleveland, D. W. (1983) Mol. Cell. Biol. 3, 1738-1745 21. Harpold, M. M., Evans, R. M., Salditt-Georgieff, M., and Darnell,

J. E. (1979) Cell 17, 1025-1035 22. Douglas, J. G., Saltman, S., Williams, C., Bartley, P., Kondo, T., and Catt, K. (1978) Endocr. Res. Commun. 5 , 173-188 23. Philippe, J., Drucker, D. J., and Habener, J. F. (1987) J. Biol. Chem. 262,1823-1828 24. Nishizuka, Y.(1986) Science 233,305-312 25. Bell, J. D., Buxton, I. L. O.,and Brunton, L. L. (1985) J. Biol. Chem. 260, 2625-2628 26. Summers, S. T., and Cronin, M. J. (1986) Biochem. Biophys. Res. Commun. 135,276-281 27. Seamon, K. B., and Daly, J. W. (1981) J. Cyclic Nucleotide Res. 7,201-224 28. Rasmussen, H. (1986) N. Engl. J. Med. 314, 1094-1101, 11641170 29. Darnell, R. B., and Boime I. (1985) Mol. Cell. Biol. 5 , 3157-3167 30. Burnside, J., Nagelberg, S. B., Lippman, S. S., and Weintraub, B. D. (1985) J. Biol. Chem. 260, 12705-12709 31. deBustros, A., Baylin, S. B., Levine, M. A., and Nelkin, B. D. (1986) J. Biol. Chem. 261,8036-8041 32. Silver, B. J., Bokar, J. A., Virgin, J. B., Vallen, E. A., Milsted, A., and Nilson, J. H. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 2198-2202 33. Deutsch, P. J., Jameson, J. L., and Habener, J. F. (1987) J. Biol. Chem. 262,12169-12174 34. Jameson, J. L., Deutsch, P. J., Gallagher, G. D., Jaffe, R. C., and Habener, J. F.(1987) Mol. Cell. Biol. 7,3032-3040 35, Delegeane, A. M., Ferland, L. H., and Mellon, P. L. (1987) Mol. Cell. Biol. 7, 3994-4002 36. Comb, M., Birnberg, N. C., Seasholtz, A., Herbert, E., and Goodman, H. M. (1986) Nature 3 2 3 , 353-356

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