DEVELOPMENTAL GENETICS 1 2 : 2 5 3 4 (1991)

Control of CAMP-Induced Gene Expression by Divergent Signal Transduction Pathways DORIEN J.M. PETERS, MARISKA CAMMANS, STEVEN SMIT, WOUTER SPEK, MICHIEL M. VAN LOOKEREN CAMPAGNE, AND PAULINE SCHAAP Cell Biology and Genetics Unit, Zoological Laboratory, Leiden University, Leiden, The Netherlands. M.M.V.L.C.’s present address is Department of Anatomy and Cell Biology, College of Physicians and Surgeons, Columbia Uniuersity, 630 West 168th Street, New York, N Y 10032. -

ABSTRACT A compilation of literature data and recent experiments led to the following conclusions regarding cyclic adenosine 3’:5’ monophosphate (CAMP) regulation of gene expression. Several classes of CAMP-induced gene expression can be discriminated by sensitivity to stimulation kinetics. The aggregation-related genes respond only to nanomolar cAMP pulses. The prestalk-related genes respond both to nanomolar pulses and persistent micromolar stimulation. The prespore specific genes respond only to persistent micromolar stimulation. The induction of the aggregation- and prestalkrelated genes by nanomolar cAMP pulses may share a common transduction pathway, which does not involve CAMP, while involvement of the inositol 1,4,5-trisphosphate ( IP,)/Ca2+ pathway is unlikely. Induction of the expression of prespore and prestalk-related genes by micromolar cAMP stimuli utilizes divergent signal processing mechanisms. CAMP-induced prespore gene expression does not involve cAMP and probably also not cyclic guanosine 3’S’ monophosphate (cGMP) as intracellular intermediate. Involvement of CAMP-induced phospholipase C (PLC) activation in this pathway is suggested by the observation that IP, and 1,2-diacylglycerol (DAG)can induce prespore gene expression, albeit in a somewhat indirect manner and by the observation that ti+ and Ca2+ antagonists inhibit prespore gene expression. Cyclic AMP induction of prestalk-related gene expression is inhibited by IP, and DAG and promoted by L i t , and is relatively insensitive to Ca2+ antagonists, which indicates that PLC activation does not mediate prestalk-related gene expression. Neither prespore nor prestalk-related gene expression utilizes the sustained CAMP-induced pH, increase as intracellular intermediate. Key words: Dictyostelium, stimulation kinetics, aggregation-related genes, genes, prespore genes

0 1991 WILEY-LISS, INC.

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INTRODUCTION

Exhaustion of the food source triggers the developmental program in Dictyostelium discoideum. Cells start to aggregate, using cyclic adenosine 3 ‘ 5 ’ monophosphate (CAMP)as chemoattractant. A multicellular slug is formed, which enters a period of migration and ultimately culminates to form a fruiting body consisting of stalk cells and spores. The changes in morphology and differentiation are reflected by a complex pattern of stage and cell-type specific gene expression. Cyclic AMP and the Differentiation Inducing Factor (DIF) control expression of the major classes of developmentally regulated genes [see review Schaap, 1986; Williams, 19881. Starvation initiates rapid expression of a number of early genes such as the I-genes, discoidin I, and K5 [Grabel and Loomis, 1978; Williams et al., 1979; Cardelli et al., 1985; Singleton et al., 19881. Expression of these genes peaks within a few hours and then declines. Besides starvation, a critical cell density, signaled by as yet unidentified cellular secretion products is required for induction of transcription [Grabel and Loomis, 1978; Margolskee et al., 1980 Clarke et al., 1987; Mann and Firtel, 19891. The decline of transcription can be prematurely induced by cAMP [Williams et al., 1979; Mann and Firtel, 1987; Singleton et al., 19881. Several genes, which are specifically expressed during the growth phase, also decrease during starvation [Cardelli et al., 1985; Kopachik et al., 1985; Singleton et al., 19871 and at least two of these genes, M4-1 and pcd-D2, are turned off by cAMP pulses [Kimmel and Carlisle, 1986; Hassanain and Kopachik, 19891. After a few hours of starvation, a number of genes associated with the aggregation process are being tran-

Received for publication January 25, 1990. Address reprint requests to Dr. Pauline Schaap, Cell Biology and Genetics Unit, Zoological Laboratory, Leiden University, Kaiserstraat 63, NL 2311 GP Leiden, The Netherlands.

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scribed, such a s cAMP receptors, CAMP-PDE, contact sites A (csA) [Malchow et al., 1972; Henderson, 1975; Gerisch et al., 1975; Lacombe e t al., 1986; Noegel e t al., 1986; Klein et al., 19881, the G-protein a-subunit, G,2 [Kumagai et al., 19891, as well a s genes D2 and M3 [Mann et al., 19881. Expression of these genes is accelerated by the chemotactic signal, i.e., nanomolar cAMP pulses [Darmon et al., 1975; Gerisch et al., 1975; Mann et al., 19881, and decreases when aggregation is completed. During aggregation a second set of cAMP responsive genes comes to expression, whose products are later preferentially associated with the anterior prestalk cells. Examples of this class are the cysteine proteinase 2 gene (CP-21, and some genes of unknown function such a s D11, D14, and 2H6 [Barklis and Lodish, 1983; Mehdy et al., 1983; Pears et al., 19851. These genes should be distinguished from two prestalk specific genes, pDd63 and pDd56, which are expressed after aggregation in response to the stalk inducing morphogen DIF [Jermyn et al., 19871. About coinciding with the formation of tips on late aggregates, a major class of spore specific genes is expressed. Expression of these genes, which include D19, 2H3, 14E6, and PL3, is also cAMP dependent and is restricted to the posterior region of migrating slugs [Barklis and Lodish, 1983; Mehdy et al., 1983; Morrissey et al., 1984; Wang and Schaap, 19881. Cyclic AMP is evidently a n ubiquitous signal controlling gene expression at different stages of development. An overview of the developmental regulation of the different classes of CAMP-induced genes as well a s the DIF-induced gene pDd63 is summarized in Figure 1. In the past 5 years research efforts in several laboratories have been aimed to identify the signal transduction chain which links the extracellular cAMP signal to gene transcription. In this study, we combine previous data with recent experiments to indicate the number of putative transduction chains involved in CAMP-induced transcription. We furthermore present a n inventory of the involvement of known signal transduction components in the control of specific classes of cAMP regulated genes.

cAMP Signal Transduction Dictyostelium cells contain several cAMP binding proteins: A cAMP dependent protein kinase (cAK) [De Fig. 1. Developmental regulation of different genes. NC-4 cells were starved on non-nutrient agar. Every 2 hours cells were harvested, RNA isolated, and Northern blots hybridised to PDE, csA, cA-R (6B), D14,CP-2 (pDd8), D19, and pDd63 cDNA. Values of densitometric scans were expressed as percentage of maximal induction. An autoradiogram of a Northern transfer probed with cA-R cDNA is inserted to show that during development bands with higher molecular weight appear. The densitometric scan incorporates all bands. PDE data represent a scan of the 2.2 Kb developmentally regulated mRNA.

Gunzburg and Veron, 19821, a n intracellular cAMP binding protein (CABP1) [Tsang and Tasaka, 1986), a CAMP-PDE, and cell surface cAMP receptors (cA-R). 0

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Haribabu and Dottin, 1986; Oyama and Blumberg, 19861. The transduction of cAMP signals to chemotaxis has been extensively investigated, and a number of mutants with specific defects have been identified (Fig. 2). The interaction of cAMP with cell surface receptors induces activation of adenylate cyclase (AC) [Roos and Gerisch, 19761, guanylate cyclase (GC) [Mato et al., 19771, and phospholipase C (PLC) [Europe-Finner and Newell, 1987; Van Haastert, 19891, resulting in a rapid increase of the intracellulair concentrations of, respectively, CAMP,cGMP, and IP,. IP, subsequently induces the release of Ca2 from intracellular stores, resulting in a n increase of cytosolic Ca2 levels [Europe-Finner and Newell, 19861. Intracellular targets for Ca2+ are a recently identified protein kinase C [Luderus et al., 19891 and calmodulin [Clarke et al., 19801. The activation of AC and PLC is mediated by different G-proteins [Theibert and Devreotes, 1986; Van Haastert et al., 1987, 19891. The a-subunit of the Gprotein linking cA-R to PLC may be the recently cloned Ga2, which is inactive in fgd A mutants [Kesbeke et al., 1988; Snaar-Jagalska et al., 1988; Kumagai et al., 1989; Pupillo et al., 1989; Van Haastert et al., 19901. Besides a G-protein, AC activation also requires a cytosolic factor, which is absent in mutant synag 7 [Theibert and Devreotes, 1986; Snaar-Jagalska and Van Haastert, 19881. cGMP is detected by a n intracellular cGMP binding protein [Mato et al., 1978; Parissenti and Coukell, 19891 and degraded by a substrate stimulated cGMPPDE [Van Haastert et al., 19821. This enzyme is defective in the stmF mutants [Ross and Newell, 19811. cAMP can potentially activate cAK and cABP1, but is mainly secreted in order to relay the chemotactic signal. Secreted cAMP is hydrolysed by CAMP-PDE. Cyclic AMP secretion is blocked in mutant HB3 [Kesbeke and Van Haastert, 19881. IP, is degraded to inositol by several different phosphatases [Van Lookeren Campagne et al., 1988al. Cyclic AMP-induced responses involved in chemotaxis, such as CAMP,cGMP, and IP, accumulation are transient and desensitize during persistent stimulation. Desensitization occurs via different mechanisms, which involve both receptor phosphorylation [Knox et al., 19861 and receptor downregulation and internalization [Klein and Juliani, 1977; Van Haastert, 1987; Wang et al., 19881. Besides the above mentioned responses, cAMP induces a n influx of Ca2 and a n efflux of H [Malchow et al., 19781 and K+-ions [Aeckerle et al., 19851 via unknown mechanisms and with unknown intracellular effects. The CAMP-induced Ca2+ influx does not adapt during persistent stimulation [Bumann et al., 19841, while the CAMP-induced H + efflux shows both a rapid transient a s a more slow sustained component [Aerts et al., 19871. With respect to cAMP regulation of gene expression, +

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Fig. 2. Signal transduction in Dictyostelium. Signal transduction pathways activated after occupation of cell surface cA-R. Mutants interfering with specific pathways are indicated. R, receptor; G, Gprotein (G2: G-protein with G,2 subunit); AC, adenylate cyclase; GC, guanylate cyclase; PLC, phospholipase C; PKC, protein kinase C; cGBP, cGMP-binding protein; CAM, calmoduline; cABP1: CAMPbinding protein.

Both kinetic [Van Haastert et al., 19861 as well as genetic studies (Saxe et al., unpublished data) indicate t h a t different subclasses of surface cAMP receptors exist (see also mRNA heterogeneity in Fig. 1). Both CABPl and cAK levels increase during development and after aggregation both proteins appear to be translocated from the cytosol to the nucleus [Woffendin et al., 1986; Kay et al., 19871, which is suggestive of a function in gene transcription. However, nucleotide specificity studies have indicated that cell surface cAMP receptors are the initial target for cAMP induction of prespore and prestalk associated gene expression [Schaap and Van Driel, 1985; Gomer et al., 1986;

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Fig. 3. Effects of stimulus modulation on gene expression in vegetative and aggregation-competent cells. Vegetative NC4 cells, freed from bacteria (A) or aggregation-competent cells, which had been starved for 16 hours at 6°C (B) were shaken at 22°C in phosphate buffer a t 5.106 cellsiml in the absence (Pb) or presence of the following cAMP stimuli: 30 nM pulses of cAMP at 6 minute intervals (PI, conM tinuous influx of 5 nM cAMPiminute (F), or a single dose of cAMP added every hour (M). Every 2 hours mRNA was isolated, sizefractionated, and hybridised with cA-R, csA, CP-2, D14, or D19 cDNA. Except for csA-rnRNA in aggregation-competent cells, the different

mRNAs usually increased gradually during the 6 hour incubation period. Optical density levels of specific mRNA bands at t = 6 hours are expressed as percentage of levels induced by nanornolar pulses (cA-R, CP-2, D14, and csA in vegetative cells) or by M CAMP/ hour (D19). In aggregation-competent cells, csA levels decreased after 2 hours of incubation. mRNA levels a t t = 2 hours (light shading) and t = 6 hours (darker shading) are expressed as percentage of levels induced by pulses at t = 2 hours. Means and SEM of two to six experiments are presented.

a primary question is whether gene regulation a t the different stages of development makes use of the chemotactic signaling system or uses a completely separate mechanism of transduction.

cAMP influx (Fig. 3A). Nanomolar pulses stimulate mRNA accumulation efficiently, and micromolar concentrations are virtually ineffective. The prestalk-related genes CP-2 and D14 are also expressed in response to pulses. Some CP-2 induction also occurs in response to micromolar stimuli. No induction of the prespore gene D19 was observed by any stimulus in vegetative cells. In aggregation-competent cells (about 6 hours of starvation), different patterns are evident (Fig. 3B). cA-R mRNA is already present a t this stage and decreases during persistent stimulation with CAMP.Nanomolar pulses induce a further increase. csA mRNA increases during the first 2 hours, regardless of stimulation and thereafter decreases. This decrease is somewhat enhanced by constant stimulation. The prestalk-

Effects of Signal Modulation on cAMP Regulated Gene Expression In order to analyse sensitivity of CAMP-induced gene expression to adaptation mechanisms, we compared the effects of stimulation with either nanomolar cAMP pulses, nanomolar influx, or 100 pM of cAMP on the expression of different cAMP regulated genes at two stages of development. In vegetative cells, incubated for 6 hours, some accumulation of cA-R and csA mRNA is evident in the absence of stimuli, which is inhibited by a nanomolar

CAMP-INDUCED GENE EXPRESSION 30 pM CAMP

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D19 Fig. 4. The effect of Li' ions on CAMP-induced prespore and prestalk-related gene expression. Aggregation-competent cells were incubated at various Li' concentrations in the presence of 30 pM CAMP.After 3 hours mRNA was isolated for the analysis of D14 and after 5 hours for the analysis of D19 mRNA levels [see Van Lookeren Campagne et al., 1988133.

related CP-2 and D14 mRNAs are present at low levels in aggregation-competent cells and accumulate strongly in response to nanomolar pulses and a continuous micromolar stimulus. However, a nanomolar influx is ineffective. The prespore mRNA D19 is absent from aggregation-competent cells, and can only be induced by micromolar stimuli. To conclude, although every investigated gene shows a more or less unique pattern of expression in response to different stimuli at different stages, a subdivision in roughly three classes can be made. Class 1, the aggregation-related genes are expressed in response to nanomolar pulses; class 2, the prestalk-related genes are induced both by nanomolar pulses and by micromolar continuous stimulation; and class 3, the prespore genes are only induced by micromolar stimulation. It appears furthermore that induction by micromolar stimuli is exclusively (D14, D19) or more (CP-2) effective in aggregation-competent cells compared with vegetative cells.

Do the Different Classes Of Genes Share Transduction Pathways? The observation that prestalk-related and prespore gene expression can both be induced by constant cAMP stimulation is suggestive of similar signal processing. The following experiments suggest that this is not the case. LiC1, a n inhibitor of CAMP-induced IP, accumulation [Peters et al., 19891, inhibits CAMP-induced prespore gene expression and promotes prestalk-related gene expression (Fig. 4). The cAMP antagonist adeno-

Fig. 5. Effects of adenosine on prespore and prestalk-related gene expression. Aggregation-competent cells were incubated in phosphate buffer with various adenosine concentrations in the presence of 30 pM CAMP.After 5 hours RNA was isolated, size-fractionated, and probed with D14 and D19 cDNA [see Spek et al., 19881.

sine, which inhibits cAMP binding to chemotactic receptors [Newell, 19821, inhibits cAMP induction of prespore gene expression, but slightly promotes cAMP induction of prestalk-related gene expression (Fig. 5). Prespore induction is furthermore strongly sensitive to inhibition by Ca2+ antagonists [Schaap et al., 19861 while prestalk-related gene expression is much less sensitive [Blumberg et al., 19881. These data indicate that the two classes of genes use divergent transduction pathways. Divergence may already start at the receptor level, since specificity studies, using adenosine derivatives, showed that inhibition of prespore induction is most likely caused by adenosine inhibition of cAMP binding [Van Lookeren Campagne et al., 19861. Cyclic AMP induction of prestalk-related gene expression apparently makes use of a n adenosine insensitive cAMP receptor. The sensitivity of prespore induction to Ca2 antagonists and LiCl is suggestive of involvement of intracellular IP,/Ca2+ in the transduction chain. Ginsburg and Kimmel [ 19891 recently showed that IP, combined with 1,2-diacylglycerol (DAG) can induce prespore gene expression. However, stimulation with these agents was required prior to the stage where cAMP mediated induction occurs, which makes i t doubtful whether the two agents are really intermediates for CAMP-induced transcription. It has also not yet been shown that Dersistent stimulation with CAMP.which is required for prespore gene expression, induces persistent IP, accumulation. Cyclic AMP not only induces the transient IP,/Ca2 response, but also induces a sustained influx of Ca2 , which may mediate prespore induction. A second sustained response is the CAMP-induced increase in intracellular pH, which is another candidate for control of either prespore or prestalk induction. The CAMP-in+

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Fig. 6. Effect of pH, on prestalk-associated and prespore gene expression. Aggregation- competent cells were shaken at 5.106 cellsiml in 10 mM phosphate buffer pH 6.6 or pH 7.4 in the presence or absence of 30 pM cAMP added every hour, and in different concentrations of

duced pH, increase can be bypassed with the weak base methylamine and inhibited with the weak acid DMO [Van Lookeren Campagne et al., 19891. Figure 6 shows that neither methylamine nor DMO induces or inhibits prespore (D19) or prestalk-related (CP-2) gene expression. This strongly suggests that the CAMP-induced increase in pH, does not mediate cAMP induction of prespore or prestalk-related gene expression. Prestalk-related gene expression can be induced by nanomolar cAMP pulses as well constant micromolar stimuli, suggesting that this response is insensitive to adaptation. This is however not the case since constant nanomolar stimulation is ineffective. It therefore appears t h a t the cAMP signal has two modes of entry into the cell: a low affinity adaptation insensitive mode and a high affinity adaptation sensitive mode. The latter mode is shared with the aggregation-related genes. We found that pulse-induced expression of both types of genes is stimulated by Li+ ions (Fig. 7), which is some evidence suggestive of a single pathway.

Analysis of Signal Transduction Mutants Mutants with specific defects in signal transduction components (see Fig. 2) are particularly useful to analyse involvement of these components in gene regulation. Involvement of CAMP as a n intracellular message was investigated using mutant synag 7, which lacks a factor required for AC activation [Frantz, 1980; Theibert and Devreotes, 1986; Snaar-Jagalska and Van Haastert, 19881. The mutant exhibits a low level of cAMP receptors, but cannot produce cAMP pulses and therefore does not aggregate. Exogenous cAMP pulses induce high levels of cAMP receptors [Schaap et al.,

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19861as well as csA (Fig. 8) and G,2, M3, and D2, other genes expressed during aggregation [Mann and Firtel, 19891. If cAMP pulses are followed up by stimulation with micromolar cAMP concentration, the D19 gene product psA is synthesized similarly as in wild-type cells, and cells can even be induced to form spores [Wang and Schaap, 1985; Schaap et al., 19861. Without pretreatment with cAMP pulses, no cAMP induction of prespore gene expression takes place. These data indicate that cAMP does not act as a n intracellular messenger for either aggregation-related or prespore gene expression. They furthermore show that a period of exposure to cAMP pulses is required to make cells competent for subsequent prespore induction. This is most

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P b P M A Fig. 8. The effect of stimulus modulation on the expression of cA-R and csA in synag 7. Synag 7 cells were incubated on non-nutrientagar (A) or shaken in phosphate buffer without stimulation (Pb) with 30 nM cAMPi6 minutes (PI or with M cAMP added every h r (M). After 6 hours mRNA was isolated and probed with cA-R and csA cDNA.

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Fig. 9. The effect of stimulus modulation on the expression of PDE, csA, and CP-2 in frigid C. NC-4 and fgd C (HC 317) cells incubated in phosphate buffer (Pb) with 30 nM CAMPIG minutes (PI or with a continuous influx of 5 nM cAMPiminute (F) or with M cAMP added as a single dose every hour (M). After 6 hours mRNA was isolated and probed with PDE, csA, and CP-2 cDNA.

likely the reason why prespore gene expression cannot be induced in vegetative cells (see Fig. 3). Streamer F mutants are defective in cGMP-PDE [Ross and Newell, 19811 resulting in a strongly en- pulses and persistent micromolar stimulation; and hanced and prolonged cGMP accumulation in response class 3, the prespore genes, respond only to persistent to CAMP. Both the CAMP-induced expression of pre- micromolar stimulation. Control of the CAMP-PDE spore [Schaap et al., 19861 a s well as aggregation-re- gene may represent a fourth class, regulated both by lated genes [Mann et al., 19881 are similar in this mu- pulses and persistent stimulation. Regulation of this tant and its parent strain, which suggests that cGMP gene involves additonal complexity as multiple prodoes not mediate either of these responses. moters [Podgorski et d., 19891 and is therefore not disThe fgd A mutants are defective in the a subunit of cussed here. the G-protein, which supposedly links the cAMP recepThe induction of the aggregation- and prestalk-retor to PLC [Coukell et al., 1983; Kesbeke et al., 1988; lated genes by nanomolar cAMP pulses may share a Kumagai et al., 19893. These mutants exhibit low lev- common transduction pathway, which does not involve els of the different subpopulations of cAMP receptors cAMP as second messenger. Involvement of cGMP and no IP, accumulation in response to CAMP, but seems unlikely, but cannot be excluded a t this moment. more remarkably also no cAMP and cGMP accumula- Involvement of the IP,/Ca2 pathway seems implition. Nanomolar cAMP pulses do not induce expression cated, because the fgd A mutants, which lack this pathof the M3 and D2 genes [Mann et al., 19881. These data way, do not show expression of these genes in response imply that Ga2 is essential for all types of chemotactic to pulses. However, Li ions, which inhibit CAMP-incAMP signaling a s well as cAMP regulated gene ex- duced IP, accumulation, promote pulse-induced gene pression. However, since all cAMP activated second expression. messenger systems are blocked, the fgd A mutants The induction of the prestalk-related genes by microyield no insight in involvement of a specific pathway. molar cAMP stimuli uses a transduction mechanism The fgd C mutant HC317 does not aggregate. In this distinct from induction of prespore gene expression by mutant PDE-mRNA is expressed to a moderate level. the same stimulus. Prespore induction does not involve Prestalk and aggregation-associated mRNAs accumu- cAMP and probably also not cGMP as intracellular inlate to very low levels during starvation, but expres- termediate. Involvement of CAMP-induced inositolsion does not respond to nanomolar pulses or micromo- phospholipid degradation in this pathway is indicated lar CAMP-stimuli (Fig. 9). The defect in this mutant by the fact that IP, and DAG can induce prespore gene has not yet been identified but it shows normal chemo- expression, albeit in a somewhat indirect manner and taxis, cAMP and cGMP accumulation [Coukell et al., by the fact that Li and Ca2 antagonists inhibit pre1983; Kesbeke and Van Haastert, unpublished re- spore gene expression. sults]. The induction of prestalk-related gene expression is inhibited by IP, and DAG and promoted by L i + , and is CONCLUSION relatively insensitive to Ca2+ antagonists. This shows We combined literature data with recent experi- that CAMP-induced PLC activation is clearly not inments in order to elucidate mechanisms involved in the volved in prestalk-related gene expression. Apart from data obtained by using drugs and mucontrol of cAMP regulated genes. During development different classes of CAMP-induced genes are expressed, tants, involvement of adaptation sensitive responses as which can be discriminated by responsiveness to cAMP GC and AC activation in prespore and prestalk gene stimulus modulation. Class 1, the aggregation-related regulation by persistent cAMP stimuli is unlikely. genes, respond only to nanomolar cAMP pulses; class 2, Candidates for these types of gene expression are the the prestalk-related genes, respond both to nanomolar adaptation insensitive responses a s the CAMP-induced +

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mulation of inositol triphosphate in Dictyostelium. J Cell Sci 87: 221-229. Frantz CE (1980): Phenotype analysis of aggregation mutants of Dictyostelium discoideum. PhD thesis, University of Chicago, Chicago. Gerisch G, Fromm H, Huesgen A, Wick U (1975): Control of cellcontact sites by cyclic AMP pulses in differentiating Dictyostelium cells. Nature 255: 547-549. Ginsburg G, Kimmel AR (1989):Inositol triphosphate and diacylglycerol can differentially modulate gene expression in Dictyostelium. Proc Natl Acad Sci USA 86: 9332-9336. Gomer RH, Armstrong D, Leichtling BH, Firtel RA (1986): cAMP induction of prespore and prestalk gene expression in Dictyostelium is mediated by the cell-surface CAMP-receptor. Proc Natl Acad Sci USA 83: 8624-8628. Grabel L, Loomis WF (1978): Effector controlling accumulation of N-acetylglucosaminidase during development of Dictyostelium discoideum. Dev Biol 64: 203-209. Haribabu H, Dottin RP (1986): Pharmacological characterization of cyclic AMP receptors mediating gene regulation in Dictyostelium discoideum. Mol Cell Biol 6: 2402-2408. Hassanain HH, Kopachik W (1989): Regulatory signals affecting seACKNOWLEDGMENTS lective loss of mRNA in Dictyostelium discoideurn. J Cell Sci 94: 501-509. We thank R. Brandt for technical assistance. We are grateful to Dr. J.G. Williams, Dr. A. Noegel, Dr. P.N. Henderson E J (1975): The cyclic adenosine 3’:5’-monophosphate receptor of Dictyostelium discoideum. J Biol Chem 250: 4730-4736. Devreotes, Dr. S.N. Cohen, and Dr. R. Kessin for their Jermyn KA, Berks M, Kay RR, Williams J G (1987): Two distinct respective gifts of pDd8, csA, cA-R, D19, and PDE pathways of prestalk-enriched mRNA sequences in Dictyostelium cDNA. discoideum. Development 100: 745-755. Kay CA, Noce T, Tsang AS (1987):Translocation of an unusual cAMP receptor to the nucleus during development of Dictyostelium discozREFERENCES deum. 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Proc Natl Acad Sci Blumberg DD, Comer J F , Higinbotham KG (1988): A Ca2+-dependent USA 83: 2506-2510. signal transduction system participates in coupling expression of Klein C, Juliani MH (1977): CAMP-induced changes in CAMP-binding some CAMP-dependent prespore genes to the cell surface receptor. sites on D. discoideum amoebae. Cell 10: 329-335. Dev Genet 9: 359-369. Klein PS, Sun TJ, Saxe I11 CL, Kimmel AR, Johnson RL, Devreotes Bumann J , Wurster B, Malchow D (1984): Attractant-induced PN (1988): A chemoattractant receptor controls development in changes in oscillations of the extracellular Ca concentration in Dictyostelium discoideum. Science 241: 1467-1472. suspensions of differentiating Dictyostelium cells. J Cell Biol 98: Knox BE, Devreotes PN, Goldbeter A, Segel LA (1986): A molecular 173-178. mechanism for sensory adaptation based on ligand-induced receptor Cardelli JA, Knecht DA, Wunderlich R, Dimond RL (1985): Major modification. Proc Natl Acad Sci USA 83: 2345-2349. changes in gene expression occur during at least four stages of development of Dictyostelium discoideum. Dev Biol 110: 147-156. Kopachik W, Bergen LG, Barclay SL (1985): Genes selectively expressed in proliferating Dictyostelium amoebae. Proc Natl Acad Sci Clarke M, Bazari WI, Kayman SC (1980): Isolation and properties of USA 85: 8540-8544. calmodulin from Dictyostelzum discoideum. J Bacteriol 141: 397400. Kumagai A, Pupillo M, Gundersen R, Miake-Lye R, Devreotes PN, Firtel RA (1989):Regulation and function of Ga protein subunits in Clarke M, Kayman SC, Riley K (1987): Density-dependent induction Dictyostelium. Cell 57: 265-275. of discoidin-I synthesis in exponential growing cells of Dictyostelium discoideum. Differentiation 34: 79-87. Lacombe ML, Podgorski GJ, Franke J , Kessin RH (1986): Molecular cloning and developmental expression of the cyclic nucleotide phosCoukell MB, Lappano S, Cameron AM (1983): Isolation and characphodiesterase gene of Dictyostelium discoideum. J Biol Chem 261: terization of cAMP unresponsive (frigid) aggregation-deficient mu16811-16817. tants of Dictyostelium discoideum. Dev Genet 3: 283-297. Darmon M, Brachet P, Pereira da Silva LH (1975): Chemotactic sig- Luderus MEE, Van der Most RG, Otte AP, Van Driel R: (1989): A protein kinase C-related enzyme activity in Dictyostelium discoinals induce cell differentiation in Dictyostelium dzscoideum. Proc deum. FEBS Lett 253: 71-75. Natl Acad Sci USA 72: 3163-3166. De Gunzburg J Veron M (1982):A CAMP-dependent protein kinase is Malchow D, Nagele B, Schwarz H, Gerisch G (1972): Membranebound cyclic AMP phosphodiesterase in chemotactically responding present in differentiating Dzctyostelium discoideum cells. EMBO J cells of Dzctyostelium discoideum. Eur J Biochem 28: 136-142. 1: 1063-1068. Europe-Finner GN, Newell PC (1986): Inositol 1,4,5-triphosphate in- Malchow D, Nanjundiah V, Wurster B, Eckstein F, Gerisch G (1978): Cyclic AMP-induced pH changes in Dictyostelium discoideum and duces calcium release from a non-mitochondria1 pool in amoebae of their control by calcium. Biochim Biophys Acta 538: 473-480. Dictyostelium. Biochim Biophys - . Acta 887: 335-340. Europe-Finner GN, Newell PC (1987): Cyclic AMP stimulates accu- Mann SK, Firtel RA (1987): Cyclic AMP regulation of early gene

Ca2+ influx and increase in pHi. The former may play a role in prespore gene expression. The latter does not mediate transduction to either type of gene expression. Most results obtained so far have identified putative signal transduction mechanisms, which are not involved in gene regulation. This reflects the situation that our knowledge of signal transduction in DictyosteZium though large, is still far from complete. Large gaps are especially evident in the field of inositol-phospholipid signaling, which as the mammalian systems show is much more complex than has at present been explored in Dictyostelium. Analysis of this field is a challenge of the first priority and further insight in cAMP controlled gene regulation may depend on availability of mutantsltransformants with identified defects in phospholipid signaling.

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CAMP-INDUCEDGENE EXPRESSION expression in Dictyostelium discoideum: Mediation via the cell surface cyclic AMP receptor. Mol Cell Biol 7: 458-469. Mann SK, Firtel RA (1989):Two-phase regulatory pathway controls cAMP receptor-mediated expression of early genes in Dictyostelium. Proc Natl Acad Sci USA 86: 1924-1928. Mann SK, Pinko C, Firtel RA (1988): cAMP regulation of early gene expression in signal transduction mutants of Dictyostelium. Dev Biol 130: 294-303. Margolskee J P , Froshauer S, Skrinska R, Lodish HF (1980): The effects of cell density and starvation on early developmental events in Dzctyostelium discoideum. Dev Biol 74: 409-421. Mato JM, Van Haastert PJM, Krens FA, Rijnsburger EH, Dobbe FCPM, Konijn TM (1977): Cyclic AMP and folic acid mediated cyclic GMP accumulation in Dictyostelium dzscozdeum. FEBS Lett 79: 331-336. Mato JM, Woelders H, Van Haastert PJM, Konijn TM (1978): Cyclic GMP binding activity in Dictyostelium dzscoideum. FEBS Lett 90: 26 1-264. Mehdy MC, Ratner D, Firtel RA (1983): Induction and modulation of cell-type-specific gene expression in Dictyostelium. Cell 32: 736771. Morrissey JH, Devine KM, Loomis WF (1984): The timing of celltype-specific differentiation in Dictyostelzum discoideum. Dev Biol 103: 414-424. Newell PC (1982): Cell surface binding of adenosine to Dictyostelium and inhibition of pulsatile signalling. FEMS Microbiol Lett 13: 417-421. Noegel A, Gerisch G, Stadler J , Westphal M (1986): Complete sequence and transcript regulation of a cell adhesion protein from aggregating Dictyostelium cells. EMBO J 5: 1473-1476. Oyama M, Blumberg DD (1986): Interaction with the cell-surface receptor induces cell-type-specific mRNA accumulation in Dictyostelium discoideurn. Proc Natl Acad Sci USA 83: 4819-4823. Parissenti AM, Coukell MB (1989): Identification of a nucleic acidregulated cyclic GMP-binding activity in Dictyostelium dzscozdeum. J Cell Sci 92: 291-301. Pears CJ, Mahbubani HM, Williams J G (1985): Characterization of two highly diverged but developmentally co-regulated cysteine proteinase genes in Dictyostelium discozdeum. Nucleic Acids Res 13: 8853-8866. Peters DJM, Van Lookeren Campagne MM, Van Haastert PJM, Spek W, Schaap P (1989): Litium ions induce prestalk-associated gene expression and inhibit prespore gene expression in Dictyostelium discoideum. J Cell Sci 93: 205-210. Podgorski G, Franke J , Faure M, Kessin RH (1989): The cyclic nucleotide phosphodiesterase gene of Dictyostelium discoideum utilizes alternate promotors and splicing for the synthesis of multiple mRNAs. Mol Cell Biol 9: 3938-3950. Pupillo M, Kumagai A, Pitt GS, Firtel RA, Devreotes PN (1989): Multiple a subunits of guanine nucleotide-binding proteins in Dictyostelium. Proc Natl Acad Sci USA 86: 4892-4896. Roos W, Gerisch G (1976): Receptor-mediated adenylate cyclase activation in Dictyostelium discoideum. FEBS Lett 68: 170-172. Ross FM, Newell PC (1981): Streamers: chemotactic mutants of Dictyostelium discozdeum with altered cyclic GMP metabolism. J Gen Microbiol 127: 339-350. Schaap (1986): Regulation of size and pattern in the cellular slime molds. Differentiation 33: 1-16. Schaap P, Van Driel R (1985): The induction of post-aggregative differentiation in Dictyostelium discoideum by CAMP.Evidence for the involvement of the cell surface cAMP receptor. Exp Cell Res 159: 388-398. Schaap P, Van Lookeren Campagne MM, Van Driel R, Spek W, Van Haastert PJM, Pinas J (1986): Postaggregative differentiation induction by cyclic AMP in Dictyostelium: intracellular transduction pathway and requirement for additional stimuli. Dev Biol 118: 5263. Singleton CK, Delude RL, McPherson CE (1987):Characterization of genes which are deactivated upon the onset of development on Diciyostelzum discoideum. Dev Biol 119: 433-441.

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PETERS ET AL.

Wang M, Van Haastert PJM, Devreotes PN, Schaap P (1988):Localization of chemoattractant receptors on Dictyostelium discoideum cells during aggregation and down-regulation. Dev Biol 128: 72-77. Williams J G (1988):The role of diffusable molecules in regulating the cellular differentiation of Dictyostelium discoideum. Development 103: 1-16. Williams JG, Lloyd MM, Devine JM (1979): Characterization and

transcription analysis of a cloned sequence from a developmentally regulated mRNA of Dictyostelium discoideum. Cell 17: 303-314. Woffendin C, Chamrers TC, Schaller KL, Leichtling BH, Rickenberg HV (1986):Translocation of CAMP-dependent protein kinase to the nucleus during development of Dictyostelium discoideum. Dev Biol 115: 1-8.

Control of cAMP-induced gene expression by divergent signal transduction pathways.

A compilation of literature data and recent experiments led to the following conclusions regarding cyclic adenosine 3':5' monophosphate (cAMP) regulat...
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