DEVELOPMENTAL GENETICS 122-5 (1991)

Signal Transduction and Gene Expression in Dictyostelium discoideum ROBERT P. DOTTIN, SOBHA R. BODDULURI, JACQUELINE F. DOODY, AND BODDULURI HARIBABU Department of Biological Sciences, Hunter College, City University of New York, New York

In recent years, Dictyostelium has become recognized a s a n organism well suited to study signal transduction, the mechanisms by which extracellular stimuli elicit intracellular responses. Work from several laboratories has contributed to this awareness: Bonner’s original discovery that extracellular cAMP is a n acrasin for chemotaxis [Konijin et al, 19681 led to demonstrations that the binding of cAMP to cell-surface receptors is required for many of the responses that are associated with chemotaxis [Devreotes, 1989; Dinauer et al., 1980; Gerisch, 1987; Berlot et al., 1987; Hall et al., 19891. Thus work on dissecting the mechanism of motility has proceeded in concert with studies t h a t determined how motility is connected to receptor activation. By contrast, our awareness that signal transduction induces genes in Dictyostelium has been relatively recent. As early as 1978, Town and Gross showed that exogenous cAMP induced several developmentally regulated enzymes in Dictyostelium, and later studies in Lodish’s laboratory showed changes in the patterns of accumulation of several proteins and mRNAs in response to addition of cAMP [Landfear and Lodish, 1980; Chung et al., 19811. cAMP was already known to function in mammalian cells as a n intracellular second messenger induced in response to peptide hormones. Exogenous dibutyryl cAMP could bypass peptide hormone action by entering mammalian cells. Thus by analogy exogenous cAMP might activate genes in Dictyostelium in a manner similar to dibutyryl CAMP. However, in recent years cAMP has been shown to induce genes in Dictyostelium by acting as a hormone directly binding to cell-surface receptors. Pharmacological studies using cAMP analogs could distinguish between the cell-surface cAMP receptors and the major intracellular protein that binds CAMP, the regulatory subunit of CAMP-dependent protein kinase (PKA) [Van Haastert and Kien, 19831. Studies using different cAMP analogs provided strong evidence that the primary targets for cAMP to induce or repress genes were the cell-surface receptors [Schaap and Van Driel, 1985; Haribabu and Dottin, 1986; Oyama and Blumberg, 1986; Gomer et al., 1986; Kimmel, 19871. No other known intracellular cAMP binding proteins have the required specificity or affinity for the low concentrations of agonist that induce the genes [Van Ments-

0 1991 WILEY-LISS, INC.

Cohen and Van Haastert, 19891. Thus extracellular cAMP induces genes in a manner similar to a peptide hormone, i.e., via cell-surface receptors. Studies from a number of laboratories have shown that cell-surface cAMP receptors are coupled via G proteins to adenyl cyclase and to phospholipase C [Theibert and Devreotes, 1986; Van Haastert, 1984; Europe-Finner and Newell, 19871. In addition to pharmacological studies, experiments with antisense CAMP-receptor-RNA and with mutants show that induction of most genes is mediated through receptor activation of specific G proteins coupled, not to adenyl cyclase, but to phospholipase C [Klein et al., 1988; Kumagai et al., 19891. Synag and frg mutants fail to aggregate or complete the developmental cycle. Synag 7 mutants are unable to activate adenyl cyclase in vivo and in vitro but can be complemented in vitro by addition of wild-type soluble extracts [Theibert and Devreotes, 19861. Nevertheless, synag 7 mutants induce many genes in response to exogenous CAMP, indicating that the pathways between the receptors and the inducible gene are essentially intact [Mann et al., 19881. Because adenyl cyclase is not activated in synag mutants, the result suggests t h a t intracellular cAMP is not required for induction of these genes. This result was also demonstrated by pharmacological experiments in which caffeine was used to block activation of adenyl cyclase [Kimmel, 1987; Haribabu et al., 1989; see Haribabu et al, this volume]. An exception to this generalization on intracellular messengers is the M4 gene, which appears to require cAMP as a n intracellular messenger [Kimmel, 19871. Recent studies by Barclay (personal communication) also suggest that intracellular cAMP may be involved in the regulation of the cyclic nucleotide phosphodistease gene. In contrast, with synag mutants, fgd A mutants are totally defective in signal transduction. They fail to respond to CAMP, to activate adenyl cyclase or phospholipase C,

Received for publication May 28, 1990. Address reprint requests to Dr. Robert P. Dottin, Department of Biological Sciences, Hunter College, CUNY, Rm. 937N, 695 Park Avenue, New York, NY 10021.

SIGNAL TRANSDUCTION IN D. DZSCOZDEUM and are defective in chemotaxis and gene induction [Kesbeke et al., 1988; Kumagai et al., 19891. They are defective in a G protein, Ga2, required for activation of phospholipase C and gene induction. Thus both pharmacological and genetic experiments argue that cAMP induces most genes by cell-surface cAMP receptors via Ga2 proteins and phospholipase C and that in most cases adenyl cyclase activation is not required to provide second messengers. Previous studies had suggested that the cAMP receptors now designated (CAR1) are present a t highest levels early (5 hr) during development [Klein et al., 19881. Although they respond to pulsatile applications of nanomolar concentrations of CAMP,they are desensitized by, or adapt to, constant high (micromolar) levels of CAMP. Thus i t was puzzling that late genes could be induced in response to high and constant levels of cAMP even when the number of detectable receptors was severely reduced. The identification by Saxe et al. (this volume) of additional cell-surface cAMP receptor genes CAR2 and CAR3 could explain this discrepancy in that the different receptors may induce different genes. The deduced amino acid sequence of the CAR13 proteins are similar. They contain seven conserved hydrophobic regions that appear to make up transmembrane domains similar to those of P-adrenergic receptors. They also contain hydrophilic carboxy-terminal segments that are significantly different in their distribution of serine residues. The mechanism of desensitization of receptors is poorly understood, but, by analogy with the mammalian P-adrenergic receptors, phosphorylation by receptor kinases may accompany its desensitization and may be required for i t [Vaughan and Devreotes, 1989; Benovic et al., 19891. Therefore, it is tempting t h a t the serine residues abundant in CARl are sites for phosphorylation that may induce adaptation. If so, their absence in CAR2 and CAR3 may explain the lack of adaptation in late genes that can be induced by constant high doses of CAMP.The mRNA of the CARl receptor is expressed early, when the cAMP relay system is being established, and declines rapidly to a low level late in development. CAR2 mRNA is expressed only late, and the CAR3 mRNA, though detectable at 5 hr, is also found late in development. The temporal expression of these receptor genes could also account for the ability of the cells to respond to cAMP at different stages. These assumptions imply that the pharmacological specificities of CAR1-3 for cAMP analogs are all similar. To complicate matters, no receptor mRNA seems to be preferentially distributed in prespore cells. Rather, all three receptors are preferentially expressed in prestalk cells, with CAR2 showing the strongest bias. The accompanying paper from Klein’s laboratory shows that a CAR kinase is required for phosphorylation of a 45-47 Kd protein that appears to be the receptor. The CARl kinase is active only when the cells are stimulated by CAMP. Membranes isolated from

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cells lacking cAMP binding activity cannot be phosphorylated by CAR kinase, and a ligand occupied receptor may be required for CAR kinase activity. The significance of a second membrane-associated protein, p36, that is phosphorylated is unknown. Although receptors become insensitive to the ligand CAMP, other mechanisms are also involved in the attenuation of the stimulus-response reactions. cAMP induces downregulation of cAMP receptors [Wang et al., 19881. Competent cells synthesize and secrete a cyclic nucleotide phosphodiesterase that degrades extracellular CAMP.The activity of the phosphodiesterase is also inhibited by a heat-stable inhibitor so that the kinetics of synthesis and secretion of these proteins play a n important role in the establishment and extinction of the signal relay system [Podgorski et al., 19881. This topic is discussed extensively in a n earlier section (Franke et al., this volume). Receptor activation of phospholipase C results in the synthesis of Ins(1,4,5)P3 and diacylglycerol from phosphoinositol bisphosphate. The metabolism of Ins(1,4,5)P, is being rapidly elucidated by Van Haastert’s lab (see Bominaar et al., this volume). The inositol cycle is similar to that of mammalian cells, but Ins(1,3,4,5)P4is not found, and the dephosphorylation of Ins(1,4,5)P3 in Dictyostelium is more complex. It involves three more enzymes and the production of a n additional form of IP2, Ins(4,5)P2,a s well as Ins( 1,4)P,, which is also present in mammalian cells. At least six enzymes are required for dephosphorylation of IP, in all, and although none has been purified, their activities can be distinguished in factions from DEAE cellulose chromatography. This report also provides evidence for cross-talk between the pathways involved in the production of cAMP and Ins(1,4,5)P3. Treatment of permeabilized fgd A mutants with dcAMP or IP3 alone did not activate adenyl cyclase but together they did. In addition, GTP stimulation of adenyl cyclase is still normal, suggesting that another G protein activates adenyl cyclase and that phospholipase C activates adenyl cyclase indirectly. Recent work from Irvine’s laboratory (personal communication) has shown that the highly polar InsP,, the most abundant inositide phosphate in nature, is the major inositide phosphate in Dictyostelium (1 mM) and is synthesized from Ins3P by addition of phosphates sequentially to positions 6,4,1,5,2 on the inositol residue. Which of the many inositide phosphates are the intracellular messengers that are required for gene induction remain to be determined. Identification of the pathways between the receptors and responsive genes is the major focus in a number of laboratories (Kimmel, Schaap, Chisholm, Firtel, Nellen, Blumberg, personal communications). Work is presented here from Schaap’s laboratory (Peters et al., this volume). Their work distinguishes among three classes of genes: aggregation-related, pulse-inducible genes; prestalk-related genes inducible both by pulses

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and persistent levels of CAMP; and prespore-specific genes, represented by D19, that respond only to persistent micromolar CAMP.The first two classes appear to share a pathway that is distinguishable from the third by Li' and by Ca2+ antagonists. LiC1, a n inhibitor of CAMP-induced IP3 accumulation, inhibits CAMP-induced prespore gene expression and promotes prestalkrelated gene expression. The authors suggest that the point of divergence may be a t the receptors themselves. Fgd C mutants may affect events after phospholipase C activation, because they induce IP3 but express reduced levels of prestalk- and aggregation-associated mRNAs. Their results suggest that the CAMP-induced increases in pHi or cGMP do not affect gene expression. Likely intracellular messengers include Ins( 1,4,5)P, and diacylglycerol, a t least for prespore-specific genes. Related experiments are described by Haribabu et al. (this volume). Focusing on the pathways that culminate in the expression of a UDPglucose pyrophosphorylase gene (UDPGPl), they propose t h a t inositide phosphates and not cAMP are intracellular messengers. They observed that LiCl distinguishes two divergent pathways, those affecting prestalk genes and those affecting prespore and the UDPGPl genes. Cascades of protein synthesis are required to induce the UDPGPl gene and, in fact, most CAMP-dependent genes. Two regulatory elements have been defined in the UDPGPl gene by deletion analysis, one of which is essential for CAMP-mediated induction of the gene. These elements interact with nuclear proteins that appear to be regulated by reversible phosphorylation. Reversible protein phosphorylation is obviously important in several stages of the signal transduction pathways. The phosphorylation of cAMP receptors, G proteins [Gundersen and Devreotes, 19901, and the DNA binding proteins discussed above illustrate this point. Thus several laboratories have initiated studies on the protein kinase genes [Mutzel et al., 1987; Williams, Reymond, Veron, Devreotes, Firtel and Spudich personal communications]. The paper by Haribabu and Dottin (this volume) describes a n approach that identified and cloned fragments encoding five putative protein kinases and two putative phosphoprotein phosphatases. Some of these kinases are differentially regulated both during development and in response to CAMP. At least a dozen different putative kinase fragments have been cloned in the labs mentioned above, and more are likely to be found. Cyclophilins are abundant ubiquitous peptidylprolyl cis-trans isomerases t h a t are inhibited by cyclosporin A, a n immunosuppressive agent which blocks T-cell responses. It is intriguing to speculate that cyclophilins may be involved in the stimulus response pathways that induce T cells to proliferate. Cyclophilin-like proteins are essential for visual transduction in Drosophila [Shieh et al., 19691. The importance of cyclophilins in signal transduction in Dictyostelium remains to be determined, but the isolation of cDNA

clone (Barisic et al., this volume) that encodes a cyclophilin should help in elucidating its role. The final paper in this section includes a n analysis of a phenomenon that is likely related to signal transduction by CAMP. Based on disaggregation and reaggregation experiments, cell-cell contact has been implicated in the induction of genes in Dictyostelium [Newell et al., 1971; Chisholm et al., 19841. The paper by Fontana et al. (this volume) shows that cell-cell contact elicits the secretion of cAMP and independently alters the amount secreted in a subsequent signalling response. The interactions are specific, because amoebae can differentiate between contact with beads, bacteria, and other amoebae. The relatedness between receptor activation of signal transduction pathways and cell-cell contact induction of genes remains to be determined. The pharmacological, biochemical, and genetic experiments have improved our understanding of signal transduction in Dictyostelium. The availability of transformation techniques to inactivate mRNA and to disrupt or overexpress specific genes promises to provide novel insights into the mechanisms of gene regulation by signal transduction.

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