Dictyostelium cells use extracellular cyclic AMP both as a chemoattractant and as a morphogen inducing cell-type-specific gene expression. Cyclic AMP binds to surface receptors, activates one or more G-proteins, and stimulates adenylate cyclase, guanylate cyclase and phosphoinositidase C. Mutant fgdC showed aberrant chemotaxis, and was devoid of cyclic AMP-induced gene expression and differentiation. Both the receptor- and G-protein-mediated stimulation of adenylate cyclase and guanylate cyclase were unaltered in mutant fgdC as compared to wild-type cells. In wild-type cells phosphoinositi dase C was activated about twofold by the cyclic AMP receptor. In mutant fgdC cells, however, the enzyme was inhibited by about 60%. These results suggest that phosphoinositidase C is regulated by a receptor-operated activation/inhibition switch that is defective in mutant fgdC. We conclude that activation of phosphoinositidase C is essential for Dictyostelium development.

Dictyostelium is used as a model organism in which to study the regulation of cell locomotion, chemotaxis and gene expression by extracellular signal molecules (Janssens and Van Haastert, 1987). In aggregation-competent cells these functions are controlled by extracellular cyclic AMP, which binds to surface receptors that have a predicted structure with seven transmembrane spanning domains like many G-protein coupled receptors (Klein et al. 1988). Activation of the receptor leads to the G-protein-mediated stimulation of adenylate cyclase, guanylate cyclase and phosphoinositidase C (Europe-Finner et al. 1989; Janssens et al. 1989; Mato et al. 1977; Schaffer, 1975; Theibert and Devreotes, 1986; Van Haastert, 1989; Van Haastert et al. 1989; Van Haastert et al. 1987). These enzymes produce the second messengers cyclic AMP, cyclic GMP, Ins(l,4,5)P3 and diacylglycerol, respectively.

In eukaryotes the mechanism by which extracellular signals induce chemotaxis and gene expression, and the role of the different second messengers in these processes, are still largely unknown. Dictyostelium mutants and transformants are potentially useful in elucidating these sensory transduction pathways. Transformed cells with reduced cyclic AMP receptor expression show aberrant chemotaxis and differentiation (Klein et al. 1988; Sun et al. 1990). Experiments with mutants stmY and synagl demonstrate that activation of guanylate cyclase is important for chemotaxis but possibly not for differentiation, whereas activation of adenylate cyclase is not directly involved in either of these responses (Ross and Newell, 1981; Schaap et al. 1986; Snaar-Jagalska and Van Haastert, 1988). Frigid mutants are insensitive to extracellular cyclic AMP for the induction of contact sites (Coukell et al. 1983). Mutant fgdA is defective in the gene coding for the G-protein a-subunit Ga2 and is defective in all known second-messenger functions, chemotaxis and cyclic AMP-induced gene expression (Coukell et al. 1983; Kesbeke et al. 1988; Kumagai et al. 1989; Snaar-Jagalska et al. 1988a,b). Here we describe mutant fgdC, which is specifically defective in the regulation of phosphoinositidase C activity.

Materials

Cyclic AMP and GTP[S3 were from Boehringer-Mannheim, dithiothreitol and EGTA were obtained from Sigma. [2,8-3H]cyclic AMP (40 Ci mmol−1), [8-3H]cyclic GMP (25 Ci mmol−1) and [3H]Ins(l,4,5)P3 (60 Ci mmol−1) were from Amersham, Buckinghamshire. The Ins(l,4,5)P3 binding protein was isolated from bovine adrenal glands as described (Baukal et al. 1985).

Culture conditions and chemotaxis

Dictyostelium cells (wild-type strains NC4 and XP-55 and mutant fgdC strain HC317) were grown in association with Escherichia coli as described (Kesbeke et al. 1988). Cells were harvested in 10 mM KH2PO4/Na2HPO4, pH 6.5 (PB). Since no significant differences could be observed between XP-55 and NC-4 the data presented are those of the more generally used wild-type strain NC-4. Chemotaxis towards different cyclic AMP concentrations was determined using the small-population assay (Konijn, 1970) the Ins(l,4,5)P3 produced was determined by isotope dilution assay (Van Haastert, 1989).

Cyclic AMP-binding to surface receptors

Cells were starved in PB at a density of 107 cells ml−1 for 5h, washed and resuspended in PB at a density of 108 cells ml’1. Cyclic AMP binding by cells was measured in incubations containing different concentrations of [3H]cyclic AMP, 10 mM dithiothreitol and 8×107 cells. After 5 min at 0°C cells were centrifuged through silicon oil, and cell-associated radioactivity was determined (Kesbeke et al. 1988). Non-specific binding was determined in the presence of 10∼4M unlabeled cyclic AMP. Cyclic AMP binding towards membranes was measured at 5 nM [3H]cyclic AMP in the presence or absense of 30, UM GTPfS] as described (Kesbeke et al. 1988).

Second messenger responses in vivo

Cells were starved for 5h in PB by shaking at a density of 107 cells ml−1. Cells were collected, resuspended to a density of 108 cells ml−1 in PB and stimulated. For cyclic AMP response cells were stimulated with 5UM 2’deoxy-cyclic AMP and 5IHM dithiothreitol; the stimulus for cyclic GMP and Ins(l,4,5)P3 responses was 0.1 μM cyclic AMP. At the times indicated in the figure legends cells were lysed by the addition of perchloric acid. The levels of cyclic AMP, cyclic GMP or Ins(l,4,5)P3 were determined in the neutralysed lysates using specific isotope dilution assays (Kesbeke et al. 1988; Van Haastert, 1989).

Regulation of phosphoinositidase C by cyclic AMP in vivo

Wild-type (strain NC4) and mutant fgdC cells (strain HC317) were starved for 5h, washed and resuspended in 40 mM Hepes/NaOH, pH 6.5, 5.9 mM EGTA (PIC-buffer). Cells were stimulated at t0 with 0.1 μM cyclic AMP, and lysed at the times indicated in the figure legends by rapid elution through a Nuclepore polycarbonate filter (pore size 3 μm). Phosphoinositidase C activity was measured immediately in a reaction mixture of 100 μl containing 6.9 mM CaC12 and 90 /d of lysate. The reaction was terminated after 20 s by the addition of 100 μl perchloric acid (3.5%, v/v), basal Ins(l,4,5)P3 levels were determined in samples to which perchloric acid was added prior to addition of CaC12. Ins(l,4,5)P3 levels were measured in the neutralized lysate by isotope-dilution assay (Van Haastert, 1989). The data presented are the Ins(l,4,5)P3 levels found in the presence of CaCl2 minus the basal level, hence they represent the amount produced during the incubation. A more-detailed description of the assay and the characteristics of PIC will be published elsewhere (A. A. Bominaar and P. J. M. Van Haastert, manuscript in preparation).

Regulation of effector enzymes by GTP[S] in cell-free systems

Wild-type NC4 and fgdC cells were starved for 5h, washed and resuspended with the indicated buffer. Cells were lysed by rapid filtration through a Nuclepore polycarbonate filter (pore size 3/on). Adenylate cyclase: lysis was in 40 mM Hepes, 0.5 mM EDTA, 250mM sucrose, pH 7.5 (AC-buffer). The lysate was incubated for 5 min at 0 °C in the absence or presence of 100 pM GTP[S] (guanosine 5′-O-(3-thiotriphosphate). Adenylate cyclase activity was then determined at 20 “C in a reaction containing lysate, AC-buffer, 2 mM MgCl2, 0.5 mM ATP and 10 mM dithiothreitol; the cyclic AMP produced was determind by isotope dilution assay as described (Theibert and Devreotes, 1986; Van Haastert et al. 1987). Guanylate cyclase: lysis was in 20 mM Hepes, 1.5 mM MgCl2, 0.5 mM EGTA, pH 7.0 (GC-buffer). Guanylate cyclase activity was determined immediately in a reaction containing lysate, GC-buffer, 5mM dithiothreitol and 0.3 mM GTP in the presence or absence of 25 μM GTPfS]; the cyclic GMP produced was determined by radioimmunoassay (Janssens et al. 1989). Phosphoinositidase C: lysis was in 40 mM Hepes, 5.9 mM EGTA, pH 6.5 (PIC-buffer), in the presence or absence of 10 μM GTP[S]. Phosphoinositidase C activity was determined within 1 min after lysis in a reaction containing lysate PIC-buffer and 6.9 mM CaCl2;

Expression of aggregation-associated genes

Vegetative wild-type NC4 and mutant fgdC cells were freed from bacteria, resuspended to 107 cells ml−r in PB and shaken at 150 revs min−1 and 21 °C. Cells were treated with either 30 nM cyclic AMP pulses delivered at 6 min intervals, a cyclic AMP influx of 5 nM min−1, pulses of 100 pM cyclic AMP added at 60 min intervals or without further additions. At the time periods indicated in the figure legends total RNA was isolated, size fractionated on formaldehyde-containing agarose gels, transferred to nylon membranes and hybridized with a 32P-labeled cDNA complementary to the contact site A gene.

Expression of prespore and prestalk genes

Vegetative wild-type NC4 and mutant fgdC cells were freed from bacteria, shaken in PB at 107 cells ml−1 and treated during 6h with 30 nM cyclic AMP for 6 min. Cells were washed once with PB, resuspended to the same density and shaken for an additional 8 h period with either 100 pM cyclic AMP (60 min), 30 nM cyclic AMP (6 min), or without further additions. At the time periods indicated in the figure legends total RNA was isolated and hybridized with 32P-labeled cDNAs, complementary to the prestalk gene CP2 or the prespore gene D19.

Cyclic AMP surface receptors

The binding of cyclic AMP to Dictyostelium wild-type and mutant cells was measured at different cyclic AMP concentrations, and is presented as a Scatchard plot (Fig. 1). Wild-type cells possess about 54000 binding sites per cell, which may exist in two affinity states with Kdl=45nM and Kd2=450nM, respectively. Mutant fgdC has a lower level of cyclic AMP binding (about 20000 sites/cell). The two affinity states have similar Kd values to those of wild-type cells (Kdl=28nM and Kd2=400nM).

Fig. 1.

Cyclic AMP-binding to surface receptors. The binding of different concentrations of [3H]cyclic AMP to NC4 wild-type (○) or fgdC mutant (•) cells was determined. The data are presented as a Scatchard plot.

Fig. 1.

Cyclic AMP-binding to surface receptors. The binding of different concentrations of [3H]cyclic AMP to NC4 wild-type (○) or fgdC mutant (•) cells was determined. The data are presented as a Scatchard plot.

Cyclic AMP-induced second messenger responses

Wild-type and mutant cells were stimulated with cyclic AMP or 2′-deoxy-cyclic AMP and the levels of accumulation of cyclic AMP, cyclic GMP and Ins(l,4,5)P3 were measured. The 2′-deoxy-cyclic AMP-induced level of accumulation of cyclic AMP in mutant fgdC was similar in respect of kinetics and magnitude to that in wild-type cells (Fig. 2A). Cyclic AMP also induced the accumulation of cyclic GMP although at a slightly lower rate than in wildtype cells (Fig. 2B). Cyclic AMP stimulation of wild-type cells resulted in a 22±11% (n=3, P<0.05) increase in the Ins(l,4,5)P3 concentration at 4 s after stimulation. In contrast to the receptor-stimulated formation of Ins(l,4,5)P3 in wild-type cells, cyclic AMP induced a 20±12% (n=3, P<0.05) decrease in Ins(l,4,5)P3 levels in mutant fgdC (Fig. 1C). The difference between wild-type and HC-317, at t=4s, is significant, with P<0.02. Although these responses are significant they are relatively small, therefore phosphoinositidase C activity was determined more directly, the results of which are presented below.

Fig. 2.

Cyclic AMP-induced responses. Cells of wild-type strain NC4 (○) and mutant fgdC strain (•) were starved for 5 h, washed and stimulated with cyclic AMP. The accumulation of cyclic AMP (A), cyclic GMP (B) or Ins(l,4,5)P3 (C) was determined by specific isotope-dilution assays. * Significantly above control with P<0.05; ** significantly below control with P<0.05. The results are the mean (±S.E.M. for C) of three independent experiments in triplicate.

Fig. 2.

Cyclic AMP-induced responses. Cells of wild-type strain NC4 (○) and mutant fgdC strain (•) were starved for 5 h, washed and stimulated with cyclic AMP. The accumulation of cyclic AMP (A), cyclic GMP (B) or Ins(l,4,5)P3 (C) was determined by specific isotope-dilution assays. * Significantly above control with P<0.05; ** significantly below control with P<0.05. The results are the mean (±S.E.M. for C) of three independent experiments in triplicate.

Receptor-G-protein-effector interactions

The interaction between surface receptors and GTP-binding proteins is most easily studied as the GTP[S]-mediated decrease in cyclic AMP binding to membranes. The results presented in Table 1 indicate that GTP[S] induced the same inhibition of cyclic AMP binding in membranes derived from wild-type cells and mutant cells.

Table 1.

Receptor-G-protein-effector interactions

Receptor-G-protein-effector interactions
Receptor-G-protein-effector interactions

Interactions between G-proteins and effector enzymes can be studied by the activation of these effector enzymes by GTP[S]. Adenylate cyclase, guanylate cyclase and phosphoinositidase C can be activated by GTP[S] in lysates from wild-type cells (Janssens et al. 1989; Theibert and Devreotes, 1986; Van Haastert et al. 1987; A. A. Bominaar and P. J. M. Van Haastert, manuscript in preparation). Although basal levels of most effector enzyme activities are lower in fgdC cells compared to wildtype cells the relative activation of these enzymes by GTP[S] in lysates of fgdC was not significantly different from that in lysates from wild-type cells (Table 1). Thus a normal interaction between G-proteins and effector enzymes exists.

Assays for cyclic AMP stimulation of phosphoinositidase C in cell-free systems are not available for Dictyostelium. Therefore, the regulation of phosphoinositidase C by the cyclic AMP receptor was investigated in vivo. Cells were stimulated with cyclic AMP, rapidly lysed and phosphoinositidase C activity was determined (Fig. 3). In wildtype cells cyclic AMP induced a twofold activation of phosphoinositidase C activity when cells were lysed 20 s after stimulation. Stimulation of mutant fgdC cells with cyclic AMP did not result in the activation of phosphoinositidase C; in contrast, basal phosphoinositidase C activity was inhibited by 60% (Fig. 3). Since under these assay conditions Ins(l,4,5)P3 phosphatases are not active (data not shown), the decrease found must reflect a decrease in the enzyme activity.

Fig. 3.

Regulation of phosphoinositidase C. Cells of wild-type strain NC4 and mutant fgdC strain HC317 were starved for 5 h, washed and resuspended in PIC-buffer. Cells were stimulated with 0.1 μm cyclic AMP (hatched bars) and lysed after 20 s, or lysed in the presence of 10 μM GTP[S] (dotted bars). Phosphoinositidase C activity was measured. Data are presented as the mean±S.D. of 2 independent experiments in quadruplicate. * Significantly larger than control (open bars) with P<0.02; ** significantly lower than control with P<0.001; *** significantly larger than control with P<0.05.

Fig. 3.

Regulation of phosphoinositidase C. Cells of wild-type strain NC4 and mutant fgdC strain HC317 were starved for 5 h, washed and resuspended in PIC-buffer. Cells were stimulated with 0.1 μm cyclic AMP (hatched bars) and lysed after 20 s, or lysed in the presence of 10 μM GTP[S] (dotted bars). Phosphoinositidase C activity was measured. Data are presented as the mean±S.D. of 2 independent experiments in quadruplicate. * Significantly larger than control (open bars) with P<0.02; ** significantly lower than control with P<0.001; *** significantly larger than control with P<0.05.

Summarizing transmembrane signal transduction in fgdC’. the results show normal cyclic AMP- and GTP[S]-mediated activation of adenylate and guanylate cyclase. Regulation of phosphoinositidase C by GTP[S] is also unaltered, but cyclic AMP inhibits the enzyme in fgdC, whereas it stimulates the enzyme in wild-type cells. Thus the defect in fgdC appears to be localized between the receptor and the G-protein.

Chemotaxis

Under physiological conditions mutant fgdC cells do not aggregate. However, chemotaxis was not absent, but about 100-fold higher cyclic AMP concentrations were required as compared to wild-type cells (Fig. 4). This low chemotactic efficiency towards cyclic AMP could explain why this mutant is aggregation-deficient (Coukell et al. 1983).

Fig. 4.

Chemotaxis. The chemotactic activity of wild-type (○) and fgdC mutant (•) cells towards different cyclic AMP concentrations was determined with the small-population assay.

Fig. 4.

Chemotaxis. The chemotactic activity of wild-type (○) and fgdC mutant (•) cells towards different cyclic AMP concentrations was determined with the small-population assay.

Gene-expression

Cyclic AMP regulates the expression of several classes of genes at different, stages of development (Mann and Firtel, 1989). Pulses of nanomolar cyclic AMP concentrations accelerate expression of aggregation-associated genes, such as those of cyclic AMP receptors and adhesive contact sites A (Gerisch et al. 1975). Constant stimulation of aggregation-competent cells with micromolar cyclic AMP concentrations induces the expression of prespore genes, and both nanomolar pulses and micromolar stimuli induce the expression of a subclass of prestalk genes.

The induction of the different classes of cyclic AMP-regulated genes was investigated in mutant fgdC and in wild-type strain NC4. Fig. 5 shows that in NC4 nanomolar cyclic AMP pulses strongly enhanced contact site A gene expression, while a nanomolar cyclic AMP influx or stimulation with micromolar cyclic AMP concentrations inhibited expression of this gene. In the fgdC mutant, a low basal expression of contact site A gene was evident, but its expression was not regulated by cyclic AMP (Fig. 5).

Fig. 5.

Cyclic AMP-regulated expression of aggregation-associated genes. Vegetative wild-type NC4 and mutant fgdC cells were stimulated with either 30 nM cyclic AMP pulses delivered at 6 min intervals, a cyclic AMP influx of finiumin’1, pulses of 100 μM cyclic AMP added at 60 min intervals or without further additions. At the indicated time periods total RNA was isolated and probed with a 32P-labeled cDNA complementary to the contact site A gene.

Fig. 5.

Cyclic AMP-regulated expression of aggregation-associated genes. Vegetative wild-type NC4 and mutant fgdC cells were stimulated with either 30 nM cyclic AMP pulses delivered at 6 min intervals, a cyclic AMP influx of finiumin’1, pulses of 100 μM cyclic AMP added at 60 min intervals or without further additions. At the indicated time periods total RNA was isolated and probed with a 32P-labeled cDNA complementary to the contact site A gene.

In NC4 cells, which were sensitized during 6h with nanomolar cyclic AMP pulses, expression of the prestalk gene CP2 was nearly maximal and increased only slightly by further treatment with pulses or micromolar cyclic AMP concentrations. In mutant fgdC neither pulses nor micromolar cyclic AMP stimuli could induce expression of this gene (Fig. 6). Micromolar cyclic AMP stimuli effectively induced expression of the D19 prespore gene in wildtype cells that were made competent by a 6 h pretreatment with cyclic AMP pulses. No expression of the D19 prespore gene was observed in the fgdC mutant (Fig. 6).

Fig. 6.

Cyclic AMP-regulated expression of prespore and prestalk genes. Vegetative wild-type NC4 and mutant fgdC cells were treated during 6h with pulses of 30 nM cyclic AMP for 6 min. Cells were washed and shaken for an additional 8h period with either 100 μM cyclic AMP (60 min) or 30 nM cyclic AMP (6min), or without further additions. At the indicated time periods total RNA was isolated and probed with 32P-labeled cDNAs, complementary to the prestalk gene CP2 or the prespore gene D19.

Fig. 6.

Cyclic AMP-regulated expression of prespore and prestalk genes. Vegetative wild-type NC4 and mutant fgdC cells were treated during 6h with pulses of 30 nM cyclic AMP for 6 min. Cells were washed and shaken for an additional 8h period with either 100 μM cyclic AMP (60 min) or 30 nM cyclic AMP (6min), or without further additions. At the indicated time periods total RNA was isolated and probed with 32P-labeled cDNAs, complementary to the prestalk gene CP2 or the prespore gene D19.

Transduction of extracellular cyclic AMP signals in Dictyostelium is mediated by surface receptors, G-proteins and effector enzymes. The final cellular responses induced by extracellular cyclic AMP are chemotaxis and cell differentiation. The signal transduction pathways probably involve activation of adenylate cyclase, guanylate cyclase and phosphoinositidase C, and the formation of the second messengers cyclic AMP, cyclic GMP, Ins(l,4,5)P3, Ca2+ and diacylglycerol. These second messengers may transduce the signal further towards the final cellular responses.

Mutants with a defect in specific parts of the signal transduction pathways are very useful in unravelling the role of each of these pathways in the different responses. Mutant fgdC, which was selected for the lack of contact sites A (Coukell et al. 1983), a cell surface protein expressed early in development and involved in the aggregation process, shows the following characteristics: (i) surface cyclic AMP receptors are present, although at a reduced level, (ii) The receptor-G-protein interaction is essentially normal, (iii) G-protein-effector interactions are essentially normal for adenylate cyclase, guanylate cyclase and phosphoinositidase C. (iv) Cyclic AMP stimulates adenylate cyclase and guanylate cyclase as in wildtype. (v) Cyclic AMP inhibits phosphoinositidase C versus stimulation in wild-type, (vi) Mutant fgdC has low chemotactic activity towards cyclic AMP. (vii) Cyclic AMP does not induce expression of genes associated with the formation of cell aggregates, prespore cells and prestalk cells.

The biochemical defect of mutant fgdC is not exactly known. Genetic and biochemical evidence indicates that mutant fgdC is not defective in either the receptor or Ga2: cARl, the gene for Ga2 and the fgdC locus are on different complementation groups (Coukell et al. 1983; Klein et al. 1988). Furthermore, mutants with a defect in Ga2 or cARl are defective in all sensory transduction processes (Kesbeke et al. 1988; Klein et al. 1988), whereas cyclic AMP activates adenylate and guanylate cyclase in mutant fgdC. Basal Ins(l,4,5)P3 levels and both basal and GTP[S]-stimulated phosphoinositidase C activity are identical in fgdC and wild-type cells, excluding a defect in phosphoinositidase C. The only biochemical defect observed in mutant fgdC is a receptor-mediated inhibition of phosphoinositidase C, compared to stimulation of enzymes activity in wild-type cells. Apparently, sensory transduction from receptor to phosphoinositidase C contains an activation/inhibition switch between the receptor and the G-protein, which is malfunctioning in mutant fgdC.

The present results with mutant fgdC imply that activation of guanylate cyclase can occur in the ahsense of stimulation of phosphoinositidase C. This conclusion is in conflict with the hypothesis that guanylate cyclase is stimulated by the sequential pathway consisting of surface receptor, phosphoinositidase C, Ins(l,4,5)P3 and cytosolic Ca2+ (Europe-Finner and Newell, 1985). The present observations also imply that activation of phosphoinositidase C is a not required for the activation of adenylate cyclase as we suggested previously (Snaar-Jagalska et al. 1988b).

The defect in signal transduction in mutant fgdC is associated with the absence of cyclic AMP-induced prespore and prestalk gene expression, and aberrant differentiation and chemotaxis. These results suggest that the activation of phosphoinositidase C is an early response required for cyclic AMP-induced differentiation in Dictyostelium. This view is supported by the observations that lithium inhibits prespore gene expression, possibly by inhibiting the activation of phosphoinositidase C (Peters et al. 1989), and that certain combinations of Ins(l,4,5)P3 and diacylglycerol induce prespore gene expression in per-meabilized Dictyostelium cells (Ginsburg and Kimmel, 1989). However, we cannot exclude the possibility that the blockade of all early differentiation represents the primary defect of mutant fgdC, and that the aberrant regulation of phosphoinositidase C is the consequence of this developmental defect. Nevertheless, mutant fgdC is the first Dictyostelium mutant that is specifically defective in the phosphoinositidase C pathway with no discernible defects in the adenylate and guanylate cyclase pathways. Mutant synagl is specifically defective in the activation of adenylate cyclase, whereas mutant stmF shows specifically alteration of the cyclic GMP response. The combined use of these mutants should be very useful in studying the role of each second messenger in complex regulatory processes.

We gratefully thank Barry Coukell for mutant strains, Peter Devreotes for anti-receptor antiserum, and Maureen Pupillo for probing Northern blot with G<r cDNA clones. This work was supported by the NWO Council for Medical Research and by the Pieter Langenhuizen Fund.

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