DEVELOPMENTAL

BIOLOGY

145,110-118 (1991)

involvement of Cyclic AMP Cell Surface Receptors and G-Proteins in Signal Transduction during Slug Migration of Dictyostelium discoideum PAUL W. SCHENK,~ASKIA Cell Biology

and Genetics

Unit,

Zoological

VANES, Luboratory,

FANJAKESBEKE, Leiden

Accepted

U~riversity,

January

ANDB.EWASNAAR-JAGALSKA Kuiserstraat

63, Bll

GP Leiden,

The Netherlands

2.9, 1991

The presence of G-proteins, interacting with CAMP surface receptors, was investigated in vegetative cells, aggregation-competent cells, and migrating slugs of Dictyostelium discoideum. Our results indicate that G-proteins are present in all stages. In vegetative cells there is a limited number of CAMP receptors but no effect of GTPTS on CAMP binding could be detected; in addition, no effect of CAMP on GTPTS binding or GTPase activity was observed. In both aggregation-competent cells and slugs GTPTS inhibits CAMP binding, while CAMP stimulates GTPTS binding and high-affinity GTPase. Since the presence of G-proteins coupled to CAMP receptors could be demonstrated in slugs, the involvement of the effector enzymes adenylate cyclase and phospholipase C was investigated. The results show that adenylate cyclase activity is stimulated by GTPrS in both stages and that in cells from migrating slugs the Ins(1,4,5)P, production is increased upon stimulation with CAMP. The possible involvement of G-proteins in signal transduction during the slug 8~cj1991 Academic PESS, IN stage of D. discoideunc is discussed. INTRODUCTION

Single cells of the slime mold Dictyostelium discoideum live in the soil, where they feed on bacteria. The cells grow and divide as long as food is available. Upon starvation D. discoideum cells form multicellular aggregates by means of chemotaxis. The chemoattractant is CAMP (Konijn et al., 1967), which is secreted by the cells in a pulsatile manner and is detected by cell surface CAMP receptors (Devreotes, 1983). CAMP oscillations control the aggregation process. An aggregate differentiates into a migrating slug or pseudoplasmodium, which develops into a fruiting body with stalk and spore cells (Gerisch, 1987). In postaggregative stages CAMP acts as a morphogen, inducing cell type-specific gene expression (Kay, 1982; Mehdy and Firtel, 1985; Schaap and Van Driel, 1985; Wang et al., 1988). The CAMP-receptor interaction induces a number of intracellular responses, including the activation of adenylate cyclase, guanylate cyclase, and phospholipase C (Janssens and Van Haastert, 1987; Europe-Finner and Newell, 1987a). During D. discoideum development, the levels of [3H]cAMP binding to intact cells and immunologically detectable CAMP receptor protein rise to a maximum of lo- to 20-fold over those in the vegetative stage during aggregation and then decline to less than 20% of the maximum (Klein et al., 1987). The level of [3H]cAMP binding to the cell surface was found to be as low as 20% during the slug stage, to increase significantly at the onset of fruiting body formation, and to decrease again when the fruiting body is mature (Schaap and Spek, 1984). D. discoideum CAMP receptor 0012-1606/91 Copyright All rights

$3.00

c’ 1991 by Academic Press, Inc. of reproduction in any form reserved.

110

cDNA has been obtained and the amino acid sequence has been deduced. The receptor structure appears to resemble that of a family of receptors which bear seven transmembrane domains and which interact with Gproteins (Klein et al., 1988). Several lines of evidence suggest that surface CAMP receptors are coupled to intracellular effecters via Gproteins in aggregation-competent cells of D. discoideum. It has been shown that CAMP binding to isolated membranes is modulated by guanine nucleotides (Van Haastert, 1984). Alternatively, CAMP increases binding of [3H]GTP and [35S]GTP~S’ to membranes (De Wit and Snaar-Jagalska, 1985; Snaar-Jagalska et al., 1988a). CAMP also stimulates high-affinity GTPase activity in membranes (Snaar-Jagalska et al., 1988b). Moreover, adenylate cyclase is stimulated or inhibited by guanine nucleotides, depending on the conditions used (Theibert and Devreotes, 1986; Van Haastert et ah, 1987). The signal transduction from receptors to adenylate cyclase is affected in vitro and in vivo by treatment of aggregation-competent cells with pertussis toxin (Van Haastert et al., 1987; Snaar-Jagalska and Van Haastert, 1990). Finally, guanine nucleotides have been shown to stimulate

r Abbreviations used: ATPTS, adenosine 5’.0-(3-thiotriphosphate); CABPl, CAMP binding protein; CAK, CAMP-dependent protein kinase; 8-Br-CAMP, X-bromoadenosine 3’:5’-monophosphate; 6-UPuRMP, 6-chloropurineriboside 3’5.monophosphate; ~-H-CAMP, 2’deoxyadenosine 3’:5’-monophosphate; DTT, dithiothreitol; GTPTS, guanosine Y-O-(3.thiotriphosphate); Hepes, N-2-hydroxyethylpiperazine-W-2-ethanesulfonic acid; Ins(1,4,5)P,, inositol 1,4,5-trisphosphate; PB, 10 mM KH,PO,/Na,HPO,, pH 6.5.

SCHENKETAL.

cAMP

Receptors

and G-Proteins

Ins(I,4,5)P, formation in permeabilized cells (EuropeFinner and Newell, 1987b). Until now, direct evidence for the coupling of G-proteins to guanylate cyclase could not be found (e.g., Janssens et al., 1989). Genes for two G-protein a-subunits (designated G,l and G,2), as well as one P-subunit, have been cloned from D. discoideum (Pupillo et ah, 1989; Kumagai et al., 1989; Johnson et ak, 1989). The present data in mutant AqdA of D. discoideum suggest that G,2 is associated with the CAMP receptors that activate phospholipase C and is essential for nearly all CAMP-induced signal transduction pathways in D. discoideum (Kumagai et al., 1989; Kesbeke et al., 1988; Snaar-Jagalska ef al., 1988c). The 38 kDa protein G,l was found to be present at detectable levels in vegetative cells, during aggregation, and in loose aggregates. The 40 kDa protein G,2 is expressed at very low levels in vegetative cells and is maximally present during aggregation (Kumagai et al., 1989). Simultaneously with maximal expression of CAMP receptors and G-proteins during aggregation, CAMP-induced cGMP and CAMP responses are maximal in aggregation-competent cells (Kesbeke et al., 1986). In the present study we investigated the extent to which CAMP cell surface receptors and G-proteins are possibly involved in biochemical pathways in vegetative cells, aggregation-competent cells, and slugs of D. discoideum. Where evidence for the presence of interacting receptors and G-proteins could be obtained, the extent to which adenylate cyclase and phospholipase C are possibly activated via CAMP receptors and/or G-proteins was also investigated. The results indicate that G-proteins are present in all stages considered; they interact with CAMP receptors in both aggregation-competent cells and pseudoplasmodia. MATERIALS AND METHODS Materials [5’,8-3H]cAMP (1.65 TBq/mmole) and the Ins( 1,4,5)P, assay kit (TRK 1000) were obtained from Amersham. [35S]GTP7S (50.0 TBq/mmole) and [T-~~P]GTP (1.30 TBq/mmole) were purchased from New England Nuclear. CAMP, CAMP derivatives, DTT, and purified BSA were obtained from Sigma. Cellulase (from Trichoderma viride), ATP, adenosine 5’-(2,3-imido)triphosphate, ATPTS, GTP, GTPTS, creatine phosphate (Trissalt), and creatine kinase were from Boehringer. Czdfu re Conditions D. discoideum NC-4 cells were grown in association with Esch,erichia Coli 281 as described (Van Haastert and Van der Heijden, 1983), harvested with cold PB, and

in Cells c$L? discoideu

rn

111

washed three times. One part of these cells was used immediately as vegetative cells, whereas other cells were used to obtain developmental stages. Aggregationcompetent cells were obtained by 16 hr of incubation at 6°C on nonnutrient agar (1.5% agar in PB) at a density of 3.5 x lo6 cells/cm2 or by 5 hr of starvation at 22°C in shaking suspension in PB at a density of lo7 cells/ml. These cells were harvested or collected by centrifugation and washed twice with PB. Migrating slugs were obtained by 16 hr of incubation at 22°C on nonnutrient agar at a density of 6.5 x lo6 cells/cm2. The pseudoplasmodia were rinsed from the plates with PB and transferred to a nylon sieve (pore size 20 pm), to remove single cells. The multicellular structures (at a density of 10’ cell equivalents/ml PB) were dissociated into single cells by treatment with 50 mg/ml cellulase. Cellulase was removed by washing three times with PB. Membrane Isolation Cell pellets (150g) were resuspended in 40 mM Hepes/ NaOH, 0.5 mM EDTA, 250 mM sucrose, pH 7.7, to a density of 10’ or 2 X 10’ cells/ml. Homogenization was performed by pressing the cell suspension through a Nuclepore filter (pore size 3 pm) at 0°C. The lysate was centrifuged at 10,OOOgand 4°C for 3 min; if not otherwise indicated, the pellet was washed once in PB and finally resuspended in this buffer to a density of lo8 or 2 X 10’ cell equivalents/ml. CAMP Binding Assay The association of [3H]cAMP to D. discoideum membranes was detected in a total volume of 100 ~1, containing 4 nM [3H]cAMP, 5 mM DTT, PB, and 50 ~1 membranes (final density 10’ cell equivalents/ml). Binding was measured after a 5-min incubation at 0°C. Samples were centrifuged at 10,OOOgand 4°C for 3 min; the supernatant was aspirated and the pellet dissolved in 80 ~1 1% SDS; 1.1 ml scintillation liquid was added and radioactivity was determined. Nonspecific binding was determined in the presence of 0.1 mM unlabeled CAMP and subtracted from all data shown. Inhibition oz [‘HIcAMP Derizat ives

Binding by CAMP and CAMP

The inhibition of [3H]cAMP binding to membranes by unlabeled CAMP and three of its analogues was determined in a volume of 100 ~1, containing 20 nM[3H]cAMP, varying concentration of CAMP or CAMP derivative, 5 mMDTT, PB, and 80 ~1membranes (final density 8 X lo7 cell equivalents/ml). Binding was measured as described under CAMP Binding Assay. Nonspecific binding was determined in the presence of 1 mM unlabeled

112

DEVELOPMENTALBIOLOGY

CAMP and subtracted from the data shown. The derivatives used were 6-Cl-PuRMP, 8-Br-CAMP, and Z-HCAMP. GTPrS Binding Assay

The binding of [35S]GTP7S to D. discoideum membranes was detected in a total volume of 100 ~1, containing 0.2 nM[35S]GTPTS, 3 mM MgCl,, 1 mM ATP, PB, and 50 ~1 membranes (final density lo8 cell equivalents/ml). Association was measured after a 30-min incubation at 0°C. Samples were centrifuged for 3 min at 10,OOOgand 4°C; the supernatant was aspirated. The pellet was dissolved in 80 ~1 1% SDS; 1.1 ml scintillation liquid was added and radioactivity was determined. Nonspecific binding was measured in the presence of 0.1 mM GTP and subtracted from all data shown. Measurement of High-Afinity

GTPase Activity

Crude membranes were washed and resuspended in 10 mMtriethanolamine/HCl, pH 7.4, to a density of 10’ cell equivalents/ml and GTPase activity was measured (Snaar-Jagalska et ah, 1988b). The reaction mixture was preincubated at 25°C for 5 min and contained 3.7 kBq [T-~‘P]GTP, 2 mM MgCl,, 0.1 mM EGTA, 0.2 mM adenosine 5’-(2,3-imido)triphosphate, 0.1 mM ATPTS, 10 mM DTT, 5 mM creatine phosphate, 0.4 mg/ml creatine kinase, and 2 mg/ml BSA in 50 mM triethanolamine/HCl, pH 7.4, in a total volume of 100 ~1. The reaction was started by the addition of 30 ~1 membranes to 70 ~1 reaction mixture and conducted for 3 min. The reaction was terminated by the addition of 0.5 ml of 50 mMphosphate buffer, pH 2.0, containing 5% (w/v) activated charcoal. Samples were centrifuged at 10,OOOg and 4°C for 5 min; radioactivity of the supernatants was determined using Cerenkov radiation.

V0~~~~145.1991

bation at 20°C the reaction was terminated by the addition of 10 ~1 of 0.1 M EDTA, pH 8.0, and immediate boiling of the samples for 2 min. Produced CAMP was determined by using the isotope dilution assay. Stimulation by 30 pM GTPTS or 50 pM2’-H-CAMP was measured by including these compounds during lysis (Theibert and Devreotes, 1986). Protein Determination

Protein concentrations were determined the method of Bradford (1976).

according to

Determination of Ins(l,.&5)P, Formation

After cellulase treatment, 150g slug cell pellets were resuspended in PB to a density of lo8 cells/ml. Air was bubbled through the suspension at a rate of about 15 ml air. mini’ . ml cell suspension-‘. After lo-30 min 900 ~1 of the cell suspension was added to either 100 ~1 of 10 pM CAMP (stimulation) or 100 ~1 H,O (control). At the times indicated 100 ~1 of the final suspension was added to 100 ~1 of 3.5% perchloric acid. After a 15-min incubation at 0°C and storage at -2O”C, the lysates were neutralized with 50 ~1 KHCO, (50% of a saturated solution at 22°C); CO, was allowed to evaporate and samples were centrifuged at 10,OOOg and 4°C for 2 min. Ins(1,4,5)P, levels were determined in the supernatants with the isotope dilution assay as described by Van Haastert (1989). RESULTS

To study the coupling between CAMP surface receptors and G-proteins in three different developmental stages of D. discoideum we measured GTPTS-induced inhibition of CAMP binding to cell surface receptors and CAMP-stimulated GTP7S binding and high-affinity GTPase activity.

Measurement of Adenylate Cyclase Activity

Adenylate cyclase activity was measured in lysates of aggregation-competent cells and cells obtained from migrating slugs. Cells were collected by centrifugation, resuspended in PB, adjusted to a density of 1.5 X 10’ cells/ml, and kept at 0°C. Lysis was performed according to Theibert and Devreotes (1986). Four hundred microliters of cells was mixed with 400 ~1 2~ lysis buffer (20 mM Tris, pH 8.0, 4 mM MgCl,) at 0°C and immediately lysed by pressing them through two Nuclepore filters with 3-pm pores. The lysate was collected in a tube at 0°C and kept on ice for 5 min. Enzyme activity was measured, using the method of Van Haastert et al. (1987). Twenty microliters of lysate was incubated with 10 mM Tris, pH 8.0, 2 mM MgCl,, 0.5 mM ATP, and 10 mM DTT in a total volume of 40 ~1. After 5 min of incu-

CAMP Binding and Its Modulation by GTPrS

GTPrS

affects CAMP binding to membranes of D. disthe affinity of CAMP cell surface receptors. At binding equilibrium, this results in a 70 to 80% inhibition of CAMP binding (Van Haastert, 1984). We compared the effects of GTPTS on CAMP binding to membranes of vegetative, aggregation-competent, and slug cells. Figure 1 shows that basal binding activity is optimal in aggregation-competent cells and is inhibited at least 85% by GTP7S. Basal binding in vegetative cells is less than 10% of that in aggregation-competent cells and is not affected by GTP7S. In slugs considerable basal binding levels are found, which are only 40% reduced compared to those in aggregation-competent cells; GTPTS causes &75% inhibition of binding (see also coideum by reducing

a0 00 FIG. 1. r3H]cAMP binding to membranes from D. discoideum vegetative cells, aggregation-competent cells, and pseudoplasmodia in the absence (black bars) or presence (prey bars) of 0.1 mM GTPTS. Data shown are the means and SDS of six (slugs) or seven (other stages) independent experiments, carried out in triplicate.

Table 1). The results indicate that in aggregation-competent cells and slugs CAMP binding proteins are present which interact with G-proteins. Speci3city of CAMP Binding in Membrane Fractions

Activity

It was previously found that surface CAMP binding activity in slugs was only 20% of binding activity in aggregation-competent cells. Figure 1 shows that binding to slug cell membranes is more than 60% of binding to aggregation-competent cell membranes. One reason for this discrepancy could be a contamination of our membrane preparation with the intracellular proteins CABPl or CAK (Tsang and Tasaka, 1986; Leichtling et ub, 1982) or CAMP-PDE. Cell surface receptors can be distinguished from CABP, CAK, and PDE by different

MODULATION

OF [3H]cAMP

BINDING,

nucleotide specificity (Van Ments-Cohen and Van Haastert, 1989). 6-Cl-PuRMP and 8-Br-CAMP bind to CAK and CABPl with a higher specificity than they bind to the cell surface CAMP receptors; ~-H-CAMP binds more specifically to surface receptors than it binds to CAK and CABPl. D, discoideum membranes from aggregation-competent cells and slugs were incubated for 5 min at 0°C with 20 nM [3H]cAMP in the presence of varying concentrations of CAMP or one of its derivatives 6-Cl-PuRMP, 8-Br-CAMP, or 2’-H-CAMP. Figure 2 shows the inhibition of [3H]cAMP binding to membranes of slug and aggregation-competent cells by CAMP and the analogues. CAMP and 2’-H-CAMP inhibit [3H]cAMP binding to membranes in a monophasic way which is similar for both stages. CAMP inhibits binding about lo-fold more specifically than 2’-H-CAMP does (Van Ments-Cohen and Van Haastert, 1989; Van Ments-Cohen, 1990). 6-ClPuRMP and 8-Br-CAMP inhibit [3H]cAMP binding to both membranes in a biphasic way (with K,,,l and K,,Z), indicating the involvement of two different populations of CAMP binding proteins. With respect to the binding specificities, competition for CAK and CABPl mainly takes place in the nanomolar &,J concentration ranges of derivative, and competition for the cell surface receptor sites takes place in the micromolar and millimolar K,,52 concentration ranges of analogue. The results of these binding studies show that about 70% of CAMP binding to membranes from aggregation-competent cells is due to cell surface CAMP receptors. The estimated contribution of surface receptor binding in CAMP binding to membranes from pseudoplasmodia is about 80% in this assay. To prevent r3H]cAMP binding to intracellular CAMP binding proteins, 10 nM 6-Cl-PuRMP was included in

TABLE 1 [%]GTPTS BINDING,

AND HIGH-AFFINITY Modulation

GTPTS inhibition binding GTPTS inhibition binding (+lO nM CAMP stimulation binding CAMP stimulation GTPase activity

GTPASE

ACTIVITY

(%’ to basal)

Vegetative

Aggregation

Slugs

33 k 55 (7)

87+-

2 (7)*

76 i

18 (6)*

93 f

5 (5)*

7x t

g(5)*

of [3H]cAMP of [3H]cAMP 6-Cl-PuRMP) of [‘%]GTPTS 17 f 26 (4)

‘71 f 65 (5)*

50 + 31(5)*

3 i 20 (3)

41 * 22 (3)*

26 k 15 (3)*

of high-affinity

Nofe. Data shown are the means ? SDS of (n) independent experiments. These experiments Materials and Methods. Inhibition was calculated as (1 ~ modulated binding/unmodulated (modulated binding/unmodulated binding - 1) x 100% * The modulation is significant compared to basal (P < 0.05, T test).

were performed in triplicate binding) X 100%. Stimulation

as described under was calculated as

114

DEVELOPMENTAL

BIOLOGY

VOLUME

145, 1991

cells and 65% for migrating slugs (data not shown). GTPTS inhibition of CAMP binding to membranes from both stages seemsto be slightly stronger in the presence of 10 nil! 6-Cl-PuRMP (Table 1). It therefore appears that the relatively high level of CAMP binding sites in slugs represents surface CAMP receptors, which are modulated by G-proteins. 60

GTPrS Binding

B

0

60

60

40

20

I,

0

10-g

10-e cyclic

10-7 nucleotide

10-6 concentration

10-s

by CAMP

GTPTS binding activity to membranes of aggregation-competent cells is heterogeneous and composed of high- and low-affinity components. In the presence of CAMP, GTPTS binding was enhanced as the result of an increase in affinity and the number of high-affinity binding sites (Snaar-Jagalska et al., 1988a). The effect of CAMP on GTPTS binding was measured in different stages of development and the results are presented in Fig. 3. Specific GTPKS binding to membranes of vegetative cells and aggregation-competent cells is not different, while binding to membranes of migrating slugs is two- to threefold higher. CAMP significantly stimulates the binding of [35S]GTP& to membranes from aggregation-competent cells and pseudoplasmodia. In vegetative cells CAMP has no significant effect on GTPrS binding to membranes (Table 1). This suggests that G-proteins are present in all stages considered, but interact only with CAMP receptors in aggregation-competent cells and migrating slugs. High-afinity by CAMP

0

and Its Stimulation

GTPase Activity

and Its Stimulation

104

(M)

FIG. 2. Inhibition of the binding of [3H]cAMP to D. discoideum metnbranes by CAMP and CAMP derivatives. Membranes were from aggregation-competent cells (A) or migrating slugs (B). The binding of 20 nM [3H]cAMP in the absence of CAMP or its derivatives was set at 100% in each experiment. [aH]cAMP binding was determined in the presence of varying concentrations of CAMP (O), 6-Cl-PuRMP (a), 8-Br-CAMP (A), and ~-H-CAMP (0). The biphasic appearance of the 6-Cl-PuRMP and 8-Br-CAMP curves is not due to averaging of the data, but found in each separate experiment. Data shown are the means of three (A) or four (B) independent experiments, carried out in triplicate. K,, is the concentration of derivative that results in a 50% inhibition of [3H]cAMP binding. (A) J&s CAMP = 0.02 PM, K,,J 6-ClPuRMP = 0.6 nM; Z&,2 6-Cl-PuRMP = 0.03 mM, K,,,l 8-Br-CAMP = 1 nM; &,2 8-Br-CAMP = 8 PM, Ko.5 ~-H-CAMP = 0.6 ).&f. (B) K,, CAMP = 0.02 &$ K,,J 6-Cl-PuRMP = 0.4 nM, K,,2 6-Cl-PuRMP = 0.04 mM; K,,l 8-Br-CAMP = 3 nM; K,,,2 8Br-CAMP = 6 PM, Ko,5 ~-H-CAMP = 2 pilf.

the CAMP binding assay to estimate CAMP binding and its modulation by GTPTS, as described above. The contribution of receptor binding to specific [3H]cAMP association was found to be 73% for aggregation-competent

G-proteins have GTPase activity that can be stimulated by receptor agonists. In membranes of aggregation-competent cells, the high-affinity GTPase is stimu-

-+

0,35

k K

0.30

r

0.25

0 E Q

0,20

5 D z

0.15 O,lO

F 0

0,05

" ?L

0,oo

FIG. 3. [?S]GTPTS binding to membranes from D. discddeum vegetative cells, aggregation-competent cells, and migrating slugs in the absence (open bars) or presence (solid bars) of 0.1 mM CAMP. Data shown are the means and SDS of four (vegetative cells) or five (other stages) independent experiments, carried out in triplicate.

crease in enzyme activity is also shown by the addition of GTPLS and 2’-H-CAMP together. Stimulation of activity by ~-H-CAMP alone is not significant. These results suggest that possibly a G-protein is involved in the adenylate cyclase signal transduction pathway during slug migration. A role for CAMP receptors in this process cannot be excluded.

Stimulation

FIG. 4. High-affinity GTPase activity in membranes from D. discoi&,?Ltt/ vegetative cells, aggregation-competent cells, and slugs. GTP hpdrolysis by high-affinity GTPase was determined in the absence (open bars) or presence (solid bars) of 10 PM cAMP at a GTP concentration of 10 nM GTP; hydrolysis bg low-affinity GTPase was determined in the presence of 100 fitM GTP. High-affinity is defined as the difference between total GTPase and low-affinity GTPase. Data shown are the means and SDS of three independent experiments with triplicate determinations.

lated by CAMP, with half-maximal effects at a CAMP et al., 1988b). concentration of 3 PM (Snaar-Jagalska High-affinity GTPase activity and its stimulation by CAMP were measured in membranes of aggregationcompetent, slug, and vegetative cells. Figure 4 shows that high-affinity GTPase activity in aggregation-competent cells and migrating slugs is, respectively, 1.5- and 3-fold higher than in vegetative cells. CAMP significantly stimulates high-affinity GTPase activity in membranes from aggregation-competent cells and slugs (Table 1). Presumably aggregation-competent cells and pseudoplasmodia contain G-proteins, with GTPase activity which can be stimulated via CAMP receptors.

Regulution of Adenylate $-H-CAMP

Cyclase Activity

by GTPrS

and

It was previously shown that in aggregation-competent cell lysates adenylate cyclase is modulated by GTPTS and/or CAMP (Theibert and Devreotes, 1986; et al., 1987). Van Haastert In the present study we investigated the possible interaction between the effector enzyme adenylate cyclase and G-proteins and/or CAMP receptors, in different stages of development. In vegetative cells basal activity is very low and no detectable stimulation by GTPTS is observed. In Table 2 results are shown of adenylate cyclase measurements in lysates of aggregation-competent cells and cells from migrating slugs. As found before, in aggregation-competent cells both GTPTS and ~-H-CAMP induce a significant stimulation of enzyme activity. In migrating slugs a small, but significant activation of adenylate cyclase by GTPrS is found; an in-

of Ins(l,i,5)P,,

Production

by cAMP

It was recently shown that in intact aggregation-comrise in petent cells 1 pM CAMP induces a transient Ins(1,4,5)P, levels, with a maximum at about 6 see after stimulation (Van Haastert, 1989). We measured Ins(1,4,5)P, production in intact slug cells in the presence or absence of 1 pMcAMP. As shown in Fig. 5, Ins(1,4,5)P, production is stimulated by CAMP in cells of migrating slugs. Addition of CAMP induces at least one transient rise of the Ins(1,4,5)P, levels, compared to the levels of control (H,O). The maximal Ins(1,4,5)P, level is reached at about 12 see after stimulation. In some experiments a second transient rise of the Ins(1,4,5)P, levels was found, with a maximum at about 30-40 see after stimulation. The Ins(1,4,5)P, control levels strongly varied between the individual experiments, probably due to slight differences in progress of development. The results suggest an activation of phospholipase C by CAMP-receptor interactions during the slug stage. DISCIJSSION

The number of D. discoideum CAMP surface receptors has been found to reach a maximum during aggregation (Schaap and Spek, 1984; Klein et al, 1987). Therefore, up till now possible interactions between CAMP receptors, putative G-proteins, and effector enzymes were investi-

REGULATION

OF ADENYLATE

TABLE 2 CYCLASE BY GTPTS

Ratio

+ GTPiS aggregation slugs

8.02 i- 2.19* 1.32 * 0.27*

(fraction

of basal

+ ~-H-CAMP 4.14 k 1.17* 1.20 f 0.34

AND ~-H-CAMP activity) + GTPTS + ~-H-CAMP 13.62 i X98* 1.61 ?I 0.49*

Arote. Experiments were performed as described under Materials and Methods. GTPTS and Z-H-CAMP were present at concentrations of 30 and 50 PM, respectively. Data shown are means + SDS from (11) independent experiments with triplicate determinations. Basal activities in aggregation-competent cells and slugs are 3.71 * 0.66 and 4.80 + 0.98 pmoles . min-’ . mg protein ‘. * The difference is significant compared to basal activity (P < 6.05; tested according to Z’test).

116

DEVELOPMENTAL 200

BIOLOGY

T

“Ib 0

40

50

seconds

FIG. 5. CAMP stimulation of Ins(1,4,5)P, formation in D. discoideum slug cells. At 0 set 900 ~1 cell suspension was added to either 100 ~1 of 10 PM CAMP or 100 ~1 H,O. Ins(1,4,5)P, was extracted from 100 ~1 incubation mixture at 4,8,12,20,30,40, and 50 sec. CAMP-stimulated Ins(1,4,5)P, formation was calculated for each experiment as a percentage of control levels at the indicated time points. Data shown are the means and SDS of five independent experiments. The control levels varied in the different experiments from 161 to 1387 pmoles/lO’ cells. Ins(1,4,5)P, formation at 12, 20, and 30 set significantly differs from Ins(1,4,5)P, formation at 4 set, which is 100 + 18% (P < 0.05; 2’ test).

gated in aggregation-competent cells (Van Haastert, 1984; De Wit and Snaar-Jagalska, 1985; Snaar-Jagalska et al., 1988a,b; Theibert and Devreotes, 1986; Van Haastert et al., 1987; Europe-Finner and Newell, 198’7a,b). Recent results indicate that several D. discoideum signal transduction components appear in multiple forms, which are probably regulated differently during development. The G-protein a-subunits G,l and G,2 are expressed in distinct developmental patterns (Kumagai et al., 1989). Both G,l and G,2 cDNA hybridize to multiple mRNAs, which show variable developmental expression (Pupillo et al., 1989). D. discoideum contains two ras genes (Ddras and DdrasG), which code for a guanine nucleotide binding protein. Ddras is maximally expressed during slug migration; DdrasG is only present during growth and early development (Reymond et al., 1986; Robbins et al, 1989). A CAMP receptor gene has been cloned from D. discoideum by Klein et al. (1988). Recently, low stringency hybridization led to the cloning of two additional CAMP receptor genes with strong homology in the putative transmembrane domains (P. N. Devreotes, C. L. Saxe, III, and A. R. Kimmel, personal communication). The distinct receptor genes may be the genetic basis for the presence of the receptor subtypes A, B, and C (Van Ments-Cohen, 1990) and could

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possibly be differentially expressed during development. In the present study we compared the possible interactions between CAMP receptors, G-proteins, and potential effecters in vegetative cells, aggregation-competent cells, and migrating slugs of D. discoideum. The major findings are: (1) [3H]cAMP binding to membranes from vegetative cells and slugs is, respectively, 6 and 61% of the binding to membranes from aggregationcompetent cells; GTPTS strongly inhibits CAMP binding to membranes of aggregation-competent cells and slugs but does not affect binding in vegetative membranes. (2) Membranes from all stages show GTPTS binding activity. Surprisingly, [35S]GTP~S binding to membranes from migrating slugs is 2 to 3-fold higher than to those from vegetative cells and aggregation competent cells. In membranes from aggregation-competent cells and pseudoplasmodia GTPTS binding is strongly stimulated by 0.1 mM CAMP; however, in vegetative membranes, GTPTS binding is not CAMP stimulated. (3) High-affinity GTPase activity in membranes from aggregationcompetent cells and slugs is, respectively, 1.5- and 3-fold higher than in membranes from vegetative cells. Highaffinity GTPase in membranes of aggregation-competent cells and migrating slugs, but not in those from vegetative cells, is stimulated by 10 pM CAMP. Our data indicate that vegetative cells contain G-proteins and a small number of CAMP receptors. However, in this stage no functional coupling seems to exist between these signal transduction components. G-proteins in vegetative cells most likely function in folate signal transduction, since folate stimulated GTPTS binding and GTPase activity (Kesbeke et al., 1990). This strongly suggests that different G-proteins mediate transduction of the two chemoattractants. In aggregation-competent cells CAMP receptors interact with G-proteins as expected (Van Haastert, 1984; De Wit and Snaar-Jagalska, 1985; Snaar-Jagalska et al., 1988a,b). Previously CAMP binding to slug cell surface was found to be only 20% of the binding to aggregationcompetent cell surface (Schaap and Spek, 1984). Surprisingly, CAMP binding to slug membranes is found to be 61% of the binding to membranes from aggregationcompetent cells in this study. [3H]cAMP binding to slug membranes in the presence of 10 nM 6-Cl-PuRMP is 65% of this binding in the absence of 6-Cl-PuRMP. At this concentration the CAMP derivative largely inhibits binding to the intracellular CAMP binding proteins; therefore 65% of specific CAMP binding to slug membranes is probably due to binding to cell surface receptors. The significant inhibition of [3H]cAMP binding to slug membranes by GTP7S in the absence and presence of 10 nM 6-Cl-PuRMP suggests coupling between CAMP

surface receptors and G-proteins in slugs. The presence of G-proteins, interacting with CAMP receptors, is also indicated by the results from the [3”S]GTP~S binding assay and the high-affinity GTPase activity measurement. Only one minor G,l mRNA and two minor G,2 mRNAs are expressed in multicellular stages (Pupillo et al., 1989). So, it is not presumable that G,l and G,2 are responsible for the GTPTS binding and GTPase activity measured in slugs to a great extent; probably, a part of the guanine nucleotide binding is due to binding to the protein coded by Ddra.s and possibly other additional G-proteins are involved. Since evidence for the presence of interacting CAMP cell surface receptors and G-proteins in both aggregation-competent cells and slugs from D. discoideum could be obtained, the extent to which the effector enzyme adenylate cyclase is possibly activated by CAMP receptors via G-proteins in both stages was investigated. Also, CAMP stimulation of Ins(1,4,5)P, formation during the slug stage was determined in vivo. As expected, GTPTS and 2’-H-CAMP significantly stimulate in vitro adenylate cyclase activity in aggregation-competent cells. GTP7S also significantly activates the enzyme during the slug stage. This suggests that G-proteins are involved in the signal transduction pathway from receptors to adenylate cyclase not only in aggregation-competent cells, but also in pseudoplasmodia. It is not clear whether CAMP receptors are involved in this signal transduction in slugs. Previously, CAMP relay in intact slugs was determined to be less than 10% of the relay at the onset of aggregation (Kesbeke et al., 1986). In this study 2’-H-CAMP does not significantly stimulate in vitro adenylate cyclase activity during slug migration. This suggests that CAMP surface receptors do not contribute to a great extent to adenylate cyclase stimulation in slugs; however, both prestalk and prespore genes of the migrating slug are regulated through cell surface CAMP receptors (Mehdy and Firtel, 1985; Schaap and van Driel, 1985). These results give no indication for the involvement of the adenylate cyclase signal transduction pathway in the control of late gene expression. It was found that 1 PLMCAMP stimulates Ins(1,4,5)P, formation in aggregation-competent cells, with a maximal Ins(1,4,5)P, level at about 6 see after stimulation (Van Haastert, 1989). In this study it is observed that at least one transient rise in Ins(1,4,5)P, levels, with a maximum at about 12 see after stimulation, is induced by 1 pM CAMP in pseudoplasmodial cells. This suggests that phospholipase C is activated by CAMP-receptor interactions not only in aggregation-competent cells, but also in slugs. The Ins(1,4,5)P, that is formed may act as a second messenger in Ca2+ mobilization, guanylate cyclase stimulation, and adenylate cyclase stimulation in

the D. discoideum cells (Europe-Finner and Newell, 1985; Snaar-Jagalska et al., 1986c). Recently, it has been observed that neither class of late genes (prestalk and prespore) involves a rise in intracellular CAMP, but each is induced at high levels in permeabilized cells treated with DAG and Ins(1,4,5)P, (Ginsberg and Kimmel, 1989), which suggests that protein kinase C and/or Ca2+ may be involved in regulating the expression of these genes. The present observations implicate the functional interaction between CAMP cell surface receptors and Gproteins in the late development of D. discoideum. The function of G-protein-mediated CAMP signal transduction in D. discoideum slugs requires further investigation. We thank Dr. P. Schaap for stimulating discussion and practical suggestions and Professor T. M. Konijn for critical reading of the manuscript. REFERENCES BRADFORD, M. M. (19’76). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Ad Biochem. 72,248-254. DEVREOTES, P. N. (1983). Cyclic nucleotides and cell-cell communication in Dict~oatelium discoidrum. A&u. Cyclic Nuclsofide Res 15,5596. DE WIT, R. J. W., and SNAAR-JAGALSKA, B. E. (1985). Folate and CAMP modulate GTP binding to isolated membranes of Dictpxsfelium discoiclezcm. Functional coupling between cell surface receptors and G-proteins. Biochem. Biophys. Res. Commun. 129, 11-1’7. EUROPE-FINNER, G. N., and NEWELL, P. C. (1985). Inositol 1,4,5-trisphosphate induces cyclic GMP formation in Dicfyostelium discoideum. Biochem. Biophys. Res. Conrnmx 130, 1115-1122. EUROPE-FINNER, G. N., and NEWELL, P. C. (1987a). Cyclic AMP stimulates accumulation of inositol trisphosphate in DictUosteliu~rr,. J Cell Sci. 87, 221-229. EUROPE-FINNER, G. N., and NEWELL, P. C. (1987b). GTP analogues stimulate inositol trisphosphate formation transiently in Dictyostelium. .I Cell Sci. 87, 513-518. GERISCH, G. (1987). Cyclic AMP and other signals controlling cell development and differentiation in Dictyoatelium. Annu. Rev. Bioeh~m. 56, X53-879. GINSBURG, G., and KIMMEL, A. R. (1989). Inositol trisphosphate and diacylglycerol can differentially modulate gene expression in Dictgtxtelium

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