Ziegler et al. : Control of GTP cyclohydrolase I activity during aggregation of Dicryostelium discoideum

Pteridines Vol. 6, 1995, pp. 116-118

Short Communication

Control of GTP Cyc1ohydrolase I Activity during Aggregation of Dictyostelium discoideum

Irmgard Ziegler§ and Markus Gutlich

GSF-Institut fur Klin.Molekularbiologie und Tumorgenetik, Munchen

(Received June 15, 1995)

Introduction dihydroxypropyl)-pterin (L-biopterin) (7) suggested that as in higher eukaryotes a de novo pteridine Upon nutrient depletion, Dictyostelium discoideum synthesis starts from GTP also in Dictyostelium. In amoeba cease growth and a rapid developmental this study we analyzed the regulation of GTP cy­ program is intiated which culminates in the for­ clohydrolase I (GTP-CH) activity and of tetrahydro­ mation of a fruiting body. Its first stage consists of dictyopterin (DH4) levels during cAMP directed the aggregation on -lOS individual amoeba to form aggregation of Dictyostelium discoideum. a multicellular mound. This aggregation is regulated by cyclic AMP. The cAMP signal is detected by Materials and Methods. surface receptors and is transduced by G proteins to effector enzymes, e.g. adenylyl cyclase. The re­ Growth of the vegetative cells and induction of leased cAMP in turn, can activate receptors on the morphogenesis was performed as described in (9). same or on adjacent cells, thus relaying the signal Preparation of membranes and G protein activation (for review see 3,4). The trimeric G protein linked was done according to (2). The activity of GTP-CH signal transduction system in the cellular slime was determined after iodine oxidation of the reac­ mold Ditcyostelium is highly homologous to that tion product to neopterin phosphates. They were used by higher eukaryotes and may serve as a model directly analyzed by ion-pair HPLC as well as after system (for review see 4,11). treatment with phosphatase by reverse-phase HPLC. In vegetatively growing Dictyostelium amoeba, fol­ DH4, DH2 and dictyopterin respectively, were de­ ate is an essential vitamin. Secreted by bacteria, it termined after acidic/alkaline iodine oxidation by serves as a chemoattractant and acts as a food-seek­ reverse-phase HPLC. The methods are outlined in ing device (8). A deaminase that uses folic acid as more detail in (13). RNA isolation and Northern well as pterin as substrate occurs as extracellular blot analysis of GTP-CH mRNA are described in and membrane bound forms and may serve to (6). cDNA probes specific for Dictyostelium GTP-CH steepen the gradient for chemotaxis (12). Fluores­ were obtained by RT-PCR using primers from the cent substances in the culture medium were clas­ highly conserved region (6). sified with pteridines and lumazines and were con­ sidered as folate decomposition products (10). The Results and Discussion identification of dictyopterin (D-threo-l,2-dihydro­ xypropyl)-pterin, a new isomer of (L-erythro-I,2- The induction of morphogenesis by starvation of the cells initiates a transient release of dictyopterin § Author to whom correspondence should be addressed. into the medium (Figure IA). Their detection needs Dr.Irmgard Ziegler, Institut fur Klinische Molekularbiologie denaturating conditions. Oxidation under alkline und Tumorgenetik, Marchioninistr 25, D-8l377 Munchen instead of acidic conditions degrades the pteridine

Pteridines / Vol. 6 / No.3 Ziegler et al. : Control of GTP cyclohydrolase I activity during aggregation of Dictyostelium discoideum I17

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control +GTP-S + cAMP +ATP-S O;------______~, Figure 2. In vitro activation of GTP cyclohydrolase I in mem­ branes of Dictyostelium discoideum. n for control cells =6. Membranes were pre-incubated with GTP-yS (20 J.1M) (n= 8; P= < 0.05) or ATP-yS (20 11M) plus cAMP (20 11M) (n=4; P= <0.05). The endogenous concentration of GDP was sufficient for supply with GTP-yS by recepotor stimulated nucleoside diphosphate kinase. Additional GDP was inhi­ bitory for the subsequent determination of GTP cyclohy­ drolase 1. No stimulation occurs when Mg++ (5 f..lM) is om­ itted during pre-incubation. Mg++ was chelated with EDTA before GTP-CH activity was assayed.

o 1 2 3 " 5 6 7 8 9 10 time (hI Figure LA : Time course of DH. levels in the medium du­ active in the plasma fraction for 6-7h (spec. activity :ing starvation induced aggregation of Dictyostelium disco i­ 50-65 pmol mg- 1min-1). Then it decreases to levels Jeum. .; Control cells, .; cells cultivated with 2,4-di­ averaging 15 pmol mg- 1min- 1 after IOh. The DH. amino-6-hydroxypyrimidine (3,3 mM). B: Time course of concentrations in the plasma fraction correlate with GTP cyclohydrolase I activity in the membrane fraction of this decline. They decrease from 450 pmol mg- 1 at Dictyostelium discoideum during starvation induced aggrega­ 4 to 80 pmol mg- 1 after 10h. The activities of 6- tion pyruvoyl-H.pterin synthase and of sepiapterin re­ ductase remain unchanged (data not shown). The into non-fluorescent products. This indictaes that epimerisation reaction which results in the D-threo DH. is bound to protein in the culture medium and instead of the L-erythro isomer, was not addressed that it exists in the tetrahydro form. Inhibition of in this study. GTP-CH activity strongly reduces the release of The data show that the steady state mRNA levels DH. (Figure lA) and points to a de novo synthesis specific for GTP-CH immediately decrease after from GTP. The decline of DH. levels in the medium onset of morphogenesis. The discrepancy between after a period of 4h - 5h can be explained by a mRNA levels and specific activity of GTP-CH du­ marked increase of pterin deaminase activity during ring onset of morphogenesis points to a post-trans­ the aggregation phase (1). lational regulation of the membrane associated The specific activities of GTP-CH in the mem­ enzyme fraction. brane fraction of Dictyostelium cells were followed The non-hydrolysable GTP analogue GTP-yS during the aggregation phase (Figure IB). They activates the G-proteins and subsequently leads to closely correspond to the kinetics of DH. levels in activation of target enzymes (5). Figure 2 demon­ the membrane fraction (data not shown) and to the strates that GTP loading of G protein causes in­ accumulation of DH. in the medium. creased activity of membrane associated GTP-CH. Nothern blot analysis of mRNA levels specific for Also GTP-yS which is produced from ATP-yS by Dictyostelium GTP-CH demonstrate that they steeply receptor stimulated nucleoside diphosphate kinase decline upon onset of morphogenesis. After 2h (2) enhances the activity of GTP-CH (Figure 2). only 10% of expression at 10 are left, staying fur­ From the data presented above it is to conclude ther at the same low level. The enzyme remains that the transient activation of DH. synthesis during

Pteridines / Vol. 6 / No.3 118 Ziegler et al. : Control of GTP cyclohydrolase I activity during aggregation of Dictyostelium discoideum cAMP controlled aggregation of Dictyostelium is 5. Gilman AG. G Proteins: Transducers of receptor-gen­ not caused by enhanced expression of the enzyme erated signals. Ann.Rev.Biochem. 1987; 56: 615-649. but rather is due to a post-translational activation 6. Gutlich M, Schott K, Werner Th, Bacher A, Ziegler I. of membrane associated GTP-CH. This activation Species and tissue specificity of mammalian GTP cyc­ lohydrolase I messenger RNA. Biochim.Biophys. Acta appears to be tied into the G protein-linked signal­ 1992; 1171: 133-140. ing pathway. The immediate mechanisms and the 7. Klein R, Thiery R, Tatischeff I. Dietyopterin, 6-(D­ location of GTP-CH upregulation remain to be threo-l,2-dihydroxypropyl)-pterin, a new natural isomer defined. of L-biopterin. Isolation from vegetative cells of Dietyo­ stelium discoideum and identifiaction. Eur. J. Biochem. Acknowledgements 1990; 187: 665-669. 8. Pan P, Gall EM, Bonner JT. Folic acid as a secondary We are wery grateful to Dr. G. Gerisch and Mrs. chemotaxis substance in the cellular slime mold. Nature Ch. Heizer, MPI fur Biochemie, Martinsried, for (New BioI). 1972; 237: 181-182. 9. Snaar-Jagalska BE, van Haastert PJM. G-Protein Assays kind introduction into Dictyostelium techniques. in Dietyostelium. Methods. In: Methods in Enzymology, We thank Dr.MVeron, Institut Pasteur, Paris, for vol. 237, New York: Academic Press, New York 1994; stimulationg discussions. 387-408. 10. Tatischeff I, Klein R. Fluorescent products secreted by References Dictyostelium discoideum cells which are able to agg­ regate. FEBS Letters 1982; 138: 265-269. 1. Berrnstein RL, VAm Driel R. Control of folate deami­ II. Van Haastert PJM, Janssens PWJ, Erneux Ch. Sensory nase activity of Dictyostelium discoidcum by cyclic transduction in eudaryotes. A comparison between Die­ AMP. FEBS Letters 1980; 119: 249-253. tyostelium and vertebrate cells. Eurl.Biochem. 1991; 195: 2. Bominaar AA et al. Activation of G-proteins by recep­ 289-303. tor-stimulated nucleoside diphosphate kinase in Dietyo­ 12. Wurster B, Bek F, Butz U Folic Acid and Pterin De­ stelium. The EMBO Journal 1993; 12: 2275-2279. aminases in Dictyostelium discoideum: Kinetic Properties 3. Devereotes PN. G Protein-Linked Signaling Pathways and Regulation by Folic Acid, Pterin, And Adenosine Control the Developmental Program of Dictyostelium. 3'5'-Phophate. J. Bact. 1981; 148: 183-192. Neuron 1994; 12: 235-241. 13. Ziegler I. et al. In a Concerted Action Kit Ligand and 4. Firtel, RA, Signal transduction pathways controlling Interleukin 3 Control the Synthesis of Serotonin in Mu­ multicellular development in Dictyostelium. Trends in rine Bone Marrow-derived Mast Cells. J. BioI. Chern. Gen. 1991; 7: 381-388. 1993; 268: 12544-12551.

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