ATP increases chemoattractant induced cyclic GMP accumulation in Dictyostelium discoideum

408

Biochimica et Biophysica Acta, 540 (1978) 408--411 © Elsevier/North-Holland Biomedical Press

BBA 28540 ATP INCREASES CHEMOATTRACTANT INDUCED CYCLIC GMP ACCUMULATION IN DICTYOSTELIUM DISCOIDEUM

JOSE M. MATO

Laboratory of Zoology, Cell Biology and Morphogenesis Unit, Kaiserstraat 63, University of Leiden, Leiden (The Netherlands) (Received November 2nd, 1977)

Summary Changes in guanosine cyclic 3',5'-monophosphate associated with adenosine cyclic 3',5'-monophosphate and folic acid addition in the presence of ATP have been examined in Dictyostelium discoideum. Preincubation with 1 mM ATP had no effect on the basal cyclic GMP level but increased the cyclic GMP accumulation in response to cyclic AMP (5 • 10 -s M) or folic acid (5 • 10 -6 M) 40-50%. ATP could n o t be replaced by ADP or 5'-adenylyliminodiphosphate. Because ATP has no effect on cyclic AMP receptor binding these results indicate that structural membrane alterations (e.g. membrane phosphorylation) may control the transduction of a chemotactic signal.

Introduction In Dictyostelium discoideum vegetative cells are attracted by folic acid [1] and aggregative cells by cyclic AMP [2]. Based on the following observations it has been proposed that the transduction of a chemotactic signal in the cellular slime molds involves changes in the levels of cGMP: (i) all known attractants increase at physiological concentrations the cellular content of cGMP [3--8]; (ii) the increase of the cGMP c o n t e n t precedes pseudopod formation [3--5]; and (iii) the same receptor is involved in cAMP mediated chemotaxis and cAMP mediated cGMP accumulation [5,8]. While a regulatory role for cGMP during chemotaxis seems to be well established nothing is known about the molecular mechanism of transduction of a chemical signal through the plasma membrane. In the present study evidence is given, which indicates that ATP increases,

Abbreviations: App(NH)p, 5S-adenylylL-ninodiphosphate;AMPMe, 5S-adenosine m o n o p h o s p h a t e methyl ester.

409 probably by plasma m e m b r a n e p h o s p h o r y l a t i o n , cGMP accumulation in response to a c h e m o t a c t i c signal in D. discoideum. Methods

D. discoideum, NC-4(H), was used for all experiments. Cells were grown on a solid med iu m and harvested as described by Konijn and Raper [9]. After centrifugation, cells were suspended in 10 mM phosphate buffer, pH 6.0, at a density of 107 cells per ml. Starvation was induced by shaking [10]. After shaking, cells were centrifuged, washed twice in cold phosphate buffer, and adjusted to 1 • 108 cells per ml. Air was bubbled through for 15 min in the presence or absence of ATP (1 mM final c o n c e n t r a t i o n in phosphate buffer pH adjusted to 6.0). Samples of 100 gl were stimulated with 20 pl of cAMP (5 • 10 -8 M final c o n c e n t r a t i o n ) or folic acid (5 • 10-6 M final c o n c e n t r a t i o n ) at time zero. At the time indicated 200 pl ice-cold ethanol/HC1 was pipetted into the tubes and the cGMP c o n t e n t measured by radioimmunoassay as described [3,5]. The a m o u n t of ATP added to the cells did n o t show cross reactivity with the antibody. The m e t h y l ester of 5'-AMP, AMPMe, was a gift of Dr. F. Eckstein. ATP, ADP, App(NH)p, cAMP and cGMP were purchased from Boehringer. Folic acid was supplied by Sigma. ]8-3H]cGMP and a n t i b o d y were from Amersham. Results

Cells shaken for a short period behave like vegetative cells and are chemotactically sensitive to folic acid. Fig. I shows the effect of ATP on the time course of cGMP accumulation when cells shaken for 1.5 h are stimulated by folic acid. ATP has no effect on the basal level of cGMP but increased 40--50% cGMP accumulation in response to this at t ract ant (Fig. 1). As shown in Figs. 2 and 3 cGMP accumulation induced by cAMP or AMPMe was also increased in the presence o f ATP. The cells used were starved for 5 h and t herefore sensitive to these attractants. Again, ATP had no effect on the basal level of cGMP

_~ 10

1C

u

~8

%

z 6

6 4

o E "

o

E Q.

2

0

20

40

60

sec

2

o

20 40 6Osec

F i g . 1. T i m e c o u r s e o f f o l i c a c i d - d e p e n d e n t c G M P a c c u m u l a t i o n i n t h e p r e s e n c e a n d a b s e n c e o f 1 m M ATP. Cells were starved for 1,5 h, w a s h e d , suspended at a density of 1 • 10 8 cells per ml, and after 15 m i n of e q u i l i b r a t i o n i n the presence (o) or a b s e n c e (e) o f 1 m M A T P , s t i m u l a t e d w i t h 5 • 10 -6 M folic acid. F i g . 2. T i m e c o u r s e o f c G M P f o r m a t i o n a f t e r D. discoideurn c e l l s w e r e t r i g g e r e d w i t h 5 • 1 0 - 8 M c A M P i n t h e p r e s e n c e (Q) o r a b s e n c e ( e ) o f 1 m M A T P . C e l l s w e r e s t a r v e d f o r 5 h.

410

6

I(3 o

8 (D u o E f~

6 4' 2 0

20

40

60 sec

0

20

40

60

F i g . 3. A t z e r o t i m e c e l l s s t a r v e d f o r 5 h w e r e s t i m u l a t e d w i t h 1 0 - 4 M A M P M e absence ($) of 1 mM ATP and the time course of cGMP formation was followed.

sec

in the presence

(3) or

F i g . 4. E f f e c t o f d i f f e r e n t n u c l e o t i d e s o n c A M P - m e d i a t e d cGMP accumulation. Cells were starved for 5 h and stimulated a t t i m e z e r o w i t h 5 . 1 0 - 8 M c A M P in t h e p r e s e n c e o f (A) 1 m M A D P ; (~) 1 m M App(NH)p; and $) control.

of these cells. In the presence of ATP, cGMP peaks lasted for about 5 s and basal levels were recovered later; controls showed the typical spike response (Figs. 1--3). The enhancing effect of ATP on attractant mediated cGMP accumulation could not be mimiced by ADP or App(NH)p (Fig. 4). ADP and App(NH)p slightly decreased the magnitude of the cGMP peak in response to cAMP (Fig. 4). Discussion

ATP increased the magnitude of attractant mediated cGMP accumulation in D. discoideum independently of the developmental stage of the cells and with-

out affecting the basal content of cGMP. ADP and App(NH)p did not increase cGMP peaks suggesting that ATP acts via plasma membrane phosphorylation. A cAMP independent protein kinase assayable with intact cells has been described in D. discoideum [11--14] which phosphorylates certain proteins [12--14] and phospholipids (unpublished) of the plasma membrane. Whether ATP acts only extracellularly or is taken up by the cells is not yet known. ATP has no effect on cAMP binding to cell surface receptors [12,15] indicating that it affects the transduction of the signal rather than its detection. The 5'-AMP derivative, AMPMe, is chemotactically and as agonist of cAMP in inducing cGMP accumulation 100-fold less active than cAMP [3,5,16]. This derivative is resistant to phosphodiesterase from brain, beef-heart [17] and D. discoideurn (unpublished). At the concentration of AMPMe used in Fig. 3 the cGMP response is saturated [3,5]; nevertheless, ATP increased cGMP accumulation 30--40%. These results suggest that structural plasma membrane alterations induced by ATP enhance the efficiency of "coupling" the chemoreceptor with, presumably, the gnanylate cyclase. There is an apparent paradox on the effect of ATP during cell aggregation in D. discoideum. On the one hand, ATP speeds up cell aggregation [12,13] and on the other hand it decreases chemotactic sensitivity of the cells [ 13 ]. Yet, these results are compatible. In the presence of

411

ATP the temporal pattern of cGMP accumulation in response to a chemoattractant was different from the control (Figs. 1--3): cGMP peaks lasted for about 5 s and basal levels were recovered later. We have previously shown that basal levels are n o t recovered later when cGMP peaks are higher [3,4,6]. It has been suggested that an intracellular gradient of cGMP may control pseudopod formation [6]. This assumes that any large change in the temporal pattern of cGMP accumulation in response to an attractant would lead to a defective intracellular distribution of this nucleotide and therefore to a decreased chemotactic sensitivity. The changes in the cGMP pattern observed in the presence of ATP would explain the previous observation [ 13] that in the presence of I mM ATP the chemotactic sensitivity of D. discoideum to cAMP is decreased. The enhanced aggregation in the presence of ATP might be due to a speeding up of cell differentiation independently of the ATP effect on the efficiency of coupling. The hypothesis that plasma membrane phosphorylation regulates signal transduction in vivo is under investigation. If this is so, it would agree with the general finding for neurotransmitters where the effect on postsynaptic membranes may also be mediated by changes in protein phosphorylation [ 18--21].

Acknowledgement I thank Theo Konijn for helpful comments. References 1 P a n , P., Hall, E.M. a n d B o n n e r , J . T . ( 1 9 7 2 ) N a t u r e N e w Biol. 2 3 7 , 1 8 1 - - 1 8 2 2 K o n i j n , T . M . , B a r k l e y , D . S . , C h a n g , Y.Y. a n d B o n n e t , J . T . ( 1 9 6 8 ) A m . N a t u r a l i s t 1 0 2 , 2 2 5 - - 2 3 4 3 M a t o , J . M . , K r e n s , F . A . , v a n H a a s t e r t , P.J.M. a n d K o n i j n , T.M. ( 1 9 7 7 ) P r o c . N a t l . A c a d . Sci. U.S. 74, 2 3 4 8 - - 2 3 5 1 4 W u r s t e r , B., S c h u b i g e r , K., W i c k , U. a n d G e r i s c h , G. ( 1 9 7 7 ) F E B S L e t t . 76, 1 4 1 - - 1 4 4 5 M a t o , J . M . , v a n H a a s t e r t , P . J . M . , K r e n s , F . A . , R h i j n s b u r g e r , E . H . , D o b b e , F . C . P . M . a n d K o n i j n , T.M. (1977) FEBS Lett. 79, 331--336 6 M a t o , J . M . a n d K o n i j n , T.M. ( 1 9 7 7 ) D e v e l o p m e n t a n d D i f f e r e n t i a t i o n in t h e C e l l u l a r S l i m e M o u l d s ( C a p p u c c i n e l l i , P. a n d A s h w o r t h , J . M . , eds.), D e v e l o p m e n t s in Cell B i o l o g y , vol. 1, p p . 9 3 - - 1 0 3 , Elsevier, N o r t h - H o l l a n d 7 W u r s t e r , B., B o z z a r o , S. a n d G e r i s c h , G. ( 1 9 7 7 ) Cell Biol. in p r e s s 8 M a t o , J . M . , K r e n s , F . A . , v a n H a a s t e r t , P.J.M. a n d K o n i j n , T.M. ( 1 9 7 7 ) B i o e h e m . B i o p h y s . Res. C o m mun. 77, 399--402 9 K o n i j n , T.M. a n d R a p e r , K . B . ( 1 9 6 1 ) D e v e l o p . Biol. 3, 7 2 5 - - - 7 5 6 1 0 G e r i s c h , G. ( 1 9 6 2 ) W i l h e l m R o u x A r c h . E n t w i c k l u n g s m e c h . O r g . 1 5 3 , 6 0 3 - - - 6 2 0 11 W e i n s t e i n , B.I. a n d K o r i t z , S.B. ( 1 9 7 3 ) D e v e l o p . Biol. 3 4 , 1 5 9 - - 1 6 2 1 2 M a t o , J . M . a n d K o n i j n , T.M. ( 1 9 7 5 ) D e v e l o p . Biol. 4 7 , 2 3 3 - - 2 3 5 13 M a t o , J . M . a n d K o n i j n , T.M. ( 1 9 7 6 ) E x p . Cell Res. 9 9 , 3 2 8 - - 3 3 2 1 4 Parish, R., Mfiller, U. a n d S c h m i d l i n , S. ( 1 9 7 7 ) F E B S L e t t . 7 9 , 3 9 3 - - 3 9 5 1 5 J u l i a n i , M.H. a n d K l e i n , C. ( 1 9 7 7 ) B i o e h i m . B i o p h y s . A c t a 4 9 7 , 3 6 9 - - 3 7 6 16 M a t o , J . M . a n d K o n i j n , T.M. ( 1 9 7 7 ) F E B S L e t t . 7 5 , 1 7 3 - - 1 7 6 17 E c k s t e i n , F. a n d B~[r, H.P. ( 1 9 6 9 ) B i o e h i m . B i o p h y s . A c t a 1 9 1 , 3 1 6 - - 3 2 1 1 8 G r e e n g a r d , P., M e A f e e , D . A . a n d K e b a b i a n , J.W. ( 1 9 7 2 ) A d v a n c e s in C y e U c N u c l e o t i d e R e s e a r c h ( G r e e n g a r d , P., P a o l e t t i , R . a n d R o b i s o n , G . A . , eds.), Vol. 1, p p . 3 3 7 - - 3 5 5 . R a v e n Press, N e w Y o r k 19 G r e e n g a r d , P. ( 1 9 7 6 ) N a t u r e 2 0 6 , 1 0 1 - - 1 0 8 2 0 G o r d o n , A . S . , Davis, C . G . , M i l l a y , D. a n d D i a m o n d , I. ( 1 9 7 7 ) N a t u r e 2 6 7 , 5 3 9 - - 5 4 0 21 T e i c h b e r g , V.I., S o b e l , A. a n d C h a n g e u x , J.P. ( 1 9 7 7 ) N a t u r e 2 6 7 , 5 4 0 - - 5 4 2