A model for cyclic AMP-chemoreceptor interaction in Dictyostelium discoideum

309

Biochimica et Biophysica Acta, 544 (1978) 309--314 © Elsevier/North-Holland Biomedical Press

BBA 28728

A MODEL FOR CYCLIC AMP-CHEMORECEPTOR INTERACTION IN DICTYOSTELIUNI DISCOIDEUM

JOSE M. MATO a, BERND JASTORFF

b, MICHAEL MORR

c and THEO M. KONIJN a

a Cell Biology and Morphogenesis Unit, Zoological Laboratory, University of Leiden, Kaiserstraat 63, Leiden (The Netherlands), and b Department of Biology and Chemistry, University of Bremen and c Department of Biotechnical Research, St6ckheim-Braunschweig (F.R.G.) (Received April 17th, 1978)

Summary Based on the chemotactic activity of approximately 50 different adenosine 3',5-cyclic-monophosphate (cyclic AMP) derivatives with substitutions at the phosphate, ribose and adenine moieties, a model for the cyclic AMP-chemoreceptor interaction in Dictyostelium discoideum is proposed. In this model the cyclic AMP molecule is bound to the receptor by three hydrogen bonds at, respectively, the 3'-oxygen of the ribose and the 6-amino and the 7-nitrogen of the base, and possibly by one ionic interaction of the negatively charged phosphate group. The conformation of the adenine moiety is in the anti range and binds additionally to the receptor by hydrophobic interactions between its r-electron system and a corresponding acceptor at the active site. Although this receptor clearly differs from that involved in protein kinase activation in higher organisms, the existence of striking similarities suggests a basic mechanism for cyclic AMP interaction conserved during evolution. Introduction Starved cells of Dictyostelium discoideum aggregate into centers by a chemotactic response to adenosine 3',5'-cyclicmonophosphate (cyclic AMP) [1]. The chemotactic response in D. discoideum can be outlined as follows: (i) the concentration of cyclic AMP is measured by cell surface receptors [2]; (ii) the cyclic AMP-receptor interaction generates a chemical signal, probably cyclic GMP [3--5], which is then processed by the cell; and (iii) the cell responds to this signal by moving in the direction of the attractant source. The cyclic AMP-receptor interaction can be studied by testing the chemotactic activity of different cyclic AMP derivatives [6]. By means of this technique it has been concluded that the cyclic AMP chemoreceptor in D. discoideum interacts

310 directly with the 3'-oxygen of the phosphate moiety [7,8]. The chemoreceptor is also sensitive to structural changes at the base moiety [9,10] and the results with different AMP derivatives indicate that the receptor is not a phosphodiesterase [8]. In this paper several new cyclic AMP derivatives have been tested and the results combined with those previously reported [7--10]. Based on the chemotactic activity of approx. 50 different cyclic AMP derivatives a model is proposed for the cyclic AMP-receptor interaction in D. discoideum. This model has been compared with the available information for cyclic AMP dependent protein kinase activation. Although both receptors appear to be different, the existence of several binding sites conserved during evolution point to a basic mechanism of interaction for cyclic AMP. Materials and Methods Amoebae of D. discoideum, NC-4(H), were grown and harvested as previously described [11]. The chemotactic activity of the various cyclic AMP derivatives was assayed with small populations of amoebae [6]. With this technique small drops of an amoebae suspension are placed on a hydrophobic agar surface. The amoebae remain homogeneously spread within the boundaries of the drop. When the cells are in their sensitive stage chemotaxis is assayed by placing the test solution (0.1 gl) three times at 5-min intervals close to the amoebae drops. The accumulation of amoebae at the edge of the drop closest to the attractant is observed microscopically 5 min after the last deposition. This test becomes quantitative by determining the concentration at which cells react positively, that is, at least twice as many amoebae pressed against the side closest to the attractant as against the opposite side. The t w o dilutions of a nucleotide between which 50% of the amoebae drops react positively were taken as the threshold concentration. All derivatives were tested three to five times at different concentrations, in all cases giving the same threshold values for chemotaxis. No detrimental effect on the amoebal behavior was observed with those derivatives which were used at very high concentrations. All the nucleotides mentioned in this paper have been tested in our laboratory during the last 9 years under standardized conditions. The threshold concentration for chemotaxis by cyclic AMP is 1 • 10-9--1 • 10 -~ M [6]. Results and Discussion To define the binding sites which occur between distinct atoms or atom groups of cyclic AMP and distinct amino acid side chains or the backbone of the receptor, the following assumption has been made: when the chemotactic activity of cyclic AMP is reduced at least 1000-fold by substitution of a distinct atom or atom group, this part of the molecule is considered to bind directly to the receptor. Such a part of the molecule is termed "essential". Drops in activity from 10- to 100-fold are considered to depend on general stereochemical or electronic features which have been changed by the modification of the ligand. Table I summarizes the effect of c o m p o u n d s with modifications in the purine ring on the ability of the resulting derivatives to trigger chemotaxis. Substitution of one of the hydrogens of the 6-amino group b y an alkyl of carboxyl

311 group decreases the chemotactive activity by a factor of 10- to 100-fold. However, the substitution of the whole amino group by a chloro or a h y d r o x y group yields an essential drop in activity, indicating the existence of ahydrogen bond interaction between the 6-amino group and an acceptor group on the receptor. It should be noted that the replacement of the 6-amino by a h y d r o x y group will n o t provide a hydrogen bond at this position because this derivative will take the more stable keto conformation. The replacement of N-7, which can act as a hydrogen bond acceptor, by a CH-group (tubercidin cyclic monophosphate) decreases chemotactic activity essentially [9], indicating the existence of a second hydrogen bond interaction between the base moiety and the receptor. All C-8 substituted cyclic AMP derivatives tested are 10- to 100-fold less active than cyclic AMP (with the only exception of 8-Br cyclic AMP) (Table I). This is in contrast with the results obtained with protein kinase activation, where the same derivatives are as active or even more active than cyclic AMP (for a review s e e ref. 12, 13). It is known that cyclic AMP in solution exists of an 1 : 1 ratio of syn and anti forms [14--16] and substitution of the 8-hydrogen in the cyclic AMP molecule switches the anti-syn equilibrium of the base to the syn range [17]. If the receptor binds the base in the anti conformation (anti-type receptor) an equilibrium in the syn range should hinder the optimal binding process of ligand and receptor, which means that higher concentrations are required to achieve the same biological effect as with cyclic AMP. On the other hand, if the receptor binds in the syn conformation (syntype receptor) an equilibrium in the syn range should enforce the binding process resulting in an even lower threshold concentration for inducing the biological response than with cyclic AMP. To explain the chemotactic activity TABLE I C H E M O T A C T I C A C T I V I T Y O F C Y C L I C AMP D E R I V A T I V E S M O D I F I E D A T T H E C-2, C-6 A N D C-8 POSITIONS

R

R2

R6

R8

Activity

Reference

H CI Br H H H H H NH 2 OH H H H H H

NH 2 NH 2 NH 2 N H - C H 2-C 6 H 5 NH-CO-C6H 5 NH-CO-C3H 7 =O CI =O =O NH 2 NH 2 NH 2 NH 2 NH 2

H H H H H H H H H H SH OH NH 2 Br piperidino

1 0 - 9 - - 1 0 -8 1 0 - 8 - - 1 0 -7 1 0 - 7 - - 1 0 -6 1 0 - 7 - - 1 0 -6 10-8--10-7 10-7--10-6 1 0 - 5 - - 1 0 -4 10-6--10-5 10-6--10-5 1 0 - 5 - - 1 0 --4 10-7--10-6 10-?--10-6 10-8--10-7 10-6--10-5 10-8--10-7

[9]

[ 10] [10] [18] [9] [9] [18]

[10] [10]

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observed with different 8-derivatives, we propose that the amoebal receptor is an anti-type receptor, while the protein kinase receptor seems to be a syn-type

receptor. Table II summarizes the ability to trigger chemotaxis of several compounds with modifications in the phosphate ring and ribose moieties. We have previously shown that there is no direct interaction between the 5'-oxygen and the receptor [ 7,8]. Furthermore, because the 5'-oxygen does not admit bulky substituents [7] it is proposed that there is only a narrow channel separating this atom from the receptor. Previously [8] the c o m p o u n d 5'-deoxy-5'-thio-cyclic AMP was surprisingly inactive. Later we observed that this same c o m p o u n d was phosphodiesterase resistant, had an ultraviolet scan which did n o t correspond to an adenine derivative and did n o t behave chromatographically as a Cyclic nucleotide (unpublished). Therefore the results for chemotaxis observed with this c o m p o u n d have n o t been included in the present model. Contrary to what" happens at the 5'-oxygen, in all compounds in which the 3'-oxygen has been substituted by a methylene or amino group the chemotactic activity decreased 1 • 104- to 1 • 106-fold. These results confirm our earlier hypothesis [7,8] that the 3'-oxygen is involved as a hydrogen bond acceptor in an essential interaction with the receptor. Obviously the 2'-position of the ribose is not involved in an essential interaction by hydrogen bonding to the receptor (Table II), which contrasts with the results observed with protein kinase activation [12,13]. The lack of activity found with adenosine when compared with the results observed with various AMP derivatives [8] suggests that the negative charge of the phosphate group might also be essentially b o u n d to the receptor by a salt-like bonding. Finally, several compounds with modifications of the purine ring (N-l, C-2)

T A B L E II CHEMOTACTIC ACTIVITY PHATE MOIETIES

OF SEVERAL

DERIVATIVES

V

W

X

Y

O OH 2 NH O O S O NH O

O O O NH CH2 NH NH NH O

O O O O O 0 S S O

OH OH OH OH OH OH OH OH H

O

O

O

O~NO

MODIFIED AT THE RIBOSE OR PHOS-

Activity

F NO2

10-9--10 -8 10-9--10 -8 10-9--10 -8 10-5--10 -4. <10-4 < 1 0 -4 <10-4 <10-4 10-8--10-7

2

10-"/--10-6

Reference

[9] [71 [81 [91

313 (see Table I and ref. 18) decrease the chemotactic activity 10- to 100-fold, probably without affecting the hydrogen bonds of the base. To explain these results it is proposed that the base moiety also binds by interaction between its r-electron system and a corresponding acceptor at the active site. This could be either an unspecific hydrophobic interaction with a hydrophobic pocket as has been established for the adenine moiety of NAD ÷ at the binding site of several dehydrogenases [19] or a more specific interaction between the base and an aromatic amino acid side chain yielding a type of stacking [20]. A plausible model for the molecular interaction of cYClic AMP and the chemoreceptor in D. discoideum is presented in Fig. 1. We propose that the interaction cyclic AMP-receptor is biphasic. In the first phase the cyclic AMP molecule binds to the receptor by means of three hydrogen bonds and possibly one ionic interaction. In the second phase the purine moiety interacts with an acceptor at the active site and generates the chemotactic signal. The cyclic AMP receptor involved in D. discoideum chemotaxis is clearly different from that involved in protein kinase activation in higher organisms. All 6-substituted cyclic AMP derivatives are good activators of protein kinase (for a review see refs. 12 and 13). The replacement of N 7 of the purine ring with a C-H or C-CN group has only a slight effect on protein kinase activity

Fig. 1. A m o d e l f o r t h e c y c l i c A M P - r e c e p t o r i n t e r a c t i o n in D. discoideum. T h e c y c l i c A M P m o l e c u l e b i n d s first t o t h e r e c e p t o r b y t h r e e h y d r o g e n b o n d s a t r e s p e c t i v e l y t h e 6 - a m i n o , t h e N-7 a n d t h e 3 t - o x y g e n a n d possibly one ionic interaction by the negative charge at the phosphorous atom. Then the base moiety b i n d s b y i n t e r a c t i o n b e t w e e n its y - e l e c t r o n s a n d a c o r r e s p o n d i n g a c c e p t o r (Phe, T y r , T r p , His) a t t h e active site ( n o t s h o w n ) . A, a m i n o a c i d side c h a i n d o n a t o r o f t h e h y d r o g e n b o n d (Set, T h r , L y s , T y r ) ; B, a m i n o a c i d side c h a i n a c c e p t o r o f t h e h y d r o g e n b o n d ( A s p , His o r a n a c c e p t o r a t t h e p r o t e i n b a c k b o n e - - ( - - C - - N - - ) ) . X , p o s i t i v e l y c h a r g e d a m i n o a c i d side c h a i n ( L y s , A r g ) .

314

[12,13]. Also, contrary to D. discoideum, any change in the 2'-oxygen and the 5'-oxygen lowers essentially the activity of the resulting derivative on protein kinase [12,13]. Despite these differences, three binding sites seem to be comm o n to D. discoideum and protein kinase: the hydrogen bond at the 3'-oxygen, the ionic interaction with the negative charge and the interaction between the 1r-electrons of the base and an acceptor at the active site [21,22]. These results suggest a general mechanism of action for cyclic AMP preserved during evolution that involves the recognition of the 3'-oxygen and hydrophobic interactions between the base and the active site of the receptor. In conclusion, we realize that this model may have to be modified when new derivatives are tested. Nevertheless, some of the essential components of the cyclic AMP-receptor interaction in D. discoideum seem to have been identified. References 1 Konijn, T.M., Barkley, D.S., Chang, Y.Y. and Bonnet, J.T. (1968) Am. Nat. 102, 225--234 2 Malchow, D. and Geriseh, G. (1974) Proc. Natl. Acad. Sci. U.S. 71, 2423--2427 3 Mato, J.M., Krens, F.A., van Haastert, P.J.M. and Konijn, T.M. (1977) Proc. Natl. Acad. Sci. U.S. 74, 2348--2351 4 Wurster, B., Schubiger, K., Wick~ U. and Geriseh, G. (1977) FEBS Lett. 76, 141--144 5 Mato, J.M. and Malchow, D. (1978) FEBS Lett. 9 0 , 1 1 9 - - 1 2 2 6 Konijn, T.M. (1970) Experientia 2 6 , 3 6 7 - - 3 6 9 7 Konijn, T.M. and Jastorff, B. (1973) Biochim. Biophys. Acta 3 0 4 , 7 7 4 - - 7 8 0 8 Mato, J.M. and Konijn, T.M. (1977) FEBS Lett. 75, 173--176 9 Konijn, T.M. (1972) Adv. Cyclic Nucleotldc Res. 1, 17--31 10 Konijn, T.M. (1973) FEBS Lett. 34, 263--266 11 Konijn, T.M. and Raper, K.B. (1961) Develop. Biol. 3, 725--756 12 Simon, L.N., Shuman, D.A. and Robins, R.K. (1973) Adv. Cyclic Nucleotide Res. 3 , 2 2 5 - - 2 5 3 13 Miller, J.P. (1977) in Cyclic 3r,5~-nueleotides Mechanism of Action (Cramer, H. and Sehultz, J., eds.), pp. 77--105, Wiley, New York 14 Heroines, P., Oppenheimer, L. and Jordan, J. (1976) J.G.S. Chem. Comm. 926 15 Fazakerlay, G.V., Russell, J.C. and Wolfe, M.A. (1975) J.G.S. Chem. Comm, 527 16 Lespinasse, J.N, and Vasilescu, D. (1974) Biopolymers 13, 63 17 Schweizer, N.P. and Robins, R.K. (1973) in C o n f o r m a t i o n of Biological Molecules and Polymers (Pullman, B. and Bergmann, E., eds.), pp. 329--343, Academic Press, New York 18 Konijn, T.M. (1974) in A n t i b i o t i e a and Chemotherapy 19, (Sorkin, E., ed.), pp. 96--110, Karger, Basel 19 Rossman, M.G., Lilias, A., B r ~ d e n , G.I. and Banaszak, L.J. (1975) in The Enzymes, Vol. XI, (Boyer, P.D., ed.), pp. 62--102, Academic Press, New York 20 Lawaczeck, R. and Wagner, K.G. (1974) Biopolymers 13, 2 0 0 3 - - 2 0 1 4 21 Panitz, N., Rieke, E., Mort, M., Wagner, K.G., Roesler, G. and Jastorff, B. (1975) Eur. J. Biochem. 55, 415--422 22 Jastorff, B. (1978) Adv. Cyclic Nucleotide Res., 9 , 7 6 0