Changes in cyclic AMP receptor properties during adaptation in Dictyostelium discoideum M. E. E. LUDERUS', M. J. SPIJKERS and R. VAN DRIEL E.C. Slater Institute for Biochemical Research, University of Amsterdam, Plantage Muidergracht 12, 1018 TVAmsterdam, The \etliet1ands •Author for correspondence Summary In developing Dictyostelium discoideum cells, receptor binding completely, presumably by acting binding of cyclic AMP to the chemotactic receptor via a G protein. The guanine nucleotides reduced has been shown to oscillate. These oscillations the affinity of the receptor at all time-points of the represent cycles of activation, adaptation and de- oscillation cycle to the minimal, i.e. adapted, level. adaptation of the cyclic AMP receptor system. We conclude that the cyclic process of activation, We studied the molecular basis of these oscilla- adaptation and de-adaptation in D. discoideum, at tory changes in cyclic AMP receptor binding. We cyclic AMP receptor level, involves changes in developed a rapid method of lysing cells during the receptor-G protein interaction. During adaptation, course of the oscillations. This method guaranteed the affinity of the cyclic AMP receptor decreases good preservation of ligand binding properties of and the receptor becomes insensitive to guanine the cyclic AMP receptor. nucleo tides. We found that oscillations in cyclic AMP binding resulted from changes in receptor affinity. The total number of receptors did not significantly change during oscillations. Our experiments also showed Key words: chemotactic cyclic AMP receptor, adaptation, D. that both GTP and GDP abolished oscillations in discoideum, G protein, oscillations. Introduction phosphodiesterase (Malchow et al. 1972). The resulting periodic changes in extracellular cyclic AMP concen- Early in development, Dictyostelium discoideum cells tration synchronize various cyclic AMP-induced cellular acquire the capacity to detect, synthesize and secrete responses. Many cellular responses have in fact been cyclic AMP. This cyclic AMP signal-relay system is the shown to oscillate with the same frequency as the cyclic basis of an intercellular communication system that AMP signal (Aeckerle et al. 1985; Bumann et al. 1986; controls chemotaxis, cell aggregation, differentiation and Gerisch and Hess, 1974; Klein et al. 1977; Malchow et pattern formation (Devreotes, 1982; Sussman, 1982). al. 1978; Wurster et al. 1977). These oscillations are the The signal molecule in this system is extracellular cyclic result of repeated cycles of activation, adaptation and AMP. It is detected by highly specific receptors that are de-adaptation. present on the surface of the cell. Binding of cyclic AMP Regulation of receptor-mediated signal transduction in to these receptors induces several rapid responses, such D. discoideum is similar to that in higher eukaryotes as synthesis and secretion of cyclic AMP (Gerisch and (Janssens, 1987). The cyclic AMP receptor of D. discoid- Wick, 1975), synthesis of cyclic GMP (Mato et al. 1977), eum is thought to have the classical seven transmembrane influx of Ca2+ (Wick et al. 1978) and efflux of K+ and H+ spanning domains that are characteristic of all eukaryotic (Aeckerle et al. 1985; Malchow e< a/. 1978). Most of these G protein-coupled receptors studied so far (Klein et al. responses are subject to adaptation. Adaptation refers to 1988). The cyclic AMP receptor is coupled to two or the mechanism by which the response to extracellular more G proteins (Janssens et al. 1985; Kumagai et al. cyclic AMP is terminated. Adaptation is induced by 1989; Van Haastert et al. 1986). These G proteins prolonged exposure of cells to a constant level of cyclic mediate the activation (Janssens and De Jong, 1988; Van AMP; de-adaptation occurs as the cyclic AMP concen- Haastert et al. 1987), and possibly adaptation (Small et tration declines. al. 1987; Van Haastert et al. 1986), of the enzymes During development, D. discoideum cells secrete adenylate cyclase and guanylate cyclase. Furthermore, pulses of cyclic AMP with a frequency of one pulse per evidence has been presented for a role for the phospho- 5-8 min (Klein et al. 1977). Secreted cyclic AMP is inositol cycle in the signal transduction system of D. hydrolyzed by the extracellular enzyme cyclic AMP- discoideum (Europe-Finner and Newell, 1985; Van Journal of Cell Science 95, 623-629 (1990) Printed in Great Britain © The Company of Biologists Limited 1990 623 Lookeren Campagne et al. 1988). Despite detailed in Dictyostelium; Green and Newell, 1975), 20fm S'AMP), 3 knowledge of the physiology of receptor-mediated re- containing [ H]cyclic AMP at concentrations given in the text. If indicated, 0.1 mM GTPyS, GDP or GMP were present. sponses in both D. discoideum and higher eukaryotes, 3 little is known about the molecular mechanism of acti- Subsequently, the amount of [ H]cyclic AMP bound to the membranes was determined by measuring the radioactivity in vation, adaptation and de-adaptation of these responses. the pellet after 2 min of centrifugation at 10 000 # in a microfuge The synchronous oscillations of the cyclic AMP receptor (sedimentation assay). Non-specific binding was determined by system of D. discoideum make this organism a convenient equilibration in the presence of a large excess (0.5 mM) of model system in which to study these mechanisms. unlabeled cyclic AMP. In the present work we studied the repetitive cycle of Measurements of equilibrium [3H]cyclic AMP binding to the activation, adaptation and de-adaptation in D. discoid- different receptor forms were done according to Van Haastert eum at the level of the cyclic AMP receptor. In cell lysates et al. (1986). Cyclic AMP receptors occur in a limited number prepared at various time-points during the oscillation of well-defined receptor types, called Fast, S and SS (Janssens cycle, we investigated ligand binding and receptor-G et al. 1986; Van Haastert and De Wit, 1984). Each of these protein interaction. Lysates were prepared by a newly receptor types is characterized by its own dissociation rate constant. In isolated membranes, at 20°C, the following dis- developed, simple procedure based on freezing cells in sociation rate constants (£-i) have been measured (Van Haas- liquid nitrogen. The procedure was rapid and guaranteed x tert, 1984): Fast type, k-x=G.2b±QM%~ \ S type, £_,= good preservation of cyclic AMP receptor properties. 22 1 1 333 (4.3±0.6)Xl0" s- ; SS type, ^_, = (3.9±0.9)Xl0-- s-s '' Our results indicate that adaptation in D. discoideum, Since no interconversion of these receptor forms occurs during measured at receptor level, involves changes in recep- the course of dissociation, it can be calculated that after a tor-G protein coupling. In the adapted state, the cyclic dissociation period of 10 s essentially no occupied Fast sites are AMP receptor has a reduced affinity for cyclic AMP and present, whereas binding to S sites is reduced to 64%, and is insensitive to guanine nucleotides. binding to SS is reduced to 97 %. After a dissociation period of 120 s, essentially all Fast and S sites are empty, while binding to SS is reduced to 61 %. This is described by the following set of Materials and methods equations: 6(0) = Fast(0) + S(0) + SS(0) Materials 6(10) = (0.64) S(0) + (0.97) SS(0) [5',8-3H]cyclic AMP (l.SSTBqmmol"1) was purchased from 6(120) = (0.61) SS(0) Amersham International (UK), cyclic AMP and dithiothreitol Thus, from measuring the amount of [3H]cyclic AMP bound at from Serva (Heidelberg, Federal Republic of Germany), and equilibrium, 6(0), and at 10s and 120s after the onset of S'AMP, GDP, GMP, and GTPyS from Boehringer (Mann- dissociation, 6(10) and 6(120), it is possible to calculate the heim, Federal Republic of Germany). Nitrocellulose filters occupancy of the three receptor forms at equilibrium (Fast(0), (type BA 85) were from Schleicher & Schiill (Dassel, Federal S(0) and SS(0)). Republic of Germany). Measurement of 6(0), 6(10) and 6(120) was performed as follows. Washed paniculate fraction (200 j/g protein) was equi- Culture conditions and membrane isolation librated in 100 fi\ BM with 10 nM [3H]cyclic AMP for 5 min at D. discoideum cells (strain AX2) were grown in HL-5 medium 20cC. Subsequently, either the total amount of [3H]cyclic AMP (Watts and Ashworth, 1970), that contained maltose instead of bound to the membranes, 6(0), was determined directly by the glucose. Cells were harvested in the late logarithmic growth 6 sedimentation assay (see above), or dissociation of the recep- phase at a density of approximately 5xl0 cellsml~ . For tor-[3H]cyclic AMP complexes was initiated at 20°C by 100- development in suspension, cells were washed once with 10 mM fold dilution with P, buffer. After 10 s and 120 s of dissociation, 3 KH2PO4/Na2HPO4, pH6.5 (P, buffer), resuspended in the 7 1 [ H]cyclic AMP that remained bound to the membranes (6(10) same buffer at 2xl0 cellsml~', and shaken at ISO revs min" at and 6(120), respectively) was determined by filtration through 22°C. When indicated in the text, every 6min a cyclic AMP 0.45 um pore-size nitrocellulose filters, measuring the [3H]cyc- pulse of 5 nM was given, starting after 4 h of development. After lic AMP retained by the membranes on the filters (nitrocellulose 5 h of development, 500-/^1 samples were taken from the filtration assay). suspension every minute, and mixed with 20% (w/v) sucrose plus the following protease inhibitors: 5 mM benzamidine, 100;Ugml~ aprotinin, 50/igml~ trypsin inhibitor and Results 20^gml~' antipain (final concentrations). The cells were lysed immediately by rapid freezing in liquid nitrogen. The sucrose in Lysis method the medium was essential for the preservation of cyclic AMP In synchronously developing D. discoideum cells, cyclic receptor binding.