Specific binding proteins for cyclic AMP and cyclic GMP in Dictyostelium discoideum.

In Dictyostelium discoideum both cyclic AMP and cyclic GMP are regulated by chemotactic stimuli. Binding proteins specific for cAMP and cGMP have been found in aggregation competent cells as well as in cells harvested during growth. The activity of binding proteins was, on the average, lower in the growth phase cells. cAMP binding proteins were separated into 3 fractions, whereas the cGMP binding activity appeared in 1 major peak both on DEAE-cellulose and Sephadex G-200. Protein kinase activity was present in most but not all cyclic necleotide binding fractions; evidence for a relationship is however missing.     

Dictyostelium discoideum lipids modulate cell-cell cohesion and cyclic AMP signaling.

During Dictyostelium discoideum development, cell-cell communication is mediated through cyclic AMP (cAMP)-induced cAMP synthesis and secretion (cAMP signaling) and cell-cell contact. Cell-cell contact elicits cAMP secretion and modulates the magnitude of a subsequent cAMP signaling response (D. R. Fontana and P. L. Price, Differentiation 41:184-192, 1989), demonstrating that cell-cell contact and cAMP signaling are not independent events. To identify components involved in the contact-mediated modulation of cAMP signaling, amoebal membranes were added to aggregation-competent amoebae in suspension. The membranes from aggregation-competent amoebae inhibited cAMP signaling at all concentrations tested, while the membranes from vegetative amoebae exhibited a concentration-dependent enhancement or inhibition of cAMP signaling. Membrane lipids inhibited cAMP signaling at all concentrations tested. The lipids abolished cAMP signaling by blocking cAMP-induced adenylyl cyclase activation. The membrane lipids also inhibited amoeba-amoeba cohesion at concentrations comparable to those which inhibited cAMP signaling. The phospholipids and neutral lipids decreased cohesion and inhibited the cAMP signaling response. The glycolipid/sulfolipid fraction enhanced cohesion and cAMP signaling. Caffeine, a known inhibitor of cAMP-induced adenylyl cyclase activation, inhibited amoeba-amoeba cohesion. These studies demonstrate that endogenous lipids are capable of modulating amoeba-amoeba cohesion and cAMP-induced activation of the adenylyl cyclase. These results suggest that cohesion may modulate cAMP-induced adenylyl cyclase activation. Because the complete elimination of cohesion is accompanied by the complete elimination of cAMP signaling, these results further suggest that cohesion may be necessary for cAMP-induced adenylyl cyclase activation in D. discoideum.     

A cyclic AMP dependent protein kinase in Dictyostelium discoideum

A cyclic AMP-dependent protein kinase was found to appear during the time course of development of $\text{Dictyostelium}\text{discoideum}$. No cyclic AMP dependency was observed at any stage of development in crude 110,000 X G soluble extracts. After partial purification, however, extracts from post-aggregation stages contained enzyme that was activated up to 6-fold by cyclic AMP, whereas protein kinase from earlier stages was not affected by cyclic AMP. Likewise, cyclic AMP binding activity increased from the aggregation to the slug stage of development. Approximately one-half of the total cyclic AMP binding activity co-purified with the cyclic AMP dependent protein kinase. The enzyme from $\text{Dictyostelium}$ showed similarities to mammalian protein kinases with respect to its kinetic properties but differed in its behavior on ion-exchange chromatography.     

Identification and cyclic AMP-induced modification of the cyclic AMP receptor in Dictyostelium discoideum

We have recently identified a cell surface cAMP-binding protein by specific photoaffinity labeling of intact Dictyostelium discoideum cells with 8-N3-[32P] cAMP. The major photolabeled protein appears as a doublet (Mr = 40,000-43,000) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiography. In this study, the doublet is shown to have the characteristics of the cAMP receptor responsible for chemotaxis and cAMP signaling. Both specific photoaffinity labeling of the doublet and binding of 8-N3-[32P]cAMP are saturable (KD = 0.3 microM), the levels of both peak at 5 h, and both are inhibited by cAMP and several cAMP analogs in the same order of potency and with K1 values similar to those measured for inhibition of [3H]cAMP binding. When cAMP-binding activity was partially purified (40-fold) and then photoaffinity labeled, the same bands (Mr = 40,000-43,000) were observed. The relative intensities of the upper and lower bands of the doublet alternated at the same frequency as the spontaneous oscillations in cAMP synthesis. When oscillations were suppressed, the lower band of the doublet predominated. Following addition of cAMP, the relative intensity gradually shifted to the upper band. When cAMP was removed, there was a gradual restoration of the lower band form. We propose that the lower band form of the receptor activates chemotaxis and cAMP signaling and that the upper band form does not. This reversible receptor modification may then be the mechanism of adaptation, the process by which the physiological responses cease to be stimulated by persistent cAMP. Several developmentally regulated genes in D. discoideum have been reported to be induced or suppressed by pulses of cAMP (adaptive regulation) and others by continuous cAMP (nonadaptive regulation). These observations may be explained by the receptor modification reported here if the two forms of the receptor, which bind cAMP with the same affinity, independently influence gene expression.     

Pharmacological characterization of cyclic AMP receptors mediating gene regulation in Dictyostelium discoideum.

Extracellular molecules regulate gene expression in eucaryotes. Exogenous cyclic AMP (cAMP) affects the expression of a large number of developmentally regulated genes in Dictyostelium discoideum. Here, we determine the specificity of the receptor(s) which mediates gene expression by using analogs of cAMP. The order of potency with which these analogs affect the expression of specific genes is consistent with the specificity of their binding to a cell surface receptor and is distinct from their affinity for intracellular cAMP-dependent protein kinase. Dose-response curves with cAMP and adenosine 3',5'-monophosphorothioate, a nonhydrolyzable analog, revealed that the requirement for high concentrations of exogenous cAMP for regulating gene expression is due to the rapid degradation of cAMP by phosphodiesterase. The addition of low concentrations of cAMP (100 nM) or analogs in pulses also regulates gene expression. Both the genes that are positively regulated by exogenous cAMP and the discoidin gene, which is negatively regulated, respond to cAMP analogs to the same degree. Genes expressed in prespore or prestalk cells are also similarly regulated. These data suggest that the effects are mediated through the same receptor. The specificity of this receptor is indistinguishable from that of the well-characterized cell surface cAMP receptor.     

Localization and levels of cyclic AMP during development of Dictyostelium discoideum

Cyclic AMP functions as the chemotactic signal during aggregation of amoebae of the cellular slime mold Dictyostelium discoideum. Evidence suggests that cAMP also acts as a regulatory molecule during Dictyostelium multicellular differentiation. We have used ultramicrotechniques and a sensitive radioimmunoassay to measure the levels of cAMP within the culmination stage individual. We show that there is a peak of cAMP at the culmination stage of development and that in the individual at this stage the molecule is localized in a gradient within the spore mass.     

Quantitative analysis of cyclic AMP waves mediating aggregation in Dictyostelium discoideum

We have previously reported the detection of cAMP waves within monolayers of aggregating Dictyostelium discoideum cells (K. J. Tomchik and P.N. Devreotes, 1981, Science 212, 443-446). The computer-assisted analysis presented here of the fluorographic images of the cAMP waves reveals (1) all the waves have a consistent width and height; (2) cAMP concentrations within centers of concentric aggregation territories oscillate periodically while at spiral centers the concentration builds up to a plateau value within 2 mm; (3) cells within the region of intersection of two oppositely directed cAMP waves are stimulated to produce more cAMP than those responding to a single wave; (4) cells start to move when the cAMP level begins to increase and cease movement when the peak cAMP concentration reaches the cell.     

Independent regulation of the extracellular cyclic AMP phosphodiesterase-inhibitor system and membrane differentiation by exogenous cyclic AMP in Dictyostelium discoideum.

When amoebae of Dictyostelium discoideum, suspended in buffer, were treated with 100 nM pulses of cAMP, the extracellular cAMP phosphodiesterase (ePD) activity increased dramatically and the synthesis of the phosphodiesterase inhibitor (PDI) was repressed. In addition, the time of appearance on the cell surface of contact sites A, membrane-bound cAMP phosphodiesterase, and cAMP binding sites was accelerated by 3–4 hr and the concentration of intracellular cAMP increased ?20-fold. When the concentration of the cAMP pulse was reduced to 1 nM, the effect of the pulses on membrane differentiation and on the cAMP pool was virtually the same, while the effect on the ePD-PDI system was reduced. When cAMP was added to the suspension continuously, the nucleotide had no effect on membrane differentiation and failed to stimulate the intracellular cAMP pool, however, the ePD-PDI system was regulated normally. When the developmental mutant, HC112, was treated with cAMP pulses, membrane differentiation and the level of the cAMP pool were unaffected, while the ePD-PDI system responded to the exogenous cAMP. In another mutant, HC53, membrane differentiation was stimulated by cAMP pulses and this response was accompanied by a sharp increase in the concentration of the cAMP pool. These results suggest that the ePD-PDI system and membrane differentiation are regulated independently by exogenous cAMP and that regulation of the ePD-PDI system does not require activation of the adenylyl cyclase.     

Cyclic AMP relay and cyclic AMP-induced cyclic GMP accumulation during development of Dictyostelium discoideum

Abstract Cyclic AMP-induced cAMP and cGMP responses during development of Dictyostelium discoideum were investigated. The cAMP-induced cGMP response is maximal when aggregation is in full progress, and then decreases to about 10% of the maximal level during further multicellular development. The cAMP response increases upon starvation, reaches its maximum at the onset of aggregation, and then decreases to about 8% of the maximum level. The dynamics of the post-aggregative cAMP response are in qualitative agreement with the dynamics of the cAMP relay response in aggregation-competent cells.     

Adenylate cyclase activity and cyclic AMP levels during the development of Dictyostelium discoideum.

Adenylate cyclase activity and endogenous cyclic AMP levels were measured using a highly sensitive radioimmunoassay and protein binding assay during 24 h of development of Dictyostelium discoideum. Adenylate cyclase activity was not detected until the aggregation stage of development (10 h) when a sudden peak of activity was found. The enzyme was active at all subsequent stages, although a slow decline in activity was observed. Similarly, cyclic AMP levels were not detectable through the first 7 h of development and then showed a sudden peak at aggregation. Following aggregation the cyclic AMP levels decreased to approximately 1/2 the peak value and maintained that level throughout the remainder of the developmental cycle. Adenylate cyclase had a narrow range of substrate saturation with a maximum velocity at 1 to 4 mM ATP at both the aggregation stage (10 h) and the sorocarp stage (24 h). At levels of ATP higher than 6 mM the enzyme from both stages was strongly inhibited. No activity was observed in the absence of Mg2+ or dithiothreitol. The activity from 10-, 14-, and 20-h stages was found bound to a 25,000 x g pellet fraction. The sudden appearance of adenylate cyclase and its product cyclic AMP at aggregation provides additional evidence of a role for this nucleotide in chemotaxis, and the retention of enzyme activity and nucleotide level during the subsequent stages may reflect a further function of cyclic AMP during formation of the two cell types.     

Purification and characterization of the extracellular cyclic AMP phosphodiesterase of Dictyostelium discoideum

Extracellular phosphodiesterase for adenosine 3':5'-monophosphate [EC 3.1.4.17] was purified from the supernatant of aggregation phase culture of Dictyostelium discoideum, and two types (type I and type II) of the enzyme were found. The type I enzyme was not absorbed on DEAE-Sephacel at pH 8.5 and had an apparent molecular weight of about 67,000 daltons. In contrast, the type II enzyme was adsorbed on DEAE-Sephacel and had an apparent molecular weight of about 120,000 daltons. The Km values of the two types were similar (2-4 microM). Upon SDS polyacrylamide gel electrophoresis analyses, however, both types produced the same bands with molecular weights of 55,000 and 57,000, indicating that they are two different forms composed of common constituents. During the growth phase, the two types of the enzyme were present in culture supernatant in roughly equal amounts, but type II accumulated predominantly in the aggregation phase, suggesting that the ratio of activity of the two forms is under developmental control. Rabbit antiserum prepared against purified type II enzyme cross-reacted with type I as well as membrane-bound enzyme, indicating that the three classes of the enzyme possess some common sequence.     

Cellular responsiveness to cyclic AMP in a phosphodiesterase-defective mutant of Dictyostelium discoideum

Responsiveness of Dictyostelium discoideum amoebae to cAMP, a chemotactic mediator, was investigated in a strain defective in cAMP-phosphodiesterase production. Cells were subjected to a high cAMP signal (10(-6) M) in the presence or absence of exogenous phosphodiesterase, and the changes of intracellular cAMP and cGMP concentrations and of adenylate cyclase activity were measured. In the presence of cAMP hydrolysis, both adenylate and guanylate cyclases are transiently activated. In the absence of hydrolysis, the high and constant extracellular cAMP concentration is sufficient to elicit a re-activation of adenylate cyclase a few minutes after the first transient response. In contrast, levels of cGMP remain basal for at least 20 min after termination of the initial response to the cAMP addition.