Binding of adenosine 3':5'-monophosphate to plasma membranes of Dictyostelium discoideum amoebae

A plasma membrane preparation from Dictyostelium discoideum amoebae which contains the high affinity cAMP receptor is described. Ligand specificity and the kinetics of cAMP association and dissociation using isolated plasma membranes were similar to those of intact cells. The changes in cAMP binding activity which occur as cells proceed through their aggregation program were also reflected in the membrane preparations. However, neither the low affinity cAMP binding site nor the oscillatory cAMP binding behavior observed on intact cells was detected with the membrane preparations.     

Guanidine hydrochloride-induced shedding of a Dictyostelium discoideum plasma membrane fraction enriched in the cyclic adenosine 3',5'-monophosphate receptor

The cell surface cyclic AMP receptor of Dictyostelium discoideum is under study in a number of laboratories with respect to both its role in development of the organism and the physiology of excitation-response coupling. We report here that when starved amoebae are exposed to the chaotrope guanidine hydrochloride at 1.8 M, they shed a particulate cyclic AMP binding activity into the medium. This activity is due to membrane vesicles which originate from the cell surface. The vesicles are enriched up to 150-fold in cyclic AMP binding activity and up to 14-fold in phospholipid content when compared to the starting amoebae. The cyclic AMP binding activity of the membrane vesicles is identical to that of the cell surface receptor with respect to the following properties; (i) it is lacking in preparations from unstarved, vegetative amoebae; (ii) it is not inhibited by cyclic GMP and is stimulated by calcium ions; (iii) it has very rapid rates of association and dissociation of bound cyclic AMP; (iv) it has two classes of binding sites with dissociation constants similar to those of the surface receptors of whole amoebae. The binding activity of the isolated membranes is stable for several days at 4 degrees C and the lower affinity binding sites are stable up to several months when stored at -80 degrees C. Due to enrichment and stability of the receptor in this preparation, it should be highly suitable for many types of studies. The usefulness is enhanced by the fact that the preparation does not contain detectable cyclic AMP phosphodiesterase activity.     

Induction of phosphodiesterase by cyclic adenosine 3':5'-monophosphate in differentiating Dictyostelium discoideum amoebae.

Cyclic adenosine 3':5'-monophosphate added to the starvation media of Dictyostelium discoideum amoebae induces both intracellular and extracellular phosphodiesterase activities of these cells. The induced enzyme activity appears several hours earlier than that in starved cells which have not been induced with cyclic nucleotide. In both cases, the appearance of enzyme is inhibited by cycloheximide, and actinomycin D, and daunomycin. The KmS for the extracellular enzyme(s) of nucleotide-induced and uninduced control cells are identical. The induction of enzyme activity seems specific for cyclic adenosine 3':5'-monophosphate since cyclic guanosine 3':5'-monophosphate, as well as other nucleotides, have no effect. No differences in the activity or excretion of either N-acetylglucosaminidase or the inhibitory of the extracellular phosphodiesterase are observed between cyclic adenosine 3':5'-monophosphate-induced and control cells. A direct activation of phosphodiesterase by cyclic adenosine 3':5'-monophosphate can be excluded, since the addition of this nucleotide to cell lysates has no effect on the enzyme activity.     

Intracellular localization of phosphodiesterase induced by cyclic adenosine 3',5'-monophosphate in Dictyostelium discoideum

The intracellular distribution of phosphodiesterase [EC 3.1.4.17] induced by cyclic adenosine 3',5'-monophosphate (cAMP) in Dictyostelium discoideum was studied. When cAMP-treated cells were homogenized and fractionated according to the method of de Duve et al. ((1955) Biochem, J. 60, 604), the specific activity of phosphodiesterase was highest in the light mitochondrial fraction. Peaks of specific activities of alkaline phosphatase (marker enzyme of membrane) and catalase (marker enzyme of peroxisomes) also appeared in the same fraction as phosphodiesterase. However, after centrifugation of the light mitochondrial fraction in a sucrose density gradient, the activity of phosphodiesterase was clearly separated with that of catalase (density 1.19 g/ml) and showed three peaks at lower density (1.10, 1.13, 1.17 g/ml) with good reproducibility. Some parts (1.13, 1.17 g/ml) of the activity in the gradient overlapped with alkaline phosphatase activity, but in the density fraction of 1.10 g/ml the activity of alkaline phosphatase was hardly detectable. When the light mitochondrial fraction was treated with Emulgen 108, or sonicated, phosphodiesterase was more easily solubilized than alkaline phosphatase and catalase, and was found in supernate after centrifugation at 20,000 X g for 30 min. In order to distinguish the locations of the three enzymes, the supernatant of the light mitochondrial fraction treated with Emulgen 108 was subjected to charge shift electrophoresis. The electrophoretic mobilities of phosphodiesterase and catalase were unaffected by ionic detergent. However, alkaline phosphatase shifted towards the anode in the presence of anionic detergent (sodium deoxycholate), and shifted towards the cathode in cationic detergent (cetyltrimethylammonium bromide), relative to nonionic detergent (Emulgen 108) alone. Thus, some part of the phosphodiesterase induced by cAMP may be associated with the plasma membrane, but the remainder is localized in some kind of intracellular particle of lower density. Moreover, the association with the membrane or particle is more easily dissociated than that of alkaline phosphatase, and the liberated phosphodiesterase is rather hydrophilic.     

A slowly dissociating form of the cell surface cyclic adenosine 3':5'-monophosphate receptor of Dictyostelium discoideum.

Experiments using a phosphodiesterase-minus mutant of Dictyostelium discoideum indicate that ligand-induced loss of cell surface cyclic adenosine 3':5'-monophosphate binding sites (down regulation) can be evoked with concentrations of cyclic adenosine 3':5'-monophosphate as low as 10(-8) M. The loss of receptor sites is observed after 5 min of cell preincubation with cyclic adenosine 3':5'-monophosphate and can be as extensive as 75 to 80%. This decrease in binding sites is correlated with the appearance of a slowly dissociating cyclic adenosine 3':5'-monophosphate binding component. Radioactive cyclic adenosine 3':5'-monophosphate bound to this form of receptor cannot be competed for by nonradioactive cyclic adenosine 3':5'-monophosphate or adenosine 5'-monophosphate and is not accessible to hydrolysis by cyclic adenosine 3':5'-monophosphate phosphodiesterase. The extent of appearance of this binding component is dependent upon the concentration of cyclic adenosine 3':5'-monophosphate used to elicit the down regulation response and the temperature of the incubation medium.     

Participation of calcium in the induction of phosphodiesterase by cyclic adenosine 3',5'-monophosphate in Dictyostelium discoideum

The effects of divalent cations on the induction of phosphodiesterase [EC 3.1.4.17] by cyclic adenosine 3',5'-monophosphate (cyclic AMP) were studied in Dictyostelium discoideum. When cells were incubated with 1 mM ethylene glycol-bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) in 20 mM Tris-HCl buffer, pH 7.5, for 2 h, the induction of cellular phosphodiesterase was inhibited by about 80%, and that of extracellular phosphodiesterase by about 65%. When cells were incubated with 1 mM EGTA for 1 h, 2 mM CaCl2 was added and the cells were further incubated for 1 h, the activities of cellular and extracellular phosphodiesterases were increased about 5 and 2.5 times, respectively, compared with those in the EGTA-inhibited cells. Although various other kinds of divalent cations were also studied, Ca2+ had the greatest effect on the induction. These results suggest that Ca2+ may participate in the induction of phosphodiesterase, and thus in the regulation of the development of the cellular slime mold.     

Comparison of properties of the cellular and extracellular phosphodiesterases induced by cyclic adenosine 3',5'-monophosphate in Dictyostelium discoideum

The kinetic properties and susceptibilities to various agents of intracellular (particulate and soluble) and extracellular phosphodiesterases [EC 3.1.4.17] of Dictyostelium discoideum induced by cyclic adenosine 3',5'-monophosphate (cyclic AMP) were studied and compared. Intracellular particulate phosphodiesterase was obtained by solubilization of the light mitochondrial fraction with Emulgen. The Michaelis constants of this enzyme were 4.5 +/- 0.7 and 10 +/- 0.7 microM, while those of the intracellular soluble phosphodiesterase were 4.6 +/- 0.3 and 13 +/- 2.8 microM. However, the Michaelis constant of the extracellular phosphodiesterase was 6.8 +/- 0.9 microM, differing from the values of the two intracellular enzymes. Susceptibilities of the enzyme activity to various agents (theophylline, caffeine, dithiothreitol, glutathione, etc.) were essentially the same among these three phosphodiesterases. In the presence of 10 mM ethylenediaminetetraacetate, the activities of the particulate and the soluble enzymes were both decreased to about 60%, while that of the extracellular enzyme remained at 90%. The inhibition constants of cyclic inosine monophosphate for the cellular enzymes (35 and 100 microM for the particulate enzyme, and 37 and 90 microM for the soluble one) were considerably different from the value for the extracellular enzyme (48 microM). These results suggest that the characteristics of these three phosphodiesterases are substantially similar, but that the affinity of the intracellular (particulate and soluble) enzymes for the substrate is somewhat different from that of the extracellular enzyme.     

Effect of treadmill exercise on plasma and urinary cyclic adenosine 3'5'-monophosphate.

The effect of treadmill exercise on plasma and urinary cyclic adenosine 3'5' monophosphate levels (cyclic AMP) was studied in twelve healthy subjects. Plasma cyclic AMP levels were found to be markedly elevated without significant changes in urinary cyclic AMP or cyclic AMP/creatine ratio. Most likely altered plasma glucagon and catecholamine levels were responsible for these changes.     

The relation of endogenous adenosine cyclic 3':5'-monophosphate to the antagonistic effects of adenosine and colchicine on cell shape.

Adenosine and colchicine have antagonistic effects on cell shape. When Chinese Hamster lung fibroblasts (CHE36-6) or SV40 transformed 3T3 (SV3T3) cells are incubated with colchicine (1 muM) for one hour at 37 degrees C, they round up into spheres with short spikes. Cells treated with adenosine (1 muM-minus 4 mM) for one hour become refractile and develop spindly processes. However, when the two compounds are added simultaneously, the characteristic responses to either drug are abolished and the cells appear normal. The counteraction is specific for adenine and its derivatives, adenosine being the most effective of the compounds we tested. Accumulation of colchicine or adenosine is not altered significantly by the presence of the other drug, ruling out decreases in uptake as the basis of the mutual antagonism. The morphological changes can be observed under conditions where there are no changes in intracellular cAMP levels (such as incubation with low concentrations of adenosine or cordycepin, an adenosine analog that cannot be directly converted to cAMP). Colchicine does not alter cAMP content of control or adenosine-treated cells. These data show that adenine compounds have potent effects on cell shape, and the antagonistic effects of adenosine and colchicine on cell shape are not mediated through changes in intracellular cAMP levels.     

The cyclic adenosine 3':5'-monophosphate receptor of Dictyostelium discoideum. Binding characteristics of aggregation-competent cells and variation of binding levels during the life cycle.

Both cyclic guanosine 3':5'-monophosphate and dithiothreitol stimulate binding of cyclic adenosine 3':5'-monophosphate (cAMP) to aggregation-competent amoebae. Both compounds appear to function solely by preventing the hydrolysis of cAMP by the cell-bound phosphodiesterase. The dissociation constant for binding of cAMP is 36 nM. Both cAMP binding and membrane-bound phosphodiesterase activities increase dramatically as cells develop aggregation competence, reach a maximum at about 11 hours, and remain at high levels for up to 48 hours if cells are maintained in shaken suspension. When amoebae are allowed to aggregate and develop naturally, binding of cAMP increases during aggregation, decreases during tip formation, and disappears during culmination. Phosphodiesterase activity parallels binding activity except that the decreased level after tip formation is retained throughout culmination. Two N-6-modified cAMP derivatives compete with cAMP for binding sites. One derivative is fluorescent (1,N-6-etheno-cAMP); the other is photolyzable [N-6(ethyl-2-diazomalonyl)cAMP]. This result opens the possibilities of using fluorescence quenching for assay of in vitro binding and of affinity labeling of binding sites. Competition by the derivatives is only partial, indicating possible heterogeneity of binding sites. Both compounds inhibit hydrolysis of cAMP by the membrane-bound phosphodiesterase.     

Protein inhibitor of cyclic adenosine 3':5'-monophosphate phosphodiesterase in retina.

A protein acting as inhibitor of cyclic 3':5'-nucleotide phosphodiesterase (EC 3.1.4.1.) activity was found in the ox retina tissue. An inhibitor from one tissue (ox retina) effectively cross-inhibited a phosphodiesterase from another tissue (rat brain), indicating a lack of tissue specificity. Kinetic analysis showed that inhibition was independent of the time of preliminary incubation of the inhibitor with enzyme but dependent on its concentration in the reaction mixture. An inhibitor decreased the V of the enzyme and had no effect on its Km for cyclic adenosine-3':5'-monophosphate. The inhibitory effect was more pronounced with cyclic adenosine-3':5'-monophosphate than with cyclic guanosine-3':5'-monophosphate used as substrates of the reaction. The extractable form of the phosphodiesterase of the retina rod outer segments was much more sensitive to the inhibitory action than the membrane-bound one. The binding of labeled cyclic adenosine-3':5'-monophosphate to the inhibitory protein was shown not to occur. The inhibitor was sensitive to trypsin treatment, indicating that it was a proten attempt was mode to purify the inhibitory factor. Gel filtration indicated that the inhibitor had a molecular weight of 38 000.     

Fluctuations in cyclic adenosine 3':5'-monophosphate and cyclic guanosine 3':5'-monophosphate during the mitotic cycle of the acellular slime mould Physarum polycephalum.

Cyclic adenosine 3′:5′-monophosphate (cyclic AMP) and cyclic guanosine 3′:5′-monophosphate (cyclic GMP) have been determined at half-hourly intervals throughout the mitotic cycle of Physarum polycephalum. Cyclic AMP was constant at 1pmole/mg protein throughout except for a transient peak of 17pmoles/mg protein in the last quarter of G2. Cyclic GMP was more variable (2–4pmole/mg protein) rising to 9.5pmole/mg protein during the 3 hour S period and to 7pmole/mg protein during the last hour of G2. The significance of these changes is discussed.     

Compartmentation of cyclic adenosine 3',5'-monophosphate signaling in caveolae.

The cAMP-signaling pathway is composed of multiple components ranging from receptors, G proteins, and adenylyl cyclase to protein kinase A. A common view of the molecular interaction between them is that these molecules are disseminated on the plasma lipid membrane and random collide with each other to transmit signals. A limitation to this idea, however, is that a signaling cascade involving multiple components may not occur rapidly. Caveolae and their principal component, caveolin, have been implicated in transmembrane signaling, particularly in G protein-coupled signaling. We examined whether caveolin interacts with adenylyl cyclase, the membrane-bound enzyme that catalyzes the conversion of ATP to cAMP. When overexpressed in insect cells, types III, IV, and V adenylyl cyclase were localized in caveolin-enriched membrane fractions. Caveolin was coimmunoprecipitated with adenylyl cyclase in tissue homogenates and copurified with a polyhistidine-tagged form of adenylyl cyclase by Ninitrilotriacetic acid resin chromatography in insect cells, suggesting the colocalization of adenylyl cyclase and caveolin in the same microdomain. Further, the regulatory subunit of protein kinase A (RIIalpha, but not RIalpha) was also enriched in the same fraction as caveolin. Gsalpha was found in both caveolin-enriched and non-caveolin-enriched membrane fractions. Our data suggest that the cAMP-signaling cascade occurs within a restricted microdomain of the plasma membrane in a highly organized manner.     

Photoaffinity labeling of the cell surface adenosine 3':5'-monophosphate receptor of Dictyostelium discoideum and its modification in down-regulated cells

The cAMP cell surface receptor of Dictyostelium discoideum amoebae was identified by the use of the photoaffinity analogue 8-N3-[32P]cAMP. Labeling by intact cells of one component, identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography, could be specifically inhibited by the presence of nonradioactive cAMP. The component, P45 (apparent molecular weight of 45,000), was not identified on vegetative cells but was labeled with increasing intensity as cells differentiated and increased their levels of surface cAMP binding sites. Developmental mutants, starved under conditions where they do not express significant levels of cAMP binding sites, did not incorporate radioactivity into this protein. These mutants did label P45 when starved under differentiation-inducing conditions such that their levels of surface cAMP binding sites increased. P45 co-purified with the plasma membrane fraction isolated from cells to which 8-N3-[32p]cAMP had been covalently bound. Down-regulated amoebae, which displayed approximately 25% of the binding activity of untreated cells, did not label P45. These cells did, however, label a new component with an apparent molecular weight of 47,000 (P47).l The appearance of this component represented the only discernible difference in labeling profile under these conditions. As in the case of P45, radioactive incorporation into P47 did not occur if the photoactivation of 8-N3-[32P]cAMP was performed in the presence of nonradioactive cAMP.     

Studies on the presence of adenosine cyclic 3':5'-monophosphate in oat coleoptiles

The incorporation of adenosine-8-¹⁴C into adenosine cyclic 3′:5′-monophosphate in coleoptile-first leaf segments of Avena sativa L. was investigated. Homogenates of segments incubated in adenosine-8-¹⁴C for either 4 or 10 hours were partially purified by thin layer chromatography followed by paper electrophoresis. A radioactive fraction, less than 0.06% of the ¹⁴C present in the original homogenate, migrated as adenosine cyclic 3′:5′-monophosphate during electrophoresis. Upon treatment with cyclic nucleotide phosphodiesterase, however, less than 10% of this radioactive fraction appeared as 5′-AMP. Deamination with NaNO₂ as well as further chromatographical purification also suggested that only a small fraction of the ¹⁴C in the partially purified samples could be in adenosine cyclic 3′:5′-monophosphate. The data suggest that levels of this nucleotide can probably be no greater than 7 to 11 picomoles per gram of fresh weight in oat coleoptiles. Treatment of such coleoptiles with physiologically active concentrations of indoleacetic acid, furthermore, had no significant effect on the ¹⁴C radioactivity in marker adenosine cyclic 3′:5′-monophosphate-containing fractions at any stage of purification during several experiments.