cAMP signaling in Dictyostelium (original) (raw)
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CAMP signaling in Dictyostelium - Complexity of cAMP synthesis, degradation and detection
Journal of Muscle Research and Cell Motility, 2002
cAMP plays a pivotal role in control of cell movement, differentiation and response to stress in all phases of the Dictyostelium life cycle. The multitudinous functions of cAMP require precise spatial and temporal control of its production, degradation and detection. Many novel proteins have recently been identified that critically modulate the cAMP signal. We focus in this review on the properties and functions of the three adenylyl cyclases and the three cAMP-phosphodiesterases that are present in Dictyostelium, and the network of proteins that regulate the activity of these enzymes. We also briefly discuss the two modes of detection of cAMP.
Molecular Biology of the Cell, 1996
In Dictyostelium, cAMP plays a role as an intracellular second messenger and in addition, as an extracellular first messenger. Both functions are thought to be tightly linked because adenylyl cyclase is coupled via G-proteins to the cell surface cAMP receptor cAR 1. Using the discoidin I gene family as a molecular marker for the first stages of development, we show here that induction of transcription requires the G-protein subunit a2 and thus an as yet unidentified surface receptor, CRAC (cytosolic regulator of adenylyl cyclase), and PKA. Induction can be conferred by an increase in intracellular cAMP. In contrast, transcriptional down-regulation occurs by stimulation of cAR 1 with extracellular cAMP and a subsequent, G-protein-independent Ca2+ influx. In a Ga2 gene disruption mutant, discoidin I expression can be efficiently modulated by analogues simulating intracellular cAMP (discoidin induction) and extracellular cAMP (discoidin down-regulation). We thus demonstrate possible antagonistic functions of intra-and extracellular cAMP.
Characterization of a cAMP-stimulated cAMP Phosphodiesterase in Dictyostelium discoideum
Journal of Biological Chemistry, 2003
A cyclic nucleotide phosphodiesterase, PdeE, that harbors two cyclic nucleotide binding motifs and a binuclear Zn 2؉-binding domain was characterized in Dictyostelium. In other eukaryotes, the Dictyostelium domain shows greatest homology to the 73-kDa subunit of the pre-mRNA cleavage and polyadenylation specificity factor. The Dictyostelium PdeE gene is expressed at its highest levels during aggregation, and its disruption causes the loss of a cAMP-phosphodiesterase activity. The pdeE null mutants show a normal cAMP-induced cGMP response and a 1.5-fold increase of cAMP-induced cAMP relay. Overexpression of a PdeE-yellow fluorescent protein (YFP) fusion construct causes inhibition of aggregation and loss of the cAMP relay response, but the cells can aggregate in synergy with wild-type cells. The PdeE-YFP fusion protein was partially purified by immunoprecipitation and biochemically characterized. PdeE and its Dictyostelium ortholog, PdeD, are both maximally active at pH 7.0. Both enzymes require bivalent cations for activity. The common cofactors Zn 2؉ and Mg 2؉ activated PdeE and PdeD maximally at 10 mM, whereas Mn 2؉ activated the enzymes to 4-fold higher levels, with half-maximal activation between 10 and 100 M. PdeE is an allosteric enzyme, which is ϳ4-fold activated by cAMP, with half-maximal activation occurring at about 10 M and an apparent K m of ϳ1 mM. cGMP is degraded at a 6-fold lower rate than cAMP. Neither cGMP nor 8-Br-cAMP are efficient activators of PdeE activity.
Expression of a cAMP receptor gene of Dictyostelium and evidence for a multigene family
Genes & Development, 1991
We have previously reported the cloning of cDNAs for a Dictyostelium cell-surface cAMP receptor that is a member of the family of G-protein-linked receptors. Here, we report the organization and the developmental expression of this cAMP receptor gene, designated CARl. CARl is a single copy gene that contains two intervening sequences. CARl mRNA levels are low in growing cells, rise to peak expression at 5-10 hr of development when the cAMP signaling system is maximally active, and decrease as development proceeds. At 5 hr the predominant mRNA species is -1.9 kb, by 10 hr the mRNA is heterogeneous with sizes of -1.9-2.1 kb, but during culmination only the 2.1 kb mRNA is detected. The variety of mRNA sizes results from differences in 5'-untranslated regions. Studies using developmental mutants with aberrant cAMP-signaling patterns indicate that pulsatile action of cAMP promotes maximal expression of CARl during early development. Low stringency hybridization of CARl probes to genomic DNA detects additional, related sequences, suggesting that there are several genes that encode a family of structurally similar receptors. Multiple functions previously attributed to the cAMP receptor instead may be fulfilled by distinct receptor subtypes encoded by specific genes.
G alpha 3 regulates the cAMP signaling system in Dictyostelium
Molecular Biology of the Cell, 1997
The Dictyostelium discoideum developmental program is initiated by starvation and its progress depends on G-protein-regulated transmembrane signaling. Disruption of the Dictyostelium G-protein a-subunit Ga3 (ga3-) blocks development unless the mutant is starved in the presence of artificial cAMP pulses. The function of Ga3 was investigated by examining the expression of several components of the cAMP transmembrane signaling system in the ga3mutant. cAMP receptor 1 protein, cyclic nucleotide phosphodiesterase, phosphodiesterase inhibitor, and aggregation-stage adenylyl cyclase mRNA expression were absent or greatly reduced when cells were starved without exogenously applied pulses of cAMP. However, cAMP receptor 1 protein and aggregation-stage adenylyl cyclase mRNA expression were restored by starving the ga3-cells in the presence of exogenous cAMP pulses. Adenylyl cyclase activity was also reduced in ga3cells starved without exogenous cAMP pulses compared with similarly treated wild-type cells but was elevated to a level twofold greater than wild-type cells in ga3cells starved in the presence of exogenous cAMP pulses. These results suggest that Ga3 is essential in early development because it controls the expression of components of the transmembrane signaling system.
Ligand-induced phosphorylation of the cAMP receptor from Dictyostelium discoideum
The Journal of biological chemistry, 1988
The cell surface cAMP receptor of Dictyostelium discoideum exists as a doublet of low (D) and high (R) electrophoretic mobility forms, both of which are phosphorylated in vivo. The R form is phosphorylated in a ligand-independent manner, while conversion of the R to D forms, induced by the chemoattractant, is accompanied by at least a 4-fold increase in the level of phosphorylation. When cells are stimulated with saturating levels of cAMP, increased phosphorylation is detectable within 5 s and reaches maximum levels by 5 min with a t1/2 of 45 s. Dephosphorylation of receptor, initiated by removal of the stimulus, is detectable within 30 s, has a half-time of 2 min, and reaches a plateau by 20 min. At half-maximal occupancy, phosphorylation occurred more slowly than at saturation, t1/2 = 1.5 min, and remained at intermediate levels until the cAMP concentration was increased. Accompanying electrophoretic mobility shifts occurred in all cases with similar, though not identical, kinetic...
Overexpression of the cAMP receptor 1 in growing Dictyostelium cells
Biochemistry, 1991
cAR1, the cAMP receptor expressed normally during the early aggregation stage of the Dictyostelium developmental program, has been expressed during the growth stage, when only low amounts of endogenous receptors are present. Transformants expressing CAR1 have 7-40 times over growth stage and 3-5-fold over aggregation stage levels of endogenous receptors. The high amounts of CAR1 protein expressed constitutively throughout early development did not drastically disrupt the developmental program; the onset of aggregation was delayed by 1-3 h, and then subsequent stages proceeded normally. The affinity of the expressed CAR1 was similar to that of the endogenous receptors in aggregation stage cells when measured either in phosphate buffer (two affinity states with Kd's of approximately 30 and 300 nM) or
An intersection of the cAMP/PKA and two-component signal transduction systems in Dictyostelium
The EMBO Journal, 1998
contributed equally to this work Terminal differentiation of both stalk and spore cells in Dictyostelium can be triggered by activation of cAMP-dependent protein kinase (PKA). A screen for mutants where stalk and spore cells mature in isolation produced three genes which may act as negative regulators of PKA: rdeC (encoding the PKA regulatory subunit), regA and rdeA. The biochemical properties of RegA were studied in detail. One domain is a cAMP phosphodiesterase (K m~5 µM); the other is homologous to response regulators (RRs) of two-component signal transduction systems. It can accept phosphate from acetyl phosphate in a reaction typical of RRs, with transfer dependent on Asp212, the predicted phosphoacceptor. RegA phosphodiesterase activity is stimulated up to 8-fold by the phosphodonor phosphoramidate, with stimulation again dependent on Asp212. This indicates that phosphorylation of the RR domain activates the phosphodiesterase domain. Overexpression of the RR domain in wild-type cells phenocopies a regA null. We interpret this dominantnegative effect as due to a diversion of the normal flow of phosphates from RegA, thus preventing its activation. Mutation of rdeA is known to produce elevated cAMP levels. We propose that cAMP breakdown is controlled by a phosphorelay system which activates RegA, and may include RdeA. Cell maturation should be triggered when this system is inhibited.
Genes & Development, 1993
A cAMP receptor (cAR1), which is expressed during early aggregation, has been cloned and sequenced previously. We have identified a new receptor subtype, cAR3, that has -56% and 69% amino acid identity with cAR1 and cAR2, respectively, cAR1, cAR2, or cAR3 expressed from plasmid in growing Dictyostelinm cells can be photoaffinity labeled with 8-N3132p]cAMP and phosphorylated when stimulated with cAMP. cAR3 RNA was not present during growth but appeared during late aggregation. Its expression peaked at 9 hr and then fell to a reduced level that was maintained until culmination. The expression of cAR3 protein followed a similar pattern, but with a 3-hr lag, and reached a maximum at the mound stage. In contrast, cAR1 protein was expressed predominantly during early aggregation and at low levels during later stages. At their respective peaks of expression, there were -5 x 103 cAR3 sites per cell compared with -7 x 104 cAR1 sites per cell. The cAR3 gene was disrupted by homologous recombination in several different parental cell lines. Surprisingly, the car3cell lines display no obvious phenotype.