Intracellular localization of secretable cAMP in relaying Dictyostelium discoideum cells (original) (raw)
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Proceedings of the National Academy of Sciences, 1987
cAMP has been implicated in the control of the expression of developmental genes in Dictyostelium discoideum. To determine the potential role of cAMP receptors as regulators of gene expression, we have used immunocytochemical and immunoblotting techniques to reveal the subcellular localization of a cAMP binding protein CABP1. Most of the CABP1 antigen in early developing cells is localized near the cell periphery, with a small amount found in the nucleus. The level of CABP1 in the nucleus increases approximately 30-fold during development. Moreover, immunofluorescence studies showed that CABP1 can also be detected on the cell surface. Binding of anti-CABP1 to intact cells followed by reaction with 125I-labeled secondary antibody revealed that the cell-surface CABP1 activity peaks during aggregation and culmination. In addition, several proteins related to CABP1 are found mainly in the nuclear fraction of developing cells. The possible role of these proteins in the regulation of deve...
cAMP signaling in Dictyostelium
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.
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
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.
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.
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...
Proceedings of the National Academy of Sciences, 1993
Dictyostelium discoideum is among the best characterized organisms for the study of receptor/guanine nucleotide binding protein-mediated control of differentiation. Dictyostelium grow unicellularly but form fully differentiated multicellular organisms through a developmental program regulated by secreted cAMP activating specific cell-surface receptors. Dictyostelium respond differentially to cAMP at different developmental stages. During early development, expression of certain genes is induced by low-level oscillations of extracellular cAMP. Later, continuous, high cAMP concentrations will promote expression of specific genes in multicellular structures. Here, we show that the cAMP receptor gene CARI, which is essential for development, utilizes two promoters that are activated at distinct stages of development and respond to different extracellular cAMP conditions. One promoter is active with low-level oscillations of cAMP; exposure to high cAMP concentrations will repress this promoter and induce a second promoter. The CARI mRNAs are alternatively spliced but encode identical proteins. Thus, through differential sensitivity to its own ligand, cAMP, two promoters and alternative splicing regulate CARI expression during Dictyostelium development.
The Journal of Cell Biology, 1980
In Dictyostelium discoideum, an increase in extracellular CAMP activates adenylate cyclase, leading to an increase in intracellular CAMP and the rate of CAMP secretion . Cells adapt to any constant CAMP stimulus after several minutes, but still respond to an increase in the concentration of the stimulus . We have now characterized the decay of adaptation (deadaptation) after the removal of CAMP stimuli . Levels of adaptation were established by the perfusion of [3 H]adenosine-labeled amoebae with a defined CAMP stimulus . After a variable recovery period, the magnitude of the signaling response to a second stimulus was measured ; its attenuation was taken as a measure of residual adaptation to the first stimulus . The level of adaptation established by the first stimulus depended on both its magnitude and duration . Deadaptation began as soon as the first stimulus was removed . The magnitude of the response to the second stimulus increased with the recovery time in a first-order fashion, with a t1 /2 = 3-4 min for stimuli of 10 -8 M to 10 -5 M CAMP .
Journal of Biological Chemistry
Dictyostelium discoideum cell membranes contain masked chemotactic receptors for cyclic AMP
FEBS Letters, 1984
Aggregating Dictyostelium dz3coideum cells possess highly specific receptors for the chemoattractant CAMP on their cell surface. Isolated membranes as well as intact cells are shown to contain a large number of latent CAMP receptors. These are reversibly unmasked in the presence of a high salt concentration (0. 1-2 M) or in the presence of millimolar concentrations of CaZ+.