scholarly journals Cyclic 3',5' AMP relay in Dictyostelium discoideum. II. Requirements for the initiation and termination of the response.

1979 ◽  
Vol 80 (2) ◽  
pp. 300-309 ◽  
Author(s):  
P N Devreotes ◽  
T L Steck
Keyword(s):  
Dictyostelium Discoideum ◽  
Recovery Period ◽  
Stimulus Level ◽  
Secretion Rate ◽  
Rapid Decline ◽  
Duration Magnitude ◽  
Total Magnitude ◽  
Integrated Response ◽  
Extracellular Camp ◽  
Camp Stimulation

The secretion of 3H-cyclic adenosine 3',5'-monophosphate (cAMP) by prelabeled and suitably differentiated Dictyostelium discoideum amoebae was elicited in a perfusion apparatus by 10(-10) to 10(-5) M [14C]cAMP stimuli of defined magnitude and duration. Exogenous stimuli evoked an immediate increase in the rate of [3H]cAMP secretion which accelerated continuously to reach a peak of up to 100 times the unstimulated rate after 2--3 min of stimulation. Withdrawal of the stimulus at any time during the response led to a rapid decline to basal levels. Furthermore, a spontaneous decline in secretion rate was observed during prolonged cAMP stimulation, with a return to basal levels after 3--8 min of stimulation. After the initial secretory event, cells did not respond further to the continued presence of external [14C]cAMP unless (a) it was interrupted by a brief recovery period or (b) the level of the stimulus was increased sufficiently. Since the second increment could follow the first at any time, continuous secretion of [3H]cAMP could be sustained for up to 30 min by progressively increasing the stimulus between 10(-10) and 10(-5) M cAMP. The total magnitude of spontaneously terminated responses depended on the size of the increment in applied cAMP, larger stimuli evoking both a more rapid acceleration and a slower deceleration in [3H]cAMP secretion rate. The integrated response to a given increment in stimulus level was apparently independent of its "shape" - i.e., the duration, magnitude, and number of sub-steps in the increment. These data support two mechanistic inferences: that amoebae respond in proportion to relative increases in extracellular cAMP concentration, but adapt to the concentration of cAMP itself. The data further suggest that the initiation and termination of the response are mediated by cellular component(s) beyond cAMP-occupied receptors.

scholarly journals Cyclic 3', 5'-AMP relay dictyostelium discoideum. V. Adaptation of the cAMP signaling response during cAMP stimulation

1980 ◽  
Vol 86 (2) ◽  
pp. 554-561 ◽  
Author(s):  
M Dinauer ◽  
TL Steck ◽  
P Devreotes
Keyword(s):  
Adenylate Cyclase ◽  
Test Stimulus ◽  
Dictyostelium Discoideum ◽  
Time Course ◽  
Intracellular Camp ◽  
Perfusion Technique ◽  
Adaptation Mechanism ◽  
Cellular Processes ◽  
Extracellular Camp ◽  
Camp Stimulation

In dictyostelium discoideum, extracellular cAMP activates adenylate cyclase, which leads to an increase in intracellular cAMP and the rate of cAMP secretion. The signaling response to a constant cAMP stimulus is terminated after several minutes by an adaptation mechanism. The time- course of adaptation stimuli of 10(-6) or 10(-7) M cAMP was assessed. We used a perfusion technique to deliver defined cAMP stimuli to [(3)H]adenosine-labeled amoebae and monitored their secretion of [(3)H]cAMP. Amoebae were pretreated with 10(-6) or 10(-7) M cAMP to periods of 0.33-12 minutes, and then immediately given test stimuli of 10(-8) M to 2.5 x 10(-7) M cAMP. The response to a given test stimulus was progressively attenuated and finally extinguished as the duration of the pretreatment stimulus increased. During concentration of the test stimulus. The responses to test stimuli of 10(-8), 5 x 10(-8), 10(-7), or 2.5 x 10(-7) M cAMP were extinguished after approximately 1, 2.25,2.5, and 10 min, respectively. 1.5 min of stimulation with 10(-7) M cAMP was necessary to extinguish the response of a test stimulus of 10(-8) M cAMP. Our data suggest that adaptation begins within 20 s of stimulation, rises rapidly for approximately 2.5 min, and reaches a plateau after approximately 10 min. The absolute rate of rise was faster during pretreatment with 10(-6) than with 10(-7) M cAMP. These results support a working hypothesis in which the occupancy of surface cAMP receptors leads to changes in two opposing cellular processes, excitation and adaptation, that control the activity of D. discoideum adenylate cyclase.

scholarly journals Functional Promiscuity of Gene Regulation by Serpentine Receptors in Dictyostelium discoideum

1998 ◽  
Vol 18 (10) ◽  
pp. 5744-5749 ◽  
Author(s):  
Irene Verkerke-Van Wijk ◽  
Ji-Yun Kim ◽  
Raymond Brandt ◽  
Peter N. Devreotes ◽  
Pauline Schaap
Keyword(s):  
Gene Expression ◽  
Cell Fate ◽  
Dictyostelium Discoideum ◽  
Developmental Regulation ◽  
Mutant Cell ◽  
Gene Induction ◽  
Specific Gene ◽  
Wild Type ◽  
Animal Development ◽  
Camp Stimulation

ABSTRACT Serpentine receptors such as smoothened and frizzled play important roles in cell fate determination during animal development. InDictyostelium discoideum, four serpentine cyclic AMP (cAMP) receptors (cARs) regulate expression of multiple classes of developmental genes. To understand their function, it is essential to know whether each cAR is coupled to a specific gene regulatory pathway or whether specificity results from the different developmental regulation of individual cARs. To distinguish between these possibilities, we measured gene induction in car1 car3 double mutant cell lines that express equal levels of either cAR1, cAR2, or cAR3 under a constitutive promoter. We found that all cARs efficiently mediate both aggregative gene induction by cAMP pulses and induction of postaggregative and prespore genes by persistent cAMP stimulation. Two exceptions to this functional promiscuity were observed. (i) Only cAR1 can mediate adenosine inhibition of cAMP-induced prespore gene expression, a phenomenon that was found earlier in wild-type cells. cAR1’s mediation of adenosine inhibition suggests that cAR1 normally mediates prespore gene induction. (ii) Only cAR2 allows entry into the prestalk pathway. Prestalk gene expression is induced by differentiation-inducing factor (DIF) but only after cells have been prestimulated with cAMP. We found that DIF-induced prestalk gene expression is 10 times higher in constitutive cAR2 expressors than in constitutive cAR1 or cAR3 expressors (which still have endogenous cAR2), suggesting that cAR2 mediates induction of DIF competence. Since in wild-type slugs cAR2 is expressed only in anterior cells, this could explain the so far puzzling observations that prestalk cells differentiate at the anterior region but that DIF levels are actually higher at the posterior region. After the initial induction of DIF competence, cAMP becomes a repressor of prestalk gene expression. This function can again be mediated by cAR1, cAR2, and cAR3.

scholarly journals cAMP induces a rapid and reversible modification of the chemotactic receptor in Dictyostelium discoideum.

1985 ◽  
Vol 100 (3) ◽  
pp. 715-720 ◽  
Author(s):  
C Klein ◽  
J Lubs-Haukeness ◽  
S Simons
Keyword(s):  
Dictyostelium Discoideum ◽  
Concentration Dependence ◽  
Time Course ◽  
Camp Synthesis ◽  
Sds Page ◽  
Reversible Modification ◽  
Camp Receptor ◽  
Camp Stimulation ◽  
Stimulation Of

Stimulation, within 1 min after cAMP stimulation, of aggregation-competent Dictyostelium discoideum amebae was found to cause a rapid (within 1 min) modification of the cell's surface cAMP receptor. The modified receptor migrated on SDS PAGE as a 47,000-mol-wt protein, as opposed to a 45,000-mol-wt protein labeled on unstimulated cells. The length of time this modified receptor could be detected depended upon the strength of the cAMP stimulus: 3-4 min after treatment with 10(-7) M cAMP, cells no longer possessed the 47,000-mol-wt form of the cAMP receptor. Instead, the 45,000-mol-wt form was present. Stimulation of cells with 10(-5) M cAMP, however, resulted in the persistent (over 15 min) expression of the modified receptor. The time course, concentration dependence, and specificity of stimulus for this cAMP-induced shift in the cAMP receptor were found to parallel the cAMP-stimulated phosphorylation of a 47,000-mol-wt protein. In addition, both phenomena were shown to occur in the absence of endogenous cAMP synthesis. The possibility that the cAMP receptor is phosphorylated in response to cAMP stimulation, and the role of this event in cell desensitization, are discussed.

scholarly journals Independent control of locomotion and orientation during Dictyostelium discoideum chemotaxis

1992 ◽  
Vol 102 (4) ◽  
pp. 763-768
Author(s):  
B. Van Duijn ◽  
P.J. Van Haastert
Keyword(s):  
Dictyostelium Discoideum ◽  
High Voltage ◽  
Cell Movement ◽  
Cell Length ◽  
Independent Control ◽  
Motile Cells ◽  
Two Component ◽  
Extracellular Camp

Chemotaxis is cell movement in the direction of a chemical and is composed of two component: movement and directionality. The directionality of eukaryotic chemotaxis is probably derived from orientation: the detection of the spacial gradient of chemoattractant over the cell length. Chemotaxis was investigated in eukaryotic Dictyostelium discoideum cells that were permeabilized by high-voltage discharges. These permeable cells respond chemotactically to extracellular cAMP. However, locomotion is impaired if the Ca2+ concentration is clamped at submicromolar concentrations; interestingly, these non-motile cells still form pseudopodia and elongate in the direction of the cAMP gradient. These results imply that locomotion and orientation during Dictyostelium chemotaxis are independently regulated.

scholarly journals Excitation, adaption, and deadaptation of the cAMP-mediated cGMP Response in dictyostelium discoideum

1983 ◽  
Vol 96 (2) ◽  
pp. 347-353 ◽  
Author(s):  
PJM Van Haaster ◽  
PR Van Der Heijden
Keyword(s):  
Cell Suspension ◽  
Dictyostelium Discoideum ◽  
Half Life ◽  
Cell Aggregation ◽  
First Order ◽  
Intracellular Cgmp ◽  
First Order Kinetics ◽  
A Cell ◽  
Stimulation Period ◽  
Extracellular Camp

Extracellular cAMP induces chemotaxis and cell aggregation in dictyostelium discoideum cells. cAMP added to a cell suspension is rapidly hydrolyzed (half-life of 10 s) and induces a rapid increase of intracellular cGMP levels, which reach a peak at 10 s and recover prestimulated levels at about 30 s. This recovery is not due to removal of the stimulus because the nonhydrolyzable analogue adenosine 3',5'-monophosphorothioate-Sp- stereoisomer (cAMPS) induced a comparable cGMP response, which peaked at 10 s, even at subsaturating cAMPS concentrations. When cells were stimulated twice with the same cAMP concentration at a 30-s interval, only the first stimulus produced a cGMP response. Cells did respond to the second stimulus when the concentration of the second stimulus was higher than that of the first stimulus. By increasing the interval between two identical stimuli, the response to the second stimulus gradually increased. Recovery from the first stimulus showed first-order kinetics with a half-life of 1-2 min. The stimulation period was shortened by adding phosphodieterase to the cell suspension. The cGMP response was unaltered if the half-life of cAMP was reduced to 2 S. The peak of the transient cGMP accumulation still appeared at 10 s even when the half- life of cAMP was 0.4 s; however, the height of the cGMP peak was reduced. The cGMP response at 10 s after stimulation was diminished by 50 percent when the half-life of 10(-7) M cAMP was 0.5 s or when the half-life of 10(-8) M cAMP was 3.0 s. These results show that the cAMP signal is transduced to two opposing processes: excitation and adaptation. Within 10 s after addition of cAMP to a cell suspension the level of adaptation reaches the level of excitation, which causes the extinction of the transduction of the signal. Deadaptation starts as soon as the signal is removed, and it has first-order kinetics with a half-life of 1-2 min.

Ammonia promotes accumulation of intracellular cAMP in differentiating amoebae of Dictyostelium discoideum

Development ◽  
1990 ◽  
Vol 109 (3) ◽  
pp. 715-722
Author(s):  
B.B. Riley ◽  
S.L. Barclay
Keyword(s):  
Dictyostelium Discoideum ◽  
Active Species ◽  
Spore Formation ◽  
Intracellular Camp ◽  
Simultaneous Exposure ◽  
High Ph ◽  
Camp Hydrolysis ◽  
Extracellular Camp ◽  
Camp Levels

We used sporogenous mutants of Dictyostelium discoideum to investigate the mechanism(s) by which exogenous NH4Cl and high ambient pH promote spore formation during in vitro differentiation. The level of NH4Cl required to optimize spore formation is correlated inversely with pH, indicating that NH3 rather than NH4+ is the active species. The spore-promoting activity of high ambient pH (without exogenous NH4Cl) was eliminated by the addition of an NH3-scavenging cocktail, suggesting that high pH promotes spore differentiation by increasing the ratio of NH3:NH4+ secreted into the medium by developing cells. High ammonia levels and high pH stimulated precocious accumulation of intracellular cAMP in both sporogenous and wild-type cells. In both treatments, peak cAMP levels equaled or exceeded control levels and were maintained for longer periods than in control cells. In contrast, ammonia strongly inhibited accumulation of extracellular cAMP without increasing the rate of extracellular cAMP hydrolysis, indicating that ammonia promotes accumulation of intracellular cAMP by inhibiting cAMP secretion. These results are consistent with previous observations that factors that raise intracellular cAMP levels increase spore formation. Lowering intracellular cAMP levels with caffeine or progesterone inhibited spore formation, but simultaneous exposure to these drugs and optimal concentrations of NH4Cl restored both cAMP accumulation and spore formation to normal levels. These data suggest that ammonia, which is a natural Dictyostelium morphogen, favors spore formation by promoting accumulation or maintenance of high intracellular cAMP levels.

A cis-acting element responsible for early gene induction by extracellular cAMP in Dictyostelium discoideum

1991 ◽  
Vol 33 (2) ◽  
pp. 147-155 ◽  
Author(s):  
Thomas May ◽  
Jürgen Blusch ◽  
Astrid Sachse ◽  
Nellen Wolfgang
Keyword(s):  
Dictyostelium Discoideum ◽  
Gene Induction ◽  
Early Gene ◽  
Cis Acting ◽  
Extracellular Camp

scholarly journals Arrestins function in cAR1 GPCR-mediated signaling and cAR1 internalization in the development of Dictyostelium discoideum

2014 ◽  
Vol 25 (20) ◽  
pp. 3210-3221 ◽  
Author(s):  
Xiumei Cao ◽  
Jianshe Yan ◽  
Shi Shu ◽  
Joseph A. Brzostowski ◽  
Tian Jin
Keyword(s):  
Dictyostelium Discoideum ◽  
Protein C ◽  
Wild Type ◽  
Multicellular Development ◽  
Membrane Recruitment ◽  
Camp Receptor ◽  
Erk2 Activation ◽  
G Protein Coupled ◽  
Camp Stimulation ◽  
Signaling Circuit

Oscillation of chemical signals is a common biological phenomenon, but its regulation is poorly understood. At the aggregation stage of Dictyostelium discoideum development, the chemoattractant cAMP is synthesized and released at 6-min intervals, directing cell migration. Although the G protein–coupled cAMP receptor cAR1 and ERK2 are both implicated in regulating the oscillation, the signaling circuit remains unknown. Here we report that D. discoideum arrestins regulate the frequency of cAMP oscillation and may link cAR1 signaling to oscillatory ERK2 activity. Cells lacking arrestins (adcB−C−) display cAMP oscillations during the aggregation stage that are twice as frequent as for wild- type cells. The adcB−C− cells also have a shorter period of transient ERK2 activity and precociously reactivate ERK2 in response to cAMP stimulation. We show that arrestin domain–containing protein C (AdcC) associates with ERK2 and that activation of cAR1 promotes the transient membrane recruitment of AdcC and interaction with cAR1, indicating that arrestins function in cAR1-controlled periodic ERK2 activation and oscillatory cAMP signaling in the aggregation stage of D. discoideum development. In addition, ligand-induced cAR1 internalization is compromised in adcB−C− cells, suggesting that arrestins are involved in elimination of high-affinity cAR1 receptors from cell surface after the aggregation stage of multicellular development.

scholarly journals EppA, a Putative Substrate of DdERK2, Regulates Cyclic AMP Relay and Chemotaxis in Dictyostelium discoideum

2006 ◽  
Vol 5 (7) ◽  
pp. 1136-1146 ◽  
Author(s):  
Songyang Chen ◽  
Jeffrey E. Segall
Keyword(s):  
Cyclic Amp ◽  
Dictyostelium Discoideum ◽  
Mitogen Activated Protein Kinase ◽  
Intracellular Camp ◽  
Mitogen Activated Protein ◽  
Delayed Development ◽  
Induced Signal ◽  
Extracellular Camp ◽  
Intracellular Camp Accumulation ◽  
Dimensional Electrophoresis

ABSTRACT The mitogen-activated protein kinase DdERK2 is critical for cyclic AMP (cAMP) relay and chemotaxis to cAMP and folate, but the details downstream of DdERK2 are unclear. To search for targets of DdERK2 in Dictyostelium discoideum,32PO4 3−-labeled protein samples from wild-type and Dderk2 − cells were resolved by 2-dimensional electrophoresis. Mass spectrometry was used to identify a novel 45-kDa protein, named EppA (ERK2-dependent phosphoprotein A), as a substrate of DdERK2 in Dictyostelium. Mutation of potential DdERK2 phosphorylation sites demonstrated that phosphorylation on serine 250 of EppA is DdERK2 dependent. Changing serine 250 to alanine delayed development of Dictyostelium and reduced Dictyostelium chemotaxis to cAMP. Although overexpression of EppA had no significant effect on the development or chemotaxis of Dictyostelium, disruption of the eppA gene led to delayed development and reduced chemotactic responses to both cAMP and folate. Both eppA gene disruption and overexpression of EppA carrying the serine 250-to-alanine mutation led to inhibition of intracellular cAMP accumulation in response to chemoattractant cAMP, a pivotal process in Dictyostelium chemotaxis and development. Our studies indicate that EppA regulates extracellular cAMP-induced signal relay and chemotaxis of Dictyostelium.

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