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 Spatial and temporal expression of the Dictyostelium discoideum G alpha protein subunit G alpha 2: expression of a dominant negative protein inhibits proper prestalk to stalk differentiation.

1994 ◽  
Vol 5 (1) ◽  
pp. 7-16 ◽  
Author(s):  
F Carrel ◽  
S Dharmawardhane ◽  
A M Clark ◽  
J A Powell-Coffman ◽  
R A Firtel
Keyword(s):  
Dictyostelium Discoideum ◽  
Receptor Activation ◽  
Dominant Negative ◽  
Wild Type ◽  
Protein Subunit ◽  
Multicellular Development ◽  
Stage Of Development ◽  
Downstream Effectors ◽  
Camp Receptor

Previous results have shown that the G alpha protein subunit G alpha 2 is required for aggregation in Dictyostelium discoideum and is essential for coupling cell-surface cAMP receptors to downstream effectors in vivo during this stage of development. G alpha 2 expresses at least four distinct transcripts that are differentially regulated during development; two of the transcripts are expressed exclusively in the multicellular stages and their expression is restricted to prestalk cells. We partially dissected the G alpha 2 promoter and identified a component that is expressed exclusively during the multicellular stages using luciferase gene fusions. When this promoter region is coupled to lacZ, beta-gal expression is restricted to the multicellular stages and localized in prestalk cells with a pattern similar to that of the ecmA prestalk-specific promoter. We show that expression in wild-type cells of the G alpha 2 mutant protein [G alpha 2(G206T)] during the early stages of development blocks aggregation and cAMP-mediated activation of adenylyl cyclase and guanylyl cyclase, suggesting it functions as a dominant negatively active G alpha subunit. When this mutant G alpha protein is expressed from the ecmA prestalk-specific promoter, abnormal stalk differentiation during culmination is observed. Expression of the mutant G alpha 2 from the SP60 prespore promoter or wild-type G alpha 2 from either the ecmA or the SP60 promoter results in no detectable phenotype. The results suggest that G alpha 2 plays an essential role during the culmination stage in prestalk cells and may mediate cAMP receptor activation of these processes during multicellular development.

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 CbfA, the C-Module DNA-Binding Factor, Plays an Essential Role in the Initiation of Dictyostelium discoideum Development

2004 ◽  
Vol 3 (5) ◽  
pp. 1349-1358 ◽  
Author(s):  
Thomas Winckler ◽  
Negin Iranfar ◽  
Peter Beck ◽  
Ingo Jennes ◽  
Oliver Siol ◽  
...  
Keyword(s):  
Dna Binding ◽  
Cell Density ◽  
Dictyostelium Discoideum ◽  
Ectopic Expression ◽  
Development Program ◽  
Cell Physiology ◽  
Dependent Manner ◽  
Wild Type ◽  
Gene Encoding ◽  
Multicellular Development

ABSTRACT We recently isolated from Dictyostelium discoideum cells a DNA-binding protein, CbfA, that interacts in vitro with a regulatory element in retrotransposon TRE5-A. We have generated a mutant strain that expresses CbfA at <5% of the wild-type level to characterize the consequences for D. discoideum cell physiology. We found that the multicellular development program leading to fruiting body formation is highly compromised in the mutant. The cells cannot aggregate and stay as a monolayer almost indefinitely. The cells respond properly to prestarvation conditions by expressing discoidin in a cell density-dependent manner. A genomewide microarray-assisted expression analysis combined with Northern blot analyses revealed a failure of CbfA-depleted cells to induce the gene encoding aggregation-specific adenylyl cyclase ACA and other genes required for cyclic AMP (cAMP) signal relay, which is necessary for aggregation and subsequent multicellular development. However, the cbfA mutant aggregated efficiently when mixed with as few as 5% wild-type cells. Moreover, pulsing cbfA mutant cells developing in suspension with nanomolar levels of cAMP resulted in induction of acaA and other early developmental genes. Although the response was less efficient and slower than in wild-type cells, it showed that cells depleted of CbfA are able to initiate development if given exogenous cAMP signals. Ectopic expression of the gene encoding the catalytic subunit of protein kinase A restored multicellular development of the mutant. We conclude that sensing of cell density and starvation are independent of CbfA, whereas CbfA is essential for the pattern of gene expression which establishes the genetic network leading to aggregation and multicellular development of D. discoideum.

scholarly journals The ROCO Kinase QkgA Is Necessary for Proliferation Inhibition by Autocrine Signals in Dictyostelium discoideum

2010 ◽  
Vol 9 (10) ◽  
pp. 1557-1565 ◽  
Author(s):  
Jonathan E. Phillips ◽  
Richard H. Gomer
Keyword(s):  
Cell Proliferation ◽  
Dictyostelium Discoideum ◽  
Fluorescent Protein ◽  
Signal Transduction Pathway ◽  
Protein Accumulation ◽  
Proliferation Inhibition ◽  
Wild Type ◽  
Content Type ◽  
Multicellular Development ◽  
Green Fluorescent

ABSTRACT AprA and CfaD are secreted proteins that function as autocrine signals to inhibit cell proliferation in Dictyostelium discoideum. Cells lacking AprA or CfaD proliferate rapidly, and adding AprA or CfaD to cells slows proliferation. Cells lacking the ROCO kinase QkgA proliferate rapidly, with a doubling time 83% of that of the wild type, and overexpression of a QkgA-green fluorescent protein (GFP) fusion protein slows cell proliferation. We found that qkgA − cells accumulate normal levels of extracellular AprA and CfaD. Exogenous AprA or CfaD does not slow the proliferation of cells lacking qkgA, and expression of QkgA-GFP in qkgA − cells rescues this insensitivity. Like cells lacking AprA or CfaD, cells lacking QkgA tend to be multinucleate, accumulate nuclei rapidly, and show a mass and protein accumulation per nucleus like those of the wild type, suggesting that QkgA negatively regulates proliferation but not growth. Despite their rapid proliferation, cells lacking AprA, CfaD, or QkgA expand as a colony on bacteria less rapidly than the wild type. Unlike AprA and CfaD, QkgA does not affect spore viability following multicellular development. Together, these results indicate that QkgA is necessary for proliferation inhibition by AprA and CfaD, that QkgA mediates some but not all of the effects of AprA and CfaD, and that QkgA may function downstream of these proteins in a signal transduction pathway regulating proliferation.

scholarly journals The surface cyclic AMP receptors, cAR1, cAR2, and cAR3, promote Ca2+ influx in Dictyostelium discoideum by a G alpha 2-independent mechanism.

1993 ◽  
Vol 4 (3) ◽  
pp. 283-292 ◽  
Author(s):  
J L Milne ◽  
P N Devreotes
Keyword(s):  
Cyclic Amp ◽  
G Proteins ◽  
Ion Selectivity ◽  
Wild Type ◽  
Folate Receptors ◽  
Multicellular Development ◽  
Alpha Subunits ◽  
Independent Mechanism ◽  
Alpha 7 ◽  
Camp Receptor

Activation of surface folate receptors or cyclic AMP (cAMP) receptor (cAR) 1 in Dictyostelium triggers within 5-10 s an influx of extracellular Ca2+ that continues for 20 s. To further characterize the receptor-mediated Ca2+ entry, we analyzed 45Ca2+ uptake in amoebas overexpressing cAR2 or cAR3, cARs present during multicellular development. Both receptors induced a cAMP-dependent Ca2+ uptake that had comparable kinetics, ion selectivity, and inhibitor profiles as folate- and cAR1-mediated Ca2+ uptake. Analysis of mutants indicated that receptor-induced Ca2+ entry does not require G protein alpha subunits G alpha 1, G alpha 2, G alpha 3, G alpha 4, G alpha 7, or G alpha 8. Overexpression of cAR1 or cAR3 in g alpha 2- cells did not restore certain G alpha 2-dependent events, such as aggregation, or cAMP-mediated activation of adenylate and guanylate cyclases, but these strains displayed a cAMP-mediated Ca2+ influx with kinetics comparable to wild-type aggregation-competent cells. These results suggest that a plasma membrane-associated Ca(2+)-influx system may be activated by at least four distinct chemoreceptors during Dictyostelium development and that the response may be independent of G proteins.

scholarly journals A Retinoblastoma Orthologue Is Required for the Sensing of a Chalone in Dictyostelium discoideum

2014 ◽  
Vol 13 (3) ◽  
pp. 376-382 ◽  
Author(s):  
Deenadayalan Bakthavatsalam ◽  
Michael J. V. White ◽  
Sarah E. Herlihy ◽  
Jonathan E. Phillips ◽  
Richard H. Gomer
Keyword(s):  
Cell Proliferation ◽  
Dictyostelium Discoideum ◽  
Accumulation Rate ◽  
Protein Accumulation ◽  
Wild Type ◽  
Spore Viability ◽  
Content Type ◽  
Multicellular Development ◽  
Normal Mass ◽  
Inhibit Cell Proliferation

ABSTRACT Retinoblastoma-like proteins regulate cell differentiation and inhibit cell proliferation. The Dictyostelium discoideum retinoblastoma orthologue RblA affects the differentiation of cells during multicellular development, but it is unclear whether RblA has a significant effect on Dictyostelium cell proliferation, which is inhibited by the secreted proteins AprA and CfaD. We found that rblA − cells in shaking culture proliferate to a higher density, die faster after reaching stationary density, and, after starvation, have a lower spore viability than wild-type cells, possibly because in shaking culture, rblA − cells have both increased cytokinesis and lower extracellular accumulation of CfaD. However, rblA − cells have abnormally slow proliferation on bacterial lawns. Recombinant AprA inhibits the proliferation of wild-type cells but not that of rblA − cells, whereas CfaD inhibits the proliferation of both wild-type cells and rblA − cells. Similar to aprA − cells, rblA − cells have a normal mass and protein accumulation rate on a per-nucleus basis, indicating that RblA affects cell proliferation but not cell growth. AprA also functions as a chemorepellent, and RblA is required for proper AprA chemorepellent activity despite the fact that RblA does not affect cell speed. Together, our data indicate that an autocrine proliferation-inhibiting factor acts through RblA to regulate cell density in Dictyostelium , suggesting that such factors may signal through retinoblastoma-like proteins to control the sizes of structures such as developing organs or tumors.

scholarly journals PakD, a Putative p21-Activated Protein Kinase in Dictyostelium discoideum, Regulates Actin

2013 ◽  
Vol 13 (1) ◽  
pp. 119-126 ◽  
Author(s):  
Miguel Garcia ◽  
Sibnath Ray ◽  
Isaiah Brown ◽  
Jon Irom ◽  
Derrick Brazill
Keyword(s):  
Protein Kinase ◽  
Actin Cytoskeleton ◽  
Dictyostelium Discoideum ◽  
Cell Function ◽  
Actin Dynamics ◽  
Wild Type ◽  
Content Type ◽  
Multicellular Development ◽  
Actin Organization ◽  
Cellular Processes

ABSTRACT Proper regulation of the actin cytoskeleton is essential for cell function and ultimately for survival. Tight control of actin dynamics is required for many cellular processes, including differentiation, proliferation, adhesion, chemotaxis, endocytosis, exocytosis, and multicellular development. Here we describe a putative p21-activated protein kinase, PakD, that regulates the actin cytoskeleton in Dictyostelium discoideum . We found that cells lacking pakD are unable to aggregate and thus unable to develop. Compared to the wild type, cells lacking PakD have decreased membrane extensions, suggesting defective regulation of the actin cytoskeleton. pakD − cells show poor chemotaxis toward cyclic AMP (cAMP) but normal chemotaxis toward folate, suggesting that PakD mediates some but not all chemotaxis responses. pakD − cells have decreased polarity when placed in a cAMP gradient, indicating that the chemotactic defects of the pakD − cells may be due to an impaired cytoskeletal response to cAMP. In addition, while wild-type cells polymerize actin in response to global stimulation by cAMP, pakD − cells exhibit F-actin depolymerization under the same conditions. Taken together, the results suggest that PakD is part of a pathway coordinating F-actin organization during development.

Roux-en-Y Gastrointestinal Bypass Promotes Activation of TGR5 and Peptide YY

2020 ◽  
Vol 20 (8) ◽  
pp. 1262-1267
Author(s):  
Haojun Yang ◽  
Hanyang Liu ◽  
YuWen Jiao ◽  
Jun Qian
Keyword(s):  
Body Weight ◽  
Food Intake ◽  
Metabolic Diseases ◽  
Peptide Yy ◽  
The Body ◽  
Wild Type ◽  
L Cells ◽  
Body Weights ◽  
Gastrointestinal Bypass ◽  
G Protein Coupled

Background: G protein-coupled bile acid receptor (TGR5) is involved in a number of metabolic diseases. The aim of this study was to identify the role of TGR5 after Roux-en-Y gastric bypass (GBP). Methods: Wild type and TGR5 knockout mice (tgr5-/-) were fed a high-fat diet (HFD) to establish the obesity model. GBP was performed. The changes in body weight and food intake were measured. The levels of TGR5 and peptide YY (PYY) were evaluated by RT-PCR, Western blot, and ELISA. Moreover, the L-cells were separated from wild type and tgr5-/- mice. The levels of PYY in L-cells were evaluated by ELISA. Results: The body weights were significantly decreased after GBP in wild type mice (p<0.05), but not tgr5-/- mice (p>0.05). Food intake was reduced after GBP in wild type mice, but also not significantly affected in tgr5-/- mice (p>0.05). The levels of PYY were significantly increased after GBP compared with the sham group (p<0.05); however, in tgr5-/- mice the expression of PYY was not significantly affected (p>0.05). After INT-777 stimulation in L-cells obtained from murine intestines, the levels of PYY were significantly increased in L-cells tgr5+/+ (p<0.05). Conclusion: Our study suggests that GBP up-regulated the expression of TGR5 in murine intestines, and increased the levels of PYY, which further reduced food intake and decreased the body weight.

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