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 Phospholipase C in Dictyostelium discoideum. Identification of stimulatory and inhibitory surface receptors and G-proteins

1994 ◽  
Vol 297 (1) ◽  
pp. 189-193 ◽  
Author(s):  
A A Bominaar ◽  
P J M Van Haastert
Keyword(s):  
Phospholipase C ◽  
G Protein ◽  
G Proteins ◽  
Alpha Subunit ◽  
Wild Type ◽  
Inhibitory Pathway ◽  
Surface Receptors ◽  
G Protein Alpha Subunit ◽  
Camp Receptor ◽  
Protein Alpha Subunit

A combined biochemical and genetic approach was used to show that phospholipase C in the cellular slime mould Dictyostelium is under dual regulation by the chemoattractant cyclic AMP (cAMP). This dual regulation involves stimulatory and inhibitory surface receptors and G-proteins. In wild-type cells both cAMP and guanosine 5′-[gamma-thio]triphosphate (GTP[S]) stimulated phospholipase C. In contrast, mutant fgd A, lacking the G-protein alpha-subunit G alpha 2, showed no stimulation by either cAMP or GTP[S], indicating that G alpha 2 is the stimulatory G-protein. In mutant fgd C cAMP did not stimulate phospholipase C, but stimulation by GTP[S] was normal, suggesting that the defect in this mutant is upstream of the stimulatory G alpha 2. Inhibition of phospholipase C was achieved in wild-type cells by the partial antagonist 3′-deoxy-3′-aminoadenosine 3′,5′-phosphate (3′NH-cAMP). This inhibition was no longer observed in transformed cell lines lacking either the surface cAMP receptor cAR1 or the G-protein alpha-subunit G alpha 1; in these cells the agonist cAMP still activated phospholipase C. These results indicate that Dictyostelium phospholipase C is regulated via a stimulatory and an inhibitory pathway. The inhibitory pathway is composed of the surface receptor cAR1 and the G-protein G1. The stimulatory pathway consists of an unknown cAMP receptor (possibly the fgd C gene product) and the G-protein G2.

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 The Cyclic AMP Receptor Protein Is Dependent on GcvA for Regulation of the gcv Operon

1999 ◽  
Vol 181 (6) ◽  
pp. 1912-1919 ◽  
Author(s):  
Laura D. Wonderling ◽  
George V. Stauffer
Keyword(s):  
Cyclic Amp ◽  
Transcription Initiation ◽  
Mutational Analysis ◽  
Initiation Site ◽  
Receptor Protein ◽  
Single Mutation ◽  
Wild Type ◽  
Double Deletion ◽  
Footprint Analysis ◽  
Camp Receptor

ABSTRACT The Escherichia coli gcv operon is transcriptionally regulated by the GcvA, GcvR, Lrp, and PurR proteins. In this study, the cyclic AMP (cAMP) receptor protein (CRP) is shown to be involved in positive regulation of the gcv operon. A crpdeletion reduced expression of a gcvT-lacZ fusion almost fourfold in glucose minimal (GM) medium. The phenotype was complemented by both the wild-type crp gene and four crpalleles that encode proteins with amino acid substitutions in known activating regions of CRP. A cyaA deletion also resulted in a fourfold decrease in gcvT-lacZ expression, and wild-type expression was restored by the addition of cAMP to the growth medium. AcyaA crp double deletion resulted in levels ofgcvT-lacZ expression identical to those observed with either single mutation, showing that CRP and cAMP regulate through the same mechanism. Growth in GM medium plus cAMP or glycerol minimal medium did not result in a significant increase ingcvT-lacZ expression. Thus, the level of cAMP present in GM medium appears to be sufficient for regulation by CRP. DNase I footprint analysis showed that CRP binds and protects two sites centered at bp −313 (site 1) and bp −140 (site 2) relative to the transcription initiation site, but a mutational analysis demonstrated that only site 1 is required for CRP-mediated regulation ofgcvT-lacZ expression. Expression of thegcvT-lacZ fusion in a crp gcvA double mutant suggested that CRP’s role is dependent on the GcvA protein.

scholarly journals G-Alpha Subunit Abundance and Activity Differentially Regulate β-Catenin Signaling

2018 ◽  
Vol 39 (5) ◽  
Author(s):  
Arshiya Banu ◽  
Karen J. Liu ◽  
Alistair J. Lax ◽  
Agamemnon E. Grigoriadis
Keyword(s):  
G Proteins ◽  
Pasteurella Multocida ◽  
Alpha Subunit ◽  
Dependent Manner ◽  
Heterotrimeric G Proteins ◽  
Wild Type ◽  
Content Type ◽  
Alpha Subunits ◽  
Licl Treatment ◽  
Signal Transduction Proteins

ABSTRACT Heterotrimeric G proteins are signal transduction proteins involved in regulating numerous signaling events. In particular, previous studies have demonstrated a role for G-proteins in regulating β-catenin signaling. However, the link between G-proteins and β-catenin signaling is controversial and appears to depend on G-protein specificity. We describe a detailed analysis of a link between specific G-alpha subunits and β-catenin using G-alpha subunit genetic knockout and knockdown approaches. The Pasteurella multocida toxin was utilized as a unique tool to activate G-proteins, with LiCl treatment serving as a β-catenin signaling agonist. The results show that Pasteurella multocida toxin (PMT) significantly enhanced LiCl-induced active β-catenin levels in HEK293T cells and mouse embryo fibroblasts. Evaluation of the effect of specific G-alpha proteins on the regulation of β-catenin showed that Gq/11 and G12/13 knockout cells had significantly higher levels of active and total β-catenin than wild-type cells. The stimulation of active β-catenin by PMT and LiCl was lost upon both constitutive and transient knockdown of G12 and G13 but not Gq. Based on our results, we conclude that endogenous G-alpha proteins are negative regulators of active β-catenin; however, PMT-activated G-alpha subunits positively regulate LiCl-induced β-catenin expression in a G12/13-dependent manner. Hence, G-alpha subunit regulation of β-catenin is context dependent.

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 Expression of wild-type and mutated rabbit osteopontin in Escherichia coli, and their effects on adhesion and migration of P388D1 cells

1995 ◽  
Vol 307 (1) ◽  
pp. 257-265 ◽  
Author(s):  
K Nasu ◽  
T Ishida ◽  
M Setoguchi ◽  
Y Higuchi ◽  
S Akizuki ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Fluid Phase ◽  
Intradermal Injection ◽  
Polymorphonuclear Leucocyte ◽  
Alpha Subunit ◽  
Wild Type ◽  
Alpha Subunits ◽  
Leucocyte Migration ◽  
And Migration ◽  
Chemotactic Effect

Recombinant wild-type rabbit osteopontin (rOP) and the protein with an aspartate-to-glutamate transposition induced by a point mutation in the rabbit OP cDNA within the Gly-Arg-Gly-Asp-Ser (GRGDS) sequence were expressed in Escherichia coli and purified to homogeneity. P388D1 cells bound rOP in a saturable manner. rOP induced adhesion and haptotaxis of P388D1 cells, whereas mutated rabbit OP (rOPmut) did not. Anti-rOP IgG F(ab′)2 and synthetic GRGDS peptide inhibited rOP-mediated adhesion and haptotaxis of P388D1 cells. Fibronectin (FN)-mediated adhesion of P388D1 cells was markedly inhibited in the presence of fluid-phase rOP. Adhesion of P388D1 cells to rOP was significantly inhibited by anti-[alpha-subunits of VLA4 (alpha 4) and VLA5 (alpha 5)] monoclonal antibodies (mAbs), but not by anti-[alpha-subunit of vitronectin (VN) receptor (alpha V) or Mac-1 (alpha M)] mAb. Adhesion of P388D1 cells to FN and VN was significantly inhibited by anti-alpha V mAb but not anti-alpha 4, -alpha 5 or -alpha M mAb. Haptotaxis of P388D1 cells to rOP was significantly inhibited by anti-alpha V mAb, but not by anti-alpha 4, -alpha 5 and alpha M mAbs, whereas that to FN showed no inhibition with all three mAbs. Haptotaxis of P388D1 cells to VN was significantly inhibited by anti-alpha 5 and -alpha V mAbs but not by anti-alpha 4 and -alpha M mAbs. Similar features of inhibition of adhesion and haptotaxis of P388D1 cells to human OP were observed by mAbs. rOP had no chemotactic effect on P388D1 cells. Significant polymorphonuclear leucocyte migration was observed 3-12 h after intradermal injection of rOP into rabbits.

scholarly journals Heat treatment induces an increase in intracellular cyclic AMP content in human epidermoid A-431 cells

1991 ◽  
Vol 276 (3) ◽  
pp. 683-689 ◽  
Author(s):  
J G Kiang ◽  
Y Y Wu ◽  
M C Lin
Keyword(s):  
Heat Treatment ◽  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
G Proteins ◽  
Phosphodiesterase Inhibitors ◽  
Metabolic Inhibitors ◽  
Thermally Induced ◽  
Heat Treated ◽  
Atp Breakdown ◽  
In Cells

The basal level of intracellular cyclic AMP (cAMPi) in A-431 cells incubated at 37 degrees C in Na(+)-containing Hanks solution is 2086 +/- 139 fmol/10(6) cells. When cells are exposed to 45 degrees C for 10 min, cAMPi increases by 40 +/- 4%, and then returns to basal levels within 30 min. Incubating cells in Ca(2+)-free or Mg(2+)-free Hanks solution has no effect on the heat-induced increase in cAMPi, but the increase is inhibited by acid-loading cells to intracellular pH 7.0 or 6.8. In unheated cells, cAMPi increases by 16 +/- 8%, 53 +/- 7%, or 39 +/- 8%, when incubated with isobutyl-1-methylxanthine (1 mM), Ro 20-1724 (0.5 mM), or theophylline (1 mM) respectively. However, heat treatment further elevates cAMPi in cells treated with phosphodiesterase inhibitors, indicating that heat treatment and phosphodiesterase inhibitors elevate cAMPi by a different pathway(s). Heat treatment increases adenylate cyclase activity 2.5-fold. When forskolin (150 microM), an adenylate cyclase stimulator, is applied to cells, the basal cAMPi increases 28 +/- 6-fold compared with controls. Subsequent heating of these cells lowers cAMPi levels to 7.0 +/- 0.5 times that in control cells. This decrease is prevented by pretreatment with pertussis toxin (30 ng/ml, 24 h), suggesting that G-proteins are involved in the process of heat-induced cAMPi increase. 2-Deoxy-D-glucose (10 mM), NaN3 (10 mM) and 2,4-dinitrophenol (1 mM) also increase cAMPi in A-431 cells. However, application of these metabolic inhibitors to cells before heat treatment does not result in cAMPi levels greater than that observed in cells with heat alone. Similar observations are obtained in heat-treated cells previously exposed to adenosine, but not to AMP or ADP. These data are the first to suggest that thermally induced increase in cAMPi is due to a combination of activation of adenylate cyclase and G-proteins, and an increase in adenosine owing to ATP breakdown caused by hyperthermia.

scholarly journals Lipid modifications of G proteins: alpha subunits are palmitoylated.

1993 ◽  
Vol 90 (8) ◽  
pp. 3675-3679 ◽  
Author(s):  
M. E. Linder ◽  
P. Middleton ◽  
J. R. Hepler ◽  
R. Taussig ◽  
A. G. Gilman ◽  
...  
Keyword(s):  
G Proteins ◽  
Lipid Modifications ◽  
Alpha Subunits
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