cAMP Receptor and G-protein Interactions Control Development in Dictyostelium

Pupillo, M.; Klein, Peter S.; Vaughan, Roxanne A.; Pitt, G.; Lilly, Pamela J.; Sun, Tzeli J.; Devreotes, Peter N.; Kumagai, Akiko; Firtel, Richard A.

doi:10.1101/sqb.1988.053.01.075

Cited by 16 publications

(7 citation statements)

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“…However, we do not favor this idea because the eight a subunits do not appear to group into distinct subclasses (Jelsema and Axelrod, 1987;Logothetis et al, 1987;Tang and Gilman, 1991;Katz et al, 1992). Dictyostelium amoebas possess a single G,B subunit that is highly homologous to those of mammalian cells (Pupillo et al, 1988). We are currently determining whether receptormediated Ca2' entry requires G proteins using recently constructed g3 null cells (Lilly, Wu, Welker, and Devreotes, personal communication …”

Section: Western Blot Analysismentioning

confidence: 99%

“…More recently, genes encoding three related cAMP receptors, which are homologous to cAR1 and expressed during multicellular development (cAR2, cAR3, and cAR4), have been isolated (Saxe et al, 1991(Saxe et al, , 1992Johnson et al, 1992a;Louis, Ginsburg, and Kimmel, personal communication). Eight distinct G protein a subunits (Pupillo et al, 1989;Hadwiger et al, 1991;Wu and Devreotes, 1991;Pupillo and Devreotes, un-published data) and a single G protein : subunit (Pupillo et al, 1988), which display considerable homology with their respective mammalian counterparts, also have been cloned. Taken together, these findings strongly implicate the importance of G protein-mediated signaling pathways during Dictyostelium development.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

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

Milne

Devreotes

1993

MBoC

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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.

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Section: Western Blot Analysismentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Milne

Devreotes

1993

MBoC

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“…Caffeine was found to only marginally affect cAMP binding to its cell surface receptors but to efficiently inhibit adenylyl cyclase activity and cAMP production in an indirect and reversible fashion [4,6]. Efforts at defining the mechanism through which caffeine inhibits cAMP synthesis in Dictyostelium lead to the suggestion that there are at least two different targets of caffeine, with at least one of them downstream from the heterotrimeric G protein Gα2, which couples to the main cAMP receptor, cAR1 [7][8][9][10]. Although the targets of caffeine in Dictyostelium remain unknown, caffeine continues to be widely used by Dictyostelium researchers to inhibit cAMP synthesis and, thereby, prevent the autocrine stimulation of cells in studies of cAMP chemoattractant signaling.…”

Section: Introductionmentioning

confidence: 99%

Caffeine inhibits PI3K and mTORC2 in Dictyostelium and differentially affects multiple other cAMP chemoattractant signaling effectors

et al. 2019

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Caffeine is commonly used in Dictyostelium to inhibit the synthesis of the chemoattractant cAMP and, therefore, its secretion and the autocrine stimulation of cells, in order to prevent its interference with the study of chemoattractant-induced responses.However, the mechanism through which caffeine inhibits cAMP synthesis in Dictyostelium has not been characterized. Here, we report the effects of caffeine on the cAMP chemoattractant signaling network. We found that caffeine inhibits phosphatidylinositol 3-kinase (PI3K) and mechanistic target of rapamycin complex 2 (mTORC2). Both PI3K and mTORC2 are essential for the chemoattractant-stimulated cAMP production, thereby providing a mechanism for the caffeine-mediated inhibition of cAMP synthesis. Our results also reveal that caffeine treatment of cells leads to an increase in cAMP-induced RasG and Rap1 activation, and inhibition of the PKA, cGMP, MyoII, and ERK1 responses. Finally, we observed that caffeine has opposite effects on Factin and ERK2 depending on the assay and Dictyostelium strain used, respectively.Altogether, our findings reveal that caffeine considerably affects the cAMP-induced chemotactic signaling pathways in Dictyostelium, most likely acting through multiple targets that include PI3K and mTORC2.3

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“…Although the two receptors display similar cAMP binding affinities in phosphate buffer, cAR3 is ~100 times less efficient than cAR1 in inducing cAMP-stimulated responses and cAR1 is essential for chemotaxis-driven aggregation through Gα2βγ [4,9,10]. Whereas there are twelve G alpha protein subunits in Dictyostelium , Gα2 (gene gpab ) is the main G alpha subunit responsible for the chemotactic responses to cAMP and there is only one G beta (gene gpba ) and gamma subunit (gene gpga ) [11–16]. …”

Section: Introductionmentioning

confidence: 99%

The cAMP-induced G protein subunits dissociation monitored in live Dictyostelium cells by BRET reveals two activation rates, a positive effect of caffeine and potential role of microtubules

Islam

Yue

Scavello

et al. 2018

Cellular Signalling

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To study the dynamics and mechanisms controlling activation of the heterotrimeric G protein Gα2βγ in Dictyostelium in response to stimulation by the chemoattractant cyclic AMP (cAMP), we monitored the G protein subunit interaction in live cells using bioluminescence resonance energy transfer (BRET). We found that cAMP induces the cAR1-mediated dissociation of the G protein subunits to a similar extent in both undifferentiated and differentiated cells, suggesting that only a small number of cAR1 (as expressed in undifferentiated cells) is necessary to induce the full activation of Gα2βγ. In addition, we found that treating cells with caffeine increases the potency of cAMP-induced Gα2βγ activation; and that disrupting the microtubule network but not F-actin inhibits the cAMP-induced dissociation of Gα2βγ. Thus, microtubules are necessary for efficient cAR1-mediated activation of the heterotrimeric G protein. Finally, kinetics analyses of Gα2βγ subunit dissociation induced by different cAMP concentrations indicate that there are two distinct rates at which the heterotrimeric G protein subunits dissociate when cells are stimulated with cAMP concentrations above 500 nM versus only one rate at lower cAMP concentrations. Quantitative modeling suggests that the kinetics profile of Gα2βγ subunit dissociation results from the presence of both uncoupled and G protein pre-coupled cAR1 that have differential affinities for cAMP and, consequently, induce G protein subunit dissociation through different rates. We suggest that these different signaling kinetic profiles may play an important role in initial chemoattractant gradient sensing.

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cAMP Receptor and G-protein Interactions Control Development in Dictyostelium

Cited by 16 publications

References 0 publications

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

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

Caffeine inhibits PI3K and mTORC2 in Dictyostelium and differentially affects multiple other cAMP chemoattractant signaling effectors

The cAMP-induced G protein subunits dissociation monitored in live Dictyostelium cells by BRET reveals two activation rates, a positive effect of caffeine and potential role of microtubules

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