Genetic Analysis of a Dictyostelium discoideum Mutant Resistant to Adenosine 3':5'-Cyclic Phosphorothioate, an Inhibitor of Wild-type Development

Wallraff, E.; Welker, Dennis L.; Williams, Kristi; Gerisch, Günther

doi:10.1099/00221287-130-8-2103

Cited by 12 publications

(17 citation statements)

References 8 publications

(10 reference statements)

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“…Cell lines that develop in the presence or absence of high concentrations of cAMP are of particular interest since they do not respond normally to cAMP pulses but will form small aggregates that develop into fruiting bodies (27). A dedifferentiation deficient cell line also exists, which abnormally retains its ability to reaggregate rapidly (28).…”

Section: Resultsmentioning

confidence: 99%

A gene expressed in undifferentiated vegetative Dictyostelium is repressed by developmental pulses of cAMP and reinduced during dedifferentiation.

Kimmel¹,

Carlisle²

1986

Proc. Natl. Acad. Sci. U.S.A.

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We describe the gene M4-1, whose unique pattern of developmental expression will allow us to study the molecular mechanisms controlling expression in undifferentiated cells in addition to repression in response to cAMP during development and reinduction during dedifferentiation. M4-1 is a Dictyostelium gene expressed in the undifferentiated cell. We have shown that M4-1 continues to be expressed very early during the developmental cycle but is repressed at a later stage of development, at a time coincident with the establishment of oscillations in the cAMP pool. Studies on the expression of the M4-1 gene in shaking culture, under conditions that mimic early development, have established that pulsatile stimulation of cells with cAMP is sufficient to repress M4-1 expression. Consistent with this, cells that are exposed to high levels of cAMP are unable to respond normally to cAMP oscillations and continue to express M4-1 at vegetative levels. These data indicate that low-level oscillations of cAMP are required for the repression of M4-1 expression rather than the continuous high levels ofcAMP responsible for the regulation of a different class of Dictyostelium genes. We suggest that cAMP may mediate developmental expression of the Dictyostelium genome by different mechanisms. We also show that cell-cell interaction, a developmental event that occurs subsequent to the cAMP pulse, does not normally influence the regulation of M4-1.Finally, we have shown that when cAMP-pulsed cells are induced to dedifferentiate, M4-1 RNA sequences rapidly reappear in nuclei and cytoplasm, suggesting that regulation of M4-1 expression is primarily mediated at the level of transcription.Dictyostelium discoideum grows as undifferentiated ameboid single cells. Development can be initiated by plating the cells on filter pads in a low-salt buffer in the absence of a food source. Synchronous development then proceeds at the air-water interface; gradients of cAMP are established and amebae move toward regions with high cAMP concentrations (1, 2). Stimulation of amebae by extracellular cAMP results in a rise in intracellular cAMP concentrations. cAMP is then secreted, thus, amplifying and propagating the original cAMP concentration wave (3-5). After relaying the cAMP signal, cells become transiently refractory to further stimulation (6, 7). During this period of adaptation or desensitization, extracellular cAMP concentrations decrease and cells recover their sensitivity to extracellular cAMP stimulation. This adaptation/deadaptation cycle repeats itself with cAMP pulses initiating from aggregation centers with a periodicity of -6 min. The result is the assemblage of groups of 105 cells into multicellular aggregates. Individual aggregates then develop into mature fruiting bodies consisting of predominantly spore and stalk cells. The addition of high (mM) concentrations of cAMP early in development inhibits aggregation and continued development (8).Specific

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Section: Resultsmentioning

confidence: 99%

A gene expressed in undifferentiated vegetative Dictyostelium is repressed by developmental pulses of cAMP and reinduced during dedifferentiation.

Kimmel¹,

Carlisle²

1986

Proc. Natl. Acad. Sci. U.S.A.

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“…Because of the multiplicity of adapting responses mediated by cAR1, slowly hydrolyzed cAMP analogs or high concentrations of cAMP block aggregation and subsequent development (Wallraff et al, 1984). We hypothesized that constitutively active cAR1 mutants would similarly promote adaptation and thus interfere with wild-type cAR1 function and development.…”

Section: Introductionmentioning

confidence: 99%

“…In chemotaxis, adaptation prevents cells from chasing after passing cAMP waves and is thought to facilitate the perception of shallow chemoattractant gradients and restrict pseudopod extension and uropod retraction to the poles of cells undergoing chemotaxis (Chung and Firtel, 2002;Iijima et al, 2002). Because of the multiplicity of adapting responses mediated by cAR1, slowly hydrolyzed cAMP analogs or high concentrations of cAMP block aggregation and subsequent development (Wallraff et al, 1984). We hypothesized that constitutively active cAR1 mutants would similarly promote adaptation and thus interfere with wild-type cAR1 function and development.…”

mentioning

confidence: 99%

Constitutively Active G Protein-coupled Receptor Mutants BlockDictyosteliumDevelopment

Zhang¹,

Goswami²,

Hereld³

2005

MBoC

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cAR1, a G protein-coupled receptor (GPCR) for cAMP, is required for the multicellular development of Dictyostelium. The activation of multiple pathways by cAR1 is transient because of poorly defined adaptation mechanisms. To investigate this, we used a genetic screen for impaired development to isolate four dominant-negative cAR1 mutants, designated DN1-4. The mutant receptors inhibit multiple cAR1-mediated responses known to undergo adaptation. Reduced in vitro adenylyl cyclase activation by GTP␥S suggests that they cause constitutive adaptation of this and perhaps other pathways. In addition, the DN mutants are constitutively phosphorylated, which normally requires cAMP binding and possess cAMP affinities that are ϳ100-fold higher than that of wild-type cAR1. Two independent activating mutations, L100H and I104N, were identified. These residues occupy adjacent positions near the cytoplasmic end of the receptor's third transmembrane helix and correspond to the (E/D)RY motif of numerous mammalian GPCRs, which is believed to regulate their activation. Taken together, these findings suggest that the DN mutants are constitutively activated and block development by turning on natural adaptation mechanisms. INTRODUCTIONG protein-coupled receptors (GPCRs) number in the thousands in humans and are responsible for physiological responses to diverse extracellular signals including light, odorants, hormones, chemoattractants, and neurotransmitters. On activation by their ligands (or photons in the case of rhodopsin), these seven transmembrane domain-containing receptors typically activate heterotrimeric G proteins that, in turn, regulate the activities of a variety of downstream effectors. Ligand binding also triggers various desensitization mechanisms which diminish the cell's responsiveness to subsequent stimulation (Strader et al., 1995;Bourne, 1997;Ferguson and Caron, 1998).These systems are highly conserved and found in all eukaryotes including the social amoeba Dictyostelium discoideum, which uses extracellular cAMP signaling and cAMPspecific GPCRs to orchestrate its multicellular development. When deprived of nutrients, this free-living soil amoeba ceases growth and embarks on a 24-h program in which some 10 5 cells aggregate and differentiate to yield a sporeladen fruiting body. The first of four highly homologous cAMP receptors (cARs) expressed during development, cAR1, is essential for aggregation and subsequent development for three principal reasons. First, by transiently activating an adenylyl cyclase (ACA), cAR1 mediates "cAMP relay," the amplification and propagation of cAMP waves, which arise spontaneously every 6 min at aggregation centers. Second, cAR1 plays a direct role in aggregation as it mediates chemotaxis of cells up the cAMP gradient inherent in each oncoming wave. Lastly, cAR1 is responsible for the induction of various aggregation-stage genes including those encoding cAR1 itself, ACA, and G␣2, the G protein ␣-subunit through which cAR1 signals (Parent and Devreotes, 1996). cAR1 governs these di...

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“…Because cAMPS is a phosphodiesterase-resistant agonist of the cyclic-AMP receptors it inhibits wild-type development, as does a low steady-state concentration of cyclic AMP (Rossier et al, 1978). Mutants which aggregate in the presence of cAMPS were collected and one of them, HG302, was chosen for further analysis (Wallraff et al, 1984). In that mutant the csA-glycoprotein is still under stringent developmental control.…”

Section: Introductionmentioning

confidence: 99%

Probing an adhesion mutant of Dictyostelium discoideum with cDNA clones and monoclonal antibodies indicates a specific defect in the contact site A glycoprotein

et al. 1985

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Genetic Analysis of a Dictyostelium discoideum Mutant Resistant to Adenosine 3':5'-Cyclic Phosphorothioate, an Inhibitor of Wild-type Development

Cited by 12 publications

References 8 publications

A gene expressed in undifferentiated vegetative Dictyostelium is repressed by developmental pulses of cAMP and reinduced during dedifferentiation.

A gene expressed in undifferentiated vegetative Dictyostelium is repressed by developmental pulses of cAMP and reinduced during dedifferentiation.

Constitutively Active G Protein-coupled Receptor Mutants BlockDictyosteliumDevelopment

Probing an adhesion mutant of Dictyostelium discoideum with cDNA clones and monoclonal antibodies indicates a specific defect in the contact site A glycoprotein

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