Identification and targeted gene disruption of cAR3, a cAMP receptor subtype expressed during multicellular stages of Dictyostelium development.

Johnson, Ronald L.; Saxe, Charles L.; Gollop, Rachel; Kimmel, Alan R.; Devreotes, Peter N.

doi:10.1101/gad.7.2.273

Cited by 90 publications

(58 citation statements)

References 49 publications

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“…In these cells, caffeine pretreatment that inhibits ACA activation (39), resulted in a complete dephosphorylation of the receptor. In contrast, in 7-h WT cells (40) and 5-h regA Ϫ and pkaR Ϫ cells, a large proportion of cAR1 is found in its phosphorylated form, even before cAMP addition. These results show that as cAMP levels increase during development, the extent of P-cAR1 is enhanced to a point where P-cAR1 levels become insensitive to external increases in cAMP levels.…”

Section: Basal Car1 Phosphorylation Increases During Dictyosteliummentioning

confidence: 88%

Direct Biochemical Measurements of Signal Relay during Dictyostelium Development

Das

Rericha

Bagorda

et al. 2011

Journal of Biological Chemistry

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Upon starvation, individual Dictyostelium discoideum cells enter a developmental program that leads to collective migration and the formation of a multicellular organism. The process is mediated by extracellular cAMP binding to the G proteincoupled cAMP receptor 1, which initiates a signaling cascade leading to the activation of adenylyl cyclase A (ACA), the synthesis and secretion of additional cAMP, and an autocrine and paracrine activation loop. The release of cAMP allows neighboring cells to polarize and migrate directionally and form characteristic chains of cells called streams. We now report that cAMP relay can be measured biochemically by assessing ACA, ERK2, and TORC2 activities at successive time points in development after stimulating cells with subsaturating concentrations of cAMP. We also find that the activation profiles of ACA, ERK2, and TORC2 change in the course of development, with later developed cells showing a loss of sensitivity to the relayed signal. We examined mutants in PKA activity that have been associated with precocious development and find that this loss in responsiveness occurs earlier in these mutants. Remarkably, we show that this loss in sensitivity correlates with a switch in migration patterns as cells transition from streams to aggregates. We propose that as cells proceed through development, the cAMP-induced desensitization and down-regulation of cAMP receptor 1 impacts the sensitivities of chemotactic signaling cascades leading to changes in migration patterns.The directed migration of individual cells or chains or sheets of cells requires cells to transduce external cues into internal signals that regulate cytoskeletal reorganization and cell polarity. Signal transduction is important during development where the transition from single to collective cell migration is regulated by multiple external cues (1). Cell-cell communication, a process that can be manifested through the relay of external chemical signals (2, 3), is critical for a wide range of developmental processes including the survival of the social amoebae Dictyostelium discoideum.Upon starvation, Dictyostelium cells enter a developmental program leading to the transition from cells operating individually to groups of cells operating as a population. Aggregation of starving cells is controlled by signaling centers that emit periodic pulses of 3Ј-5Ј-cAMP. This process is mediated by the binding of extracellular cAMP to the G protein-coupled receptor cAR1, 3 which initiates a signaling cascade leading to, among other things, the activation of ACA (4). When ACA is first activated, rapid cAMP synthesis leads to a burst in internal cAMP levels. Although part of the synthesized cAMP remains inside cells to activate PKA and regulate gene expression, the majority of it is rapidly secreted (5-7). The secreted cAMP binds to cAR1 and, in an autocrine and paracrine amplification step, results in the production and secretion of more cAMP (2, 3). For a spatially extended cell population, the stimulated release of cAMP result...

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Section: Basal Car1 Phosphorylation Increases During Dictyosteliummentioning

confidence: 88%

Direct Biochemical Measurements of Signal Relay during Dictyostelium Development

Das

Rericha

Bagorda

et al. 2011

Journal of Biological Chemistry

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“…cAMP receptor 1 (cAR1) is expressed in the ¢rst 10 h of development, cAR3 appears between 5 and 15 h, and cAR2 and cAR4 are expressed after 10 h of development [19]. In our assay condition with 5 h starved cells, a double knock-out mutant (car1 3 /car3 3 ) ensures that no cAMP receptors are present on the cell surface.…”

Section: Surface Camp Receptors and G Proteinsmentioning

confidence: 99%

“…The single GL subunit is essential for all chemotactic signals [14], whereas GK2 and GK4 speci¢cally transduce signals from the cAMP and folic acid receptors, respectively [19]. Knock-out mutants of GK2 and GL were examined for their responses to osmotic stress.…”

Section: Surface Camp Receptors and G Proteinsmentioning

confidence: 99%

Chemotactic and osmotic signals share a cGMP transduction pathway in Dictyostelium discoideum

Kuwayama

Haastert

1998

FEBS Letters

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In the ameboid eukaryote Dictyostelium discoideum, chemotactic stimulation by cAMP induces an increase of intracellular cGMP and subsequently the phosphorylation of myosin heavy chain II. Resistance to high osmotic stress also requires transient increases of intracellular cGMP and phosphorylation of myosin heavy chain II, although the kinetics is much slower than for chemotaxis. To examine if chemotaxis and osmotic stress share common signaling components we systematically analyzed the osmotic cGMP response and survival in chemotactic mutants with altered cGMP signaling. Null mutants with deletions of cell surface cAMP receptors or the associated GTP-binding proteins GK K2 and GL L show no cAMP-induced cGMP response and chemotaxis; in contrast, osmotic stress induces the normal cGMP accumulation and survival. The same result was obtained with the non-chemotactic mutant KI-10, which lacks the activation of guanylyl cyclase by cAMP. This indicates that these components are required for chemotaxis but not osmotic cGMP signaling and survival. The potential guanylyl cyclase null mutant KI-8 shows no chemotaxis, no osmotic cGMP increase and reduced survival in high osmolarity. Two types of cGMP-binding protein mutants, also show reduced tolerance during high osmotic stress. Taken together, these observations clarify that chemotactic and osmotic signals are detected by different mechanisms, but share a cGMP signaling pathway.z 1998 Federation of European Biochemical Societies.

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“…15), yielding the car1 Ϫ uracil auxotroph, JB5. Finally, cAR3 was disrupted in JB5 cells to yield RI9 cells using the prototrophy-restoring construct 5cAR3ura as described previously (16). All homologous recombination events were confirmed by Southern analysis.…”

Section: Generation Growth and Development Of Cell Lines Expressingmentioning

confidence: 99%

Phosphorylation of Chemoattractant Receptors Is Not Essential for Chemotaxis or Termination of G-protein-mediated Responses

Kim

Soede

Schaap

et al. 1997

Journal of Biological Chemistry

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In several G-protein-coupled signaling systems, ligandinduced receptor phosphorylation by specific kinases is suggested to lead to desensitization via mechanisms including receptor/G-protein uncoupling, receptor internalization, and receptor down-regulation. We report here that elimination of phosphorylation of a chemoattractant receptor of Dictyostelium, either by site-directed substitution of the serines or by truncation of the C-terminal cytoplasmic domain, completely prevented agonist-induced loss of ligand binding but did not impair the adaptation of several receptor-mediated responses including the activation of adenylyl and guanylyl cyclases and actin polymerization. In addition, the phosphorylation-deficient receptors were capable of mediating chemotaxis, aggregation, and differentiation. We propose that for chemoattractant receptors agonistinduced phosphorylation regulates surface binding activity but other phosphorylation-independent mechanisms mediate response adaptation.

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Identification and targeted gene disruption of cAR3, a cAMP receptor subtype expressed during multicellular stages of Dictyostelium development.

Cited by 90 publications

References 49 publications

Direct Biochemical Measurements of Signal Relay during Dictyostelium Development

Direct Biochemical Measurements of Signal Relay during Dictyostelium Development

Chemotactic and osmotic signals share a cGMP transduction pathway in Dictyostelium discoideum

Phosphorylation of Chemoattractant Receptors Is Not Essential for Chemotaxis or Termination of G-protein-mediated Responses

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