cAMP-induced desensitization of surface cAMP receptors in Dictyostelium: different second messengers mediate receptor phosphorylation, loss of ligand binding, degradation of receptor, and reduction of receptor mRNA levels.

Surface cAMP receptors on Dictyostelium cells are linked to several second messenger systems and mediate multiple physiological responses, including chemotaxis and differentiation. Activation of the receptor also triggers events which desensitize signal transduction. These events include the following: 1) loss of ligand binding without loss of receptor protein; 2) phosphorylation of the receptor protein, which may lead to impaired signal transduction; 3) redistribution and degradation of the receptor protein; and 4) decrease of cyclic AMP (cAMP) receptor mRNA levels. These mechanisms of desensitization were investigated with the use of mutant synag7, with no activation of adenylyl cyclase; fgdC, with no activation of phospholipase C; and fgdA, with defects in both pathways. cAMP-induced receptor phosphorylation and loss of ligand binding activity was normal in all mutants. In contrast, cAMP-induced degradation of the receptor was absent in all mutants. The cAMP-induced decrease of cAMP-receptor mRNA levels was normal in mutant synag7, but absent in mutant fgdC. Finally, the cAMP analogue (Rp)-cAMPS induced loss of ligand binding without inducing second messenger responses or phosphorylation, redistribution, and degradation of the receptor. We conclude that 1) loss of ligand binding can occur in the absence of receptor phosphorylation; 2) loss of ligand binding and receptor phosphorylation do not require the activation of second messenger systems; 3) cAMP-induced degradation of the receptor may require the phosphorylation of the receptor as well as the activation of at least the synag7 and fgdC gene products; and 4) cAMP-induced decrease of receptor mRNA levels requires the activation of the fgdC gene product and not the synag7 gene product. These results imply that desensitization is composed of multiple components that are regulated by different but partly overlapping sensory transduction pathways.     

G alpha 3 regulates the cAMP signaling system in Dictyostelium.

The Dictyostelium discoideum developmental program is initiated by starvation and its progress depends on G-protein-regulated transmembrane signaling. Disruption of the Dictyostelium G-protein alpha-subunit G alpha 3 (g alpha 3-) blocks development unless the mutant is starved in the presence of artificial cAMP pulses. The function of G alpha 3 was investigated by examining the expression of several components of the cAMP transmembrane signaling system in the g alpha 3- mutant. cAMP receptor 1 protein, cyclic nucleotide phosphodiesterase, phosphodiesterase inhibitor, and aggregation-stage adenylyl cyclase mRNA expression were absent or greatly reduced when cells were starved without exogenously applied pulses of cAMP. However, cAMP receptor 1 protein and aggregation-stage adenylyl cyclase mRNA expression were restored by starving the g alpha 3- cells in the presence of exogenous cAMP pulses. Adenylyl cyclase activity was also reduced in g alpha 3- cells starved without exogenous cAMP pulses compared with similarly treated wild-type cells but was elevated to a level twofold greater than wild-type cells in g alpha 3- cells starved in the presence of exogenous cAMP pulses. These results suggest that G alpha 3 is essential in early development because it controls the expression of components of the transmembrane signaling system.     

Genome-wide expression analyses of gene regulation during early development of Dictyostelium discoideum

Using genome-wide microarrays, we recognized 172 genes that are highly expressed at one stage or another during multicellular development of Dictyostelium discoideum. When developed in shaken suspension, 125 of these genes were expressed if the cells were treated with cyclic AMP (cAMP) pulses at 6-min intervals between 2 and 6 h of development followed by high levels of exogenous cAMP. In the absence of cAMP treatment, only three genes, carA, gbaB, and pdsA, were consistently expressed. Surprisingly, 14 other genes were induced by cAMP treatment of mutant cells lacking the activatable adenylyl cyclase, ACA. However, these genes were not cAMP induced if both of the developmental adenylyl cyclases, ACA and ACR, were disrupted, showing that they depend on an internal source of cAMP. Constitutive activity of the cAMP-dependent protein kinase PKA was found to bypass the requirement of these genes for adenylyl cyclase and cAMP pulses, demonstrating the critical role of PKA in transducing the cAMP signal to early gene expression. In the absence of constitutive PKA activity, expression of later genes was strictly dependent on ACA in pulsed cells.     

Lithium respecifies cyclic AMP-induced cell-type specific gene expression in Dictyostelium

We investigated the effect of LiCl on pattern formation and cAMP-regulated gene expression in Dictyostelium discoideum. In intact slugs, 5 mM LiCl induces an almost complete redifferentiation of prespore into prestalk cells. We found that LiCl acts by interfering with the transduction of extracellular cAMP to cell-type-specific gene expression; LiCl inhibits the induction of prespore-specific gene expression by cAMP, while it promotes the induction of prestalk-associated gene expression by cAMP. Our results indicate that two divergent pathways transduce the extracellular cAMP signal to, respectively, prestalk and prespore gene expression.     

Transcriptional regulation of post-aggregation genes in Dictyostelium by a feed-forward loop involving GBF and LagC

Expression profiles of developmental genes in Dictyostelium were determined on microarrays during development of wild type cells and mutant cells lacking either the DNA binding protein GBF or the signaling protein LagC. We found that the mutant strains developed in suspension with added cAMP expressed the pulse-induced and early adenylyl cyclase (ACA)-dependent genes, but not the later ACA-dependent, post-aggregation genes. Since expression of lagC itself is dependent on GBF, expression of the post-aggregation genes might be controlled only by signaling from LagC. However, expression of lagC in a GBF-independent manner in a gbfA- null strain did not result in expression of the post-aggregation genes. Since GBF is necessary for accumulation of LagC and both the DNA binding protein and the LagC signal transduction pathway are necessary for expression of post-aggregation genes, GBF and LagC form a feed-forward loop. Such network architecture is a common motif in diverse organisms and can act as a filter for noisy inputs. Breaking the feed-forward loop by expressing lagC in a GBF-independent manner in a gbfA+ strain does not significantly affect the patterns of gene expression for cells developed in suspension with added cAMP, but results in a significant delay at the mound stage and asynchronous development on solid supports. This feed-forward loop can integrate temporal information with morphological signals to ensure that post-aggregation genes are only expressed after cell contacts have been made.     

CbfA, the C-module DNA-binding factor, plays an essential role in the initiation of Dictyostelium discoideum development

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.     

Dictyostelium discoideum: a model system for differentiation and patterning

In Dictyostelium, development begins with the aggregation of free living amoebae, which soon become organized into a relatively simple organism with a few different cell types. Coordinated cell type differentiation and morphogenesis lead to a final fruiting body that allows the dispersal of spores. The study of these processes is having increasing impact on our understanding of general developmental mechanisms. The availability of biochemical and molecular genetics techniques has allowed the discovery of complex signaling networks which are essential for Dictyostelium development and are also conserved in other organisms. The levels of cAMP (both intracellular and extracellular) play essential roles in every stage of Dictyostelium development, regulating many different signal transduction pathways. Two-component systems, involving histidine kinases and response regulators, have been found to regulate intracellular cAMP levels and PKA during terminal differentiation. The sequence of the Dictyostelium genome is expected to be completed in less than two years. Nevertheless, the available sequences that are already being released, together with the results of expressed sequence tags (ESTs), are providing invaluable tools to identify new and interesting genes for further functional analysis. Global expression studies, using DNA microarrays in synchronous development to study temporal changes in gene expression, are presently being developed. In the near future, the application of this type of technology to the complete set of Dictyostelium genes (approximately 10,000) will facilitate the discovery of the effects of mutation of components of the signaling networks that regulate Dictyostelium development on changes in gene expression.     

The Regulation of Dictyostelium Development by Transmembrane Signalling

ABSTRACT. Dictyostelium discoideum has a well characterized life cycle where unicellular growth and multicellular development are separated events. Development is dependent upon signal transduction mediated by cell surface, cAMP receptor/G protein linkages. Secreted cAMP acts extracellularly as a primary signal and chemoattractant. There are 4 genes for the distinct cAMP receptor subtypes, CAR1, CAR2, CAR3 and CAR4. These subtypes are expressed with temporally and spatially specific patterns and cells carrying null mutations for each gene have distinct developmental phenotypes. These results indicate an essential role for cAMP signalling throughout Dictyostelium development to regulate such diverse pathways as cell motility, aggregation (multicellularity), cytodifferentiation, pattern formation and cell type-specific gene expression.     

Requirements for the adenylyl cyclases in the development of Dictyostelium

It has been suggested that all intracellular signaling by cAMP during development of Dictyostelium is mediated by the cAMP-dependent protein kinase, PKA, since cells carrying null mutations in the acaA gene that encodes adenylyl cyclase can develop so as to form fruiting bodies under some conditions if PKA is made constitutive by overexpressing the catalytic subunit. However, a second adenylyl cyclase encoded by acrA has recently been found that functions in a cell autonomous fashion during late development. We have found that expression of a modified acaA gene rescues acrA- mutant cells indicating that the only role played by ACR is to produce cAMP. To determine whether cells lacking both adenylyl cyclase genes can develop when PKA is constitutive we disrupted acrA in a acaA- PKA-C(over) strain. When developed at high cell densities, acrA- acaA- PKA-C(over) cells form mounds, express cell type-specific genes at reduced levels and secrete cellulose coats but do not form fruiting bodies or significant numbers of viable spores. Thus, it appears that synthesis of cAMP is required for spore differentiation in Dictyostelium even if PKA activity is high.     

An adenylyl cyclase that functions during late development of Dictyostelium.

A variety of extracellular signals lead to the accumulation of cAMP which can act as a second message within cells by activating protein kinase A (PKA). Expression of many of the essential developmental genes in Dictyostelium discoideum are known to depend on PKA activity. Cells in which the receptor-coupled adenylyl cyclase gene, acaA, is genetically inactivated grow well but are unable to develop. Surprisingly, acaA(-) mutant cells can be rescued by developing them in mixtures with wild-type cells, suggesting that another adenylyl cyclase is present in developing cells that can provide the internal cAMP necessary to activate PKA. However, the only other known adenylyl cyclase gene in Dictyostelium, acgA, is only expressed during germination of spores and plays no role in the formation of fruiting bodies. By screening morphological mutants generated by Restriction Enzyme Mediated Integration (REMI) we discovered a novel adenylyl cyclase gene, acrA, that is expressed at low levels in growing cells and at more than 25-fold higher levels during development. Growth and development up to the slug stage are unaffected in acrA(-) mutant strains but the cells make almost no viable spores and produce unnaturally long stalks. Adenylyl cyclase activity increases during aggregation, plateaus during the slug stage and then increases considerably during terminal differentiation. The increase in activity following aggregation fails to occur in acrA(-) cells. As long as ACA is fully active, ACR is not required until culmination but then plays a critical role in sporulation and construction of the stalk.     

Initiation of multicellular differentiation in Dictyostelium discoideum is regulated by coronin A

Many biological systems respond to environmental changes by activating intracellular signaling cascades, resulting in an appropriate response. One such system is represented by the social amoeba Dictyostelium discoideum. When food sources become scarce, these unicellular cells can initiate a cAMP-driven multicellular aggregation program to ensure long-term survival. On starvation, the cells secrete conditioned medium factors that initiate cAMP signal transduction by inducing expression of genes such as cAMP receptors and adenylate cyclase. The mechanisms involved in the activation of the first pulses of cAMP release have been unclear. We here show a crucial role for the evolutionarily conserved protein coronin A in the initiation of the cAMP response. On starvation, coronin A–deficient cells failed to up-regulate the expression of cAMP-regulated genes, thereby failing to initiate development, despite a normal prestarvation response. Of importance, external addition of cAMP to coronin A–deficient cells resulted in normal chemotaxis and aggregate formation, thereby restoring the developmental program and suggesting a functional cAMP relay in the absence of coronin A. These results suggest that coronin A is dispensable for cAMP sensing, chemotaxis, and development per se but is part of a signal transduction cascade essential for system initiation leading to multicellular development in Dictyostelium.     

Changes during differentiation in requirements for cAMP for expression of cell-type-specific mRNAs in the cellular slime mold, Dictyostelium discoideum

A number of genes encoding developmentally regulated mRNAs in the cellular slime mold, Dictyostelium discoideum, have been described. Many of these are regulated by cAMP. Analysis of the earliest time at which elevated levels of cAMP can induce the expression of these mRNAs reveals a more complex pattern of regulation in which genes change in their ability to be induced in response to cAMP with developmental stage. A prestalk mRNA (C1/D11) previously thought not be regulated by elevated levels of cAMP is inducible by cAMP between aggregation and loose mound stage; later in development its expression becomes independent of elevated cAMP. The early prespore genes (prespore class I) also show two modes of regulation; early in development they are induced independently of continuous elevated levels of cAMP, while later in development their expression is dependent upon elevated cAMP. The period during development when the prestalk genes are cAMP inducible precedes by 2 hr the first time at which either the early prespore class I or late prespore class II mRNAs are inducible by continuous elevated levels of cAMP. Previous analysis of these mRNAs has been carried out using Dictyostelium cells grown axenically. In this report we have studied the developmental expression of these mRNAs in cells grown on bacteria. A substantial shutoff of the class I prestalk and early prespore (class I) mRNAs not seen in axenically grown cells is observed when bacterially grown cells are plated for development. Less than 10% of the maximal level of these mRNAs remains in the cells at the time of mature spore and stalk differentiation. Additionally, in the bacterially grown cells two distinct patterns of developmental regulation are observed for mRNAs which in axenically growing cells appear to be constitutively expressed throughout growth and development.     

Different molecular mechanisms for cAMP regulation of gene expression during Dictyostelium development

Alterations in cAMP concentrations have been implicated in developmentally regulated gene expression in Dictyostelium. Using a variety of culture conditions to control the metabolism of cAMP during cytodifferentiation, I have examined the role of the cyclic nucleotide in development. Conditions which allow intracellular synthesis of cAMP promote the normal developmental repression of gene M4-1 by a mechanism which is completely independent of the formation of multicellular aggregates. If, however, cells are inhibited in their ability to activate adenylate cyclase and, thus, intracellular cAMP signaling, they prove unable to repress M4-1, even in the presence of exogenous cAMP. In contrast, expression of genes which exhibit maximal activity after aggregate formation depends upon accumulation of extracellular cAMP. Inhibition of intracellular cAMP signaling does not prevent the expression of these genes if cultures are simultaneously exposed to high levels of exogenously added extracellular cAMP. These results indicate that there are at least two independent mechanisms involved in the developmental regulation of gene expression by cAMP in Dictyostelium. I discuss plausible molecular mechanisms through which cAMP might alter gene expression.