Mini-theme Critical Review on Nucleotidyl Cyclases

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1 IUBMB Life, 56(9): , September 2004 Mini-theme Critical Review on Nucleotidyl Cyclases Adenylyl Cyclase Expression and Regulation During the Differentiation of Dictyostelium Discoideum Paul W. Kriebel and Carole A. Parent Laboratory of Cellular and Molecular Biology, National Cancer Institute, NIH, Bethesda, MD, USA Summary Cyclic AMP metabolism is essential for the survival of the social amoebae Dictyostelium discoideum. Three distinct adenylyl cyclases are expressed and required for the normal development of this simple eukaryote. The adenylyl cyclase expressed during aggregation, ACA, is related to the mammalian and Drosophila G protein-coupled enzymes and is responsible for the synthesis of camp that is required for cell-cell signaling in early development. ACB harbors histidine kinase and response-regulator domains and is required for terminal differentiation. Finally, the adenylyl cyclase expressed during germination, ACG, acts as an osmosensor and is involved in controlling spore germination. Together, these enzymes generate the various levels of camp that are required for D. discoideum to transition from uni- to multi-cellularity. This review will highlight the properties of these enzymes and describe the signaling cascades that lead to their activation. IUBMB Life, 56: , 2004 Keywords Chemotaxis; development; camp; G proteins; PH domains; osmolarity; histidine kinase. INTRODUCTION Cyclic AMP metabolism is an integral part of Dictyostelium discoideum development. In the presence of nutrients, these social amoebae live a solitary life, independently hunting and ingesting various food sources. Upon starvation however, this egoistic behavior is drastically changed and the cells start to communicate and aggregate, eventually forming mounds that differentiate into a fruiting body composed of resistant spores atop a stalk of vacuolated cells (1, 2). This transition is mediated by a variety of signaling molecules, one of them being camp. D. discoideum cells start to synthesize and secrete camp a few hours after the initiation of starvation. The secreted camp binds Received 16 September 2004; accepted 16 September 2004 Address correspondence to: Carole A. Parent, Laboratory of Cellular and Molecular Biology, National Cancer Institute, National Institutes of Health, 37 Convent Drive, Bldg.37/Rm2066, Bethesda MD , USA. Tel: Fax: parentc@helix.nih.gov and activates a family of G protein-coupled receptors (camp receptors or cars) leading to the activation of multiple downstream effectors, which eventually give rise to chemotaxis, the control of gene expression, and the synthesis and secretion of additional camp. This signal relay loop allows cells to aggregate by chemotaxis and align in a head to tail fashion to form streams that later coalesce into mounds (Fig. 1). Because it is highly accessible at the biochemical and genetic levels, D. discoideum has been invaluable to study signal transduction mechanisms in the context of a variety of fundamental cellular processes, including cytokinesis, motility and chemotaxis, as well as tissue formation and cell-fate determination. Throughout the developmental program of D. discoideum, a multitude of signaling components are expressed with specific temporal and spatial characteristics (3). Among them, four cars subtypes (car1-car4) and 11 Ga subunits are sequentially expressed throughout development. Each of the 11 Ga subunits are thought to be coupled to the same Gbg complex. Of the multiple effectors present, a single PLC, at least five PI3Ks and seven PTENs (one of which displaying all the domains found in the mammalian PTEN), more than 20 Ras superfamily homologues, two guanylyl cyclases, multiple members of the MAPK pathway, and four STATs have been identified. As camp plays a key role in the development of D. discoideum, the components involved in its synthesis and breakdown as well as its main cytosolic effector, PKA, have been studied in great detail. Three class III adenylyl cyclases, ACA, ACB, and ACG, with different topologies and regulatory mechanisms, account for the synthesis of camp. The degradation of extracellular camp is mediated by membrane-bound and secreted forms of phosphodiesterase (Pde), both encoded by a single gene, PdsA. Conversely, the breakdown of intracellular camp is mainly achieved by two distinct Pdes: RegA and PdeE. Finally, all of the known effects of cytosolic camp are mediated by a unique PKA, which, in contrast to the mammalian enzyme, is composed of single regulatory and catalytic subunits (4 6). In this review, we will focus on the recent progress that has been made in the cloning and characterization of the three ISSN print/issn online # 2004 IUBMB DOI: /

2 542 KRIEBEL AND PARENT Figure 1. Adenylyl cyclase regulation during the development of D. discoideum. The top panel shows a montage of pictures depicting the morphological changes that take place during the development of D. discoideum. The mature fruiting body is *2mm in height. The time after the initiation of starvation is written for each picture. The cartoon below the pictures illustrates the signaling cascades that regulate the three adenylyl cyclases expressed in D. discoideum, ACA, ACB, and ACG (see text for details). ACB and ACG are drawn as dimers. The picture on the bottom left corner depicts the cellular distribution of ACA in differentiated chemotaxing cells. The picture was taken as 6-h stage ACA-YFP/aca 7 cells are forming streams during chemotaxis. The inset shows a high magnification image of one cell the red star depicts the front of the migrating cell. adenylyl cyclases expressed throughout D. discoideum s development. As most of the signaling components listed above have been characterized at the biochemical level and through the analyses of deleted cell lines, the reader is referred to excellent recent reviews that describe pathways that use these other components (7 12). ACA IS REGULATED BY HETEROTRIMERIC G PROTEINS The adenylyl cyclase gene expressed during aggregation (ACA) was cloned using a fragment of the catalytic domain of ACG (adenylyl cyclase expressed during germination, see below) to screen a cdna library made from aggregating cells (13). The ACA gene encodes a protein of 1407 amino acids that shares homology and is topologically related to the mammalian and Drosophila G protein-coupled adenylyl cyclases, which are predicted to be composed of two sets of six transmembrane domains each followed by a large cytosolic loop where the catalytic and part of the regulatory domains reside (Fig. 1). The regions of homology between ACA and the Type I mammalian enzyme are restricted to the two catalytic loops, showing *50% similarity. Furthermore, random mutagenesis screens of ACA identified highly conserved residues, within the catalytic loops, that are essential for activity and regulation by G proteins (14 16). ACA is maximally expressed in aggregating cells, showing peak levels *6 h after the initiation of starvation. Growing cells are devoid of measurable levels of ACA (13). ACA is Regulated by Gbg-subunits and Cytosolic Regulators The most interesting aspect of ACA lies in its mode of regulation (Fig. 1). As observed for the mammalian adenylyl

3 ADENYLYL CYCLASE FUNCTION AND REGULATION IN DICTYOSTELIUM 543 cyclases, the enzymatic activity of ACA is tightly regulated by heterotrimeric G proteins. The activation of car1 following the addition of exogenous camp, gives rise to a rapid but transient activation of ACA, peaking * 1 min after receptor stimulation, followed by a return to basal levels within 7 10 min (13). Cells lacking Ga2 (which couples to car1) or Gb subunits, lose any measurable agonist-mediated adenylyl cyclase activity (17, 18). Interestingly however, while GTPgSmediated activation, which bypasses the receptor, is lost in gb 7 cells, it still occurs in ga2 7 cells (18, 19). This finding suggests that, in contrast to the mammalian adenylyl cyclases, Ga subunits do not directly activate ACA. Instead, in D. discoideum, the activation of ACA requires, in addition to the Gb subunit, two novel cytosolic regulators: CRAC (cytosolic regulator of adenylyl cyclase) and Pianissimo (Pia). CRAC was identified from a chemical mutagenesis screen and shown to be essential for receptor- and GTPgS-mediated activation of adenylyl cyclase (20). It was also established that the loss of adenylyl cyclase activity in crac 7 lysates could be restored by the addition of cytosolic fractions harvested from wild type cells (21). Not surprisingly, cells lacking CRAC do not enter development and do not aggregate when starved. Aside from harboring an N-terminal pleckstrin homology (PH) domain, CRAC does not contain other known sequence motifs (22). It has been determined that receptor stimulation induces a rapid (peak at *5 s) and transient (back to basal levels at *2 min) translocation of CRAC from the cytosol to the plasma membrane. Remarkably, this translocation occurs specifically at the leading edge of chemotaxing cells, thereby spatially sequestering the signaling cascade (23). The PH domain of CRAC, through its 3-phosphoinositide binding capacity, has been shown to be sufficient and required to mediate plasma membrane recruitment (24). Indeed, the concerted action of the 3-phosphoinositide metabolizing enzymes, PI3K and PTEN, is involved in tightly controlling the translocation of CRAC as well as PKB, another PH domain-containing protein that specifically associates with the leading edge of chemotaxing D. discoideum and neutrophils (25 28). As in mammalian cells, the D. discoideum PI3K harbors a Ras binding domain that is essential for its activation and cells lacking AleA, a Ras exchange factor, RIP3, a Ras interacting protein, or RasC show a decreased ability to activate ACA following receptor stimulation (26, 29 31). Moreover, cells lacking PTEN show a dramatically higher peak of ACA activation compared to wild type cells (27). These results suggest that the recruitment of CRAC to the plasma membrane is somehow involved in the activation of ACA. However, there must be additional steps between CRAC translocation and ACA activation as these two proteins have distinct spatial and temporal behaviors. Indeed, in sharp contrast to the translocation of CRAC at the front of chemotaxing cells, ACA is highly enriched at the back of polarized cells (32). In addition, as mentioned, the kinetics of the receptor-mediated ACA activation are slower than CRAC translocation (23). Pia was isolated from an insertional mutagenesis screen designed to identify proteins required in the early aggregation of D. discoideum (33). Pia is a 130 kda cytosolic protein that harbors no known sequence motifs. Cells lacking Pia are defective in both receptor- and GTPgS-mediated activation of ACA. As shown for CRAC, the activation of ACA in pia 7 lysates can also be recovered by the addition of wild type cytosolic fractions. Furthermore, studies of cell lines lacking CRAC and Pia have established that the activation of ACA requires both cytosolic regulators (33). In contrast to CRAC, Pia does not redistribute following receptor stimulation and the mechanism by which it activates ACA remains to be established (34). Interestingly, while CRAC homologues have yet to be found, several Pia homologues in organisms ranging from yeast to mammals have been identified. The fact that the activity of ACA shuts down in the presence of constant, saturating doses of camp implies that inhibitory signals also control its activity. The mechanisms by which this adaptation pathway works are just beginning to be understood with the identification and characterization of Ga9, which inhibits a variety of camp-dependent signals (35, 36). Cells lacking Ga9 show a dramatic delay in the adaptation of ACA as well as PI3K and GC. Conversely, wild type cells overexpressing a constitutively active mutant of Ga9, have the opposite effect displaying much more transient responses. The target of Ga9 has yet to be identified. In addition, as ACA (as well as PI3K and GC) still shows residual adaptation in the absence of Ga9, other components are clearly required. ACA is Required for Signal Relay and Streaming Experiments with cells in which ACA has been deleted by homologous recombination clearly established that ACA is responsible for the synthesis of camp that is required for cellcell signaling in early development (13). While wild type cells show a robust stimulation of adenylyl cyclase activity in response to exogenously added camp, cells lacking ACA are specifically defective in this response. Moreover, aca 7 cells do not aggregate and remain as smooth monolayers when plated on non-nutrient agar. Intriguingly, while ACA is topologically related to ABC transporters, it is not involved in the secretion of camp, as aca 7 cells exogenously expressing the unrelated adenylyl cyclase ACG from a constitutive promoter still secrete camp (13). The cellular distribution of ACA revealed a novel mechanism explaining how cells respond to secreted camp. Analysis of aca 7 cells showed that while these cells can sense and move chemotactically in exogenous gradients of camp, they do not align in a head to tail fashion and form streams (32). As mentioned above, the plasma membrane labeling of ACA is highly enriched at the back of polarized cells (32) (Fig. 1). Since aca 7 cells expressing a constitutively active mutant of ACA display dramatically reduced enrichment at the back and show serious streaming defects, it was proposed that the asymmetric distribution of ACA provides a compartment

4 544 KRIEBEL AND PARENT from which camp is specifically secreted and attracts surrounding cells that then stream during chemotaxis. The exact mechanisms that control the asymmetric cellular distribution of ACA remain to be determined, although it has been shown to require the actin cytoskeleton and to be independent of CRAC and PKA (32). ACB IS A MULTIDOMAIN ADENYLYL CYCLASE The adenylyl cyclase B gene, AcrA, was serendipitously cloned by screening for morphological mutants, although clever biochemical analyses had previously established its existence. It was first demonstrated that the addition of exogenous camp pulses to aca 7 cells restores normal gene expression and development (37). Since camp does not cross the plasma membrane, these experiments suggested that another adenylyl cyclase is present to generate the intracellular camp required for the activation of PKA. More conclusive evidence came later from studies in cells lacking RdeA. RdeA is a phosphotransferase that relays a phosphoryl group to RegA, a phosphodiesterase. Phosphorylation of RegA leads to a 20- fold increase in its phosphodiesterase activity (Fig. 1) (38 41). High levels of camp were measured in cells lacking either RdeA or RegA and, most notably, in aca 7 /rdea 7 as well as in aca 7 /acg 7 double null cell lines (42). These findings conclusively established the existence of a third adenylyl cyclase. ACB is a large 2124 amino acid protein that reportedly harbors two hydrophobic domains at its N-terminus followed by a pseudo-histidine kinase domain (Fig. 1) (43). Histidine kinase domains autophosphorylate to initiate two-component phosphorelay signaling cascades. However, in ACB, the histidine kinase domain lacks an essential histidine residue required for autophosphorylation and it is therefore believed to be inactive. C-terminal to the pseudo-histidine kinase domain is a response-regulator domain, which in two component phosphorelay signaling systems harbors an aspartate that accepts a phosphoryl group from a histidine kinase domain. The single adenylyl cyclase catalytic domain is located at the C-terminus and is most similar to the adenylyl cyclase domains found in CyaA of Anabena spirulensis. It also shares significant similarity with the catalytic domains from CyaC of Spirulena platensis and ACA, although ACA is more related to ACG than to ACB. ACB mrna levels are present at low levels in vegetative cells. In differentiating cells high levels are detected starting 4 h after the initiation of starvation. These levels remain high until culmination. ACB Shows Distinctive Activity By measuring camp accumulation in cells lacking ACA and RdeA, Kim and collaborators were able to characterize ACB activity in D. discoideum cells and lysates (42). They demonstrated that in contrast to ACA and other class III adenylyl cyclases, ACB s activity is higher in the presence of magnesium compared with manganese. Moreover, studies by Meima and Schaap later established that ACB is insensitive to the exogenous addition of chemoattractants, ammonia, bicarbonate, or to the morphogen DIF and that it is unresponsive to G protein activation (44). Taken together, these findings suggest that ACB is constitutively active and that the camp produced by ACB is controlled by the activity of the phosphodiesterase RegA. ACB is Required for Terminal Differentiation By comparing adenylyl cyclase activity throughout the development of wild type cells with the activity in cells lacking ACB, Soderbom and collaborators showed that ACB is mainly responsible for the increase in adenylyl cyclase activity that occurs after aggregation (43). In accordance with this, acb 7 cells show normal development until the slug stage after which abnormal fruiting bodies with long stalks and unstable spores are formed. This defect is reversed by the expression of a constitutively active ACA mutant that displays high basal activity. These findings strongly suggest that the main role of ACB is to synthesize camp in late development (45). ACG IS REGULATED BY HIGH OSMOLARITY The ACG gene was the first adenylyl cyclase cloned in D. discoideum. A fragment of the catalytic domain of ACG was amplified using a degenerate primer based on the second catalytic domain of the Type I and II mammalian adenylyl cyclases (13). The full-length gene was subsequently obtained by screening a genomic library. ACG is an 858 amino acid protein composed of two N-terminal hydrophobic spans the first of which is proposed to represent a signal sequence for insertion into the endoplasmic reticulum followed by a single C-terminal catalytic domain (Fig. 1). The predicted topology of ACG, with a single transmembrane domain connecting a large extracellular N-terminal domain to a unique cytoplasmic catalytic loop, is similar to the membrane-bound guanylyl cyclases. The regions of homology between ACG and the Type I mammalian adenylyl cyclase are restricted to the catalytic loops showing 53 and 60% similarity for the first and second loops. Interestingly, the similarity between the catalytic domains of ACA and ACG is also *60%. ACG is exclusively expressed after the fruiting body has formed during germination. ACG is an Osmosensor Chemoattractants or heterotrimeric G proteins do not regulate ACG (13). Chemoattractant addition to aca 7 cells expressing ACG from of a constitutive promoter (ACG/aca 7 ) does not increase adenylyl cyclase activity. Moreover, lysates derived from ACG/aca 7 cells are insensitive to the addition of GTPgS. Although, the camp synthesized by ACG can rescue the developmental defect of aca 7 cells, it does so by bypassing the signal relay loop and directly activating PKA, which leads to the regulation of downstream gene expression. Indeed, in contrast to wild type cells, which recruit neighboring cells by

5 ADENYLYL CYCLASE FUNCTION AND REGULATION IN DICTYOSTELIUM 545 signal relay and streaming to form large aggregates, ACG/ aca 7 cells form very small aggregates that give rise to undersized fruiting bodies. van Es and colleagues established that ACG activity is regulated by high osmolarity (46). They showed that the adenylyl cyclase activity of ACG/aca 7 cells is robustly stimulated following a pre-incubation with NaCl or sucrose, showing a bell-shape response peaking at 100 mm for both agents and persisting for *10 min before it returns to basal levels after the stimulus is removed. Through a series of elegant studies, Saran and Schaap later showed that determinants within ACG are responsible for sensing the change in osmolarity and that dimerization of ACG, although required for catalytic activity, does not mediate the activation process (47). ACG is Required for Spore Germination Cells lacking ACG develop into normal fruiting bodies that harbor viable spores, but display abnormalities in the germination process (46). In wild type cells, heat shockstimulated germination occurs synchronously at low osmolarity and is inhibited in the presence of 250 mm sucrose. In contrast, spores from acg 7 cells will germinate spontaneously even in high osmolarity such as 250 mm sucrose. Dormant spores have high camp levels that decrease just before germination. It has been established that high spore osmolarity, maintained by the presence of 100 mm ammonium phosphate, maintains high levels of camp and inhibits germination (48, 49). During germination ACG therefore acts as an osmosensor. In conditions of high osmolarity, activation of ACG gives rise to high levels of camp that lead to the activation of PKA and to the maintenance of dormancy. Indeed, cells exhibiting constitutive PKA activity germinate very poorly independently of the level of osmolarity (46). PERSPECTIVES D. discoideum cells express three distinct, yet related, adenylyl cyclases that are required for the transition of freeliving amoebae into healthy and viable spores. This underscores the essential and unique role of camp throughout the developmental program of D. discoideum. As the organism transitions from uni- to multi-cellularity, the amount of camp synthesized changes significantly. Before the aggregation stage, the camp signal is presented as pulses at *6 min intervals, allowing cells to migrate directionally, stream, and form tight aggregates. In aggregative cells, the signal appears in constant nm concentrations and regulates the expression of post-aggregative genes and the repression of growth and early genes. At the mound stage, mm camp levels are detected and, together with the morphogen DIF, these high levels are responsible for spore and stalk specific genes expression. In parallel to these changes, four cars are sequentially expressed throughout development, each exhibiting different affinities allowing cells to sense and respond to the various camp concentrations. ACA, ACB, and ACG are therefore part of a specialized and highly regulated signaling cascade that is essential for the survival of D. discoideum. While the molecular genetic analyses of adenylyl cyclase function in D. discoideum have established novel pathways by which these enzymes are activated and regulated, many facets of these signaling cascades remain unclear. How is ACA regulated by the cytosolic regulators CRAC and Pia and are such regulators required for the activation of the mammalian adenylyl cyclases? How is ACA targeted to the back of polarized cells? 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