Inositol phosphate-containing molecules play an important role in a broad range of cellular processes. Inositol 5-phosphatases participate in the regulation of these signaling molecules. We have identified four inositol 5-phosphatases in Dictyostelium discoideum, Dd5P1-4, showing a high diversity in domain composition. Dd5P1 possesses only a inositol 5-phosphatase catalytic domain. An unique domain composition is present in Dd5P2 containing a RCC1-like domain. RCC1 has a seven-bladed propeller structure and interacts with Gproteins. Dd5P3 and Dd5P4 have a domain composition similar to human Synaptojanin with a SacI domain and OCRL with a RhoGAP domain, respectively. We have expressed the catalytic domains and show that these inositol 5-phosphatases have different substrate preferences. Single and double gene inactivation suggest a functional redundancy for Dd5P1, Dd5P2, and Dd5P3. Inactivation of the gene coding for Dd5P4 leads to defects in growth and development. These defects are restored by the expression of the complete protein but not by the 5-phosphatase catalytic domain.Inositol phosphates play a role in a variety of eukaryotic cellular processes, including chemotaxis and membrane trafficking. They are regulated by a number of enzymes. The group of phosphatidylinositol 3-kinases (PI3K) 1 phosphorylates the lipid substrates PI, PI(4)P, and PI(4,5)P 2 at the 3-position of the inositol ring (1). The lipid product PI(3,4,5)P 3 has been strongly implicated to be important in chemotaxis in neutrophils and fibroblasts (2, 3). PTEN, identified as a tumor suppressor gene (4), reverses the action of PI3K by dephosphorylation of PI(3,4,5)P 3 and PI(3,4)P 2 at the 3-position (5). Another group of enzymes, the inositol 5-phosphatases, can remove the phosphate group at the 5-position of the inositol ring (6 -8). The importance of inositol 5-phosphatase activity in PI(3,4,5)P 3 regulation is demonstrated by SHIP1. In stimulated B-cells, SHIP1 accounts for the major phosphatase activity toward PI(3,4,5)P 3 , and inactivation of SHIP1 leads to an increased and prolonged PI(3,4,5)P 3 production (9). Other inositol 5-phosphatases have been shown to play important roles in a number of cellular processes. Mutations in the inositol 5-phosphatase OCRL are responsible for Lowe syndrome in human (10), and deletion of the presynaptic inositol 5-phosphatase Synaptojanin leads to neurological abnormalities and early death of mice (11).In the social amoeba, Dictyostelium discoideum chemotaxis toward folic acid and cAMP is an essential strategy for survival (12). Several observations suggest that phosphatidylinositol phosphates mediate chemotaxis and, in particular, the localization of the signal inside D. discoideum cells. The PH domains of a number of proteins involved in chemotaxis, including CRAC, Akt/PKB, and PhdA, have been shown to transiently localize at the leading edge of cells moving in a chemotactic gradient (13-15). As these PH domains bind to PI(3,4,5)P 3 and PI(3,4)P 2, an asymmetric lipid distribution is implicated by...

The core of adenylate and guanylate cyclases is formed by an intramolecular or intermolecular dimer of two cyclase domains arranged in an antiparallel fashion. Metazoan membrane-bound adenylate cyclases are composed of 12 transmembrane spanning regions, and two cyclase domains which function as a heterodimer and are activated by G-proteins. In contrast, membrane-bound guanylate cyclases have only one transmembrane spanning region and one cyclase domain, and are activated by extracellular ligands to form a homodimer. In the cellular slime mould, Dictyosteliumdiscoideum, membrane-bound guanylate cyclase activity is induced after cAMP stimulation; a G-protein-coupled cAMP receptor and G-proteins are essential for this activation. We have cloned a Dictyostelium gene, DdGCA, encoding a protein with 12 transmembrane spanning regions and two cyclase domains. Sequence alignment demonstrates that the two cyclase domains are transposed, relative to these domains in adenylate cyclases. DdGCA expressed in Dictyostelium exhibits high guanylate cyclase activity and no detectable adenylate cyclase activity. Deletion of the gene indicates that DdGCA is not essential for chemotaxis or osmo-regulation. The knock-out strain still exhibits substantial guanylate cyclase activity, demonstrating that Dictyostelium contains at least one other guanylate cyclase.

Recently, we recognized two genes, gbpA and gbpB, encoding putative cGMP-binding proteins with a Zn(2+)-hydrolase domain and two cyclic nucleotide binding domains. The Zn(2+)-hydrolase domains belong to the superfamily of beta-lactamases, also harboring a small family of class II phosphodiesterases from bacteria and lower eukaryotes. Gene inactivation and overexpression studies demonstrate that gbpA encodes the cGMP-stimulated cGMP-phosphodiesterase that was characterized biochemically previously and was shown to be involved in chemotaxis. cAMP neither activates nor is a substrate of GbpA. The gbpB gene is expressed mainly in the multicellular stage and seems to encode a dual specificity phosphodiesterase with preference for cAMP. The enzyme hydrolyses cAMP approximately 9-fold faster than cGMP and is activated by cAMP and cGMP with a K(A) value of approximately 0.7 and 2.3 microM, respectively. Cells with a deletion of the gbpB gene have increased basal and receptor stimulated cAMP levels and are sporogeneous. We propose that GbpA and GbpB hydrolyze the substrate in the Zn(2+)-hydrolase domain, whereas the cyclic nucleotide binding domains mediate activation. The human cGMP-stimulated cAMP/cGMP phosphodiesterase has similar biochemical properties, but a completely different topology: hydrolysis takes place by a class I catalytic domain and GAF domains mediate cGMP activation.

Dictyostelium discoideum cells possess multiple cyclic nucleotide phosphodiesterases that belong either to class I enzymes that are present in all eukaryotes or to the rare ␤-lactamase class II. We describe here the identification and characterization of DdPDE4, the third class I enzyme of Dictyostelium. The deduced amino acid sequence predicts that DdPDE4 has a leader sequence, two transmembrane segments, and an extracellular catalytic domain that exhibits a high degree of homology with human cAMP-specific PDE8. Expression of the catalytic domain of DdPDE4 shows that the enzyme is a cAMP-specific phosphodiesterase with a K m of 10 M; cGMP is hydrolyzed at least 100-fold more slowly. The full-length protein is shown to be membrane-bound with catalytic activity exposed to the extracellular medium. Northern blots and activity measurements reveal that expression of DdPDE4 is low during single cell stages and increases at 9 h of starvation, corresponding with mound stage. A function during multicellular development is confirmed by the phenotype of ddpde4 ؊ knock-out strains, showing normal aggregation but impaired development from the mound stage on. These results demonstrate that DdPDE4 is a unique membranebound phosphodiesterase with an extracellular catalytic domain regulating intercellular cAMP during multicellular development.During different developmental stages of Dictyostelium discoideum the cyclic nucleotides cAMP and cGMP play a central role in diverse signal transduction processes. cAMP mediates chemotaxis during cell aggregation and controls gene expression during development. cGMP regulates cytoskeletal organization affecting shape, stability, and motility of single cells. The intracellular concentration of the second messengers cAMP and cGMP is determined by the combined action of production and removal. Production directly depends on the enzymatic activity of the adenylyl cyclases and guanylyl cyclases to form cAMP and cGMP, respectively. Removal of intracellular cAMP or cGMP depends on the activity of phosphodiesterases (PDEs) 3 that hydrolyze cAMP to form 5Ј-AMP and cGMP to form 5Ј-GMP and on the ability of D. discoideum cells to secrete cAMP. This extrusion mechanism is pivotal in the formation of extracellular cAMP waves that mediate chemotaxis during aggregation, mound formation, and slug movement.In D. discoideum three different adenylyl cyclases and two guanylyl cyclases have been identified (see Refs. 1 and 2). In addition, five different phosphodiesterases have been reported and characterized in D. discoideum. These PDEs belong to two classes that exhibit distinct differences in the amino acid sequence of the putative catalytic domains, namely class I, which is ubiquitous in eukaryotes, and class II, which predominantly occurs in lower eukaryotes.PdsA (or DdPDE1) is a class II dual specificity PDE that degrades cAMP as well as cGMP and is exposed on the cell surface or secreted in the medium. It is the main PDE that degrades extracellular cAMP and thereby essential for shaping cAMP waves (3...

In Dictyostelium cAMP and cGMP have important functions as first and second messengers in chemotaxis and development. Two cyclic-nucleotide phosphodiesterases (DdPDE 1 and 2) have been identified previously, an extracellular dual-specificity enzyme and an intracellular cAMP-specific enzyme (encoded by the psdA and regA genes respectively). Biochemical data suggest the presence of at least one cGMP-specific phosphodiesterase (PDE) that is activated by cGMP. Using bioinformatics we identified a partial sequence in the Dictyostelium expressed sequence tag database that shows a high degree of amino acid sequence identity with mammalian PDE catalytic domains (DdPDE3). The deduced amino acid sequence of a full-length DdPDE3 cDNA isolated in this study predicts a 60 kDa protein with a 300-residue C-terminal PDE catalytic domain, which is preceded by approx. 200 residues rich in asparagine and glutamine residues. Expression of the DdPDE3 catalytic domain in Escherichia coli shows that the enzyme has Michaelis-Menten kinetics and a higher affinity for cGMP (K(m)=0.22 microM) than for cAMP (K(m)=145 microM); cGMP does not stimulate enzyme activity. The enzyme requires bivalent cations for activity; Mn(2+) is preferred to Mg(2+), whereas Ca(2+) yields no activity. DdPDE3 is inhibited by 3-isobutyl-1-methylxanthine with an IC(50) of approx. 60 microM. Overexpression of the DdPDE3 catalytic domain in Dictyostelium confirms these kinetic properties without indications of its activation by cGMP. The properties of DdPDE3 resemble those of mammalian PDE9, which also shows the highest sequence similarity within the catalytic domains. DdPDE3 is the first cGMP-selective PDE identified in lower eukaryotes.