DdPDE4, a Novel cAMP-specific Phosphodiesterase at the Surface of Dictyostelium Cells

Abstract

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...

Computational modelling suggests dynamic interactions between Ca2+, IP3 and G protein-coupled modules are key to robust Dictyostelium aggregation

“…Recent experimental results have also shown that a PDE isoform is expressed on the outer surface of the Dictyostelium cell membrane. 58 Fig. 6A reveals the emergence followed by the disappearance of ACA oscillations predicted by our model in response to a gradual increase and decrease in the extracellular PDE concentration (Fig.…”

A Protein with Similarity to PTEN Regulates Aggregation Territory Size by Decreasing Cyclic AMP Pulse Size during Dictyostelium discoideum Development

“…Cells were then stimulated with an oversaturating dose of 2Ј-deoxy-cAMP to eliminate the responses to the intracellular as well as newly synthesized cAMP (11). In the presence of DTT, which inhibits most PDE activity (2,33), cells accumulate newly produced cAMP upon 2Ј-deoxy-cAMP stimulation (11). We observed that wild-type cells accumulated cAMP (Fig.…”

“…Secreted PDE activities in CM and intracellular PDE activities in cell lysates were determined by measuring the hydrolysis of [ 3 H]cAMP, as previously described (2). PIP 3 levels were measured as described previously (35).…”

Characterization of Renal Ecto-Phosphodiesterase

“…For example, the slime mold Dictyostelium discoideum secretes cAMP as a signaling molecule. Importantly, a recent study identified and characterized an ecto-PDE in D. discoideum that has a catalytic domain with a high degree of homology with that of mammalian PDE8 (Bader et al, 2006). A mammalian enzyme similar to the D. discoideum ecto-PDE remains to be identified.…”