DdPDE4, a novel cAMP-specific phosphodiesterase at the surface of dictyostelium cells.
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 beta-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 microm; 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 membrane-bound phosphodiesterase with an extracellular catalytic domain regulating intercellular cAMP during multicellular development.
Bader
Sonya
S
Department of Molecular Cell Biology, University of Groningen, Kerklaan 30, 9751NN Haren, the Netherlands.
Kortholt
Arjan
A
Snippe
Helena
H
Van Haastert
Peter J M
PJ
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology*; D017124:Conserved Sequence; D000242:Cyclic AMP; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D017931:DNA Primers; D004023:Dictyostelium / Q000201:enzymology* / Q000235:genetics; D017353:Gene Deletion; D006801:Humans; D008969:Molecular Sequence Data; D010641:Phenotype; D016133:Polymerase Chain Reaction; D015800:Protozoan Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D015800:Protozoan Proteins; D000242:Cyclic AMP; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4