title: Characterization of the tandem GAF domain of human phosphodiesterase 5 using a cyanobacterial adenylyl cyclase as a reporter enzyme.
abstract: We analyzed cGMP signaling by the human phosphodiesterase 5 (hPDE5) tandem GAF domain based on a functional activation assay. The C-terminal catalytic domain of the cyanobacterial adenylyl cyclase (AC) cyaB1 was used as a reporter enzyme. We demonstrate functional coupling between the hPDE5 GAF ensemble and the AC resulting in a chimera stimulated 10-fold by cGMP. The hPDE5 GAF domain has an inhibitory effect on AC activity, which is released upon cGMP activation. Removal of 109 amino acids from the N terminus resulted in partial disengagement of the GAF domain and AC, i.e. in a 10-fold increase in basal activity, and affected cGMP affinity. The Ser-102 phosphorylation site of hPDE5 increased cGMP affinity, as shown by a 5-fold lower K(D) for cGMP in a S102D mutant, which mimicked complete modification. The function of the NKFDE motif, which is a signature of all GAF domains with known cyclic nucleotide binding capacity, was elucidated by targeted mutations. Data with either single and double mutants in either GAF A or GAF B or a quadruple mutant affecting both subdomains simultaneously indicated that it is impossible to functionally assign cGMP binding and intramolecular signaling to either GAF A or B of hPDE5. Both subdomains are structurally and functionally interdependent and act in concert in regulating cycaB1 AC and, most likely, also hPDE5.
authors.affiliation: Abteilung Pharmazeutische Biochemie, Fakultät für Chemie und Pharmazie, Universität Tübingen, Morgenstelle 8, 72076 Tübingen, Germany.
authors.forename: Sandra
authors.initials: S
authors.lastname: Bruder
authors.forename: Anita
authors.initials: A
authors.lastname: Schultz
authors.forename: Joachim E
authors.initials: JE
authors.lastname: Schultz
mesh_terms: D015106:3',5'-Cyclic-GMP Phosphodiesterases; D000262:Adenylyl Cyclases / Q000235:genetics* / Q000378:metabolism*; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D020134:Catalytic Domain; D000458:Cyanobacteria / Q000201:enzymology / Q000235:genetics; D000242:Cyclic AMP / Q000494:pharmacology; D006152:Cyclic GMP / Q000494:pharmacology; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; D017930:Genes, Reporter; D006801:Humans; D007700:Kinetics; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D000242:Cyclic AMP; D010727:Phosphoric Diester Hydrolases; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; C517381:PDE5A protein, human; D000262:Adenylyl Cyclases; D006152:Cyclic GMP