cAMP is a ligand for the tandem GAF domain of human phosphodiesterase 10 and cGMP for the tandem GAF domain of phosphodiesterase 11.
N-terminal tandem GAF domains are present in 5 out of 11 mammalian phosphodiesterase (PDE) families. The ligand for the GAF domains of PDEs 2, 5, and 6 is cGMP, whereas those for PDEs 10 and 11 remained enigmatic for years. Here we used the cyanobacterial cyaB1 adenylyl cyclase, which has an N-terminal tandem GAF domain closely related to those of the mammalian PDEs, as an assay system to identify the ligands for the human PDEs 10 and 11 GAF domains. We report that a chimera between the PDE10 GAF domain and the cyanobacterial cyclase was 9-fold stimulated by cAMP (EC50= 19.8 microm), whereas cGMP had only low activity. cAMP increased Vmax in a non-cooperative manner and did not affect the Km for ATP of 27 microm. In an analogous chimeric construct with the tandem GAF domain of human PDE11A4, cGMP was identified as an allosteric activator (EC50 = 72.5 microm) that increased Vmax of the cyclase non-cooperatively 4-fold. GAF-B of PDE10 and GAF-A of PDE11A4 contain an invariant NKFDE motif present in all mammalian PDE GAF ensembles. We mutated the aspartates within this motif in both regions and found that intramolecular signaling was considerably reduced or abolished. This was in line with all data concerning GAF domains with an NKFDE motif as far as they have been tested. The data appeared to define those GAF domains as a distinct subclass within the >3100 annotated GAF domains for which we propose a tentative classification scheme.
Gross-Langenhoff
Marco
M
Abteilung Pharmazeutische Biochemie, Fakultät für Chemie und Pharmazie, Universität Tübingen, Morgenstelle 8, 72076 Tübingen, Germany.
Hofbauer
Karina
K
Weber
Jost
J
Schultz
Anita
A
Schultz
Joachim E
JE
D015106:3',5'-Cyclic-GMP Phosphodiesterases; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins; D001665:Binding Sites; D000242:Cyclic AMP / Q000378:metabolism* / Q000494:pharmacology; D006152:Cyclic GMP / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008667:Metalloproteins; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins; D015398:Signal Transduction
D001426:Bacterial Proteins; D008024:Ligands; D008667:Metalloproteins; D011993:Recombinant Fusion Proteins; C073757:auracyanin B-1 protein, bacteria; D000242:Cyclic AMP; C119843:PDE10A protein, human; D010727:Phosphoric Diester Hydrolases; D015106:3',5'-Cyclic-GMP Phosphodiesterases; C406737:PDE11A protein, human; D006152:Cyclic GMP