title: PDE5 is converted to an activated state upon cGMP binding to the GAF A domain.
abstract: cGMP-specific, cGMP-binding phosphodiesterase (PDE5) regulates such physiological processes as smooth muscle relaxation and neuronal survival. PDE5 contains two N-terminal domains (GAF A and GAF B), but the functional roles of these domains have not been determined. Here we show that recombinant PDE5 is activated directly upon cGMP binding to the GAF A domain, and this effect does not require PDE5 phosphorylation. PDE5 exhibited time- and concentration-dependent reversible activation in response to cGMP, with the highest activation (9- to 11-fold) observed at low substrate concentrations (0.1 micro M cGMP). A monoclonal antibody directed against GAF A blocked cGMP binding, prevented PDE5 activation and decreased basal activity, revealing that PDE5 in its non-activated state has low intrinsic catalytic activity. Activated PDE5 showed higher sensitivity towards sildenafil than non-activated PDE5. The stimulatory effect of cGMP binding on the catalytic activity of PDE5 suggests that this mechanism of enzyme activation may be common among other GAF domain-containing proteins. The data also suggest that development of agonists and antagonists of PDE5 activity based on binding to this site might be possible.
authors.affiliation: Department of Pharmacology, University of Washington, Seattle, WA 98195-7280, USA.
authors.forename: Sergei D
authors.initials: SD
authors.lastname: Rybalkin
authors.forename: Irina G
authors.initials: IG
authors.lastname: Rybalkina
authors.forename: Masami
authors.initials: M
authors.lastname: Shimizu-Albergine
authors.forename: Xiao-Bo
authors.initials: XB
authors.lastname: Tang
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Beavo
mesh_terms: D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D002460:Cell Line; D006152:Cyclic GMP / Q000378:metabolism*; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; D004789:Enzyme Activation; D006801:Humans; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010726:Phosphodiesterase Inhibitors / Q000378:metabolism; D010766:Phosphorylation; D010879:Piperazines / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011687:Purines; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000068677:Sildenafil Citrate; D013450:Sulfones
chemical_list: D000911:Antibodies, Monoclonal; D010726:Phosphodiesterase Inhibitors; D010879:Piperazines; D011687:Purines; D011994:Recombinant Proteins; D013450:Sulfones; D000068677:Sildenafil Citrate; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; C517381:PDE5A protein, human; C517382:Pde5a protein, mouse; D006152:Cyclic GMP