Rybalkin Sergei D, Rybalkina Irina G, Shimizu-Albergine Masami, Tang Xiao-Bo, Beavo Joseph A
Department of Pharmacology, University of Washington, Seattle, WA 98195-7280, USA.
EMBO J. 2003 Feb 3;22(3):469-78. doi: 10.1093/emboj/cdg051.
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.
环磷酸鸟苷（cGMP）特异性、结合cGMP的磷酸二酯酶（PDE5）调节诸如平滑肌舒张和神经元存活等生理过程。PDE5含有两个N端结构域（GAF A和GAF B），但这些结构域的功能作用尚未确定。在此我们表明，重组PDE5在cGMP与GAF A结构域结合时直接被激活，且这种效应不需要PDE5磷酸化。PDE5对cGMP表现出时间和浓度依赖性的可逆激活，在低底物浓度（0.1微摩尔/升cGMP）下观察到最高激活（9至11倍）。一种针对GAF A的单克隆抗体阻断cGMP结合，阻止PDE5激活并降低基础活性，表明处于未激活状态的PDE5具有低内在催化活性。激活的PDE5对西地那非的敏感性高于未激活的PDE5。cGMP结合对PDE5催化活性的刺激作用表明，这种酶激活机制可能在其他含GAF结构域的蛋白质中普遍存在。数据还表明，基于与该位点结合开发PDE5活性的激动剂和拮抗剂可能是可行的。
