Molecular Control of cGMP Signaling by PKGs and PDEs

描述（由申请人提供）： 在哺乳动物中归因于cGMP的作用数量迅速增加，包括平滑肌松弛、血小板抑制、中性粒细胞脱粒、视觉、基因表达、离子和水转运、骨吸收、皮肤变黑、长期神经抑制和阿片作用。cGMP介导天然信号如一氧化氮、利钠肽和鸟苷酸的作用，以及药物如硝酸甘油和西地那非（Viagra/TM）的作用。鸟苷酸环化酶催化cGMP的合成，磷酸二酯酶（PDE）催化cGMP降解;这两种活性的平衡决定了cGMP的组织水平。据信介导cGMP效应的已知细胞内受体是cGMP依赖性蛋白激酶（PKG）、cGMP门控离子通道蛋白、cGMP结合PDE（PDE 2、PDE 5、PDE 6、PDE 10、PDE 11）和通过交叉激活的cAMP依赖性蛋白激酶（PKA）。本申请的主要主题是PKG和PDE 5/PDE 11的调节，特别强调这两种酶之间的功能关系。这表明存在多达六种机制，PDE 5通过cGMP升高介导PKG激活后cGMP的下降，这代表cGMP通路的负反馈调节。这些机制中的一些增加了PDE 5催化位点的亲和力，使得药物如Viagra TM对cGMP的升高会导致其自身作用的增强。假设这些机制涉及cGMP结合对PDE 5的变构调节以及PKG对PDE 5的磷酸化。还将检查组织中cGMP升高延长诱导鸟苷酸环化酶、PDE 5和PKG水平代偿性调整的可能性。研究PKG被自抑制的基本机制，以及自抑制被解除的基本机制。定点诱变将用于研究与PKG的假底物位点并列的保守丝氨酸的分子作用，其对自身抑制和cGMP结合的抑制都有很大贡献。Arg- 59在假底物位点中的作用也将被仔细研究。小角X射线散射，三维电子显微镜，氘/氢交换将被用来检查PKG的四级结构和域的拓扑结构，并破译cGMP结合产生的这些参数的变化。三种潜在商业化的PDE 5抑制剂（西地那非、伐地那非、他达拉非）已标记有氚。它们将用于鉴定和定量粗组织提取物中的PDE 5和PDE 11，并在这些提取物中寻找其他PDE受体结合蛋白。它们还将用于解决PDE 5和PDE 11的未知催化位点特征，如催化位点异质性、结合亲和力和二价阳离子对亲和力的影响。还将研究cGMP与变构位点（GAF结构域）结合的影响以及PKG对PDE 5磷酸化对放射性标记PDE 5抑制剂催化位点亲和力的影响。PDE 11的物理和生物化学特性将进行彻底的梳理，并将探讨通过配体结合其GAF结构域或通过酶磷酸化来调节这种酶的可能性。

DESCRIPTION (provided by applicant): The rapidly increasing number of effects ascribed to cGMP in mammals include smooth muscle relaxation, platelet inhibition, neutrophil degranulation, vision, gene expression, ion and water transport, bone resorption, skin darkening, long-term nerve depression, and opiod action, cGMP mediates effects of natural signals such as nitric oxide, natriuretic peptides, and guanylins, as well as effects of medications such as nitroglycerin and sildenafil (Viagra/TM). Guanylyl cyclases catalyze synthesis of cGMP, and phosphodiesterases (PDE) catalyze cGMP degradation; the balance of these two activities determines the tissue level of cGMP. The known intracellular receptors that are believed to mediate cGMP effects are cGMP-dependent protein kinase (PKG), cGMP-gated ion channel proteins, cGMP-binding PDEs (PDE2, PDE5, PDE6, PDE 10, PDE11), and cAMP-dependent protein kinase (PKA) by cross-activation. The main subjects of this application are regulation of PKG and PDE5/PDE11, with particular emphasis on functional relationships between these two enzyme classes. It is suggested that up to six mechanisms exist by which PDE5 mediates decline of cGMP after PKG activation by cGMP elevation, which represents negative feedback regulation of the cGMP pathway. Some of these mechanisms increase PDE5 catalytic site affinity, so that elevation of cGMP by drugs such as Viagra TM would cause potentiation of their own effects. It is hypothesized that these mechanisms involve both allosteric regulation of PDE5 by cGMP binding as well as phosphorylation of PDE5 by PKG. The probability that prolonged cGMP elevation in tissues induces compensatory adjustment in levels of guanylyl cyclase, PDE5, and PKG will also be inspected. Investigation of the fundamental mechanisms by which PKG is autoinhibited, and by which autoinhibition is relieved will be carded out. Site-directed mutagenesis will be used to study the molecular roles of a conserved serine juxtaposed to the pseudosubstrate site of PKG, which contributes strongly to both autoinhibition and inhibition of cGMP binding. The roles of Arg- 59 in the pseudosubstrate site will also be scrutinized. Small angle x-ray scattering, 3D electron microscopy, and deuterium/hydrogen exchange will be used to examine the quaternary structure and domain topography of PKG, and to decipher the changes in these parameters produced by cGMP binding. Three potentially commercialized PDE5 inhibitors (sildenafil, vardenafil, tadalafil) have been labeled with tritium. They will be used to identify and quantify PDE5 and PDE11 in crude tissue extracts, and to search for other PDE inhibitor-binding proteins in these extracts. They will also be used to address unknown catalytic site features of PDE5 and PDE11 such as catalytic site heterogeneity, binding affinity, and effects of divalent cations on affinity. Effects of cGMP binding to allosteric sites (GAF domains) and effects of PDE5 phosphorylation by PKG on catalytic site affinity for radiolabeled PDE5 inhibitors will also be studied. A thorough physical and biochemical characterization of PDE11 will be carded out, and the likelihood of regulation of this enzyme by ligand binding to its GAF domains or by enzyme phosphorylation will be explored.