Molecular Control of cGMP Signaling by PKGs and PDEs

描述(由申请人提供)： 由于cGMP在哺乳动物中的作用迅速增加，包括平滑肌松弛、血小板抑制、中性粒细胞脱颗粒、视觉、基因表达、离子和水运输、骨吸收、皮肤变暗、长期神经抑制和阿片作用，cGMP介导自然信号如一氧化氮、利钠肽和鸟苷素的作用，以及硝酸甘油和西地那非(Viagra/TM)等药物的作用。鸟苷酸环化酶催化cGMP的合成，磷酸二酯酶(PDE)催化cGMP的降解，两者的平衡决定了cGMP的组织水平。已知的介导cGMP效应的细胞内受体有cGMP依赖的蛋白激酶(PKG)、cGMP门控离子通道蛋白、cGMP结合的PDE(PDE2、PDE5、PDE6、PDE10、PDE11)和交叉激活的cAMP依赖的蛋白激酶(PKA)。这一应用的主要主题是PKG和PDE5/PDE11的调节，特别强调这两个酶类之间的功能关系。提示PDE5可能通过上调cGMP来调节PKG激活后cGMP的下降，这代表了cGMP途径的负反馈调节。其中一些机制增加了PDE5催化位点的亲和力，因此药物如伟哥TM上调cGMP将导致其自身作用的增强。推测这些机制既包括cGMP结合对PDE5的变构调节，也包括PKG对PDE5的磷酸化。延长组织中cGMP升高导致鸟苷酸环化酶、PDE5和PKG水平的代偿性调整的概率也将被检查。对PKG自我抑制的基本机制以及解除自身抑制的基本机制的研究将展开。定点突变将被用来研究保守的丝氨酸并列在PKG的假底物位置上的分子作用，它对自身抑制和cGMP结合的抑制都有很大的贡献。Arg-59在假底物位点中的作用也将被仔细研究。用小角X射线散射、三维电子显微镜和氢/氢交换等手段研究了PKG的四级结构和磁区形貌，并解释了cGMP结合对这些参数的影响。三种可能商业化的PDE5抑制剂(西地那非、伐地那非、他达拉非)已经被氚标记。它们将被用来鉴定和定量组织粗提物中的PDE5和PDE11，并在这些提取物中寻找其他PDE抑制物结合蛋白。它们还将被用来研究PDE5和PDE11的未知催化中心特征，如催化中心的异质性、结合亲和力以及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)的结合以及PKG磷酸化PDE5对放射性标记PDE5抑制剂催化位点亲和力的影响。将对PDE11进行全面的物理和生化表征，并探索通过配体与其GAF结构域结合或通过酶磷酸化来调节该酶的可能性。

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.