Molecular Mechanisms of PDE5 Regulation

描述(申请人摘要)：对哺乳动物cGMP级联反应的兴趣 最近很紧张。这一领域的许多研究都集中在信号上。 调节cGMP形成的受体和鸟苷酸环化酶，以及 CGMP的细胞作用。天然信号(利钠肽、鸟苷类和 一氧化氮)，除了许多药物外，还通过以下方式刺激cGMP 激活这些循环系统。环核苷酸磷酸二酯酶(PDE)，它 也是高度调节的酶，参与调节细胞cAMP和 CGMP水平通过催化cAMP和cGMP的分解。这个的主题是 应用程序是cGMP结合的cGMP特定的PDE(PDE5)，它特定于 CGMP位于cAMP的变构cGMP结合部位和催化部位。 长期目标是确定作用机制和细胞 PDE5的调节，这是许多组织中cGMP水平的主要决定因素。 CGMP的经典作用包括松弛平滑肌、抑制血管紧张素转换酶 血小板活化、中性粒细胞脱颗粒和视觉调节。新的 这些发现扩大了这一清单，将基因表达的调控包括在内， 氯在肠道和肾脏的转运，水的转运，骨吸收， 皮肤中的黑色素生成、长期的神经抑制和阿片类药物效应。 升高cGMP的治疗剂包括PDE抑制剂(例如，咖啡因， 罂粟碱和西地那非)和硝基血管扩张剂(例如硝酸甘油)。 某些肠毒素通过升高cGMP而引起分泌性腹泻。科尔宾博士 最近证实，在某些情况下，cGMP通过“交叉激活”起作用。 CAMP受体。然而，PDE5是cGMP的特异性细胞内受体。 目前的建议代表了一种彻底的生化和生理学 PDE5调节机制的研究。这种酶是一种特殊的 西地那非(伟哥)，用于治疗男性勃起功能障碍 与糖尿病、衰老、脊髓损伤和其他病理有关。 环核苷酸依赖蛋白对PDE5磷酸化的影响 本课程将探讨激酶对cGMP的催化和变构结合，以及对cGMP结合的影响 将磷酸化位点Ser-102(人)突变为丙氨酸、谷氨酸和天冬氨酸 将会被检查。首席调查员将研究PDE5调控 通过表达截断突变体来表达结构域结构和功能 两个变构的CUMP结合位点和 酶的磷酸化部位，但不包括催化区。小的 角x射线散射和傅里叶变换红外光谱将 用于研究cGMP和磷酸化对这些构象的影响 域名。某些被截断的调控结构域的结晶学将 已尝试。磷酸化蛋白磷酸酶的特异性和有效性 血管平滑肌提取物催化Ser-102 Will去磷酸化 被调查。PDE5在完整血管平滑肌中的短期调节 通过cGMP类似物、磷酸蛋白磷酸酶抑制剂和升高 对cGMP进行研究。这些制剂可以改变酶的可能性 活性、亚细胞定位或cGMP水平(反馈机制) 检查过了。除cGMP外，PDE5的潜在非共价调节剂将是 被追寻。PdE5变构中心代表封存的可能性 将探索cGMP在细胞中的位置。这些研究的结果将提供 了解与cGMP信令有关的基本问题的基础 很多纸巾。

DESCRIPTION (Applicant's abstract): Interest in mammalian cGMP cascades has recently been intense. Much research in this area has centered on the signal receptors and guanylyl cyclases that modulate cGMP formation, as well as on cellular actions of cGMP. Natural signals (natriuretic peptides, guanylins and nitric oxide), in addition to many medications, stimulate cGMP cascades by activating these cyclases. Cyclic nucleotide phosphodiesterases (PDEs), which are also highly modulated enzymes, participate in regulating cellular cAMP and cGMP levels by catalyzing breakdown of cAMP and cGMP. The subject of this application is the cGMP-binding cGMP-specific PDE (PDE5), which is specific for cGMP over cAMP at its allosteric cGMP-binding sites and at its catalytic site. The long-term goal is to determine the mechanism of action and cellular regulation of PDE5, a major determinant of cGMP level in many tissues. Classical effects of cGMP include relaxation of smooth muscle, inhibition of platelet activation, neutrophil degranulation, and mediation of vision. New discoveries have expanded this list to include regulation of gene expression, chloride transport in intestine and kidney, watertransport, bone resorption, melanogenesis in skin, long-term nerve depression, and opioid effects. Therapeutic agents that elevate cGMP include PDE inhibitors (e.g., caffeine, papaverine, and sildenafil) and nitrovasodilators (e.g., nitroglycerin). Certain enterotoxins cause secretory diarrhea by elevating cGMP. Dr. Corbin recently demonstrated that in some instances cGMP acts by "crossactivating" cAMP receptors. However, PDE5 is a specific intracellular receptor for cGMP. The present proposal represents a thorough biochemical and physiological investigation of PDE5 regulation. This enzyme is the specific target of sildenafil (Viagra.), which is used in treatment of male erectile dysfunction associated with diabetes, aging, spinal cord injuries and other pathologies. Effects of phosphorylation of PDE5 by cyclic nucleotide-dependent protein kinases on catalysis and allosteric cGMP binding will be explored, and effects of mutating the phosphorylation site, Ser- 102 (human), to Ala, Glu, and Asp will be examined. The principal investigator will study the PDE5 regulatory domain structure and function by expressing truncation mutants that include various combinations of the two allosteric cUMP-binding sites and the phosphorylation site of the enzyme but exclude the catalytic domain. Small angle x-ray scattering and Fourier transformed infra-red spectroscopy will be used to study effects of cGMP and phosphorylation on conformation of these domains. Crystallography of certain truncated regulatory domains will be attempted. The specificity and efficacy with which phosphoprotein phosphatases of vascular smooth muscle extracts catalyze dephosphorylation of Ser-102 will be investigated. Short-term regulation of PDE5 in intact vascular smooth muscle by cGMP analogs, phosphoprotein phosphatase inhibitors, and agents that elevate cGMP will be studied. The possibility that these agents could alter enzyme activity, subcellular localization, or cGMP levels (feedback mechanism) will be examined. Potential non-covalent modulators of PDE5 other than cGMP will be sought. The possibility that PDE5 allosteric sites represent a sequestration site for cGMP in cells will be explored. Results of these studies will provide a basis for understanding fundamental questions relating to cGMP signaling in many tissues.