REGULATION OF CGMP DEPENDENT PROTEIN KINASE

cGMP作为第二信使的兴趣在2010年急剧上升， 近年 cGMP在哺乳动物组织中的作用列表现在是 相当大，而且还在增长。 cGMP依赖性蛋白激酶（PKG） 是cGMP的主要细胞内受体。阐明 PKG的生理调节是本发明的长期目标。 调查 除了PKG的经典角色之外， 利钠肽、一氧化氮或鸟苷酸的介导作用， 舒张气道和血管平滑肌，抑制血小板 聚集和中性粒细胞脱粒，PKG也可能介导 这些和其他试剂对基因表达的cGMP依赖性作用， 肠和肾脏中的氯离子转运，心脏收缩力，水 通过血管内皮的运输，骨吸收， 皮肤中的黑素生成、长期神经抑制和阿片样物质作用。 PKG激活被认为是许多药理学作用的原因。 药物如“PDE抑制剂”（例如，咖啡因， 罂粟碱）和硝基血管扩张剂（例如，硝酸甘油）， 缓解胸痛、哮喘、男性阳痿、高血压 压力PKG也可能介导某些激素引起的分泌性腹泻。 细菌肠毒素 PKG的重要性最近被 由于认识到cAMP的某些作用是由 PKG的交叉激活。 PKG-I-α和PKG-I-β的二聚化机制将是 用诱变和蛋白水解的方法研究。 天然和突变PKG-I- α和PKG-I-β将用于定义 自身抑制结构域和研究自身磷酸化。 的 负责激活每种亚型的自磷酸化位点 将被识别。与cGMP结合相关的构象变化 和自磷酸化将使用小角度X射线测量 散射、凝胶过滤和天然凝胶电泳。 PKG将是 用作其他丝氨酸/苏氨酸和酪氨酸特异性 通过两种配体结合激活的蛋白激酶（例如，环状 核苷酸Ca 2 +/钙调蛋白、胰岛素、生长因子）和 自身磷酸化通过确定是否激活cGMP- 结合或自磷酸化产生相同的酶构象。 活化过程的分子机制将被检查。 天然凝胶电泳，分离不同的 PKG的自磷酸化物质和液相色谱-质谱联用 光谱法将用于确定这些物质是否存在于 完整的组织 PKG蛋白和mRNA水平的生理调节 将被探索。这些调查的结果将解决重大问题。 通过PKG的cGMP信号传导方面。

Interest in cGMP as a second messenger has dramatically escalated in recent years. The list of cGMP actions in mammalian tissues is now quite large, and it is growing. The cGMP-dependent protein kinase (PKG) is a major intracellular receptor for cGMP. Elucidation of the physiological regulation of PKG is the long-term objective of this investigation. In addition to the classical roles ascribed to PKG in mediating effects of natriuretic peptides, nitric oxide or guanylins on airway and vascular smooth muscle relaxation, inhibition of platelet aggregation, and neutrophil degranulation, PKG may also mediate the cGMP-dependent effects of these and other agents on gene expression, chloride transport in intestine and kidney, heart contractility, water transport through the vascular endothelium, bone resorption, melanogenesis in skin, long-term nerve depression and opioid effects. PKG activation is believed to account for many of the pharmacological actions of medications such as "PDE inhibitors" (e.g., caffeine, papaverine) and nitrovasodilators (e.g., nitroglycerin) which are used for relief of chest pain, asthma, male impotence, and high blood pressure. PKG may also mediate the secretory diarrhea caused by certain bacterial enterotoxins. The importance of PKG has recently been enhanced by the realization that some effects of cAMP are mediated by cross-activation of PKG. The mechanism of dimerizaiton of PKG-I-alpha and PKG-I-beta will be studied using mutagenesis and proteolysis. Native and mutant PKG-I- alpha and PKG-I-beta will be utilized to define functional elements of the autoinhibitory domain and to study autophosphorylation. The autophosphorylation site(s) responsible for activation of each isoform will be identified. Conformational changes associated with cGMP binding and autophosphorylation will be measured using small angle X-ray scattering, gel filtration and native gel electrophoresis. PKG will be used as a model for other serine/threonine- and tyrosine-specific protein kinases that are activated by both ligand-binding (e.g., cyclic nucleotides Ca2+/calmodulin, insulin, growth factors) and autophosphorylation by determining whether or not activation by cGMP- binding or autophosphorylation produces the same enzyme conformation. The molecular mechanism of the activation processes will be examined. Native gel electrophoresis, which separates the different autophosphorylated species of the PKGs, and liquid chromatography-mass spectrometry will be used to determine if these species are present in intact tissues. Physiological regulation of PKG protein and mRNA levels will be explored. Results of these investigations will address major aspects of cGMP signaling through PKG.