REGULATION OF CGMP DEPENDENT PROTEIN KINASE

对cGMP作为第二信使的兴趣在#年急剧升级 最近几年。哺乳动物组织中cGMP的作用清单现在是 规模相当大，而且还在不断增长。CGMP依赖的蛋白激酶(PKG) 是cGMP的主要细胞内受体。澄清： 对PKG的生理调节是其长期目标 调查。除了被归因于PKG在 利钠肽、一氧化氮或鸟苷的调节作用 呼吸道和血管平滑肌松弛，抑制血小板 聚集和中性粒细胞脱颗粒，PKG也可能介导 这些药物和其他药物对基因表达的cGMP依赖效应， 氯化物在肠道和肾脏的转运，心脏的收缩能力，水 通过血管内皮细胞运输，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。 PKG的激活被认为是许多药理作用的原因 药物的作用，例如“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) 用于缓解胸痛、哮喘、男性阳萎和高血 压力。PKG也可能介导某些特定因素引起的分泌性腹泻。 细菌肠毒素。PKG的重要性最近被认为是 通过认识到cAMP的一些作用是通过 PKG的交叉激活。 PKG-I-α和PKG-I-β的二聚化机制将是 采用诱变和蛋白水解法进行研究。原生和突变型PKG-I- 将使用Alpha和PKG-I-beta来定义 自我抑制结构域，并研究自我磷酸化。这个 负责激活每种异构体的自磷酸化位点(S) 将会被确认。与cGMP结合相关的构象变化 并将使用小角X射线测量自动磷酸化 散射、凝胶过滤和天然凝胶电泳法。PKG将成为 用作其他丝氨酸/苏氨酸和酪氨酸专一性的模型 由配体结合(例如，环)激活的蛋白激酶 核苷酸钙/钙调蛋白、胰岛素、生长因子)和 通过确定是否被cGMP-激活而实现自动磷酸化 结合或自动磷酸化会产生相同的酶构象。 我们将研究激活过程的分子机制。 天然凝胶电泳法，将不同的 PKG的自磷酸化物种和液-质联用 光谱将被用来确定这些物种是否存在于 完整的组织。PKG蛋白和mRNA水平的生理调节 将会被探索。这些调查的结果将涉及主要 通过PKG的cGMP信号转导方面。

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.