REGULATION OF CYCLIC GMP DEPENDENT PROTEIN KINASE

第二个信使，环状GMP(CGMP)被认为是介导 一氧化氮和心钠素在细胞内的作用 关于平滑肌的松弛。这种同样的肌肉松弛是 被认为是硝基血管扩张剂在调解中的作用机制 对心绞痛的治疗作用。在这份提案中， 实验被用来检验cGMP导致平滑的假设。 激活特定的cGMP依赖的蛋白激酶实现肌肉松弛 (CGK)。由于先前已经表征了CGK的多种异构体， 我们将确定这些异构体中的哪些在不同类型的 使用分子和免疫学技术的平滑肌肉。一旦CGK 与平滑肌松弛有关的亚型已被确定，然后 将对这些cGK进行生化表征，以确定关键特性 与其调节平滑肌收缩有关的酶的种类。 体外诱变将被用来产生结构性的活性和 这些cGKs的显性阴性形式用于以后的活体分析 这些酶在调节平滑肌松弛中所起的作用。在……里面 此外，还将筛选随机多肽文库，以开发 CGKs的特异性假底物抑制剂。然后这些试剂就会 用来研究底物蛋白在体内的磷酸化 转染腺病毒的平滑肌细胞系及原代细胞 文化。双向凝胶电泳法将用于鉴定 并将这些底物的磷酸化与调节 平滑肌细胞的功能，如细胞内钙的调节， ANF受体的致敏和抑制有丝分裂的发生。这个 显性负性和结构性活性突变体以及 伪亚州抑制剂将被用来最终证明一个强制性的 CGK在血管松弛中的作用。最后，重要的是在体内 底物蛋白将使用一种新的表达方式进行鉴定和克隆- 基于筛选的方法。这些研究完成后，将为 我们在关于平稳运行的机制的认识上存在着重大差距 肌肉松弛受心钠素、一氧化氮和硝基血管扩张剂的调节。

The second messenger, cyclic GMP (cGMP) is believed to mediate the intracellular effects of nitric oxide and atrial natriuretic factor (ANF) on the relaxation of smooth muscle. This same smooth muscle relaxation is thought to be the mechanism of action of nitrovasodilators in mediating their therapeutic effects on angina pectoris. In this proposal, experiments are described to test the hypothesis that cGMP causes smooth muscle relaxation by activation of a specific cGMP-dependent protein kinase (cGK). Since multiple isoforms of cGK have been characterized previously, we will determine which of these isoforms are expressed in various types of smooth muscle using molecular and immunological techniques. Once the cGK isoforms relevant to smooth muscle relaxation have been identified, then these cGKs will be characterized biochemically to determine key properties of the enzymes relevant to their regulation of smooth muscle contraction. In vitro mutagenesis will be used to generate constitutively active and dominant negative forms of these cGKs for later in vivo analysis of the role that these enzymes play in regulation of smooth muscle relaxation. In addition, random peptide libraries will be screened in order to develop a specific pseudosubstrate inhibitor for the cGKs. These reagents will then be used to study the in vivo phosphorylation of substrate proteins in transfected smooth muscle cell lines as well as primary smooth muscle cell cultures. Two-dimensional gel electrophoresis will be used to characterize these substrates and to correlate their phosphorylation with regulation of smooth muscle cell functions such as regulation of intracellular calcium, densensitization of ANF receptors, and inhibition of mitogenesis. The dominant negative and constitutively active mutants as well as the pseudosubstate inhibitor will be used to definitively prove an obligatory role of cGK in relaxation of smooth muscle. Finally, important in vivo substrate proteins will be identified and cloned using a novel expression- based screening method. When completed, these studies will bridge a crucial gap in our knowledge concerning the mechanisms by which smooth muscle relaxation is regulated by ANF, nitric oxide, and nitrovasodilators.