STRUCTURE /FUNCTION OF PHOSPHODIESTERASE 3 ISOFORMS

磷酸二酯酶 3 异构体的结构/功能

• 批准号: 2576751
• 负责人: V MANGANIELLO
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/2576751
• 关键词: 3'5' cyclic nucleotide phosphodiesterase; Sf9 cell line; adipocytes; cyclic AMP; cyclic GMP; enzyme activity; enzyme structure; fluorescent in situ hybridization; isozymes; laboratory rat; protein isoforms; recombinant proteins

By catalyzing hydrolysis of cAMP and cGMP, cyclic nucleotide phosphodiesterases (PDEs) are critical regulators of intracellular concentrations of and biological processes mediated by cyclic nucleotides. Seven PDE gene families have been identified. We have focussed on PDE3s or cGMP-inhibited PDEs, which are characterized by their high affinity for cAMP and cGMP, their specific inhibition by drugs that increase myocardial contractility, relax airway and vascular smooth muscle and stimulate insulin secretion, and their rapid activation in response to insulin and agents that increase cAMP. We have identified two PDE3 subfamilies, PDE3A and B, which are products of distinct but related genes, located by fluorescent in situ hybridization to human chromosomes 12p12 and 11p15, respectively. The domain organization of PDE3A and B isoforms is identical with predicted homologous catalytic domains in the C-terminal half of the PDE molecules and divergent N-terminal regulatory domains which contain large hydrophobic regions with several predicted transmembrane segments and downstream consensus sites for phosphorylation by protein kinase A. Activation of a rat adipocyte PDE3B is important in the antilipolytic action of insulin; in collaborative studies, we found that in intact rat adipocytes serine 302 of a membrane associated PDE3B (based on its deduced sequence) is phosphorylated by A-kinase and an insulin-stimulated kinase. Expression of "full-length" and truncated recombinant PDE3s in fibroblasts, COS cells, Sf9 insect cells and E. coli demonstrated that the catalytic core of PDE3s includes the catalytic domain conserved among all mammalian PDEs plus some additional N- and C-terminal sequences, and that the N-terminal half of the molecules was not required for catalytic activity, low Km for cAMP and cGMP or sensitivity to specific inhibitors. Biochemical studies with PDE3 N-terminal deletion recombinants and immunofluorescent localization of FLAG-epitope-tagged PDE3 recombinants indicate that the N-terminal hydrophobic domain is important in association of PDE3s with intracellular membranes.

通过催化cAMP和cGMP的水解，环核苷酸 磷酸二酯酶 (PDE) 是细胞内的重要调节剂 循环介导的浓度和生物过程 核苷酸。 已鉴定出七个 PDE 基因家族。 我们有 专注于 PDE3 或 cGMP 抑制的 PDE，其特点是 对 cAMP 和 cGMP 的高亲和力，药物的特异性抑制 增加心肌收缩力，舒张气道，血管畅通 肌肉并刺激胰岛素分泌，及其快速激活 对胰岛素和增加 cAMP 的药物的反应。 我们已经确定了两个 PDE3 亚家族：PDE3A 和 B，它们是产品 不同但相关的基因，通过荧光原位定位 分别与人类染色体 12p12 和 11p15 杂交。 这 PDE3A 和 B 亚型的结构域结构与预测相同 PDE 分子 C 端一半的同源催化结构域 和不同的 N 端调控域，其中包含大 具有多个预测的跨膜片段的疏水区域和 蛋白激酶 A 磷酸化的下游共有位点。 大鼠脂肪细胞 PDE3B 的激活在抗脂解中很重要 胰岛素的作用；在合作研究中，我们发现在完整的大鼠中 膜相关 PDE3B 的脂肪细胞丝氨酸 302（基于其 推导的序列）被 A-激酶和胰岛素刺激的磷酸化 激酶。 “全长”和截短的重组 PDE3 的表达 成纤维细胞、COS 细胞、Sf9 昆虫细胞和大肠杆菌证明 PDE3 的催化核心包括保守的催化结构域 所有哺乳动物 PDE 加上一些额外的 N 端和 C 端序列，以及 催化作用不需要分子的 N 端部分 活性、cAMP 和 cGMP 的低 Km 或对特定抑制剂的敏感性。 PDE3 N 端缺失重组体的生化研究 FLAG 表位标记的 PDE3 重组体的免疫荧光定位 表明 N 端疏水结构域在 PDE3 与细胞内膜的关联。