STRUCTURE /FUNCTION OF PHOSPHODIESTERASE 3 ISOFORMS

磷酸二酯酶 3 异构体的结构/功能

• 批准号: 6290382
• 负责人: VINCENT 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/6290382
• 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

Ten cyclic nucleotide phosphodiesterase gene families (PDE1-10) have been identified. PDE3 isoforms are characterized by their high affinity for cAMP and cGMP; their specific inhibition by certain drugs that increase myocardial contractility, relax airway and vascular smooth muscle, inhibit platelet aggregation and stimulate insulin secretion; and their rapid activation in response to insulin, IGF-1, IL-4, and agents that increase cAMP. A possible pre-translational variant of PDE3A was cloned from a cultured porcine aorta smooth muscle (VSM) cDNA library. The predicted molecular weight of this cardiovascular short PDE3A isoform is similar to that (~110-115 kDa) of a PDE3 purified from bovine aorta. Although we were unable to identify 5 UTR sequence upstream from the putative translation initiation (ATG) codon by 5- RACE, RT-PCR, or primer extension, RNase protection assays indicated the presence of mRNAs corresponding to both short and full-length PDE3A isoforms in myocardium, but only short forms in VSM. To continue our structure/function studies of the N-terminal portion of PDE3, the N- terminal portion was arbitrarily divided into region 1 (aa1-300) and region 2 (aa301-500). Region 1 contains a large hydrophobic domain (of ~200aa) with six predicted transmembrane helical segments. Region 2 contains a smaller hydrophobic domain (of ~50aa). N-terminal truncation mutants, with different amounts of region 1 and region 2 sequence removed, were FLAG-tagged at their C-termini and expressed in COS-7 and Sf9 cells. Subcellular distribution of mutant PDE3 activities in Sf9 cells and immunofluorescent localization in COS-7 cells indicated that the large hydrophobic domain in region 1 contains structural determinants responsible for insertion of PDE3 into, or strong association with, ER membranes. Although region 2, especially the small hydrophobic domain, contains information for targeting of PDE3 to membranous structures, the molecules associate as non-integral membrane proteins that do not anchor efficiently. After removal of regions 1 and 2, PDE3 isoforms were virtually completely localized in the cytosol. We are attempting to learn more of PDE3A and 3B functions by disruption of PDE3A and B genes in mice. Homozygous null PDE3B mice have been generated; functional studies will soon be initiated. The PDE3A gene has been successfully targeted in ES cells and, in collaborative studies, we are attempting to generate PDE3A chimeric mice. In collaborative studies, the human (H) PDE3A gene was cloned; its structural organization is quite similar to that of the HPDE3B gene, which we previously characterized. The intron/exon structure of the catalytic domains of H and M (mouse) PDE3A genes is highly conserved. - PDE3, structure/function, immunofluorescence, membrane association domains, endoplasmic reticulum localization - Human Subjects

已鉴定出十个环核苷酸磷酸二酯酶基因家族（PDE1-10）。 PDE3 同工型的特点是对 cAMP 和 cGMP 具有高亲和力；它们被某些增加心肌收缩力、松弛气道和血管平滑肌、抑制血小板聚集和刺激胰岛素分泌的药物特异性抑制；它们响应胰岛素、IGF-1、IL-4 和增加 cAMP 的药物而快速激活。 PDE3A 的一个可能的翻译前变体是从培养的猪主动脉平滑肌 (VSM) cDNA 文库中克隆出来的。这种心血管短 PDE3A 亚型的预测分子量与从牛主动脉纯化的 PDE3 的分子量 (~110-115 kDa) 相似。尽管我们无法通过 5-RACE、RT-PCR 或引物延伸鉴定推定翻译起始 (ATG) 密码子上游的 5 UTR 序列，但 RNase 保护测定表明存在与短和全长 PDE3A 亚型相对应的 mRNA存在于心肌中，但仅存在于 VSM 中的简短形式。为了继续我们对 PDE3 N 端部分的结构/功能研究，N 端部分被任意分为区域 1 (aa1-300) 和区域 2 (aa301-500)。区域 1 包含一个大的疏水结构域（约 200 个氨基酸），具有六个预测的跨膜螺旋片段。区域 2 包含较小的疏水结构域（约 50 个氨基酸）。 N 端截短突变体（去除了不同量的区域 1 和区域 2 序列）在其 C 端带有 FLAG 标签，并在 COS-7 和 Sf9 细胞中表达。 Sf9 细胞中突变 PDE3 活性的亚细胞分布和 COS-7 细胞中的免疫荧光定位表明，区域 1 中的大疏水结构域包含负责将 PDE3 插入 ER 膜或与 ER 膜强关联的结构决定簇。尽管区域 2，尤其是小的疏水结构域，包含 PDE3 靶向膜结构的信息，但这些分子以非完整膜蛋白的形式结合，无法有效锚定。去除区域 1 和区域 2 后，PDE3 同工型几乎完全定位于细胞质中。我们正试图通过破坏小鼠 PDE3A 和 B 基因来了解更多 PDE3A 和 3B 功能。已产生 PDE3B 纯合无效小鼠；功能研究将很快启动。 PDE3A 基因已成功靶向 ES 细胞，在合作研究中，我们正在尝试生成 PDE3A 嵌合小鼠。在合作研究中，克隆了人类（H）PDE3A基因；它的结构组织与我们之前表征的 HPDE3B 基因非常相似。 H 和 M（小鼠）PDE3A 基因催化结构域的内含子/外显子结构高度保守。 - PDE3、结构/功能、免疫荧光、膜关联域、内质网定位 - 人类受试者