STRUCTURE /FUNCTION OF PHOSPHODIESTERASE 3 ISOFORMS

磷酸二酯酶 3 异构体的结构/功能

• 批准号: 6109177
• 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/6109177
• 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

Nine cyclic nucleotide phosphodiesterase gene families (PDE1-9) 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. Two PDE3 families, PDE3A and PDE3B, have been identified. A second human (H) PDE3B isoform, HPDE3B2, was cloned from a human Jurkat cell cDNA library. The open reading frame of HPDE3B2 is virtually identical to that of HPDE3B1; these isoforms differ in the sequences of their 3' UTRs and may reflect the presence of 2 mRNA species observed in Northern Blots of multiple human tissue mRNAs with HPDE3B probes. A possible splice variant of PDE3A was cloned from a porcine aorta smooth muscle cDNA library; its predicted molecular weight was similar to that of a PDE3 purified from bovine aorta. Structure/function studies with wild type and N- and C-terminal truncated recombinants of PDE3A and B indicated that 1) the PDE3 catalytic core includes the C-terminal domain [approx. 270 aa (amino acids)]conserved among all mammalian PDEs plus some additional upstream and downstream sequences; 2) the N-terminal half of PDE3 is not required for catalytic activity or sensitivity to specific inhibitors. HPDE3Adel190(in which the first 190 aa were deleted) exhibited an IC50 value 4 fold lower than that for wild type PDE3A, suggesting that the N-terminal portion of PDE3A can regulate responsiveness to at least this specific PDE3 inhibitor; and 3) the N-terminal hydrophobic region of PDE3 which contains 5-6 predicted transmembrane helices is important in association of PDE3 with, or targeting to, intracellular membranes. Immunofluorescent microscopy using an antibody raised against the N-terminal sequence of PDE3B indicated that in cultured 3T3-L1 adipocytes, PDE3B exhibited strong reticular staining which co-localized with the endoplasmic reticulum (ER) marker protein BIP. These studies are consistent with those reported last year with N-terminal truncated recombinants expressed in both COS cells and Sf9 insect cells which suggested that the hydrophobic region (and transmembrane segments) is important in association of PDE3 with the ER. Gel filtration studies also indicated that whereas the N-terminal hydrophobic region promoted formation of large PDE3 aggregates, it was not necessary for oligomerization since recombinants in which the first 511 aa of HPDE3A were deleted eluted as a dimer during chromatography on Ultrogel AcA54. To learn more of PDE3 function we are attempting to disrupt PDE3A and B genes by homologous recombination in mice. Heterozygous chimeric mice in which the PDE3B gene has been successfully disrupted have been generated and we are now attempting to produce homozygous mice. Promoter activity in the approx. 5 kb of the 5' flanking region of the mouse PDE3B gene has been identified with a strong upstream promoter separated from a much weaker downstream promoter by at least 2-3 kb of an inhibitory or suppressor region.

九个环状核苷酸磷酸二酯酶基因 已经确定了家庭（PDE1-9）。 PDE3同工型为 以他们对营地和CGMP的高亲和力为特征 某些增加心肌的药物的特定抑制作用 收缩力，放松气道和血管平滑肌，抑制 血小板聚集并刺激胰岛素分泌及其快速 响应胰岛素，IGF-1，IL-4和剂的激活， 增加营地。两个PDE3家族PDE3A和PDE3B有 已确定。第二个人（H）PDE3B同工型HPDE3B2， 从人类的Jurkat细胞cDNA库中克隆。开放 HPDE3B2的阅读框实际上与 HPDE3B1;这些同工型在其3'UTR的序列上有所不同 并可能反映出在 用HPDE3B的多个人体组织mRNA的北印迹 探针。从一个pde3a的可能的剪接变体是从一个克隆的 猪主动脉平滑肌cDNA库；它的预测分子 重量与从牛主动脉纯化的PDE3相似。 野生型以及N-和C末端的结构/功能研究 PDE3A和B的截短重组者表明1） PDE3催化核包括C末端结构域[大约。 270 AA（氨基酸）]在所有哺乳动物PDES中保守 附加上游和下游序列； 2）N末端 对催化活性或敏感性不需要一半的PDE3 特定抑制剂。 HPDE3Adel190（第一个190 AA是 删除）IC50值比野生低4倍 PDE3A类型，表明PDE3A的N末端部分可以 调节至少对该特定PDE3抑制剂的响应能力；和 3）PDE3的N末端疏水区，其中包含5-6 预测的跨膜螺旋在关联中很重要 PDE3带有或靶向细胞内膜。 使用针对该抗体提出的抗体免疫荧光显微镜 PDE3B的N末端序列表明，在培养的3T3-L1中 脂肪细胞，PDE3B表现出强烈的网状染色 与内质网（ER）标记蛋白共定位 bip。这些研究与去年报道的研究一致 N末端截短的重组剂在COS细胞和 SF9昆虫细胞表明疏水区（和 跨膜段）在PDE3与 急诊室。凝胶过滤研究还表明，尽管 N末端疏水区促进了大型PDE3的形成 骨料，由于 删除了第一个511 aa的重组者 在Ultrogel ACA54上色谱期间洗脱为二聚体。到 了解更多的PDE3功能，我们正在尝试破坏PDE3A 和B基因通过小鼠同源重组。杂合子 PDE3B基因已成功的嵌合小鼠 被破坏已经产生，我们现在正在尝试 产生纯合小鼠。启动子活性大约5 kb的 已经鉴定出小鼠PDE3B基因的5'侧翼区域 与强大的上游启动子与弱弱分开 抑制性至少2-3 kb的下游启动子或 抑制区域。