STRUCTURE /FUNCTION OF PHOSPHODIESTERASE 3 ISOFORMS

九环核苷酸磷酸二酯酶基因 家族（PDE 1 -9）已被鉴定。PDE 3亚型是 其特征在于它们对cAMP和cGMP的高亲和力， 某些药物的特异性抑制， 收缩，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， 对胰岛素、IGF-1、IL-4和 增加cAMP。两个PDE 3家族，PDE 3A和PDE 3B， 被识别。第二种人（H）PDE 3B同种型HPDE 3B 2， 从人Jurkat细胞cDNA文库中克隆。开放 HPDE 3B 2的阅读帧实际上与 HPDE 3B 1;这些同种型在它们的3'UTR序列上不同 并可能反映了存在的2种mRNA的观察， 用HPDE 3B对多种人类组织mRNA进行北方印迹 probes. PDE 3A的一个可能的剪接变体是从一个 猪主动脉平滑肌cDNA文库;预测分子 重量与从牛主动脉纯化的PDE 3的重量相似。 野生型和N-和C-末端的结构/功能研究 PDE 3A和B的截短重组体表明：1） PDE 3催化核心包括C-末端结构域[约270 aa（氨基酸）]，在所有哺乳动物PDE中保守， 额外的上游和下游序列; 2）N-末端 PDE 3的一半不需要催化活性或对 特异性抑制剂HPDE 3Adel 190（其中前190个aa为 缺失的）表现出比野生型低4倍的IC 50值 PDE 3A型，这表明PDE 3A的N-末端部分可以 调节对至少该特异性PDE 3抑制剂的响应性;以及 3)PDE 3的N-末端疏水区含有5-6个 预测的跨膜螺旋是重要的关联， 具有或靶向细胞内膜的PDE 3。 免疫荧光显微镜检查使用的抗体提出了对 PDE 3B的N-末端序列表明，在培养的3 T3-L1中， PDE 3B呈强网状染色， 与内质网（ER）标记蛋白共定位 BIP。这些研究与去年报告的研究一致， 在COS细胞中表达的N-末端截短重组体， Sf 9昆虫细胞，这表明疏水区（和 跨膜片段）在PDE 3与 急诊室凝胶过滤研究还表明， N-末端疏水区促进大PDE 3的形成 聚集体，它不是低聚所必需的，因为 HPDE 3A的前511个氨基酸缺失的重组体 在Ultrogel AcA 54上层析期间作为二聚体洗脱。到 了解更多PDE 3功能，我们正试图破坏PDE 3A 和B基因的同源重组。杂合 PDE 3B基因已经成功地 已经产生了破坏，我们现在正试图 产生纯合子小鼠。启动子活性在约。5 kb的 已经鉴定了小鼠PDE 3B基因的5'侧翼区 强上游启动子与弱得多的 下游启动子通过至少2-3 kb的抑制性或 抑制区

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.