STRUCTURE /FUNCTION OF PHOSPHODIESTERASE 3 ISOFORMS

已鉴定出10个环核苷酸磷酸二酯酶基因家族（PDE 1 -10）。PDE 3同种型的特征在于其对cAMP和cGMP的高亲和力;其被某些增加心肌收缩性、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;以及其响应于胰岛素、IGF-1、IL-4和增加cAMP的试剂而快速活化。从培养的猪主动脉平滑肌（VSM）cDNA文库中克隆了PDE 3A的一个可能的翻译前变体。该心血管短PDE 3A同种型的预测分子量与从牛主动脉纯化的PDE 3的分子量（约110-115 kDa）相似。虽然我们无法通过5- RACE、RT-PCR或引物延伸鉴定推定的翻译起始（ATG）密码子上游的5个UTR序列，但RNA酶保护试验表明，心肌中存在与短和全长PDE 3A同种型相对应的mRNA，但在VSM中仅存在短形式。为了继续我们对PDE 3的N-末端部分的结构/功能研究，将N-末端部分任意地分为区域1（aa 1 -300）和区域2（aa 301 -500）。区域1含有一个大的疏水结构域（约200 aa），具有六个预测的跨膜螺旋片段。区域2含有较小的疏水结构域（约50 aa）。N-末端截短突变体，与不同量的区域1和区域2序列删除，FLAG标记在其C-末端和COS-7和Sf 9细胞中表达。亚细胞分布的突变体PDE 3活性在Sf 9细胞和免疫荧光定位在COS-7细胞中表明，在区域1中的大的疏水结构域包含的结构决定因素负责插入PDE 3到，或强关联，ER膜。尽管区域2，特别是小的疏水结构域，包含用于将PDE 3靶向膜结构的信息，但是分子作为不能有效地锚的非整合膜蛋白缔合。去除区域1和2后，PDE 3亚型几乎完全定位于胞质溶胶中。我们正试图通过破坏小鼠中的PDE 3A和B基因来了解更多的PDE 3A和3 B功能。已经产生了纯合子无效PDE 3B小鼠;功能研究将很快开始。PDE 3A基因已成功地在ES细胞中靶向，在合作研究中，我们正试图产生PDE 3A嵌合小鼠。在合作研究中，克隆了人类（H）PDE 3A基因;其结构组织与我们先前表征的HPDE 3B基因非常相似。H和M（小鼠）PDE 3A基因催化结构域的内含子/外显子结构高度保守。- PDE 3，结构/功能，免疫荧光，膜结合结构域，内质网定位-人类受试者

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