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（PDE1-10）。PDE3亚型的特点是对cAMP和cGMP具有高亲和力；它们被某些药物特异性抑制，增加心肌收缩力，放松气道和血管平滑肌，抑制血小板聚集，刺激胰岛素分泌；以及它们对胰岛素、IGF-1、IL-4和增加cAMP的药物的快速激活。从培养的猪主动脉平滑肌（VSM） cDNA文库中克隆了一个可能的PDE3A翻译前变异。该心血管短PDE3A异构体的预测分子量与从牛主动脉纯化的PDE3相似（~110-115 kDa）。虽然我们无法通过5- RACE、RT-PCR或引物延伸来鉴定推测的翻译起始（ATG）密码子上游的5个UTR序列，但RNase保护实验表明，心肌中存在与短长度和全长PDE3A同种异构体相对应的mrna，而在VSM中只存在短长度的PDE3A同种异构体。为了继续我们对PDE3 N端部分的结构/功能研究，我们将N端部分任意划分为区域1 （aa1-300）和区域2 （aa301-500）。区域1包含一个大的疏水结构域（约200aa），有6个预测的跨膜螺旋片段。区域2包含一个较小的疏水结构域（约50aa）。n端截断突变体去除不同数量的1区和2区序列，在其c端进行flag标记，并在COS-7和Sf9细胞中表达。突变体PDE3活性在Sf9细胞中的亚细胞分布和COS-7细胞中的免疫荧光定位表明，1区大疏水结构域含有结构决定因素，负责PDE3插入ER膜或与ER膜强关联。尽管2区，尤其是小的疏水结构域，包含了PDE3靶向膜结构的信息，但这些分子作为非完整的膜蛋白结合在一起，不能有效地锚定。在去除1区和2区后，PDE3异构体几乎完全定位在细胞质中。我们正试图通过破坏小鼠PDE3A和B基因来了解更多PDE3A和3B的功能。纯合子无PDE3B小鼠已经产生；功能研究将很快开始。PDE3A基因已经在胚胎干细胞中成功靶向，在合作研究中，我们正在尝试产生PDE3A嵌合小鼠。在合作研究中，克隆了人类(H) PDE3A基因；其结构组织与我们之前描述的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

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