STRUCTURE /FUNCTION OF PHOSPHODIESTERASE 3 ISOFORMS

磷酸二酯酶 3 异构体的结构/功能

基本信息

批准号：
6432648
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
项目类别：
财政年份：
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/6432648
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

项目摘要

Eleven cyclic nucleotide phosphodiesterase gene families (PDE1-11) 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 (lacking the N-terminal 145 amino acids of PDE3A1,originally cloned by us from a myocardial cDNA library) was cloned from a cultured porcine aorta smooth muscle (VSM) cDNA library. 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.Although the predicted molecular weight of this cardiovascular "short" PDE3A isoform is~118kDa, in Sf21-cell lysates,its Mr(SDS-PAGE) is~131kDa, similar to that of a PDE3A isoform detected with specific anti-PDE3A peptide antibodies only in microsomal fractions from human myocardial samples. Other anti-PDE3A peptide antibodies detected ~115-120 kDa and ~90kDa forms in both cytosol and microsomal fractions from human myocardium. Experiments are underway to determine if expression of the latter isoform(~90kDa)is regulated by both transcriptional and translational mechanisms. 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.

已经鉴定出11个环核苷酸磷酸二酯酶基因家族（PDE1-11）。 PDE3同工型的特征是它们对CAMP和CGMP的高亲和力。它们通过某些药物的特定抑制作用，这些药物增加心肌收缩力，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；以及它们响应胰岛素，IGF-1，IL-4和增加营地的药物的快速激活。从培养的猪主动脉平滑肌（VSM）cDNA库中克隆了一种可能的PDE3A的前翻译变体（最初由我们从心肌cDNA库中克隆的N末端145氨基酸，最初是从心肌cDNA库中克隆的）。尽管我们无法通过5'-race，RT-PCR或底漆扩展从推定的翻译启动（ATG）密码子上游识别5'UTR序列，但RNase保护分析表明存在与“短”和Full的mRNA相对应 - 长度PDE3A同工型在心肌中，但仅在VSm中“短”形式。类似于与特异性抗PDE3A肽抗体检测到的PDE3A同工型，仅在人类心肌样品的微粒体级分中。其他抗PDE3A肽抗体在人体心肌中检测到〜115-120 kDa和〜90KDA形成。正在进行实验以确定后者同工型（〜90KDA）的表达是否受转录和翻译机制的调节。为了继续我们对PDE3的N末端部分的结构/功能研究，将N端部分任意分为区域1（AA1-300）和2区（AA301-500）。区域1包含一个较大的疏水结构域（约为200AA），其中有六个预测的跨膜螺旋段。区域2包含一个较小的疏水结构域（约为50AA）。在其C-termini上标记了标记为flag标记的N端截断突变体，其中有不同量的区域1和区域2序列，并在COS-7和SF9细胞中表达。 SF9细胞中突变体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中的较大疏水结构域包含负责将PDE3插入或与ER膜牢固关联的结构决定因素。尽管区域2，尤其是小的疏水结构域，其中包含用于靶向PDE3到膜结构的信息，但该分子将无法有效锚定的非整合膜蛋白缔合。去除区域1和2后，PDE3同工型几乎完全定位在细胞质中。我们试图通过中断小鼠的PDE3A和B基因来了解更多的PDE3A和3B功能。已经产生了纯合子null PDE3B小鼠；功能研究将很快开始。 PDE3A基因已成功地针对ES细胞，在协作研究中，我们正在尝试生成PDE3A嵌合小鼠。在协作研究中，人（H）PDE3A基因被克隆。它的结构组织与我们先前表征的HPDE3B基因非常相似。 H和M（小鼠）PDE3A基因的催化结构域的内含子/外显子结构是高度保守的。

项目成果

期刊论文数量（7）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Evidence for the presence of several phosphodiesterase isoforms in brown adipose tissue of Zucker rats: modulation of PDE2 by the fa gene expression.

Zucker 大鼠棕色脂肪组织中存在多种磷酸二酯酶亚型的证据：fa 基因表达对 PDE2 的调节。

DOI：
10.1016/s0014-5793(99)00934-5
发表时间：
1999
期刊：
FEBS letters
影响因子：
3.5
作者：
Coudray,C;Charon,C;Komas,N;Mory,G;Diot-Dupuy,F;Manganiello,V;Ferre,P;Bazin,R
通讯作者：
Bazin,R

Cardiac type cGMP-inhibited phosphodiesterase (PDE3A) gene structure: similarity and difference to adipocyte type PDE3B gene.

心脏型 cGMP 抑制磷酸二酯酶 (PDE3A) 基因结构：与脂肪细胞型 PDE3B 基因的相似性和差异。

DOI：
10.1006/bbrc.2000.2226
发表时间：
2000
期刊：
Biochemical and biophysical research communications
影响因子：
3.1
作者：
Kasuya,J;Liang,SJ;Goko,H;Park,SH;Kato,K;Xu,ZD;Hockman,S;Manganiello,VC;Fujita-Yamaguchi,Y
通讯作者：
Fujita-Yamaguchi,Y

Membrane localization of cyclic nucleotide phosphodiesterase 3 (PDE3). Two N-terminal domains are required for the efficient targeting to, and association of, PDE3 with endoplasmic reticulum.

环核苷酸磷酸二酯酶 3 (PDE3) 的膜定位。

DOI：
10.1074/jbc.m001734200
发表时间：
2000
期刊：
The Journal of biological chemistry
影响因子：
0
作者：
Shakur,Y;Takeda,K;Kenan,Y;Yu,ZX;Rena,G;Brandt,D;Houslay,MD;Degerman,E;Ferrans,VJ;Manganiello,VC
通讯作者：
Manganiello,VC

Phosphorylation and activation of phosphodiesterase type 3B (PDE3B) in adipocytes in response to serine/threonine phosphatase inhibitors: deactivation of PDE3B in vitro by protein phosphatase type 2A.