STRUCTURE /FUNCTION OF PHOSPHODIESTERASE 3 ISOFORMS

磷酸二酯酶 3 异构体的结构/功能

• 批准号: 6162669
• 负责人: V 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/6162669
• 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

Seven cyclic nucleotide phosphodiesterase gene families (PDE1-7) 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. The N-terminal portions of PDE3A and PDE3B isoforms contain a large hydrophobic region with 5-6 predicted transmembrane segments, and several downstream consensus phosphorylation sites. To study the role of the hydrophobic region in the membrane-association of PDE3, a human cardiac HPDE3A cDNA and several truncated recombinants (delta 190, delta 398, delta 511 which lack coding sequence for the first 190, 398, and 511 amino acids, respectively) and mouse adipocyte MPDE3B and delta 604MPDE3B cDNA, lacking coding sequence for the first 604 amino acids, were tagged at their C-terminal ends with the FLAG epitope, expressed transiently in COS-7 cells and localized by immunofluorescence microscopy. Both HPDE3A and MPDE3B showed strong staining of a reticular network which co-localized with the ER-marker BIP (not with alpha-tubulin) and intense perinuclear staining similar to that observed for Golgi proteins beta-COP and p58. A rat PDE3B recombinant permanently expressed in NIH 3006 fibroblasts exhibited reticular staining virtually identical to HPDE3A and MPDE3B in COS cells when detected with an anti-peptide antibody raised against the N-terminus of MPDE3B. delta 190HPDE3A (which includes part of the hydrophobic region) fluorescence was identical to that for PDE3A and PDE3B. delta 398HPDE3A (lacking the hydrophobic domain) fluorescence was more diffuse than intact HPDE3A with some reticular staining, indicating a difference in the distribution of this recombinant between membrane and cytosol. Staining of delta 511HPDE3A and MPDE3B delta 604 was diffuse, with little or no reticular staining, suggesting cytosolic localization of these recombinants. delta 511HPDE3A and delta 604MPDE3B activities were almost exclusively cytosolic when expressed in Sf9 cells. These results suggest that sequences in the hydrophobic domain plus additional downstream sequences are involved in the association of PDE3 with intracellular membranes. Future studies will attempt to further define the domains involved in and regulation of membrane targeting as well as effects of subcellular location on PDE3 regulation and function and the importance of PDE3 localization and compartmentalization in cell function. Although PDE3B may be involved in the regulation of several physiological processes, including insulin secretion and insulin action, little is known of its role in development. To understand more of PDE3B function, we are attempting to disrupt the mouse PDE3B gene by homologous recombination. An about 16 kb Sal1 genomic fragment containing mouse PDE3B 5' flanking sequences and exon 1 was cloned from a mouse genomic library. Both mouse and human PDE3B exon 1 encode the N-terminal portion of PDE3B that includes the putative membrane association domain and the downstream serine residue (analogous to rat PDE3B Ser 302) phosphorylated in response to insulin and isoproterenol in intact adipocytes. We are attempting to analyze promoter elements in the 5' flanking region of the MPDE3B gene using MPDE3B-Luciferase expression chimeras. A replacement vector targeting MPDE3B was constructed with about 6.0 kb Xba1 fragment (subcloned from the about 16 kb Sal1 genomic clone). We have recently successfully introduced the replacement vector into mouse ES cells by homologous recombination and then into pseudopregnant mice who have produced a number of chimeric agouti pups.

七个环核苷酸磷酸二酯酶基因家族（PDE1-7）具有 已被识别。 PDE3 同工型的特点是具有高亲和力 对于 cAMP 和 cGMP，它们被某些药物特异性抑制 增加心肌收缩力，舒张气道，血管畅通 肌肉，抑制血小板聚集，刺激胰岛素分泌， 及其对胰岛素、IGF-1、IL-4 和胰岛素的快速激活 增加 cAMP 的药物。 PDE3A 和 PDE3B 的 N 端部分 同工型包含一个大的疏水区域，预计有 5-6 个 跨膜片段和几个下游共有磷酸化 网站。研究疏水区域的作用 PDE3、人类心脏 HPDE3A cDNA 和几种的膜关联 截短的重组体（缺少 delta 190、delta 398、delta 511） 前190、398和511个氨基酸的编码序列， 分别）和小鼠脂肪细胞 MPDE3B 和 delta 604MPDE3B cDNA， 缺乏前 604 个氨基酸的编码序列，被标记为 它们的 C 末端带有 FLAG 表位，瞬时表达于 COS-7 细胞并通过免疫荧光显微镜定位。 均为 HPDE3A MPDE3B 显示出网状网络的强染色， 与 ER 标记 BIP（不与 α-微管蛋白）共定位并且强烈 核周染色与高尔基体蛋白观察到的相似 β-COP 和 p58。 在 NIH 中永久表达的大鼠 PDE3B 重组体 [第 3006 章] 用抗肽检测时 COS 细胞中的 HPDE3A 和 MPDE3B 针对 MPDE3B N 末端产生的抗体。 台达 190HPDE3A （包括部分疏水区域）荧光 与 PDE3A 和 PDE3B 相同。 Delta 398HPDE3A（缺少 疏水结构域）荧光比完整的 HPDE3A 更弥散 带有一些网状染色，表明存在差异 该重组体在膜和细胞质之间的分布。 染色 δ 511HPDE3A 和 MPDE3B δ 604 是弥漫性的，很少或没有 网状染色，表明这些细胞质定位 重组体。 Delta 511HPDE3A 和 Delta 604MPDE3B 活性 当在 Sf9 细胞中表达时，几乎完全在胞质中表达。 这些结果 表明疏水结构域中的序列加上额外的 下游序列参与 PDE3 与 细胞内膜。 未来的研究将尝试进一步定义 涉及膜靶向和调节的领域以及 亚细胞位置对 PDE3 调节和功能的影响以及 PDE3 在细胞中定位和区室化的重要性 功能。 尽管 PDE3B 可能参与多种调控 生理过程，包括胰岛素分泌和胰岛素作用， 人们对其在发展中的作用知之甚少。 了解更多 PDE3B 功能，我们试图通过以下方式破坏小鼠 PDE3B 基因 同源重组。 约 16 kb Sal1 基因组片段 含有小鼠 PDE3B 5' 侧翼序列和外显子 1 克隆自 小鼠基因组文库。 小鼠和人类 PDE3B 外显子 1 均编码 PDE3B 的 N 末端部分，包括假定的膜 关联结构域和下游丝氨酸残基（类似于大鼠 PDE3B Ser 302) 响应胰岛素和异丙肾上腺素而磷酸化 在完整的脂肪细胞中。 我们正在尝试分析启动子元件 使用 MPDE3B-荧光素酶在 MPDE3B 基因的 5' 侧翼区域中 表达嵌合体。 靶向 MPDE3B 的替代载体是 用约 6.0 kb Xba1 片段构建（从约 16 kb Sal1 基因组克隆）。 我们最近成功推出了 通过同源重组将替换载体导入小鼠 ES 细胞并 然后进入假孕小鼠体内，这些小鼠产生了许多嵌合体 刺豚鼠幼崽。