• 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Cellular Signalling >Occupancy of the catalytic site of the PDE4A4 cyclic AMP phosphodiesterase by rolipram triggers the dynamic redistribution of this specific isoform in living cells through a cyclic AMP independent process
【24h】

Occupancy of the catalytic site of the PDE4A4 cyclic AMP phosphodiesterase by rolipram triggers the dynamic redistribution of this specific isoform in living cells through a cyclic AMP independent process

机译：咯利普兰对PDE4A4环状AMP磷酸二酯酶催化位点的占据触发了这种特定同工型通过环状AMP独立过程在活细胞中的动态重新分布。

获取原文
获取原文并翻译 | 示例
           
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

In cells transfected to express wild-type PDE4A4 cAMP phosphodiesterase (PDE), the PDE4 selective inhibitor rolipram caused PDE4A4 to relocalise so as to form accretion foci. This process was followed in detail in living cells using a PDE4A4 chimera formed with Green Fluorescent Protein (GFP). The same pattern of behaviour was also seen in chimeras of PDE4A4 formed with various proteins and peptides, including LimK, RhoC, FRB and the V5-6His tag. Maximal PDE4A4 foci formation, occurred over a period of about 10 h, was dose-dependent on rolipram and was reversible upon washout of rolipram. Inhibition of protein synthesis, using cycloheximide, but not PKA activity with H89, inhibited foci generation. Foci formation was elicited by Ro20-1724 and RS25344 but not by either Ariflo((R)) or RP73401, showing that not all PDE4 selective inhibitors had this effect. Ariflo R and RP73401 dose-dependently antagonised rolipram-induced foci formation and dispersed rolipram pre-formed foci as did the adenylyl cyclase activator, forskolin. Foci formation showed specificity for PDE4A4 and its rodent homologue, PDE4A5, as it was not triggered in living cells expressing the PDE4B2, PDE4C2, PDE4D3 and PDE4D5 isoforms as GFP chimeras. Altered foci formation was seen in the Deltab-LR2-PDE4A4 construct, which deleted a region within LRZ, showing that appropriate linkage between the N-tenninal portion of PDE4A4 and the catalytic unit of PDE4A4 was needed for foci formation. Certain single point mutations within the PDE4A4 catalytic site (His505Asn, His506Asn and Val475Asp) were shown to ablate foci formation but still allow rolipram inhibition of PDE4A4 catalytic activity. We suggest that the binding of certain, but not all, PDE4 selective inhibitors to PDE4A4 induces a conformational change in this isciform by 'inside-out' signalling that causes it to redistribute in the cell. Displacing foci-forming inhibitors with either cAMP or inhibitors that do not form foci can antagonise this effect. Specificity of this effect for PDE4A4 and its homologue PDE4A5 suggests that interplay between the catalytic site and the unique N-terminal region of these isoforms is required. Thus. certain PDE4 selective inhibitors may exert effects on PDE4A4 that extend beyond simple catalytic inhibition. These require protein synthesis and may lead to redistribution of PDE4A4 and any associated proteins. Foci formation of PDE4A4 may be of use in probing for conformational changes in this isciform and for sub-categorising PDE4 selective inhibitors. (C) 2003 Elsevier Inc. All rights reserved. [References: 53]
机译：在转染表达野生型PDE4A4 cAMP磷酸二酯酶（PDE）的细胞中，PDE4选择性抑制剂rolipram导致PDE4A4重新定位，从而形成增生灶。使用与绿色荧光蛋白（GFP）形成的PDE4A4嵌合体，在活细胞中详细跟踪此过程。在由各种蛋白质和肽（包括LimK，RhoC，FRB和V5-6His标签）形成的PDE4A4嵌合体中，也观察到了相同的行为模式。 PDE4A4的最大病灶形成发生在约10小时的时间内，其剂量依赖于咯利普兰，并且在冲洗掉咯利普兰后可逆。使用环己酰亚胺抑制蛋白质合成，但不抑制H89的PKA活性，可抑制病灶的产生。 Ro20-1724和RS25344引发了灶形成，但Ariflo（R）或RP73401却未引起灶形成，这表明并非所有PDE4选择性抑制剂都具有这种作用。 Ariflo R和RP73401与腺苷酸环化酶激活剂forskolin一样，剂量依赖性地拮抗rolipram诱导的病灶形成，并分散了rolipram预形成的病灶。焦点形成对PDE4A4及其啮齿动物同系物PDE4A5表现出特异性，因为它在表达PDE4B2，PDE4C2，PDE4D3和PDE4D5同种型为GFP嵌合体的活细胞中未被触发。在Deltab-LR2-PDE4A4构建体中发现了改变的焦点形成，该结构删除了LRZ内的一个区域，这表明焦点形成需要PDE4A4的N末端部分和PDE4A4的催化单元之间的适当连接。 PDE4A4催化位点内的某些单点突变（His505Asn，His506Asn和Val475Asp）显示可减少病灶形成，但仍然允许咯利普兰抑制PDE4A4催化活性。我们建议某些（但不是全部）PDE4选择性抑制剂与PDE4A4的结合通过“由内而外”的信号转导方式在该异构体中诱导构象变化，从而导致其在细胞中重新分布。用cAMP或不形成病灶的抑制剂代替病灶形成抑制剂可拮抗这种作用。这种作用对PDE4A4及其同系物PDE4A5的特异性表明，催化位点与这些同工型的独特N端区域之间需要相互作用。从而。某些PDE4选择性抑制剂可能对PDE4A4产生超出简单催化抑制作用的作用。这些需要蛋白质合成，并可能导致PDE4A4和任何相关蛋白质的重新分布。 PDE4A4的病灶形成可用于探索该isciform的构象变化以及将PDE4选择性抑制剂亚分类。 （C）2003 Elsevier Inc.保留所有权利。 [参考：53]

著录项

相似文献

  • 外文文献
  • 专利
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号