Terry Robert, Cheung York-Fong, Praestegaard Morten, Baillie George S, Huston Elaine, Gall Irene, Adams David R, Houslay Miles D
BioImage A/S, Moerkhoej Bygade 28, Soeborg DK-2860, Denmark.
Cell Signal. 2003 Oct;15(10):955-71. doi: 10.1016/s0898-6568(03)00092-5.
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 or RP73401, showing that not all PDE4 selective inhibitors had this effect. Ariflo 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-terminal 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 isoform 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 isoform and for sub-categorising PDE4 selective inhibitors.
在转染以表达野生型磷酸二酯酶4A4（PDE4A4）环磷酸腺苷磷酸二酯酶（PDE）的细胞中，PDE4选择性抑制剂咯利普兰使PDE4A4重新定位，从而形成积聚灶。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了这一过程。在与各种蛋白质和肽（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最大灶形成在约10小时内发生，呈咯利普兰剂量依赖性，且在洗去咯利普兰后可逆。使用环己酰亚胺抑制蛋白质合成，但不使用H89抑制PKA活性，可抑制灶的产生。Ro20 - 1724和RS25344可引发灶形成，但阿立氟和RP73401则不能，这表明并非所有PDE4选择性抑制剂都有此作用。阿立氟和RP73401呈剂量依赖性拮抗咯利普兰诱导的灶形成，并使咯利普兰预先形成的灶分散，腺苷酸环化酶激活剂福斯可林也有此作用。灶形成对PDE4A4及其啮齿动物同源物PDE4A5具有特异性，因为在表达PDE4B2、PDE4C2、PDE4D3和PDE4D5同工型作为GFP嵌合体的活细胞中未触发此现象。在缺失LRZ内一个区域的Deltab - LR2 - PDE4A4构建体中观察到灶形成改变，这表明PDE4A4的N端部分与PDE4A4的催化单元之间需要适当连接才能形成灶。PDE4A4催化位点内的某些单点突变（His505Asn、His506Asn和Val475Asp）显示可消除灶形成，但仍允许咯利普兰抑制PDE4A4催化活性。我们认为，某些但并非所有PDE4选择性抑制剂与PDE4A4的结合通过“由内而外”信号传导诱导该同工型构象变化，导致其在细胞内重新分布。用环磷酸腺苷或不形成灶的抑制剂取代形成灶的抑制剂可拮抗此效应。此效应对PDE4A4及其同源物PDE4A5的特异性表明，这些同工型的催化位点与独特的N端区域之间需要相互作用。因此，某些PDE4选择性抑制剂对PDE4A4的作用可能超出简单的催化抑制。这些作用需要蛋白质合成，并可能导致PDE4A4和任何相关蛋白质的重新分布。PDE4A4的灶形成可能有助于探究该同工型的构象变化以及对PDE4选择性抑制剂进行亚分类。
