RNA silencing identifies PDE4D5 as the functionally relevant cAMP phosphodiesterase interacting with βarrestin to control the protein kinase A/AKAP79-mediated switching of the β2-adrenergic receptor to activation of ERK in HEK293B2 cells
RNA silencing identifies PDE4D5 as the functionally relevant cAMP phosphodiesterase interacting with βarrestin to control the protein kinase A/AKAP79-mediated switching of the β2-adrenergic receptor to activation of ERK in HEK293B2 cells
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DOI：
10.1074/jbc.m414316200
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发表时间：
2005-09-30
影响因子：
4.8
通讯作者：
Houslay, MD
中科院分区：
文献类型：
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作者：
Lynch, MJ;Baillie, GS;Houslay, MD
PDE 4 B和PDE 4D在人胚肾（HEK 293 B2）细胞中提供> 90%的PDE 4 cAMP磷酸二酯酶活性。它们的选择性小干扰RNA（siRNA）介导的敲低增强异丙肾上腺素刺激的蛋白激酶A（PKA）活化。尽管内源性PDE 4D与β抑制蛋白共免疫沉淀，但内源性PDE 4 B不与β抑制蛋白共免疫沉淀，即使在PDE 4D敲低后也是如此。PDE 4 B2的异位过表达赋予与β抑制蛋白的免疫共沉淀。PDE 4D而非PDE 4 B的敲低通过PKA放大了异丙肾上腺素刺激的β（2）-肾上腺素能受体（β（2）-AR）的磷酸化和通过G（i）激活细胞外信号调节激酶（ERK）。同种型选择性敲低鉴定PDE 4D 5作为调节这两个过程的异丙肾上腺素刺激的功能重要的物种。Ht 31介导的PKA与AKAP支架蛋白的束缚的破坏减弱了ERK的异丙肾上腺素激活，即使在PDE 4D敲低后。选择性siRNA介导的敲低鉴定AKAP 79，其与β（2）-AR组成性相关，而不是异丙肾上腺素募集的gravin，作为该过程中功能相关的AKAP。异丙肾上腺素刺激的PDE 4D膜募集在β抑制蛋白敲低后消除。一个突变，妥协与β抑制蛋白的相互作用，防止催化失活的PDE 4D 5从执行一个显性负作用，在增强异丙肾上腺素刺激的ERK激活。β抑制蛋白募集的PDE 4D 5使异丙肾上腺素刺激的β（2）-AR的PKA磷酸化和其信号传导到ERK的相应转换脱敏。在PDE 4D 5敲低后观察到细胞表型的能力证明，在内源性水平表达的其他PDE 4同种型不能在HEK 293 B2细胞中提供拯救。
PDE4B and PDE4D provide > 90% of PDE4 cAMP phosphodiesterase activity in human embryonic kidney (HEK293B2) cells. Their selective small interference RNA ( siRNA)-mediated knockdown potentiates isoprenaline-stimulated protein kinase A (PKA) activation. Whereas endogenous PDE4D co-immunoprecipitates with beta arrestin, endogenous PDE4B does not, even upon PDE4D knockdown. Ectopic overexpression of PDE4B2 confers co-immunoprecipitation with beta arrestin. Knockdown of PDE4D, but not PDE4B, amplifies isoprenaline-stimulated phosphorylation of the beta(2)-adrenergic receptor (beta(2)-AR) by PKA and activation of extracellular signal-regulated kinase (ERK) through G(i). Isoform-selective knockdown identifies PDE4D5 as the functionally important species regulating isoprenaline stimulation of both these processes. Ht31-mediated disruption of the tethering of PKA to AKAP scaffold proteins attenuates isoprenaline activation of ERK, even upon PDE4D knockdown. Selective siRNA-mediated knockdown identifies AKAP79, which is constitutively associated with the beta(2)-AR, rather than isoprenaline-recruited gravin, as being the functionally relevant AKAP in this process. Isoprenaline-stimulated membrane recruitment of PDE4D is ablated upon beta arrestin knockdown. A mutation that compromises interactions with beta arrestin prevents catalytically inactive PDE4D5 from performing a dominant negative role in potentiating isoprenaline-stimulated ERK activation. beta arrestin-recruited PDE4D5 desensitizes isoprenaline-stimulated PKA phosphorylation of the beta(2)-AR and the consequential switching of its signaling to ERK. The ability to observe a cellular phenotype upon PDE4D5 knockdown demonstrates that other PDE4 isoforms, expressed at endogenous levels, are unable to afford rescue in HEK293B2 cells.