β-Arrestin-mediated PDE4 cAMP phosphodiesterase recruitment regulates β-adrenoceptor switching from Gs to Gi (Retracted Article)
β-Arrestin-mediated PDE4 cAMP phosphodiesterase recruitment regulates β-adrenoceptor switching from Gs to Gi (Retracted Article)
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DOI：
10.1073/pnas.262787199
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发表时间：
2003-02-04
影响因子：
11.1
通讯作者：
Houslay, MD
中科院分区：
文献类型：
--
作者：
Baillie, GS;Sood, A;Houslay, MD
cAMP激活的蛋白激酶A（PKA）使β（2）肾上腺素受体（β（2）AR）磷酸化，使其主要偶联从刺激性鸟嘌呤核苷酸调节蛋白（G（s））转变为抑制性鸟嘌呤核苷酸调节蛋白（G（i））。β-抑制蛋白将cAMP降解PDE 4磷酸二酯酶募集到β 2 AR，从而控制膜上的PKA活性。在这里，我们研究了PDE 4募集在β 2 AR调节G蛋白转换中的作用。在过表达重组β 2 AR的人胚肾293细胞中，异丙肾上腺素刺激可募集β-抑制蛋白1和2以及PDE 4D 3和PDE 4D 5至受体，并通过PKA刺激受体磷酸化。当用选择性PDE 4抑制剂咯利普兰处理细胞时，或当用竞争性抑制异丙肾上腺素刺激的天然PDE 4向β 2 AR募集的无催化活性PDE 4D突变体（PDE 4D 5-D556 A）转染细胞时，β 2 AR的PKA磷酸化状态显著增强。咯利普兰和PDE 4D 5-D556 A还增强β（2）AR介导的细胞外信号调节激酶ERK 1/2的活化。这与受体从G（s）到G（i）偶联的转换一致，因为ERK 1/2激活对PKA（H89）和G（i）（百日咳毒素）的抑制剂都敏感。在心肌细胞中，β（2）AR也从G（s）偶联转换为G（i）偶联。用异丙肾上腺素处理原代心肌细胞诱导PDE 4D 3和PDE 4D 5募集到膜并激活ERK 1/2。咯利普兰以对百日咳毒素和H89敏感的方式强烈增强这种活化。腺病毒介导的PDE 4D 5-D556 A的表达也增强ERK 1/2活化。因此，受体刺激的β-抑制蛋白介导的PDE 4募集在生理系统（心肌细胞）中通过β（2）AR调节G蛋白转换中发挥重要作用。
Phosphorylation of the beta(2) adrenoreceptor (beta(2)AR) by cAMP-activated protein kinase A (PKA) switches its predominant coupling from stimulatory guanine nucleotide regulatory protein (G(s)) to inhibitory guanine nucleotide regulatory protein (G(i)). beta-Arrestins recruit the cAMP-degrading PDE4 phosphodiesterases to the beta(2)AR, thus controlling PKA activity at the membrane. Here we investigate a role for PDE4 recruitment in regulating G protein switching by the beta(2)AR. In human embryonic kidney 293 cells overexpressing a recombinant beta(2)AR, stimulation with isoprenaline recruits beta-arrestins 1 and 2 as well as both PDE4D3 and PDE4D5 to the receptor and stimulates receptor phosphorylation by PKA. The PKA phosphorylation status of the beta(2)AR is enhanced markedly when cells are treated with the selective PDE4-inhibitor rolipram or when they are transfected with a catalytically inactive PDE4D mutant (PDE4D5-D556A) that competitively inhibits isoprenaline-stimulated recruitment of native PDE4 to the beta(2)AR. Rolipram and PDE4D5-D556A also enhance beta(2)AR-mediated activation of extracellular signal-regulated kinases ERK1/2. This is consistent with a switch in coupling of the receptor from G(s) to G(i), because the ERK1/2 activation is sensitive to both inhibitors of PKA (H89) and G(i) (pertussis toxin). In cardiac myocytes, the beta(2)AR also switches from G(s) to G(i) coupling. Treating primary cardiac myocytes with isoprenaline induces recruitment of PDE4D3 and PDE4D5 to membranes and activates ERK1/2. Rolipram robustly enhances this activation in a manner sensitive to both pertussis toxin and H89. Adenovirus-mediated expression of PDE4D5-D556A also potentiates ERK1/2 activation. Thus, receptor-stimulated beta-arrestin-mediated recruitment of PDE4 plays a central role in the regulation of G protein switching by the Beta(2)AR in a physiological system, the cardiac myocyte.