Bruss Matthew D, Richter Wito, Horner Kathleen, Jin S-L Catherine, Conti Marco
Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California, San Francisco, California 94143, USA.
J Biol Chem. 2008 Aug 15;283(33):22430-42. doi: 10.1074/jbc.M803306200. Epub 2008 May 28.
One of the defining properties of beta2-adrenergic receptor (beta(2)AR) signaling is the transient and rapidly reversed accumulation of cAMP. Here we have investigated the contribution of different PDE4 proteins to the generation of this transient response. To this aim, mouse embryonic fibroblasts deficient in PDE4A, PDE4B, or PDE4D were generated, and the regulation of PDE activity, the accumulation of cAMP, and CREB phosphorylation in response to isoproterenol were monitored. Ablation of PDE4D, but not PDE4A or PDE4B, had a major effect on the beta-agonist-induced PDE activation, with only a minimal increase in PDE activity being retained in PDE4D knock-out (KO) cells. Accumulation of cAMP was markedly enhanced, and the kinetics of cAMP accumulation were altered in their properties in PDE4DKO but not PDE4BKO cells. Modest effects were observed in PDE4AKO mouse embryonic fibroblasts. The return to basal levels of both cAMP accumulation and CREB phosphorylation was greatly delayed in the PDE4DKO cells, suggesting that PDE4D is critical for dissipation of the beta2AR stimulus. This effect of PDE4D ablation was in large part due to inactivation of a negative feedback mechanism consisting of the PKA-mediated activation of PDE4D in response to elevated cAMP levels, as indicated by experiments using the cAMP-dependent protein kinase inhibitors H89 and PKI. Finally, PDE4D ablation affected the kinetics of beta2AR desensitization as well as the interaction of the receptor with Galphai. These findings demonstrate that PDE4D plays a major role in shaping the beta2AR signal.
β2 - 肾上腺素能受体（β(2)AR）信号传导的一个决定性特性是cAMP的瞬时且迅速逆转的积累。在此，我们研究了不同的磷酸二酯酶4（PDE4）蛋白对这种瞬时反应产生的作用。为此，我们构建了缺乏PDE4A、PDE4B或PDE4D的小鼠胚胎成纤维细胞，并监测了PDE活性的调节、cAMP的积累以及异丙肾上腺素刺激下cAMP反应元件结合蛋白（CREB）的磷酸化情况。敲除PDE4D，但不是PDE4A或PDE4B，对β - 激动剂诱导的PDE激活有重大影响，在PDE4D基因敲除（KO）细胞中，PDE活性仅保留了极小的增加。cAMP的积累显著增强，并且在PDE4D基因敲除细胞中cAMP积累的动力学特性发生了改变，但在PDE4B基因敲除细胞中未出现这种情况。在PDE4A基因敲除的小鼠胚胎成纤维细胞中观察到了适度的影响。在PDE4D基因敲除细胞中，cAMP积累和CREB磷酸化恢复到基础水平的过程大大延迟，这表明PDE4D对于β2AR刺激的消散至关重要。PDE4D基因敲除的这种作用在很大程度上是由于一种负反馈机制的失活，该负反馈机制由PKA介导的PDE4D在cAMP水平升高时的激活组成，这是通过使用cAMP依赖性蛋白激酶抑制剂H89和PKI的实验表明的。最后，PDE4D基因敲除影响了β2AR脱敏的动力学以及受体与Gαi的相互作用。这些发现表明PDE4D在塑造β2AR信号中起主要作用。
