β2-adrenergic receptor signaling and desensitization elucidated by quantitative modeling of real time cAMP dynamics
β2-adrenergic receptor signaling and desensitization elucidated by quantitative modeling of real time cAMP dynamics
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DOI：
10.1074/jbc.m707009200
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发表时间：
2008-02-01
影响因子：
4.8
通讯作者：
Lefkowitz, Robert J.
中科院分区：
文献类型：
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作者：
Violin, Jonathan D.;DiPilato, Lisa M.;Lefkowitz, Robert J.
G蛋白偶联受体信号受多种反馈机制的动态调节，这些反馈机制迅速减弱配体刺激引起的信号，导致脱敏。然而，人们对这些机制的个别贡献知之甚少。在这里，我们使用一种改进的cAMP荧光生物传感器来测量活细胞中由内源性β(2)-肾上腺素能受体(β(2)AR)刺激的第二信使动力学。用异丙肾上腺素刺激β(2)AR产生一过性的cAMP脉冲，最大浓度接近10微米，持续时间不到5分钟。我们通过使用小分子抑制剂、小干扰RNA和小鼠胚胎成纤维细胞，研究了cAMP依赖的激酶、G蛋白偶联受体激酶和β-arrestin在调节β(2)AR信号动力学中的作用。我们发现，在HEK-293细胞中，cAMP反应受到两种不同机制的限制：G蛋白偶联受体激酶(GRK6)介导的受体磷酸化导致β-arrestin介导的受体失活和cAMP依赖的激酶介导的磷酸二酯酶诱导cAMP代谢。符合这些数据的β(2)AR信号动力学数学模型表明，cAMP依赖的激酶直接使受体失活并不显著，但GRK6/β-arrestin介导的失活是迅速而深刻的，发生的半衰期为70 S。这一定量系统分析代表了在量化受体信号生理调节机制方面的重要进展。
G protein-coupled receptor signaling is dynamically regulated by multiple feedback mechanisms, which rapidly attenuate signals elicited by ligand stimulation, causing desensitization. The individual contributions of these mechanisms, however, are poorly understood. Here, we use an improved fluorescent biosensor for cAMP to measure second messenger dynamics stimulated by endogenous beta(2)-adrenergic receptor (beta(2)AR) in living cells. beta(2)AR stimulation with isoproterenol results in a transient pulse of cAMP, reaching a maximal concentration of similar to 10 mu M and persisting for less than 5 min. We investigated the contributions of cAMP-dependent kinase, G protein-coupled receptor kinases, and beta-arrestin to the regulation of beta(2)AR signal kinetics by using small molecule inhibitors, small interfering RNAs, and mouse embryonic fibroblasts. We found that the cAMP response is restricted in duration by two distinct mechanisms in HEK-293 cells: G protein-coupled receptor kinase (GRK6)-mediated receptor phosphorylation leading to beta-arrestin mediated receptor inactivation and cAMP-dependent kinase-mediated induction of cAMP metabolism by phosphodiesterases. A mathematical model of beta(2)AR signal kinetics, fit to these data, revealed that direct receptor inactivation by cAMP-dependent kinase is insignificant but that GRK6/beta-arrestin-mediated inactivation is rapid and profound, occurring with a half-time of 70 s. This quantitative system analysis represents an important advance toward quantifying mechanisms contributing to the physiological regulation of receptor signaling.