Roles of GRK and PDE4 activities in the regulation of beta2 adrenergic signaling.
Roles of GRK and PDE4 activities in the regulation of beta2 adrenergic signaling.
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DOI：
10.1085/jgp.200709881
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发表时间：
2008-04
期刊：
影响因子：
--
通讯作者：
Rich TC
中科院分区：
文献类型：
--
作者：
Xin W;Tran TM;Richter W;Clark RB;Rich TC
细胞生物学的一个重要焦点是了解不同的反馈机制如何调节 G 蛋白偶联受体系统。为此，我们通过测量单个 HEK-293 细胞中的 cAMP 信号来研究内源性 β2 肾上腺素受体 (β2AR) 和磷酸二酯酶 (PDE) 的调节。我们使用基因编码的环核苷酸门控 (CNG) 通道监测 cAMP 信号。这种高分辨率方法使我们能够进行一些观察。 (a) 将细胞暴露于 1 μM 异丙肾上腺素会触发质膜附近的 cAMP 水平短暂增加。用 10 μM 咯利普兰（一种 PDE4 抑制剂）预处理细胞，可以防止异丙肾上腺素诱导的 cAMP 信号下降。 (b) 1 μM 异丙肾上腺素引发 4 型磷酸二酯酶 (PDE4) 活性持续双倍增加。 (c) 在移液管溶液中添加 20 nM PKI（一种 PKA 抑制剂）或 3 μM 59-74E（一种 GRK 抑制剂）不会显着改变异丙肾上腺素依赖性 cAMP 水平的下降；然而，当移液器溶液中同时包含 PKI 和 59-74E 时，cAMP 水平的下降被阻止。 (d) 在用异丙肾上腺素进行初始 5 分钟刺激和 5 分钟冲洗后，在用异丙肾上腺素进行第二次 5 分钟刺激期间几乎没有或没有观察到信号恢复。 (e) 当移液管溶液中包含 PKI 时，响应第二次异丙肾上腺素刺激的信号幅度没有改变，但当包含 59-74E 时，信号幅度显着增加。综上所述，这些数据表明，GRK 介导的 β2AR 脱敏或 PKA 介导的 PDE4 活性刺激足以导致 cAMP 信号下降。此外，数据表明GRK介导的脱敏主要负责β2AR信号传导的持续抑制。为了更好地理解受体脱敏和 PDE4 活性在控制 cAMP 信号中的相互作用，我们开发了该系统的数学模型。使用该模型对 cAMP 信号进行的模拟与实验数据一致，并证明了受体水平、受体脱敏、基础腺苷酸环化酶活性和 PDE 活性调节在控制 cAMP 信号中的重要性，从而影响了系统的整体灵敏度。
An important focus in cell biology is understanding how different feedback mechanisms regulate G protein–coupled receptor systems. Toward this end we investigated the regulation of endogenous β2 adrenergic receptors (β2ARs) and phosphodiesterases (PDEs) by measuring cAMP signals in single HEK-293 cells. We monitored cAMP signals using genetically encoded cyclic nucleotide-gated (CNG) channels. This high resolution approach allowed us to make several observations. (a) Exposure of cells to 1 μM isoproterenol triggered transient increases in cAMP levels near the plasma membrane. Pretreatment of cells with 10 μM rolipram, a PDE4 inhibitor, prevented the decline in the isoproterenol-induced cAMP signals. (b) 1 μM isoproterenol triggered a sustained, twofold increase in phosphodiesterase type 4 (PDE4) activity. (c) The decline in isoproterenol-dependent cAMP levels was not significantly altered by including 20 nM PKI, a PKA inhibitor, or 3 μM 59-74E, a GRK inhibitor, in the pipette solution; however, the decline in the cAMP levels was prevented when both PKI and 59-74E were included in the pipette solution. (d) After an initial 5-min stimulation with isoproterenol and a 5-min washout, little or no recovery of the signal was observed during a second 5-min stimulation with isoproterenol. (e) The amplitude of the signal in response to the second isoproterenol stimulation was not altered when PKI was included in the pipette solution, but was significantly increased when 59-74E was included. Taken together, these data indicate that either GRK-mediated desensitization of β2ARs or PKA-mediated stimulation of PDE4 activity is sufficient to cause declines in cAMP signals. In addition, the data indicate that GRK-mediated desensitization is primarily responsible for a sustained suppression of β2AR signaling. To better understand the interplay between receptor desensitization and PDE4 activity in controlling cAMP signals, we developed a mathematical model of this system. Simulations of cAMP signals using this model are consistent with the experimental data and demonstrate the importance of receptor levels, receptor desensitization, basal adenylyl cyclase activity, and regulation of PDE activity in controlling cAMP signals, and hence, on the overall sensitivity of the system.
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影响因子：
4.1
作者：
BARBER, R
通讯作者：
BARBER, R
影响因子：
3.6
作者：
Friedman, J;Babu, B;Clark, RB
通讯作者：
Clark, RB
影响因子：
4.1
作者：
BARBER, R;GOKA, TJ;BUTCHER, RW
通讯作者：
BUTCHER, RW
DOI：
10.1073/pnas.97.12.6751
发表时间：
2000-06-06
影响因子：
11.1
作者：
Hansen，G;Jin，SLC;Conti，M
通讯作者：
Conti，M
影响因子：
64.8
作者：
Daaka, Y;Luttrell, LM;Lefkowitz, RJ
通讯作者：
Lefkowitz, RJ