Trans-Golgi Network (TGN) as a Regulatory Node for β1-Adrenergic Receptor (β1AR) Down-modulation and Recycling
Trans-Golgi Network (TGN) as a Regulatory Node for β1-Adrenergic Receptor (β1AR) Down-modulation and Recycling
复制标题
DOI：
10.1074/jbc.m111.323782
复制
发表时间：
2012-04-20
影响因子：
4.8
通讯作者：
Filardo, Edward J.
中科院分区：
文献类型：
--
作者：
Cheng, Shi-Bin;Filardo, Edward J.
受体下调是G蛋白偶联受体（GPCR）防止受体过度信号转导的关键机制。最近，trans-Golgi网络（TGN）被认为是受体内吞和降解的关键检查点。在这里，我们研究了持续的异丙肾上腺素刺激的β 1-肾上腺素能受体的下调中TGN的参与。免疫荧光染色显示，5 h时，类似于50%的内吞β 1 AR与TGN-46共定位。布雷菲德菌素A（Brefeldin A，BFA）对TGN的破坏导致内吞的β 1AR在Rab 11（+）再循环内体中的大量积累，抑制β 1AR进入LAMP 1（+）溶酶体，从而增强β 1AR再循环至质膜。促溶酶体剂氯喹在4 h内可使TGN内吞的β 1 AR大部分阻滞。免疫印迹分析表明，无论是破坏TGN或阻断溶酶体阻止β 1 AR降解。GFP-arrestin-3在β 1AR细胞中的共表达增加了β 1AR的内吞作用，并促进其进入TGN，但抑制再循环至质膜。BFA处理可逆转Arrestin-3诱导的β 1AR再循环抑制，而氯喹可诱导arrestin-3与β 1AR在TGN中蓄积。这些结果首次证明，TGN作为一个检查点的回收和下调的β 1 AR和arrestin-3不仅介导β 1 AR内吞作用，但也通过TGN回收。
Receptor down-modulation is the key mechanism by which G protein-coupled receptors (GPCRs) prevent excessive receptor signaling in response to agonist stimulation. Recently, the trans-Golgi network (TGN) has been implicated as a key checkpoint for receptor endocytosis and degradation. Here, we investigated the involvement of the TGN in down-modulation of beta 1-adrenergic receptor in response to persistent isoprotenerol stimulation. Immunofluorescent staining showed that similar to 50% of endocytosed beta 1AR colocalized with TGN-46 at 5 h. Disruption of the TGN by brefeldin A (BFA) led to the robust accumulation of endocytosed beta 1AR in Rab11(+) recycling endosomes, inhibited beta 1AR entry into LAMP1(+) lysosomes, and as a result enhanced beta 1AR recycling to the plasma membrane. The lysosomotropic agent, chloroquine arrested the majority of endocytosed beta 1AR in the TGN by 4 h. Immunoblot analysis showed that either disruption of the TGN or blockage of the lysosome prevented beta 1AR degradation. Co-expression of GFP-arrestin-3 in beta 1AR cells increased the endocytosis of beta 1AR and facilitated its entry to the TGN but inhibited recycling to the plasma membrane. Arrestin-3-induced inhibition of beta 1AR recycling was reversed by BFA treatment, whereas chloroquine induced the accumulation of arrestin-3 with beta 1AR in the TGN. These results demonstrate for the first time that the TGN acts as a checkpoint for both the recycling and down-regulation of beta 1AR and that arrestin-3 not only mediates beta 1AR endocytosis but also its recycling through the TGN.