Trans-Golgi Network (TGN) as a Regulatory Node for β1-Adrenergic Receptor (β1AR) Down-modulation and Recycling

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 β1-adrenergic receptor in response to persistent isoprotenerol stimulation. Immunofluorescent staining showed that ~50% of endocytosed β1AR colocalized with TGN-46 at 5 h. Disruption of the TGN by brefeldin A (BFA) led to the robust accumulation of endocytosed β1AR in Rab11(+) recycling endosomes, inhibited β1AR entry into LAMP1(+) lysosomes, and as a result enhanced β1AR recycling to the plasma membrane. The lysosomotropic agent, chloroquine, arrested the majority of endocytosed β1AR in the TGN by 4 h. Immunoblot analysis showed that either disruption of the TGN or blockage of the lysosome prevented β1AR degradation. Co-expression of GFP-arrestin-3 in β1AR cells increased the endocytosis of β1AR and facilitated its entry to the TGN but inhibited recycling to the plasma membrane. Arrestin-3-induced inhibition of β1AR recycling was reversed by BFA treatment, whereas chloroquine induced the accumulation of arrestin-3 with β1AR 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 β1AR and that arrestin-3 not only mediates β1AR endocytosis but also its recycling through the TGN.