title: Trans-Golgi Network (TGN) as a regulatory node for β1-adrenergic receptor (β1AR) down-modulation and recycling.
abstract: 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.
authors.affiliation: Division of Hematology and Oncology, Rhode Island Hospital and Brown University, Providence, Rhode Island 02903, USA.
authors.forename: Shi-Bin
authors.initials: SB
authors.lastname: Cheng
authors.forename: Edward J
authors.initials: EJ
authors.lastname: Filardo
mesh_terms: D019390:Arrestins / Q000378:metabolism; D020126:Brefeldin A / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D015536:Down-Regulation; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008247:Lysosomes / Q000378:metabolism; D008954:Models, Biological; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D018342:Receptors, Adrenergic, beta-1 / Q000378:metabolism*; D021601:trans-Golgi Network / Q000378:metabolism*
chemical_list: D019390:Arrestins; D018342:Receptors, Adrenergic, beta-1; C082333:arrestin3; D049452:Green Fluorescent Proteins; D020126:Brefeldin A; D046988:Proteasome Endopeptidase Complex