The functional roles of arrestin domain-containing protein 3 in regulating G protein-coupled receptor trafficking and signaling
Arrestin domain-containing protein 3 (ARRDC3) is a member of the mammalian β-arrestin family, which is predicted to share similar tertiary structure with visual-/β-arrestins and also contains C-terminal PPxY motifs that mediate interaction with E3 ubiquitin ligases. Recently, ARRDC3 has been proposed to play a role in regulating the trafficking of G protein-coupled receptors, although mechanistic insight into this process is lacking. Here we focused on characterizing the role of ARRDC3 in regulating the trafficking of the β2-adrenergic receptor (β2AR). Using confocal and total internal reflection fluorescence microscopy (TIR-FM) in fixed and live cells, we find that both overexpressed and endogenous ARRDC3 primarily localizes to EEA1-positive early endosomes, where it also interacts with the ESCRT-0 complex. While the PPxY motifs of ARRDC3 are essential for its endosomal localization, only one PPxY motif as well as the arrestin-like domains are needed for proper localization. Using three approaches, both in vitro and in cells, we found that ARRDC3 directly interacts with the β2AR in a ligand-independent manner. Functionally, while ARRDC3 has no effect on β2AR endocytosis or degradation, it negatively regulates agonist-promoted β2AR recycling from early endosomes to the plasma membrane. Further mechanistic studies, using fluorescent microscopy in both fixed and live cells, suggest that ARRDC3 negatively modulates β2AR entry into SNX27-occupied endosomal tubules by regulating the association between the β2AR and SNX27. Concomitantly, by modulating the endosomal residence time of the β2AR, ARRDC3 regulates the recruitment of Gαs to the receptor-occupied endosomes and the β2AR–dependent signaling from the endosomes. Thus, ARRDC3 functions as a switch to modulate the endosomal residence time and subsequent intracellular signaling of the β2AR. Future studies should focus on identifying the interacting motifs of the β2AR and ARRDC3, as well as expanding additional interacting partners of ARRDC3, such as additional GPCRs.
Tian, Xufan, "The functional roles of arrestin domain-containing protein 3 in regulating G protein-coupled receptor trafficking and signaling" (2016). ETD Collection for Thomas Jefferson University. AAI10124154.