| Arrestin domain-containing protein 3 (ARRDC3) is a member of the mammalian beta-arrestin family, which is predicted to share similar tertiary structure with visual-/beta-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 beta2-adrenergic receptor (beta2AR).;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 beta2AR in a ligand-independent manner. Functionally, while ARRDC3 has no effect on beta2AR endocytosis or degradation, it negatively regulates agonist-promoted beta2AR 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 beta2AR entry into SNX27-occupied endosomal tubules by regulating the association between the beta2AR and SNX27.;Concomitantly, by modulating the endosomal residence time of the beta2AR, ARRDC3 regulates the recruitment of Galphas to the receptor-occupied endosomes and the beta2AR--dependent signaling from the endosomes. Thus, ARRDC3 functions as a switch to modulate the endosomal residence time and subsequent intracellular signaling of the beta2AR. Future studies should focus on identifying the interacting motifs of the beta2AR and ARRDC3, as well as expanding additional interacting partners of ARRDC3, such as additional GPCRs. |
