Agonist-stimulated ␤ 2 -adrenergic receptor (␤ 2 AR) ubiquitination is a major factor that governs both lysosomal trafficking and degradation of internalized receptors, but the identity of the E3 ubiquitin ligase regulating this process was unknown. Among the various catalytically inactive E3 ubiquitin ligase mutants that we tested, a dominant negative Nedd4 specifically inhibited isoproterenol-induced ubiquitination and degradation of the ␤ 2 AR in HEK-293 cells. Moreover, siRNA that downregulates Nedd4 expression inhibited ␤ 2 AR ubiquitination and lysosomal degradation, whereas siRNA targeting the closely related E3 ligases Nedd4-2 or AIP4 did not. Interestingly, ␤ 2 AR as well as ␤-arrestin2, the endocytic and signaling adaptor for the ␤ 2 AR, interact robustly with Nedd4 upon agonist stimulation. However, ␤ 2 AR-Nedd4 interaction is ablated when ␤-arrestin2 expression is knocked down by siRNA transfection, implicating an essential E3 ubiquitin ligase adaptor role for ␤-arrestin2 in mediating ␤ 2 AR ubiquitination. Notably, ␤-arrestin2 interacts with two different E3 ubiquitin ligases, namely, Mdm2 and Nedd4 to regulate distinct steps in ␤ 2 AR trafficking. Collectively, our findings indicate that the degradative fate of the ␤ 2 AR in the lysosomal compartments is dependent upon ␤-arrestin2-mediated recruitment of Nedd4 to the activated receptor and Nedd4-catalyzed ubiquitination.

Agonist-induced ubiquitination of the beta(2) adrenergic receptor (beta(2)AR) functions as an important post-translational modification to sort internalized receptors to the lysosomes for degradation. We now show that this ubiquitination is reversed by two deubiquitinating enzymes, ubiquitin-specific proteases (USPs) 20 and 33, thus, inhibiting lysosomal trafficking when concomitantly promoting receptor recycling from the late-endosomal compartments as well as resensitization of recycled receptors at the cell surface. Dissociation of constitutively bound endogenously expressed USPs 20 and 33 from the beta(2)AR immediately after agonist stimulation and reassociation on prolonged agonist treatment allows receptors to first become ubiquitinated and then deubiquitinated, thus, providing a 'trip switch' between degradative and recycling pathways at the late-endosomal compartments. Thus, USPs 20 and 33 serve as novel regulators that dictate both post-endocytic sorting as well as the intensity and extent of beta(2)AR signalling from the cell surface.

ObjectiveTo investigate the roles of the 7TMR adaptor β‐arrestin2 (βarr2) and the related arrestin domain containing α‐arrestins (ARRDC 2, 3, and 4) in β2 adrenergic receptor (β2AR) trafficking and signaling.Resultsβarr2 binding to activated β2ARs is rapid and detected at the plasma‐membrane, whereas GFP‐tagged ARRDC binding is delayed and detected on endosomes. Gene‐silencing of βarr2, but not of ARRDC3 blocks internalization, MAP Kinase activation, receptor ubiquitination and lysosomal trafficking of activated β2AR. βarr2 binds clathrin, AP‐2 and pERK robustly upon agonist‐activation of the β2AR whereas ARRDC3 does not. ARRDC3 has canonical poly‐proline domains, which are binding regions for WW‐domain containing E3 ubiquitin ligase Nedd4 that ubiquitinates agonist‐activated β2AR. Mutations within the poly‐proline domains in ARRDC3 not only ablate its binding with Nedd4, but also prevent its localization on endosomes and eliminate the interaction with internalized β2AR.ConclusionsThe role of βarr2 in β2AR internalization, ubiquitination, lysosomal trafficking and ERK signaling cannot be substituted by ARRDC3. The apparent association of ARRDC3 with the β2AR results from ARRDC3/Nedd4 primarily and Nedd4/β2AR association secondarily. ARRDC2 or ARRDC4 can substitute for ARRDC3 in associating with Nedd4 during β2AR post‐endocytic trafficking. (Supported by HL 080525 to SKS).