β‐arrestin2 and ARRDC proteins have distinct roles in β₂AR trafficking and signaling

Abstract

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).