Receptor-specific ubiquitination of β-arrestin directs assembly and targeting of seven-transmembrane receptor signalosomes

Abstract

Angiotensin II type 1a (AT1a), vasopressin V2, and neurokinin 1 (NK1) receptors are seven-transmembrane receptors (7TMRs) that bind and co-internalize with the multifunctional adaptor protein, β-arrestin. These receptors also lead to robust and persistent activation of extracellular-signal regulated kinase 1/2 (ERK1/2) localized on endosomes. Recently, the co-trafficking of receptor-β-arrestin complexes to endosomes was demonstrated to require stable β-arrestin ubiquitination (Shenoy, S. K., and Lefkowitz, R. J. (2003) J. Biol. Chem. 278, 14498-14506). We now report that lysines at positions 11 and 12 in β-arrestin2 are specific and required sites for its AngII-mediated sustained ubiquitination. Thus, upon AngII stimulation the mutant β-arrestin2K11,12R is only transiently ubiquitinated, does not form stable endocytic complexes with the AT1aR, and is impaired in scaffolding-activated ERK1/2. Fusion of a ubiquitin moiety in-frame to β-arrestin2K11,12R restores AngII-mediated trafficking and signaling. Wild type β-arrestin2 and β-arrestin2K11R,K12R- Ub, but not β-arrestin2K11R,K12R, prevent nuclear translocation of pERK. These findings imply that sustained β-arrestin ubiquitination not only directs co-trafficking of receptor-β-arrestin complexes but also orchestrates the targeting of "7TMR signalosomes" to microcompartments within the cell. Surprisingly, binding of β-arrestin2K11R,K12R to V2R and NK1R is indistinguishable from that of wild type β-arrestin2. Moreover, ubiquitination patterns and ERK scaffolding of β-arrestin2 K11,12R are unimpaired with respect to V2R stimulation. In contrast, a quintuple lysine mutant (β-arrestin2 K18R,K107R,K108R,K207R,K296R) is impaired in endosomal trafficking in response to V2R but not AT1aR stimulation. Our findings delineate a novel regulatory mechanism for 7TMR signaling, dictated by the ubiquitination of β-arrestin on specific lysines that become accessible for modification due to the specific receptor-bound conformational states of β-arrestin2. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.