Regulation of receptor fate by ubiquitination of activated β2-adrenergic receptor and β-arrestin

Although trafficking and degradation of several membrane proteins are regulated by ubiquitination catalyzed by E3 ubiquitin ligases, there has been little evidence connecting ubiquitination with regulation of mammalian G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor (GPCR) function. Agonist stimulation of endogenous or transfected beta (2)-adrenergic receptors (beta (2)ARs) led to rapid ubiquitination of both the receptors and the receptor regulatory protein, beta -arrestin. Moreover, proteasome inhibitors reduced receptor internalization and degradation, thus implicating a role for the ubiquitination machinery in the trafficking of the beta (2)AR. Receptor ubiquitination required beta -arrestin, which bound to the E3 ubiquitin ligase Mdm2. Abrogation of beta -arrestin ubiquitination, either by expression in Mdm2-null cells or by dominant-negative forms of Mdm2 lacking E3 ligase activity, inhibited receptor internalization with marginal effects on receptor degradation. However, a beta (2)AR mutant tacking lysine residues, which was not ubiquitinated, was internalized normally but was degraded ineffectively. These findings delineate an adapter role of beta -arrestin in mediating the ubiquitination of the beta (2)AR and indicate that ubiquitination of the receptor and of beta -arrestin have distinct and obligatory roles in the trafficking and degradation of this prototypic GPCR.