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

Abstract

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 stimutation of endogenous or transfected β2-adrenergic receptors (β2ARs) led to rapid ubiquitination of both the receptors and the receptor regulatory protein, β-arrestin. Moreover, proteasome inhibitors reduced receptor internalization and degradation, thus implicating a role for the ubiquitination machinery in the trafficking of the β2AR. Receptor ubiquitination required β-arrestin, which bound to the E3 ubiquitin ligase Mdm2. Abrogation of β-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 β2AR mutant lacking lysine residues, which was not ubiquitinated, was internalized normally but was degraded ineffectively. These findings delineate an adapter role of β-arrestin in mediating the ubiquitination of the β2AR and indicate that ubiquitination of the receptor and of β-arrestin have distinct and obligatory roles in the trafficking and degradation of this prototypic GPCR.