Regulation of Receptor Fate by Ubiquitination of Activated β ₂ -Adrenergic Receptor and β-Arrestin

TLDR

Ubiquitination by E3 ligases regulates trafficking of many membrane proteins, yet its role in mammalian GPCR function has been poorly understood. Agonist stimulation triggers rapid ubiquitination of β2‑adrenergic receptors and β‑arrestin, and β‑arrestin‑dependent ubiquitination via Mdm2 is essential for receptor internalization, while receptor ubiquitination is required for efficient degradation, revealing distinct roles for ubiquitination of the receptor and its regulatory protein in GPCR trafficking.

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 stimulation of endogenous or transfected β 2 -adrenergic receptors (β 2 ARs) 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 β 2 AR. 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 β 2 AR 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 β 2 AR 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.