Trafficking patterns of β-arrestin and G protein-coupled receptors determined by the kinetics of β-arrestin deubiquitination

Abstract

Agonist-dependent internalization of G protein-coupled receptors via clathrin-coated pits is dependent on the adaptor protein β-arrestin, which interacts with elements of the endocytic machinery such as AP2 and clathrin. For the β2-adrenergic receptor (β2AR) this requires ubiquitination of β-arrestin by E3 ubiquitin ligase, Mdm2. Based on trafficking patterns and affinity of β-arrestin, G protein-coupled receptors are categorized into two classes. For class A receptors (e.g. β2AR), which recycle rapidly, β-arrestin directs the receptors to clathrin-coated pits but does not internalize with them. For class B receptors (e.g. V2 vasopressin receptors), which recycle slowly, β-arrestin internalizes with the receptor into endosomes. In COS-7 and human embryonic kidney (HEK)-293 cells, stimulation of the β2AR or V2 vasopressin receptor leads, respectively, to transient or stable β-arrestin ubiquitination. The time course of ubiquitination and deubiquitination of β-arrestin correlates with its association with and dissociation from each type of receptor. Chimeric receptors, constructed by switching the cytoplasmic tails of the two classes of receptors (β2AR and V2 vasopressin receptors), demonstrate reversal of the patterns of both β-arrestin trafficking and β-arrestin ubiquitination. To explore the functional consequences of β-arrestin ubiquitination we constructed a yellow fluorescent protein-tagged β-arrestin2-ubiquitin chimera that cannot be deubiquitinated by cellular deubiquitinases. This "permanently ubiquitinated" β-arrestin did not dissociate from the β2AR but rather internalized with it into endosomes, thus transforming this class A receptor into a class B receptor with respect to its trafficking pattern. Overexpression of this β-arrestin ubiquitin chimera in HEK-293 cells also results in enhancement of β2AR internalization and degradation. In the presence of N-ethylmaleimide (an inhibitor of deubiquitinating enzymes), coimmunoprecipitation of the receptor and β-arrestin was increased dramatically, suggesting that deubiquitination of β-arrestin triggers its dissociation from the receptor. Thus the ubiquitination status of β-arrestin determines the stability of the receptor-β-arrestin complex as well as the trafficking pattern of β-arrestin.