Arrestin domain-containing protein 3 recruits the NEDD4 E3 ligase to mediate ubiquitination of the beta2-adrenergic receptor

Abstract

Prolonged stimulation of the beta2-adrenergic receptor (beta2AR) leads to receptor ubiquitination and downregulation. Using a genome-wide RNA interference screen, we identified arrestin domain-containing 3 (ARRDC3) as a gene required for beta2AR regulation. The ARRDC3 protein interacts with ubiquitin ligase neural precursor development downregulated protein 4 (NEDD4) through two conserved PPXY motifs and recruits NEDD4 to the activated receptor. The ARRDC3 protein also interacts and co-localizes with activated beta2AR. Knockdown of ARRDC3 expression abolishes the association between NEDD4 and beta2AR. Furthermore, functional inactivation of ARRDC3, either through small interfering RNA (siRNA)-mediated knockdown or overexpression of a mutant that does not interact with NEDD4, blocks receptor ubiquitination and degradation. Our results establish ARRDC3 as an essential adaptor for beta2AR ubiquitination.

Figure 1

Identification of ARRDC3 as a new gene required for β2AR degradation and ubiquitination. (A) Schematic diagram of the RNAi-based screen to identify regulators of β2AR. (B) ARRDC3 knockdown enhances β2ARt membrane levels. FLAG–β2ARt cells transduced with lentiviral particles carrying a non-targeting (NT) shRNA or an ARRDC3-specific shRNA and treated as indicated, before immunostaining and FACS analysis. The mean fluorescence intensity of NT or ARRDC3 shRNA-expressing cells is indicated. (C,D) Effects of siRNA-mediated ARRDC3 knockdown on (C) β2ARt and (D) β2AR degradation. Normalized pixel densitometry values, shown as a bar graph, and s.e. values are averages of three independent experiments; ^**P<0.01 and ^***P<0.001. (E,F) Ubiquitination (Ub) of (E) β2ARt and (F) β2AR in ARRDC1 and ARRDC3 knockdown cells. β2AR, β2-adrenergic receptor; β2ARt, truncated β2-adrenergic receptor; ARRDC3, arrestin domain-containing 3; FACS, fluorescence-activated cell sorting; FICT, fluorescein isothiocyanate; IP, immunoprecipitation; ISO, isoproterenol; RNAi, RNA-mediated interference; shRNA, short hairpin RNA; WCE, whole cell extract.

Figure 2

ARRDC3 interacts with and recruits NEDD4 E3 ligase to the activated β2AR to mediate receptor ubiquitination. (A) Alignment of the PPXY-containing domains of ARRDC3 orthologues. PPXY motifs are highlighted in red in the alignment and were mutated as indicated to generate individual PPXY motif mutants ARRDC3–AASA and ARRDC3–AALA, or the double PPXY motif mutant ARRDC3ΔΔPPXY. (B) ARRDC3 interaction with NEDD4 requires the PPXY motifs. FLAG-tagged NEDD4 was co-transfected into 293T cells with HA-tagged ARRDC3 or its mutants. FLAG immunoprecipitated complexes were immunoblotted with the indicated antibodies. (C) Co-localization of ARRDC3 with NEDD4. GFP–ARRDC3- and mCherry–NEDD4-transfected β2AR-expressing cells were treated as indicated and fixed with paraformaldehyde before visualization by confocal microscopy. White arrowheads indicate NEDD4 and ARRDC3 co-localization. (D) Effects of ARRDC3 or ARRDC3ΔΔPPXY expression on β2AR degradation. GFP or GFP-tagged ARRDC3 (or the double PPXY mutant) was co-transfected with FLAG–β2AR expression vector into 293T cells. Cells were treated with vehicle (water) or ISO. β2AR degradation was assayed by anti-FLAG immunoblotting. Anti-GFP was used to assess the expression of the GFP constructs. The asterisk indicates non-specific bands. (E) Effect of ARRDC3 or ARRDC3 PPXY mutant overexpression on β2AR ubiquitination. HA-tagged ARRDC3 (or the double PPXY mutant) was transfected into β2ARt-expressing cells. β2AR ubiquitination was determined by FLAG IP followed by anti-ubiquitin (Ub) immunoblotting. All results are representative of duplicate independent experiments. β2AR, β2-adrenergic receptor; β2ARt, truncated β2-adrenergic receptor; ARRDC3, arrestin domain-containing 3; GFP, green fluorescent protein; HA, haemagglutinin; IP, immunoprecipitation; ISO, isoproterenol; NEDD4, neural precursor development downregulated protein 4; WCE, whole cell extract; wt, wild type.

Figure 3

ARRDC3 interacts with activated β2AR. (A) Association of ARRDC3 with activated β2AR. Cells stably expressing FLAG–β2ARt were transfected with the indicated plasmids then subjected to a treatment with vehicle or ISO. Anti-FLAG immunoprecipitates from the corresponding lysates and WCEs were analysed with the indicated antibodies. (B) ARRDC3 co-localizes with β2AR and EEA1 after agonist stimulation. β2AR-expressing cells transfected with GFP (control) or ARRDC3–GFP were incubated with a rabbit-raised FLAG antibody and subjected to treatment with ISO (or vehicle) as indicated. Cells were fixed and permeabilized as detailed in the ‘Methods' section then incubated with a mouse-raised EEA1 antibody. Cells were later stained with a rabbit IgG TRITC-conjugated antibody and a mouse IgG Alexa 647-conjugated antibody before mounting on slides and visualization. White arrowheads indicate regions of co-localization. β2AR, β2-adrenergic receptor; β2ARt, truncated β2-adrenergic receptor; ARRDC3, arrestin domain-containing 3; EEA1, early endosomal antigen 1; GFP, green fluorescent protein; ISO, isoproterenol; TRITC, tetramethylrhodamine isothiocyanate; WCE, whole cell extract.

Figure 4

ARRDC3 is an essential adaptor for β2AR ubiquitination. (A) ARRDC3 is required for the NEDD4–β2AR association. Cells stably expressing FLAG–β2ARt were transfected with siRNAs targeting nothing (scrambled), ARRDC3 or β-arrestin 2 and then subjected to a treatment with vehicle or ISO. Anti-FLAG immunoprecipitates from the corresponding lysates and WCE were analysed by immunoblotting with the indicated antibodies. (B) A schematic model of ARRDC3 functioning as an adaptor for β2AR ubiquitination (Ub). β2AR, β2-adrenergic receptor; β2ARt, truncated β2-adrenergic receptor; ARRDC3, arrestin domain-containing 3; IP, immunoprecipitation; ISO, isoproterenol; NEDD4, neural precursor development downregulated protein 4; siRNA, small interfering RNA; WCE, whole cell extract.