Desensitization, internalization, and signaling functions of beta-arrestins demonstrated by RNA interference

Abstract

Beta-arrestins bind to activated G protein-coupled receptor kinase-phosphorylated receptors, which leads to their desensitization with respect to G proteins, internalization via clathrin-coated pits, and signaling via a growing list of "scaffolded" pathways. To facilitate the discovery of novel adaptor and signaling roles of beta-arrestins, we have developed and validated a generally applicable interfering RNA approach for selectively suppressing beta-arrestins 1 or 2 expression by up to 95%. Beta-arrestin depletion in HEK293 cells leads to enhanced cAMP generation in response to beta(2)-adrenergic receptor stimulation, markedly reduced beta(2)-adrenergic receptor and angiotensin II receptor internalization and impaired activation of the MAP kinases ERK 1 and 2 by angiotensin II. This approach should allow discovery of novel signaling and regulatory roles for the beta-arrestins in many seven-membrane-spanning receptor systems.

Figure 1

Analysis of β-arrestin expression in siRNA-transfected HEK293 cells. Whole-cell lysates were prepared from the indicated RNA-transfected HEK293 cells as described. Equal amounts of proteins (20 μg) in each sample were used to determine expression of β-arrestins 1 and 2 by immunoblotting with the polyclonal anti-β-arrestin antibody A1CT. Values shown are expressed as percent of the level of each β-arrestin in mock (No RNA)-transfected cells and represents the mean ± SE from seven independent experiments. Statistical significance was determined by using a one-way ANOVA to correct for multiple comparisons (PRISM software) between β-arrestin siRNA-transfected cells and control cells [both mock and nonsilencing, control RNA (CTL)-transfected] (*, P < 0.001). A representative immunoblot is shown (Upper).

Figure 2

Effect of siRNA-mediated β-arrestin depletion on cAMP accumulation by endogenously expressed β₂-AR. HEK293 cells were transfected with the indicated RNA oligonucleotides as described and subsequently stimulated with 10 μM isoproterenol for 2 min. Accumulation of cAMP was determined by competition with [³H]cAMP for PKA binding sites and then normalized to total forskolin (10 μM)-stimulated cAMP levels for each treatment. Data are expressed as percent of agonist-induced cAMP accumulation in control RNA-treated (CTL) cells and represent the mean ± SE of six independent experiments. NS, nonstimulated. Statistical significance was determined by using a one-way ANOVA to correct for multiple comparisons between β-arrestin siRNA-transfected cells and control cells. The asterisk indicates P < 0.05 vs. both control RNA- and mock (No RNA)-transfected cells.

Figure 3

Effect of siRNA-mediated suppression of β-arrestin levels on 7MS receptor internalization. (A) HEK293 cells were transfected with either control RNA (CTL) or the indicated β-arrestin siRNA and subsequently transfected again with plasmids encoding Flag epitope-tagged β₂-AR as described. Serum-starved cells were exposed to 10 μM isoproterenol for 30 min at 37°C and analyzed for their plasma membrane content of the Flag epitope-tagged β₂-AR by flow cytometry. Values shown are expressed as percentage of loss of agonist-induced cell surface receptors over nonstimulated cells. (B) HEK293 cells were transfected with appropriate amounts of AT_1A-R encoding plasmids and the indicated siRNAs simultaneously. Receptor expression was equivalent (200–300 fmol per mg of protein) among all transfected cells. Cells were cooled down in ice-cold, serum-free media and subsequently stimulated with 0.2 nM ¹²⁵I-labeled AngII for 5 min at 37°C. Percent of agonist-stimulated AT_1A-R sequestration was determined as acid-resistant cpm divided by the total cpm bound. Data represent the mean ± SE from three independent experiments done in triplicate. Statistical significance was determined by using a one-way ANOVA to correct for multiple comparisons between β-arrestin siRNA-transfected cells and control cells [both receptor-transfected cells without RNA (Rc alone) and control RNA-treated cells] (*, P < 0.05; **, P < 0.01).

Figure 4

Effect of siRNA-inhibited expression of β-arrestin on ERK activation in response to AT_1A-R stimulation. HEK293 cells transiently expressing AT_1A-R (≈300 fmol per mg of protein) by cotransfection with either control RNA (CTL) or the β-arrestin 2 siRNA were serum-starved for 1–2 h and stimulated with the indicated concentrations of AngII for 5 min at 37°C. Phosphorylation of ERK in whole-cell lysates, normalized to equal amounts of proteins in each sample, was detected by immunoblotting with an antiphospho-ERK 1/2 antibody as described. Equal loading of total ERK proteins were also confirmed by immunoblotting with an anti-ERK 1/2 antibody (data not shown). Each data point is expressed as percent of the maximal phosphorylation of ERK in response to 10⁻⁶ M AngII in control cells and represents the mean ± SE from seven independent experiments. Dose–response curves and EC₅₀s between control RNA- and β-arrestin 2 siRNA-treated samples were obtained and analyzed by a two-way ANOVA to determine statistical significance for shift of the curve (P < 0.001) by using PRISM software. A representative immunoblot is shown (Upper).