Distinct phosphorylation sites on the β(2)-adrenergic receptor establish a barcode that encodes differential functions of β-arrestin

Abstract

Phosphorylation of G protein-coupled receptors (GPCRs, which are also known as seven-transmembrane spanning receptors) by GPCR kinases (GRKs) plays essential roles in the regulation of receptor function by promoting interactions of the receptors with β-arrestins. These multifunctional adaptor proteins desensitize GPCRs, by reducing receptor coupling to G proteins and facilitating receptor internalization, and mediate GPCR signaling through β-arrestin-specific pathways. Detailed mapping of the phosphorylation sites on GPCRs targeted by individual GRKs and an understanding of how these sites regulate the specific functional consequences of β-arrestin engagement may aid in the discovery of therapeutic agents targeting individual β-arrestin functions. The β(2)-adrenergic receptor (β(2)AR) has many serine and threonine residues in the carboxyl-terminal tail and the intracellular loops, which are potential sites of phosphorylation. We monitored the phosphorylation of the β(2)AR at specific sites upon stimulation with an agonist that promotes signaling by both G protein-mediated and β-arrestin-mediated pathways or with a biased ligand that promotes signaling only through β-arrestin-mediated events in the presence of the full complement of GRKs or when either GRK2 or GRK6 was depleted. We correlated the specific and distinct patterns of receptor phosphorylation by individual GRKs with the functions of β-arrestins and propose that the distinct phosphorylation patterns established by different GRKs establish a "barcode" that imparts distinct conformations to the recruited β-arrestin, thus regulating its functional activities.

Fig. 1

The effect of knockdown of GRK2, GRK6, or both GRK2 and GRK6 on desensitization and internalization of the β₂AR. (A) HEK293 cells stably transfected with the GloSensor reporter and the indicated siRNAs were pretreated with 100 nM isoproterenol for 5 min, washed two to three times over 10 min, and then assayed for cAMP accumulation after restimulation with isoproterenol at the indicated concentrations (10⁻⁹ to 10⁻⁴ M). The fits of the data are sigmoidal, and the amounts of cAMP were normalized within each group and plotted as percent of the maximal response achieved. (B) The data from (A) are shown here as the baseline-corrected changes in maximal response upon pretreatment with isoproterenol. All data shown are the means ± SEM on dose-response data performed in duplicate or triplicate for at least n = 6. Statistical significance was assessed by one-way analysis of variance (ANOVA) with Bonferroni post-test to compare each group versus CTL siRNA–treated cells (**P < 0.01). (C) Representative immunoblot (IB) for silencing of GRK2, GRK6, or both demonstrates the siRNA transfection efficiency. Quantification (SE ± SEM) of at least six independent siRNA transfections used for various experiment types is shown. (D) Effect of the indicated siRNAs on the internalization of the β₂AR. Internalization was determined with ³H-CGP-12177 and ICI-118551 to detect nonspecific binding as described in Materials and Methods. The percent receptor internalized is shown as a loss of ³H-CGP-12177 binding, and the values represent the means ± SEM from at least three independent experiments. Statistical comparison of the curves was determined with two-way ANOVA between GRK siRNA–transfected cells and control cells (***P < 0.001).

Fig. 2

The roles of GRK2 and GRK6 in β₂AR-mediated ERK activation. (A) siRNA-transfected cells stably carrying the β₂AR were serum-starved and then stimulated with either 10 µM isoproterenol (left panel) or 10 µM carvedilol (right panel) at 37°C for the indicated time points. Both phosphorylated ERK (pERK) and total ERK (tERK) were visualized by Western blotting. (B) Signals from (A) were quantified by densitometry and expressed as the percent of phosphorylated ERK1/2 obtained at the 5-min stimulation in control (CTL) siRNA–transfected cells. Data represent the means ± SEM from at least three independent experiments. Statistical significance was determined with a one-way ANOVA with Bonferroni post-test to correct for multiple comparisons between each GRK siRNA–transfected and control-transfected cells (*P < 0.05; **P < 0.01).

Fig. 3

β₂AR phosphorylation dictates β-arrestin conformation without affecting recruitment. (A) HEK293 cells transiently cotransfected with β₂AR and the β-arrestin2 BRET biosensor (RLuc–β-arr-YFP) were transfected with either control (CTL), GRK2, or GRK6 siRNAs. Changes in intramolecular BRET upon stimulation of β₂AR by isoproterenol (10 µM for 15 min) were measured. Data are the means ± SEM of six independent experiments, each performed in quadruplicate. ***P < 0.001; NS, not significant between basal and stimulated conditions as determined by one-way ANOVA with Bonferroni post-test. (B) Immunoprecipitation of stably transfected FLAG-β₂AR followed by Western blot analysis shows that agonist-induced β-arrestin association after 15-min stimulation with 10 µM isoproterenol was unaffected by knockdown of GRK2 or GRK6. Data shown are the means ± SEM from three independent experiments. One-way ANOVA and Bonferroni post-tests indicated no significant difference between the groups of data compared to control (CTL). The antibody used for the Western analysis detects both β-arrestin1 and β-arrestin2 (βarr1/2).

Fig. 4

Quantitative analysis of β₂AR phosphorylation. (A) Summary of phosphorylation changes in response to agonist stimulation as determined by quantitative LC-MS/MS. HEK293 cells stably transfected with the β₂AR were treated with 10 µM isoproterenol (Iso) or carvedilol (Carv) for 5 min before harvesting. Fold changes in phosphorylation were calculated relative to samples without agonist treatment. *, singly phosphorylated peptides were detected when either one of the listed sites was phosphorylated; **, the listed two sites were identified as a cluster when both sites were phosphorylated; CTL, control; NC, no change; ND, not detected. (B) Summary of phosphorylation changes in response to GRK2 or GRK6 knock-down as determined by quantitative LC-MS/MS. GRK2 siRNA or GRK6 siRNA was used to knock down the individual kinases in HEK293 cells stably transfected with the β₂AR. Fold changes were calculated relative to samples with CTL siRNA treatment. Both GRK siRNA– and CTL siRNA–treated cells were stimulated with 10 µM isoproterenol for 5 min before harvesting. (C) Mapping GRK2 and GRK6 phosphorylation sites on the β₂AR. GRKs responsible for phosphorylation of individual residues were determined by siRNA treatment combined with quantitative MS. Significant fold changes of the phosphorylated residue clusters are shown and indicated by filled circles. Residues that exhibited a decrease in the extent of phosphorylation upon GRK2 knockdown are indicated in red and those that exhibited a decreasewithGRK6 knockdown are shown in blue. PKA consensus sites are shown in gray and the site phosphorylated by ATM is shown in yellow.

Fig. 5

Confirmation of site-specific β₂AR phosphorylation by GRK2 and GRK6 with phosphorylation site–specific antibodies. β₂ARs were purified from HEK293 cells stably transfected with the β₂AR that were transfected with CTL, GRK2, or GRK6 siRNA. Cells were stimulated with or without 10 µM isoproterenol for 5 min before the receptors were pulled down using an alprenolol-agarose affinity purification procedure. Western blot analyses of the purified β₂ARs were then performed with phosphorylation site–specific antibodies. At least three independent experiments were quantified in (A) to (E) and data shown are the means ± SEM. Statistical significance was assessed by one-way ANOVA with Bonferroni post-test comparing CTL siRNA–treated cells versus either GRK2 or GRK6 siRNA–treated cells (***P < 0.001). (A to E) β₂AR phosphorylation (A) at pSer³⁵⁵ and pSer³⁵⁶, (B) at pSer³⁹⁶, C) at pSer⁴⁰⁷ and pSer⁴¹¹, D) at the PKA consensus sites pSer²⁶¹ and pSer²⁶², and (E) at the PKA consensus sites pSer³⁴⁵ and pSer³⁴⁶. Representative Western blots for the data shown in (A) are shown in fig. S4C, and the data quantified are nonstimulated (0 min) and the 5-min data points. Representative Western blots for the remaining panels are shown in fig. S5.