A unique mechanism of beta-blocker action: carvedilol stimulates beta-arrestin signaling

Abstract

For many years, beta-adrenergic receptor antagonists (beta-blockers or betaAR antagonists) have provided significant morbidity and mortality benefits in patients who have sustained acute myocardial infarction. More recently, beta-adrenergic receptor antagonists have been found to provide survival benefits in patients suffering from heart failure, although the efficacy of different beta-blockers varies widely in this condition. One drug, carvedilol, a nonsubtype-selective betaAR antagonist, has proven particularly effective in the treatment of heart failure, although the mechanism(s) responsible for this are controversial. Here, we report that among 16 clinically relevant betaAR antagonists, carvedilol displays a unique profile of in vitro signaling characteristics. We observed that in beta2 adrenergic receptor (beta2AR)-expressing HEK-293 cells, carvedilol has inverse efficacy for stimulating G(s)-dependent adenylyl cyclase but, nonetheless, stimulates (i) phosphorylation of the receptor's cytoplasmic tail on previously documented G protein-coupled receptor kinase sites; (ii) recruitment of beta-arrestin to the beta2AR; (iii) receptor internalization; and (iv) activation of extracellular regulated kinase 1/2 (ERK 1/2), which is maintained in the G protein-uncoupled mutant beta2AR(T68F,Y132G,Y219A) (beta2AR(TYY)) and abolished by beta-arrestin2 siRNA. Taken together, these data indicate that carvedilol is able to stabilize a receptor conformation which, although uncoupled from G(s), is nonetheless able to stimulate beta-arrestin-mediated signaling. We hypothesize that such signaling may contribute to the special efficacy of carvedilol in the treatment of heart failure and may serve as a prototype for a new generation of therapeutic beta2AR ligands.

Fig. 1.

cAMP responses monitored by ICUE2. HEK-293 cells stably expressing β2AR and the cAMP biosensor ICUE2 were treated for 2 min with a panel of ligands described as β2AR antagonists. (A) cAMP agonism was measured as the rate of change of the ICUE2 FRET ratio corresponding to the rate of cAMP accumulation. Ligands that did not induce cAMP generation were tested for inverse agonism in the same cells. (B) These cells exhibit constitutive β2AR activity that, although too weak to generate high basal cAMP, causes a rapid increase in cAMP when phosphodiesterases are inhibited with isoxybutylmethylxanthine (IBMX). This effect is receptor-specific, because there is no IBMX-induced cAMP increase in cells lacking overexpressed receptor (data not shown). We measured inverse agonism by pretreating cells with ligand for 5 min and quantifying the rate of cAMP increase for 30 sec after IBMX treatment. Inverse agonists are those ligands that do not stimulate cAMP accumulation on their own and decrease the rate of IBMX-induced cAMP accumulation caused by constitutive receptor activity. Data represent mean ± SE from five independent experiments. **, P < 0.001 vs. nonstimulated cells (NS).

Fig. 2.

ERK activation in β2AR and β2AR^TYY stable cells. HEK-293 cells stably expressing β2AR (A) or β2AR^TYY (B) were stimulated with the panel of β2AR ligands used in Fig. 1 at 10 μM for 5 min, and cell lysates were analyzed for pERK and ERK by Western blot. pERK was normalized to total ERK protein. Data represent mean ± SE of at least three independent experiments done in duplicate. Quantification of pERK bands is as a percentage of maximal activity observed for isoproterenol. *, P < 0.05 vs. NS, **, P < 0.001 vs. NS.

Fig. 3.

β2AR phosphorylation stimulated by carvedilol. HEK-293 cells stably expressing β2AR were stimulated with 10 μM of ligand for 30 min, and cell lysates were either analyzed for receptor phosphorylation by Western blot (A) or immunoprecipitated with anti-FLAG beads and analyzed by ³²P metabolic labeling (B). Data represent mean ± SE of at least five independent experiments. ***, P < 0.0001 vs. NS.

Fig. 4.

β-arrestin2-GFP translocation to the β2AR-V2R and receptor internalization stimulated by carvedilol. HEK-293 cells transiently expressing the β2AR-V2R chimera were stimulated for 2 min with either isoproterenol (Iso), carvedilol (Carv), or propranolol (Prop). (A) β-arrestin2-GFP translocation to the β2AR-V2R was then monitored by confocal microscopy. Images are representative of six independent experiments. (B) HEK-293 cells stably expressing β2AR were stimulated with 10 μM of ligand for 30 min and assayed for internalization by fluorescence-activated cell sorting. Data represent mean ± SE of five independent experiments done in duplicate. *, P < 0.05 vs. NS, ***, P < 0.0001 vs. NS.

Fig. 5.

Carvedilol-stimulated ERK 1/2 phosphorylation is abolished by siRNA targeting β-arrestin2. HEK-293 cells stably expressing β2AR (A) or β2AR^TYY (B) were stimulated with 10 μM isoproterenol (Iso) or carvedilol (Carv) in the presence of either control siRNA (CTL) or siRNA targeting β-arrestin2 (βarr2) for 5 min, and cell lysates were analyzed for pERK, ERK, and β-arrestin 1/2 by Western blot. Data represent mean ± SE of four independent experiments done in duplicate. Quantification of pERK bands is as a percentage of maximal activity observed for isoproterenol. **, P < 0.01; *** P < 0.001.