Beta-blockers alprenolol and carvedilol stimulate beta-arrestin-mediated EGFR transactivation

Abstract

Recent evidence suggests that binding of agonist to its cognate receptor initiates not only classical G protein-mediated signaling, but also beta-arrestin-dependent signaling. One such beta-arrestin-mediated pathway uses the beta(1)-adrenergic receptor (beta(1)AR) to transactivate the EGFR. To determine whether beta-adrenergic ligands that do not activate G protein signaling (i.e., beta-blockers) can stabilize the beta(1)AR in a signaling conformation, we screened 20 beta-blockers for their ability to stimulate beta-arrestin-mediated EGFR transactivation. Here we show that only alprenolol (Alp) and carvedilol (Car) induce beta(1)AR-mediated transactivation of the EGFR and downstream ERK activation. By using mutants of the beta(1)AR lacking G protein-coupled receptor kinase phosphorylation sites and siRNA directed against beta-arrestin, we show that Alp- and Car-stimulated EGFR transactivation requires beta(1)AR phosphorylation at consensus G protein-coupled receptor kinase sites and beta-arrestin recruitment to the ligand-occupied receptor. Moreover, pharmacological inhibition of Src and EGFR blocked Alp- and Car-stimulated EGFR transactivation. Our findings demonstrate that Alp and Car are ligands that not only act as classical receptor antagonists, but can also stimulate signaling pathways in a G protein-independent, beta-arrestin-dependent fashion.

Fig. 1.

Alp and Car induce EGFR internalization. (A) HEK293 cells stably expressing WTβ₁AR are transfected with EGFR-GFP. EGFR internalization following agonists (Dob, Iso, and EGF) or β-blocker administration is visualized using confocal microscopy. In the absence of agonist, EGFR-GFP is visualized on the membrane, whereas Dob, Iso, and EGF stimulation results in the redistribution of EGFR-GFP into cellular aggregates. Cells stimulated with Iso were pretreated with the β₂AR-selective antagonist ICI-118551. Among 20 β-blockers, only Alp and Car induce redistribution of EGFR into cellular aggregates. (B) HEK293 cells stably expressing WTβ₁AR with transient transfection of EGFR-GFP are pretreated with Prop and stimulated with Dob, Alp, or Car. Dob-, Alp-, or Car-induced EGFR internalization is blocked by Prop. Each confocal image is a composite of representative images.

Fig. 2.

Alp- or Car-β₁AR-stimulated EGFR transactivation and ERK1/2 activation are sensitive to EGFR inhibition. (A) HEK293 cells stably expressing WTβ₁ARs with transient transfection of FLAG-EGFR are treated with Dob, Alp, Car, or EGF with or without AG1478. Both Alp and Car induced EGFR transactivation and ERK activation, which are sensitive to EGFR inhibition. (B) HEK293 cells stably expressing WTβ₁AR with transient transfection of FLAG-EGFRs are pretreated with Prop and stimulated with Dob, Alp, or Car. Dob-, Alp-, or Car-induced ERK activation is blocked by Prop. (C) Dose-dependent ERK responses by Alp or Car. HEK293 cells stably expressing WTβ₁ARs with transient transfection of FLAG-EGFR are treated with the indicated concentrations of Dob, Alp, or Car for 5 min. (D) WT mice are pretreated with erlotinib or DMSO followed by infusion with ligands. Myocardial lysates are immunoblotted with anti-phospho-ERK1/2 and anti-total ERK1/2 antibodies. Data represent mean ± SE of at least four independent experiments. *, P < 0.05 vs. without AG1478; †, P < 0.01 vs. without AG1478 or Prop; ‡, P < 0.001 vs. without AG1478.

Fig. 3.

Alp- or Car-induced β₁AR-mediated transactivation of EGFR requires GRK phosphorylation. HEK293 cells stably expressing WTβ₁AR (A), or GRK⁻ β₁AR (B) are transfected with FLAG-EGFR and then treated with Dob, Alp, or Car. As indicated, WTβ₁AR induces an increase in phospho-EGFR in response to treatment with Dob, Alp, and Car, whereas GRK⁻ β₁AR lacks this effect. (C) Metabolically labeled HEK293 cells stably expressing PKA⁻ β₁AR are stimulated with various ligands. Alp and Car stimulate a significant increase in β1AR phosphorylation at the GRK sites. Data represent mean ± SE of at least four independent experiments. *, P < 0.05 vs. NS; †, P < 0.01 vs. NS; ‡, P < 0.001 vs. ICI or NS.

Fig. 4.

β-Arrestin is recruited to β₁AR by Alp or Car and is required for Alp- or Car-induced ERK activation. (A) HEK293 cells stably expressing FLAG-WTβ₁AR are stimulated with Iso, Alp, Car, or Prop. Alp or Car induces β-arrestin recruitment to the β₁AR. (B) HEK293 cells stably expressing WTβ₁AR are transfected with either FLAG-EGFR and scrambled siRNA (si-Con); or FLAG-EGFR and siRNAs targeting β-arrestin 1 (si-βarr1), β-arrestin 2 (si-βarr2), or β-arrestin1/2 (si-βarr1/2), respectively. Alp- or Car-induced phospho-ERK1/2 is diminished in cells transfected with siRNA targeting the β-arrestins. (C) β₁AR agonists enhance cardiac contractility by G protein-dependent signaling, and they also induce β-arrestin-dependent signaling (Left). However, Alp and Car selectively induce β-arrestin-mediated β₁AR transactivation of EGFR (Right). Alp and Car induce GRK-mediated phosphorylation of β₁AR, and recruitment of β-arrestin and Src. This leads to MMP activation to promote HB-EGF shedding, and subsequent activation of EGFR and its downstream signaling such as ERK. Data represent mean ± SE of at least five independent experiments. *, P < 0.05 vs. NS and Prop or si-Con; †, P < 0.01 vs. si-Con; ‡, P < 0.001 vs. NS and Prop.