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PNAS 105 (38): 14555-14560

Copyright © 2008 by the National Academy of Sciences.

β-Blockers alprenolol and carvedilol stimulate β-arrestin-mediated EGFR transactivation

Il-Man Kim*, Douglas G. Tilley*, Juhsien Chen*, Natasha C. Salazar*, Erin J. Whalen*, Jonathan D. Violin*, and Howard A. Rockman*,{dagger},{ddagger},§

Departments of *Medicine, {dagger}Cell Biology, and {ddagger}Molecular Genetics, Duke University Medical Center, Durham, NC 27710


Figure 1
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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.

 

Figure 2
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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; {dagger}, P < 0.01 vs. without AG1478 or Prop; {ddagger}, P < 0.001 vs. without AG1478.

 

Figure 3
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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; {dagger}, P < 0.01 vs. NS; {ddagger}, P < 0.001 vs. ICI or NS.

 

Figure 4
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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; {dagger}, P < 0.01 vs. si-Con; {ddagger}, P < 0.001 vs. NS and Prop.

 


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