Technical Reference #1711

1711.

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*,†,‡,§, Duke University Medical Center, PNAS, 105(38), Supporting Information, (2008)
Link To Paper

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

Keywords:
Beta-adrenergic receptor, G protein-coupled receptor, signaling

Materials & Methods:
Confocal Laser Microscopy. Confocal microscopy was performed as previously described (1). Briefly, HEK293 cells stably expressing FLAG-tagged WT 1AR and 1AR mutants were transfected with cDNA encoding fluorescently labeled EGFR (i.e., EGFRGFP, 2 g). Following transfection, cells were trypsinized and plated onto 35-mm collagen-coated (5 g/ml) glass-bottomed culture dishes (MatTek). Following stimulation, cells were washed once with PBS solution and fixed in 4% paraformaldehyde for 30 min. EGFR receptor internalization following stimulation was visualized by green fluorescence using a single sequential line of excitation filters. EGFR-GFP internalization was visualized using a combination of excitation of 488 nm and emission filters between 499 and 520 nm.

Microscopic Technique
Confocal Microscopy, Laser

Cell Type(s)
HEK-293