Effect of angiotensin II on the cellular distribution of HA-AT1aR, GFP-β-arrestin-2, and RFP-ERK2. (a) HEK-293 cells were transiently transfected with plasmid DNA encoding HA-AT1aR and GFP-β-arrestin-2. Serum-starved cells were prestained with anti-HA rhodamine to label cell surface HA-AT1aR, treated with vehicle (NS) or angiotensin II (Ang II, 1 μM) for 15 min, fixed with paraformaldehyde, and examined by confocal microscopy. The distribution of rhodamine-labeled HA-AT1aR (red) and GFP-β-arrestin-2 (green) are shown in the single channel images. Colocalization of HA-AT1aR and GFP-β-arrestin-2 is shown in the overlay images (yellow; arrows). (b) Cells were transiently transfected with plasmid DNA encoding HA-AT1aR, GFP-β-arrestin-2, and RFP-ERK2. Serum-starved cells were treated with vehicle (NS) or PMA (100 nM) or angiotensin II (Ang II) for 15 min, and fixed cells were examined by confocal microscopy. The distribution of GFP-β-arrestin-2 (green) and RFP-ERK2 (red) are shown in the single channel images. Colocalization of GFP-β-arrestin-2 and RFP-ERK2 is shown in the overlay images (yellow; arrows). Each image depicts a representative confocal microscopic image from one of three separate experiments.

Effect of angiotensin II on the cellular distribution of RFP-ERK2 and phospho-ERK1/2. HEK-293 cells were transiently transfected with plasmid DNA encoding HA-AT1aR, Flag-β-arrestin-2, and RFP-ERK2. Serum-starved cells were treated with vehicle (NS), PMA, or angiotensin II (Ang II) for 15 min, fixed with paraformaldehyde, permeabilized, and stained with FITC-conjugated monoclonal anti-phospho-ERK1/2 before examination by confocal microscopy. The distribution RFP-ERK2 (red) and FITC-stained phospho-ERK1/2 (green) are shown in the single channel images. Colocalization of RFP-ERK2 and phospho-ERK1/2 is shown in the overlay images (yellow; arrows). Each image depicts a representative confocal microscopic image from one of three separate experiments.

Binding of myc-cRaf-1, MEK1 and activated GFP-ERK2 to wild-type and mutant Flag-β-arrestin-2. (a) COS-7 cells were transiently transfected with plasmid DNA encoding myc-cRaf-1, MEK1, GFP-ERK2, and Flag-β-arrestin-2 in various combinations as indicated. GFP-ERK2, which undergoes agonist-stimulated phosphorylation and nuclear translocation like the wild-type kinase (10; not shown), was used in these studies to allow each overexpressed protein to have a unique epitope tag. Anti-Flag immunoprecipitates were probed for coprecipitated cRaf-1, MEK1, ERK2, and β-arrestin-2. Equivalent expression of each construct was confirmed by immunoblotting an aliquot of each whole cell lysate in parallel (not shown). Shown are representative immunoblots from one of six separate experiments. (b) Bar graphs depict the amount of myc-cRaf-1 (Left) and GFP-ERK2 (Right) present in Flag-β-arrestin-2 immunoprecipitates from cells coexpressing myc-cRaf-1 only, GFP-ERK2 only, or both myc-cRaf-1 and GFP-ERK2. Data are presented in arbitrary units where the amount of myc-cRaf-1 or GFP-ERK2 binding to Flag-β-arrestin-2 when singly expressed is defined as 1. Data shown represent the mean ± SD from four separate experiments. (c) Cells were transiently transfected with plasmid DNA encoding GFP-ERK2 and increasing amounts of myc-cRaf-1, in the presence and absence of Flag-β-arrestin-2 and MEK(K97A) as indicated. The amount of GFP-ERK2 and phospho-GFP-ERK2 present in Flag-β-arrestin-2 immunoprecipitates was determined by immunoblotting (Upper). Graphs depict the amount of GFP-ERK2 and phospho-GFP-ERK2 present in Flag-β-arrestin-2 immunoprecipitates (Left) and the effect of MEK(K97A) expression on the amount of phospho-GFP-ERK2 present in Flag-β-arrestin-2 immunoprecipitates (Right). Data are presented in arbitrary units where the amount of GFP-ERK2 or phospho-GFP-ERK2 present in Flag-β-arrestin-2 immunoprecipitates in the absence of overexpressed myc-cRaf-1 is defined as 1. Data shown represent the mean ± SD from three separate experiments.

Effect of angiotensin II stimulation on the assembly of complexes containing HA-AT1aR, Flag-β-arrestin-2, myc-cRaf-1, and GFP-ERK2. (a) COS-7 cells were transiently transfected with plasmid cDNA encoding HA-AT1aR, Flag-β-arrestin-2, myc-cRaf-1, and GFP-ERK2. Serum-starved cells were stimulated with angiotensin II (Ang II) for the time indicated. The amount of myc-cRaf-1 and GFP-ERK2 present in Flag-β-arrestin-2 immunoprecipitates was determined by immunoblotting. (b) Serum-starved cells were stimulated with angiotensin II (Ang II) for 5 min before covalent crosslinking of receptor-associated proteins with DSP. The amount of myc-cRaf-1, GFP-ERK2, and Flag-β-arrestin-2 present in HA-AT1aR immunoprecipitates was determined by immunoblotting. Immunoblots shown are representative of three to five separate experiments.

Model of ERK activation and targeting by β-arrestin scaffolds. Agonist (H) binding to heptahelical receptors results in dissociation of heterotrimeric G proteins into Gα-GTP and Gβγ subunits, which activate G protein effectors (E). One consequence of Gβγ subunit release is enhanced GRK-mediated phosphorylation of the agonist-occupied receptor. β-Arrestins 1 (β-arr) bind to both GRK-phosphorylated receptor and to the component kinases of the ERK cascade, resulting in assembly of an ERK activation complex that is targeted into endosomal vesicles.