Activation and Targeting of Extracellular Signal-Regulated Kinases by β-Arrestin Scaffolds

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 98; no. 5; pp. 2449 - 2454
• Main Authors: Luttrell, Louis M., Roudabush, Francine L., Choy, Eric W., Miller, William E., Field, Michael E., Pierce, Kristen L., Lefkowitz, Robert J.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 27.02.2001; National Acad Sciences; The National Academy of Sciences
• Subjects: Angiotensin II - pharmacology; Animals; Arrestins - metabolism; B lymphocytes; beta-Arrestin 2; beta-Arrestins; Biological Sciences; Cell Line; Cellular biology; COS cells; Enzyme Activation; Fluorescence; Humans; Immunoblotting; Microscopy; Microscopy, Confocal; Mitogen-Activated Protein Kinase 1 - metabolism; Phosphorylation; Physiological regulation; Plasmids; Proteins; Receptors; Scaffolds
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.041604898

Using both confocal immunofluorescence microscopy and biochemical approaches, we have examined the role of β-arrestins in the activation and targeting of extracellular signal-regulated kinase 2 (ERK2) following stimulation of angiotensin II type 1a receptors (AT1aR). In HEK-293 cells expressing hemagglutinin-tagged AT1aR, angiotensin stimulation triggered β-arrestin-2 binding to the receptor and internalization of AT1aR-β-arrestin complexes. Using red fluorescent protein-tagged ERK2 to track the subcellular distribution of ERK2, we found that angiotensin treatment caused the redistribution of activated ERK2 into endosomal vesicles that also contained AT1aR-β-arrestin complexes. This targeting of ERK2 reflects the formation of multi-protein complexes containing AT1aR, β-arrestin-2, and the component kinases of the ERK cascade, cRaf-1, MEK1, and ERK2. Myc-tagged cRaf-1, MEK1, and green fluorescent protein-tagged ERK2 coprecipitated with Flag-tagged β-arrestin-2 from transfected COS-7 cells. Coprecipitation of cRaf-1 with β-arrestin-2 was independent of MEK1 and ERK2, whereas the coprecipitation of MEK1 and ERK2 with β-arrestin-2 was significantly enhanced in the presence of overexpressed cRaf-1, suggesting that binding of cRaf-1 to β-arrestin facilitates the assembly of a cRaf-1, MEK1, ERK2 complex. The phosphorylation of ERK2 in β-arrestin complexes was markedly enhanced by coexpression of cRaf-1, and this effect is blocked by expression of a catalytically inactive dominant inhibitory mutant of MEK1. Stimulation with angiotensin increased the binding of both cRaf-1 and ERK2 to β-arrestin-2, and the association of β-arrestin-2, cRaf-1, and ERK2 with AT1aR. These data suggest that β-arrestins function both as scaffolds to enhance cRaf-1 and MEK-dependent activation of ERK2, and as targeting proteins that direct activated ERK to specific subcellular locations.