期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2001
卷号：98
期号：5
页码：2449-2454
DOI：10.1073/pnas.041604898
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Using both confocal immunofluorescence microscopy and biochemical approaches, we have examined the role of {beta}-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 {beta}-arrestin-2 binding to the receptor and internalization of AT1aR-{beta}-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-{beta}-arrestin complexes. This targeting of ERK2 reflects the formation of multiprotein complexes containing AT1aR, {beta}-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 {beta}-arrestin-2 from transfected COS-7 cells. Coprecipitation of cRaf-1 with {beta}-arrestin-2 was independent of MEK1 and ERK2, whereas the coprecipitation of MEK1 and ERK2 with {beta}-arrestin-2 was significantly enhanced in the presence of overexpressed cRaf-1, suggesting that binding of cRaf-1 to {beta}-arrestin facilitates the assembly of a cRaf-1, MEK1, ERK2 complex. The phosphorylation of ERK2 in {beta}-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 {beta}-arrestin-2, and the association of {beta}-arrestin-2, cRaf-1, and ERK2 with AT1aR. These data suggest that {beta}-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.
