Activation and targeting of extracellular signal-regulated kinases by beta-arrestin scaffolds

Using both confocal immunofluorescence microscopy and biochemical approache s, we have examined the role of beta -arrestins in the activation and targe ting of extracellular signal-regulated kinase 2 (ERK2) following stimulatio n of angiotensin II type 1a receptors (AT1aR). In HEK-293 cells expressing hemagglutinin-tagged AT1aR, angiotensin stimulation triggered beta -arresti n-2 binding to the receptor and internalization of AT1aR-beta -arrestin com plexes. Using red fluorescent protein-tagged ERK2 to track the subcellular distribution of ERK2, we found that angiotensin treatment caused the redist ribution of activated ERK2 into endosomal vesicles that also contained AT1a R-beta -arrestin complexes. This targeting of ERK2 reflects the formation o f multiprotein complexes containing AT1aR, beta -arrestin-2, and the compon ent kinases of the ERK cascade, cRaf-1, MEK1, and ERK2. Myc-tagged cRaf-1, MEK1, and green fluorescent protein-tagged ERK2 coprecipitated with Flag-ta gged beta -arrestin-2 from transfected COS-7 cells. Coprecipitation of cRaf -1 with beta -arrestin-2 was independent of MEK1 and ERK2, whereas the copr ecipitation of MEK1 and ERK2 with beta -arrestin-2 was significantly enhanc ed 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 compl ex. The phosphorylation of ERK2 in beta -arrestin complexes was markedly en hanced 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 -ar restin-2, and the association of beta -arrestin-2, cRaf-1, and ERK2 with AT 1aR. These data suggest that beta -arrestins function both as scaffolds to enhance cRaf-1 and MEK-dependent activation of ERK2, and as targeting prote ins that direct activated ERK to specific subcellular locations.