Activation and targeting of extracellular signal-regulated kinases by beta-arrestin scaffolds.
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.
The Geriatrics Research, Education and Clinical Center, Durham Veterans Affairs Medical Center, Durham, NC 27705, USA. luttrell@receptor-biol.duke.edu
L M
LM
Luttrell
F L
FL
Roudabush
E W
EW
Choy
W E
WE
Miller
M E
ME
Field
K L
KL
Pierce
R J
RJ
Lefkowitz
D000804:Angiotensin II / Q000494:pharmacology; D000818:Animals; D019390:Arrestins / Q000378:metabolism*; D002460:Cell Line; D004789:Enzyme Activation; D006801:Humans; D018613:Microscopy, Confocal; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins
C000605254:ARRB2 protein, human; D019390:Arrestins; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins; D000804:Angiotensin II; D019950:Mitogen-Activated Protein Kinase 1