TY - JOUR
T1 - β-arrestin-dependent, G protein-independent ERK1/2 activation by the β2 adrenergic receptor
AU - Shenoy, Sudha K.
AU - Drake, Matthew T.
AU - Nelson, Christopher D.
AU - Houtz, Daniel A.
AU - Xiao, Kunhong
AU - Madabushi, Srinivasan
AU - Reiter, Eric
AU - Premont, Richard T.
AU - Lichtarge, Olivier
AU - Lefkowitz, Robert J.
PY - 2006/1/13
Y1 - 2006/1/13
N2 - Physiological effects of β adrenergic receptor (β2AR) stimulation have been classically shown to result from G s-dependent adenylyl cyclase activation. Here we demonstrate a novel signaling mechanism wherein β-arrestins mediate β2AR signaling to extracellular-signal regulated kinases 1/2 (ERK 1/2) independent of G protein activation. Activation of ERK1/2 by the β2AR expressed in HEK-293 cells was resolved into two components dependent, respectively, on G s-G i/protein kinase A (PKA) or β-arrestins. G protein-dependent activity was rapid, peaking within 2-5 min, was quite transient, was blocked by pertussis toxin (G i inhibitor) and H-89 (PKA inhibitor), and was insensitive to depletion of endogenous β-arrestins by siRNA. β-Arrestin-dependent activation was slower in onset (peak 5-10 min), less robust, but more sustained and showed little decrement over 30 min. It was insensitive to pertussis toxin and H-89 and sensitive to depletion of either β-arrestin1 or -2 by small interfering RNA. In G s knock-out mouse embryonic fibroblasts, wild-type β2AR recruited β-arrestin2-green fluorescent protein and activated pertussis toxin-insensitive ERK1/2. Furthermore, a novel β2AR mutant (β2AR T68F,Y132G,Y219A or β2AR TYY), rationally designed based on Evolutionary Trace analysis, was incapable of G protein activation but could recruit β-arrestins, undergo β-arrestin-dependent internalization, and activate β-arrestin-dependent ERK. Interestingly, overexpression of GRK5 or -6 increased mutant receptor phosphorylation and β-arrestin recruitment, led to the formation of stable receptor-β- arrestin complexes on endosomes, and increased agonist-stimulated phospho-ERK1/2. In contrast, GRK2, membrane translocation of which requires Gβγ release upon G protein activation, was ineffective unless it was constitutively targeted to the plasma membrane by a prenylation signal (CAAX). These findings demonstrate that the β2AR can signal to ERK via a GRK5/6-β-arrestin-dependent pathway, which is independent of G protein coupling.
AB - Physiological effects of β adrenergic receptor (β2AR) stimulation have been classically shown to result from G s-dependent adenylyl cyclase activation. Here we demonstrate a novel signaling mechanism wherein β-arrestins mediate β2AR signaling to extracellular-signal regulated kinases 1/2 (ERK 1/2) independent of G protein activation. Activation of ERK1/2 by the β2AR expressed in HEK-293 cells was resolved into two components dependent, respectively, on G s-G i/protein kinase A (PKA) or β-arrestins. G protein-dependent activity was rapid, peaking within 2-5 min, was quite transient, was blocked by pertussis toxin (G i inhibitor) and H-89 (PKA inhibitor), and was insensitive to depletion of endogenous β-arrestins by siRNA. β-Arrestin-dependent activation was slower in onset (peak 5-10 min), less robust, but more sustained and showed little decrement over 30 min. It was insensitive to pertussis toxin and H-89 and sensitive to depletion of either β-arrestin1 or -2 by small interfering RNA. In G s knock-out mouse embryonic fibroblasts, wild-type β2AR recruited β-arrestin2-green fluorescent protein and activated pertussis toxin-insensitive ERK1/2. Furthermore, a novel β2AR mutant (β2AR T68F,Y132G,Y219A or β2AR TYY), rationally designed based on Evolutionary Trace analysis, was incapable of G protein activation but could recruit β-arrestins, undergo β-arrestin-dependent internalization, and activate β-arrestin-dependent ERK. Interestingly, overexpression of GRK5 or -6 increased mutant receptor phosphorylation and β-arrestin recruitment, led to the formation of stable receptor-β- arrestin complexes on endosomes, and increased agonist-stimulated phospho-ERK1/2. In contrast, GRK2, membrane translocation of which requires Gβγ release upon G protein activation, was ineffective unless it was constitutively targeted to the plasma membrane by a prenylation signal (CAAX). These findings demonstrate that the β2AR can signal to ERK via a GRK5/6-β-arrestin-dependent pathway, which is independent of G protein coupling.
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U2 - 10.1074/jbc.M506576200
DO - 10.1074/jbc.M506576200
M3 - Article
C2 - 16280323
AN - SCOPUS:33644857279
SN - 0021-9258
VL - 281
SP - 1261
EP - 1273
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 2
ER -