β-arrestin-dependent, G protein-independent ERK1/2 activation by the β2 adrenergic receptor

被引:613
作者
Shenoy, SK
Drake, MT
Nelson, CD
Houtz, DA
Xiao, KH
Madabushi, S
Reiter, E
Premont, RT
Lichtarge, O
Lefkowitz, RJ
机构
[1] Duke Univ, Med Ctr, Howard Hughes Med Inst, Dept Med, Durham, NC 27710 USA
[2] Duke Univ, Dept Biochem, Med Ctr, Durham, NC 27710 USA
[3] INRA, F-37380 Nouzilly, France
[4] Baylor Coll Med, Program Struct & Computat Biol & Mol Biophys, Houston, TX 77030 USA
[5] Baylor Coll Med, Mol & Human Genet Dept, Houston, TX 77030 USA
关键词
D O I
10.1074/jbc.M506576200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Physiological effects of beta adrenergic receptor (beta 2AR) stimulation have been classically shown to result from G(s)-dependent adenylyl cyclase activation. Here we demonstrate a novel signaling mechanism wherein beta-arrestins mediate beta 2AR signaling to extracellular-signal regulated kinases 1/2 (ERK 1/2) independent of G protein activation. Activation of ERK1/2 by the beta 2AR expressed in HEK-293 cells was resolved into two components dependent, respectively, on G(s)-G(i)/protein kinase A (PKA) or beta-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 beta-arrestins by siRNA. beta-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 beta-arrestin1 or -2 by small interfering RNA. In G(s) knock-out mouse embryonic fibroblasts, wild-type beta 2AR recruited beta-arrestin2-green fluorescent protein and activated pertussis toxin-insensitive ERK1/2. Furthermore, a novel beta 2AR mutant (beta 2AR(T68F,Y132G,Y219A) or beta 2AR(TYY)), rationally designed based on Evolutionary Trace analysis, was incapable of G protein activation but could recruit beta-arrestins, undergo beta-arrestin-dependent internalization, and activate beta-arrestin-dependent ERK. Interestingly, overexpression of GRK5 or -6 increased mutant receptor phosphorylation and beta-arrestin recruitment, led to the formation of stable receptor-beta-arrestin complexes on endosomes, and increased agonist-stimulated phospho-ERK1/2. In contrast, GRK2, membrane translocation of which requires G beta gamma 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 beta 2AR can signal to ERK via a GRK5/6-beta-arrestin-dependent pathway, which is independent of G protein coupling.
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页码:1261 / 1273
页数:13
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