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

Sudha K. Shenoy, Matthew T. Drake, Christopher D. Nelson, Daniel A. Houtz, Kunhong Xiao, Srinivasan Madabushi, Eric Reiter, Richard T. Premont, Olivier Lichtarge, Robert J. Lefkowitz

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512 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)1261-1273
Number of pages13
JournalJournal of Biological Chemistry
Volume281
Issue number2
DOIs
StatePublished - Jan 13 2006

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Shenoy, S. K., Drake, M. T., Nelson, C. D., Houtz, D. A., Xiao, K., Madabushi, S., Reiter, E., Premont, R. T., Lichtarge, O., & Lefkowitz, R. J. (2006). β-arrestin-dependent, G protein-independent ERK1/2 activation by the β2 adrenergic receptor. Journal of Biological Chemistry, 281(2), 1261-1273. https://doi.org/10.1074/jbc.M506576200