β-Arrestin-dependent, G Protein-independent ERK1/2 Activation by the β2 Adrenergic Receptor

Abstract

Physiological effects of ␤ adrenergic receptor (␤2AR) stimulation have been classically shown to result from G s -dependent adenylyl cyclase activation. Here we demonstrate a novel signaling mechanism wherein ␤-arrestins mediate ␤2AR signaling to extracellularsignal regulated kinases 1/2 (ERK 1/2) independent of G protein activation. Activation of ERK1/2 by the ␤2AR expressed in HEK-293 cells was resolved into two components dependent, respectively, on G s -G i /protein kinase A (PKA) or ␤-arrestins. G proteindependent 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 ␤-arrestins by siRNA. ␤-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 ␤-arrestin1 or -2 by small interfering RNA. In G s knock-out mouse embryonic fibroblasts, wild-type ␤2AR recruited ␤-arrestin2-green fluorescent protein and activated pertussis toxin-insensitive ERK1/2. Furthermore, a novel ␤2AR mutant (␤2AR T68F,Y132G,Y219A or ␤2AR TYY ), rationally designed based on Evolutionary Trace analysis, was incapable of G protein activation but could recruit ␤-arrestins, undergo ␤-arrestin-dependent internalization, and activate ␤-arrestin-dependent ERK. Interestingly, overexpression of GRK5 or -6 increased mutant receptor phosphorylation and ␤-arrestin recruitment, led to the formation of stable receptor-␤-arrestin complexes on endosomes, and increased agonist-stimulated phospho-ERK1/2. In contrast, GRK2, membrane translocation of which requires G␤␥ 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 ␤2AR can signal to ERK via a GRK5/6-␤-arrestin-dependent pathway, which is independent of G protein coupling.The ␤2-adrenergic receptor (␤2AR) 4 is a well studied member of the large and diverse group of seven transmembrane receptors (7TMRs), which have been shown classically to exert their intracellular effects through G protein activation (1-3). Agonist stimulation of the ␤2AR leads to G s -mediated activation of adenylyl cyclase, resulting in the production of cAMP and subsequent downstream signaling events. Moreover, additional studies both in cultured cell lines and in vitro have demonstrated that, in response to agonist, the ␤2AR can undergo PKAdependent phosphorylation leading to activation of G i (a process referred to as G protein "switching"), thereby effectively changing the signaling specificity of the receptor (4).Cessation of agonist-activated ␤2AR-G s -mediated signaling occurs via recruitment of modulatory proteins, ␤-arrestins, to the cytoplasmic surface of the receptor, a process that is enhanced by receptor phosphorylation by G protein-coupled receptor kinases (GRKs) (5). ␤-arrestin binding physically pre...

Physical Interaction of Calmodulin with the 5-Hydroxytryptamine2C Receptor C-Terminus Is Essential for G Protein-independent, Arrestin-dependent Receptor Signaling

“…Although ␤-arrestin 2 was identified as the predominant ␤-arrestin isoform interacting with 5-HT 2C receptors (Marion et al, 2004), depletion of either ␤-arrestin 1 or ␤-arrestin 2 by RNA interference strongly reduced 5-HT 2C receptor-mediated ERK1,2 phosphorylation. These results are coherent with several reports indicating that both ␤-arrestin isoforms are required for G protein-independent ERK1,2 signaling mediated by various GPCRs such as ␤ 2 -adrenergic and parathyroid hormone type 1 receptors (Gesty-Palmer et al, 2006;Shenoy et al, 2006). Nonetheless, activation of ERK1,2 by other GPCRs, including angiotensin II type 1a and V2 receptors, is critically dependent on ␤-arrestin 2, whereas ␤-arrestin 1 has a minor influence or an antagonistic action on ERK1,2 signaling (Ahn et al, 2004;Ren et al, 2005), indicating that specificity of ␤-arrestin isoforms for ERK signaling is dependent on the GPCR.…”

Distinct conformational changes in β-arrestin report biased agonism at seven-transmembrane receptors

“…Although there are some indications of ␤-arrestin-biased ligands for ␤2AR (15,16), their weak efficacy for ␤-arrestin recruitment and signaling limits their usefulness in our BRET assay system. However, based on evolutionary trace analysis, a mutant ␤2AR (T68F-Y132G-Y219A, referred to as ␤2AR TYY ) has been designed (17) that, similar to the DRY/AAY mutant of AT1aR, does not couple to G proteins but exhibits only ␤-arrestin-mediated signaling (17). As shown in Fig.…”

Fenoterol, a β2-adrenoceptor agonist, inhibits LPS-induced membrane-bound CD14, TLR4/CD14 complex, and inflammatory cytokines production through β-arrestin-2 in THP-1 cell line

“…However, β-arrestins have been considered as novel non-G proteindependent signaling molecules and play functional roles in the regulation of a variety of signaling pathways and in the mediation of cross-talk between receptors [21][22][23] . For example, β-arrestin-2-dependent stabilization of cytosolic IκBα and inhibition of NF-κB activation following LPS stimulation are essential for rapid and sufficient production of NO in response to microbial attack [14] .…”