Constitutive ERK1/2 Activation by a Chimeric Neurokinin 1 Receptor-β-Arrestin1 Fusion Protein

Abstract

The ␤-arrestins, a small family of G protein-coupled receptor (GPCR)-binding proteins involved in receptor desensitization, have been shown to bind extracellular signal-regulated kinases 1 and 2 (ERK1/2) and function as scaffolds for GPCR-stimulated ERK1/2 activation. To better understand the mechanism of ␤-arrestin-mediated ERK1/2 activation, we compared ERK1/2 activation by the wild-type neurokinin 1 (NK1) receptor with a chimeric NK1 receptor having ␤-arrestin1 fused to the receptor C terminus (NK1-␤Arr1). The NK1 receptor couples to both G s and G q/11 , resides on the plasma membrane, and mediates rapid ERK1/2 activation and nuclear translocation in response to neurokinin A. In contrast, NK1-␤Arr1 is a G protein-uncoupled "constitutively desensitized" receptor that resides almost entirely in an intracellular endosomal compartment. Despite its inability to respond to neurokinin A, we found that NK1-␤Arr1 expression caused robust constitutive activation of cytosolic ERK1/2 and that endogenous Raf, MEK1/2, and ERK1/2 coprecipitated in a complex with NK1-␤Arr1. While agonist-dependent ERK1/2 activation by the NK1 receptor was independent of protein kinase A (PKA) or PKC activity, NK1-␤Arr1-mediated ERK1/2 activation was completely inhibited when basal PKA and PKC activity were blocked. In addition, the rate of ERK1/2 dephosphorylation was slowed in NK1-␤Arr1-expressing cells, suggesting that ␤-arrestinbound ERK1/2 is protected from mitogen-activated protein kinase phosphatase activity. These data suggest that ␤-arrestin binding to GPCRs nucleates the formation of a stable "signalsome" that functions as a passive scaffold for the ERK1/2 cascade while confining ERK1/2 activity to an extranuclear compartment.

Ubiquitination of β-Arrestin Links Seven-transmembrane Receptor Endocytosis and ERK Activation

“…Moreover, a subsequent stable interaction of receptor and ␤-arrestin is required for sustaining and targeting this activity to subcellular compartments. Interestingly, previous studies have shown that the membrane recruitment of ␤-arrestin2 itself can lead to ERK signaling and that a 7TMR-␤-arrestin1 chimeric protein can act as a constitutive signalosome (44,45). Our findings indicate that ␤-arrestin ubiquitination controls not only receptor trafficking but the nature, stability, and subcellular localization of active ERK signals.…”

Manifold roles of β-arrestins in GPCR signaling elucidated with siRNA and CRISPR/Cas9

“…In cells in which basal Ras signaling is high, simple βArr1/2-dependent assembly of the cRaf1-MEK1/2-ERK1/2 complex on a membrane surface may be sufficient to exceed the pathway activation threshold. Indeed, GPCR-independent recruitment of cyclophilin FRB domain-tagged βArr2 to a myristolyated-cyclophilin FKBP domain expressed on the plasma membrane is sufficient to activate ERK1/2, as is expression of a constitutively internalized neurokinin NK1 receptor-βArr1 chimera (28,87).…”

“…As a result, recruitment of βArr1/2 to activated GPCRs promotes assembly of the cRaf1-MEK1/2-ERK1/2 complex, supporting activation of ERK1/2 and its retention in cytosolic GPCR-arrestin “signalsome” complexes (19,24). In their role as signaling scaffolds, βArr1/2 affect the kinetics of GPCR-stimulated ERK1/2 activation, favoring prolonged activation, because βArr1/2-bound ERK1/2 is protected from rapid dephosphorylation by nuclear and cytosolic MAPK phosphatases (25–28), and determining its function, because the spatial constraints imposed on arrestin-bound ERK1/2 favor phosphorylation of cytosolic substrates but inhibit its nuclear functions. As a result, β-arrestin–bound ERK1/2 is implicated in the regulation of GPCR internalization and trafficking (29,30), cytoskeletal rearrangement and chemotaxis (31,32), matrix metalloproteinase–dependent ectodomain shedding (33–35), and protein synthesis (36,37), but dampens Elk-1 dependent transcription (38,39).…”

Posttranslation Modification of G Protein-Coupled Receptor in Relationship to Biased Agonism

“…Binding of β-arrestin to receptor complex uncouples Gαi2, which desensitizes Gα2-mediated signaling and induces the translocation of OPRM1 out of lipid raft microdomains (Lefkowitz & Shenoy, 2005; Lefkowitz & Whalen, 2004; Zheng, Chu, et al, 2008). Binding of β-arrestin to receptor complex also initiates the signaling transduction through β-arrestin-pathway, such as the β-arrestin-mediated ERK phosphorylation (Caunt et al, 2006; Jafri et al, 2006; Wang, Lu, Zhao, & Limbird, 2006). As β-arrestin can interact with phosphorylated ERK and translocate into the nucleus (Ma & Pei, 2007), the Elk-1 in nucleus may be activated (Janknecht, Zinck, Ernst, & Nordheim, 1994).…”