How Does Arrestin Assemble MAPKs into a Signaling Complex?

Abstract

Arrestins bind active phosphorylated G protein-coupled receptors, precluding G protein activation and channeling signaling to alternative pathways. Arrestins also function as mitogen-activated protein kinase (MAPK) scaffolds, bringing together three components of MAPK signaling modules. Here we have demonstrated that all four vertebrate arrestins interact with JNK3, MKK4, and ASK1, but only arrestin3 facilitates JNK3 activation. Thus, the functional specificity of arrestins is not determined by differential binding of the kinases. Using receptor binding-impaired mutant, we have shown that free arrestin3 readily promotes JNK3 phosphorylation. We identified key arrestin-binding elements in JNK3 and ASK1 and investigated the molecular interactions of arrestin2 and arrestin3 and their individual domains with the components of the two MAPK cascades, ASK1-MKK4-JNK3 and c-Raf-1-MEK1-ERK2. We found that both arrestin domains interact with all six kinases. These findings shed new light on the mechanism of arrestin-mediated MAPK activation and the spatial arrangement of the three kinases on arrestin molecule.Arrestins are multifunctional regulators of cell signaling (1, 2). Arrestins, which bind active phosphorylated G protein-coupled receptors (GPCRs), 2 which play a major role in receptor desensitization and internalization (3, 4). With the identification of numerous non-receptor binding partners, the classical paradigm of arrestin function has been expanded, implicating arrestins in mitogen-activated protein kinase (MAPK) activation, protein ubiquitination, chemotaxis, apoptosis, and other cellular functions (2, 5-11).The first indication that arrestins function as signaling adapters came from the studies of arrestin-dependent c-Src recruitment to the receptors, which results in the activation of extracellular signal-regulated kinases (ERK1/2) (10, 12, 13).Subsequently, arrestin2 and arrestin3 in complex with different receptors were reported to scaffold JNK3 (9), ERK1/2 (8, 14), and p38 (15, 16) activation cascades. Although arrestins play an important role in regulating different MAPK pathways, the mechanism of arrestin-dependent assembly of MAP kinases into a signaling complex remains largely unexplored. Existing models have limited predictive value. For example, the idea that JNK3 is activated solely by arrestin3 because this arrestin subtype has unique ability to bind JNK3 (9, 17) was not supported by further experimentation (18 -20). Similarly, the hypothesis that only receptor-bound arrestins interact with MAP kinases (8, 9) was not confirmed (17-20).Here we addressed several key mechanistic issues in arrestindependent MAPK signaling. First, we show that the scaffolding function is not limited to receptor-bound arrestin; free arrestin3 facilitates ASK1-mediated JNK3 activation, indicating that arrestins are not exclusively receptor-regulated adapters as thought previously. Second, we show that all four mammalian arrestins bind each component of the JNK3 cascade with comparable affinity, demonstrating that bindin...

Arrestin-3 Binds c-Jun N-terminal Kinase 1 (JNK1) and JNK2 and Facilitates the Activation of These Ubiquitous JNK Isoforms in Cells via Scaffolding

“…Collectively, the data in Figs. 3-5 show that arrestin-3 scaffolds ASK1-MKK4/7-JNK1/2 signaling modules, similar to the reported scaffolding of ASK1-MKK4/7-JNK3␣2 modules (6,8,9,11).…”

“…Cells-The binding of arrestin-3 to JNK3 was inferred from co-immunoprecipitation of these two co-expressed proteins from intact cells (6,8,9,11) as well as from the ability of arrestin-3 to remove JNK3 from the nucleus (26,27). Only recently was this interaction unambiguously demonstrated using two purified proteins in vitro in the absence of any potential helpers and/or intermediaries (10,12).…”

“…Subsequent work showed that arrestin-3 promotes JNK3␣2 phosphorylation independently of GPCRs (7)(8)(9)11), in agreement with the finding that conformationally biased arrestin mutants, both the pre-activated 3A form with enhanced propensity to bind GPCRs (43-47) and the one "frozen" in basal-like conformation with impaired ability to bind receptors (11,48,49), interact with JNK3␣2 similarly to WT arrestin-3 (26,49). Arrestin-3 was also shown to co-immunoprecipitate with MKK4 essentially as efficiently as with ASK1 and JNK3␣2 (8,9,11). The construction of mutants deficient in either GPCR binding or the ability to promote JNK activation showed that these functions do not correlate (11).…”

“…The ability of overexpressed arrestin-3 5 to promote the activation of exogenous c-Jun N-terminal kinase 3 (JNK3) in cells was demonstrated more than a decade ago (6). Numerous subsequent studies showed that this function does not depend on arrestin-3 association with GPCRs (7)(8)(9)(10)(11)(12). In all these studies JNK3, which is predominantly expressed in neurons, heart, and testes (13), was either exogenously expressed in cultured cells that normally do not contain this isoform (6 -9, 11, 12) or used in purified form for in vitro reconstitution experiments (10,12).…”

Evidence that Behavioral Phenotypes of Morphine in β-arr2−/− Mice Are Due to the Unmasking of JNK Signaling

“…In the absence of b-arrestin 2, these agonists recruit b-arrestin 1, PKC is not recruited and the JNK cascade is not significantly activated. Arrestin and JNK regulation of mu receptor function N Mittal et al b-Arrestin 2 has been shown to bind with JNK3, but not JNK1 or 2, to control GPCR activation of the JNK cascade (McDonald et al, 2000;Miller et al, 2001;Song et al, 2006Song et al, , 2009Willoughby and Collins, 2005). Removing b-arrestin 2 may therefore alter both the location and control of JNK3 to allow sustained JNK activation in b-arr2À/À mice.…”