Scaffolding mechanism of arrestin-2 in the cRaf/MEK1/ERK signaling cascade

Abstract

Arrestins were initially identified for their role in homologous desensitization and internalization of G protein–coupled receptors. Receptor-bound arrestins also initiate signaling by interacting with other signaling proteins. Arrestins scaffold MAPK signaling cascades, MAPK kinase kinase (MAP3K), MAPK kinase (MAP2K), and MAPK. In particular, arrestins facilitate ERK1/2 activation by scaffolding ERK1/2 (MAPK), MEK1 (MAP2K), and Raf (MAPK3). However, the structural mechanism underlying this scaffolding remains unknown. Here, we investigated the mechanism of arrestin-2 scaffolding of cRaf, MEK1, and ERK2 using hydrogen/deuterium exchange–mass spectrometry, tryptophan-induced bimane fluorescence quenching, and NMR. We found that basal and active arrestin-2 interacted with cRaf, while only active arrestin-2 interacted with MEK1 and ERK2. The ATP binding status of MEK1 or ERK2 affected arrestin-2 binding; ATP-bound MEK1 interacted with arrestin-2, whereas only empty ERK2 bound arrestin-2. Analysis of the binding interfaces suggested that the relative positions of cRaf, MEK1, and ERK2 on arrestin-2 likely facilitate sequential phosphorylation in the signal transduction cascade.

Scaffolding of Mitogen-Activated Protein Kinase Signaling by β-Arrestins

“…Whether β-arrestin 1 needs to be activated by a phosphorylated receptor for cRaf binding remains controversial. Our results, with HDX-MS and in cell BRET assays, suggest that activated receptors are not required for β-arrestin 1 and cRaf interaction, while a recent study using a GST pull-down assay showed an increased β-arrestin 1 and cRaf interaction when V2Rpp, a phosphorylated C-terminal peptide of the V2 vasopressin receptor, was co-incubated [44,55]. Therefore, further studies are needed to fully understand whether receptor-mediated β-arrestin 1 activation is required for cRaf binding.…”

“…cRaf is composed of a Ras-binding domain (RBD), C1 domain (CRD), hinge region, and kinase domain (Figure 2C, left) [53,54], and K84 is located within the RBD. A recent hydrogen/deuterium exchange mass spectrometry (HDX-MS) study with purified β-arrestin 1 and cRaf RBD confirmed that the back loop of β-arrestin 1 (i.e., near R307) interacts with cRaf RBD (Figure 2D) [44]. It is interesting to note that the kinase activity of the Raf family is auto-inhibited by the N-terminal domains, including RBD and CRD, and the binding of Ras to RBD releases the N-terminal domain, leading to exposure of the kinase domain (Figure 2C, right) [53].…”

“…The molecular mechanism of β-arrestin-mediated scaffolding of the ERK1/2 signaling components is a long-standing mystery. Several studies have suggested the structural mechanism of scaffolding using biochemical and biophysical tools such as mutagenesis, peptide array, molecular simulation, and hydrogen/deuterium exchange mass spectrometry (HDX-MS) [40][41][42][43][44]. Although not completely understood, these studies provide insight into how β-arrestins scaffold ERK1/2 signaling components.…”

“…The binding interface between β-arrestin and MEK1 has been suggested in a few studies, and these studies have shown different results [40,41,43,44]. Meng et al suggested that residues 46-70 in the N-terminal region of MEK1 and D26 and D29 in the N-domain of β-arrestin 1 are critical for the interaction [40], and a molecular simulation study suggested that D29 and E35 of β-arrestin 1 interact with a region near E272 of MEK1 [43].…”

Follicle-Stimulating Hormone Induces Lipid Droplets via Gαi/o and β-Arrestin in an Endometrial Cancer Cell Line

“…In addition, many receptors recruit a member of the key GPCR scaffolding protein family, the arrestins/b-arrestins (the non-visual arrestins comprised of b-arrestin 1 and barrestin 2) following receptor activation and phosphorylation. The b-arrestins play numerous roles in regulating GPCR activity in part due to its ability to adopt multiple distinct conformations in complex with the receptor (7)(8)(9)(10)(11), although its ability to associate with other signal proteins, including G proteins, have also been reported (12)(13)(14)(15)(16)(17). Thus, in addition to classical roles in G protein-uncoupling and receptor internalization, b-arrestins can form signalosomes that interact with multiple partners and induce diverse downstream signaling pathways.…”