The GPCR–β-arrestin complex allosterically activates C-Raf by binding its amino terminus

Abstract

G protein–coupled receptors (GPCRs) convert external stimuli into cellular signals through heterotrimeric guanine nucleotide-binding proteins (G-proteins) and β-arrestins (βarrs). In a βarr-dependent signaling pathway, βarrs link GPCRs to various downstream signaling partners, such as the Raf–mitogen-activated protein kinase extracellular signal–regulated kinase–extracellular signal-regulated kinase cascade. Agonist-stimulated GPCR–βarr complexes have been shown to interact with C-Raf and are thought to initiate the mitogen-activated protein kinase pathway through simple tethering of these signaling partners. However, recent evidence shows that in addition to canonical scaffolding functions, βarrs can allosterically activate downstream targets, such as the nonreceptor tyrosine kinase Src. Here, we demonstrate the direct allosteric activation of C-Raf by GPCR–βarr1 complexes in vitro . Furthermore, we show that βarr1 in complex with a synthetic phosphopeptide mimicking the human V2 vasopressin receptor tail that binds and functionally activates βarrs also allosterically activates C-Raf. We reveal that the interaction between the phosphorylated GPCR C terminus and βarr1 is necessary and sufficient for C-Raf activation. Interestingly, the interaction between βarr1 and C-Raf was considerably reduced in the presence of excess activated H-Ras, a small GTPase known to activate C-Raf, suggesting that H-Ras and βarr1 bind to the same region on C-Raf. Furthermore, we found that βarr1 interacts with the Ras-binding domain of C-Raf. Taken together, these data suggest that in addition to canonical scaffolding functions, GPCR–βarr complexes directly allosterically activate C-Raf by binding to its amino terminus. This work provides novel insights into how βarrs regulate effector molecules to activate downstream signaling pathways.

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.…”

“…The observation that β-arrestin 1 interacts with the RBD of cRaf prompted the hypothesis that β-arrestin 1 binding would release the N-terminal domains from the kinase domain, resulting in kinase domain activation (Figure 2D). This hypothesis was confirmed by a recent study in which the kinase activity of cRaf increased upon β-arrestin 1 binding [55]. Whether β-arrestin 1 needs to be activated by a phosphorylated receptor for cRaf binding remains controversial.…”

β−Arrestins: Structure, Function, Physiology, and Pharmacological Perspectives

“…The molecular mechanisms underlying β-arrestin scaffolding of the Raf1-MEK1-ERK1/2 cascade (Qu et al, 2021) and likely differences between βarr1 and βarr2 in mediating this process (Perry-Hauser et al, 2022a) are currently the subject of intense investigation. Interestingly, recent data suggest that different GPCR/βarr1 complexes, besides acting as scaffolding proteins, can directly activate, in an allosteric fashion, the protein kinases Src and C-Raf (Pakharukova et al, 2020;Zang et al, 2021).…”

Targeting CRAF kinase in anti-cancer therapy: progress and opportunities

“…The activation of PAK1 and CHK2 is triggered through the p.S338 site of CRAF via a mechanism independent of its kinase activity [ 197 ]. It was reported that G protein-coupled receptors (GPCRs) activate CRAF through guanine nucleotide-binding G-proteins and the β-arrestins signaling pathway [ 198 ]. Interestingly, β-arrestins specifically bind to the Ras-binding domain of CRAF to balance CRAF activation due to stimuli from G-protein coupled receptors (GPCRs) and the EGFR-RAS signaling cascade.…”