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The GPCR-β-arrestin complex allosterically activates C-Raf by binding its amino terminus
- Source :
- The Journal of Biological Chemistry
- Publication Year :
- 2021
-
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.
- Subjects :
- V2Rpp, V2 vasopressin receptor phosphopeptide
Accelerated Communication
MBP, myelin basic protein
SEC, size-exclusion chromatography
Allosteric regulation
allosteric activation
ERK, extracellular signal–regulated kinase
Biochemistry
Receptors, G-Protein-Coupled
MEK, Raf–MAPK extracellular signal–regulated kinase
Allosteric Regulation
Heterotrimeric G protein
GST, glutathione-S-transferase
Humans
c-Raf
Phosphorylation
Protein kinase A
RBD, Ras-binding domain
Molecular Biology
βarr, β-arrestin
beta-Arrestins
CRD, cysteine-rich domain
G protein-coupled receptor
MSP, membrane scaffold protein
biology
Chemistry
arrestin
G protein–coupled receptors
M2V2R, M2 muscarinic receptor with phosphorylated tail of V2 vasopressin receptor
Cell Biology
Cell biology
Proto-Oncogene Proteins c-raf
C-Raf
CR, conserved region
Mitogen-activated protein kinase
biology.protein
GPCR, G protein–coupled receptor
Signal transduction
signal transduction
MAPK, mitogen-activated protein kinase
Proto-oncogene tyrosine-protein kinase Src
Protein Binding
Subjects
Details
- ISSN :
- 1083351X
- Volume :
- 297
- Issue :
- 6
- Database :
- OpenAIRE
- Journal :
- The Journal of biological chemistry
- Accession number :
- edsair.doi.dedup.....cbaf4416497c7699797de531ddaae175