title: A single mutation in arrestin-2 prevents ERK1/2 activation by reducing c-Raf1 binding.
abstract: Arrestins regulate the signaling and trafficking of G protein-coupled receptors (GPCRs). GPCR complexes with both nonvisual arrestins channel signaling to G protein-independent pathways, one of which is the activation of extracellular signal regulated kinase 1/2 (ERK1/2). Here we used alanine-scanning mutagenesis of residues on the nonreceptor-binding surface conserved between arrestin-2 and arrestin-3. We show that an Arg307Ala mutation significantly reduced arrestin-2 binding to c-Raf1, whereas the binding of the mutant to active phosphorylated receptor and downstream kinases MEK1 and ERK2 was not affected. In contrast to wild-type arrestin-2, the Arg307Ala mutant failed to rescue arrestin-dependent ERK1/2 activation via β2-adrenergic receptor in arrestin-2/3 double knockout mouse embryonic fibroblasts. Thus, Arg307 plays a specific role in arrestin-2 binding to c-Raf1 and is indispensable in the productive scaffolding of c-Raf1-MEK1-ERK1/2 signaling cascade. Arg307Ala mutation specifically eliminates arrestin-2 signaling through ERK, which makes arrestin-2-Arg307Ala the first signaling-biased arrestin mutant constructed. In the crystal structure the side chain of homologous arrestin-3 residue Lys308 points in a different direction. Alanine substitution of Lys308 does not significantly affect c-Raf1 binding to arrestin-3 and its ability to promote ERK1/2 activation, suggesting that the two nonvisual arrestins perform the same function via distinct molecular mechanisms.
authors: Department of Pharmacology, Vanderbilt University, Nashville, Tennessee 37232, United States.
authors: Sergio
authors: S
authors: Coffa
authors: Maya
authors: M
authors: Breitman
authors: Benjamin W
authors: BW
authors: Spiller
authors: Vsevolod V
authors: VV
authors: Gurevich
mesh_terms: D019390:Arrestins / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D015153:Blotting, Western; D002460:Cell Line; D004789:Enzyme Activation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism*
chemical_list: D019390:Arrestins; D019908:Proto-Oncogene Proteins c-raf; D048049:Extracellular Signal-Regulated MAP Kinases