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.