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 both non-visual 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 non-receptor-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 non-visual arrestins perform the same function via distinct molecular mechanisms.

JNK3 Enzyme Binding to Arrestin-3 Differentially Affects the Recruitment of Upstream Mitogen-activated Protein (MAP) Kinase Kinases

“…MAPK scaffolds are extremely diverse, including kinase suppressor of Ras (KSR), JNK-interacting protein (JIP), paxillin involved in cell adhesion, MAPKK kinase (MEKKK1), etc. Non-visual arrestins were also reported to scaffold MAPK pathways leading to the activation of JNK3 (10, 19 -21, 23), ERK1/2 (11,37,44), and p38 (12,45). Most scaffolds are believed to use a simple tethering mechanism to facilitate the efficiency of signal transduction (38,39,42), although this model is rarely proved experimentally.…”

Silent Scaffolds

“…1, E, F, I, J, and K). Although agonist activation of ␤2AR can promote MAPK activation via G protein-mediated signaling (45), the binding of the biased agonist ICI118551, which acts as an inverse agonist for G protein coupling, selectively facilitates arrestin-mediated signaling to MAPKs (27,28,46). To test whether receptor binding plays any role in JNK3 activation, we transfected COS-7 cells with different forms of arrestin-3, JNK3, and ASK1, activated endogenous ␤2AR with the classical agonist isoproterenol or ICI118551, and measured the levels of phosphorylation of ERK1/2, which is known to be specifically activated by the receptor-bound arrestin (28,46) and of JNK3 in the same cells (Fig.…”

Arrestin-Dependent Activation of ERK and Src Family Kinases

“…Like ERK1/2, c-Raf1 prefers the receptorbound conformation, although the difference is less dramatic, while MEK1 binds equivalently to both free arrestin (Meng et al 2009;Coffa et al 2011b) and all three mutationally constrained conformations (Coffa et al 2011a). Perhaps significantly, cRaf1 and ERK1/2 binding to the microtubule-bound pool of arrestin may provide a mechanism for dampening basal ERK1/2 activity in the absence of receptor stimulation.…”