The effect of arrestin conformation on the recruitment of c-Raf1, MEK1, and ERK1/2 activation.

Coffa S, Breitman M, Hanson SM, Callaway K, Kook S, Dalby KN, Gurevich VV
PLoS One. 2011 6 (12): e28723

PMID: 22174878 · PMCID: PMC3236217 · DOI:10.1371/journal.pone.0028723

Arrestins are multifunctional signaling adaptors originally discovered as proteins that "arrest" G protein activation by G protein-coupled receptors (GPCRs). Recently GPCR complexes with arrestins have been proposed to activate G protein-independent signaling pathways. In particular, arrestin-dependent activation of extracellular signal-regulated kinase 1/2 (ERK1/2) has been demonstrated. Here we have performed in vitro binding assays with pure proteins to demonstrate for the first time that ERK2 directly binds free arrestin-2 and -3, as well as receptor-associated arrestins-1, -2, and -3. In addition, we showed that in COS-7 cells arrestin-2 and -3 association with β(2)-adrenergic receptor (β2AR) significantly enhanced ERK2 binding, but showed little effect on arrestin interactions with the upstream kinases c-Raf1 and MEK1. Arrestins exist in three conformational states: free, receptor-bound, and microtubule-associated. Using conformationally biased arrestin mutants we found that ERK2 preferentially binds two of these: the "constitutively inactive" arrestin-Δ7 mimicking microtubule-bound state and arrestin-3A, a mimic of the receptor-bound conformation. Both rescue arrestin-mediated ERK1/2/activation in arrestin-2/3 double knockout fibroblasts. We also found that arrestin-2-c-Raf1 interaction is enhanced by receptor binding, whereas arrestin-3-c-Raf1 interaction is not.

MeSH Terms (25)

Animals Arrestin Arrestins beta-Arrestins Cattle Cercopithecus aethiops COS Cells Embryo, Mammalian Enzyme Activation Fibroblasts HEK293 Cells Humans Ligands MAP Kinase Kinase 1 Mice Mice, Knockout Mitogen-Activated Protein Kinase 1 Mitogen-Activated Protein Kinase 3 Mutant Proteins Phosphorylation Protein Binding Protein Conformation Proto-Oncogene Proteins c-raf Receptors, Adrenergic, beta-2 Structure-Activity Relationship

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