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

• Published in: PloS one Vol. 6; no. 12; p. e28723
• Main Authors: Coffa, Sergio, Breitman, Maya, Hanson, Susan M, Callaway, Kari, Kook, Seunghyi, Dalby, Kevin N, Gurevich, Vsevolod V
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 12.12.2011; Public Library of Science (PLoS)
• Subjects: Activation; Adapters; Adaptor proteins; Adrenergic receptors; Animals; Arrestin; Arrestin - chemistry; Arrestin - metabolism; Arrestins - chemistry; Arrestins - metabolism; beta-Arrestins; Binding; Biochemistry; Biology; Cattle; Chlorocebus aethiops; COS Cells; Embryo, Mammalian - cytology; Enzyme Activation; Extracellular signal-regulated kinase; Fibroblasts; Fibroblasts - enzymology; G protein-coupled receptors; G proteins; HEK293 Cells; Humans; Kinases; Ligands; MAP Kinase Kinase 1 - metabolism; Mice; Mice, Knockout; Mimicry; Mitogen-Activated Protein Kinase 1 - metabolism; Mitogen-Activated Protein Kinase 3 - metabolism; Mutagenesis; Mutant Proteins - chemistry; Mutant Proteins - metabolism; Mutants; Pharmaceutical sciences; Pharmacology; Phosphorylation; Phosphotransferases; Photoreceptors; Protein Binding; Protein Conformation; Proteins; Proto-Oncogene Proteins c-raf - metabolism; Receptors; Receptors, Adrenergic, beta-2 - metabolism; Recruitment; Signaling; Structure-Activity Relationship; United States > US; Texas; Tennessee; Nashville Tennessee
• DOI: 10.1371/journal.pone.0028723
• ISSN: 1932-6203

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.