title: The effect of arrestin conformation on the recruitment of c-Raf1, MEK1, and ERK1/2 activation.
abstract: 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.
authors.affiliation: Department of Pharmacology, Vanderbilt University, Nashville, Tennessee, United States of America.
authors.forename: Sergio
authors.initials: S
authors.lastname: Coffa
authors.forename: Maya
authors.initials: M
authors.lastname: Breitman
authors.forename: Susan M
authors.initials: SM
authors.lastname: Hanson
authors.forename: Kari
authors.initials: K
authors.lastname: Callaway
authors.forename: Seunghyi
authors.initials: S
authors.lastname: Kook
authors.forename: Kevin N
authors.initials: KN
authors.lastname: Dalby
authors.forename: Vsevolod V
authors.initials: VV
authors.lastname: Gurevich
mesh_terms: D000818:Animals; D019393:Arrestin / Q000737:chemistry* / Q000378:metabolism*; D019390:Arrestins / Q000737:chemistry / Q000378:metabolism; D019556:COS Cells; D002417:Cattle; D002522:Chlorocebus aethiops; D004622:Embryo, Mammalian / Q000166:cytology; D004789:Enzyme Activation; D005347:Fibroblasts / Q000201:enzymology; D057809:HEK293 Cells; D006801:Humans; D008024:Ligands; D048369:MAP Kinase Kinase 1 / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism*; D018343:Receptors, Adrenergic, beta-2 / Q000378:metabolism; D013329:Structure-Activity Relationship; D000071557:beta-Arrestins
chemical_list: D019393:Arrestin; D019390:Arrestins; D008024:Ligands; D050505:Mutant Proteins; D018343:Receptors, Adrenergic, beta-2; C082333:arrestin3; D000071557:beta-Arrestins; D019908:Proto-Oncogene Proteins c-raf; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D048369:MAP Kinase Kinase 1; C482127:Map2k1 protein, mouse