The Effect of Arrestin Conformation on the Recruitment of c-Raf1, MEK1, and ERK1/2 Activation




Coffa, Sergio
Breitman, Maya
Hanson, Susan M.
Callaway, Kari
Kook, Seunghyi
Dalby, Kevin N.
Gurevich, Vsevolod V.

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Public Library of Science


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 β2-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.


Sergio Coffa is with Vanderbilt University; Maya Breitman is with Vanderbilt University; Susan M. Hanson is with Vanderbilt University; Seunghyi Kook is with Vanderbilt University; Vsevolod V. Gurevich is with Vanderbilt University; Kari Callaway is with UT Austin; Kevin N. Dalby is with UT Austin.

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Coffa S, Breitman M, Hanson SM, Callaway K, Kook S, et al. (2011) The Effect of Arrestin Conformation on the Recruitment of c-Raf1, MEK1, and ERK1/2 Activation. PLoS ONE 6(12): e28723. doi:10.1371/journal.pone.0028723