the effect of arrestin conformation on the recruitment of c-raf1, mek1, and erk12 activationarrestin构象的影响在c-raf1招聘,mek1,erk12激活.pdfVIP

The Effect of Arrestin Conformation on the Recruitment of c-Raf1, MEK1, and ERK1/2 Activation 1 1 1¤ 2 1 2 Sergio Coffa , Maya Breitman , Susan M. Hanson , Kari Callaway , Seunghyi Kook , Kevin N. Dalby , Vsevolod V. Gurevich1* 1 Department of Pharmacology, Vanderbilt University, Nashville, Tennessee, United States of America, 2 Division of Medicinal Chemistry, The University of Texas at Austin, Austin, Texas, United States of America 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 b2-adrenergic receptor (b2AR) 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-D7 mimicking microtubule- bound state and arrestin-3A, a mimic of the receptor-bound conformation. Both rescue arrestin-mediated ERK1/2/activation in a