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

PLoS One. 2011;6(12):e28723. doi: 10.1371/journal.pone.0028723. Epub 2011 Dec 12.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

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

Substances

  • Arrestin
  • Arrestins
  • Ligands
  • Mutant Proteins
  • Receptors, Adrenergic, beta-2
  • arrestin3
  • beta-Arrestins
  • Proto-Oncogene Proteins c-raf
  • Mitogen-Activated Protein Kinase 1
  • Mitogen-Activated Protein Kinase 3
  • MAP Kinase Kinase 1
  • Map2k1 protein, mouse