Mutations in arrestin-3 differentially affect binding to neuropeptide Y receptor subtypes

Cell Signal. 2014 Jul;26(7):1523-31. doi: 10.1016/j.cellsig.2014.03.019. Epub 2014 Mar 29.


Based on the identification of residues that determine receptor selectivity in arrestins and the phylogenetic analysis of the arrestin (arr) family, we introduced fifteen mutations of receptor-discriminator residues in arr-3, which were identified previously using mutagenesis, in vitro binding, and BRET-based recruitment assay in intact cells. The effects of these mutations were tested using neuropeptide Y receptors Y1R and Y2R. NPY-elicited arr-3 recruitment to Y1R was not affected by these mutations, or even alanine substitution of all ten residues (arr-3-NCA), which prevented arr-3 binding to other receptors tested so far. However, NCA and two other mutations prevented agonist-independent arr-3 pre-docking to Y1R. In contrast, eight out of 15 mutations significantly reduced agonist-dependent arr-3 recruitment to Y2R. NCA eliminated arr-3 binding to active Y2R, whereas Tyr239Thr reduced it ~7-fold. Thus, manipulation of key residues on the receptor-binding surface generates arr-3 with high preference for Y1R over Y2R. Several mutations differentially affect arr-3 pre-docking and agonist-induced recruitment. Thus, arr-3 recruitment to the receptor involves several mechanistically distinct steps. Targeted mutagenesis can fine-tune arrestins directing them to specific receptors and particular activation states of the same receptor.

Keywords: Arrestins; Bioluminescence resonance energy transfer (BRET); GPCRs; Neuropeptide Y receptors; Protein engineering; Signal transduction.

Publication types

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

MeSH terms

  • Animals
  • Arrestins / genetics*
  • Arrestins / metabolism*
  • COS Cells
  • Cell Line
  • Chlorocebus aethiops
  • Mutation
  • Protein Binding / genetics
  • Receptors, Adrenergic, beta / metabolism
  • Receptors, Dopamine / metabolism
  • Receptors, Muscarinic / metabolism
  • Receptors, Neuropeptide Y / metabolism*


  • Arrestins
  • Receptors, Adrenergic, beta
  • Receptors, Dopamine
  • Receptors, Muscarinic
  • Receptors, Neuropeptide Y
  • arrestin3
  • neuropeptide Y-Y1 receptor
  • neuropeptide Y2 receptor