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

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

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Department of Pharmacology, Vanderbilt University, Nashville, TN 37232, USA.
Institute of Biochemistry, Faculty of Biosciences, Pharmacy and Psychology, Leipzig University, Brüderstraße 34, D-04103 Leipzig, Germany.
Department of Pharmacology, Vanderbilt University, Nashville, TN 37232, USA. Electronic address:


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.


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

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