title: Mutations in arrestin-3 differentially affect binding to neuropeptide Y receptor subtypes.
abstract: 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.
authors.affiliation: Department of Pharmacology, Vanderbilt University, Nashville, TN 37232, USA.
authors.forename: Luis E
authors.initials: LE
authors.lastname: Gimenez
authors.affiliation: Institute of Biochemistry, Faculty of Biosciences, Pharmacy and Psychology, Leipzig University, Brüderstraße 34, D-04103 Leipzig, Germany.
authors.forename: Stefanie
authors.initials: S
authors.lastname: Babilon
authors.affiliation: Institute of Biochemistry, Faculty of Biosciences, Pharmacy and Psychology, Leipzig University, Brüderstraße 34, D-04103 Leipzig, Germany.
authors.forename: Lizzy
authors.initials: L
authors.lastname: Wanka
authors.affiliation: Institute of Biochemistry, Faculty of Biosciences, Pharmacy and Psychology, Leipzig University, Brüderstraße 34, D-04103 Leipzig, Germany.
authors.forename: Annette G
authors.initials: AG
authors.lastname: Beck-Sickinger
authors.affiliation: Department of Pharmacology, Vanderbilt University, Nashville, TN 37232, USA. Electronic address: vsevolod.gurevich@vanderbilt.edu.
authors.forename: Vsevolod V
authors.initials: VV
authors.lastname: Gurevich
mesh_terms: D000818:Animals; D019390:Arrestins / Q000235:genetics* / Q000378:metabolism*; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D009154:Mutation; D011485:Protein Binding / Q000235:genetics; D011943:Receptors, Adrenergic, beta / Q000378:metabolism; D011954:Receptors, Dopamine / Q000378:metabolism; D011976:Receptors, Muscarinic / Q000378:metabolism; D017476:Receptors, Neuropeptide Y / Q000378:metabolism*
chemical_list: D019390:Arrestins; D011943:Receptors, Adrenergic, beta; D011954:Receptors, Dopamine; D011976:Receptors, Muscarinic; D017476:Receptors, Neuropeptide Y; C082333:arrestin3; C097212:neuropeptide Y-Y1 receptor; C095889:neuropeptide Y2 receptor
keywords: Arrestins; Bioluminescence resonance energy transfer (BRET); GPCRs; Neuropeptide Y receptors; Protein engineering; Signal transduction