title: Role of receptor-attached phosphates in binding of visual and non-visual arrestins to G protein-coupled receptors.
abstract: Arrestins are a small family of proteins that regulate G protein-coupled receptors (GPCRs). Arrestins specifically bind to phosphorylated active receptors, terminating G protein coupling, targeting receptors to endocytic vesicles, and initiating G protein-independent signaling. The interaction of rhodopsin-attached phosphates with Lys-14 and Lys-15 in β-strand I was shown to disrupt the interaction of α-helix I, β-strand I, and the C-tail of visual arrestin-1, facilitating its transition into an active receptor-binding state. Here we tested the role of conserved lysines in homologous positions of non-visual arrestins by generating K2A mutants in which both lysines were replaced with alanines. K2A mutations in arrestin-1, -2, and -3 significantly reduced their binding to active phosphorhodopsin in vitro. The interaction of arrestins with several GPCRs in intact cells was monitored by a bioluminescence resonance energy transfer (BRET)-based assay. BRET data confirmed the role of Lys-14 and Lys-15 in arrestin-1 binding to non-cognate receptors. However, this was not the case for non-visual arrestins in which the K2A mutations had little effect on net BRET(max) values for the M2 muscarinic acetylcholine (M2R), β(2)-adrenergic (β(2)AR), or D2 dopamine receptors. Moreover, a phosphorylation-deficient mutant of M2R interacted with wild type non-visual arrestins normally, whereas phosphorylation-deficient β(2)AR mutants bound arrestins at 20-50% of the level of wild type β(2)AR. Thus, the contribution of receptor-attached phosphates to arrestin binding varies depending on the receptor-arrestin pair. Although arrestin-1 always depends on receptor phosphorylation, its role in the recruitment of arrestin-2 and -3 is much greater in the case of β(2)AR than M2R and D2 dopamine receptor.
authors.affiliation: Department of Pharmacology, Vanderbilt University, Nashville, Tennessee 37232, USA.
authors.forename: Luis E
authors.initials: LE
authors.lastname: Gimenez
authors.forename: Seunghyi
authors.initials: S
authors.lastname: Kook
authors.forename: Sergey A
authors.initials: SA
authors.lastname: Vishnivetskiy
authors.forename: M Rafiuddin
authors.initials: MR
authors.lastname: Ahmed
authors.forename: Eugenia V
authors.initials: EV
authors.lastname: Gurevich
authors.forename: Vsevolod V
authors.initials: VV
authors.lastname: Gurevich
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D019390:Arrestins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D006801:Humans; D010710:Phosphates / Q000378:metabolism*; D011485:Protein Binding; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism*; D012243:Rhodopsin / Q000235:genetics / Q000378:metabolism
chemical_list: D019390:Arrestins; D010710:Phosphates; D043562:Receptors, G-Protein-Coupled; D012243:Rhodopsin