title: The sustainability of interactions between the orexin-1 receptor and beta-arrestin-2 is defined by a single C-terminal cluster of hydroxy amino acids and modulates the kinetics of ERK MAPK regulation.
abstract: The orexin-1 receptor interacts with beta-arrestin-2 in an agonist-dependent manner. In HEK-293T cells, these two proteins became co-internalized into acidic endosomes. Truncations from the C-terminal tail did not prevent agonist-induced internalization of the orexin-1 receptor or alter the pathway of internalization, although such mutants failed to interact with beta-arrestin-2 in a sustained manner or produce its co-internalization. Mutation of a cluster of three threonine and one serine residue at the extreme C-terminus of the receptor greatly reduced interaction and abolished co-internalization of beta-arrestin-2-GFP (green fluorescent protein). Despite the weak interactions of this C-terminally mutated form of the receptor with beta-arrestin-2, studies in wild-type and beta-arrestin-deficient mouse embryo fibroblasts confirmed that agonist-induced internalization of this mutant required expression of a beta-arrestin. Although without effect on agonist-mediated elevation of intracellular Ca2+ levels, the C-terminally mutated form of the orexin-1 receptor was unable to sustain phosphorylation of the MAPKs (mitogen-activated protein kinases) ERK1 and ERK2 (extracellular-signal-regulated kinases 1 and 2) to the same extent as the wild-type receptor. These studies indicate that a single cluster of hydroxy amino acids within the C-terminal seven amino acids of the orexin-1 receptor determine the sustainability of interaction with beta-arrestin-2, and indicate an important role of beta-arrestin scaffolding in defining the kinetics of orexin-1 receptor-mediated ERK MAPK activation.
authors.affiliation: Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, UK.
authors.forename: Sandra
authors.initials: S
authors.lastname: Milasta
authors.forename: Nicholas A
authors.initials: NA
authors.lastname: Evans
authors.forename: Laura
authors.initials: L
authors.lastname: Ormiston
authors.forename: Shelagh
authors.initials: S
authors.lastname: Wilson
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Lefkowitz
authors.forename: Graeme
authors.initials: G
authors.lastname: Milligan
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D019390:Arrestins / Q000737:chemistry*; D002460:Cell Line; D004789:Enzyme Activation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000737:chemistry / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D064446:Orexin Receptors; D017354:Point Mutation; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry / Q000502:physiology; D018013:Receptors, Neuropeptide / Q000737:chemistry* / Q000235:genetics; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins
chemical_list: C000605254:ARRB2 protein, human; C000605258:Arrb2 protein, mouse; D019390:Arrestins; D064446:Orexin Receptors; D043562:Receptors, G-Protein-Coupled; D018013:Receptors, Neuropeptide; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins; D048049:Extracellular Signal-Regulated MAP Kinases