title: Mutation of three residues in the third intracellular loop of the dopamine D2 receptor creates an internalization-defective receptor.
abstract: Arrestins mediate desensitization and internalization of G protein-coupled receptors and also direct receptor signaling toward heterotrimeric G protein-independent signaling pathways. We previously identified a four-residue segment (residues 212-215) of the dopamine D2 receptor that is necessary for arrestin binding in an in vitro heterologous expression system but that also impairs receptor expression. We now describe the characterization of additional mutations at that arrestin binding site in the third intracellular loop. Mutating two (residues 214 and 215) or three (residues 213-215) of the four residues to alanine partially decreased agonist-induced recruitment of arrestin3 without altering activation of a G protein. Arrestin-dependent receptor internalization, which requires arrestin binding to β2-adaptin (the β2 subunit of the clathrin-associated adaptor protein AP2) and clathrin, was disproportionately affected by the three-residue mutation, with no agonist-induced internalization observed even in the presence of overexpressed arrestin or G protein-coupled receptor kinase 2. The disjunction between arrestin recruitment and internalization could not be explained by alterations in the time course of the receptor-arrestin interaction, the recruitment of G protein-coupled receptor kinase 2, or the receptor-induced interaction between arrestin and β2-adaptin, suggesting that the mutation impairs a property of the internalization complex that has not yet been identified.
authors.affiliation: the Department of Behavioral Neuroscience, Oregon Health & Science University, Portland, Oregon 97239.
authors.forename: Cecilea C
authors.initials: CC
authors.lastname: Clayton
authors.affiliation: the Departments of Psychiatry and Pharmacology, Columbia University College of Physicians and Surgeons, New York, New York 10032, the Division of Molecular Therapeutics, New York State Psychiatric Institute, New York, New York 10032, and.
authors.forename: Prashant
authors.initials: P
authors.lastname: Donthamsetti
authors.affiliation: the Department of Pharmacology and Toxicology, Medical College of Georgia, Georgia Regents University, Augusta, Georgia 30912.
authors.forename: Nevin A
authors.initials: NA
authors.lastname: Lambert
authors.affiliation: the Departments of Psychiatry and Pharmacology, Columbia University College of Physicians and Surgeons, New York, New York 10032, the Division of Molecular Therapeutics, New York State Psychiatric Institute, New York, New York 10032, and jaj2@columbia.edu.
authors.forename: Jonathan A
authors.initials: JA
authors.lastname: Javitch
authors.affiliation: the Department of Behavioral Neuroscience, Oregon Health & Science University, Portland, Oregon 97239, From the Research Service, Department of Veterans Affairs Medical Center, Portland, Oregon 97239, nevek@ohsu.edu.
authors.forename: Kim A
authors.initials: KA
authors.lastname: Neve
mesh_terms: D033966:Adaptor Protein Complex beta Subunits / Q000235:genetics / Q000378:metabolism; D054769:G-Protein-Coupled Receptor Kinase 2 / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D009154:Mutation; D017433:Protein Structure, Secondary; D021381:Protein Transport / Q000502:physiology; D017448:Receptors, Dopamine D2 / Q000235:genetics / Q000378:metabolism*
chemical_list: D033966:Adaptor Protein Complex beta Subunits; C581293:DRD2 protein, human; D017448:Receptors, Dopamine D2; D054769:G-Protein-Coupled Receptor Kinase 2
keywords: Adenylate Cyclase (Adenylyl Cyclase); Arrestin; Bioluminescence Resonance Energy Transfer (BRET); Dopamine Receptor; G Protein; beta2-Adaptin