Mutation of three residues in the third intracellular loop of the dopamine D2 receptor creates an internalization-defective receptor

Cecilea C. Clayton, Prashant Donthamsetti, Nevin A. Lambert, Jonathan A. Javitch, Kim A. Neve

Research output: Contribution to journalArticlepeer-review

15 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)33663-33675
Number of pages13
JournalJournal of Biological Chemistry
Volume289
Issue number48
DOIs
StatePublished - Nov 28 2014

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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