Mutation of Three Residues in the Third Intracellular Loop of the Dopamine D-2 Receptor Creates an Internalization-defective Receptor

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 receptorexpression. 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 aGprotein. Arrestin-dependent receptor internalization, which requires arrestin binding to beta 2-adaptin (the beta 2 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 over-expressed 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 beta 2-adaptin, suggesting that the mutation impairs a property of the internalization complex that has not yet been identified.