Received for publication April 26, 2004.
Revised September 9, 2004.
Accepted for publication September 10, 2004.

Preferential Interaction between the Dopamine D2 Receptor and Arrestin2 in Neostriatal Neurons

Abstract

Dopamine D2 receptor interactions with arrestins and arrestin-dependent internalization have been characterized using heterologously expressed D2 receptor and arrestins. The purpose of this study was to investigate D2 receptor interaction with endogenous arrestins. Arrestin2 and 3 in striatal homogenates bound to the third cytoplasmic loop of the D2 receptor, and purified arrestin2 and 3 bound to the second and third loops and C-terminus of the D2 receptor, in a GST pull-down assay. In NS20Y neuroblastoma cells expressing an enhanced green fluorescent protein-tagged D2 receptor (D2-EGFP), 2-hr D2 agonist stimulation enhanced the colocalization of D2-EGFP with endogenous arrestin2 and 3. These results suggest that the D2 receptor has the intrinsic ability to bind both non-visual arrestins. Agonist treatment of D2-EGFP NS20Y cells induced D2 receptor internalization (36-46%) that was maximal within 20 min, but that was prevented by siRNA-induced depletion of arrestin2 and 3. In neostriatal neurons, 2-hr agonist treatment selectively increased the colocalization of the endogenous D2 receptor with arrestin2, whereas receptor colocalization with arrestin3 was reduced. Agonist stimulation caused translocation of arrestin2, but not arrestin3, to the membrane in neurons, and selectively enhanced the co-immunoprecipitation of the D2 receptor and arrestin2. All three measures of receptor:arrestin interaction (colocalization, translocation, and coprecipitation) demonstrated selective agonist-induced interaction between the D2 receptor and arrestin2 in neurons.

Key words: Dopamine, Gi family, Sequestration/Internalization, GRKs, barrestins, Fluorescence techniques, Immunocytochemistry