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
arrestin3 in striatal homogenates bound to the third cytoplasmic loop of the D2 receptor, and purified arrestin2 and
arrestin3 bound to the second and third loops and C terminus of the D2 receptor, in a
glutathione S-transferase pull-down assay. In NS20Y
neuroblastoma cells expressing an
enhanced green-fluorescent protein-tagged D2 receptor (D2-EGFP), 2-h D2 agonist stimulation enhanced the colocalization of D2-EGFP with endogenous arrestin2 and
arrestin3. These results suggest that the D2 receptor has the intrinsic ability to bind both nonvisual
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
small interfering RNA-induced depletion of arrestin2 and
arrestin3. In neostriatal neurons, 2-h 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 coimmunoprecipitation 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.