FIGURE 4.

PICK1-binding chimeric receptor β₂DAT8 clusters eYFP-PICK1 and traffics to PICK1-positive clusters upon agonist-induced internalization. a, schematic representation of β₂DAT8 and β₂DAT8 +Ala showing the N-terminal FLAG tag sequence followed by the full-length β₂ adrenergic receptor and the last 8 PICK1-binding C-terminal residues of DAT. In β₂DAT8 +Ala, PICK1 binding is disrupted due to the addition of an extra C-terminal alanine. b, confocal laser scanning micrographs of Flp-In T-REx 293 eYFP-PICK1 cells transiently expressing β₂DAT8. The pictures show agonist (isoproterenol)-induced internalization of β₂DAT8 that was surface-labeled with Alexa Fluor 568-conjugated anti-FLAG M1 antibody at 4 °C without (−Tet) and with (+Tet) induced expression of eYFP-PICK1 (green). Internalization was stimulated by 10 μm Iso for 25 min at 37 °C or by 10 μm Iso for 25 min followed by a 60-min incubation at 37 °C with the antagonist Alp to allow recycling of receptors. Panels starting from the left show the β₂DAT8 signal in non-induced cells, β₂DAT8 signal in tetracycline-induced cells (red), eYFP-PICK1 signal (green), and merge of red and green signals. c, confocal laser scanning micrographs of Flp-In T-REx 293 eYFP-PICK1 cells transiently expressing β₂DAT8 +Ala. The pictures show conditions similar to those described for β₂DAT8 above. Notably, β₂DAT8 +Ala does not promote formation of perinuclear eYFP-PICK1 clusters. Pictures are representative of ∼25 cells visualized per condition in each experiment and over six separate experiments. The small squares mark areas that are shown enlarged inside the large squares. Scale bars, 15 μm.