FIGURE 7.

Impairment of β₂DAT8 recycling by PICK1 is dependent on both intact PDZ binding crevice and BAR domain. a, confocal laser scanning micrographs of Flp-In T-REx 293 eYFP-PICK1 3KE cells induced with tetracycline and transiently expressing β₂DAT8. The pictures show untreated cells (top) and agonist (isoproterenol at 10 μm)-induced internalization of β₂DAT8 that was surface-labeled with Alexa Fluor 568-conjugated anti-FLAG M1 antibody at 4 °C prior to stimulation of internalization at 37 °C by 10 μm Iso for 25 min (middle) or with 10 μm Iso for 25 min followed by a 60-min incubation with the antagonist Alp (bottom) to allow recycling of receptors. Left panels, β₂DAT8 signal (red); middle panels, eYFP-PICK1 3KE signal (green); right panels, merge of red and green signals. b, confocal laser scanning micrographs of Flp-In T-REx 293 eYFP-PICK1 A87L cells induced with tetracycline and transiently expressing β₂DAT8. The pictures show conditions similar to those described for eYFP-PICK1 3KE above. Pictures are representative of ≈20 cells visualized per condition in each experiment and over four separate experiments. Scale bars, 15 μm. Small white squares mark areas that are shown enlarged inside large white squares. c, Flp-In T-REx 293 eYFP-PICK1 cells, Flp-In T-REx 293 eYFP-PICK1 3KE cells, or Flp-In T-REx HEK293 eYFP-PICK1 A87L cells transiently expressing β₂DAT8 with (black bars; +Tet) and without (white bars; −Tet) tetracycline induction were surface-labeled with anti-FLAG M1 antibody prior to stimulation of internalization with agonist (10 μm Iso for 25 min). Subsequently, cells were treated with the antagonist Alp (60 min at 10 μm) to allow recycling. Surface receptor immunoreactivity was determined by surface ELISA as described under “Experimental Procedures.” Data are shown as recycling expressed as the fractional recovery of surface receptor following antagonist incubation for 1 h. Neither PICK1 3KE nor PICK1 A87L retained the ability to impede recycling of β₂DAT8. Data represent means ± S.E. from four independent experiments. *, p < 0.05, unpaired t test.