An intact PDZ-binding ligand is required for arrestin- but not clathrin-dependent internalization of the P2Y₁₂ receptor. (A) Cells stably expressing receptor constructs were transfected with arrestin-2–GFP. Before imaging, coverslips were mounted in an imaging chamber at 37°C. The initial diffuse cytoplasmic distribution of arrestin-2–GFP is shown before agonist stimulation (0 second). ADP (10μM) was added, and the redistribution of arrestin-2 was monitored in real time. The images shown were collected at 10, 60, and 240 seconds after agonist addition. Data shown are representative of 3 independent experiments. (B) Cells stably expressing receptor construct were transiently transfected with arrestin-2–GFP. Cells were stimulated with ADP (10μM; 5 and 10 minutes). Receptor was immunoprecipitated from cell lysates with the use of an anti-HA Ab (HA-11), and arrestin-2 association was assessed with an anti-GFP Ab. Equal loading of arrestin-2–GFP was confirmed in cell lysates taken before receptor immunoprecipitation. As shown arrestin-2/receptor association was only found in cells expressing the full-length P2Y₁₂ purinergic receptor. Like E³³⁹stop and the P341A variant, T³²⁰stop did not associate with arrestin (data not shown). Data shown are representative of 3 independent experiments. (C) Receptor-expressing cells were transiently transfected with DNM forms of arrestin-2 (319-418; arrestin-DNM), eps-15 (E95-295; eps-15–DNM), dynamin (K44A; dynamin-DNM), or vector (pcDNA3) alone. Cells were subsequently challenged with ADP (10μM; 30 minutes), and surface receptor loss was assessed by ELISA. The data represent means ± SEMs of 7 independent experiments. *P < .05 compared with respective pcDNA3 vector transfected controls (Mann-Whitney U test).