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. 2017 Oct 2;216(10):3323–3338. doi: 10.1083/jcb.201701034

Figure 7.

Figure 7.

PICK1–AP2 interaction is required for AMPAR endocytosis after NMDAR activation. (A) PICK1–AP2 interaction is involved in constitutive AMPAR trafficking. Neurons infected with lentivirus expressing GFP, shPICK1 + GFP, shPICK1 + sh-resistant GFP-WT-PICK1, or shPICK1 + sh-resistant GFP-D189A-PICK1 were subjected to surface biotinylation. Representative blots show surface and 5% of the total GluA2 in lysates. (B) PICK1–AP2 interaction is required for NMDA-induced reduction in surface GluA2 containing AMPARs. Neurons infected with lentivirus expressing GFP, shPICK1 + GFP, shPICK1 + sh-resistant GFP-WT-PICK1, or shPICK1 + sh-resistant GFP-DDAA-PICK1 were exposed to NMDA for 3 min, returned to normal medium for 7 min, and subjected to surface biotinylation. Representative blots show surface and 5% of the total GluA2 in lysates. (A and B) Graph shows surface/total ratio (n = 5 independent experiments; *, P < 0.05; one-way ANOVA followed by Tukey’s test; values are means ± SEMs). (C) PICK1–AP2 interaction is required for NMDA-induced endocytosis of GluA2-containing AMPARs. Hippocampal neurons transfected with GFP, shPICK1 + GFP, shPICK1 + sh-resistant GFP-WT-PICK1, or shPICK1 + sh-resistant GFP-DDAA-PICK1 were surface labeled with GluA2 antibody before application of NMDA for 3 min. 7 min later, cells were fixed and stained for the internal and surface pools of GluA2 using different secondary antibodies. Bar, 5 µm. Graph shows internalization index (internalized/total; n = 5 independent experiments [35–45 cells per condition in total]; *, P < 0.05; one-way ANOVA followed by Tukey’s test; values are means ± SEMs). (D) PICK1 is not required for the endocytosis of transferrin receptors. Hippocampal neurons transfected with GFP, shPICK1 + GFP, shPICK1 + GFP-WT-PICK1, or shPICK1 + GFP-DDAA-PICK1 were surface labeled with Alexa Fluor 568–conjugated transferrin for 12 min. After acid wash, cells were fixed and imaged. Bar, 2 µm. Graph shows quantification of internalized transferrin (n = 3 independent experiments [12–14 cells per condition in total]).