Figure 2.

PICK1 binds AP2 via FxDxF and DxF motifs. (A) Schematic of PICK1 indicating the FxDxF and DxF AP2-binding motifs and the DD189,356AA mutations. (B) Endogenous PICK1 binds endogenous AP2 in neurons. Extracts of cortical neurons were immunoprecipitated with PICK1 antibody or control IgG. Proteins were detected by Western blotting. Input is 5% of offered protein. Blots shown are representative of more than five independent experiments. (C) PICK1 directly interacts with the appendage domain of α-adaptin. GST, GST-α-appendage, GST-β-appendage, or GST-μ cargo-recognition domain were immobilized on glutathione agarose and incubated with purified his₆-PICK1. Proteins were detected by Western blotting. Blots shown are representative of five independent experiments. (D) DDAA mutant does not bind AP2. GST or GST-α-appendage were immobilized on glutathione agarose and incubated with purified his₆-WT-PICK1 or mutants. Graph shows quantification of PICK1 binding to α-adaptin (n = 6 independent experiments; **, P < 0.01; ***, P < 0.001; one-way ANOVA followed by Tukey’s test; values are means ± SEMs).