Figure 9.

ICA69 reduces formation of homomeric PICK1 BAR domain complexes. A, MBP-tagged ICA69 fusion proteins were mixed with liposomes and subjected to high-speed centrifugation. After centrifugation, substantial amounts of ICA69 were found to come down with liposomes and showed up in the pellet (first panel, left two lanes; S, supernatant; P, pellet.). Without liposomes, ICA69 was not found in the pellet (first panel, right two lanes). As a control, MBP itself was not found in the liposome-containing pellet (second panel). The BAR domain (ICA-BAR) shows a similar lipid-binding capability as full-length ICA69 (third panel), whereas the C-terminal domain (ICAC) does not bind to lipid (fourth panel). B, ICA69 does not change PICK1's lipid-binding capability. MBP-PICK1 was used for the liposome sedimentation assay with or without MBP-ICA69. Equal amounts of pellet and supernatant were loaded and resolved by SDS-PAGE and stained by Coomassie blue. The level of PICK1 in the liposome-associated pellet fraction is similar with or without ICA69. MBP-ICA69 itself also binds well to liposomes in the presence of PICK1. C, ICA69 does not bind to GluR2 directly. GluR2, myc-PICK1, and myc-ICA69 were expressed in 293T cells as indicated and immunoprecipitated by an anti-myc antibody. GluR2 was only pulled down together with myc-PICK1 but not with myc-ICA69. The cell lysates used for the coimmunoprecipitation were loaded as INPUT to show comparable expression levels of proteins in each experiment. D, ICA69 does not reduce PICK1–GluR2 interaction. GFP-PICK1 and GluR2 were transfected into 293T cells with or without myc-ICA69, and then GFP-PICK1 was immunoprecipitated down with an anti-GFP antibody. The level of GluR2 coimmunoprecipitated with PICK1 is not reduced in the presence of ICA69, whereas ICA69 itself was robustly coimmunoprecipitated with PICK1 and GluR2 (bottom). E, ICA69 disrupts PICK1 self-association. GFP-PICK1 and myc-PICK1 were transfected into 293T cells with or without myc-ICA69. GFP-PICK1 was immunoprecipitated using an anti-GFP antibody. The level of coimmunoprecipitated myc-PICK1 with GFP-PICK1 was significantly reduced in the presence of ICA69. In contrast, ICA69 was robustly coimmunoprecipitated with GFP-PICK1, indicating that ICA69 forms heteromeric BAR domain complexes at the expense of homomeric PICK1 BAR domain complexes. F, This model illustrates the role of ICA69 and PICK1 in synaptic targeting of GluR2. There are two pools of GluR2-containing AMPA receptors. The heteromeric BAR domain complexes of ICA69 and PICK1 maintain a pool of AMPA receptors at the dendrites and cell bodies. The homomeric BAR domain complexes of PICK1 maintain a pool of AMPA receptors at the synapses.