Fig. 2.

Fbx2-dependent ubiquitination of NR1 mediated interaction with the glycosylated ectodomain of NR1. (a) Structure of Fbx2 and deletion mutants. The F-box and F-box-associated (FBA) domains are indicated. (b) The FBA region mediates NR1 binding. FLAG-tagged Fbx2 variants were transfected into COS7 cells. The cell lysates were immunoprecipitated with anti-FLAG antibody and then immunoblotted with anti-NR1. Wild-type Fbx2 and Fbx2(ΔF) but not Fbx(ΔC) were coimmunoprecipitated with Fbx2. (c) Fbx2 protein interacts with NR1 in vivo. Rat brain lysates were immunoprecipitated with two independent anti-Fbx2 antibodies or control IgG and immunoblotted for NR1, GluR2, and Fbx2. (d) Fbx2 interacts with high-mannose glycans on NR1. Proteins from COS7 cells expressing Myc-tagged NTD of NR1 were treated with EndoH or peptide N-glycosidase F (PNGaseF). Immunoblotting demonstrates deglycosylation (Left). Far Western blotting shows that EndoH or PNGaseF treatment abolished interaction of NTD(NR1)-AP with Fbx2. (e) Both NR1 alternatively spliced variants (NR1a and NR1b), but not GluR2, bind to Fbx2. COS7 cells expressing Fbx2 were fixed, permeabilized, and probed with NTD of NR1a, NR1b, or GluR2 (GR2). Bound AP was quantitated enzymatically by using p-nitrophenylphosphate as substrate. Error bar, SEM. (f) Fbx2 constructs or GFP were cotransfected with full-length NR1 and ubiquitin into COS7 cells. MG132 (10 μM) was applied 10 h before harvesting. Cell lysates were immunoprecipitated with anti-NR and blotted with antiubiquitin or anti-NR1 antibodies.