Journal: bioRxiv
Article Title: Dynamic extracellular interactions with AMPA receptors
doi: 10.1101/2025.07.11.664166
Figure Lengend Snippet: (A) Differential biotinylation of IgLON proteins by APEX2-AMPARs identified by BioSITe-MS. Four Iglons identified are listed. Number of unique peptides identified and total number of BxxP-modified peptides (No. shown in parentheses) are shown for each gene, as well as the site of biotinylation (tyrosine, Y). *LTP/Ctrl, P≤0.05, # LTP/AP5 P≤0.05. (B) Western blot analysis of streptavidin-enriched lysates after APEX labeling by APEX2-AMPARs in neurons. Left, Immunoblotting with antibodies against IgLON proteins OBCAM, NTM and Kilon/NEGR1 detects bands in streptavidin-enriched fractions only in samples where APEX2-AMPARs were expressed (lane 2). No IgLONs were detected in fractions isolated from control labeled neurons where APEX2 was not expressed (lane 1). Immunoblots for GluA2 and GluA1 are shown as positive controls. Right, immunoblotting with anti-biotin antibodies shows robust APEX labeling only when APEX2-AMPARs are expressed (right two lanes). (C) AMPARs interact with OBCAM in HEK cells. HA-tagged OBCAM was expressed in HEK cells along with individual, Myc-tagged AMPAR subunits (GluA1-4). AMPAR complexes were immunoprecipitated (co-IP) from whole-cell lysates using anti-myc antibodies. Immunoblotting with anti-HA antibodies show OBCAM is present in AMPAR precipitates, but not in controls (wild-type or cells empty pCAG vector). Immunoblotting of whole-cell lysates (lower panels) shows relative expression of each AMPAR subunit and OBCAM prior to co-IP. HA-NTM and HA-Kilon were also co-immunoprecipitated by Myc-AMPARs (data not shown). (D) OBCAM directly interacts with GluA2-containing AMPARs. Co-IP experiments in HEK cells show HA-OBCAM is present in GluA2 precipitates (lane 4). To demonstrate specificity of this interaction we used a CRISPR KO guide targeting OBCAM cDNA. Expression of OBCAM is successfully depleted by expression of CRISPR knockout guides targeting the cDNA (OBCAM KO g#2) (lane 2) and thereby depletes OBCAM from GluA2 precipitates (lane 5). (E) IgLONs interact with endogenous AMPAR complexes in neurons. Neurons expressing HA-tagged OBCAM or NTM were fractionated and endogenous AMPARs were purified from crude membrane fractions using anti-GluA1 antibodies. Immunoblotting with anti-HA antibodies shows HA-tagged IgLONs precipitate with GluA1 AMPARs, but not in control neurons. HA-IgLONs were found in AMPAR complexes isolated using anti-GluA2 antibodies specific for the N- and C-terminus (Supplemental Figure 2C-D) . (F) Schematic of IgLON-AMPAR complexes. IgLONs are GPI-anchored proteins with all 3 protein domains expressed on the cell surface. Picture shows IgLONs interacting with AMPARs in cis (on the same side of the membrane). It is possible that they interact with AMPARs in cis or trans (across the synapse/from another membrane).
Article Snippet: CRISPR KO plasmids were subcloned into pU6-(Bbsl)-CBh-Cas9-T2A-mCherry (Addgene #64324).
Techniques: Modification, Western Blot, Labeling, Isolation, Control, Immunoprecipitation, Co-Immunoprecipitation Assay, Plasmid Preparation, Expressing, CRISPR, Knock-Out, Purification, Membrane
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