Figure 4.

NRXN3 AS5+ proteoforms recruit specific synaptic ligands

(A) Volcano plot of protein abundance (iBAQ, log₂ scale fold change knockin versus WT mice and q value) in anti-HA immunoprecipitates from hippocampi from Nrxn3-AS5^HA/HA and wild-type (negative control) P25-30 mice (N = 5 mice per genotype). Selected known Neurexin ligands detected in the analysis are marked in orange. See Table S1 for detailed data.

(B) Heatmap of protein abundance (iBAQ, log₂ scale) of known Neurexin ligands recovered from wild-type and Nrxn3-AS5^HA/HA mice in anti-HA immunoprecipitates and recovered from wild-type mice in control IgG and anti-NRXN immunoprecipitates, respectively. The anti-NRXN antibody is raised against the cytoplasmic tail of NRXN1 nd cross-reacts with all transmembrane NRXNs (Muhammad et al., 2015). Nrxn3 AS5^HA/HA versus wild type: q < 0.001 for NRXN3, NXPH1, FAM19A1, and FAM19A2; q > 0.05 for NRXN1, NLGN3, and DAG1. Anti-NRXN versus control IgG: q < 0.001 for all except C1QL2 (q = 0.0066) and C1QL3 (q = 0.08) (multiple t test with Benjamini, Krieger, and Yekutieli correction). See Table S2 for NRXN immunoprecipitates, and Table S3 for details on selected ligands. Nondetectable proteins depicted as boxes with dashed outline.

(C) Fluorescent in situ hybridization for Nrxn3 and ligands Nxph1 and Fam19A2 transcripts in dentate gyrus of P30 mice.

(D) Confirmation of differential ligand interactions by western blotting. Input (IN, 1%) and immunoprecipitates with anti-HA (left) or control IgG and anti-NRXN1 antibodies (right) probed with anti-NLGN (top) and anti-HA or anti-NRXN antibodies (bottom). Molecular weight markers indicated in kDa. # indicates heavy IgG-chains.

(E) Schematic of NRXN3 domain organization, alternatively spliced segments (blue), and proteotypic peptides (PTPs) of constitutive/common (gray) and proteoform-specific (blue) amino acids indicated, which are quantified for AS3, AS4, and AS6, normalized to recombinant protein expressing all splice isoforms (100% splice site inclusion) and to constitutive exons.

Mean and SEM, Scale bars, 100 μm (overview) and 10 μm (high-magnification images).