Fig. 4: ErbB4 interacts transcellularly with Slitrk3.

A ErbB4 was distributed to the presynaptic and postsynaptic areas. Subcellular fractions of the WT brains were blotted with anti-ErbB4, anti-GluN2A (a postsynaptic glutamate receptor), anti-gephyrin (an inhibitory postsynaptic marker), anti-PSD95 (a PSD marker), and anti-synaptophysin (SYN, a presynaptic marker). Hom, homogenate; S1, supernatant; P1, pelleted nuclear fraction; P2, crude synaptosomal fraction; P2’, washed crude synaptosomal fraction; S3, crude synaptic vesicle fraction; P3, lysed synaptosomal membrane fraction; SV, synaptic vesicle fraction; SPM, synaptosomal plasma membrane; Pre, presynaptic fraction; PSD, postsynaptic density. B, C Colocalization of ErbB4 with VGAT (B) or gephyrin (C). Primary hippocampal neurons at DIV10 were co-stained with ErbB4 and VGAT or gephyrin. Boxed areas were enlarged in the respective right images. Arrows indicate the co-localized ErbB4 with VGAT or gephyrin, and arrowheads indicate ErbB4 puncta alone. Scale bar, 20 μm for lower magnification images and 10 μm for enlarged images of boxed areas. D Schematic illustration of constructs with different ErbB4 domain structures. E, F In trans ErbB4–Slitrk3 interaction required the RLD of ErbB4. Representative images of cell aggregation assay (E). Scale bar, 1 mm. HEK392T cells co-transfected with DsRed and K751M, ΔRLD, NRXN1β or Mock mixed with HEK392T cells co-transfected with EGFP and Slitrk3, NLGN1 or Mock. In K751M + Slitrk3 + RLD-IgG group, recombinant RLD-IgG protein was added to the mixture of K751M cells and Slitrk3 cells. Quantitative analysis of cell aggregates in E (F). Data were from three independent experiments and shown as mean ± SEM. ***p < 0.001. One-way ANOVA. G ErbB4 interacted with Slitrk3 through RLD domain. HEK293T cells were co-transfected with myc-tagged Slitr3-ΔICD plus Flag-tagged different ECD domains of ErbB4 and subjected to co-IP with Flag-beads.