Histidine-tagged FLRT3 and ecto-LPHN3-Fc or control Fc proteins were mixed in solution, and the Fc proteins were precipitated with bead-coupled protein A/G and assessed by western blot. We found that FLRT3-His coprecipitated with ecto-LPHN3-Fc, but not with control Fc or NRXN1β(-S4)-Fc (Figure 1L), confirming a direct

interaction between the ectodomains of FLRT3 and LPHN3. To quantitatively characterize the affinity of the FLRT3-LPHN3 interaction, we employed a surface plasmon resonance (SPR) bioassay to measure specific ligand-receptor binding (Figure 1M). JNK inhibitor molecular weight Plotting the maximum relative response versus the ecto-LPHN3-Fc concentrations, we calculated the dissociation constant (Kd) of the LPHN3-FLRT3 interaction to be 14.7 nM (Figure 1N), indicating a high-affinity interaction. To identify brain regions where FLRT3 is likely to function, we examined Flrt3 expression in the developing brain and found that Flrt3 was highly expressed in specific neuronal populations during the first 2 postnatal weeks ( Figures 2A and S2A). In the hippocampus, the principal cell layers of the dentate gyrus (DG) and CA3 showed strong signal, whereas Flrt3 expression was not detected in CA1. Given its interaction with the extracellular domain of LPHNs, we hypothesized Smad inhibitor that FLRT3 might be a postsynaptic protein. We first employed a subcellular fractionation approach to examine the distribution of FLRT3 across different synaptic fractions (Figure 2B)

and found FLRT3 to be enriched in synaptosome and postsynaptic density (PSD) fractions, mirroring the distribution of PSD95 and in contrast to synaptophysin, which is excluded from PSD fractions. Next, because endogenous FLRT3 could not be detected by immunofluorescence with currently available antibodies, we expressed FLRT3-myc in dissociated hippocampal neurons and examined its subcellular distribution. FLRT3-myc was found in dendrites in puncta that partially colocalized with glutamatergic but not GABAergic synapses (Figure 2C and S2C). Together, these results suggest that FLRT3 is a postsynaptic protein of glutamatergic synapses. As a putative trans-synaptic

complex, FLRT3 and LPHN3 must be able to interact across sites of cell-cell contact. We tested whether LPHN3 and FLRT3 can interact in trans by overexpressing LPHN3-GFP in dissociated hippocampal neurons and coculturing them with HEK293 cells expressing Selleckchem Etoposide FLRT3-myc or a control construct. Strong axonal clustering of the GPCR and NTF fragments of LPHN3, as well as enrichment of FLRT3-myc, were observed at sites of contact with FLRT3-myc-expressing HEK293 cells ( Figure 2D). No clustering of LPHN3-GFP was observed when axons contacted control cells (data not shown). The accumulation of FLRT3 at sites of contact with LPHN3-expressing axons ( Figure 2D) demonstrates that FLRT3 is capable of interacting in trans with axonal LPHN3 and that the interaction can mediate mutual recruitment or retention.