To determine which domain of FLRT3 and LPHN3 is directly involved in the association, we engineered different constructs for each protein (Figure 1A). The extracellular domain of LPHN3 was divided into three portions: the N-terminal half including the RBL, OLF, and the O-linked carbohydrate domains, the OLF domain alone, and the C-terminal half containing the HR-GAIN-GPS domains. The extracellular domain of FLRT3 was also split into two constructs, one containing the FN3 domain alone (FLRT3-FN3) and the other containing the ten LRR repeats between amino acids 29–386 (FLRT3-LRR). ITC experiments were performed to assess which construct retained the affinity of the full extracellular domain. Our data show that binding occurred between FLRT3-LRR and LPHN3-OLF (LPHN3 amino acids 199–495) (Figure 1B and Table 1) with an affinity similar to the one previously reported for the full length extracellular domains (O’Sullivan et al., 2012). Because no association was recorded using either the LPHN3-HR-GAINGPS construct or FLRT3-FN3 domains, we conclude that LPHN3-OLF and FLRT3-LRR represent the complete binding cassette of these proteins. ITC experiments in the presence of 1 mM EDTA or 2
