A highly conserved arginine (R) residue that is mutated in human NLG-3 R451C linked to ASD ( Jamain et al., 2003) is present in the ApNLG ( Figure 1B). We next determined the subcellular localization of endogenous ApNLG by immunocytochemical analysis using an affinity-purified polyclonal antibody generated against the extracellular region of ApNLG (Figure S2). In sensory-to-motor neuron cocultures, immunostaining with ApNLG Anti-cancer Compound Library order antibody showed clustering of ApNLG at the initial segment and the proximal regions of major axons of the postsynaptic motor neuron

where the majority of functionally competent synaptic connections are found in sensory-to-motor neuron cocultures (Figure 1C). Immunostaining in nonpermeabilized condition showed a similar ApNLG staining pattern suggesting that they are clusters at the cell surface (data

not shown). The subcellular localization of endogenous ApNLG is consistent with the exclusive localization of mammalian neuroligins in the postsynaptic density (Song et al., 1999). When GFP was expressed in sensory neurons as a whole-cell marker, it became readily evident that presynaptic sensory neuron varicosities, especially the ones in contact with the initial segment and major axons of postsynaptic motor neurons and thus containing functional presynaptic compartments (Kim et al., 2003), partially or completely overlap with ApNLG clusters (Figure 1C). Employing a PCR-based strategy and

using the partial sequence homologous to known neurexin sequences found in the Aplysia EST database ( Moroz Z-VAD-FMK et al., 2006), we cloned a single Aplysia homolog of neurexin (ApNRX). ApNRX also shares a high degree of sequence conservation with other invertebrate and with mammalian neurexins (35% identity and 52% similarity with human neurexin-1α) ( Figure S1). The domain organization of ApNRX is very similar Carnitine dehydrogenase to mammalian α -neurexins. It has a cleavable signal peptide, a large extracellular domain that contains three repeats consisting of two LNS (Laminin-Neurexin-Sex hormone globulin) motifs flanking an EFG motif, followed by single transmembrane domain, and then a short cytoplasmic tail ( Figure 2A). Furthermore, four out of the five alternative splice sites present in mammalian α-neurexin, including splice site 4, which is common to both α- and β-neurexins and determines binding affinity to neuroligin ( Ichtchenko et al., 1995), are also present in equivalent locations in ApNRX, suggesting a high degree of functional conservation ( Figure S1 and Table S1). The high degree of conservation extends to the PDZ binding motif at the C-terminal end, which is conserved and characteristic for “true” neurexins as opposed to the related but different neurexin IV, which shares a number of the other domains of neurexin ( Figure 2B).