In this regard, the activity-dependent proteolytic cleavage of NRX at the presynapse (Bot et al., 2011; Saura et al., 2011) may be functionally linked to the processing of NLG. It has been reported that Fc-fused recombinant NRX extracellular domain inhibited the synaptogenic activity of NLG (Scheiffele et al., 2000; Levinson et al., 2005); it is possible that soluble NRX functions as a negative regulator of NLG1 via induction of shedding.

Ligand-induced shedding has been reported in several γ-secretase substrates (Mumm et al., 2000; Sugahara et al., 2003; Kenchappa et al., 2006; Findley et al., 2007) too, although the molecular mechanisms whereby the ligands activate the processing remain unknown. Ligand/receptor complex formation has been shown to increase the ADAM activity in the ephrin/Eph receptor system (Janes et al., 2009). In the case of CP-690550 mouse Notch, “pulling” movement induced by the endocytosis of bound ligand is thought to cause a structural change leading to cleavage (Gordon et al., 2007). Notably, mucin-like O-linked glycosylation, which might create steric hindrance against ADAM10, was identified in the juxtamembranous stalk region of NLG1 (i.e., Ser683 and Ser686, respectively; Hoffman

et al., 2004). We also have found that amino acid substitutions including the O-glycosylation sites (i.e., PSPF/AAAA and SVDQ/AAAA mutants) increased the shedding of NLG1 ( Figure 4C). Thus, it is possible that the binding of soluble NRX induces structural changes in the stalk region of NLG1 in a way to expose the cleavage Thalidomide site and/or activate the ADAM10 activity. Further ALK inhibitor studies on the mechanism of ADAM10 activation as well as structural analyses of the cleavage site would clarify the mechanism of NLG1 shedding. What, then, is the physiological function of the NLG1 fragments? Extracellular domain of NLG1 is sufficient for binding its ligands (Ichtchenko et al., 1995). Intriguingly, soluble form of NLG1 has been shown to inhibit the synaptogenic effect of TSP1 in immature neurons (Xu et al., 2010). Moreover, clustering of NRXs by recombinant NLG1 extracellular domain mediated the

assembly of presynaptic terminals (Dean et al., 2003), raising the possibility that sNLG1 may bind to soluble as well as membrane-tethered forms of ligands and modulate their functions. Unexpectedly, however, overexpression of NLG1-ΔE that lacks the extracellular domain retained the capacity to induce dendritic spines in granule cells. Our observation is consistent with the previous data showing that the conserved cytoplasmic domain, rather than NRX binding, is necessary and sufficient for the induction of dendritic spines by NLG1 in transfected neurons (Ko et al., 2009; Shipman et al., 2011). Importantly, however, overexpression of NLG1-ICD, which retains the intact intracellular domain, failed to increase the spine numbers. Moreover, NLG1-ICD was highly labile and degraded by proteasomal activity.