NLG1 knockout (KO) or transgenic mice showed synaptic dysfunctions and ASD-like behaviors (Varoqueaux et al., 2006; Chubykin et al., 2007; Blundell et al., 2010; Dahlhaus et al., 2010). Thus, the levels of NLGs within the synaptic membranes are presumed to directly modulate the synaptic functions in vivo. Although several reports indicated that the surface see more levels of NLG1 are regulated by synaptic activities through membrane trafficking (Schapitz et al., 2010; Thyagarajan and Ting, 2010), the regulatory mechanisms to control protein levels of NLG remains unclear. Here, we show that NLG1 is sequentially cleaved by ADAM10 and γ-secretase to release its extra- and intracellular domain fragments,
respectively. Proteolytic processing of NLG1 resulted in the elimination of NLG1 on the cell surface, thereby causing a decrease in the synaptogenic activity of NLG1. We further show that ADAM10-mediated shedding is regulated in an activity-dependent manner through NMDA receptor (NMDAR) activation or by binding to secreted forms of NRXs. Our present results suggest that neuronal activity and interaction with NRXs regulate the levels of NLG1 via proteolytic processing to modulate the adhesion
system as well as the functions of synapses. NLGs are synaptogenic type 1 transmembrane proteins that harbor INCB024360 concentration large extracellular domains (Ichtchenko et al., 1995). While the levels of NLGs are presumed to be correlated with their physiological and pathological functions (Varoqueaux et al., 2006; Chubykin et al., 2007; Glessner et al., Resminostat 2009; Blundell et al., 2010; Dahlhaus et al., 2010), little information is available on the proteolytic mechanism of NLGs. Several lines of evidence have indicated that a subset of type 1 transmembrane proteins are processed by
sequential cleavages by ectodomain shedding and intramembrane cleavage, the latter being executed by γ-secretase (Beel and Sanders, 2008; Bai and Pfaff, 2011). To test whether the levels of NLGs are regulated by proteolytic processing, we analyzed endogenous NLG polypeptides in adult rat brains (Figure 1A). Immunoblot analysis using antibodies that specifically recognize the cytoplasmic region of NLG1 and NLG2 (see Figure S1 available online) revealed immunopositive bands at ∼20–25 kDa, in addition to full-length (FL) protein that migrated at ∼120 kDa. Because the predicted sizes of the cytoplasmic domain of NLGs were within the range of 120–165 amino acid (aa) lengths (NLG1, 125 aa; NLG2, 137 aa), we reasoned that the ∼20–25 kDa polypeptides represent the membrane-tethered C-terminal fragment (CTF) of endogenous NLGs. Multiple bands corresponding to CTFs may represent different posttranslational modifications (e.g., glycosylation, see below). To examine whether these CTFs are processed by the γ-secretase activity, we incubated the membrane fractions of rat brains at 37°C and detected the appearance of additional bands that migrate faster than the CTFs with each NLG.