Figure 5. Soluble Ig-Neurexin Impairs Synaptogenic Activity of LRRTM2: Model of LRRTM2 Action.
A. & B. Representative images (A) and quantitation (B) of artificial synapse formation assays to measure the effect of Ig-neurexin fusion protein lacking (Nrx1βSS4−) or containing an insert in splice site #4 (Nrx1βSS4+) on the synaptogenic activities of LRRTM2 and neuroligin-1. Cultured hippocampal neurons (DIV 7) were co-cultured for 12 h with COS cells expressing mVenus-fusion proteins of LRRTM2 or of neuroligin-1 lacking inserts in splice sites A and B (NL1ΔAB). Co-cultures were performed in the presence of 50 µg/ml of IgC (control), IgNrx1βSS4−, or IgNrx1βSS4+. Panels in A show representative immunofluorescence images of the co-cultures stained with antibodies to mVenus (GFP; green) and synapsin (red; coincident signals = yellow; scale bar = 25 µm, applies to all images). In B, the immunofluorescence intensities for synapsin and for mVenus were measured over the same transfected COS cells (left and middle bar graphs), and the ratios of synapsin to mVenus fluorescence signal were determined (right bar graph). Dashed lines correspond to the IgC-treated values as the baseline. Data shown are means ± SEMs (n=3 independent experiments, *=p<0.05).
C. Model for the trans-synaptic interactions of neurexins with neuroligins and LRRTM2. Both bind to neurexins via the 6th LNS-domain that is shared by α- and β-neurexins, but exhibit distinct specificities: neuroligins bind to neurexins in a manner that is modulated by alternative splicing at site #4 without being absolutely dependent on this splice site, whereas LRRTM2 binds to neurexins only if splice site #4 lacks an insert.