Figure 4.

LRRTM1, the family member linked to schizophrenia, also binds and is recruited by (−S4) neurexins. A, Recombinant Nrx1β(−S4)-Fc, Nrx1β(+S4)-Fc, and Nrx1β(ΔLNS)-Fc were incubated with COS7 cells transfected with Myc-LRRTM1, Myc-LRRTM2, or Myc-SALM2. Both Myc-LRRTM1 and Myc-LRRTM2, detected by surface staining for Myc, bound Nrx1β(−S4)-Fc but not Nrx1β(+S4)-Fc or Nrx1β(ΔLNS)-Fc. Myc-SALM2 was the negative control for binding. Scale bar, 10 μm. B, Quantitation of the data in A (ANOVA, p < 0.0001; Tukey's post hoc test, *p < 0.001 compared to Myc-SALM2; n = 20; results are expressed as mean ± SEM). C, Hemisynaptogenesis assay as described in Figure 3. COS7 cells expressing Nrx1β(−S4)-CFP or Nrx1α(−S4)-CFP but not Nrx1β(+S4)-CFP or Nrx1α(+S4)-CFP clustered YFP-LRRTM1 expressed in neurons. Scale bar, 10 μm. D, The assay in C was also performed for +S4 and −S4 variants of neurexins 2β′, 3β′, 2α, and 3α. All (−S4) neurexin variants clustered YFP-LRRTM1 expressed in neurons. These clusters contained PSD-95 but not synapsin. (++++, 80–95% contact sites were positive; −, 0–5% contact sites were positive; n > 30 contact sites).