Neurexin mutations that reduce synaptogenic activity reduce binding affinity to neuroligins. A–H, Compared with wild-type soluble neurexin-Fc (nrx −S4) binding to HA-neuroligins (NLG) expressed in COS cells (A, E), neurexin mutations that reduced synaptogenic activity reduced binding to HA-neuroligins (B, C, F, G), and mutations that abolished synaptogenic activity abolished binding to HA-neuroligins (D, H). The mutations reduced binding to neuroligin-1 (B, C) to a greater extent than to neuroligin-2 (F, G). HA staining was performed live to reveal only surface neuroligin. I, Quantification of the average intensity of bound neurexin-Fc normalized to average intensity of surface HA-neuroligin and expressed as a percentage of binding of wild-type neurexin-Fc to HA-neuroligin-1 or -2. All mutations significantly reduced binding of neurexin to neuroligin-1 and -2 (p < 0.005; n ≥ 40 cells each), except that the β2β3mid mutation did not affect binding to neuroligin-2 (p > 0.1). Scale bar, 20 μm.