In situ screening for postsynaptic cell adhesion molecules during synapse formation

Neuronal synapse formation is regulated by pre- and postsynaptic cell adhesion molecules. Presynaptic neurexins (NRXNs) and receptor protein tyrosine phosphatases (RPTPs; PTPδ, PTPσ and LAR in mammals) can induce postsynaptic differentiation through the interaction with various postsynaptic cell adhesion molecules. Here, we developed a novel in situ screening method to identify postsynaptic membranous proteins involved in synaptogenesis. Magnetic beads coated with the extracellular domains of NRXN1β(-S4) and PTPδ-A6 variants preferentially induced excitatory postsynaptic differentiation on the beads' surface when co-cultured with cortical neurons. After inducing postsynaptic sites on these beads, protein complexes including NRXN1β (-S4)/PTPδ-A6 and their ligands on the neuronal membrane were chemically crosslinked and purified using a magnetic separator. Liquid chromatography-tandem mass spectrometry analysis of the complexes revealed two types of postsynaptic ligands for NRXN1β (-S4) and PTPδ-A6, one has an activity to induce presynaptic differentiation in a trans manner, whereas the other has no such activity. These results suggest that synapse formation is regulated by the interplay between presynaptic NRXN/PTPδ and their postsynaptic ligands with functionally different impacts on pre- and postsynaptic differentiation. Thus, our in situ screening method for identifying synapse- organizing complexes will help to understand the molecular basis for elaborate neuronal networks.