MDGA2 is an excitatory synapse-specific suppressor that uses distinct extracellular mechanisms to negatively regulate various postsynaptic properties. Here, we identify EphB2, an excitatory synapse-specific receptor tyrosine kinase, as a new binding partner for MDGA2. The first three immunoglobulin domains of MDGA2 undergo cis-binding to the ligand-binding domain of EphB2, enabling MDGA2 to compete with Ephrin-B1 for binding to EphB2. Moreover, EphB2 forms complexes with MDGA2 and GluN2B-containing NMDA receptors (NMDARs) in mouse brains. MDGA2 deletion promotes formation of the EphB2/Ephrin-B1 complex but does not alter the surface expression levels and Ephrin-stimulated activation of EphB2 receptors and downstream GluN2B-containing NMDARs in cultured neurons. AlphaFold-based molecular replacement experiments reveal that MDGA2 must bind EphB2 to suppress spontaneous synaptic transmission and NMDAR-mediated, but not AMPAR-mediated, postsynaptic responses at excitatory synapses in cultured neurons. These results collectively suggest that MDGA2 is a versatile factor that suppresses distinct excitatory postsynaptic properties via different transsynaptic pathways.
