Altas B, Tuffy LP, Patrizi A, Dimova K, Soykan T, Romanowski AJ, Robertson CD, Khim SN, Bunce GW, Ambrozkiewicz MC, Yagensky O, Krueger-Burg D, Hammer M, Hsiao HH, Laskowski PR, Dyck L, Sassoè-Pognetto M, Chua JJE, Urlaub H, Jahn O, Brose N, Poulopoulos A
Neuroligin-3 is a postsynaptic adhesion molecule involved in development, function, and pathologies of synapses in the brain. It is a genetic cause of autism and a potent component of the tumor microenvironment in gliomas. There are four Neuroligins that operate at distinct synapse types, selectively interacting with presynaptic adhesion and postsynaptic scaffold proteins. We investigated the subcellular localization and scaffold specificities of synaptic Neuroligin-3 and demonstrate an unexpected pattern of localization to excitatory synapses in cortical areas, and inhibitory synapses in subcortical areas. Using phosphoproteomics, we identify Neuroligin-3-specific serine phosphorylation in cortex and hippocampus that obstructs a key binding site for inhibitory synapse scaffolds. Using in utero CRISPR/Cas9 knockout and replacement with phosphomimetic mutants, we demonstrate that phosphorylation at this site determines excitatory versus inhibitory synapse localization of Neuroligin-3 in vivo. Our data reveal a mechanism that differentially regulates the balance of Neuroligin-3 between excitatory and inhibitory synapses, adding to our emerging understanding of their role in the development of brain connectivity and associated pathologies.