Clustering of the Adhesion-GPCR Latrophilin-3 Drives Excitatory Synapse Assembly

Precise control of synapse formation is essential for brain function. Adhesion-GPCRs combine adhesion, signaling, and scaffold recruitment to regulate synapse formation, yet their mechanisms of action remain unclear. Here, we identify nanoscale clustering of the adhesion-GPCR Latrophilin-3 as a key mechanism regulating excitatory synapse assembly. We find that endogenous Latrophilin-3 forms densely packed nanoclusters, with larger nanoclusters enriched at excitatory synapses. To probe the functional significance of Latrophilin-3 clustering, we engineered an optogenetically controlled Latrophilin-3. Light-induced Latrophilin-3 clustering triggered robust excitatory synapse formation, which required both Latrophilin-3-mediated G-protein signaling and postsynaptic scaffold recruitment. Strikingly, clustering of a mutant Latrophilin-3 lacking known ligand-binding domains still promoted synapse formation, indicating that Latrophilin-3 ligands induce synapse assembly primarily by clustering Latrophilin-3. Our findings suggest a general paradigm by which extracellular ligand-induced clustering activates adhesion-GPCRs by generating compartmentalized signaling, which in the case of Latrophilin-3, cooperates with postsynaptic scaffold recruitment to drive synapse assembly.