Wang, S.; Sudhof, T. C.; Sun, W.; Quake, S. R.; Roth, B. L.; DeLeon, C.

The assembly and specification of synapses in the brain is incompletely understood. Latrophilin-3, a postsynaptic adhesion-GPCR, mediates synapse formation in the hippocampus but the mechanisms involved remain unclear. Here we show that Latrophilin-3 organizes synapses by a novel dual-pathway mechanism: activation of GS/cAMP-signaling and recruitment of phase-separated postsynaptic protein scaffolds. We found that cell type-specific alternative splicing of Latrophilin-3 controls its G protein coupling mode, resulting in Latrophilin-3 variants that predominantly signal via Gs/cAMP or via G12/13. CRISPR-mediated manipulation of Latrophilin-3 alternative splicing from a GS- to a G12/13-coupled mode impaired synaptic connectivity to a degree similar to the overall deletion of Latrophilin-3, suggesting that GS/cAMP-signaling by Latrophilin-3 splice variants mediates synapse formation. Moreover, GS- but not G12/13-coupled splice variants of Latrophilin-3 recruit phase-transitioned postsynaptic protein scaffolds that are clustered by binding of presynaptic Latrophilin-3 ligands. Strikingly, neuronal activity promotes alternative splicing of the synaptogenic variant of Latrophilin-3. Together, these data suggest that activity-dependent alternative splicing of a key synaptic adhesion molecule controls synapse formation by parallel activation of two convergent pathways: GS/cAMP signaling and the phase separation of postsynaptic protein scaffolds.