Alternative splicing of latrophilin-3 controls synapse formation

The assembly and specification of synapses in the brain is incompletely understood^1-3. Latrophilin-3 (encoded by Adgrl3, also known as Lphn3)-a postsynaptic adhesion G-protein-coupled receptor-mediates synapse formation in the hippocampus⁴ but the mechanisms involved remain unclear. Here we show in mice that LPHN3 organizes synapses through a convergent dual-pathway mechanism: activation of Gα_s signalling and recruitment of phase-separated postsynaptic protein scaffolds. We found that cell-type-specific alternative splicing of Lphn3 controls the LPHN3 G-protein-coupling mode, resulting in LPHN3 variants that predominantly signal through Gα_s or Gα_12/13. CRISPR-mediated manipulation of Lphn3 alternative splicing that shifts LPHN3 from a Gα_s- to a Gα_12/13-coupled mode impaired synaptic connectivity as severely as the overall deletion of Lphn3, suggesting that Gα_s signalling by LPHN3 splice variants mediates synapse formation. Notably, Gα_s-coupled, but not Gα_12/13-coupled, splice variants of LPHN3 also recruit phase-transitioned postsynaptic protein scaffold condensates, such that these condensates are clustered by binding of presynaptic teneurin and FLRT ligands to LPHN3. Moreover, neuronal activity promotes alternative splicing of the synaptogenic Gα_s-coupled variant of LPHN3. 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: Gα_s signalling and clustered phase separation of postsynaptic protein scaffolds.