Structural basis and physiological significance of non-canonical Gs coupling to the prototypical Gi-coupled melatonin MT ₁ receptor

G protein-coupled receptors (GPCRs) transduce extracellular stimuli into intracellular signals by coupling to various heterotrimeric G proteins. However, the rules governing G protein preference remain largely elusive. MT ₁ and MT ₂ are prototypical G _i/o -coupled GPCRs responding to melatonin, a hormone secreted in a circadian manner. We show here that MT ₁ , but not MT ₂ , couples also to G _s proteins in vitro and activates the G _s /cAMP pathway upon long-term melatonin exposure in vivo , mimicking physiological dawn conditions. We solved the cryo–electron microscopy structure of the melatonin-MT ₁ -G _s complex at 3.0Å resolution, which revealed a strikingly distinct binding mode compared to the MT ₁ –G _i complex. The third intracellular loop of MT ₁ emerges as a key stabilizer for G _s coupling, a feature previously unrecognized. This is the first solved receptor-G _s complex of a primary G _i -coupled GPCRs, providing new structural and functional insights into G protein selectivity and circadian switch of G protein coupling.