Structural basis and physiological significance of non-canonical Gs coupling to the melatonin MT1 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. MT1 and MT2 are prototypical Gi/o-coupled GPCRs responding to melatonin, a hormone secreted in a circadian manner. We show here that MT1, but not MT2, couples also to Gs proteins in vitro and activates the Gs/cAMP pathway upon long-term melatonin exposure in vivo, mimicking physiological dawn conditions. We solve the cryo–electron microscopy structure of the melatonin-MT1-Gs complex at 3.0Å resolution, which reveals a distinct binding mode compared to the MT1–Gi complex. The third intracellular loop of MT1 emerges as a key stabilizer for Gs coupling. This structure of a GPCR primary coupling to Gi, here in complex with Gs, provides structural and functional insights into G protein selectivity and circadian switch of G protein coupling.