Identification of Key Regions Mediating Human Melatonin Type 1 Receptor Functional Selectivity Revealed by Natural Variants

[Image: see text] Melatonin is a hormone mainly produced by the pineal gland and MT(1) is one of the two G protein-coupled receptors (GPCRs) mediating its action. Despite an increasing number of available GPCR crystal structures, the molecular mechanism of activation of a large number of receptors, including MT(1), remains poorly understood. The purpose of this study is to elucidate the structural elements involved in the process of MT(1)’s activation using naturally occurring variants affecting its function. Thirty-six nonsynonymous variants, including 34 rare ones, were identified in MTNR1A (encoding MT(1)) from a cohort of 8687 individuals and their signaling profiles were characterized using Bioluminescence Resonance Energy Transfer-based sensors probing 11 different signaling pathways. Computational analysis of the experimental data allowed us to group the variants in clusters according to their signaling profiles and to analyze the position of each variant in the context of the three-dimensional structure of MT(1) to link functional selectivity to structure. MT(1) variant signaling profiles revealed three clusters characterized by (1) wild-type-like variants, (2) variants with selective defect of βarrestin-2 recruitment, and (3) severely defective variants on all pathways. Our structural analysis allows us to identify important regions for βarrestin-2 recruitment as well as for Gα12 and Gα15 activation. In addition to identifying MT(1) domains differentially controlling the activation of the various signaling effectors, this study illustrates how natural variants can be used as tools to study the molecular mechanisms of receptor activation.