Melatonin is a neurohormone secreted by the pineal gland mainly regulating circadian rhythm and binding to 2 receptors: MT1 and MT2. A recent genome‐wide association studies revealed 40 non‐synonymous polymorphisms of MT2. All these variants are normally expressed at cell surface and 4 of them show loss of melatonin binding. Given that no crystal structure is available yet for MT2 and that variants are well distributed along MT2, we took advantage of these natural variants to delineate structural features controlling MT2 functionality. By monitoring β‐arrestin 2 recruitment, ERK phosphorylation, inhibition of cAMP production, Gαi1 and Gαz activation, we generated 40 signaling signatures and similar profiles were grouped together using non‐negative matrix factorization (NMF). A total of 8 different types of signatures (clusters) were obtained. Each cluster has unique signaling features and is distinctly spatially localized along MT2. While residues important for general signal transmission are located in transmembrane regions, the residues responsible for signal selectivity are almost exclusively localized in intracellular domains. Using computational homology modeling (PDB ID: 4Zwj and 3sn6), we proposed molecular mechanisms underlying general signal transmission but also Gα/β‐arrestin and Gαz/Gαi1 selectivities. Altogether, our findings represent a proof of principle that natural variants of a given receptor can be exploited and used as a platform to learn more about its structural properties.Support or Funding InformationThis work was supported by a grant from FRQS to MB, from FRM and ANR to RJ and from NIH, NSF and DARPA to OL. Postdoctoral fellowships from CIHR and from Diabetes Canada to BP, from FRM to AK and from ETH Zurich to TF also funded this work.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.