Chimeric melatonin mt(1) and melatonin-related receptors - Identification of domains and residues participating in ligand binding and receptor activation of the melatonin mt(1) receptor

Melatonin receptors bind and become activated by melatonin. The melatonin-r elated receptor, despite sharing considerable amino acid sequence identity with melatonin receptors, does not bind melatonin and is currently an orpha n G protein-coupled receptor. To investigate the structure and function of both receptors, we engineered a series of 14 chimeric receptor constructs, allowing us to determine the relative contribution of each transmembrane do main to Ligand binding and receptor function. Results identified that when sequences encoding transmembrane domains 1, 2, 3, 5, or 7 of the melatonin mt, receptor were replaced by the corresponding domains of the melatonin-re lated receptor, the resultant chimeric receptors all displayed specific 2-[ I-125]iodomelatonin binding, Replacement of sequences incorporating transme mbrane domains 4 or 6, however, resulted in chimeric receptors that display ed no detectable 2-[I-125]iodomelatonin binding. The subsequent testing of a "reverse" chimeric receptor in which sequences encoding transmembrane dom ains 4 and 6 of the melatonin-related receptor were replaced by the corresp onding melatonin mt, receptor sequences identified specific 2-[I-125]iodome latonin binding and melatonin-mediated modulation of cyclic AMP levels. To further investigate these findings, site-directed mutagenesis was performed on residues within transmembrane domain 6 of the melatonin mt, receptor. T his identified Gly(258) (Gly(6.55)) as a critical residue required for high affinity ligand binding and receptor function.