Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 275, Issue 27, Pages 20602-20609Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M002358200
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Melatonin receptors bind and become activated by melatonin. The melatonin-related receptor, despite sharing considerable amino acid sequence identity with melatonin receptors, does not bind melatonin and is currently an orphan 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 domain 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-related receptor, the resultant chimeric receptors all displayed specific 2-[I-125]iodomelatonin binding, Replacement of sequences incorporating transmembrane domains 4 or 6, however, resulted in chimeric receptors that displayed no detectable 2-[I-125]iodomelatonin binding. The subsequent testing of a reverse chimeric receptor in which sequences encoding transmembrane domains 4 and 6 of the melatonin-related receptor were replaced by the corresponding melatonin mt, receptor sequences identified specific 2-[I-125]iodomelatonin 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. This identified Gly(258) (Gly(6.55)) as a critical residue required for high affinity ligand binding and receptor function.
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