Mutagenesis studies of the human MT2 melatonin receptor

Melatonin mediates its physiological effects through activation of high affinity G protein-coupled receptors. The vertebrate MT1, MT2 and Mel(1c), melatonin receptors are molecularly and pharmacologically distinct. Three molecular models of melatonin recognition for the MT1 and/or Mel(1c) melatonin receptors have been proposed. To determine if these models applied to the MT2 melatonin receptor, we mutated seven conserved residues to alanine in the hMT(2) melatonin receptor and expressed the receptors in HEK-293 cells. Competition of melatonin for 2-[I-125]-iodomelatonin binding revealed that mutation of Asn 16 in TM4 or His 7 in TM5 of the hMT(2) melatonin receptor significantly decreased the binding affinity for melatonin when compared with wild-type. In addition, competition of 4P-ADOT, N-acetyltryptamine, luzindole, and 5-methoxytryptophol for 2-[I-125]-iodomelatonin binding suggested Asn 16 in TM4 may facilitate binding of the 5-methoxy group of the melatonin molecule to the hMT2 melatonin receptor. Trp 13 or Phe 6 in TM6 while not critical for melatonin binding, may interact with aromatic regions of luzindole and 4P-ADOT. Mutation of Ser 8 or Ser 12 in TM3, or Ser 6 in TM7 did not affect the affinity of melatonin for competition with 2- [I-125]-iodomelatonin to the hMT(2) melatonin receptor, although equivalent serines (Ser 8 and Ser 12 in TM3) were reported to be critical for melatonin binding to the hMT(1) melatonin receptor. Thus these results are the first to identify residues within the transmembrane regions of the hMT(2) melatonin receptor critical for melatonin binding, highlighting potential structural differences between the MT1 and MT2 melatonin receptor binding pockets. (C) 2003 Elsevier Science Inc. All rights reserved.