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 hMT₂ melatonin receptor and expressed the receptors in HEK-293 cells. Competition of melatonin for 2-[¹²⁵I]-iodomelatonin binding revealed that mutation of Asn 16 in TM4 or His 7 in TM5 of the hMT₂ 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-[¹²⁵I]-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-[¹²⁵I]-iodomelatonin to the hMT₂ melatonin receptor, although equivalent serines (Ser 8 and Ser 12 in TM3) were reported to be critical for melatonin binding to the hMT₁ melatonin receptor. Thus these results are the first to identify residues within the transmembrane regions of the hMT₂ melatonin receptor critical for melatonin binding, highlighting potential structural differences between the MT₁ and MT₂ melatonin receptor binding pockets.