Identification of cysteines involved in ligand binding to the human melatonin MT₂ receptor

In mammals, melatonin activates melatonin MT₁ and MT₂ receptors. Using site-directed mutagenesis and chemical modification, we investigated the role of conserved cysteines in ligand binding. Dithiothreitol inhibited 2-[¹²⁵I]iodomelatonin binding to the FLAG-tagged human melatonin MT₂ receptor without affecting ligand affinity. Alanine substitution of Cys¹¹³ or Cys¹⁹⁰ resulted in a loss of specific 2-[¹²⁵I]iodomelatonin binding, without altering cell surface receptor expression. This suggests that a putative disulfide bond linking Cys¹¹³ and Cys¹⁹⁰ is essential to maintain a proper human melatonin MT₂ receptor conformation for melatonin binding. N-ethylmaleimide alkylation of cysteines inhibited 2-[¹²⁵I]iodomelatonin binding, decreasing both ligand affinity and receptor density. Alkylation of Cys¹⁴⁰ contributes to changes in ligand affinity, while alkylation of Cys¹⁴³ and Cys²¹⁹ reduced binding capacity. We suggest that a disulfide bridge is important for the proper structural conformation of the human melatonin MT₂ receptor to bind melatonin. Cysteines located in receptor regions near the ligand binding site and/or G protein coupling region are involved in N-ethylmaleimide-induced changes in affinity and receptor density.