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

Abstract

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.