Identification of cysteines involved in ligand binding to the human melatonin MT(2) receptor.
In mammals, melatonin activates melatonin MT(1) and MT(2) receptors. Using site-directed mutagenesis and chemical modification, we investigated the role of conserved cysteines in ligand binding. Dithiothreitol inhibited 2-[(125)I]iodomelatonin binding to the FLAG-tagged human melatonin MT(2) receptor without affecting ligand affinity. Alanine substitution of Cys(113) or Cys(190) resulted in a loss of specific 2-[(125)I]iodomelatonin binding, without altering cell surface receptor expression. This suggests that a putative disulfide bond linking Cys(113) and Cys(190) is essential to maintain a proper human melatonin MT(2) receptor conformation for melatonin binding. N-ethylmaleimide alkylation of cysteines inhibited 2-[(125)I]iodomelatonin binding, decreasing both ligand affinity and receptor density. Alkylation of Cys(140) contributes to changes in ligand affinity, while alkylation of Cys(143) and Cys(219) reduced binding capacity. We suggest that a disulfide bridge is important for the proper structural conformation of the human melatonin MT(2) 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.
Mseeh
Faika
F
Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, Chicago, IL 60611-3008, USA.
Gerdin
Matthew J
MJ
Dubocovich
Margarita I
MI
D000477:Alkylating Agents / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism*; D018076:DNA, Complementary / Q000187:drug effects / Q000235:genetics; D004229:Dithiothreitol / Q000494:pharmacology; D000939:Epitopes; D005033:Ethylmaleimide / Q000494:pharmacology; D006801:Humans; D007150:Immunohistochemistry; D008024:Ligands; D008550:Melatonin / Q000378:metabolism; D018613:Microscopy, Confocal; D016297:Mutagenesis, Site-Directed / Q000187:drug effects; D009842:Oligopeptides; D010455:Peptides; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D044094:Receptors, Melatonin; D013438:Sulfhydryl Compounds / Q000494:pharmacology; D014162:Transfection
D000477:Alkylating Agents; D018076:DNA, Complementary; D000939:Epitopes; D008024:Ligands; D009842:Oligopeptides; D010455:Peptides; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear; D044094:Receptors, Melatonin; D013438:Sulfhydryl Compounds; C085706:FLAG peptide; D008550:Melatonin; D003545:Cysteine; D005033:Ethylmaleimide; D004229:Dithiothreitol