Identification of cysteines involved in ligand binding to the human melatonin MT2 receptor
| Autor: | Matthew J. Gerdin, Faika Mseeh, Margarita L. Dubocovich |
|---|---|
| Rok vydání: | 2002 |
| Předmět: |
Alkylating Agents
DNA Complementary Stereochemistry Receptors Melatonin Receptors Cytoplasmic and Nuclear Receptors Cell Surface Ligands Transfection Dithiothreitol Melatonin Epitopes chemistry.chemical_compound Cell surface receptor medicine Humans Cysteine Sulfhydryl Compounds Receptor Pharmacology Microscopy Confocal Chemistry Ligand binding assay Ligand (biochemistry) Immunohistochemistry Biochemistry Ethylmaleimide Melatonin binding Mutagenesis Site-Directed Peptides Oligopeptides medicine.drug |
| Zdroj: | European Journal of Pharmacology. 449:29-38 |
| ISSN: | 0014-2999 |
| DOI: | 10.1016/s0014-2999(02)01903-9 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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