C-terminal domains within human MT1 and MT2 melatonin receptors are involved in internalization processes

• Published in: Journal of pineal research 2008-09, Vol.45 (2), p.212-218
• Main Authors: Sethi, Shalini, Adams, Wendy, Pollock, John, Witt-Enderby, Paula A.
• Format: Article
• Language: English
• Subjects: Animals; Arrestins - metabolism; beta-Arrestins; Binding Sites - genetics; Biological and medical sciences; Cercopithecus aethiops; COS Cells; Cyclic AMP - metabolism; desensitization; Endocytosis - physiology; Fundamental and applied biological sciences. Psychology; Humans; internalization; Melatonin - metabolism; melatonin receptors; Microscopy, Confocal; Models, Biological; mutagenesis; Mutation; Receptor, Melatonin, MT1 - genetics; Receptor, Melatonin, MT1 - physiology; Receptor, Melatonin, MT2 - genetics; Receptor, Melatonin, MT2 - physiology; Vertebrates: endocrinology
• ISSN: 0742-3098; 1600-079X
• DOI: 10.1111/j.1600-079X.2008.00579.x

:  Melatonin, a molecule implicated in a variety of diseases, including cancer, often exerts its effects through G‐protein‐coupled melatonin receptors, MT1 and MT2. In this study, we sought to understand further the domains involved in the function and desensitization patterns of these receptors through site‐directed mutagenesis. Two mutations were constructed in the cytoplasmic C‐terminal tail of each receptor subtype: (i) a cysteine residue in the C‐terminal tail was mutated to alanine, thus removing a putative palmitoylation site, and a site possibly required for normal receptor function (MT1C7.72A and MT2C7.77A) and (ii) the C‐terminal tail in the MT1 and MT2 receptors was truncated, removing the putative phosphorylation and β‐arrestin binding sites (MT1Y7.64 and MT2Y7.64). These mutations did not alter the affinity of 2‐[125I]‐iodomelatonin binding to the MT1 or MT2 receptors. Using confocal microscopy, it was determined that the putative palmitoylation site (cysteine residue) did not play a role in receptor internalization; however, this residue was essential for receptor function, as determined by 3′,5′‐cyclic adenosine monophosphate (cAMP) accumulation assays. Truncation of the C‐terminal tail of both receptors (MT1Y7.64 and MT2Y7.64) inhibited internalization as well as the cAMP response, suggesting the importance of the C‐terminal tail in these receptor functions.