Journal
JOURNAL OF PINEAL RESEARCH
Volume 45, Issue 2, Pages 212-218Publisher
WILEY
DOI: 10.1111/j.1600-079X.2008.00579.x
Keywords
desensitization; internalization; melatonin receptors; mutagenesis
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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 (MT(1)C7.72A and MT(2)C7.77A) and (ii) the C-terminal tail in the MT1 and MT2 receptors was truncated, removing the putative phosphorylation and beta-arrestin binding sites (MT(1)Y7.64 and MT(2)Y7.64). These mutations did not alter the affinity of 2-[I-125]-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 (MT(1)Y7.64 and MT(2)Y7.64) inhibited internalization as well as the cAMP response, suggesting the importance of the C-terminal tail in these receptor functions.
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