C‐terminal domains within human MT 1 and MT 2 melatonin receptors are involved in internalization processes

  Melatonin, a molecule implicated in a variety of diseases, including cancer, often exerts its effects through G‐protein‐coupled melatonin receptors, MT 1 and MT 2 . 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 MT 1 and MT 2 receptors was truncated, removing the putative phosphorylation and β‐arrestin binding sites (MT 1 Y7.64 and MT 2 Y7.64). These mutations did not alter the affinity of 2‐[ 125 I]‐iodomelatonin binding to the MT 1 or MT 2 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.