The effect of C‐terminal tail mutations on the function and internalization patterns of MT1 and MT2 melatonin receptors expressed in COS‐7 cells

Melatonin receptors belong to the family of G‐protein coupled receptors. To determine the amino acid residues important for their function and regulation, mutations were made within the C‐terminal region of MT 1 and MT 2 melatonin receptors. In the first mutation, a cysteine residue in the C‐terminal tail of each receptor gene was converted to alanine. (MT 1 C7.72A and MT 2 C7.77A). This substitution removes any possibility of palmitoylation of the C‐terminal tail, an important component for signaling in other GPCRs. In the second mutation, the C‐terminal tail of each receptor was truncated, deleting all potential phosphorylation sites (MT 1 Y7.64GFP and MT 2 Y7.64GFP). All constructs were GFP‐tagged at the C‐terminal region. The results of this study reveal that the affinity (K D ) and Bmax values of 2‐[ 125 I]‐iodomelatonin for the mutated receptors were not significantly different from those of wild type (wt) receptors. Exposure of wt MT 1 and MT 2 receptors to melatonin (1 μM, 1 h) resulted in their internalization as revealed through fluorescence microscopy. By contrast, truncation of the C‐tails on either receptor prevented their internalization by melatonin whereas the cysteine mutations did not. Both of the MT 1 receptor mutations did not impair the function of the MT 1 receptor, whereas both MT 2 receptor mutations resulted in a complete loss of receptor function. The residues within the C‐terminal tail (and perhaps phosphorylation sites) play an important role in MT 1 and MT 2 receptor internalization. The palmitoylated cysteine residue and the C‐terminal tail do not play a role in the cAMP response in the MT 1 receptor but are important in cAMP‐dependent signaling in the MT 2 receptor. This work was supported by the Pennsylvania Health Assessment Fund.