The end of a myth: cloning and characterization of the ovine melatonin MT2 receptor

Background and purpose: For many years, it was suspected that sheep expressed only one melatonin receptor (closely resembling MT 1 from other mammal species). Here we report the cloning of another melatonin receptor, MT 2 , from sheep. Experimental approach: Using a thermo-resistant reverse transcriptase and polymerase chain reaction primer set homologous to the bovine MT 2 mRNA sequence, we have cloned and characterized MT 2 receptors from sheep retina. Key results: The ovine MT 2 receptor presents 96%, 72% and 67% identity with cattle, human and rat respectively. This MT 2 receptor stably expressed in CHO-K1 cells showed high-affinity 2[ 12S I]-iodomelatonin binding (K D = 0.04 nM). The rank order of inhibition of 2[ 125 I]-iodomelatonin binding by melatonin, 4-phenyl-2-propionamidotetralin and luzindole was similar to that exhibited by MT 2 receptors of other species (melatonin > 4-phenyl-2-propionamidotetralin > luzindole). However, its pharmacological profile was closer to that of rat, rather than human MT 2 receptors. Functionally, the ovine MT 2 receptors were coupled to G i proteins leading to inhibition of adenylyl cyclase, as the other melatonin receptors. In sheep brain, MT 2 mRNA was expressed in pars tuberalis, choroid plexus and retina, and moderately in mammillary bodies. Real-time polymerase chain reaction showed that in sheep pars tuberalis, premammillary hypothalamus and mammillary bodies, the temporal pattern of expression of MT 1 and MT 2 mRNA was not parallel in the three tissues. Conclusion and implications: Co-expression of MT 1 and MT 2 receptors in all analysed sheep brain tissues suggests that MT 2 receptors may participate in melatonin regulation of seasonal anovulatory activity in ewes by modulating MT 1 receptor action.