Identification of the Melatonin-binding SiteMT 3 as the Quinone Reductase 2

Abstract

The regulation of the circadian rhythm is relayed from the central nervous system to the periphery by melatonin, a hormone synthesized at night in the pineal gland. Besides two melatonin G-coupled receptors, mt 1 and MT 2 , the existence of a novel putative melatonin receptor, MT 3 , was hypothesized from the observation of a binding site in both central and peripheral hamster tissues with an original binding profile and a very rapid kinetics of ligand exchange compared with mt 1 and MT 2 . In this report, we present the purification of MT 3 from Syrian hamster kidney and its identification as the hamster homologue of the human quinone reductase 2 (QR 2 , EC 1.6.99.2). Our purification strategy included the use of an affinity chromatography step which was crucial in purifying MT 3 to homogeneity. The protein was sequenced by tandem mass spectrometry and shown to align with 95% identity with human QR 2 . After transfection of CHO-K1 cells with the human QR 2 gene, not only did the QR 2 enzymatic activity appear, but also the melatonin-binding sites with MT 3 characteristics, both being below the limit of detection in the native cells. We further confronted inhibition data from MT 3 binding and QR 2 enzymatic activity obtained from samples of Syrian hamster kidney or QR 2 -overexpressing Chinese hamster ovary cells, and observed an overall good correlation of the data. In summary, our results provide the identification of the melatonin-binding site MT 3 as the quinone reductase QR 2 and open perspectives as to the function of this enzyme, known so far mainly for its detoxifying properties.Melatonin, a neurohormone produced at night in the pineal gland, is suspected to relay to the peripheral organs the circadian rhythm detected by the central nervous system. Several high affinity melatonin receptors have been identified to date, among which the mt 1 (1) and MT 2 (2) receptors have been cloned from human tissues. The pharmacology of these two receptors is well documented, and several compounds, including melatonin, are ligands with picomolar binding affinity (for review, see Ref.3). Another putative melatonin receptor was identified on pharmacological grounds, with lower melatonin affinity (nanomolar range), very rapid ligand association/dissociation kinetics, and an original pharmacological profile (4 -6). In line with mt 1 and MT 2 receptors, this putative receptor was named MT 3 , according to the nomenclature recommendations of the IUPHAR (7). So far, the known inhibitors of MT 3 hardly reach the nanomolar range and encompass an unusually large structural diversity of highly hydrophobic cyclic or polycyclic compounds (Refs. 5 and 6, and for review, see Ref.3).1 All pharmacological investigations on mt 1 , MT 2 , and MT 3 were performed using the radioligand [125 I]melatonin, a ligand with high affinity for mt 1 and MT 2 (K d ϭ 10 -200 pM) and with lower affinity for MT 3 (K d ϭ 3-9 nM). The hamster kidney, liver, and brain have been used as model tissues for MT 3 pharmacological studies, and our recent data con...

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