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

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, mt1 and MT2, the existence of a novel putative melatonin receptor,MT3 , 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 mt1 and MT2. In this report, we present the purification of MT3 from Syrian hamster kidney and its identification as the hamster homologue of the human quinone reductase 2 (QR2, EC 1.6.99.2). Our purification strategy included the use of an affinity chromatography step which was crucial in purifying MT3 to homogeneity. The protein was sequenced by tandem mass spectrometry and shown to align with 95% identity with human QR2. After transfection of CHO-K1 cells with the human QR2 gene, not only did the QR2 enzymatic activity appear, but also the melatonin-binding sites with MT3 characteristics, both being below the limit of detection in the native cells. We further confronted inhibition data fromMT3 binding and QR2 enzymatic activity obtained from samples of Syrian hamster kidney or QR2-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 siteMT3 as the quinone reductase QR2and open perspectives as to the function of this enzyme, known so far mainly for its detoxifying properties.