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

TLDR

Melatonin regulates circadian rhythm and a novel receptor MT3 was hypothesized from a rapidly exchanging binding site in hamster tissues. The study aimed to purify MT3 from Syrian hamster kidney and identify it as the hamster homologue of human quinone reductase 2. MT3 was purified to homogeneity using an affinity chromatography step. Sequencing showed 95 % identity to human QR2, transfection of CHO‑K1 cells expressed QR2 enzymatic activity and MT3‑like melatonin binding, and inhibition data from kidney and overexpressing cells correlated, confirming MT3 as QR2 and suggesting new functional roles.

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, mt₁ and MT₂, the existence of a novel putative melatonin receptor,<i>MT₃</i> , 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 <i>MT₃</i> 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 <i>MT₃</i> 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 <i>QR₂</i> gene, not only did the QR₂ enzymatic activity appear, but also the melatonin-binding sites with <i>MT₃</i> characteristics, both being below the limit of detection in the native cells. We further confronted inhibition data from<i>MT₃</i> 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 site<i>MT₃</i> as the quinone reductase QR₂and open perspectives as to the function of this enzyme, known so far mainly for its detoxifying properties.