Detection of recombinant and endogenous mouse melatonin receptors by monoclonal antibodies targeting the C-terminal domain.

Melatonin receptors play important roles in the regulation of circadian and seasonal rhythms, sleep, retinal functions, the immune system, depression and type 2 diabetes development. Melatonin receptors are approved drug targets for insomnia, non-24h sleep-wake disorders and major depressive disorders. In mammals, two melatonin receptors (MTRs) exist, MT1 and MT2 , belonging to the G protein-coupled receptor (GPCR) super-family. Similar to most other GPCRs, reliable antibodies recognizing melatonin receptors prooved to be difficult to obtain. Here we describe the development of the first monoclonal antibodies (mABs) for mouse MT1 and MT2 . Purified antibodies were extensively characterized for specific reactivity with mouse, rat and human MT1 and MT2 by western blot, immunoprecipitation, immunofluorescence and proximity ligation assay. Several mABs were specific for either mouse MT1 or MT2 . None of the mABs cross-reacted with rat MTRs, and some were able to react with human MTRs. The specificity of the selected mABs was validated by immunofluorescence microscopy in three established locations (retina, suprachiasmatic nuclei, pituitary gland) for MTR expression in mice using MTR KO mice as control. MT2 expression was not detected instead in mouse insulinoma MIN6 cells or pancreatic beta-cells. Collectively, we report the first monoclonal antibodies recognizing recombinant and native mouse melatonin receptors that will be valuable tools for future studies. This article is protected by copyright. All rights reserved.