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J Pineal Res. 2019 Mar;66(2):e12540. doi: 10.1111/jpi.12540. Epub 2019 Jan 17.

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

Cecon E1,2,3, Ivanova A4,5,6, Luka M1,2,3, Gbahou F1,2,3, Friederich A4,5,6, Guillaume JL1,2,3, Keller P7, Knoch K4,5,6, Ahmad R1,2,3, Delagrange P8, Solimena M4,5,6,7, Jockers R1,2,3.

Author information

1
Inserm U1016, Institut Cochin, Paris, France.
2
CNRS UMR 8104, Paris, France.
3
Sorbonne Paris Cité, Univ. Paris Descartes, Paris, France.
4
Molecular Diabetology, Faculty of Medicine, University Hospital, TU Dresden, Dresden, Germany.
5
Faculty of Medicine, Paul Langerhans Institute Dresden (PLID) of the Helmholtz Center Munich at University Hospital Carl Gustav Carus, TU Dresden, Dresden, Germany.
6
German Center for Diabetes Research (DZD), Munich Neuherberg, Germany.
7
Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG), Dresden, Germany.
8
Pôle d'Innovation Thérapeutique Neuropsychiatrie, Institut de Recherches Servier, Croissy, France.

Abstract

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-24-hour 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) superfamily. Similar to most other GPCRs, reliable antibodies recognizing melatonin receptors proved 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 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.

KEYWORDS:

G protein-coupled receptor; melatonin; melatonin receptor; monoclonal antibodies

PMID:
30475390
DOI:
10.1111/jpi.12540

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