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

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.

Pharmacological evidence for transactivation within melatonin MT 2 and serotonin 5‐HT 2C receptor heteromers in mouse brain

“…The soluble fraction was subjected to immunoprecipitation using G protein Sepharose beads (Sigma) with monoclonal antibodies (3 μg/mg proteins) against mouse MT 1 or MT 2 receptors. 20 Samples were denatured overnight at room temperature in SDS-PAGE loading buffer (125 mM Tris/HCl, pH 6.8, 10% SDS, 10% glycerol, and 0.5% bromophenol blue) and the proteins separated by SDS-PAGE. Immunoblot analysis was performed with the indicated antibodies and revealed using secondary antibodies coupled to 680 nm fluorophore using the Odyssey LI-COR infrared fluorescent scanner (ScienceTec, France).…”

Melatonin receptor ligands: A pharmaco‐chemical perspective

“…As previously mentioned, 18 the landscape of receptor molecular pharmacology—particularly melatonin receptors—became even more complex due to the lack of selective antibodies (until recently 156 ) and the complex nature of biasism 19 . The melatonin receptor crystallization works of Stauch et al on MT 1 125 and of Johansson et al on MT 2 126 open new and exciting avenues of research regarding this bicycle motto, which has already begun 135,136 …”

Melatonin promotes regeneration of injured motor axons via MT1 receptors

“…MT 1 signal is lost from presynaptic nerve terminals 24 hours after α-LTx injection, in line with the ongoing fragmentation of the MAT (shown by disappearance of NF staining), although, intriguingly, the receptor becomes expressed by terminal or perisynaptic SC (PSC) ( Figure 3C, lower panels and orthogonal projection). Validation of recently generated monoclonal anti-MT 1 and anti-MT 2 antibodies 22 was performed in brain slices, where neurons expressing MT 1 and MT 2 are detectable in the hypothalamus and in the cerebral cortex ( Figure S3 panels B and C, respectively). 23 No background signal was obtained by incubation with secondary antibodies only ( Figure S3D).…”