Abstract
The main step of classical desensitization of a receptor, by mean of its disappearance from the plasma membrane, is its internalization. This is a key factor in the regulation of agonist-mediated signaling pathways, as it most of the time stops the activation of the receptor. Internalization is thus important to evaluate, as a complementary information for a natural ligand or an alternative synthetic agonist. Enzyme fragment complementation is an elegant but delicate way to measure this phenomenon, by fusing two complementary parts of an enzyme to two partners, and to measure the activity of the reconstituted enzyme upon complexation of the partners. In the present chapter, using two parts of β-galactosidase, one fused to the C-terminus of the MT1 receptor, the other to an endosomal protein, one can measure the formation of the complex; thus, the transfer of the receptor to the endosome from which MT1 will be recirculated.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Liu L, Labani N, Cecon E et al (2019) Melatonin target proteins: too many or not enough? Front Endocrinol (Lausanne) 10:791. https://doi.org/10.3389/fendo.2019.00791
Calebiro D, Godbole A (2018) Internalization of G-protein-coupled receptors: implication in receptor function, physiology and diseases. Best Pract Res Clin Endocrinol Metab 32:83–91. https://doi.org/10.1016/j.beem.2018.01.004
von Moo E, van Senten JR, Bräuner-Osborne H et al (2021) Arrestin-dependent and -independent internalization of G protein-coupled receptors: methods, mechanisms, and implications on cell signaling. Mol Pharmacol 99:242–255. https://doi.org/10.1124/molpharm.120.000192
Kenakin T (2015) The measurement of receptor signaling bias. Methods Mol Biol 1335:163–176. https://doi.org/10.1007/978-1-4939-2914-6_11
Legros C, Dupré C, Brasseur C et al (2020) Characterization of the various functional pathways elicited by synthetic agonists or antagonists at the melatonin MT1 and MT2 receptors. Pharmacol Res Perspect 8:e00539. https://doi.org/10.1002/prp2.539
Boutin JA, Legros C (2020) The five dimensions of receptor pharmacology exemplified by melatonin receptors: an opinion. Pharmacol Res Perspect 8:e00556. https://doi.org/10.1002/prp2.556
Kenakin T (2021) Biased signaling as allosteric probe dependence. Cell Signal 79:109844. https://doi.org/10.1016/j.cellsig.2020.109844
Jackson DA, Yanofsky C (1969) Restoration of enzymic activity by complementation in vitro between mutant alpha subunits of tryptophan synthetase and between mutant subunits and fragments of the alpha subunit. J Biol Chem 244:4539–4546
Ma X, Leurs R, Vischer HF (2021) NanoLuc-based methods to measure β-Arrestin2 recruitment to G protein-coupled receptors. Methods Mol Biol 2268:233–248. https://doi.org/10.1007/978-1-0716-1221-7_16
Dupré C, Bruno O, Bonnaud A et al (2018) Assessments of cellular melatonin receptor signaling pathways: β-arrestin recruitment, receptor internalization, and impedance variations. Eur J Pharmacol 818:534–544. https://doi.org/10.1016/j.ejphar.2017.11.022
Olson KR, Eglen RM (2007) β-galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol 5:137–144. https://doi.org/10.1089/adt.2006.052
Tsvetanova NG, Irannejad R, von Zastrow M (2015) G protein-coupled receptor (GPCR) signaling via heterotrimeric G proteins from endosomes. J Biol Chem 290:6689–6696. https://doi.org/10.1074/jbc.R114.617951
Mu FT, Callaghan JM, Steele-Mortimer O et al (1995) EEA1, an early endosome-associated protein. EEA1 is a conserved alpha-helical peripheral membrane protein flanked by cysteine “fingers” and contains a calmodulin-binding IQ motif. J Biol Chem 270:13503–13511. https://doi.org/10.1074/jbc.270.22.13503
Marunaka K, Fujii N, Kimura T et al (2019) Rescue of tight junctional localization of a claudin-16 mutant D97S by antimalarial medicine primaquine in Madin-Darby canine kidney cells. Sci Rep 9:9647. https://doi.org/10.1038/s41598-019-46250-4
Cabaniols J-P, Ouvry C, Lamamy V et al (2010) Meganuclease-driven targeted integration in CHO-K1 cells for the fast generation of HTS-compatible cell-based assays. J Biomol Screen 15:956–967. https://doi.org/10.1177/1087057110375115
Acknowledgments
The authors wish to thank Dr. Philippe Delagrange for decades of melatonin-related discussions.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Dupré, C., Legros, C., Boutin, J.A. (2022). Functionality of Melatonin Receptors: Internalization. In: Jockers, R., Cecon, E. (eds) Melatonin. Methods in Molecular Biology, vol 2550. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2593-4_23
Download citation
DOI: https://doi.org/10.1007/978-1-0716-2593-4_23
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-2592-7
Online ISBN: 978-1-0716-2593-4
eBook Packages: Springer Protocols