An antibody biosensor establishes the activation of the M1 muscarinic acetylcholine receptor during learning and memory

Butcher, A. J. et al. (2016) An antibody biosensor establishes the activation of the M1 muscarinic acetylcholine receptor during learning and memory. Journal of Biological Chemistry, 291(17), pp. 8862-8875. (doi: 10.1074/jbc.M115.681726) (PMID:26826123) (PMCID:PMC4861454)

Abstract

Establishing the in vivo activation status of G protein-coupled receptors would not only indicate physiological roles of G protein-coupled receptors but would also aid drug discovery by establishing drug/receptor engagement. Here, we develop a phospho-specific antibody-based biosensor to detect activation of the M-1 muscarinic acetylcholine receptor (M-1 mAChR) in vitro and in vivo. Mass spectrometry phosphoproteomics identified 14 sites of phosphorylation on the M-1 mAChR. Phospho-specific antibodies to four of these sites established that serine at position 228 (Ser(228)) on the M-1 mAChR showed extremely low levels of basal phosphorylation that were significantly up-regulated by orthosteric agonist stimulation. In addition, the M-1 mAChR-positive allosteric modulator, 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid, enhanced acetylcholine-mediated phosphorylation at Ser(228). These data supported the hypothesis that phosphorylation at Ser(228) was an indicator of M-1 mAChR activation. This was further supported in vivo by the identification of phosphorylated Ser(228) on the M-1 mAChR in the hippocampus of mice following administration of the muscarinic ligands xanomeline and 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid. Finally, Ser(228) phosphorylation was seen to increase in the CA1 region of the hippocampus following memory acquisition, a response that correlated closely with up-regulation of CA1 neuronal activity. Thus, determining the phosphorylation status of the M-1 mAChR at Ser(228) not only provides a means of establishing receptor activation following drug treatment both in vitro and in vivo but also allows for the mapping of the activation status of the M-1 mAChR in the hippocampus following memory acquisition thereby establishing a link between M-1 mAChR activation and hippocampus-based memory and learning.

• Item Type: Articles
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Prihandoko, Dr Rudi and Bourgognon, Dr Julie-Myrtille and Tobin, Andrew and Bradley, Dr Sophie
• Authors: Butcher, A. J., Bradley, S. J., Prihandoko, R., Brooke, S. M., Mogg, A., Bourgognon, J.-M., Macedo-Hatch, T., Edwards, J. M., Bottrill, A. R., Challiss, R. A. J., Broad, L. M., Felder, C. C., and Tobin, A. B.
• College/School: College of Medical Veterinary and Life Sciences > School of Molecular Biosciences; College of Medical Veterinary and Life Sciences > School of Health & Wellbeing > Mental Health and Wellbeing
• Journal Name: Journal of Biological Chemistry
• Publisher: American Society for Biochemistry and Molecular Biology, Inc.
• ISSN: 0021-9258
• ISSN (Online): 1083-351X
• Published Online: 29 January 2016
• Copyright Holders: Copyright © 2016 The American Society for Biochemistry and Molecular Biology, Inc.
• First Published: First published in Journal of Biological Chemistry 291(17):8862-8875
• Publisher Policy: Reproduced under a Creative Commons License