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An Antibody Biosensor Establishes the Activation of the M1 Muscarinic Acetylcholine Receptor during Learning and Memory.

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posted on 2016-12-01, 15:38 authored by Adrian J. Butcher, Sophie J. Bradley, Rudi Prihandoko, Simon M. Brooke, A. Mogg, Julie-Myrtille Bourgognon, Timothy Macedo-Hatch, Jennifer M. Edwards, Andrew R. Bottrill, R. A. John Challiss, L. M. Broad, C. C. Felder, Andrew B. Tobin
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 M1 muscarinic acetylcholine receptor (M1 mAChR) in vitro and in vivo Mass spectrometry phosphoproteomics identified 14 sites of phosphorylation on the M1 mAChR. Phospho-specific antibodies to four of these sites established that serine at position 228 (Ser(228)) on the M1 mAChR showed extremely low levels of basal phosphorylation that were significantly up-regulated by orthosteric agonist stimulation. In addition, the M1 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 M1 mAChR activation. This was further supported in vivo by the identification of phosphorylated Ser(228) on the M1 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 M1 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 M1 mAChR in the hippocampus following memory acquisition thereby establishing a link between M1 mAChR activation and hippocampus-based memory and learning.

Funding

This work was supported by Lilly grants (to C. C. F., L. M. B., and A. M.), a Lilly LRAP grant (to J. M. B.), a Medical Research Council program leader grant provided by the Medical Research Council Toxicology Unit (to A. B. T., S. J. B., A. J. B., T. M. H., and J. M. E.), a Medical Research Council Integrative Toxicology Training Partnership Ph.D. studentship scheme (to S. M. B.), and the Heptares Therapeutics and Biotechnology and Biological Sciences Research Council Grant BB/L02781X/1 (to R. P.).

History

Citation

Journal of Biological Chemistry, 2016, 291 (17), pp. 8862-8875

Author affiliation

/Organisation/COLLEGE OF MEDICINE, BIOLOGICAL SCIENCES AND PSYCHOLOGY/MBSP Non-Medical Departments/Molecular & Cell Biology

Version

  • VoR (Version of Record)

Published in

Journal of Biological Chemistry

Publisher

American Society for Biochemistry and Molecular Biology

issn

0021-9258

eissn

1083-351X

Acceptance date

2016-01-27

Copyright date

2016

Available date

2016-12-01

Publisher version

http://www.jbc.org/content/291/17/8862

Language

en

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