title: An Antibody Biosensor Establishes the Activation of the M1 Muscarinic Acetylcholine Receptor during Learning and Memory.
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 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.
authors.affiliation: From the Medical Research Council Toxicology Unit and.
authors.forename: Adrian J
authors.initials: AJ
authors.lastname: Butcher
authors.affiliation: From the Medical Research Council Toxicology Unit and.
authors.forename: Sophie J
authors.initials: SJ
authors.lastname: Bradley
authors.affiliation: From the Medical Research Council Toxicology Unit and.
authors.forename: Rudi
authors.initials: R
authors.lastname: Prihandoko
authors.affiliation: From the Medical Research Council Toxicology Unit and.
authors.forename: Simon M
authors.initials: SM
authors.lastname: Brooke
authors.affiliation: Eli Lilly and Co. Neuroscience, Erl Wood Manor, Windlesham, Surrey GU20 6PH, United Kingdom.
authors.forename: Adrian
authors.initials: A
authors.lastname: Mogg
authors.affiliation: From the Medical Research Council Toxicology Unit and.
authors.forename: Julie-Myrtille
authors.initials: JM
authors.lastname: Bourgognon
authors.affiliation: From the Medical Research Council Toxicology Unit and.
authors.forename: Timothy
authors.initials: T
authors.lastname: Macedo-Hatch
authors.affiliation: From the Medical Research Council Toxicology Unit and.
authors.forename: Jennifer M
authors.initials: JM
authors.lastname: Edwards
authors.affiliation: Protein and Nucleic Acid Chemistry Laboratory, University of Leicester, Hodgkin Building, Lancaster Road, Leicester LE1 9HN, United Kingdom.
authors.forename: Andrew R
authors.initials: AR
authors.lastname: Bottrill
authors.affiliation: the Department of Molecular and Cell Biology, University of Leicester, Henry Wellcome Building, Lancaster Road, Leicester LE1 9HN, United Kingdom.
authors.forename: R A John
authors.initials: RA
authors.lastname: Challiss
authors.affiliation: Eli Lilly and Co. Neuroscience, Erl Wood Manor, Windlesham, Surrey GU20 6PH, United Kingdom.
authors.forename: Lisa M
authors.initials: LM
authors.lastname: Broad
authors.affiliation: Eli Lilly and Co. Neuroscience, Lilly Corporate Center, Indianapolis, Indiana 46285, and.
authors.forename: Christian C
authors.initials: CC
authors.lastname: Felder
authors.affiliation: From the Medical Research Council Toxicology Unit and tba@le.ac.uk.
authors.forename: Andrew B
authors.initials: AB
authors.lastname: Tobin
mesh_terms: D000818:Animals; D058846:Antibodies, Monoclonal, Murine-Derived / Q000737:chemistry*; D015374:Biosensing Techniques / Q000379:methods*; D056547:CA1 Region, Hippocampal / Q000166:cytology / Q000378:metabolism*; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D007858:Learning / Q000502:physiology*; D008568:Memory / Q000502:physiology*; D051379:Mice; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000502:physiology; D043643:Receptor, Muscarinic M1 / Q000235:genetics / Q000378:metabolism*
chemical_list: D058846:Antibodies, Monoclonal, Murine-Derived; D010750:Phosphoproteins; D043643:Receptor, Muscarinic M1
keywords: G protein-coupled receptor (GPCR); drug discovery; hippocampus; learning; mass spectrometry (MS); memory; phosphorylation
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