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

被引:30
作者
Butcher, Adrian J. [1 ,6 ]
Bradley, Sophie J. [1 ,6 ]
Prihandoko, Rudi [1 ]
Brooke, Simon M. [1 ]
Mogg, Adrian [3 ]
Bourgognon, Julie-Myrtille [1 ]
Macedo-Hatch, Timothy [1 ]
Edwards, Jennifer M. [1 ]
Bottrill, Andrew R. [2 ]
Challiss, R. A. John [5 ]
Broad, Lisa M. [3 ]
Felder, Christian C. [4 ]
Tobin, Andrew B. [1 ,6 ]
机构
[1] Univ Leicester, MRC, Toxicol Unit, Hodgkin Bldg,Lancaster Rd, Leicester LE1 9HN, Leics, England
[2] Univ Leicester, Prot & Nucle Acid Chem Lab, Hodgkin Bldg,Lancaster Rd, Leicester LE1 9HN, Leics, England
[3] Eli Lilly & Co, Neurosci, Windlesham GU20 6PH, Surrey, England
[4] Eli Lilly & Co, Lilly Corp Ctr, Neurosci, Indianapolis, IN 46285 USA
[5] Univ Leicester, Dept Mol & Cell Biol, Henry Wellcome Bldg,Lancaster Rd, Leicester LE1 9HN, Leics, England
[6] Univ Glasgow, Inst Mol Cell & Syst Biol, Glasgow G12 8QQ, Lanark, Scotland
基金
英国生物技术与生命科学研究理事会; 英国医学研究理事会;
关键词
PROTEIN-COUPLED RECEPTORS; ANTIPSYCHOTIC-LIKE ACTIVITY; ALLOSTERIC MODULATION; BETA-ARRESTIN; MUTANT MICE; AGONIST; HIPPOCAMPUS; PHOSPHORYLATION; SCOPOLAMINE; XANOMELINE;
D O I
10.1074/jbc.M115.681726
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:8862 / 8875
页数:14
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