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

Adrian J Butcher,Sophie J Bradley,Rudi Prihandoko,Simon M Brooke,Adrian Mogg,Julie-Myrtille Bourgognon,Timothy Macedo-Hatch,Jennifer M Edwards,Andrew R Bottrill,R.A.John Challiss,Lisa M Broad,Christian C Felder,Andrew B Tobin

doi:10.1074/jbc.m115.681726

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 (Ser228) 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 Ser228. These data supported the hypothesis that phosphorylation at Ser228 was an indicator of M1 mAChR activation. This was further supported in vivo by the identification of phosphorylated Ser228 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, Ser228 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 Ser228 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.

Highlights

From the ‡Medical Research Council Toxicology Unit and ¶Protein and Nucleic Acid Chemistry Laboratory, University of Leicester, Hodgkin Building, Lancaster Road, Leicester LE1 9HN, United Kingdom, §Eli Lilly and Co
Determination of the Phosphorylation Sites on the M1 mAChR—Our initial evaluation of the phosphorylation status of the M1 mAChR was conducted on Chinese hamster ovary cells expressing the mouse M1 mAChR (CHO-M1 cells) stimulated with the natural ligand acetylcholine or the M1/M4-preferring agonist xanomeline (Fig. 1A) [19, 20]
The phospho-specific nature of the antibodies generated was tested by immunoprecipitation of M1 mAChRs from CHO-M1 cells followed by treatment of the immunoprecipitate with calf intestinal alkaline phosphatase (CIAP) to remove the phosphate groups (Fig. 2A)

Summary

Introduction

From the ‡Medical Research Council Toxicology Unit and ¶Protein and Nucleic Acid Chemistry Laboratory, University of Leicester, Hodgkin Building, Lancaster Road, Leicester LE1 9HN, United Kingdom, §Eli Lilly and Co. We develop a phospho-specific antibody-based biosensor to detect activation of the M1 muscarinic acetylcholine receptor (M1 mAChR) in vitro and in vivo.

Results

Conclusion