An Antibody Biosensor Establishes the Activation of the M ₁ Muscarinic Acetylcholine Receptor during Learning and Memory * ?

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.