An Antibody Biosensor Establishes the Activation of the M1 Muscarinic Acetylcholine Receptor during Learning and Memory.
An Antibody Biosensor Establishes the Activation of the M1 Muscarinic Acetylcholine Receptor during Learning and Memory.
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DOI：
10.1074/jbc.m115.681726
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发表时间：
2016-04-22
期刊：
影响因子：
--
通讯作者：
Tobin AB
中科院分区：
文献类型：
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作者：
Butcher AJ;Bradley SJ;Prihandoko R;Brooke SM;Mogg A;Bourgognon JM;Macedo-Hatch T;Edwards JM;Bottrill AR;Challiss RA;Broad LM;Felder CC;Tobin AB
建立G蛋白偶联受体的体内活化状态不仅表明G蛋白偶联受体的生理作用，而且还将通过建立药物/受体接合来帮助药物发现。在这里，我们开发了一种磷酸化特异性抗体为基础的生物传感器来检测激活的M1毒蕈碱乙酰胆碱受体（M1 mAChR）在体外和体内。质谱磷酸蛋白质组学鉴定了M1 mAChR上的14个磷酸化位点。磷酸特异性抗体，这些网站的四个确定，丝氨酸在位置228（Ser 228）的M1 mAChR显示极低水平的基础磷酸化，正构激动剂刺激显着上调。此外，M1 mAChR阳性变构调节剂，1-（4-甲氧基苄基）-4-氧代-1，4-二氢喹啉-3-羧酸，增强乙酰胆碱介导的丝氨酸228磷酸化。这些数据支持了Ser 228磷酸化是M1 mAChR激活的指标的假设。这进一步支持了在体内的M1 mAChR的磷酸化Ser 228在小鼠海马体的毒蕈碱配体咕诺美林和1-（4-甲氧基苄基）-4-氧代-1，4-二氢喹啉-3-羧酸给药后的鉴定。最后，Ser 228磷酸化被认为是增加在海马CA 1区以下的记忆采集，与CA 1神经元活性的上调密切相关的反应。因此，确定M1 mAChR在Ser 228处的磷酸化状态不仅提供了在体外和体内药物治疗后建立受体活化的手段，而且还允许在记忆获得后映射海马中M1 mAChR的活化状态，从而建立M1 mAChR活化与基于海马的记忆和学习之间的联系。
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.