Antibody sensors of G protein-coupled receptor activation reveals M1-muscarinic receptor activity during memory acquisition

A Butcher, S Bradley, A Tobin. Medical Research Council Toxicology Unit, Leicester, UK

The canonical view of “homologous” G protein-coupled receptor (GPCR) phosphorylation states that only the agonist-occupied receptor acts as a substrate for the G protein-coupled receptor kinase (GRK) family. In this model, agonist occupation mediates a conformational change in a receptor from an inactive “R” conformation to an active “R*” state that unmasks phospho-acceptor sites, usually in the C-terminal tail or third intracellular loop, allowing for phosphorylation by one or more of the GRKs. This mechanism is likely to operate in concert with other processes, such as the localization of the GRKs to the activated receptor through, for example, interaction with liberated G protein βγ-subunits as seen for GRK2/3. There appear also to be some subtle feedback mechanisms that regulate the activity of the GRKs, which otherwise are generally considered to be constitutively active. Despite these important regulatory processes, the primary event that determines the phosphorylation status of GPCRs is the proportion of receptors that adopt an active R* conformation. In this sense, measurement of the phosphorylation status of a GPCR could act as a measure of the active conformation of a receptor and establishing a method of detecting the active state of a receptor in vivo would be highly desirable both in terms of understanding the basic biology of a receptor subtype and also in drug discovery.

Here we have examined the phosphorylation status of the M1 muscarininc receptor. Using 32Pi metabolic labelling of CHO cells expressing the M1 muscarininc receptor we found that upon stimulation with muscarinic receptor agonists acetylcholine (100 µM) or xanomeline (10 µM) M1 muscarininc receptor phosphorylation was increased 1.77 +/- 0.05 (n=4) fold-over-basal and 1.77 +/- 0.15 (n=4) fold-over-basal, respectively. To further examine M1 muscarininc receptor phosphorylation, we have employed a proteomic approach using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) to identify 14 sites of phosphorylation on the M1 receptor. Twelve of these are in the 3rd intracellular loop and 2 sites in the C-terminal tail. We have used these data to generate phospho-specific antibodies to target four of the sites and have used these to probe the phosphorylation status of these sites in M1 muscarinic receptors expressed in CHO cells. When cells were stimulated with acetylcholine or xanomeline similar increases in agonist-regulated phosphorylation were observed at each of the four sites suggesting that the pattern of phosphorylation induced by these two agonists at the M1 muscarinic receptor is the same. In addition, we have used these antibodies to probe the phosphorylation status of M1 muscarinic receptors in the hippocampus and cerebral cortex and in this manner determine if the phosphorylation profile of the M1 muscarinic receptor is dependent on the brain area and cell-type in which the receptor is expressed.

We have extended these studies by using one of these antibodies, anti-phospho-serine 228, which appears to be uniquely sensitive to agonist stimulation, and have used this antibody to determine changes in M1 muscarinic receptor phosphorylation in the hippocampus of C57BL6N mice using immunohistochemistry. In so doing we are able to determine M1 muscarinic receptor activation in the hippocampus following drug treatment and during memory acquisition.