Receptor phosphorylation is key for agonist-induced internalisation of M₁ muscarinic acetylcholine receptors

M. Scarpa, A. B. Tobin, S. J. Bradley. The Centre for Translational Pharmacology, Institute of Molecular, Cell and Systems Biology, The University of Glasgow, Glasgow, United Kingdom.

Introduction: M₁ muscarinic acetylcholine receptor (M₁ mAChR) agonists and positive allosteric modulators improve cognition and enhance cholinergic signalling in Alzheimer’s disease models^1-3. However, these ligands can show a wide range of adverse effect liabilities associated with on-target overstimulation of the receptor⁴. Recently, our lab have demonstrated that transgenic mice expressing a phoshorylation-deficient mutant of the M₁ mAChR, are more sensitive to adverse responses, such as seizures, in response to administration of M₁-selective agonists. We hypothesise that M₁-agonists stimulating increased phosphorylation and internalisation may be less likely to induce receptor overstimulation and on-target adverse effects. In this study, we aim to understand the role of phosphorylation in agonist-induced internalisation specifically.

Method: Cell monolayers of CHO cells expressing wild-type (WT) M₁ muscarinic acetylcholine receptors (mAChRs) or the phosphorylation-deficient mutant (PD) where all serine residues in the third intracellular loop and C-terminal tail are mutated to alanine, were stimulated for 0, 5, 15 and 30, 60 and 240 minutes with the muscarinic agonist carbachol (CCh; 100 μM). CCh-treated cells were transferred on ice, washed with ice-cold Krebs-Henseleit buffer and radiolabelled with saturating concentration of [³H]-NMS (4 nM) overnight at 4°C to quantify cell surface expression. All data are expressed as means ± S.E.M. of three separate experiments (n=3) and were normalised as percentage of untreated cells for each assay.

Results: In M₁ mAChR WT cells, four hours of CCh stimulation caused a reduction in cell surface receptors to to 58.4% ±2.2% compared to untreated cells, with most of the receptors (29.9% ±7.1%) internalising within the first hour (Fig. 1). In contrast, agonist treatment of cells expressing PD mutant of M₁ AChR caused no change in levels of surface receptors within the first hour of stimulation and 8.4% ±3.4% receptor internalisation after 4 hours , which may be associated with receptor desensitisation or constitutive internalisation rather than agonist-induced internalisation.

Conclusions: Agonist-induced internalisation of M₁ mAChRs is mediated by receptor phosphorylation and it was prevented when receptor phosphorylation sites were removed. Receptor phosphorylation is crucial for agonist-induced internalisation and is likely contributing to an increased adverse effect liability of M₁-selective agonists in the M₁ PD mice.

Reference:

1 Bodick, N. C. et al. (1997). Archives of Neurology 54, 465-473.

2 Puri, V., et al. (2015). Behavioural Brain Research 287, 96-99.

3 Bradley, S. J. et al. (2017). J Clin Invest. 127, 487-499.

4 Rook, J. M. et al. (2017). ACS Chemical Neuroscience 8, 866-883.