Understanding the physiological role of endogenous allosteric modulators at the muscarinic acetylcholine receptors

[Speaker] Ee Von Moo:1
[Co-author] Patrick M Sexton:1, Arthur Christopoulos:1, Celine Valant:1
1:Monash Institute of Pharmaceutical Sciences, Australia

Allosteric binding sites on G protein-coupled receptor (GPCR) can be targeted by synthetic or natural (endogenous) molecules (van der Westhuizen et al., 2015). However, the (patho)physiological role(s) of many endogenous allosteric modulators remain poorly understood. One interesting example is major basic protein (MBP), a highly basic peptide that acts as a negative allosteric modulator (NAM) of acetylcholine (ACh) at airway M2 muscarinic acetylcholine receptors (mAChR; Jacoby et al., 1993). We hypothesized that, in addition to MBP, other endogenous basic peptides, including the antimicrobial, LL-37, involved in chemotaxis, maturation of immune cells and apoptosis (Kahlenberg et al., 2013) could also interact allosterically with the M2 mAChRs and have major physiological impacts. In this study, we aim to characterise the pharmacological properties and the putative (patho)physiological roles of LL-37 at mAChRs. Using IMR-32, a native cell line endogenously expressing human M2 mAChRs and mouse tissues predominantly expressing mouse M2 mAChRs, we performed [3H]NMS radioligand binding and [35S]GTPgS turnover as a functional measure of receptor activation, to assess the allosteric effect of LL-37.
We found that LL-37 mediated a concentration-dependent partial inhibition of the antagonist [3H]NMS binding in IMR-32 cells and mouse cardiac tissues, a hallmark of allostery. Additionally, LL-37 also negatively modulated ACh-mediated G protein activation in mouse hypothalamus preparations. Our results suggest that LL-37 is a NAM of antagonist binding and agonist function at the M2 mAChR. The M2 mAChRs are highly expressed on both neuronal and non-neuronal cells, including immune cells and epithelial cells, and are known to be involved in their survival outcome. In the context of inflammation and cancer, when LL-37 is highly expressed, the antagonism of M2 mAChR activity by the peptide could therefore have unappreciated (patho)physiological consequences.

van der Westhuizen ET al. (2015) J Pharm Exp Ther 353(2):246-60.
Jacoby et al. (1993) J Clin Invest 91:1314-1318.
Kahlenberg et al. (2013) J immunol 191(10):4893-901.

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