Regulation of M₂Muscarinic Acetylcholine Receptor Expression and Signaling by Prolonged Exposure to Allosteric Modulators

Abstract

The effects of prolonged exposure of M 2 muscarinic acetylcholine receptors (mAChRs), stably expressed in Chinese hamster ovary cells, to the allosteric modulators gallamine, alcuronium, and heptane-1,7-bis (dimethyl-3Ј-phthalimidopropyl)-ammonium bromide (C 7 /3Ј-phth) were compared with the effects of the agonist carbachol (CCh) and antagonists atropine and Nmethylscopolamine (NMS). Intact cell saturation binding assays using [3 H]NMS found that pretreatment of the cells for 24 h with CCh caused a significant down-regulation of receptor number, whereas atropine, NMS, and all three allosteric modulators caused receptor up-regulation. Functional assays using a cytosensor microphysiometer to measure whole-cell metabolic rate found no acute effects of gallamine on receptor signaling, whereas atropine seemed to behave as an inverse agonist. Pretreatment of the cells with gallamine (20 M) or atropine (20 nM) resulted in a significant enhancement of the maximal effect evoked by CCh. In contrast, CCh (100 M) pretreatment resulted in a significant reduction in maximal receptor signaling capacity. Time-course experiments revealed that the effects of atropine and gallamine on receptor up-regulation are only visualized after at least 12-h ligand exposure, compared with the more rapid effects of CCh, which achieve steady-state downregulation within 90 min. Additional experiments monitoring CCh-mediated M 2 mAChR internalization in the presence of gallamine revealed that part of the mechanism underlying the effects of the modulator on receptor expression may involve a change in receptor internalization properties. These findings suggest that, like orthosteric ligands, G protein-coupled receptor allosteric modulators also are able to mediate long-term effects on receptor regulation.

Allosteric Modulation of M1 Muscarinic Acetylcholine Receptor Internalization and Subcellular Trafficking

“…In addition to BQCA, modulation of mAChR internalization has been demonstrated previously by the mAChR negative allosteric ligand gallamine at the M 2 mAChR and by LY2033298 at the M 4 mAChR. Gallamine decreases CCh-induced M 2 mAChR internalization and upregulates cell surface receptor levels (41). In contrast, LY2033298 potentiates ACh-induced M 4 mAChR internalization to a greater extent than would be predicted from acute signaling assays, effectively demonstrating a biased action toward regulatory pathways (10).…”

Probing the Molecular Mechanism of Interaction between 4-n-Butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl]-piperidine (AC-42) and the Muscarinic M1Receptor: Direct Pharmacological Evidence That AC-42 Is an Allosteric Agonist

“…Likewise, the peptide ASLW has been shown to activate the chemokine CXCR4 receptor in a manner that is likely to be allosteric (Sachpatzidis et al, 2003). For the mAChRs, Jakubik et al (1996) demonstrated that gallamine and alcuronium could promote receptor activation, but these results are not consistently reproduced May et al, 2005) and may reflect the requirement of particular stoichiometries of receptor to G protein (Jakubik et al, 1998). Earlier radioligand binding studies suggested that the partial agonist McN-A-343 was also an allosteric modulator of M 2 mAChRs , although a subsequent functional study was unable to conclude whether this compound interacted allosterically with very high negative cooperativity or via simple orthosteric competition when tested against carbachol (Christopoulos and Mitchelson, 1997).…”

Contrasting Effects of Allosteric and Orthosteric Agonists on M1Muscarinic Acetylcholine Receptor Internalization and Down-regulation

“…The effects of allosteric ligands on GPCR regulation are poorly defined at present and appear to be receptor subtype-specific. Thus, previous work exploring long-term exposure to allosteric modulators of the M 2 mACh receptor revealed an up-regulation of receptor numbers (May et al, 2005), whereas chronic exposure to AC260584, an allosteric agonist structurally related to AC-42 (Spalding et al, 2002), resulted in no significant changes in M 1 mACh receptor expression (Davis et al, 2005). In contrast, and looking beyond the mACh receptor subfamily, activation of the type 7 metabotropic glutamate receptor by the allosteric agonist AMN082 caused receptor internalization in a manner similar to that elicited by the orthosteric ligand, L-AP4 (Pelkey et al, 2007).…”

“…It is possible for this AC-42/atropine effect to be explained by a complex allosteric interaction, where AC-42 has positive cooperativity with respect to atropine in terms of efficacy, with the functional output in this case being a decrease in receptor internalization/degradation. Both AC-42 and atropine have been independently shown to increase cell-surface mACh receptor expression (May et al, 2005), indicating that both are "agonists" with respect to this endpoint. Even if AC-42 and atropine are highly negatively cooperative with respect to binding (although this has not been directly measured here), if they exhibit a sufficiently positive efficacy cooperativity, then the decrease in constitutive receptor recycling mediated by atropine may be facilitated in the presence of AC-42.…”

“…6). Allosteric modulators of the M 2 mACh receptor have been reported to cause receptor up-regulation in a CHO cell background (May et al, 2005). AC-42 and 77-LH-28-1 can bind to non-M 1 mACh receptor subtypes (Spalding et al, 2002;May et al, 2007;Langmead et al, 2008a), but generally lack efficacy (but see May et al, 2007;Langmead et al, 2008a) (Fig.…”