Allosteric Site on Muscarinic Acetylcholine Receptors: Identification of Two Amino Acids in the Muscarinic M₂Receptor That Account Entirely for the M₂/M₅Subtype Selectivities of Some Structurally Diverse Allosteric Ligands inN-Methylscopolamine-Occupied Receptors

Abstract

Two epitopes have been identified recently to be responsible for the high-affinity binding of alkane-bisammonium and caracurine V type allosteric ligands to N-methylscopolamine (NMS)-occupied M 2 muscarinic acetylcholine receptors, relative to M 5 receptors: the amino acid M 2 -Thr 423 at the top of transmembrane region (TM) 7 and an epitope comprising the second extracellular loop (o2) of the M 2 receptor including the flanking regions of TM4 and TM5. We aimed to find out whether a single amino acid could account for the contribution of this epitope to binding affinity. Allosteric interactions were investigated in wildtype and mutant receptors in which the orthosteric binding site was occupied by [ 3 H]NMS (5 mM Na,K,P i buffer, pH 7.4, 23°C were replaced by the corresponding amino acids of M 5 revealed that these two amino acids account entirely for the (approximately 100-fold) M 2 /M 5 selectivity of the alkane-bisammonium and the caracurine V type allosteric ligands. At NMSfree M 2 receptors, the caracurine V derivative also displayed approximately 100-fold M 2 /M 5 selectivity, but the double point mutation reduced the M 2 affinity by only ϳ10-fold; thus, additional epitopes may influence selectivity for the free receptors.A three-dimensional model of the M 2 receptor was used to simulate allosteric agent docking to NMS-occupied receptors. M 2 -Tyr 177 and M 2 -Thr 423 seem to be located near the junction of the allosteric and the orthosteric areas of the M 2 receptor ligand binding cavity.Muscarinic acetylcholine receptors are members of the rhodopsin-like family of G protein-coupled receptors, which share general structural motifs, including seven hydrophobic transmembrane helices connected by intracellular and extracellular loops, an extracellular amino terminus, and a cytoplasmic carboxyl terminus. Molecular cloning studies revealed the existence of five (M 1 -M 5 ) muscarinic acetylcholine receptors (Bonner et al., 1987), all of which are susceptible to allosteric modulation (Ellis et al., 1991). The orthosteric acetylcholine binding site seems to be lined by the transmembrane helices (Wess, 1993) and seems to be highly conserved among the five subtypes (Hulme et al., 1990). The allosteric binding site is located at the entrance of the ligand binding pocket (Ellis et al., 1993;Leppik et al., 1994) and is likely to be less well conserved than the orthosteric ligand binding site, thus potentially allowing the design of ligands with greater subtype selectivity (Tuček and Proška, 1995). The family of the muscarinic acetylcholine receptors has been widely studied as a model system for the interaction of allosteric modulators with G protein-coupled receptors (Christopoulos and Kenakin, 2002).It is striking that muscarinic allosteric ligands exhibit generally the highest affinity to the M 2 receptor subtype (Lee and El-Fakahany, 1991;Ellis et al., 1991;Ellis and Seidenberg, 2000). A number of studies aimed to identify receptor This article is dedicated to the late Dr. Stanislav Tuček, Academy of Scien...

Identification of multiple allosteric sites on the M1 muscarinic acetylcholine receptor

“…This site was situated very close to the EXC site, at the extracellular face of TM3 and TM4, and the extracellular loop 2. The existence of both binding sites has been described for the M 2 muscarinic acetylcholine receptor [20].…”

“…In that case, Mohr and co-workers [20] modeled the complex between diallylcaracurine V, N-methylscopolamine and the M 2 muscarinic receptor. They found that the binding site was located in a similar place as the binding site described in the present study (in this case, the M 1 subtype).…”

A Novel Multivalent Ligand That Bridges the Allosteric and Orthosteric Binding Sites of the M2 Muscarinic Receptor

“…From mutagenesis studies, amino acid residues essential for binding the well characterized allosteric modulators gallamine and W84 include the M 2 -EDGE sequence, M 2 -Tyr 177 and M 2 -Thr 423 (Leppik et al, 1994;Voigtlä nder et al, 2003). Of these, only M 2 -D 173 and M 2 -Y 177 can be found in other subtypes (Bonner et al, 1988).…”

Monoclonal anti-β1-adrenergic receptor antibodies activate G protein signaling in the absence of β-arrestin recruitment

“…34 A number of groups have described a critical role for ECL2 in the binding of orthosteric and allosteric ligands to GPCRs, 35 including ligand activation of the C5a receptor 36 and residues that contribute to ligand specificity between the muscarinic M 2 and M 5 receptor subtypes. 37 It is also thought that receptor antibodies generated by immunization can act in a different way to autoantibodies isolated from patient sera. 38 The agonist activity attributed to β 1 AR autoantibodies is less prone to induce receptor desensitization than classical agonist ligands 39 and, when bound at the same time as natural agonists, the autoantibodies are able to modulate the receptor response.…”