Transmembrane Domains 4 and 7 of the M1Muscarinic Acetylcholine Receptor Are Critical for Ligand Binding and the Receptor Activation Switch

Abstract

Activation of the muscarinic acetylcholine receptors requires agonist binding followed by a conformational change, but the ligand binding and conformationswitching residues have not been completely identified. Systematic alanine-scanning mutagenesis has been used to assess residues 142-164 in transmembrane helix 4 and 402-421 in transmembrane helix 7 of the M 1 muscarinic acetylcholine receptor. Several inward-facing amino acid side chains in the exofacial parts of transmembrane helices 4 and 7 contribute to acetylcholine binding. Alanine substitution of the aromatic residues in this group reduced signaling efficacy, suggesting that they may form part of a charge-stabilized aromatic cage, which triggers rotation and movement of the transmembrane helices. The mutation of adjacent residues modulated receptor activation, either reducing signaling or causing constitutive activation. In the buried endofacial section of transmembrane helix 7, alanine substitution mutants of the conserved NSXXNPXXY motif displayed strongly reduced signaling efficacy, despite having increased or unchanged acetylcholine affinity. These residues may have dual functions, forming intramolecular contacts that stabilize the receptor in the inactive ground state, but that are broken, allowing them to form new intramolecular bonds in the activated state. This conformational rearrangement is critical to produce a G protein binding site and may represent a key mechanism of receptor activation. Muscarinic acetylcholine receptors (mAChRs)1 belong to the rhodopsin-like family of 7-transmembrane (7-TM) receptors. Agonists bind to these receptors at the extracellular end. This leads to the binding and activation of a G protein at the intracellular face. These receptors are characterized by the possession of a particular set of evolutionarily conserved amino acids, mostly located in the 7-TM helices, implying that they may share a common mechanism of activation. However, the topography of the binding pockets and the conformational changes related to agonist-induced receptor activation are incompletely understood. In recent studies on the M 1 mAChR, we have identified ligand contact residues in TM 3 (1), 5 (2), and 6 (3) and inferred a strip of residues in TM 3 contributing to the activated state of the M 1 mAChR by using alanine-scanning mutagenesis, or cysteine-scanning mutagenesis. The three-dimensional crystal structure of the ground state of rhodopsin at 2.8 Å (4) has provided a framework for modeling the mAChRs and allowed us to interpret most of the information obtained by mutagenesis studies on the M 1 mAChR.In the initial projection map of rhodopsin (5), TM 4 appeared as an outlier of the helical bundle, with a large lipid-exposed surface and few polar residues. Consequently, its function has received little attention. However, a recent photo-activated chromophore cross-linking study of rhodopsin has shown a flip-over of the ionone ring from the neighborhood of TM 6 to TM 4 during receptor activation, implying that a substantial movement of TM ...

Generation of a homology model of the human histamine H3 receptor for ligand docking and pharmacophore-based screening

“…Based on a better overall structural preservation of the model and a more reasonable hydrogenbond pattern evolving between residues T3.37, Y4.57 and E5.46, the placement of Y4.57 into the binding site was favoured. Such a placement is also in accordance with the observation that for residue 4.57 an involvement in ligand binding or receptor activation has been reported for other GPCRs [40][41][42]. For the clash involving Y2.61, W3.28, W7.40, and W7.43 too many placements and corresponding start conformations for MD-simulations would have resulted when following this strategy.…”

“…As can be seen from Fig. 10, the mean docking score for the hH 3 R actives lay in the cluster of [40,50] and was thus significantly shifted by a value of 20 to higher docking scores, when compared to the mean value of the distribution of WDI and MDB compounds ( [20,30]), thereby indicating that by docking into the hH 3 R binding site, significant higher scores were in average obtained for validated hH 3 R ligands. At the arbitrary chosen GoldScore cut-off of 40, at which 66.5% of the validated hH 3 R antagonists would have been retrieved, 87% of the WDI and MDB compounds were filtered out, resulting in 1720 structures, which were further analysed by visual inspection.…”

Characterization of a CNS penetrant, selective M1muscarinic receptor agonist, 77‐LH‐28‐1

“…The mAChR agonists developed to date, such as xanomeline and sabcomeline, improve cognition preclinically, although evidence of clinical efficacy has been confounded by peripheral effects such as sweating, nausea and diarrhoea, which are believed to be attributed to the relatively non-selective mAChR activity profile of these compounds (Wood et al, 1999). This lack of selectivity is believed to be due to binding to the orthosteric ACh binding site that is highly conserved across the mAChR family (Spalding et al, 1994;Baldwin et al, 1997;Gether, 2000;Lu et al, 2001).…”

Identification of multiple allosteric sites on the M1 muscarinic acetylcholine receptor

“…This deletion did not modify ligand binding activity and shows good signaling activity [10][11][12]. The accuracy of the structure and the protonation states were analyzed with the program WHAT IF [13].…”