Site-directed mutagenesis of the putative human muscarinic M2 receptor binding site

Heitz, Freddy; Holzwarth, James A.; Gies, Jean-Pierre; Pruss, Rebecca M.; Trumpp-Kallmeyer, S.; Hibert, Marcel; Guenet, Chantal

doi:10.1016/s0014-2999(99)00439-2

Cited by 53 publications

(51 citation statements)

References 29 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…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.…”

Section: Resultssupporting

confidence: 88%

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

Schlegel

Laggner

Meier

et al. 2007

J Comput Aided Mol Des

View full text Add to dashboard Cite

The human histamine H 3 receptor (hH 3 R) is a G-protein coupled receptor (GPCR), which modulates the release of various neurotransmitters in the central and peripheral nervous system and therefore is a potential target in the therapy of numerous diseases. Although ligands addressing this receptor are already known, the discovery of alternative lead structures represents an important goal in drug design. The goal of this work was to study the hH 3 R and its antagonists by means of molecular modelling tools. For this purpose, a strategy was pursued in which a homology model of the hH 3 R based on the crystal structure of bovine rhodopsin was generated and refined by molecular dynamics simulations in a dipalmitoylphosphatidylcholine (DPPC)/water membrane mimic before the resulting binding pocket was used for high-throughput docking using the program GOLD. Alternatively, a pharmacophore-based procedure was carried out where the alleged bioactive conformations of three different potent hH 3 R antagonists were used as templates for the generation of pharmacophore models. A pharmacophore-based screening was then carried out using the program Catalyst. Based upon a database of 418 validated hH 3 R antagonists both strategies could be validated in respect of their performance. Seven hits obtained during this screening procedure were commercially purchased, and experimentally tested in a [ 3 H]N a -methylhistamine binding assay. The compounds tested showed affinities at hH 3 R with K i values ranging from 0.079 to 6.3 lM.

show abstract

Section: Resultssupporting

confidence: 88%

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

Schlegel

Laggner

Meier

et al. 2007

J Comput Aided Mol Des

View full text Add to dashboard Cite

show abstract

“…Several of these residues have been targeted previously (8,10,11,13,25,26), but this is the first comprehensive study that allows their relative importance to be ranked and their role in receptor function to be analyzed.…”

Section: Discussionmentioning

confidence: 99%

“…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 3 and/or 4 may accompany receptor activation (6). Site-directed mutagenesis studies have also indicated the potential importance of TM 4 in ligand binding and G protein recognition (7)(8)(9).…”

Section: Muscarinic Acetylcholine Receptors (Machrs)mentioning

confidence: 99%

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

Lu¹,

Saldanha²,

Hulme³

2001

Journal of Biological Chemistry

106

View full text Add to dashboard Cite

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 ...

show abstract

“…Point mutations (Wess, 1996;Heitz et al, 1999), particularly alanine-scanning (Hulme et al, 2003), have been particularly useful in identifying them. The putative contacts made by the inverse agonist (Ϫ)-N-methyl scopolamine (NMS) with the TM domain of the M 1 mAChR are homologous to the contacts of ground-state rhodopsin with cis-retinal (Palczewski et al, 2000;Li et al, 2004), with the exception of Trp157 in TM4 (Lu et al, 2001) (residue 4.57 using the notation of Ballesteros and Weinstein, 1995).…”

mentioning

confidence: 99%

Roof and Floor of the Muscarinic Binding Pocket: Variations in the Binding Modes of Orthosteric Ligands

Goodwin¹,

Hulme²,

Langmead³

et al. 2007

Mol Pharmacol

View full text Add to dashboard Cite

Alanine substitution mutagenesis has been used to investigate residues that make up the roof and floor of the muscarinic binding pocket and regulate ligand access. We mutated the amino acids in the second extracellular loop of the M 1 muscarinic acetylcholine receptor that are homologous to the cisretinal contact residues in rhodopsin, the disulfide-bonded Cys178 and Cys98 that anchor the loop to transmembrane helix 3, the adjoining acidic residue Asp99, and the conserved aromatic residues Phe197 and Trp378 in the transmembrane domain. The effects on ligand binding, kinetics, and receptor function suggest that the second extracellular loop does not provide primary contacts for orthosteric ligands, including acetylcholine, but that it does contribute to microdomains that are important for the conformational changes that accompany receptor activation. Kinetic studies suggest that the disulfide

show abstract

Site-directed mutagenesis of the putative human muscarinic M2 receptor binding site

Cited by 53 publications

References 29 publications

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

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

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

Roof and Floor of the Muscarinic Binding Pocket: Variations in the Binding Modes of Orthosteric Ligands

Contact Info

Product

Resources

About