The allosteric core region of the M2 muscarinic acetylcholine receptorrole for ligand selectivity and action
The allosteric core region of the M2 muscarinic acetylcholine receptor
role for ligand selectivity and action
Dokument öffnen (3MB)Art der Hochschulschrift
DissertationPrüfungsdatum
17.07.2015Datum der Veröffentlichung
23.09.2015Erstgutachter
Mohr, KlausZweitgutachter
von Kügelgen, IvarMetadaten
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Inhalt
Muscarinic acetylcholine receptors (mAChRs) are seven transmembrane domain-spanning proteins belonging to the family A of G protein-coupled receptors (GPCRs). In addition to the orthosteric binding site (i.e. the site where the endogenous ligand acetylcholine (ACh) binds and activates the receptor), mAChRs possess a common allosteric binding site which is located directly above the orthosteric binding site. The amino acids Y177, W422 and T423 located in the extracellular loop 2 and the upper part of transmembrane domain 7, respectively have been previously found to line the core region of the allosteric binding site of the M2 mAChR1
Here, the aim was to investigate the consequences caused by the loss of the three core amino acids located in the “bottleneck-region” (CHO-hM2 W422A, T423A, Y177A), between the orthosteric and the allosteric part of the ligand binding cavity of the M2 mAChRs on ligand binding, receptor activation and receptor-ligand selectivity.
Radioligand binding experiments with [3H]NMS in M2 wt and triple mutant receptors showed that the triple mutant strongly reduces the binding affinities of the M2 muscarinic full agonists. Interestingly, the partial agonist pilocarpine´s binding to M2 receptor was observed to be completely insensitive to the triple mutation. The [35S]GTPγS binding assay showed that the M2 muscarinic full agonists lose their potencies at the triple mutant but their efficacies remain unchanged compared to M2 wt receptor in Gi-signalling. However, the partial agonist pilocarpine loses efficacy at the triple mutant but its potency remains unchanged compared to M2 wt receptors. The cAMP and dynamic mass redistribution (DMR) assays showed that the full agonists acetylcholine and iperoxo displayed reduced efficacy in Gs-signalling at the M2 triple mutant. The allosteric ligand 6-naph which is a positive allosteric modulator of [3H]NMS binding at the M2 wt receptor4,5,6 becomes a negative allosteric modulator at the M2 triple mutant. Dualsteric ligands which consist of both the allosteric and orthosteric pharmacophores, e.g. iper-6-naph, switch to a predominantly dualsteric binding pose at the M2 triple mutant compared to the M2 wt receptors.
Hence, the studies show that not only is the M2 allosteric binding site important for allosteric ligand binding, but also regulates orthosteric and dualsteric ligand activity.
Here, the aim was to investigate the consequences caused by the loss of the three core amino acids located in the “bottleneck-region” (CHO-hM2 W422A, T423A, Y177A), between the orthosteric and the allosteric part of the ligand binding cavity of the M2 mAChRs on ligand binding, receptor activation and receptor-ligand selectivity.
Radioligand binding experiments with [3H]NMS in M2 wt and triple mutant receptors showed that the triple mutant strongly reduces the binding affinities of the M2 muscarinic full agonists. Interestingly, the partial agonist pilocarpine´s binding to M2 receptor was observed to be completely insensitive to the triple mutation. The [35S]GTPγS binding assay showed that the M2 muscarinic full agonists lose their potencies at the triple mutant but their efficacies remain unchanged compared to M2 wt receptor in Gi-signalling. However, the partial agonist pilocarpine loses efficacy at the triple mutant but its potency remains unchanged compared to M2 wt receptors. The cAMP and dynamic mass redistribution (DMR) assays showed that the full agonists acetylcholine and iperoxo displayed reduced efficacy in Gs-signalling at the M2 triple mutant. The allosteric ligand 6-naph which is a positive allosteric modulator of [3H]NMS binding at the M2 wt receptor4,5,6 becomes a negative allosteric modulator at the M2 triple mutant. Dualsteric ligands which consist of both the allosteric and orthosteric pharmacophores, e.g. iper-6-naph, switch to a predominantly dualsteric binding pose at the M2 triple mutant compared to the M2 wt receptors.
Hence, the studies show that not only is the M2 allosteric binding site important for allosteric ligand binding, but also regulates orthosteric and dualsteric ligand activity.
Bemerkung
B.C. was funded by the NRW- International Graduate Research School BIOTECH-PHARMA.Klassifikation (DDC)
570 Biowissenschaften, Biologie 615 Pharmakologie, TherapeutikChirinda, Brian: The allosteric core region of the M2 muscarinic acetylcholine receptor : role for ligand selectivity and action. - Bonn, 2015. - Dissertation, Rheinische Friedrich-Wilhelms-Universität Bonn.
Online-Ausgabe in bonndoc: https://nbn-resolving.org/urn:nbn:de:hbz:5n-41247
Online-Ausgabe in bonndoc: https://nbn-resolving.org/urn:nbn:de:hbz:5n-41247
@phdthesis{handle:20.500.11811/6532,
urn: https://nbn-resolving.org/urn:nbn:de:hbz:5n-41247,
author = {{Brian Chirinda}},
title = {The allosteric core region of the M2 muscarinic acetylcholine receptor : role for ligand selectivity and action},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2015,
month = sep,
note = {Muscarinic acetylcholine receptors (mAChRs) are seven transmembrane domain-spanning proteins belonging to the family A of G protein-coupled receptors (GPCRs). In addition to the orthosteric binding site (i.e. the site where the endogenous ligand acetylcholine (ACh) binds and activates the receptor), mAChRs possess a common allosteric binding site which is located directly above the orthosteric binding site. The amino acids Y177, W422 and T423 located in the extracellular loop 2 and the upper part of transmembrane domain 7, respectively have been previously found to line the core region of the allosteric binding site of the M2 mAChR1
Here, the aim was to investigate the consequences caused by the loss of the three core amino acids located in the “bottleneck-region” (CHO-hM2 W422A, T423A, Y177A), between the orthosteric and the allosteric part of the ligand binding cavity of the M2 mAChRs on ligand binding, receptor activation and receptor-ligand selectivity.
Radioligand binding experiments with [3H]NMS in M2 wt and triple mutant receptors showed that the triple mutant strongly reduces the binding affinities of the M2 muscarinic full agonists. Interestingly, the partial agonist pilocarpine´s binding to M2 receptor was observed to be completely insensitive to the triple mutation. The [35S]GTPγS binding assay showed that the M2 muscarinic full agonists lose their potencies at the triple mutant but their efficacies remain unchanged compared to M2 wt receptor in Gi-signalling. However, the partial agonist pilocarpine loses efficacy at the triple mutant but its potency remains unchanged compared to M2 wt receptors. The cAMP and dynamic mass redistribution (DMR) assays showed that the full agonists acetylcholine and iperoxo displayed reduced efficacy in Gs-signalling at the M2 triple mutant. The allosteric ligand 6-naph which is a positive allosteric modulator of [3H]NMS binding at the M2 wt receptor4,5,6 becomes a negative allosteric modulator at the M2 triple mutant. Dualsteric ligands which consist of both the allosteric and orthosteric pharmacophores, e.g. iper-6-naph, switch to a predominantly dualsteric binding pose at the M2 triple mutant compared to the M2 wt receptors.
Hence, the studies show that not only is the M2 allosteric binding site important for allosteric ligand binding, but also regulates orthosteric and dualsteric ligand activity.},
url = {https://hdl.handle.net/20.500.11811/6532}
}
urn: https://nbn-resolving.org/urn:nbn:de:hbz:5n-41247,
author = {{Brian Chirinda}},
title = {The allosteric core region of the M2 muscarinic acetylcholine receptor : role for ligand selectivity and action},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2015,
month = sep,
note = {Muscarinic acetylcholine receptors (mAChRs) are seven transmembrane domain-spanning proteins belonging to the family A of G protein-coupled receptors (GPCRs). In addition to the orthosteric binding site (i.e. the site where the endogenous ligand acetylcholine (ACh) binds and activates the receptor), mAChRs possess a common allosteric binding site which is located directly above the orthosteric binding site. The amino acids Y177, W422 and T423 located in the extracellular loop 2 and the upper part of transmembrane domain 7, respectively have been previously found to line the core region of the allosteric binding site of the M2 mAChR1
Here, the aim was to investigate the consequences caused by the loss of the three core amino acids located in the “bottleneck-region” (CHO-hM2 W422A, T423A, Y177A), between the orthosteric and the allosteric part of the ligand binding cavity of the M2 mAChRs on ligand binding, receptor activation and receptor-ligand selectivity.
Radioligand binding experiments with [3H]NMS in M2 wt and triple mutant receptors showed that the triple mutant strongly reduces the binding affinities of the M2 muscarinic full agonists. Interestingly, the partial agonist pilocarpine´s binding to M2 receptor was observed to be completely insensitive to the triple mutation. The [35S]GTPγS binding assay showed that the M2 muscarinic full agonists lose their potencies at the triple mutant but their efficacies remain unchanged compared to M2 wt receptor in Gi-signalling. However, the partial agonist pilocarpine loses efficacy at the triple mutant but its potency remains unchanged compared to M2 wt receptors. The cAMP and dynamic mass redistribution (DMR) assays showed that the full agonists acetylcholine and iperoxo displayed reduced efficacy in Gs-signalling at the M2 triple mutant. The allosteric ligand 6-naph which is a positive allosteric modulator of [3H]NMS binding at the M2 wt receptor4,5,6 becomes a negative allosteric modulator at the M2 triple mutant. Dualsteric ligands which consist of both the allosteric and orthosteric pharmacophores, e.g. iper-6-naph, switch to a predominantly dualsteric binding pose at the M2 triple mutant compared to the M2 wt receptors.
Hence, the studies show that not only is the M2 allosteric binding site important for allosteric ligand binding, but also regulates orthosteric and dualsteric ligand activity.},
url = {https://hdl.handle.net/20.500.11811/6532}
}
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