Muscarinic Acetylcholine Receptors
Muscarinic acetylcholine receptors belong to the Class A (rhodopsin-like family) of G protein-coupled receptors. The endogenous agonist acetylcholine can bind to five different subtypes of muscarinic acetylcholine receptors designated M1-M5. These five members are divided into two subtypes based on their primary coupling efficiency to G-proteins - M1, M3 and M5 receptors interact with Gq/11-proteins, while M2 and M4 interact with Gi/o-proteins. However, it is clear that muscarinic acetylcholine receptors can activate a wide range of signalling pathways (both G-protein mediated and G-protein independent). Structure of these receptors is particularly interesting due to the presence of allosteric binding sites.
Our workgroup has studied muscarinic acetylcholine receptors for a long time. For example, in his doctoral thesis professor Ago Rinken focused on how solubilisation of different muscarinic receptors can influence ligand binding. We have also developed a screening system for the characterization of agonists, partial agonists and inverse agonists by reconstitution of M2 receptor with either Gi- or Go-proteins in Sf9 insect cells (Uustare et al. 2004).
Currently, our workgroup is focusing on measuring the ligand binding in time. For this, we use various fluorescence-based assays. We are collaborating with Dr. Max Keller from the University of Regensburg to characterize novel fluorescent ligands (Gruber et al. 2020) to study muscarinic acetylcholine receptors M1, M2 , M3 and M4 in fluorescence anisotropy (FA) based assay and in live-cell fluorescence microscopy assay. We have successfully implemented the assays to perform ligand binding to M1 (manuscript in preparation), M2 (Grätz et al. 2021) and M4 (Tahk et al. 2022) receptors. We have recently described the FA assay set-up and detailed protocol in a book chapter Laasfeld et al. 2024.
Interestingly, the two structurally similar fluorescent ligands show very different ligand binding kinetics and it also for different muscarinic acetylcholine receptor subtypes (Figure 1). For more detailed information, check out the publications mentioned above.
Our workgroup has studied muscarinic acetylcholine receptors for a long time. For example, in his doctoral thesis professor Ago Rinken focused on how solubilisation of different muscarinic receptors can influence ligand binding. We have also developed a screening system for the characterization of agonists, partial agonists and inverse agonists by reconstitution of M2 receptor with either Gi- or Go-proteins in Sf9 insect cells (Uustare et al. 2004).
Currently, our workgroup is focusing on measuring the ligand binding in time. For this, we use various fluorescence-based assays. We are collaborating with Dr. Max Keller from the University of Regensburg to characterize novel fluorescent ligands (Gruber et al. 2020) to study muscarinic acetylcholine receptors M1, M2 , M3 and M4 in fluorescence anisotropy (FA) based assay and in live-cell fluorescence microscopy assay. We have successfully implemented the assays to perform ligand binding to M1 (manuscript in preparation), M2 (Grätz et al. 2021) and M4 (Tahk et al. 2022) receptors. We have recently described the FA assay set-up and detailed protocol in a book chapter Laasfeld et al. 2024.
Interestingly, the two structurally similar fluorescent ligands show very different ligand binding kinetics and it also for different muscarinic acetylcholine receptor subtypes (Figure 1). For more detailed information, check out the publications mentioned above.
UR-MK342
UR-CG072
Figure 1. Kinetics of fluorescent ligand UR-MK342 (upper graph) or UR-CG072 (lower graph) binding to muscarinic acetylcholine receptor expressing BBVs in FA assay. The reaction was started by adding BBVs expressing either muscarinic M1, M2 or M4 receptors to fluorescent ligands in the absence (total binding, black circle) or presence of scopolamine (non-specific binding, grey circle). Dissociation was initiated as indicated with a black arrow by the addition of the same final concentration of scopolamine as was used for non-specific binding (blue triangle).