Selective activation of individual subtypes of muscarinic receptors is a promising way to safely alleviate a wide range of pathological conditions in the central nervous system and the periphery as well. The flexible G-protein interface of muscarinic receptors allows them to interact with several G-proteins with various efficacy, potency, and kinetics. Agonists biased to the particular G-protein mediated pathway may result in selectivity among muscarinic subtypes and, due to the non-uniform expression of individual G-protein alpha subunits, possibly achieve tissue specificity. Here, we demonstrate that novel tetrahydropyridine-based agonists exert specific signalling profiles in coupling with individual G-protein α subunits. These signalling profiles profoundly differ from the reference agonist carbachol. Moreover, coupling with individual Gα induced by these novel agonists varies among subtypes of muscarinic receptors which may lead to subtype selectivity. Thus, the novel tetrahydropyridine-based agonist can contribute to the elucidation of the mechanism of pathway-specific activation of muscarinic receptors and serve as a starting point for the development of desired selective muscarinic agonists.

• MeSH
• Muscarinic Agonists * pharmacology   MeSH
• CHO Cells MeSH
• Cricetulus MeSH
• Carbachol pharmacology   MeSH
• Humans MeSH
• GTP-Binding Protein alpha Subunits metabolism   genetics   MeSH
• GTP-Binding Proteins metabolism   MeSH
• Pyridines pharmacology   MeSH
• Receptors, Muscarinic * metabolism   MeSH
• Signal Transduction drug effects   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Muscarinic Agonists * MeSH
• Carbachol MeSH
• GTP-Binding Protein alpha Subunits MeSH
• GTP-Binding Proteins MeSH
• Pyridines MeSH
• Receptors, Muscarinic * MeSH

A complex evaluation of agonist bias at G-protein coupled receptors at the level of G-protein classes and isoforms including non-preferential ones is essential for advanced agonist screening and drug development. Molecular crosstalk in downstream signaling and a lack of sufficiently sensitive and selective methods to study direct coupling with G-protein of interest complicates this analysis. We performed binding and functional analysis of 11 structurally different agonists on prepared fusion proteins of individual subtypes of muscarinic receptors and non-canonical promiscuous α-subunit of G16 protein to study agonist bias. We have demonstrated that fusion of muscarinic receptors with Gα16 limits access of other competitive Gα subunits to the receptor, and thus enables us to study activation of Gα16 mediated pathway more specifically. Our data demonstrated agonist-specific activation of G16 pathway among individual subtypes of muscarinic receptors and revealed signaling bias of oxotremorine towards Gα16 pathway at the M2 receptor and at the same time impaired Gα16 signaling of iperoxo at M5 receptors. Our data have shown that fusion proteins of muscarinic receptors with α-subunit of G-proteins can serve as a suitable tool for studying agonist bias, especially at non-preferential pathways.

• Keywords
• fusion proteins, muscarinic receptors, non-canonical signaling, signaling bias,
• MeSH
• Cyclic AMP metabolism   MeSH
• CHO Cells MeSH
• Cricetulus MeSH
• Inhibitory Concentration 50 MeSH
• Isoxazoles chemistry   MeSH
• Cricetinae MeSH
• Quaternary Ammonium Compounds chemistry   MeSH
• Humans MeSH
• Molecular Conformation MeSH
• Oxotremorine chemistry   MeSH
• GTP-Binding Protein alpha Subunits, Gq-G11 metabolism   MeSH
• Receptors, Muscarinic metabolism   MeSH
• Recombinant Fusion Proteins chemistry   MeSH
• Signal Transduction * MeSH
• Molecular Dynamics Simulation MeSH
• Protein Binding MeSH
• Animals MeSH
• Check Tag
• Cricetinae MeSH
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Cyclic AMP MeSH
• G protein alpha 16 MeSH Browser
• iperoxo MeSH Browser
• Isoxazoles MeSH
• Quaternary Ammonium Compounds MeSH
• Oxotremorine MeSH
• GTP-Binding Protein alpha Subunits, Gq-G11 MeSH
• Receptors, Muscarinic MeSH
• Recombinant Fusion Proteins MeSH

BACKGROUND AND PURPOSE: More than 30% of currently marketed medications act via GPCRs. Thus, GPCRs represent one of the most important pharmacotherapeutic targets. In contrast to traditional agonists activating multiple signalling pathways, agonists activating a single signalling pathway represent a new generation of drugs with increased specificity and fewer adverse effects. EXPERIMENTAL APPROACH: We have synthesized novel agonists of muscarinic ACh receptors and tested their binding and function (on levels of cAMP and inositol phosphates) in CHO cells expressing individual subtypes of muscarinic receptors, primary cultures of rat aortic smooth muscle cells and suspensions of digested native tissues from rats. Binding of the novel compounds to M2 receptors was modelled in silico. KEY RESULTS: Two of the tested new compounds (1-(thiophen-2-ylmethyl)-3,6-dihydro-2H-pyridinium and 1-methyl-1-(thiophen-2-ylmethyl)-3,6-dihydro-2H-pyridinium) only inhibited cAMP synthesis in CHO cells, primary cultures, and native tissues, with selectivity for M2 muscarinic receptors and displaying bias towards the Gi signalling pathway at all subtypes of muscarinic receptors. Molecular modelling revealed interactions with the orthosteric binding site in a way specific for a given agonist followed by agonist-specific changes in the conformation of the receptor. CONCLUSIONS AND IMPLICATIONS: The identified compounds may serve as lead structures in the search for novel non-steroidal and non-opioid analgesics acting via M2 and M4 muscarinic receptors with reduced side effects associated with activation of the phospholipase C signalling pathway.

• MeSH
• Muscarinic Agonists * pharmacology   MeSH
• Muscarinic Antagonists pharmacology   MeSH
• CHO Cells MeSH
• Cricetulus MeSH
• Cricetinae MeSH
• Rats MeSH
• Receptor, Muscarinic M2 MeSH
• Receptors, Muscarinic * MeSH
• Signal Transduction MeSH
• Animals MeSH
• Check Tag
• Cricetinae MeSH
• Rats MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Muscarinic Agonists * MeSH
• Muscarinic Antagonists MeSH
• Receptor, Muscarinic M2 MeSH
• Receptors, Muscarinic * MeSH

We have found earlier that changes in membrane cholesterol content have distinct impact on signaling via the M1, M2, or M3 receptors expressed in CHO cells (CHO-M1 through CHO-M3). Now we investigated whether gradual changes in membrane cholesterol exerts differential effects on coupling of the M1 and M3 muscarinic receptors to preferential signaling pathways through Gq/11 and non-preferential Gs G-proteins signaling. Changes in membrane cholesterol resulted in only marginal alterations of antagonist and agonist affinity of the M1 and M3 receptors, and did not influence precoupling of either subtype. Changes in membrane cholesterol did not influence parameters of carbachol-stimulated GTP-γ(35)S binding in CHO-M1 membranes while reduction as well as augmentation of membrane cholesterol lowered the efficacy but increased the potency of carbachol in CHO-M3 membranes. Gradual increase or decrease in membrane cholesterol concentration dependently attenuated agonist-induced inositolphosphates release while only cholesterol depletion increased basal values in both cell lines. Similarly, membrane cholesterol manipulation modified basal and agonist-stimulated cAMP synthesis via Gs in the same way in both cell lines. These results demonstrate that changes in membrane cholesterol concentration differentially impact preferential and non-preferential M1 and M3 receptor signaling. They point to the activated G-protein/effector protein interaction as the main site of action in alterations of M1 receptor-mediated stimulation of second messenger pathways. On the other hand, modifications in agonist-stimulated GTP-γ(35)S binding in CHO-M3 membranes indicate that in this case changes in ligand-activated receptor/G-protein interaction may also play a role.

• Keywords
• Agonist binding, Cholesterol, G-Proteins, Muscarinic receptors, Signal transduction, cAMP synthesis,
• MeSH
• CHO Cells MeSH
• Cholesterol metabolism   MeSH
• Cricetulus MeSH
• Carbachol pharmacology   MeSH
• Humans MeSH
• GTP-Binding Proteins metabolism   MeSH
• Receptor, Muscarinic M1 drug effects   metabolism   MeSH
• Receptor, Muscarinic M3 drug effects   metabolism   MeSH
• Signal Transduction MeSH
• Second Messenger Systems physiology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cholesterol MeSH
• Carbachol MeSH
• GTP-Binding Proteins MeSH
• Receptor, Muscarinic M1 MeSH
• Receptor, Muscarinic M3 MeSH

Based on the kinetics of interaction between a receptor and G-protein, a myriad of possibilities may result. Two extreme cases are represented by: 1/Collision coupling, where an agonist binds to the free receptor and then the agonist-receptor complex "collides" with the free G-protein. 2/Pre-coupling, where stable receptor/G-protein complexes exist in the absence of agonist. Pre-coupling plays an important role in the kinetics of signal transduction. Odd-numbered muscarinic acetylcholine receptors preferentially couple to G(q/11), while even-numbered receptors prefer coupling to G(i/o). We analyzed the coupling status of the various subtypes of muscarinic receptors with preferential and non-preferential G-proteins. The magnitude of receptor-G-protein coupling was determined by the proportion of receptors existing in the agonist high-affinity binding conformation. Antibodies directed against the C-terminus of the α-subunits of the individual G-proteins were used to interfere with receptor-G-protein coupling. Effects of mutations and expression level on receptor-G-protein coupling were also investigated. Tested agonists displayed biphasic competition curves with the antagonist [(3)H]-N-methylscopolamine. Antibodies directed against the C-terminus of the α-subunits of the preferential G-protein decreased the proportion of high-affinity sites, and mutations at the receptor-G-protein interface abolished agonist high-affinity binding. In contrast, mutations that prevent receptor activation had no effect. Expression level of preferential G-proteins had no effect on pre-coupling to non-preferential G-proteins. Our data show that all subtypes of muscarinic receptors pre-couple with their preferential classes of G-proteins, but only M(1) and M(3) receptors also pre-couple with non-preferential G(i/o) G-proteins. Pre-coupling is not dependent on agonist efficacy nor on receptor activation. The ultimate mode of coupling is therefore dictated by a combination of the receptor subtype and the class of G-protein.

• MeSH
• CHO Cells MeSH
• Cricetulus MeSH
• Guanosine 5'-O-(3-Thiotriphosphate) metabolism   MeSH
• Carbachol metabolism   MeSH
• Kinetics MeSH
• Binding, Competitive MeSH
• Cricetinae MeSH
• Humans MeSH
• Mutation MeSH
• N-Methylscopolamine metabolism   MeSH
• GTP-Binding Proteins metabolism   MeSH
• Receptors, Muscarinic genetics   metabolism   MeSH
• Animals MeSH
• Check Tag
• Cricetinae MeSH
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Guanosine 5'-O-(3-Thiotriphosphate) MeSH
• Carbachol MeSH
• N-Methylscopolamine MeSH
• GTP-Binding Proteins MeSH
• Receptors, Muscarinic MeSH

BACKGROUND AND PURPOSE: Conventional determination of agonist efficacy at G-protein coupled receptors is measured by stimulation of guanosine-5'-γ-thiotriphosphate (GTPγS) binding. We analysed the role of guanosine diphosphate (GDP) in the process of activation of the M₂ muscarinic acetylcholine receptor and provide evidence that negative cooperativity between agonist and GDP binding is an alternative measure of agonist efficacy. EXPERIMENTAL APPROACH: Filtration and scintillation proximity assays measured equilibrium binding as well as binding kinetics of [³⁵S]GTPγS and [³H]GDP to a mixture of G-proteins as well as individual classes of G-proteins upon binding of structurally different agonists to the M₂ muscarinic acetylcholine receptor. KEY RESULTS: Agonists displayed biphasic competition curves with the antagonist [³H]-N-methylscopolamine. GTPγS (1 µM) changed the competition curves to monophasic with low affinity and 50 µM GDP produced a similar effect. Depletion of membrane-bound GDP increased the proportion of agonist high-affinity sites. Carbachol accelerated the dissociation of [³H]GDP from membranes. The inverse agonist N-methylscopolamine slowed GDP dissociation and GTPγS binding without changing affinity for GDP. Carbachol affected both GDP association with and dissociation from G(i/o) G-proteins but only its dissociation from G(s/olf) G-proteins. CONCLUSIONS AND IMPLICATIONS: These findings suggest the existence of a low-affinity agonist-receptor conformation complexed with GDP-liganded G-protein. Also the negative cooperativity between GDP and agonist binding at the receptor/G-protein complex determines agonist efficacy. GDP binding reveals differences in action of agonists versus inverse agonists as well as differences in activation of G(i/o) versus G(s/olf) G-proteins that are not identified by conventional GTPγS binding.

• MeSH
• Muscarinic Agonists metabolism   MeSH
• Allosteric Regulation MeSH
• Muscarinic Antagonists metabolism   MeSH
• CHO Cells MeSH
• Cricetulus MeSH
• Guanosine 5'-O-(3-Thiotriphosphate) metabolism   MeSH
• Guanosine Diphosphate metabolism   MeSH
• Kinetics MeSH
• Cricetinae MeSH
• Humans MeSH
• N-Methylscopolamine metabolism   MeSH
• GTP-Binding Proteins classification   metabolism   MeSH
• Radioligand Assay MeSH
• Receptor, Muscarinic M2 genetics   metabolism   MeSH
• Recombinant Proteins genetics   metabolism   MeSH
• Transfection MeSH
• Protein Binding MeSH
• Animals MeSH
• Check Tag
• Cricetinae MeSH
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Muscarinic Agonists MeSH
• Muscarinic Antagonists MeSH
• Guanosine 5'-O-(3-Thiotriphosphate) MeSH
• Guanosine Diphosphate MeSH
• N-Methylscopolamine MeSH
• GTP-Binding Proteins MeSH
• Receptor, Muscarinic M2 MeSH
• Recombinant Proteins MeSH

We investigated the influence of membrane cholesterol content on preferential and non-preferential signaling through the M(2) muscarinic acetylcholine receptor expressed in CHO cells. Cholesterol depletion by 39% significantly decreased the affinity of M(2) receptors for [(3)H]-N-methylscopolamine ([(3)H]-NMS) binding and increased B(max) in intact cells and membranes. Membranes displayed two-affinity agonist binding sites for carbachol and cholesterol depletion doubled the fraction of high-affinity binding sites. In intact cells it also reduced the rate of agonist-induced receptor internalization and changed the profile of agonist binding from a single site to two affinity states. Cholesterol enrichment by 137% had no effects on carbachol E(max) of cAMP synthesis inhibition and on cAMP synthesis stimulation and inositolphosphates (IP) accumulation at higher agonist concentrations (non-preferred pathways). On the other hand, cholesterol depletion significantly increased E(max) of cAMP synthesis inhibition or stimulation without change in potency, and decreased E(max) of IP accumulation. Noteworthy, modifications of membrane cholesterol had no effect on membrane permeability, oxidative activity, protein content, or relative expression of G(s), G(i/o), and G(q/11) alpha subunits. These results demonstrate distinct changes of M(2) receptor signaling through both preferential and non-preferential G-proteins consequent to membrane cholesterol depletion that occur at the level of receptor/G-protein/effector protein interactions in the cell membrane. The significant decrease of IP accumulation by cholesterol depletion was also observed in cells expressing M(3) receptors and by both cholesterol depletion and enrichment in cells expressing M(1) receptors indicating relevance of reduced G(q/11) signaling for the pathogenesis of Alzheimer's disease.

• MeSH
• Acetylcholine analogs & derivatives   MeSH
• Muscarinic Antagonists pharmacology   MeSH
• beta-Cyclodextrins pharmacology   MeSH
• Cell Membrane drug effects   metabolism   MeSH
• CHO Cells MeSH
• Cholesterol metabolism   MeSH
• Cricetulus MeSH
• Carbachol analogs & derivatives   metabolism   pharmacology   MeSH
• Cricetinae MeSH
• Humans MeSH
• N-Methylscopolamine metabolism   pharmacology   MeSH
• GTP-Binding Proteins metabolism   MeSH
• Receptor, Muscarinic M2 metabolism   MeSH
• Second Messenger Systems * drug effects   MeSH
• Animals MeSH
• Check Tag
• Cricetinae MeSH
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Acetylcholine MeSH
• Muscarinic Antagonists MeSH
• beta-Cyclodextrins MeSH
• Cholesterol MeSH
• Carbachol MeSH
• methyl-beta-cyclodextrin MeSH Browser
• N-Methylscopolamine MeSH
• GTP-Binding Proteins MeSH
• Receptor, Muscarinic M2 MeSH