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

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