Amyloid β is considered a key player in the development and progression of Alzheimer's disease (AD). Many studies investigating the effect of statins on lowering cholesterol suggest that there may be a link between cholesterol levels and AD pathology. Since cholesterol is one of the most abundant lipid molecules, especially in brain tissue, it affects most membrane-related processes, including the formation of the most dangerous form of amyloid β, Aβ42. The entire Aβ production system, which includes the amyloid precursor protein (APP), β-secretase, and the complex of γ-secretase, is highly dependent on membrane cholesterol content. Moreover, cholesterol can affect amyloidogenesis in many ways. Cholesterol influences the stability and activity of secretases, but also dictates their partitioning into specific cellular compartments and cholesterol-enriched lipid rafts, where the amyloidogenic machinery is predominantly localized. The most complicated relationships have been found in the interaction between cholesterol and APP, where cholesterol affects not only APP localization but also the precise character of APP dimerization and APP processing by γ-secretase, which is important for the production of Aβ of different lengths. In this review, we describe the intricate web of interdependence between cellular cholesterol levels, cholesterol membrane distribution, and cholesterol-dependent production of Aβ, the major player in AD.

• Keywords
• Amyloid precursor protein, Amyloid β, Amyloidogenesis, Cholesterol, Secretase,
• Publication type
• Journal Article MeSH
• Review MeSH

Muscarinic acetylcholine receptors are membrane receptors involved in many physiological processes. Malfunction of muscarinic signaling is a cause of various internal diseases, as well as psychiatric and neurologic conditions. Cholesterol, neurosteroids, neuroactive steroids, and steroid hormones are molecules of steroid origin that, besides having well-known genomic effects, also modulate membrane proteins including muscarinic acetylcholine receptors. Here, we review current knowledge on the allosteric modulation of muscarinic receptors by these steroids. We give a perspective on the research on the non-genomic effects of steroidal compounds on muscarinic receptors and drug development, with an aim to ultimately exploit such knowledge.

• Keywords
• allosteric modulation, cholesterol, muscarinic receptors, neuroactive steroids, neurosteroids,
• MeSH
• Cholesterol MeSH
• Hormones metabolism   MeSH
• Neurosteroids * pharmacology   MeSH
• Receptors, Muscarinic MeSH
• Steroids pharmacology   MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Cholesterol MeSH
• Hormones MeSH
• Neurosteroids * MeSH
• Receptors, Muscarinic MeSH
• Steroids MeSH

G-protein coupled receptors (GPCRs) are membrane proteins that convey extracellular signals to the cellular milieu. They represent a target for more than 30% of currently marketed drugs. Here we review the effects of membrane cholesterol on the function of GPCRs of Class A. We review both the specific effects of cholesterol mediated via its direct high-affinity binding to the receptor and non-specific effects mediated by cholesterol-induced changes in the properties of the membrane. Cholesterol binds to many GPCRs at both canonical and non-canonical binding sites. It allosterically affects ligand binding to and activation of GPCRs. Additionally, it changes the oligomerization state of GPCRs. In this review, we consider a perspective of the potential for the development of new therapies that are targeted at manipulating the level of membrane cholesterol or modulating cholesterol binding sites on to GPCRs.

• Keywords
• GPCRs, allosteric modulation, cholesterol,
• MeSH
• Allosteric Regulation MeSH
• Anticholesteremic Agents pharmacology   therapeutic use   MeSH
• Cell Membrane metabolism   MeSH
• Cholesterol chemistry   metabolism   MeSH
• Molecular Targeted Therapy methods   MeSH
• Humans MeSH
• Ligands MeSH
• Receptors, G-Protein-Coupled chemistry   classification   metabolism   MeSH
• Protein Binding MeSH
• Binding Sites drug effects   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Anticholesteremic Agents MeSH
• Cholesterol MeSH
• Ligands MeSH
• Receptors, G-Protein-Coupled MeSH

Allosteric modulators are ligands that bind to a site on the receptor that is spatially separated from the orthosteric binding site for the endogenous neurotransmitter. Allosteric modulators modulate the binding affinity, potency, and efficacy of orthosteric ligands. Muscarinic acetylcholine receptors are prototypical allosterically-modulated G-protein-coupled receptors. They are a potential therapeutic target for the treatment of psychiatric, neurologic, and internal diseases like schizophrenia, Alzheimer's disease, Huntington disease, type 2 diabetes, or chronic pulmonary obstruction. Here, we reviewed the progress made during the last decade in our understanding of their mechanisms of binding, allosteric modulation, and in vivo actions in order to understand the translational impact of studying this important class of pharmacological agents. We overviewed newly developed allosteric modulators of muscarinic receptors as well as new spin-off ideas like bitopic ligands combining allosteric and orthosteric moieties and photo-switchable ligands based on bitopic agents.

• Keywords
• acetylcholine, allosteric modulation, muscarinic receptors,
• MeSH
• Muscarinic Agonists metabolism   MeSH
• Allosteric Regulation physiology   MeSH
• Muscarinic Antagonists metabolism   MeSH
• Humans MeSH
• Ligands MeSH
• Receptors, Muscarinic metabolism   physiology   MeSH
• Receptors, G-Protein-Coupled MeSH
• Binding Sites MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Muscarinic Agonists MeSH
• Muscarinic Antagonists MeSH
• Ligands MeSH
• Receptors, Muscarinic MeSH
• Receptors, G-Protein-Coupled MeSH

Cholesterol is a key component of cell membranes with a proven modulatory role on the function and ligand-binding properties of G-protein-coupled receptors (GPCRs). Crystal structures of prototypical GPCRs such as the adenosine A2A receptor (A2AR) have confirmed that cholesterol finds stable binding sites at the receptor surface suggesting an allosteric role of this lipid. Here we combine experimental and computational approaches to show that cholesterol can spontaneously enter the A2AR-binding pocket from the membrane milieu using the same portal gate previously suggested for opsin ligands. We confirm the presence of cholesterol inside the receptor by chemical modification of the A2AR interior in a biotinylation assay. Overall, we show that cholesterol's impact on A2AR-binding affinity goes beyond pure allosteric modulation and unveils a new interaction mode between cholesterol and the A2AR that could potentially apply to other GPCRs.

• MeSH
• Cell Membrane chemistry   metabolism   MeSH
• Cholesterol chemistry   metabolism   MeSH
• Binding, Competitive MeSH
• Rats MeSH
• Cell Line, Tumor MeSH
• Protein Domains * MeSH
• Receptor, Adenosine A2A chemistry   metabolism   MeSH
• Receptors, G-Protein-Coupled chemistry   metabolism   MeSH
• Molecular Dynamics Simulation MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cholesterol MeSH
• Receptor, Adenosine A2A MeSH
• Receptors, G-Protein-Coupled 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