Muscarinic G-protein-coupled Receptors Research Articles

In Search of Synergistic Insect Repellents: Modeling of Muscarinic GPCR Interactions with Classical and Bitopic Photoactive Ligands.

Insect vector-borne diseases pose serious health problems, so there is a high demand for efficient molecules that could reduce transmission. Using molecular docking and molecular dynamics (MD) simulation, we studied a series of compounds acting on human and insect muscarinic acetylcholine receptors (mAChRs), a novel target of synergistic agents in pest control. We characterized early conformational changes of human M1 and fruit fly type-A mAChR G protein-coupled receptors (GPCRs) in response to DEET, IR3535, and muscarine binding based on the MD analysis of the activation microswitches known to form the signal transduction pathway in class A GPCRs. We indicated groups of microswitches that are the most affected by the presence of a ligand. Moreover, to increase selectivity towards insects, we proposed a new, bitopic, photoswitchable mAChR ligand—BQCA-azo-IR353 and studied its interactions with both receptors. Modeling data showed that using a bitopic ligand may be a promising strategy in the search for better insect control.

GPCR voltage dependence controls neuronal plasticity and behavior

G-protein coupled receptors (GPCRs) play a paramount role in diverse brain functions. Almost 20 years ago, GPCR activity was shown to be regulated by membrane potential in vitro, but whether the voltage dependence of GPCRs contributes to neuronal coding and behavioral output under physiological conditions in vivo has never been demonstrated. Here we show that muscarinic GPCR mediated neuronal potentiation in vivo is voltage dependent. This voltage dependent potentiation is abolished in mutant animals expressing a voltage independent receptor. Depolarization alone, without a muscarinic agonist, results in a nicotinic ionotropic receptor potentiation that is mediated by muscarinic receptor voltage dependency. Finally, muscarinic receptor voltage independence causes a strong behavioral effect of increased odor habituation. Together, this study identifies a physiological role for the voltage dependency of GPCRs by demonstrating crucial involvement of GPCR voltage dependence in neuronal plasticity and behavior. Thus, this study suggests that GPCR voltage dependency plays a role in many diverse neuronal functions including learning and memory.

Chronic hM3Dq signaling in microglia ameliorates neuroinflammation in male mice

Microglia express muscarinic G protein-coupled receptors (GPCRs) that sense cholinergic activity and are activated by acetylcholine to potentially regulate microglial functions. Knowledge about how distinct types of muscarinic GPCR signaling regulate microglia function in vivo is still poor, partly due to the fact that some of these receptors are also present in astrocytes and neurons. We generated mice expressing the hM3Dq Designer Receptor Exclusively Activated by Designer Drugs (DREADD) selectively in microglia to investigate the role of muscarinic M3Gq-linked signaling. We show that activation of hM3Dq using clozapine N-oxide (CNO) elevated intracellular calcium levels and increased phagocytosis of FluoSpheres by microglia in vitro. Interestingly, whereas acute treatment with CNO increased synthesis of cytokine mRNA, chronic treatment attenuated LPS-induced cytokine mRNA changes in the brain. No effect of CNO on cytokine expression was observed in DREADD-negative mice. Interestingly, CNO activation of M3Dq in microglia was able to attenuate LPS-mediated decrease in social interactions. These results suggest that chronic activation of M3 muscarinic receptors (the hM3Dq progenitor) in microglia, and potentially other Gq-coupled GPCRs, can trigger an inflammatory-like response that preconditions microglia to decrease their response to further immunological challenges. Our results indicate that hM3Dq can be a useful tool to modulate neuroinflammation and study microglial immunological memory in vivo, which may be applicable for manipulations of neuroinflammation in neurodegenerative and psychiatric diseases.

Cryptic pocket formation underlies allosteric modulator selectivity at muscarinic GPCRs

Allosteric modulators are highly desirable as drugs, particularly for G-protein-coupled receptor (GPCR) targets, because allosteric drugs can achieve selectivity between closely related receptors. The mechanisms by which allosteric modulators achieve selectivity remain elusive, however, particularly given recent structures that reveal similar allosteric binding sites across receptors. Here we show that positive allosteric modulators (PAMs) of the M1 muscarinic acetylcholine receptor (mAChR) achieve exquisite selectivity by occupying a dynamic pocket absent in existing crystal structures. This cryptic pocket forms far more frequently in molecular dynamics simulations of the M1 mAChR than in those of other mAChRs. These observations reconcile mutagenesis data that previously appeared contradictory. Further mutagenesis experiments validate our prediction that preventing cryptic pocket opening decreases the affinity of M1-selective PAMs. Our findings suggest opportunities for the design of subtype-specific drugs exploiting cryptic pockets that open in certain receptors but not in other receptors with nearly identical static structures.

Enhanced Simulations and Drug Discovery of a Muscarinic G-Protein-Coupled Receptor

G-protein-coupled receptors (GPCRs) represent primary targets of about one third of currently marketed drugs. However, GPCR drug discovery has suffered from major challenges, including the highly flexible nature of the receptors and the toxicity of agonist or antagonist drugs that are designed to bind the conserved “orthosteric” site. Here, we have performed all-atom enhanced simulations using the robust Gaussian accelerated molecular dynamics (GaMD) method to investigate the ligand-dependent structural dynamics of the M2 muscarinic GPCR. The GaMD simulations revealed distinct structural flexibility and free energy profiles that depict graded activation of the GPCR. Both dissociation and binding of an orthosteric ligand were captured in a single all-atom GPCR simulation(1). In addition, we have implemented a unique structure-based approach to design allosteric modulators as selective drug leads of the M2 muscarinic receptor. Through iterative molecular docking and experimental testing, half of our 38 computationally selected lead compounds were validated as effective allosteric modulators of the M2 receptor. Our method successfully identified both positive and negative allosteric modulators of the M2 muscarinic receptor with unprecedented chemical diversity and outstanding potential for further structure-activity relationship studies(2). This approach shall be of wide applicability for drug discovery of many other GPCRs(3).

The Allosteric Site is Required for Voltage Dependence of Muscarinic GPCRs

G-protein coupled receptors (GPCRs) encompass the largest group of membrane proteins in eukaryotes and regulate numerous intracellular pathways. Class A GPCRs are activated by binding of specific agonists to an orthosteric binding site, which is formed by the seven trans-membrane helices. In contrast, so called allosteric modulators bind to a site on the extracellular surface, the allosteric site. There is an interaction of both sites to modify receptor function and allosteric modulators either enhance or attenuate receptor signaling. Previous work from our laboratory showed that muscarinic receptors (M-Rs) are voltage sensitive. Voltage dependence was only evident for active receptors and may represent allosteric modulation of M-Rs. In this study we compared the voltage dependencies of M1-Rs and M3-Rs to allosteric modulation of both receptors. Changes in receptor signaling were quantified in single HEK 293 cells with a FRET biosensor for the Gq protein cycle. In the presence of acetylcholine (ACh), a depolarization of the membrane from −90 mV to +40 mV potentiated M1-R signaling, whereas it attenuated M3-R signaling. Likewise, the positive allosteric modulator BQCA potentiated ACh/M1-R signaling, whereas the negative allosteric modulator Gallamine attenuated ACh/M3-R signaling. In terms of kinetics and amplitude, receptor modulation by voltage or by allosteric compounds was surprisingly similar. Because the orthosteric site is well conserved, but the allosteric site varies among M-R subtypes, we constructed “allosteric” M1/M3-R chimeras and subjected them to membrane depolarization. Some chimeras were almost insensitive to voltage, indicating that extracellular receptor structures affect the mechanism of voltage dependence. Moreover, a single point mutation that disrupts the cooperativity between the orthosteric and allosteric site removed the voltage sensitivity of M3-R. We conclude that the allosteric site is involved in a mechanism that connects voltage sensitivity and muscarinic receptor function.

Graded activation and free energy landscapes of a muscarinic G-protein–coupled receptor

G-protein-coupled receptors (GPCRs) recognize ligands of widely different efficacies, from inverse to partial and full agonists, which transduce cellular signals at differentiated levels. However, the mechanism of such graded activation remains unclear. Using the Gaussian accelerated molecular dynamics (GaMD) method that enables both unconstrained enhanced sampling and free energy calculation, we have performed extensive GaMD simulations (∼19 μs in total) to investigate structural dynamics of the M2 muscarinic GPCR that is bound by the full agonist iperoxo (IXO), the partial agonist arecoline (ARC), and the inverse agonist 3-quinuclidinyl-benzilate (QNB), in the presence or absence of the G-protein mimetic nanobody. In the receptor-nanobody complex, IXO binding leads to higher fluctuations in the protein-coupling interface than ARC, especially in the receptor transmembrane helix 5 (TM5), TM6, and TM7 intracellular domains that are essential elements for GPCR activation, but less flexibility in the receptor extracellular region due to stronger binding compared with ARC. Two different binding poses are revealed for ARC in the orthosteric pocket. Removal of the nanobody leads to GPCR deactivation that is characterized by inward movement of the TM6 intracellular end. Distinct low-energy intermediate conformational states are identified for the IXO- and ARC-bound M2 receptor. Both dissociation and binding of an orthosteric ligand are observed in a single all-atom GPCR simulation in the case of partial agonist ARC binding to the M2 receptor. This study demonstrates the applicability of GaMD for exploring free energy landscapes of large biomolecules and the simulations provide important insights into the GPCR functional mechanism.

Activation of a Muscarinic G-Protein Coupled Receptor and Structure-Based Design of Allosteric Modulators

G-protein coupled receptors (GPCRs) are important cell signaling membrane proteins that represent primary targets of about one third of currently marketed drugs. The M2 muscarinic receptor is a key GPCR that regulates the human heart rate and contractile forces of cardiomyocytes, and is thus targeted for treating many heart diseases. Here, we have performed long-timescale accelerated molecular dynamics (aMD) simulations and captured activation of the M2 receptor at an atomistic level [1]. The receptor activation is characterized by large-scale structural rearrangements of the transmembrane helices and conformational changes in the inter-helical salt bridge and hydrogen bond interactions [2]. Furthermore, using the aMD simulation-derived structural ensembles that account for the receptor flexibility, we have mapped the receptor surface for druggable allosteric sites [3] and targeted the extracellular vestibule for designing allosteric modulators. Retrospective docking of known ligands is first carried out to validate the simulation receptor ensembles, followed by prospective docking to predict new allosteric ligands from the National Cancer Institute compound library. The computationally selected lead compounds are finally tested using experimental binding and functional assays to identify potential allosteric modulators of the M2 muscarinic GPCR [4].1. Miao, Y., et al., Activation and dynamic network of the M2 muscarinic receptor. Proc Natl Acad Sci U S A, 2013. 110(27): p. 10982-10987.2. Miao, Y., S.E. Nichols, and J.A. McCammon, Free Energy Landscape of G-Protein Coupled Receptors, Explored by Accelerated Molecular Dynamics. Physical Chemistry Chemical Physics, 2014. 16(14): p. 6398-6406.3. Miao, Y., S.E. Nichols, and J.A. McCammon, Mapping of Allosteric Druggable Sites in Activation-Associated Conformers of the M2 Muscarinic Receptor. Chemical Biology & Drug Design, 2013. 83(2): p. 237-246.4. Miao, Y., et al., Structural-Based Design of Allosteric Modulators of the M2 Muscarinic G-protein Coupled Receptor. In Preparation, 2015.

Allosteric Effects of Sodium Ion Binding on Activation of the M3 Muscarinic G-Protein-Coupled Receptor

G-protein-coupled receptors (GPCRs) are important membrane proteins that mediate cellular signaling and represent primary targets for about one-third of currently marketed drugs. Recent x-ray crystallographic studies identified distinct conformations of GPCRs in the active and inactive states. An allosteric sodium ion was found bound to a highly conserved D2.50 residue in inactive GPCRs, whereas the D2.50 allosteric pocket became collapsed in active GPCR structures. However, the dynamic mechanisms underlying these observations remain elusive. In this study, we aimed to understand the mechanistic effects of sodium ion binding on dynamic activation of the M3 muscarinic GPCR through long-timescale accelerated molecular dynamics (aMD) simulations. Results showed that with the D2.50 residue deprotonated, the M3 receptor is bound by an allosteric sodium ion and confined mostly in the inactive state with remarkably reduced flexibility. In contrast, the D2.50-protonated receptor does not exhibit sodium ion binding to the D2.50 allosteric site and samples a significantly larger conformational space. The receptor activation is captured and characterized by large-scale structural rearrangements of the transmembrane helices via dynamic hydrogen bond and salt bridge interactions. The residue motions are highly correlated during receptor activation. Further network analysis revealed that the allosteric signaling between residue D2.50 and key residues in the intracellular, extracellular, and orthosteric pockets is significantly weakened upon sodium ion binding.

Activation and Drug Design of a Muscarinic G-Protein Coupled Receptor

The M2 muscarinic acetylcholine receptor is a key G-protein coupled receptor (GPCR) that regulates the human heart rate and contractile forces of cardiomyocytes, and is thus targeted for treating many heart diseases. Here, we have performed long-timescale accelerated molecular dynamics (aMD) simulations and captured activation of the M2 muscarinic receptor at an atomistic level(1). The receptor activation is characterized by large-scale structural rearrangements of the transmembrane helices and conformational changes in the inter-helical salt bridge and hydrogen bond interactions(1, 2). Furthermore, using the aMD simulation-derived structural ensembles that account for the receptor flexibility, we have mapped the receptor surface for druggable allosteric sites(3) and then targeted the extracellular vestibule for designing allosteric modulators. Retrospective docking of known ligands is first carried out to validate the simulation receptor ensembles, followed by prospective docking to predict new allosteric modulators from the National Cancer Institute (NCI) compound library. The computationally selected compounds will be tested using experimental binding and functional assays, which may allow us to discover selective allosteric drugs of the M2 muscarinic receptor.

21 Concomitant activation of G-protein coupled receptors (GPCR) and sigma1 receptor (Sig1R): a novel pharmacological concept in treatment of Alzheimer's disease (AD)

The M1 muscarinic agonists AF102B (Evoxac: prescribed in Sjogren’s syndrome) and AF267B {successful in Phases I and IIa (Sjogren’s syndrome)} are effective cognitive enhancers and disease modifiers with a wide safety margin. Notably, i) AF102B decreased CSF Abeta in Alzheimer’s disease (AD) patients (Nitsch et al, Ann Neurol 2000); ii) AF267B rescued cognitive deficits and decreased Abeta42 and tau pathologies in 3xTg-AD mice (Caccamo et al, Neuron, 2006); and iii) in transgenic mice overexpressing human alpha-synuclein, AF102B and AF267B decreased brain alpha-synuclein aggregates (A. Fisher, E. Mazliah, B. Hutter-Paier, D. Havas, M. Windisch, ADPD 2011). Thus the M1 muscarinic GPCR appears to be a pivotal target for treatment of AD, Parkinson’s disease (PD) and Lewy body dementia (LBD). We have also hypothesized that concomitant activation of such a GPCR and the molecular chaperone Sig1R may have broader therapeutic implications in AD and related diseases. To achieve such combined activity we designed a low MW (357.5) compound, AF710B. This compound shows a novel mechanism of enhancing neuroprotection and cognition via Sig1R activation and M1 muscarinic modulation, but not resembling sigma1, M1 muscarinic (allosteric or orthosteric) and dual sigma1/M1 agonists, respectively. The effects of AF710B at low concentrations in vitro against neurodegeneration, oxidative stress, Abeta, Tau-phosphorylation and GSK-3beta activation translate into down-regulation of the apoptotic protein Bax and mitochondrial dysfunction, up-regulation of antiapoptotic Bcl2 and possible modulations of downstream kinases (e.g. PKC, PKB, GSK-3beta). AF710B has an exceptional pharmacology being an excellent cognitive enhancer in rats (at 1-30 and 10-100 mcg/kg, po in trihexyphenidyland in MK801-induced passive avoidance impairments, respectively). Furthermore, AF710B is devoid of side effects, having an unprecedented safety margin 50000 (po). In summary, while M1 agonists can alter pathologies in AD, PD and LBD, the unique profile of AF710B indicates widespread therapeutic advantage, inter alia, also on mitochondrial dysfunctions in these and other protein-aggregation related diseases. 22 NPT002: A NOVEL APPROACH FOR TARGETING -AMYLOID AND TAU AGGREGATES IN ALZHEIMER’S DISEASE

Ligand Regulation of the Quaternary Organization of Cell Surface M3 Muscarinic Acetylcholine Receptors Analyzed by Fluorescence Resonance Energy Transfer (FRET) Imaging and Homogeneous Time-resolved FRET

Flp-In(TM) T-REx(TM) 293 cells expressing a wild type human M(3) muscarinic acetylcholine receptor construct constitutively and able to express a receptor activated solely by synthetic ligand (RASSL) form of this receptor on demand maintained response to the muscarinic agonist carbachol but developed response to clozapine N-oxide only upon induction of the RASSL. The two constructs co-localized at the plasma membrane and generated strong ratiometric fluorescence resonance energy transfer (FRET) signals consistent with direct physical interactions. Increasing levels of induction of the FRET donor RASSL did not alter wild type receptor FRET-acceptor levels substantially. However, ratiometric FRET was modulated in a bell-shaped fashion with maximal levels of the donor resulting in decreased FRET. Carbachol, but not the antagonist atropine, significantly reduced the FRET signal. Cell surface homogeneous time-resolved FRET, based on SNAP-tag technology and employing wild type and RASSL forms of the human M(3) receptor expressed stably in Flp-In(TM) TREx(TM) 293 cells, also identified cell surface dimeric/oligomeric complexes. Now, however, signals were enhanced by appropriate selective agonists. At the wild type receptor, large increases in FRET signal to carbachol and acetylcholine were concentration-dependent with EC(50) values consistent with the relative affinities of the two ligands. These studies confirm the capacity of the human M(3) muscarinic acetylcholine receptor to exist as dimeric/oligomeric complexes at the surface of cells and demonstrate that the organization of such complexes can be modified by ligand binding. However, conclusions as to the effect of ligands on such complexes may depend on the approach used.

Consequences of Direct Versus Indirect Activation of Epidermal Growth Factor Receptor in Intestinal Epithelial Cells Are Dictated by Protein-tyrosine Phosphatase 1B

The epidermal growth factor receptor (EGFR) is an integral regulator of many cellular functions. EGFR also acts as a central conduit for extracellular signals involving direct activation of the receptor by EGFR ligands or indirect activation by G protein-coupled receptor (GPCR)-stimulated transactivation of the EGFR. We have previously shown that EGFR negatively regulates epithelial chloride secretion as a result of transforming growth factor-alpha-mediated EGFR transactivation in response to muscarinic GPCR activation. Here we show that direct activation of the EGFR by EGFR ligands produces a different pattern of EGFR tyrosine phosphorylation and downstream phosphatidylinositol 3-kinase recruitment than GPCR-stimulated transactivation of the EGFR occurring via paracrine EGFR ligand release. Moreover, we demonstrate that this differential signaling and its consequences depend on protein-tyrosine phosphatase 1B activity. Thus protein-tyrosine phosphatase 1B governs differential recruitment of signaling pathways involved in EGFR regulation of epithelial ion transport. Our findings furthermore establish how divergent signaling outcomes can arise from the activation of a single receptor.

Differential Regulation of Intracellular Calcium Oscillations by Mitochondria and Gap Junctions

Fluctuations of intracellular Ca2+ ([Ca2+]i) regulate a variety of cellular functions. The classical Ca2+ transport pathways in the endoplasmic reticulum (ER) and plasma membrane are essential to [Ca2+]i oscillations. Although mitochondria have recently been shown to absorb and release Ca2+ during G protein-coupled receptor (GPCR) activation, the role of mitochondria in [Ca2+]i oscillations remains to be elucidated. Using fluo-3-loaded human teratocarcinoma NT2 cells, we investigated the regulation of [Ca2+]i oscillations by mitochondria. Both the muscarinic GPCR agonist carbachol and the ER Ca2+-adenosine triphosphate inhibitor thapsigargin (Tg) induced [Ca2+]i oscillations in NT2 cells. The [Ca2+]i oscillations induced by carbachol were unsynchronized among individual NT2 cells; in contrast, Tg-induced oscillations were synchronized. Inhibition of mitochondrial functions with either mitochondrial blockers or depletion of mitochondrial DNA eliminated carbachol--but not Tg-induced [Ca2+]i oscillations. Furthermore, carbachol-induced [Ca2+]i oscillations were partially restored to mitochondrial DNA-depleted NT2 cells by introduction of exogenous mitochondria. Treatment of NT2 cells with gap junction blockers prevented Tg-induced but not carbachol-induced [Ca2+]i oscillations. These data suggest that the distinct patterns of [Ca2+]i oscillations induced by GPCR and Tg are differentially modulated by mitochondria and gap junctions.

Roles for Gα and Gβγ in PLC Activation

There are many isoforms of phospholipase C (PLC), the enzyme that cleaves phosphatidylinositol 4,5 diphosphate (PIP 2 ). PLC-β isoforms are activated by Gα q in response to activation of G protein-coupled receptors (GPCRs). The regulation of the PLC-δ isoforms is less well understood. Guo et al . present evidence to suggest that PLC-β2 maintains PLC-δ1 in an inactive state and that increased concentrations of the Gβγ disrupt the interaction between the two proteins, allowing PLC-δ1 to become active. This provides one model to explain how PLC-δ amplifies calcium signals initiated by PLC-β. Using a fluorescent probe covalently attached to PLC-β2, a direct association between PLC-β2 and PLC-δ1 was detected in vitro, and the affinity of the interaction was increased if the enzymes were immobilized on membranes. In vitro enzymatic activity was less than additive when both isoforms were present, suggesting that one or both enzymes were being inhibited. Using several different assay conditions, the authors determined that the activity of PLC-δ1 was inhibited by the presence of PLC-β2. The addition of Gβγ to the mixture of PLC-β2 and PLC-δ1 disrupted complex formation between the two enzymes in vitro and abolished inhibition of enzyme activity when the enzymes were mixed. Experiments with endogenously expressed PLC-β2 and PLC-δ1, as well as with cells transfected with versions of the proteins for bimolecular fluorescence complementation (BiFC), indicated that both proteins localized at the plasma membrane in unstimulated cells. In the BiFC experiment, stimulation of the cells with acetylcholine, which activates muscarinic GPCRs, decreased the enzymes' interaction. In the transfected cells, inhibition of Gα with pertussis toxin, which allows Gβγ to accumulate, disrupted the interaction between PLC-β2 and PLC-δ1, and this disruption by increasing concentrations of Gβγ was reproducible in isolated membrane preparations from the BiFC-transfected cells. Thus, it appears that in cells expressing both PLC-β2 and PLC-δ1, PLC-β2 maintains PLC-δ1 in an inactive state, and this inhibition is relieved by the Gβγ subunits of the G proteins activated in response to GPCR stimulation. Thus G proteins have a dual role in producing a calcium signal: Gα stimulates PLC-β2 and Gβγ disrupts the PLC-β2 and PLC-δ1 complex, allowing PLC-δ1 to enhance the signal. Y. Guo, M. Rebecchi, S. Scarlata, Phospholipase Cβ 2 binds to and inhibits phospholipase Cδ 1 . J. Biol. Chem . 280 , 1438-1447 (2005). [Abstract] [Full Text]