Gq-coupled Muscarinic Receptor Research Articles

Obesity-associated microglial inflammatory activation paradoxically improves glucose tolerance

Hypothalamic gliosis associated with high-fat diet (HFD) feeding increases susceptibility to hyperphagia and weight gain. However, the body-weight-independent contribution of microglia to glucose regulation has not been determined. Here, we show that reducing microglial nuclear factor κB (NF-κB) signaling via cell-specific IKKβ deletion exacerbates HFD-induced glucose intolerance despite reducing body weight and adiposity. Conversely, two genetic approaches to increase microglial pro-inflammatory signaling (deletion of an NF-κB pathway inhibitor and chemogenetic activation through a modified Gq-coupled muscarinic receptor) improved glucose tolerance independently of diet in both lean and obese rodents. Microglial regulation of glucose homeostasis involves a tumor necrosis factor alpha (TNF-α)-dependent mechanism that increases activation of pro-opiomelanocortin (POMC) and other hypothalamic glucose-sensing neurons, ultimately leading to a marked amplification of first-phase insulin secretion via a parasympathetic pathway. Overall, these data indicate that microglia regulate glucose homeostasis in a body-weight-independent manner, an unexpected mechanism that limits the deterioration of glucose tolerance associated with obesity.

Palmitoylation Targets the Calcineurin Phosphatase to the Phosphatidylinositol 4‐kinase Complex at the Plasma Membrane

Calcineurin (CN), the serine/threonine protein phosphatase and the target of immunosuppressants, is a critical regulator of Ca2+ signaling. To discover novel CN‐regulated processes, we examined an understudied isoform, CNAb1, that is highly conserved across vertebrates and expressed in every human tissue. CN is a heterodimer of regulatory and catalytic subunits whose functions and regulation by Ca2+ and calmodulin are well understood for canonical CN isozymes. The few studies of CNAb1, demonstrate its unique physiological and biochemical properties, however, mechanisms that confer these properties and CNAb1‐ specific substrates remain unknown. Here, we show that the non‐canonical C‐terminus of CNAb1, generated by alternative 3’ pre‐mRNA processing, confers unique intracellular localization, enzymatic regulation and substrate specificity for this isoform. In vitro, CNAb1 displays distinct enzymatic properties, where instead of the auto‐inhibitory domain that blocks the active site of canonical CN isoforms under non‐signaling conditions, CNAβ1 is autoinhibited by a sequence motif at its C tail that blocks substrate binding. In cells, unlike canonical cytosolic CN, CNAb1 localizes to the plasma membrane and Golgi due to palmitoylation of its divergent C‐terminal tail. This palmitoylation, which is dynamically regulated by the ABHD17A depalmitoylase, targets CNAb1 to distinct set of membrane‐associated interactors including the phosphatidylinositol 4‐kinase (PI4KA) complex containing EFR3B, PI4KA, TTC7B and FAM126A. This complex recruits the cytosolic PI4KIIIA to the PM where it synthesizes phosphatidylinositol‐4‐phosphate (PI4P), a precursor of the critical signaling phospholipid, PI(4,5)P2, required for sustained Ca2+signaling through GPCRs. Using hydrogen‐deuterium exchange, we reveal multiple CN‐PI4KA complex contacts, including a calcineurin‐binding peptide motif in the disordered tail of FAM126A which we further establish as a novel calcineurin substrate. Using BRET‐based detection of phosphoinositides in live cells, we show that CN inhibitors decrease PM PI4P production during Gq‐coupled muscarinic receptor signaling, suggesting that CN dephosphorylates and promotes PI4KA complex activity. Together, this work not only discovers dynamic palmitoylation as a novel mechanism that confers unique localization, substrate specificity and regulation to CNAb1 but also uncovers a novel Ca2+/CN‐mediated, feedback loop, that promotes PI4P replenishment at the PM during GPCR signaling.

Gq-Coupled Muscarinic Receptor Enhancement of KCNQ2/3 Channels and Activation of TRPC Channels in Multimodal Control of Excitability in Dentate Gyrus Granule Cells.

KCNQ (Kv7, "M-type") K+ channels and TRPC (transient receptor potential, "canonical") cation channels are coupled to neuronal discharge properties and are regulated via Gq/11-protein-mediated signals. Stimulation of Gq/11-coupled receptors both consumes phosphatidylinositol 4,5-bisphosphate (PIP2) via phosphalipase Cβ hydrolysis and stimulates PIP2 synthesis via rises in Ca2+i and other signals. Using brain-slice electrophysiology and Ca2+ imaging from male and female mice, we characterized threshold K+ currents in dentate gyrus granule cells (DGGCs) and CA1 pyramidal cells, the effects of Gq/11-coupled muscarinic M1 acetylcholine (M1R) stimulation on M current and on neuronal discharge properties, and elucidated the intracellular signaling mechanisms involved. We observed disparate signaling cascades between DGGCs and CA1 neurons. DGGCs displayed M1R enhancement of M-current, rather than suppression, due to stimulation of PIP2 synthesis, which was paralleled by increased PIP2-gated G-protein coupled inwardly rectifying K+ currents as well. Deficiency of KCNQ2-containing M-channels ablated the M1R-induced enhancement of M-current in DGGCs. Simultaneously, M1R stimulation in DGGCs induced robust increases in [Ca2+]i, mostly due to TRPC currents, consistent with, and contributing to, neuronal depolarization and hyperexcitability. CA1 neurons did not display such multimodal signaling, but rather M current was suppressed by M1R stimulation in these cells, similar to the previously described actions of M1R stimulation on M-current in peripheral ganglia that mostly involves PIP2 depletion. Therefore, these results point to a pleiotropic network of cholinergic signals that direct cell-type-specific, precise control of hippocampal function with strong implications for hyperexcitability and epilepsy.SIGNIFICANCE STATEMENT At the neuronal membrane, protein signaling cascades consisting of ion channels and metabotropic receptors govern the electrical properties and neurotransmission of neuronal networks. Muscarinic acetylcholine receptors are G-protein-coupled metabotropic receptors that control the excitability of neurons through regulating ion channels, intracellular Ca2+ signals, and other second-messenger cascades. We have illuminated previously unknown actions of muscarinic stimulation on the excitability of hippocampal principal neurons that include M channels, TRPC (transient receptor potential, "canonical") cation channels, and powerful regulation of lipid metabolism. Our results show that these signaling pathways, and mechanisms of excitability, are starkly distinct between peripheral ganglia and brain, and even between different principal neurons in the hippocampus.

Involvement of specific neuronal populations of basal ganglia in decision-making

Event Abstract Back to Event Involvement of specific neuronal populations of basal ganglia in decision-making Elena Chaves Rodriguez1, 2*, Serge N. Schiffmann1, 2 and Alban De Kerchove D'Exaerde1, 2, 3 1 FNRS-Belgium, Belgium 2 Université Libre de Bruxelles, Belgium 3 WELBIO, Belgium The basal ganglia are a group of brain nuclei involved in decision-making processes. They receive major cortical inputs, as well as important dopaminergic projections, linking cognitive and emotional processes to an individual’s motor output. Using specific-Cre transgenic mouse lines that allowed us to target specific populations of neurons in the basal ganglia, we succeeded to manipulate their activity by means of a chemogenetic -DREADD- approach. This DREADD technology permitted us to express a human modified Gq-coupled muscarinic receptor in the neurons of interest, whose activation was dependent on the administration of an artificial ligand (CNO). Using this tool we could induce an activation of the targeted neurons, whose consequences were analyzed with a battery of behavioral tests in order to assess any motor alteration or decision-making deficit. Keywords: decision-making, Basal Ganglia, chemogenetics, Behavior, mouse models Conference: 12th National Congress of the Belgian Society for Neuroscience, Gent, Belgium, 22 May - 22 May, 2017. Presentation Type: Poster Presentation Topic: Cognition and Behavior Citation: Chaves Rodriguez E, Schiffmann SN and De Kerchove D'Exaerde A (2019). Involvement of specific neuronal populations of basal ganglia in decision-making. Front. Neurosci. Conference Abstract: 12th National Congress of the Belgian Society for Neuroscience. doi: 10.3389/conf.fnins.2017.94.00087 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 21 Apr 2017; Published Online: 25 Jan 2019. * Correspondence: Miss. Elena Chaves Rodriguez, FNRS-Belgium, Brussels, Belgium, echavesr@ulb.ac.be Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Elena Chaves Rodriguez Serge N Schiffmann Alban De Kerchove D'Exaerde Google Elena Chaves Rodriguez Serge N Schiffmann Alban De Kerchove D'Exaerde Google Scholar Elena Chaves Rodriguez Serge N Schiffmann Alban De Kerchove D'Exaerde PubMed Elena Chaves Rodriguez Serge N Schiffmann Alban De Kerchove D'Exaerde Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

ERK5 activation by Gq-coupled muscarinic receptors is independent of receptor internalization and β-arrestin recruitment.

G-protein-coupled receptors (GPCRs) are known to activate both G protein- and β-arrestin-dependent signalling cascades. The initiation of mitogen-activated protein kinase (MAPK) pathways is a key downstream event in the control of cellular functions including proliferation, differentiation, migration and apoptosis. Both G proteins and β-arrestins have been reported to mediate context-specific activation of ERK1/2, p38 and JNK MAPKs. Recently, the activation of ERK5 MAPK by Gq-coupled receptors has been described to involve a direct interaction between Gαq and two novel effectors, PKCζ and MEK5. However, the possible contribution of β-arrestin towards this pathway has not yet been addressed. In the present work we sought to investigate the role of receptor internalization processes and β-arrestin recruitment in the activation of ERK5 by Gq-coupled GPCRs. Our results show that ERK5 activation is independent of M1 or M3 muscarinic receptor internalization. Furthermore, we demonstrate that phosphorylation-deficient muscarinic M1 and M3 receptors are still able to fully activate the ERK5 pathway, despite their reported inability to recruit β-arrestins. Indeed, the overexpression of Gαq, but not that of β-arrestin1 or β-arrestin2, was found to potently enhance ERK5 activation by GPCRs, whereas silencing of β-arrestin2 expression did not affect the activation of this pathway. Finally, we show that a β-arrestin-biased mutant form of angiotensin II (SII; Sar1-Ile4-Ile8 AngII) failed to promote ERK5 phosphorylation in primary cardiac fibroblasts, as compared to the natural ligand. Overall, this study shows that the activation of ERK5 MAPK by model Gq-coupled GPCRs does not depend on receptor internalization, β-arrestin recruitment or receptor phosphorylation but rather is dependent on Gαq-signalling.

AKAP79, PKC, PKA and PDE4 participate in a Gq-linked muscarinic receptor and adenylate cyclase 2 cAMP signalling complex

AC2 (adenylate cyclase 2) is stimulated by activation of Gq-coupled muscarinic receptors through PKC (protein kinase C) to generate localized cAMP in HEK (human embryonic kidney)-293 cells. In the present study, we utilized a sensitive live-cell imaging technique to unravel the proteins that play essential roles in a Gq-coupled muscarinic receptor-mediated cAMP signalling complex. We reveal that, upon agonist binding to the Gq-coupled muscarinic receptor, AKAP79 (A-kinase-anchoring protein 79) recruits PKC to activate AC2 to produce cAMP. The cAMP formed is degraded by PDE4 (phosphodiesterase 4) activated by an AKAP-anchored PKA (protein kinase A). Calcineurin, a phosphatase bound to AKAP79, is not involved in this regulation. Overall, a transient cAMP increase is generated from AC2 by Gq-coupled muscarinic receptor activation, subject to sophisticated regulation through AKAP79, PKC, PDE4 and PKA, which significantly enhances acetylcholine-mediated signalling.

Muscarinic receptors stimulate AC2 by novel phosphorylation sites, whereas Gβγ subunits exert opposing effects depending on the G-protein source

Direct phosphorylation of AC2 (adenylyl cyclase 2) by PKC (protein kinase C) affords an opportunity for AC2 to integrate signals from non-canonical pathways to produce the second messenger, cyclic AMP. The present study shows that stimulation of AC2 by pharmacological activation of PKC or muscarinic receptor activation is primarily the result of phosphorylation of Ser490 and Ser543, as opposed to the previously proposed Thr1057. A double phosphorylation-deficient mutant (S490/543A) of AC2 was insensitive to PMA (phorbol myristic acid) and CCh (carbachol) stimulation, whereas a double phosphomimetic mutant (S490/543D) mimicked the activity of PKC-activated AC2. Putative Gβγ-interacting sites are in the immediate environment of these PKC phosphorylation sites (Ser490 and Ser543) that are located within the C1b domain of AC2, suggesting a significant regulatory importance of this domain. Consequently, we examined the effect of both Gq-coupled muscarinic and Gi-coupled somatostatin receptors. Employing pharmacological and FRET (fluorescence resonance energy transfer)-based real-time single cell imaging approaches, we found that Gβγ released from the Gq-coupled muscarinic receptor or Gi-coupled somatostatin receptors exert inhibitory or stimulatory effects respectively. These results underline the sophisticated regulatory capacities of AC2, in not only being subject to regulation by PKC, but also and in an opposite manner to Gβγ subunits, depending on their source.

Amiodarone and N‐ethyl‐amiodarone exert opposing allosteric effects via a novel site on muscarinic receptors

Allosteric modulation provides several advantages over the classical (orthosteric) approach to receptor activation; further, the characterization of new allosteric sites provides additional opportunities for the development of subtype‐selective and functionally selective compounds. We have been investigating the allosteric actions of amiodarone and N‐ethyl‐amiodarone (NEA) at muscarinic receptors. Previous studies have demonstrated that amiodarone interacts with a site that is distinct from the one that has been defined by gallamine.We have shown that amiodarone enhances stimulation of [3H]arachidonic acid release induced by agonists acting at Gq‐coupled muscarinic receptors. Evaluation of this action in light of Hall's allosteric two‐state model indicates that amiodarone's enhancement of agonist efficacy is tempered by a concomitant reduction in agonist binding affinity. The enhancement of efficacy is attenuated by NEA but not by the orthosteric antagonist N‐methylscopolamine; further evaluation of this finding using an elaboration of Hall's model suggests that amiodarone and NEA exert their functional effects through a common allosteric site.In conclusion, we have demonstrated that amiodarone and NEA interact allosterically with a novel, extracellular, site on muscarinic receptors, through which they modulate agonist potency and efficacy in a complex manner.

The Gβ5−RGS7 Complex Selectively Inhibits Muscarinic M3 Receptor Signaling via the Interaction between the Third Intracellular Loop of the Receptor and the DEP Domain of RGS7

Regulators of G protein signaling (RGS) make up a diverse family primarily known as GTPase-activating proteins (GAPs) for heterotrimeric G proteins. In addition to the RGS domain, which is responsible for GAP activity, most RGS proteins contain other distinct structural motifs. For example, members of the R7 family of RGS proteins contain a DEP, GGL, and novel DHEX domain and are obligatory dimers with G protein beta subunit Gbeta5. Here we show that the Gbeta5-RGS7 complex can inhibit Ca2+ mobilization elicited by muscarinic acetylcholine receptor type 3 (M3R), but not by other Gq-coupled receptors such as M1, M5, histamine H1, and GNRH receptors. The isolated DEP domain of RGS7 is sufficient for the inhibition of M3R signaling, whereas the deletion of the DEP domain renders the Gbeta5-RGS7 complex ineffective. Deletion of a portion of the third intracellular loop allowed the receptor (M3R-short) to signal but rendered it insensitive to the effect of the Gbeta5-RGS7 complex. Accordingly, the recombinant DEP domain bound in vitro to the GST-fused i3 loop of the M3R. These results identify a novel molecular mechanism that can impart receptor subtype selectivity on signal transduction via Gq-coupled muscarinic receptors.

Phosphorylation of Inositol 1,4,5-Trisphosphate Receptors by cAMP-dependent Protein Kinase: TYPE I, II, AND III RECEPTORS ARE DIFFERENTIALLY SUSCEPTIBLE TO PHOSPHORYLATION AND ARE PHOSPHORYLATED IN INTACT CELLS

The ability of cAMP-dependent protein kinase (PKA) to phosphorylate type I, II, and III inositol 1,4,5-trisphosphate (InsP3) receptors was examined. The receptors either were immunopurified from cell lines and then phosphorylated with purified PKA or were phosphorylated in intact cells after activating intracellular cAMP formation. The former studies showed that the type I receptor was a good substrate for PKA (0.65 mol Pi incorporated/mol receptor), whereas type II and III receptors were phosphorylated relatively weakly. The latter studies showed that despite these differences, each of the receptors was phosphorylated in intact cells in response to forskolin or activation of neurohormone receptors. Detailed examination of SH-SY5Y neuroblastoma cells, which express >/=99% type I receptor, revealed that minor increases in cAMP concentration were sufficient to cause maximal phosphorylation. Thus, VIP and pituitary adenylyl cyclase activating peptide (acting through Gs-coupled pituitary adenylyl cyclase activating peptide-I receptors) were potent stimuli of type I receptor phosphorylation, and remarkably, even slight increases in cAMP concentration induced by carbachol (acting through Gq-coupled muscarinic receptors) or other Ca2+ mobilizing agents were sufficient to cause phosphorylation. Finally, PKA enhanced InsP3-induced Ca2+ mobilization in a range of permeabilized cell types, irrespective of whether the type I, II, or III receptor was predominant. In summary, these data show that all InsP3 receptors are phosphorylated by PKA, albeit with marked differences in stoichiometry. The ability of both Gs- and Gq-coupled cell surface receptors to effect InsP3 receptor phosphorylation by PKA suggests that this process is widespread in mammalian cells and provides multiple routes by which the cAMP signaling pathway can influence Ca2+ mobilization.