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1.  Regulation of Neuronal Plasticity and Fear by a Dynamic Change in PAR1 – G Protein Coupling in the Amygdala 
Molecular psychiatry  2012;18(10):1136-1145.
Fear memories are acquired through neuronal plasticity, an orchestrated sequence of events regulated at circuit and cellular levels. The conventional model of fear acquisition assumes unimodal (e.g. excitatory or inhibitory) roles of modulatory receptors in controlling neuronal activity and learning. Contrary to this view, we show that protease-activated receptor-1 (PAR1) promotes contrasting neuronal responses depending on the emotional status of an animal by a dynamic shift between distinct G protein coupling partners. In the basolateral amygdala of fear-naïve mice PAR1 couples to Gαq/11 and Gαo proteins, while after fear conditioning coupling to Gαo increases. Concurrently, stimulation of PAR1 before conditioning enhanced, but afterwards it inhibited firing of basal amygdala neurons. An initial impairment of the long-term potentiation (LTP) in PAR1-deficient mice was transformed into an increase in LTP and enhancement of fear after conditioning. These effects correlated with more frequent AMPA receptor-mediated miniature post synaptic events and increased neuronal excitability. Our findings point to experience-specific shifts in PAR1-G protein-coupling in the amygdala as a novel mechanism regulating neuronal excitability and fear.
PMCID: PMC3690134  PMID: 23032873
2.  The regulation of M1 muscarinic acetylcholine receptor desensitization by synaptic activity in cultured hippocampal neurons1 
Journal of Neurochemistry  2007;103(6):2268-2280.
To better understand metabotropic/ionotropic integration in neurons we have examined the regulation of M1 muscarinic acetylcholine (mACh) receptor signalling in mature (> 14 days in vitro), synaptically-active hippocampal neurons in culture. Using a protocol where neurons are exposed to an EC50 concentration of the muscarinic agonist methacholine (MCh) prior to (R1), and following (R2) a desensitizing pulse of a high concentration of this agonist, we have found that the reduction in M1 mACh receptor responsiveness is decreased in quiescent (+tetrodotoxin) neurons and increased when synaptic activity is enhanced by blocking GABAA receptors with picrotoxin. The picrotoxin-mediated effect on M1 mACh receptor responsiveness was completely prevented by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor blockade. Inhibition of endogenous G protein-coupled receptor kinase 2 by transfection with the non-Gq/11α-binding, catalytically-inactive D110A,K220RG protein-coupled receptor kinase 2 mutant, decreased the extent of M1 mACh receptor desensitization under all conditions. Pharmacological inhibition of protein kinase C (PKC) activity, or chronic phorbol ester-induced PKC down-regulation had no effect on agonist-mediated receptor desensitization in quiescent or spontaneously synaptically active neurons, but significantly decreased the extent of receptor desensitization in picrotoxin-treated neurons. MCh stimulated the translocation of diacylglycerol- sensitive eGFP-PKCε, but not Ca2+/diacylglycerol-sensitive eGFP-PKCβII in both the absence, and presence of tetrodotoxin. Under these conditions, MCh-stimulated eGFP-myristoylated, alanine-rich C kinase substrate translocation was dependent on PKC activity, but not Ca2+/calmodulin. In contrast, picrotoxin-driven translocation of myristoylated, alanine-rich C kinase substrate was accompanied by translocation of PKCβII, but not PKCε, and was dependent on PKC and Ca2+/calmodulin. Taken together these data suggest that the level of synaptic activity may determine the different kinases recruited to regulate M1 mACh receptor desensitization in neurons.
PMCID: PMC2658029  PMID: 17908240
G protein-coupled receptor kinase; hippocampal neuron; muscarinic acetylcholine receptor; protein kinase C; receptor desensitization; synaptic activity
3.  Increased Nicotinamide Adenine Dinucleotide Phosphate Oxidase 4 Expression Mediates Intrinsic Airway Smooth Muscle Hypercontractility in Asthma 
Rationale: Asthma is characterized by disordered airway physiology as a consequence of increased airway smooth muscle contractility. The underlying cause of this hypercontractility is poorly understood.
Objectives: We sought to investigate whether the burden of oxidative stress in airway smooth muscle in asthma is heightened and mediated by an intrinsic abnormality promoting hypercontractility.
Methods: We examined the oxidative stress burden of airway smooth muscle in bronchial biopsies and primary cells from subjects with asthma and healthy controls. We determined the expression of targets implicated in the control of oxidative stress in airway smooth muscle and their role in contractility.
Measurements and Main Results: We found that the oxidative stress burden in the airway smooth muscle in individuals with asthma is heightened and related to the degree of airflow obstruction and airway hyperresponsiveness. This was independent of the asthmatic environment as in vitro primary airway smooth muscle from individuals with asthma compared with healthy controls demonstrated increased oxidative stress–induced DNA damage together with an increased production of reactive oxygen species. Genome-wide microarray of primary airway smooth muscle identified increased messenger RNA expression in asthma of NADPH oxidase (NOX) subtype 4. This NOX4 overexpression in asthma was supported by quantitative polymerase chain reaction, confirmed at the protein level. Airway smooth muscle from individuals with asthma exhibited increased agonist-induced contraction. This was abrogated by NOX4 small interfering RNA knockdown and the pharmacological inhibitors diphenyleneiodonium and apocynin.
Conclusions: Our findings support a critical role for NOX4 overexpression in asthma in the promotion of oxidative stress and consequent airway smooth muscle hypercontractility. This implicates NOX4 as a potential novel target for asthma therapy.
PMCID: PMC3402550  PMID: 22108207
asthma; airway smooth muscle; airway hyperresponsiveness; NOX4; SOD2
4.  FRET-Based Detection of M1 Muscarinic Acetylcholine Receptor Activation by Orthosteric and Allosteric Agonists 
PLoS ONE  2012;7(1):e29946.
Background and Objective
Muscarinic acetylcholine receptors (mAChRs) are 7-transmembrane, G protein-coupled receptors that regulate a variety of physiological processes and represent potentially important targets for therapeutic intervention. mAChRs can be stimulated by full and partial orthosteric and allosteric agonists, however the relative abilities of such ligands to induce conformational changes in the receptor remain unclear. To gain further insight into the actions of mAChR agonists, we have developed a fluorescently tagged M1 mAChR that reports ligand-induced conformational changes in real-time by changes in Förster resonance energy transfer (FRET).
Variants of CFP and YFP were inserted into the third intracellular loop and at the end of the C-terminus of the mouse M1 mAChR, respectively. The optimized FRET receptor construct (M1-cam5) was expressed stably in HEK293 cells.
The variant CFP/YFP-receptor chimera expressed predominantly at the plasma membrane of HEK293 cells and displayed ligand-binding affinities comparable with those of the wild-type receptor. It also retained an ability to interact with Gαq/11 proteins and to stimulate phosphoinositide turnover, ERK1/2 phosphorylation and undergo agonist-dependent internalization. Addition of the full agonist methacholine caused a reversible decrease in M1 FRET (FEYFP/FECFP) that was prevented by atropine pre-addition and showed concentration-dependent amplitude and kinetics. Partial orthosteric agonists, arecoline and pilocarpine, as well as allosteric agonists, AC-42 and 77-LH-28-1, also caused atropine-sensitive decreases in the FRET signal, which were smaller in amplitude and significantly slower in onset compared to those evoked by methacholine.
The M1 FRET-based receptor chimera reports that allosteric and orthosteric agonists induce similar conformational changes in the third intracellular loop and/or C-terminus, and should prove to be a valuable molecular reagent for pharmacological and structural investigations of M1 mAChR activation.
PMCID: PMC3260180  PMID: 22272263
5.  Clustered Coding Variants in the Glutamate Receptor Complexes of Individuals with Schizophrenia and Bipolar Disorder 
PLoS ONE  2011;6(4):e19011.
Current models of schizophrenia and bipolar disorder implicate multiple genes, however their biological relationships remain elusive. To test the genetic role of glutamate receptors and their interacting scaffold proteins, the exons of ten glutamatergic ‘hub’ genes in 1304 individuals were re-sequenced in case and control samples. No significant difference in the overall number of non-synonymous single nucleotide polymorphisms (nsSNPs) was observed between cases and controls. However, cluster analysis of nsSNPs identified two exons encoding the cysteine-rich domain and first transmembrane helix of GRM1 as a risk locus with five mutations highly enriched within these domains. A new splice variant lacking the transmembrane GPCR domain of GRM1 was discovered in the human brain and the GRM1 mutation cluster could perturb the regulation of this variant. The predicted effect on individuals harbouring multiple mutations distributed in their ten hub genes was also examined. Diseased individuals possessed an increased load of deleteriousness from multiple concurrent rare and common coding variants. Together, these data suggest a disease model in which the interplay of compound genetic coding variants, distributed among glutamate receptors and their interacting proteins, contribute to the pathogenesis of schizophrenia and bipolar disorders.
PMCID: PMC3084736  PMID: 21559497
6.  Temporal profiling of changes in phosphatidylinositol 4,5-bisphosphate, inositol 1,4,5-trisphosphate and diacylglycerol allows comprehensive analysis of phospholipase C-initiated signalling in single neurons1 
Journal of Neurochemistry  2008;107(3):602-615.
Phosphatidylinositol 4,5-bisphosphate (PIP2) fulfils vital signalling roles in an array of cellular processes, yet until recently it has not been possible selectively to visualize real-time changes in PIP2 levels within living cells. Green fluorescent protein (GFP)-labelled Tubby protein (GFP-Tubby) enriches to the plasma membrane at rest and translocates to the cytosol following activation of endogenous Gαq/11-coupled muscarinic acetylcholine receptors in both SH-SY5Y human neuroblastoma cells and primary rat hippocampal neurons. GFP-Tubby translocation is independent of changes in cytosolic inositol 1,4,5-trisphosphate and instead reports dynamic changes in levels of plasma membrane PIP2. In contrast, enhanced GFP (eGFP)-tagged pleckstrin homology domain of phospholipase C (PLCδ1) (eGFP-PH) translocation reports increases in cytosolic inositol 1,4,5-trisphosphate. Comparison of GFP-Tubby, eGFP-PH and the eGFP-tagged C12 domain of protein kinase C-γ [eGFP-C1(2); to detect diacylglycerol] allowed a selective and comprehensive analysis of PLC-initiated signalling in living cells. Manipulating intracellular Ca2+ concentrations in the nanomolar range established that GFP-Tubby responses to a muscarinic agonist were sensitive to intracellular Ca2+ up to 100–200 nM in SH-SY5Y cells, demonstrating the exquisite sensitivity of agonist-mediated PLC activity within the range of physiological resting Ca2+ concentrations. We have also exploited GFP-Tubby selectively to visualize, for the first time, real-time changes in PIP2 in hippocampal neurons.
PMCID: PMC2779467  PMID: 18665913
phospholipase C; phosphatidylinositol 4,5-bisphosphate; Tubby protein; SH-SY5Y; hippocampal neuron
7.  Modulation of Gq protein-coupled IP3 and Ca2+ signaling by the membrane potential 
Gq protein-coupled receptors (GqPCRs) are widely distributed in the CNS and play fundamental roles in a variety of neuronal processes. Their activation results in PIP2 hydrolysis and Ca2+ release from intracellular stores via the PLC-IP3 signaling pathway. Since early GqPCR signaling events occur at the plasma membrane of neurons, they might be influenced by changes in membrane potential. In this study we use combined patch-clamp and imaging methods to investigate whether membrane potential changes can modulate GqPCR signaling in neurons. Our results demonstrate that GqPCR signaling in the human neuronal cell line SH-SY5Y and in rat cerebellar granule neurons is directly sensitive to changes in membrane potential, even in the absence of extracellular Ca2+. Depolarization has a bi-directional effect on GqPCR signaling, potentiating thapsigargin-sensitive Ca2+ responses to muscarinic receptor activation but attenuating those mediated by bradykinin receptors. The depolarization-evoked potentiation of the muscarinic signaling is graded, bipolar, non-inactivating and with no apparent upper limit, ruling out traditional voltage-gated ion channels as the primary voltage sensors. Flash photolysis of caged IP3/GPIP2 places the voltage-sensor before the level of the Ca2+ store and measurements using the fluorescent bioprobe eGFP-PHPLCδ directly demonstrate that voltage affects muscarinic signaling at the level of the IP3 production pathway. The sensitivity of GqPCR IP3 signaling in neurons to voltage itself may represent a fundamental mechanism by which ionotropic signals can shape metabotropic receptor activity in neurons and influence processes such as synaptic plasticity in which the detection of coincident signals is crucial.
PMCID: PMC2266565  PMID: 17005862
voltage; G protein-coupled receptor; calcium; inositol 1,4,5-trisphosphate; membrane potential; muscarinic acetylcholine receptor
8.  Principal role of adenylyl cyclase 6 in K+ channel regulation and vasodilator signalling in vascular smooth muscle cells 
Cardiovascular Research  2011;91(4):694-702.
Membrane potential is a key determinant of vascular tone and many vasodilators act through the modulation of ion channel currents [e.g. the ATP-sensitive potassium channel (KATP)] involved in setting the membrane potential. Adenylyl cyclase (AC) isoenzymes are potentially important intermediaries in such vasodilator signalling pathways. Vascular smooth muscle cells (VSMCs) express multiple AC isoenzymes, but the reason for such redundancy is unknown. We investigated which of these isoenzymes are involved in vasodilator signalling and regulation of vascular ion channels important in modulating membrane potential.
Methods and results
AC isoenzymes were selectively depleted (by >75%) by transfection of cultured VSMCs with selective short interfering RNA sequences. AC6 was the predominant isoenzyme involved in vasodilator-mediated cAMP accumulation in VSMCs, accounting for ∼60% of the total response to β-adrenoceptor (β-AR) stimulation. AC3 played a minor role in β-AR signalling, whereas AC5 made no significant contribution. AC6 was also the principal isoenzyme involved in β-AR-mediated protein kinase A (PKA) signalling (determined using the fluorescent biosensor for PKA activity, AKAR3) and the substantial β-AR/PKA-dependent enhancement of KATP current. KATP current was shown to play a vital role in setting the resting membrane potential and in mediating the hyperpolarization observed upon β-AR stimulation.
AC6, but not the closely related AC5, plays a principal role in vasodilator signalling and regulation of the membrane potential in VSMCs. These findings identify AC6 as a vital component in the vasodilatory apparatus central to the control of blood pressure.
PMCID: PMC3156907  PMID: 21606183
Adenylyl cyclase; Cyclic AMP; Vascular smooth muscle; ATP-sensitive potassium channel; β-Adrenoceptor
9.  G protein-coupled receptor kinase 2 and arrestin2 regulate arterial smooth muscle P2Y-purinoceptor signalling 
Cardiovascular Research  2010;89(1):193-203.
Prolonged P2Y-receptor signalling can cause vasoconstriction leading to hypertension, vascular smooth muscle hypertrophy, and hyperplasia. G protein-coupled receptor signalling is negatively regulated by G protein-coupled receptor kinases (GRKs) and arrestin proteins, preventing prolonged or inappropriate signalling. This study investigates whether GRKs and arrestins regulate uridine 5′-triphosphate (UTP)-stimulated contractile signalling in adult Wistar rat mesenteric arterial smooth muscle cells (MSMCs).
Methods and results
Mesenteric arteries contracted in response to UTP challenge: When an EC50 UTP concentration (30 µM, 5 min) was added 5 min before (R1) and after (R2) the addition of a maximal UTP concentration (Rmax: 100 µM, 5 min), R2 responses were decreased relative to R1, indicating desensitization. UTP-induced P2Y-receptor desensitization of phospholipase C signalling was studied in isolated MSMCs transfected with an inositol 1,4,5-trisphosphate biosensor and/or loaded with Ca2+-sensitive dyes. A similar protocol (R1/R2 = 10 µM; Rmax = 100 µM, applied for 30 s) revealed markedly reduced R2 when compared with R1 responses. MSMCs were transfected with dominant-negative GRKs or siRNAs targeting specific GRK/arrestins to probe their respective roles in P2Y-receptor desensitization. GRK2 inhibition, but not GRK3, GRK5, or GRK6, attenuated P2Y-receptor desensitization. siRNA-mediated knockdown of arrestin2 attenuated UTP-stimulated P2Y-receptor desensitization, whereas arrestin3 depletion did not. Specific siRNA knockdown of the P2Y2-receptor almost completely abolished UTP-stimulated IP3/Ca2+ signalling, strongly suggesting that our study is specifically characterizing this purinoceptor subtype.
These new data highlight roles of GRK2 and arrestin2 as important regulators of UTP-stimulated P2Y2-receptor responsiveness in resistance arteries, emphasizing their potential importance in regulating vasoconstrictor signalling pathways implicated in vascular disease.
PMCID: PMC3002865  PMID: 20705669
UTP; G protein-coupled receptor kinase; Arrestin; P2Y-purinoceptor; Resistance artery
10.  Endothelin signalling in arterial smooth muscle is tightly regulated by G protein-coupled receptor kinase 2 
Cardiovascular Research  2009;85(3):424-433.
Prolonged endothelin (ET) receptor signalling causes vasoconstriction and can lead to hypertension, vascular smooth muscle hypertrophy, and hyperplasia. Usually, G protein-coupled receptor signalling is negatively regulated by G protein-coupled receptor kinases (GRKs), preventing prolonged or inappropriate signalling. This study investigated whether GRKs regulate ET receptor contractile signalling in adult Wistar rat mesenteric arterial smooth muscle cells (MSMCs).
Methods and results
ET-1-stimulated phospholipase C (PLC) activity and changes in [Ca2+]i were assessed using confocal microscopy in rat MSMCs transfected with the pleckstrin-homology domain of PLCδ1 (eGFP-PH) and loaded with Fura-Red. ET-1 applications (30 s) stimulated transient concentration-dependent eGFP-PH translocations from plasma membrane to cytoplasm and graded [Ca2+]i increases. ET-1-mediated PLC signalling was blocked by the type A endothelin receptor (ETAR) antagonist, BQ123. To characterize ETAR desensitization, cells were stimulated with a maximally effective concentration of ET-1 (50 nM, 30 s) followed by a variable washout period and a second identical application of ET-1. This brief exposure to ET-1 markedly decreased ETAR responsiveness to re-challenge, and reversal was incomplete even after increasing the time period between agonist challenges to 60 min. To assess GRK involvement in ETAR desensitization, MSMCs were co-transfected with eGFP-PH and catalytically inactive D110A,K220RGRK2, D110A,K220RGRK3, K215RGRK5, or K215RGRK6 constructs. D110A,K220RGRK2 expression significantly attenuated ETAR desensitization, whereas other constructs were ineffective. Small interfering RNA-targeted GRK2 depletion equally attenuated ETAR desensitization. Finally, immunocyotchemical data showed that ETAR activation recruited endogenous GRK2 from cytoplasm to membrane.
These studies identify GRK2 as a key regulator of ETAR responsiveness in resistance arteries, highlighting the potential importance of this GRK isoenzyme in regulating vasoconstrictor signalling pathways implicated in vascular disease.
PMCID: PMC2802200  PMID: 19748906
Endothelin-1; Endothelin-A receptor; G protein-coupled receptor kinase; Receptor desensitization; Vasoconstriction; Resistance artery; Mesenteric
11.  Regulation of cyclic AMP response-element binding-protein (CREB) by Gq/11-protein-coupled receptors in human SH-SY5Y neuroblastoma cells 
Biochemical Pharmacology  2008;75(4-3):942-955.
Human SH-SY5Y neuroblastoma cells have been used to investigate mechanisms involved in CREB phosphorylation after activation of two endogenously expressed Gq/11-protein-coupled receptors, the M3 muscarinic acetylcholine (mACh) and B2 bradykinin receptors. Stimulation with either methacholine or bradykinin resulted in maximal increases in CREB phosphorylation within 1 min, with either a rapid subsequent decrease (bradykinin) to basal levels, or a sustained response (methacholine). Inhibitor studies were performed to assess the involvement of a number of potential kinases in signalling to CREB phosphorylation. Removal of extracellular Ca2+, inhibition of Ca2+/calmodulin-dependent protein kinase II and down-regulation of protein kinase C (PKC) resulted in reduced CREB phosphorylation after both M3 mACh and B2 bradykinin receptor activation. In contrast, inhibition of MEK1/2 by U0126 resulted in significantly reduced CREB phosphorylation levels after B2 bradykinin, but not M3 mACh receptor activation. In addition, we demonstrate that maintained phosphorylation of CREB is necessary for CRE-dependent gene transcription as the M3 mACh, but not the B2 bradykinin receptor activates both a recombinant CRE-dependent reporter gene, and the endogenous c-Fos gene. These data highlight the involvement of multiple, overlapping signalling pathways linking these endogenous Gq/11-coupled metabotropic receptors to CREB and emphasize the importance of the duration of signalling pathway activation in converting a CREB phosphorylation event into a significant change in transcriptional activity.
PMCID: PMC2593902  PMID: 18036509
CREB, cyclic AMP response-element binding-protein; ERK, extracellular signal-regulated kinase; GPCR, G-protein-coupled receptor; IBMX, 3-isobutyl-1-methylxanthine; mACh, muscarinic acetylcholine; MAPK, mitogen-activated protein kinase; PDBu, phorbol 12,13-dibutyrate; PKA, cyclic AMP-dependent protein kinase; PKC, protein kinase C; PLC, phospholipase C; Cyclic AMP response-element binding-protein (CREB); Muscarinic acetylcholine receptor; Bradykinin receptor; SH-SY5Y cells; Intracellular calcium concentration [Ca2+]i; Calmodulin; Ca2+/calmodulin-dependent protein kinase; Protein kinase C; c-Fos

Results 1-11 (11)