Gq-coupled Receptors Research Articles

Diacylglycerol mediates regulation of TASK potassium channels by Gq-coupled receptors.

The two-pore domain potassium (K2P) channels TASK-1 (KCNK3) and TASK-3 (KCNK9) are important determinants of background K(+) conductance and membrane potential. TASK-1/3 activity is regulated by hormones and transmitters that act through G protein-coupled receptors (GPCR) signalling via G proteins of the Gαq/11 subclass. How the receptors inhibit channel activity has remained unclear. Here, we show that TASK-1 and -3 channels are gated by diacylglycerol (DAG). Receptor-initiated inhibition of TASK required the activity of phospholipase C, but neither depletion of the PLC substrate PI(4,5)P2 nor release of the downstream messengers IP3 and Ca(2+). Attenuation of cellular DAG transients by DAG kinase or lipase suppressed receptor-dependent inhibition, showing that the increase in cellular DAG-but not in downstream lipid metabolites-mediates channel inhibition. The findings identify DAG as the signal regulating TASK channels downstream of GPCRs and define a novel role for DAG that directly links cellular DAG dynamics to excitability.

GPR40 (FFAR1) - Combined Gs and Gq signaling invitro is associated with robust incretin secretagogue action exvivo and invivo.

ObjectivesGPR40 (FFAR1), a clinically proven anti-diabetes target, is a Gq-coupled receptor for long chain fatty acids (LCFA) stimulating insulin secretion directly and mediating a major part of the dietary triglyceride-induced secretion of the incretins GLP-1 and GIP. In phase-II studies the GPR40 agonist TAK-875 decreased blood glucose but surprisingly without stimulating incretins.Methods and resultsHere we find that GPR40 can signal through not only Gq and IP3 but also Gs and cAMP when stimulated with certain agonists such as AM-1638 and AM-5262 in contrast to the endogenous LCFA ligands and agonists such as TAK-875 and AM-837, which only signal through Gq. In competition binding against [3H]AM-1638 and [3H]L358 the Gq + Gs and the Gq-only agonists either competed for or showed positive cooperativity by increasing the binding of the two different radio-ligands, in opposite ways. Nevertheless, both the Gq-only and the Gq + Gs agonists all docked surprisingly well into the binding site for TAK-875 in the X-ray structure of GPR40. In murine intestinal primary cell-cultures the endogenous LCFAs and the Gq-only agonists stimulated GLP-1 secretion with rather poor efficacy as compared with the high efficacy Gq + Gs GPR40 agonists and a prototype GPR119 agonist. Similarly, in fasting both male and female mice the Gq + Gs agonists showed significantly higher efficacy than the Gq-only agonists in respect of increasing plasma GLP-1 and plasma GIP in a GPR40-dependent manner.ConclusionsIt is concluded that stimulation of GPR40 by endogenous LCFAs or by Gq-only synthetic agonists result in a rather limited incretin response, whereas Gq + Gs GPR40 agonists stimulate incretin secretion robustly.

Voltage-dependent regulation of CaV2.2 channels by Gq-coupled receptor is facilitated by membrane-localized β subunit.

G protein-coupled receptors (GPCRs) signal through molecular messengers, such as Gβγ, Ca(2+), and phosphatidylinositol 4,5-bisphosphate (PIP2), to modulate N-type voltage-gated Ca(2+) (CaV2.2) channels, playing a crucial role in regulating synaptic transmission. However, the cellular pathways through which GqPCRs inhibit CaV2.2 channel current are not completely understood. Here, we report that the location of CaV β subunits is key to determining the voltage dependence of CaV2.2 channel modulation by GqPCRs. Application of the muscarinic agonist oxotremorine-M to tsA-201 cells expressing M1 receptors, together with CaV N-type α1B, α2δ1, and membrane-localized β2a subunits, shifted the current-voltage relationship for CaV2.2 activation 5 mV to the right and slowed current activation. Muscarinic suppression of CaV2.2 activity was relieved by strong depolarizing prepulses. Moreover, when the C terminus of β-adrenergic receptor kinase (which binds Gβγ) was coexpressed with N-type channels, inhibition of CaV2.2 current after M1 receptor activation was markedly reduced and delayed, whereas the delay between PIP2 hydrolysis and inhibition of CaV2.2 current was decreased. When the Gβγ-insensitive CaV2.2 α1C-1B chimera was expressed, voltage-dependent inhibition of calcium current was virtually abolished, suggesting that M1 receptors act through Gβγ to inhibit CaV2.2 channels bearing membrane-localized CaV β2a subunits. Expression of cytosolic β subunits such as β2b and β3, as well as the palmitoylation-negative mutant β2a(C3,4S), reduced the voltage dependence of M1 muscarinic inhibition of CaV2.2 channels, whereas it increased inhibition mediated by PIP2 depletion. Together, our results indicate that, with membrane-localized CaV β subunits, CaV2.2 channels are subject to Gβγ-mediated voltage-dependent inhibition, whereas cytosol-localized β subunits confer more effective PIP2-mediated voltage-independent regulation. Thus, the voltage dependence of GqPCR regulation of calcium channels can be determined by the location of isotype-specific CaV β subunits.

Identification of protein kinase C activation as a novel mechanism for RGS2 protein upregulation through phenotypic screening of natural product extracts.

Biochemical high-throughput screening is widely used in drug discovery, using a variety of small molecule libraries. However, broader screening strategies may be more beneficial to identify novel biologic mechanisms. In the current study we used a β-galactosidase complementation method to screen a selection of microbial-derived pre-fractionated natural product extracts for those that increase regulator of G protein signaling 2 (RGS2) protein levels. RGS2 is a member of a large family of proteins that all regulate signaling through G protein-coupled receptors (GPCRs) by accelerating GTPase activity on active Gα as well as through other mechanisms. RGS2(-/-) mice are hypertensive, show increased anxiety, and are prone to heart failure. RGS2 has a very short protein half-life due to rapid proteasomal degradation, and we propose that enhancement of RGS2 protein levels could be a beneficial therapeutic strategy. Bioassay-guided fractionation of one of the hit strains yielded a pure compound, Indolactam V, a known protein kinase C (PKC) activator, which selectively increased RGS2 protein levels in a time- and concentration-dependent manner. Similar results were obtained with phorbol 12-myristate 13-acetate as well as activation of the Gq-coupled muscarinic M3 receptor. The effect on RGS2 protein levels was blocked by the nonselective PKC inhibitor Gö6983 (3-[1-[3-(dimethylamino)propyl]-5-methoxy-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione), the PKCβ-selective inhibitor Ruboxastaurin, as well as small interfering RNA-mediated knockdown of PKCβ. Indolactam V-mediated increases in RGS2 protein levels also had functional effects on GPCR signaling. This study provides important proof-of-concept for our screening strategy and could define a negative feedback mechanism in Gq/Phospholipase C signaling through RGS2 protein upregulation.

The phosphatidylinositol(4,5)bisphosphate-binding sequence of transient receptor potential channel canonical 4α is critical for its contribution to cardiomyocyte hypertrophy.

Cardiomyocyte hypertrophy requires a source of Ca(2+) distinct from the Ca(2+) that regulates contraction. The canonical transient receptor potential channel (TrpC) family, a family of cation channels regulated by activation of phospholipase C (PLC), has been implicated in this response. Cardiomyocyte hypertrophy downstream of Gq-coupled receptors is mediated specifically by PLCβ1b that is scaffolded onto a SH3 and ankyrin repeat protein 3 (Shank3) complex at the sarcolemma. TrpC4 exists as two splice variants (TrpC4α and TrpC4β) that differ only in an 84-residue sequence that binds to phosphatidylinositol(4,5)bisphosphate (PIP2), the substrate of PLCβ1b. In neonatal rat cardiomyocytes, TrpC4α, but not TrpC4β, coimmunoprecipitated with both PLCβ1b and Shank3. Heightened PLCβ1b expression caused TrpC4α, but not TrpC4β, translocation to the sarcolemma, where it colocalized with PLCβ1b. When overexpressed in cardiomyocytes, TrpC4α, but not TrpC4β, increased cell area (893 ± 18 to 1497 ± 29 mm(2), P < 0.01) and marker gene expression (atrial natriuretic peptide increased by 409 ± 32%, and modulatory calcineurin inhibitory protein 1 by 315 ± 28%, P < 0.01). Dominant-negative TrpC4 reduced hypertrophy initiated by PLCβ1b, or PLCβ1b-coupled receptor activation, by 72 ± 8% and 39 ± 5 %, respectively. We conclude that TrpC4α is selectively involved in mechanisms downstream of PLCβ1b culminating in cardiomyocyte hypertrophy, and that the hypertrophic response is dependent on the TrpC4α splice variant-specific sequence that binds to PIP2.

Ca2+Influx through P2X1 Receptors Amplifies P2Y1 Receptor-Evoked Ca2+Signaling and ADP-Evoked Platelet Aggregation

Many cells express both P2X cation channels and P2Y G-protein-coupled receptors that are costimulated by nucleotides released during physiologic or pathophysiologic responses. For example, during hemostasis and thrombosis, ATP-gated P2X1 channels and ADP-stimulated P2Y1 and P2Y12 G-protein coupled receptors play important roles in platelet activation. It has previously been reported that P2X1 receptors amplify P2Y1-evoked Ca(2+) responses in platelets, but the underlying mechanism and influence on function is unknown. In human platelets, we show that maximally activated P2X1 receptors failed to stimulate significant aggregation but could amplify the aggregation response to a submaximal concentration of ADP. Costimulation of P2X1 and P2Y1 receptors generated a superadditive Ca(2+) increase in both human platelets and human embryonic kidney 293 (HEK293) cells via a mechanism dependent on Ca(2+) influx rather than Na(+) influx or membrane depolarization. The potentiation, due to an enhanced P2Y1 response, was observed if ADP was added up to 60 seconds after P2X1 activation. P2X1 receptors also enhanced Ca(2+) responses when costimulated with type 1 protease activated and M1 muscarinic acetylcholine receptors. The P2X1-dependent amplification of Gq-coupled [Ca(2+)]i increase was mimicked by ionomycin and was not affected by inhibition of protein kinase C, Rho-kinase, or extracellular signal-regulated protein kinase 1/2, which suggests that it results from potentiation of inositol 1,4,5-trisphosphate receptors and/or phospholipase C. We conclude that Ca(2+) influx through P2X1 receptors amplifies Ca(2+) signaling through P2Y1 and other Gq-coupled receptors. This represents a general form of co-incidence detection of ATP and coreleased agonists, such as ADP at sites of vascular injury or synaptic transmitters acting at metabotropic Gq-coupled receptors.

Potentiation of insulin secretion and improvement of glucose intolerance by combining a novel G protein-coupled receptor 40 agonist DS-1558 with glucagon-like peptide-1 receptor agonists

G protein-coupled receptor 40 (GPR40) is a Gq-coupled receptor for free fatty acids predominantly expressed in pancreatic β-cells. In recent years, GPR40 agonists have been investigated for use as novel therapeutic agents in the treatment of type 2 diabetes. We discovered a novel small molecule GPR40 agonist, (3S)-3-ethoxy-3-(4-{[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]oxy}phenyl)propanoic acid (DS-1558). The GPR40-mediated effects of DS-1558 on glucose-stimulated insulin secretion were evaluated in isolated islets from GPR40 knock-out and wild-type (littermate) mice. The GPR40-mediated effects on glucose tolerance and insulin secretion were also confirmed by an oral glucose tolerance test in these mice. Furthermore, oral administration of DS-1558 (0.03, 0.1 and 0.3mg/kg) significantly and dose-dependently improved hyperglycemia and increased insulin secretion during the oral glucose tolerance test in Zucker fatty rats, the model of insulin resistance and glucose intolerance. Next, we examined the combination effects of DS-1558 with glucagon like peptide-1 (GLP-1). DS-1558 not only increased the glucose-stimulated insulin secretion by GLP-1 but also potentiated the maximum insulinogenic effects of GLP-1 after an intravenous glucose injection in normal Sprague Dawley rats. Furthermore, the glucose lowering effects of exendin-4, a GLP-1 receptor agonist, were markedly potentiated by the DS-1558 (3mg/kg) add-on in diabetic db/db mice during an intraperitoneal glucose tolerance test. In conclusion, our results indicate that add-on GPR40 agonists to GLP-1 related agents might be a potential treatment compared to single administration of these compounds. Therefore the combinations of these agents are a novel therapeutic option for type 2 diabetes.

Anoctamin 1 induces calcium-activated chloride secretion and proliferation of renal cyst–forming epithelial cells

Polycystic kidney diseases are characterized by multiple bilateral renal cysts that gradually enlarge and lead to a decline in renal function. Cyst enlargement is driven by transepithelial chloride secretion, stimulated by enhanced levels of cyclic adenosine monophosphate, which activates apical cystic fibrosis transmembrane conductance regulator chloride channels. However, chloride secretion by calcium-dependent chloride channels, activated through stimulation of purinergic receptors, also has a major impact. To identify the molecular basis of calcium-dependent chloride secretion in cyst expansion, we determined the role of anoctamin 1 and 6, two recently discovered calcium-activated chloride channels both of which are expressed in epithelial cells. We found that anoctamin 1, which plays a role in epithelial fluid secretion and proliferation, is strongly expressed in principal-like MDCK cells (PLCs) forming cysts within a collagen matrix, in an embryonic kidney cyst model, and in human autosomal dominant polycystic kidney disease tissue. Knockdown of anoctamin 1 but not anoctamin 6 strongly diminished the calcium-dependent chloride secretion of PLCs. Moreover, two inhibitors of anoctamin ion channels, tannic acid and a more selective inhibitor of anoctamin 1, significantly inhibited PLC cyst growth and cyst enlargement in an embryonic kidney cyst model. Knockdown of ANO1 by morpholino analogs also attenuated embryonic cyst growth. Thus, calcium-activated chloride secretion by anoctamin 1 appears to be a crucial component of renal cyst growth.

Local control of TRPV4 channels by AKAP150-targeted PKC in arterial smooth muscle.

Transient receptor potential vanilloid 4 (TRPV4) channels are Ca(2+)-permeable, nonselective cation channels expressed in multiple tissues, including smooth muscle. Although TRPV4 channels play a key role in regulating vascular tone, the mechanisms controlling Ca(2+) influx through these channels in arterial myocytes are poorly understood. Here, we tested the hypothesis that in arterial myocytes the anchoring protein AKAP150 and protein kinase C (PKC) play a critical role in the regulation of TRPV4 channels during angiotensin II (AngII) signaling. Super-resolution imaging revealed that TRPV4 channels are gathered into puncta of variable sizes along the sarcolemma of arterial myocytes. Recordings of Ca(2+) entry via single TRPV4 channels ("TRPV4 sparklets") suggested that basal TRPV4 sparklet activity was low. However, Ca(2+) entry during elementary TRPV4 sparklets was ∼ 100-fold greater than that during L-type CaV1.2 channel sparklets. Application of the TRPV4 channel agonist GSK1016790A or the vasoconstrictor AngII increased the activity of TRPV4 sparklets in specific regions of the cells. PKC and AKAP150 were required for AngII-induced increases in TRPV4 sparklet activity. AKAP150 and TRPV4 channel interactions were dynamic; activation of AngII signaling increased the proximity of AKAP150 and TRPV4 puncta in arterial myocytes. Furthermore, local stimulation of diacylglycerol and PKC signaling by laser activation of a light-sensitive Gq-coupled receptor (opto-α1AR) resulted in TRPV4-mediated Ca(2+) influx. We propose that AKAP150, PKC, and TRPV4 channels form dynamic subcellular signaling domains that control Ca(2+) influx into arterial myocytes.

β-Adrenergic Signaling Inhibits G q -Dependent Protein Kinase D Activation by Preventing Protein Kinase D Translocation

Rationale: Both β-adrenergic receptor (β-AR) and G q -coupled receptor (G q R) agonist–driven signaling play key roles in the events, leading up to and during cardiac dysfunction. How these stimuli interact at the level of protein kinase D (PKD), a nodal point in cardiac hypertrophic signaling, remains unclear. Objective: To assess the spatiotemporal dynamics of PKD activation in response to β-AR signaling alone and on coactivation with G q R-agonists. This will test our hypothesis that compartmentalized PKD signaling reconciles disparate findings of PKA facilitation and inhibition of PKD activation. Methods and Results: We report on the spatial and temporal profiles of PKD activation using green fluorescent protein-tagged PKD (wildtype or mutant S427E) and targeted fluorescence resonance energy transfer–based biosensors (D-kinase activity reporters) in adult cardiomyocytes. We find that β-AR/PKA signaling drives local nuclear activation of PKD, without preceding sarcolemmal translocation. We also discover pronounced interference of β-AR/cAMP/PKA signaling on G q R-induced translocation and activation of PKD throughout the cardiomyocyte. We attribute these effects to direct, PKA-dependent phosphorylation of PKD-S427. We also show that phosphomimetic substitution of S427 likewise impedes G q R-induced PKD translocation and activation. In neonatal myocytes, S427E inhibits G q R-evoked cell growth and expression of hypertrophic markers. Finally, we show altered S427 phosphorylation in transverse aortic constriction–induced hypertrophy. Conclusions: β-AR signaling triggers local nuclear signaling and inhibits G q R-mediated PKD activation by preventing its intracellular translocation. PKA-dependent phosphorylation of PKD-S427 fine-tunes the PKD responsiveness to G q R-agonists, serving as a key integration point for β-adrenergic and G q -coupled stimuli.

Gq/5-HT 2c receptor signals activate a local GABAergic inhibitory feedback circuit to modulate serotonergic firing and anxiety in mice

Serotonin 2c receptors (5-HT2c-Rs) are drug targets for certain mental disorders, including schizophrenia, depression, and anxiety. 5-HT2c-Rs are expressed throughout the brain, making it difficult to link behavioral changes to circuit specific receptor expression. Various 5-HT-Rs, including 5-HT2c-Rs, are found in the dorsal raphe nucleus (DRN); however, the function of 5-HT2c-Rs and their influence on the serotonergic signals mediating mood disorders remain unclear. To investigate the role of 5-HT2c-Rs in the DRN in mice, we developed a melanopsin-based optogenetic probe for activation of Gq signals in cellular domains, where 5-HT2c-Rs are localized. Our results demonstrate that precise temporal control of Gq signals in 5-HT2c-R domains in GABAergic neurons upstream of 5-HT neurons provides negative feedback regulation of serotonergic firing to modulate anxiety-like behavior in mice.

Dynamics of adenylate cyclase regulation via heterotrimeric G-proteins

A wide variety of G-protein-coupled receptors either activate or inhibit ACs (adenylate cyclases), thereby regulating cellular cAMP levels and consequently inducing proper physiological responses. Stimulatory and inhibitory G-proteins interact directly with ACs, whereas G(q)-coupled receptors exert their effects primarily via Ca2+. Using the FRET-based cAMP sensor Epac1 (exchange protein directly activated by cAMP 1)-cAMPS (adenosine 3',5'-cyclic monophosphorothioate), we studied cAMP levels in single living VSMCs (vascular smooth muscle cells) or HUVECs (human umbilical vein endothelial cells) with subsecond temporal resolution. Stimulation of purinergic (VSMCs) or thrombin (HUVECs) receptors rapidly decreased cAMP levels in the presence of the β-adrenergic agonist isoprenaline via a rise in Ca2+ and subsequent inhibition of AC5 and AC6. Specifically in HUVECs, we observed that, in the continuous presence of thrombin, cAMP levels climbed slowly after the initial decline with a delay of a little less than 1 min. The underlying mechanism includes phospholipase A2 activity and cyclo-oxygenase-mediated synthesis of prostaglandins. We studied further the dynamics of the inhibition of ACs via G(i)-proteins utilizing FRET imaging to resolve interactions between fluorescently labelled G(i)-proteins and AC5. FRET between Gα(i1) and AC5 developed at much lower concentration of agonist compared with the overall G(i)-protein activity. We found the dissociation of Gα(i1) subunits and AC5 to occur slower than the G(i)-protein deactivation. This led us to the conclusion that AC5, by binding active Gα(i1), interferes with G-protein deactivation and reassembly and thereby might sensitize its own regulation.

Activation of a Gq-coupled membrane estrogen receptor rapidly attenuates α2-adrenoceptor-induced antinociception via an ERK I/II-dependent, non-genomic mechanism in the female rat

Though sex differences in pain and analgesia are known, underlying mechanisms remain elusive. This study addresses the selective contribution of membrane estrogen receptors (mERs) and mER-initiated non-genomic signaling mechanisms in our previously reported estrogen-induced attenuation of α2-adrenoceptor-mediated antinociception. By selectively targeting spinal mERs in ovariectomized female rats using β-estradiol 6-(O-carboxy-methyl)oxime bovine serum albumin (E2BSA) (membrane impermeant estradiol analog), and ERα selective agonist 4,4′,4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT), ERβ selective agonist 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN), G-protein-coupled estrogen receptor 30 (GPR30) agonist G1 and Gq-coupled mER (Gq-mER) agonist STX, we provide strong evidence that Gq-mER activation may solely contribute to suppressing clonidine (an α2-adrenoceptor agonist)-induced antinociception, using the nociceptive tail-flick test. Increased tail-flick latencies (TFLs) by intrathecal (i.t.) clonidine were not significantly altered by i.t. PPT, DPN, or G1. In contrast, E2BSA or STX rapidly and dose-dependently attenuated clonidine-induced increase in TFL. ICI 182,780, the ER antagonist, blocked this effect. Consistent with findings with the lack of effect of ERα and ERβ agonists that modulate receptor-regulated transcription, inhibition of de novo protein synthesis using anisomycin also failed to alter the effect of E2BSA or STX, arguing against a contribution of genomic mechanisms. Immunoblotting of spinal tissue revealed that mER activation increased levels of phosphorylated extracellular signal-regulated kinase (ERK) but not of protein kinase A (PKA) or C (PKC). In vivo inhibition of ERK with U0126 blocked the effect of STX and restored clonidine antinociception. Although estrogen-induced delayed genomic mechanisms may still exist, data presented here indicate that Gq-mER may solely mediate estradiol-induced attenuation of clonidine antinociception via a rapid, reversible, and ERK-dependent, non-genomic mechanism, suggesting that Gq-mER blockade might provide improved analgesia in females.

Optogenetic activation of Gq signalling modulates pacemaker activity of cardiomyocytes

Investigation of Gq signalling with pharmacological agonists of Gq-coupled receptors lacks spatio-temporal precision. The aim of this study was to establish melanopsin, a light-sensitive Gq-coupled receptor, as a new tool for the investigation of spatial and temporal effects of Gq stimulation on pacemaking in cardiomyocytes at an early developmental stage. A vector for ubiquitous expression of melanopsin was tested in HEK293FT cells, which showed light-induced production of inositol-1,4,5-trisphosphate and elevation of intracellular Ca(2+) concentration. Mouse embryonic stem cells were stably transfected with this plasmid and differentiated into spontaneously beating embryoid bodies (EBs). Cardiomyocytes within EBs showed melanopsin expression and illumination (60 s, 308.5 nW/mm(2), 470 nm) of EBs increased beating rate within 10.2 ± 1.7 s to 317.1 ± 16.3% of baseline frequency. Illumination as short as 5 s was sufficient for generating the maximal frequency response. After termination of illumination, baseline frequency was reached with a decay constant of 27.1 ± 2.5 s. The light-induced acceleration of beating frequency showed a sigmoid dependence on light intensity with a half maximal effective light intensity of 41.7 nW/mm(2). Interestingly, EBs showed a high rate of irregular contractions after termination of high-intensity illumination. Local Gq activation by illumination of a small region in a functional syncytium of cardiomyocytes led to pacemaker activity within the illuminated area. Light-induced Gq activation in melanopsin-expressing cardiomyocytes increases beating rate and generates local pacemaker activity. We propose that melanopsin is a powerful optogenetic tool for the investigation of spatial and temporal aspects of Gq signalling in cardiovascular research.

Nonredundant function of two highly homologous octopamine receptors in food‐deprivation‐mediated signaling in Caenorhabditis elegans

It is common for neurotransmitters to possess multiple receptors that couple to the same intracellular signaling molecules. This study analyzes two highly homologous G-protein-coupled octopamine receptors using the model animal Caenorhabditis elegans. In C. elegans, the amine neurotransmitter octopamine induces activation of cAMP response element-binding protein (CREB) in the cholinergic SIA neurons in the absence of food through activation of the Gq-coupled octopamine receptor SER-3 in these neurons. We also analyzed another Gq-coupled octopamine receptor, SER-6, that is highly homologous to SER-3. As seen in ser-3 deletion mutants, octopamine- and food-deprivation-mediated CREB activation was decreased in ser-6 deletion mutants compared with wild-type animals, suggesting that both SER-3 and SER-6 are required for signal transduction. Cell-specific expression of SER-6 in the SIA neurons was sufficient to restore CREB activation in the ser-6 mutants, indicating that SER-6, like SER-3, functions in these neurons. Taken together, these results demonstrate that two similar G-protein-coupled receptors, SER-3 and SER-6, function in the same cells in a nonredundant manner.

Cross-Signaling between the Metabotropic Glutamate 2 Receptor and the Serotonin (5-Ht) 2A Receptor in Hek-293 Cells

Atypical antipsychotic drugs targeting the Gq-coupled 5-HT2A receptor (2AR) are widely used in the treatment of schizophrenia and psychosis. Recent studies point to a new class of potential antipsychotic drugs targeting the Gi-coupled metabotropic glutamate 2 receptor (mGluR2). We recently reported that a heteromeric complex formed between these two GPCRs integrates the actions of serotonergic and glutamatergic drugs, modulating the balance between Gi and Gq signaling and allowing us to predict the psychoactive properties of these drugs (Fribourg et al. 2011, Cell. 147(5):1011-23). Our results uncover a unifying mechanism of action of two families of antipsychotic drugs and pave the road for the development of assays for high-throughput screening of serotonergic and glutamatergic drugs. While developing such an assay, we needed to address a controversy raised by a recent study, in which co-expression of the two receptors in HEK-293 cells had no significant effect on either Gi or Gq signaling in response to several serotonergic and glutamatergic drugs (Delille et al., 2012, Neuropharmacology 62(7):2184-91). Since in Xenopus oocytes the degree of functional crosstalk between the two receptors depended on their expression levels, we generated several clones of HEK-293 cells expressing different levels of the two receptors in the background of the G-protein inwardly rectifying GIRK1/GIRK4 channel, which can serve as a reporter for both Gi and Gq signaling. Using fluorescence indicator and electrophysiological assays we were able to identify clones showing various degrees of functional crosstalk between the two receptors in response to single ligands. Our results have confirmed functional crosstalk of the two receptors. Even in cases where a single ligand failed to show crosstalk, like in the Delille et al. study, a combination of ligands targeting both receptors could elicit functional crosstalk.

Voltage Dependence of Muscarinic M1-,M3- and M5 Receptors

Muscarinic receptors (MR) are G protein-coupled receptors (GPCRs). Localized to the plasma membrane, they sense changes of the membrane potential and exhibit voltage-dependent signaling. Because voltage activates Gq-coupled receptors (e.g. M1R) but deactivates Gi-coupled receptors (e.g. M2R), it was proposed that GPCRs which couple to the same G protein class share common voltage-dependent properties. In this study we analyzed signaling of the Gq protein-coupled M1R, M3R and M5R with fluorescence resonance energy transfer (FRET) microscopy and voltage clamp. To test whether Gq protein coupling influences voltage sensitivity, we expressed a biosensor that reports conformations of M1R in HEK 293 cells and analyzed Carbachol (CCh)-induced receptor activation at −90 mV and at +60 mV. Depolarization caused activation of M1R, which was not abolished after uncoupling of Gq proteins with GTPγS. A FRET assay that reports activation of Gq was then used to compare wild type M1R, M3R and M5R. Application of CCh induced robust activation of Gq at −90 mV for all three receptors. Subsequent depolarization revealed subtype-specific phenotypes: at +60 mV the Gq protein was either activated (M1R) or deactivated (M3R or M5R). Voltage sensitivity of MR was also influenced by the nature of the activating agonist: M3R displayed deactivation (CCh) or activation (Pilocarpine) at +60 mV. Computational docking studies predicted different molecular binding modes for each agonist and guided us to select key point mutations in the orthosteric binding site of muscarinic receptors. Further docking of mutant M3R predicted a novel “Pilocarpine-like” binding mode for CCh. Subsequent experiments confirmed that mutant M3R were indeed activated by voltage in the presence of CCh. Together, these results imply that the molecular mechanism by which GqPCRs sense voltage resides in the receptor protein with negligible contribution of G protein coupling.

From FMRP Function to Potential Therapies for Fragile X Syndrome

Fragile X syndrome (FXS) is caused by mutations in the fragile X mental retardation 1 (FMR1) gene. Most FXS cases occur due to the expansion of the CGG trinucleotide repeats in the 5' un-translated region of FMR1, which leads to hypermethylation and in turn silences the expression of FMRP (fragile X mental retardation protein). Numerous studies have demonstrated that FMRP interacts with both coding and non-coding RNAs and represses protein synthesis at dendritic and synaptic locations. In the absence of FMRP, the basal protein translation is enhanced and not responsive to neuronal stimulation. The altered protein translation may contribute to functional abnormalities in certain aspects of synaptic plasticity and intracellular signaling triggered by Gq-coupled receptors. This review focuses on the current understanding of FMRP function and potential therapeutic strategies that are mainly based on the manipulation of FMRP targets and knowledge gained from FXS pathophysiology.

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.

G protein-coupled receptor signalling potentiates the osmo-mechanical activation of TRPC5 channels

TRPC5 is an ion channel permeable to monovalent and divalent cations that is widely expressed in different tissues. Although implicated in the control of neurite extension and in the growth cone morphology of hippocampal neurons, as well as in fear-related behaviour, the mechanisms by which TRPC5 is activated remain poorly understood. TRPC5 is known to be activated downstream of Gq-coupled receptors and by membrane stretch, and since there is evidence that mechanical stress may directly activate Gq-coupled receptors, we examined the relationship between the activation of TRPC5 by the type 1 histamine receptor and osmotic stress. Using calcium imaging and patch clamp recordings, we found that a higher proportion of cells expressing TRPC5 respond to hypoosmotic solution when they co-express H1R. This response is associated with a phospholipase C-dependent increase in the cells internal calcium concentration, which is abolished on depletion of calcium stores. We also found that the hypoosmotic stimulus that provokes mechanical stress drives the translocation of TRPC5 to the plasma membrane by a mechanism dependent on PI3K. This increase in TRPC5 at the plasma membrane augments the proportion of cells that respond to hypoosmotic stimulation. Together, these results suggest that hypoosmotic cell-swelling activates Gq-coupled receptors, which in turn enhance the activation of TRPC5 by regulating this channel membrane trafficking. Gq-coupled receptors and TPRC5 are co-expressed in several tissues such as those of the vascular system and in somatosensory neurons, suggesting that this mechanism of TRPC5 activation may have interesting and important implications in arterial pressure sensing and mechanotransduction.