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Transient receptor potential canonical 4 (TRPC4) is a receptor-operated cation channel codependent on both the Gq/11–phospholipase C signaling pathway and Gi/o proteins for activation. This makes TRPC4 an excellent coincidence sensor of neurotransmission through Gq/11- and Gi/o-coupled receptors. In whole-cell slice recordings of lateral septal neurons, TRPC4 mediates a strong depolarizing plateau that shuts down action potential firing, which may or may not be followed by a hyperpolarization that extends the firing pause to varying durations depending on the strength of Gi/o stimulation. We show that the depolarizing plateau is codependent on Gq/11-coupled group I metabotropic glutamate receptors and on Gi/o-coupled γ-aminobutyric acid type B receptors. The hyperpolarization is mediated by Gi/o activation of G protein–activated inwardly rectifying K+ (GIRK) channels. Moreover, the firing patterns, elicited by either electrical stimulation or receptor agonists, encode information about the relative strengths of Gq/11 and Gi/o inputs in the following fashion. Pure Gq/11 input produces weak depolarization accompanied by firing acceleration, whereas pure Gi/o input causes hyperpolarization that pauses firing. Although coincident Gq/11–Gi/o inputs also pause firing, the pause is preceded by a burst, and both the pause duration and firing recovery patterns reflect the relative strengths of Gq/11 versus Gi/o inputs. Computer simulations demonstrate that different combinations of TRPC4 and GIRK conductances are sufficient to produce the range of firing patterns observed experimentally. Thus, concurrent neurotransmission through the Gq/11 and Gi/o pathways is converted to discernible electrical responses by the joint actions of TRPC4 and GIRK for communication to downstream neurons.

Astrocytes express a plethora of G protein-coupled receptors (GPCRs) that are crucial for shaping synaptic activity. Upon GPCR activation, astrocytes can respond with transient variations in intracellular Ca 2+ . In addition, Ca 2+ -dependent and/or Ca 2+ -independent release of gliotransmitters can occur, allowing them to engage in bidirectional neuron-astrocyte communication. The development of designer receptors exclusively activated by designer drugs (DREADDs) has facilitated many new discoveries on the roles of astrocytes in both physiological and pathological conditions. They are an excellent tool, as they can target endogenous GPCR-mediated intracellular signal transduction pathways specifically in astrocytes. With increasing interest and accumulating research on this topic, several discrepancies on astrocytic Ca 2+ signalling and astrocyte-mediated effects on synaptic plasticity have emerged, preventing a clear-cut consensus about the downstream effects of DREADDs in astrocytes. In the present study, we performed a side-by-side evaluation of the effects of bath application of the DREADD agonist, clozapine-N-oxide (10 µM), on Gq- and Gi-DREADD activation in mouse CA1 hippocampal astrocytes. In doing so, we aimed to avoid confounding factors, such as differences in experimental procedures, and to directly compare the actions of both DREADDs on astrocytic intracellular Ca 2+ dynamics and synaptic plasticity in acute hippocampal slices. We used an adeno-associated viral vector approach to transduce dorsal hippocampi of male, 8-week-old C57BL6/J mice, to drive expression of either the Gq-DREADD or Gi-DREADD in CA1 astrocytes. A viral vector lacking the DREADD construct was used to generate controls. Here, we show that agonism of Gq-DREADDs, but not Gi-DREADDs, induced consistent increases in spontaneous astrocytic Ca 2+ events. Moreover, we demonstrate that both Gq-DREADD as well as Gi-DREADD-mediated activation of CA1 astrocytes induces long-lasting synaptic potentiation in the hippocampal CA1 Schaffer collateral pathway in the absence of a high frequency stimulus. Moreover, we report for the first time that astrocytic Gi-DREADD activation is sufficient to elicit de novo potentiation. Our data demonstrate that activation of either Gq or Gi pathways drives synaptic potentiation through Ca 2+ -dependent and Ca 2+ -independent mechanisms, respectively., (© 2021. The Author(s).)

Regulators of G protein signaling (RGS) proteins modulate signaling by G protein-coupled receptors. Using a knock-in transgenic mouse model with a mutation in G α o that does not bind RGS proteins (RGS-insensitive), we determined the effect of RGS proteins on presynaptic μ opioid receptor (MOR)-mediated inhibition of GABA release in the ventrolateral periaqueductal gray (vlPAG). The MOR agonists [d-Ala 2 , N -MePhe 4 , Gly-ol]-enkephalin (DAMGO) and met-enkephalin (ME) inhibited evoked inhibitory postsynaptic currents (eIPSCs) in the RGS-insensitive mice compared with wild-type (WT) littermates, respectively. Fentanyl inhibited eIPSCs similarly in both WT and RGS-insensitive mice. There were no differences in opioid agonist inhibition of spontaneous GABA release between the genotypes. To further probe the mechanism underlying these differences between opioid inhibition of evoked and spontaneous GABA release, specific myristoylated G α peptide inhibitors for G α o 1 and G α i 1-3 that block receptor-G protein interactions were used to test the preference of agonists for MOR-G α complexes. The G α o 1 inhibitor reduced DAMGO inhibition of eIPSCs, but G α i 1-3 inhibitors had no effect. Both G α o 1 and G α i 1-3 inhibitors separately reduced fentanyl inhibition of eIPSCs but had no effects on ME inhibition. G α i 1-3 inhibitors blocked the inhibitory effects of ME and fentanyl on miniature postsynaptic current (mIPSC) frequency, but both G α o 1 and G α i 1-3 inhibitors were needed to block the effects of DAMGO. Finally, baclofen-mediated inhibition of GABA release is unaffected in the RGS-insensitive mice and in the presence of G α o 1 and G α i 1-3 inhibitor peptides, suggesting that GABA B receptor coupling to G proteins in vlPAG presynaptic terminals is different than MOR coupling. SIGNIFICANCE STATEMENT: Presynaptic μ opioid receptors (MORs) in the ventrolateral periaqueductal gray are critical for opioid analgesia and are negatively regulated by RGS proteins. These data in RGS-insensitive mice provide evidence that MOR agonists differ in preference for Gαo versus Gαi and regulation by RGS proteins in presynaptic terminals, providing a mechanism for functional selectivity between agonists. The results further define important differences in MOR and GABA B receptor coupling to G proteins that could be exploited for new pain therapies., (Copyright © 2021 by The American Society for Pharmacology and Experimental Therapeutics.)

Neuroligin-3 (NLGN3) is necessary and sufficient to promote glioma cell growth. The recruitment of Gαi1/3 to the ligand-activated receptor tyrosine kinases (RTKs) is essential for mediating oncogenic signaling. Methods: Various genetic strategies were utilized to examine the requirement of Gαi1/3 in NLGN3-driven glioma cell growth. Results: NLGN3-induced Akt-mTORC1 and Erk activation was inhibited by decreasing Gαi1/3 expression. In contrast ectopic Gαi1/3 overexpression enhanced NLGN3-induced signaling. In glioma cells, NLGN3-induced cell growth, proliferation and migration were attenuated by Gαi1/3 depletion with shRNA, but facilitated with Gαi1/3 overexpression. Significantly, Gαi1/3 silencing inhibited orthotopic growth of patient-derived glioma xenografts in mouse brain, whereas forced Gαi1/3-overexpression in primary glioma xenografts significantly enhanced growth. The growth of brain-metastatic human lung cancer cells in mouse brain was largely inhibited with Gαi1/3 silencing. It was however expedited with ectopic Gαi1/3 overexpression. In human glioma Gαi3 upregulation was detected, correlating with poor prognosis. Conclusion: Gαi1/3 mediation of NLGN3-induced signaling is essential for neuronal-driven glioma growth ., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Chronic stress is one of the main risk factors of bone loss. While the neurons and neural circuits of the ventromedial hypothalamus (VMH) mediate bone loss induced by chronic stress, the detailed intrinsic mechanisms within the VMH nucleus still need to be explored. Astrocytes in brain regions play important roles in the regulation of metabolism and anxiety-like behavior through interactions with surrounding neurons. However, whether astrocytes in the VMH affect neuronal activity and therefore regulate chronic stress-induced anxiety and bone loss remain elusive. In this study, we found that VMH astrocytes were activated during chronic stress-induced anxiety and bone loss. Pharmacogenetic activation of the Gi and Gq pathways in VMH astrocytes reduced and increased the levels of anxiety and bone loss, respectively. Furthermore, activation of VMH astrocytes by optogenetics induced depolarization in neighboring steroidogenic factor-1 (SF-1) neurons, which was diminished by administration of N-methyl-D-aspartic acid (NMDA) receptor blocker but not by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor blocker. These results suggest that there may be a functional "glial-neuron microcircuit" in VMH nuclei that mediates anxiety and bone loss induced by chronic stress. This study not only advances our understanding of glial cell function but also provides a potential intervention target for chronic stress-induced anxiety and bone loss therapy., Competing Interests: The authors declare that no conflicts of interest exist., (Copyright © 2021 Yunhui Liu et al.)

Pathological impulsivity is a debilitating symptom of multiple psychiatric diseases with few effective treatment options. To identify druggable receptors with anti-impulsive action we developed a systematic target discovery approach combining behavioural chemogenetics and gene expression analysis. Spatially restricted inhibition of three subdivisions of the prefrontal cortex of mice revealed that the anterior cingulate cortex (ACC) regulates premature responding, a form of motor impulsivity. Probing three G-protein cascades with designer receptors, we found that the activation of G i -signalling in layer-5 pyramidal cells (L5-PCs) of the ACC strongly, reproducibly, and selectively decreased challenge-induced impulsivity. Differential gene expression analysis across murine ACC cell-types and 402 GPCRs revealed that - among G i -coupled receptor-encoding genes - Grm2 is the most selectively expressed in L5-PCs while alternative targets were scarce. Validating our approach, we confirmed that mGluR2 activation reduced premature responding. These results suggest G i -coupled receptors in ACC L5-PCs as therapeutic targets for impulse control disorders.

Platelet activation plays a pivotal role in physiological hemostasis and pathological thrombosis causing heart attack and stroke. Previous studies conclude that simultaneous activation of Gi and G 12/13 signaling pathways is sufficient to cause platelet aggregation. However, using Gq knockout mice and Gq-specific inhibitors, we here demonstrated that platelet aggregation downstream of coactivation of Gi and G 12/13 depends on agonist concentrations; coactivation of Gi and G 12/13 pathways only induces platelet aggregation under higher agonist concentrations. We confirmed Gi and G 12/13 pathway activation by showing cAMP (cyclic adenosine monophosphate) decrease and RhoA activation in platelets stimulated at both low and high agonist concentrations. Interestingly, we found that though Akt and PAK (p21-activated kinase) translocate to the platelet membrane upon both low and high agonist stimulation, membrane-translocated Akt and PAK only phosphorylate at high agonist concentrations, correlating well with platelet aggregation downstream of concomitant Gi and G 12/13 pathway activation. PAK inhibitor abolishes Akt phosphorylation, inhibits platelet aggregation in vitro and arterial thrombus formation in vivo. We propose that the PAK-PI3K/Akt pathway mediates platelet aggregation downstream of Gi and G 12/13, and PAK may represent a potential antiplatelet and antithrombotic target., Competing Interests: None declared., (Thieme. All rights reserved.)

Objectives: Typically, obesity results from an inappropriate balance between energy uptake from nutrient consumption and burning of calories, which leads to a pathological increase in fat mass. Obesity is a major cause of insulin resistance and diabetes. Inhibitory G proteins (Gα i ) form a subfamily that is involved in the regulation of adipose tissue function. Among the three Gα i members, i.e. Gα i1 , Gα i2 , Gα i3 , the Gα i2 , protein is predominantly expressed in adipose tissue. However, the functions of the Gα i2 isoform in adipose tissue and its impact on the development of obesity are poorly understood., Methods: By using AdipoqCreER T2 mice, we generated adipocyte-specific Gnai2-deficient mice to study Gα i2 function, specifically in white and brown adipocytes. These mice were fed either a control diet (CD) or a high fat diet (HFD). Mice were examined for obesity development, insulin resistance and glucose intolerance. We examined adipocyte morphology and the development of inflammation in the white adipose tissue. Finally, intracellular cAMP levels as an indicator of Gα i signaling and glycerol release as an indicator of lipolysis rates were measured to verify the impact of Gα i2 on the signaling pathway in brown and white adipocytes., Results: An adipocyte-specific deficiency of Gα i2 significantly reduced diet-induced obesity, leading to decreased fat masses, smaller adipocytes and decreased inflammation in the white adipose tissue relative to littermate controls. Concurrently, oxygen consumption of brown adipocytes and in vivo measured energy expenditure were significantly enhanced. In addition, glucose tolerance and insulin sensitivity of HFD-fed adipocyte-specific Gnai2-deficient mice were improved compared to the respective controls. In the absence of Gα i2 , adrenergic stimulation of intracellular adipocyte cAMP levels was increased, which correlated with increased lipolysis and energy expenditure., Conclusion: We conclude that adipocyte Gα i2 is a major regulator of adipocyte lipid content in diet-induced obesity by inhibiting adipocyte lipolysis in a cAMP-dependent manner resulting in increased energy expenditure., (Copyright © 2020 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Hypertension is a risk factor for cardiovascular diseases, which have been associated with dysfunction of sympathetic and purinergic neurotransmission. Therefore, herein, we evaluated whether modifications of adenosine receptor signaling may contribute to the cardiac dysfunction observed in hypertension. Isolated right atria from spontaneously hypertensive (SHR) or normotensive Wistar rats (NWR) were used to investigate the influence of adenosine receptor signaling cascade in the cardiac chronotropism. Our results showed that adenosine, the endogenous agonist of adenosine receptors, and CPA, a selective agonist of A 1 receptor, decreased the atrial chronotropism of NWR and SHR in a concentration- and time-dependent manner, culminating in cardiac arrest (0 bpm). Interestingly, a 3-fold lower concentration of adenosine was required to induce the negative chronotropic effect in SHR atria. Pre-incubation of tissues from both strains with DPCPX, a selective A 1 receptor antagonist, inhibited the negative chronotropic effect of CPA, while simultaneous inhibition of A 2 and A 3 receptors, with ZM241385 and MRS1523, did not change the adenosine chronotropic effects. Moreover, 1 μg/ml pertussis toxin, which inactivates the Gαi protein subunit, reduced by 80% the negative chronotropic effects of adenosine in the NWR atrium, with minor effects in SHR tissue. These data indicate that the negative chronotropic effect of adenosine in right atrium depends exclusively on the activation of A 1 receptors. Moreover, the distinct responsiveness of NWR and SHR atria to pertussis toxin reveals that the enhanced negative chronotropic response of SHR right atrium is probably due to an increased activity of Gαi protein-mediated.

The follicular (FO) versus marginal zone (MZ) B cell fate decision in the spleen depends upon BCR, BAFF, and Notch2 signaling. Whether or how G i signaling affects this fate decision is unknown. Here, we show that direct contact with Notch ligand expressing stromal cells (OP9-Delta-like 1) cannot promote normal MZ B cell development when progenitor B cells lack Gα i proteins, or if Gi signaling is disabled. Consistent with faulty ADAM10-dependent Notch2 processing, Gα i -deficient transitional B cells had low ADAM10 membrane expression levels and reduced Notch2 target gene expression. Immunoblotting Gα i -deficient B cell lysates revealed a reduction in mature, processed ADAM10. Suggesting that Gα i signaling promotes ADAM10 membrane expression, stimulating normal transitional B cells with CXCL12 raised it, while inhibiting Gα i nucleotide exchange blocked its upregulation. Surprisingly, inhibiting Gα i nucleotide exchange in transitional B cells also impaired the upregulation of ADAM10 that occurs following antigen receptor crosslinking. These results indicate that Gα i signaling supports ADAM10 maturation and activity in transitional B cells, and ultimately Notch2 signaling to promote MZ B cell development.

Deposition of amyloid-β (Aβ), the proteolytic product of the amyloid precursor protein (APP), might cause neurodegeneration and cognitive decline in Alzheimer's disease (AD). However, the direct involvement of APP in the mechanism of Aβ-induced degeneration in AD remains on debate. Here, we analyzed the interaction of APP with heterotrimeric Go protein in primary hippocampal cultures and found that Aβ deposition dramatically enhanced APP-Go protein interaction in dystrophic neurites. APP overexpression rendered neurons vulnerable to Aβ toxicity by a mechanism that required Go-Gβγ complex signaling and p38-mitogen-activated protein kinase activation. Gallein, a selective pharmacological inhibitor of Gβγ complex, inhibited Aβ-induced dendritic and axonal dystrophy, abnormal tau phosphorylation, synaptic loss, and neuronal cell death in hippocampal neurons expressing endogenous protein levels. In the 3xTg-AD mice, intrahippocampal application of gallein reversed memory impairment associated with early Aβ pathology. Our data provide further evidence for the involvement of APP/Go protein in Aβ-induced degeneration and reveal that Gβγ complex is a signaling target potentially relevant for developing therapies for halting Aβ degeneration in AD., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Dopaminergic modulation of prefrontal cortex (PFC) is thought to play key roles in many cognitive functions and to be disrupted in pathological conditions, such as schizophrenia. We have previously described a phenomenon whereby dopamine D2 receptor (D2R) activation elicits afterdepolarizations (ADPs) in subcortically projecting (SC) pyramidal neurons within L5 of the PFC. These D2R-induced ADPs only occur following synaptic input, which activates NMDARs, even when the delay between the synaptic input and ADPs is relatively long (e.g., several hundred milliseconds). Here, we use a combination of electrophysiological, optogenetic, pharmacological, transgenic, and chemogenetic approaches to elucidate cellular mechanisms underlying this phenomenon in male and female mice. We find that knocking out D2Rs eliminates the ADP in a cell-autonomous fashion, confirming that this ADP depends on D2Rs. Hyperpolarizing current injection, but not AMPA receptor blockade, prevents synaptic stimulation from facilitating D2R-induced ADPs, suggesting that this phenomenon depends on the recruitment of voltage-dependent currents (e.g., NMDAR-mediated Ca 2+ influx) by synaptic input. Finally, the D2R-induced ADP is blocked by inhibitors of cAMP/PKA signaling, insensitive to pertussis toxin or β-arrestin knock-out, and mimicked by G s -DREADD stimulation, suggesting that D2R activation elicits the ADP by stimulating cAMP/PKA signaling. These results show that this unusual physiological phenomenon, in which D2Rs enhance cellular excitability in a manner that depends on synaptic input, is mediated at the cellular level through the recruitment of signaling pathways associated with G s , rather than the G i/o -associated mechanisms that have classically been ascribed to D2Rs. SIGNIFICANCE STATEMENT Dopamine D2 receptors (D2Rs) in the prefrontal cortex (PFC) are thought to play important roles in behaviors, including working memory and cognitive flexibility. Variation in D2Rs has also been implicated in schizophrenia, Tourette syndrome, and bipolar disorder. Recently, we described a new mechanism through which D2R activation can enhance the excitability of pyramidal neurons in the PFC. Here, we explore the underlying cellular mechanisms. Surprisingly, although D2Rs are classically assumed to signal through G i/o -coupled G-proteins and/or scaffolding proteins, such as β-arrestin, we find that the effects of D2Rs on prefrontal pyramidal neurons are actually mediated by pathways associated with G s -mediated signaling. Furthermore, we show how, via this D2R-dependent phenomenon, synaptic input can enhance the excitability of prefrontal neurons over timescales on the order of seconds. These results elucidate cellular mechanisms underlying a novel signaling pathway downstream of D2Rs that may contribute to prefrontal function under normal and pathological conditions., (Copyright © 2017 the authors 0270-6474/17/3710063-11$15.00/0.)

G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs) act in concert to regulate cell growth, proliferation, survival, and migration. Metabotropic GABA B receptor (GABA B R) is the GPCR for the main inhibitory neurotransmitter GABA in the central nervous system. Increased expression of GABA B R has been detected in human cancer tissues and cancer cell lines, but the role of GABA B R in these cells is controversial and the underlying mechanism remains poorly understood. Here, we investigated whether GABA B R hijacks RTK signaling to modulate the fates of human prostate cancer cells. RTK array analysis revealed that the GABA B R-specific agonist baclofen selectively induced the transactivation of EGFR in PC-3 cells. EGFR transactivation resulted in the activation of ERK1/2 by a mechanism that is dependent on G i/o protein and that requires matrix metalloproteinase-mediated proligand shedding. Positive allosteric modulators (PAMs) of GABA B R, such as CGP7930, rac-BHFF, and GS39783, can function as PAM agonists to induce EGFR transactivation and subsequent ERK1/2 activation. Moreover, both baclofen and CGP7930 promoted cell migration and invasion through EGFR signaling. In summary, our observations demonstrated that GABA B R transactivated EGFR in a ligand-dependent mechanism to promote prostate cancer cell migration and invasion, thus providing new insights into developing a novel strategy for prostate cancer treatment by targeting neurotransmitter signaling., (Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.)

Age-related bone loss is an important risk factor for fractures in the elderly; it results from an imbalance in bone remodeling mainly due to decreased bone formation. We have previously demonstrated that endogenous G protein-coupled receptor (GPCR)-driven Gi signaling in osteoblasts (Obs) restrains bone formation in mice during growth. Here, we launched a longitudinal study to test the hypothesis that Gi signaling in Obs restrains bone formation in aging mice, thereby promoting bone loss. Our approach was to block Gi signaling in maturing Obs by the induced expression of the catalytic subunit of pertussis toxin (PTX) after the achievement of peak bone mass. In contrast to the progressive cancellous bone loss seen in aging sex-matched littermate control mice, aging female Col1(2.3)+/PTX+ mice showed an age-related increase in bone volume. Increased bone volume was associated with increased bone formation at both trabecular and endocortical surfaces as well as increased bending strength of the femoral middiaphyses. In contrast, male Col1(2.3)+/PTX+ mice were not protected from age-related bone loss. Our results indicate that Gi signaling markedly restrains bone formation at cancellous and endosteal bone surfaces in female mice during aging. Blockade of the relevant Gi-coupled GPCRs represents an approach for the development of osteoporosis therapies-at least in the long bones of aging women.

Sensory perception in the inner ear relies on the hair bundle, the highly polarized brush of movement detectors that crowns hair cells. We previously showed that, in the mouse cochlea, the edge of the forming bundle is defined by the 'bare zone', a microvilli-free sub-region of apical membrane specified by the Insc-LGN-Gαi protein complex. We now report that LGN and Gαi also occupy the very tip of stereocilia that directly abut the bare zone. We demonstrate that LGN and Gαi are both essential for promoting the elongation and differential identity of stereocilia across rows. Interestingly, we also reveal that total LGN-Gαi protein amounts are actively balanced between the bare zone and stereocilia tips, suggesting that early planar asymmetry of protein enrichment at the bare zone confers adjacent stereocilia their tallest identity. We propose that LGN and Gαi participate in a long-inferred signal that originates outside the bundle to model its staircase-like architecture, a property that is essential for direction sensitivity to mechanical deflection and hearing., (© 2016. Published by The Company of Biologists Ltd.)

Background: We previously showed that the levels of both Giα-2 and Giα-3 proteins were augmented in spontaneously hypertensive rats (SHRs) before the onset of hypertension. In addition, intraperitoneal injection of pertussis toxin, which inactivates both Giα proteins, prevented the development of hypertension in SHRs. The aim of the present study was to determine the specific contributions of Giα-2 and Giα-3 proteins to the development of hypertension., Methods and Results: Antisense oligodeoxynucleotide of Giα-2 and Giα-3 encapsulated in PEG/DOTAP/DOPE cationic liposomes were administrated intravenously into 3-week-old prehypertensive SHRs and Wistar Kyoto rats, whereas the control Wistar Kyoto rats and SHRs received PBS, empty liposomes, or sense. The knockdown of Giα-2 but not Giα-3 protein attenuated tachycardia and prevented the development of hypertension up to age 6 weeks; thereafter, blood pressure started increasing and reached the same level as that of untreated SHRs at 9 weeks. Furthermore, Giα-2 and Giα-3 antisense oligodeoxynucleotide treatments significantly decreased the enhanced levels of Giα-2 and Giα-3 proteins, respectively, and enhanced levels of superoxide anion and NADPH oxidase activity in heart, aorta, and kidney and hyperproliferation of vascular smooth muscle cells from SHRs aged 6 weeks. In addition, antisense oligodeoxynucleotide treatment with Giα-2 but not Giα-3 restored enhanced inhibition of adenylyl cyclase by oxotremorine to WKY levels., Conclusions: These results suggested that the enhanced expression of Giα-2 but not Giα-3 protein plays an important role in the pathogenesis of hypertension and tachycardia in SHRs., (© 2016 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.)

The heart is controlled by the sympathetic and parasympathetic limbs of the autonomic nervous system with inhibitory signaling mechanisms recruited in both limbs. The aim of this study was to determine the role of inhibitory heterotrimeric G proteins in the central nervous mechanisms underlying autonomic control of the heart and its potential role in arrhythmogenesis. Mice with conditional deletion of the inhibitory heterotrimeric G protein GαO in the presympathetic area of the rostral ventral lateral medulla (RVLM) were generated to determine the role of GαO-mediated signalling in autonomic control and electrophysiological properties of the heart. GαO deletion within the RVLM was not associated with changes in heart rate (HR) or the arterial blood pressure at rest (home cage, normal behavior). However, exposure to stressful conditions (novel environment, hypoxia, or hypercapnia) in these mice was associated with abnormal HR responses and an increased baroreflex gain when assessed under urethane anesthesia. This was associated with shortening of the ventricular effective refractory period. This phenotype was reversed by systemic beta-adrenoceptor blockade, suggesting that GαO depletion in the RVLM increases central sympathetic drive. The data obtained support the hypothesis that GαO-mediated signaling within the presympathetic circuits of the RVLM contributes to the autonomic control of the heart. GαO deficiency in the RVLM has a significant impact on cardiovascular responses to stress, cardiovascular reflexes and electrical properties of the heart., (© 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.)

Transient Receptor Potential Canonical (TRPC) proteins form nonselective cation channels commonly known to be activated downstream from receptors that signal through phospholipase C (PLC). Although TRPC3/C6/C7 can be directly activated by diacylglycerols produced by PLC breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2), the mechanism by which the PLC pathway activates TRPC4/C5 remains unclear. We show here that TRPC4 activation requires coincident stimulation of Gi/o subgroup of G proteins and PLCδ, with a preference for PLCδ1 over PLCδ3, but not necessarily the PLCβ pathway commonly thought to be involved in receptor-operated TRPC activation. In HEK293 cells coexpressing TRPC4 and Gi/o-coupled µ opioid receptor, µ agonist elicited currents biphasically, with an initial slow phase preceding a rapidly developing phase. The currents were dependent on intracellular Ca(2+) and PIP2. Reducing PIP2 through phosphatases abolished the biphasic kinetics and increased the probability of channel activation by weak Gi/o stimulation. In both HEK293 cells heterologously expressing TRPC4 and renal carcinoma-derived A-498 cells endogenously expressing TRPC4, channel activation was inhibited by knocking down PLCδ1 levels and almost completely eliminated by a dominant-negative PLCδ1 mutant and a constitutively active RhoA mutant. Conversely, the slow phase of Gi/o-mediated TRPC4 activation was diminished by inhibiting RhoA or enhancing PLCδ function. Our data reveal an integrative mechanism of TRPC4 on detection of coincident Gi/o, Ca(2+), and PLC signaling, which is further modulated by the small GTPase RhoA. This mechanism is not shared with the closely related TRPC5, implicating unique roles of TRPC4 in signal integration in brain and other systems.

Gi/o-coupled G protein-coupled receptors can exert an inhibitory effect on vesicle release through several G protein-driven mechanisms, more than one of which may be concurrently present in individual presynaptic terminals. The synaptosomal-associated protein of 25 kDa (SNAP25) is a key downstream effector of Gβγ subunits. It has previously been shown that proteolytic cleavage of SNAP25 by botulinum toxin A reduces the ability of Gβγ to compete with the calcium sensor synaptotagmin 1 (Syt1) for binding to SNAP25 in a calcium-dependent manner. These truncated SNAP25 proteins sustain a low level of exocytosis but are unable to support serotonin-mediated inhibition of exocytosis in lamprey spinal neurons. Here, we generate a SNAP25 extreme C-terminal mutant that is deficient in its ability to bind Gβγ while retaining normal calcium-dependent Syt1 binding to soluble N-ethylmaleimide attachment protein receptor (SNARE) and vesicle release. The SNAP25Δ3 mutant, in which residue G204 is replaced by a stop codon, features a partial reduction in Gβ1γ2 binding in vitro as well as a partial reduction in the ability of the lamprey 5-hydroxytryptamine1b-type serotonin receptor to reduce excitatory postsynaptic current amplitudes, an effect previously shown to be mediated through the interaction of Gβγ with SNAP25. Syt1 calcium-dependent binding to SNAP25Δ3 was reduced by a small extent compared with the wild type. We conclude that the extreme C terminus of SNAP25 is a critical region for the Gβγ-SNARE interaction., (Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.)

CXCR6, the receptor for CXCL16, is expressed on multiple cell types and can be a coreceptor for human immunodeficiency virus 1. Except for CXCR6, all human chemokine receptors contain the D(3.49)R(3.50)Y(3.51) sequence, and all but two contain A(3.53) at the cytoplasmic terminus of the third transmembrane helix (H3C), a region within class A G protein-coupled receptors that contacts G proteins. In CXCR6, H3C contains D(3.49)R(3.50)F(3.51)I(3.52)V(3.53) at positions 126-130. We investigated the importance and interdependence of the canonical D126 and the noncanonical F128 and V130 in CXCR6 by mutating D126 to Y, F128 to Y, and V130 to A singly and in combination. For comparison, we mutated the analogous positions D142, Y144, and A146 to Y, F, and V, respectively, in CCR6, a related receptor containing the canonical sequences. Mutants were analyzed in both human embryonic kidney 293T and Jurkat E6-1 cells. Our data show that for CXCR6 and/or CCR6, mutations in H3C can affect both receptor signaling and chemokine binding; noncanonical H3C sequences are functionally linked, with dual changes mitigating the effects of single mutations; mutations in H3C that compromise receptor activity show selective defects in the use of individual Gi/o proteins; and the effects of mutations in H3C on receptor function and selectivity in Gi/o protein use can be cell-type specific. Our findings indicate that the ability of CXCR6 to make promiscuous use of the available Gi/o proteins is exquisitely dependent on sequences within the H3C and suggest that the native sequence allows for preservation of this function across different cellular environments., (U.S. Government work not protected by U.S. copyright.)

Available evidence indicates that some Tie2-expressing (Tie2(+) ) cells serve as multipotent progenitors that have robust BMP-dependent osteogenic activity and mediate heterotopic ossification (HO). Since signaling through the G protein Gi is required for cell motility, we hypothesized that blockade of endogenous Gi signaling in Tie2(+) cell populations would prevent HO formation. Blockade of Gi signaling in Tie2(+) cells was accomplished in transgenic mice with expression of pertussis toxin (PTX) under the control of the Tie2 promoter (Tie2(+) /PTX(+) ). Bone formation within HOs was evaluated 2 weeks after BMP injection. Expression of PTX in Tie2(+) cells significantly reduced the bone volume (BV) of HOs in male and female mice. Orthotopic bones were assessed at the distal femur and expression of PTX significantly increased trabecular bone fractional volume and bone formation rate in females only. In adult Tie2(+) /GFP(+) mice, GFP(+) cells appeared both inside and at the surfaces of bone tissue within HOs and in orthotopic bones. In summary, blockade of Gi signaling in Tie2(+) cells reduced the accrual of HOs and stimulated osteogenesis in orthotopic bones. Targeting of Gi protein coupled receptors in Tie2(+) cells may be a novel therapeutic strategy in states of abnormal bone formation such as osteoporosis and HO., (© 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Previously, we established cell lines stably producing goldfish membrane progestin receptor α (goldfish mPRα) proteins, which mediate steroidal nongenomic actions. In this study, we transfected these cell lines (MDA-MD-231) with cDNAs encoding a recombinant luciferase gene (GloSensor). These cells can be used for monitoring the effects of ligands that bind to mPR by means of luminescence, the intensity of which reflects intracellular cyclic adenosine monophosphate (cAMP) levels. Luminescence intensity of the cells increased significantly when cells were treated with forskolin, strong activator of adenylyl cyclase. Then, we established a strategy to measure changes in luminescence that correlated with the actions of the ligands. The actions of ligands were measurable by the prevention of stimulation caused by forskolin after ligand stimulation. The studies using these cell lines indicated that cAMP concentrations were decreased specifically by the mPR ligands 17α,20β-dihydroxy-4-pregnen-3-one, diethylstilbestrol and progesterone. Furthermore, pertussis toxin inhibited the decrease in cAMP levels caused by mPR ligands. These results support evidence from previous results that mPRα is coupled to an inhibitory G protein., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Expression of the G protein subunit Goα has been shown to be prominent in the atria of the rat heart and to be significantly associated with atrial natriuretic factor (ANF)-containing atrial-specific secretory granules by immunocytochemistry. In addition, differential expression profile analysis using oligonucleotide arrays has shown that the Goα isoform 1 (Goα1) is 2.3-fold more abundant in the atria than it is in the ventricles. In the present report, we show protein-protein interaction between Goα and ANF by yeast two-hybrid and by immunoprecipitation. A cardiac conditional Goα knockout model developed for the present study showed a 90% decrease in Goα expression and decreased atrial expression and ANF and brain natriuretic peptides (BNP) content. Expression of chromogranin A, a specific atrial granule core constituent, was not affected. Morphometric assessment of atrial tissue showed a very significant decrease in atrial-specific granule density as well as granule core electron density. Atrial electrical activity was not affected. The results obtained are compatible with the suggestion that Goα plays a role in ANF sorting during intracellular vectorial transport and with the presence of a mechanism that preserves the molar relationship between cellular ANF and BNP stores in the face of the decreased production of these hormones., (© 2015 Society for Endocrinology.)

Numerous molecular components have been identified that regulate the directed migration of eukaryotic cells toward sources of chemoattractant. However, how the components of this system are wired together to coordinate multiple aspects of the response, such as directionality, speed, and sensitivity to stimulus, remains poorly understood. Here we developed a method to shape chemoattractant gradients optically and analyze cellular chemotaxis responses of hundreds of living cells per well in 96-well format by measuring speed changes and directional accuracy. We then systematically characterized migration and chemotaxis phenotypes for 285 siRNA perturbations. A key finding was that the G-protein Giα subunit selectively controls the direction of migration while the receptor and Gβ subunit proportionally control both speed and direction. Furthermore, we demonstrate that neutrophils chemotax persistently in response to gradients of fMLF but only transiently in response to gradients of ATP. The method we introduce is applicable for diverse chemical cues and systematic perturbations, can be used to measure multiple cell migration and signaling parameters, and is compatible with low- and high-resolution fluorescence microscopy., (© 2015 The Authors. Published under the terms of the CC BY 4.0 license.)

One feature of neuropathic pain is a reduced GABAergic inhibitory function. Nociceptors have been suggested to play a key role in this process. However, the mechanisms behind nociceptor-mediated modulation of GABA signaling remain to be elucidated. Here we describe the identification of GINIP, a Gαi-interacting protein expressed in two distinct subsets of nonpeptidergic nociceptors. GINIP null mice develop a selective and prolonged mechanical hypersensitivity in models of inflammation and neuropathy. GINIP null mice show impaired responsiveness to GABAB, but not to delta or mu opioid receptor agonist-mediated analgesia specifically in the spared nerve injury (SNI) model. Consistently, GINIP-deficient dorsal root ganglia neurons had lower baclofen-evoked inhibition of high-voltage-activated calcium channels and a defective presynaptic inhibition of lamina IIi interneurons. These results further support the role of unmyelinated C fibers in injury-induced modulation of spinal GABAergic inhibition and identify GINIP as a key modulator of peripherally evoked GABAB-receptors signaling., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Caveolae are a nexus for protective signaling. Trafficking of caveolin to mitochondria is essential for adaptation to cellular stress though the trafficking mechanisms remain unknown. The authors hypothesized that G protein-coupled receptor/inhibitory G protein (Gi) activation leads to caveolin trafficking to mitochondria., Methods: Mice were exposed to isoflurane or oxygen vehicle (30 min, ± 36 h pertussis toxin pretreatment, an irreversible Gi inhibitor). Caveolin trafficking, cardioprotective "survival kinase" signaling, mitochondrial function, and ultrastructure were assessed., Results: Isoflurane increased cardiac caveolae (n = 8 per group; data presented as mean ± SD for Ctrl versus isoflurane; [caveolin-1: 1.78 ± 0.12 vs. 3.53 ± 0.77; P < 0.05]; [caveolin-3: 1.68 ± 0.29 vs. 2.67 ± 0.46; P < 0.05]) and mitochondrial caveolin levels (n = 16 per group; [caveolin-1: 0.87 ± 0.18 vs. 1.89 ± .19; P < 0.05]; [caveolin-3: 1.10 ± 0.29 vs. 2.26 ± 0.28; P < 0.05]), and caveolin-enriched mitochondria exhibited improved respiratory function (n = 4 per group; [state 3/complex I: 10.67 ± 1.54 vs. 37.6 ± 7.34; P < 0.05]; [state 3/complex II: 37.19 ± 4.61 vs. 71.48 ± 15.28; P < 0.05]). Isoflurane increased phosphorylation of survival kinases (n = 8 per group; [protein kinase B: 0.63 ± 0.20 vs. 1.47 ± 0.18; P < 0.05]; [glycogen synthase kinase 3β: 1.23 ± 0.20 vs. 2.35 ± 0.20; P < 0.05]). The beneficial effects were blocked by pertussis toxin., Conclusions: Gi proteins are involved in trafficking caveolin to mitochondria to enhance stress resistance. Agents that target Gi activation and caveolin trafficking may be viable cardioprotective agents.

Asymmetric cell divisions allow stem cells to balance proliferation and differentiation. During embryogenesis, murine epidermis expands rapidly from a single layer of unspecified basal layer progenitors to a stratified, differentiated epithelium. Morphogenesis involves perpendicular (asymmetric) divisions and the spindle orientation protein LGN, but little is known about how the apical localization of LGN is regulated. Here, we combine conventional genetics and lentiviral-mediated in vivo RNAi to explore the functions of the LGN-interacting proteins Par3, mInsc and Gαi3. Whereas loss of each gene alone leads to randomized division angles, combined loss of Gnai3 and mInsc causes a phenotype of mostly planar divisions, akin to loss of LGN. These findings lend experimental support for the hitherto untested model that Par3-mInsc and Gαi3 act cooperatively to polarize LGN and promote perpendicular divisions. Finally, we uncover a developmental switch between delamination-driven early stratification and spindle-orientation-dependent differentiation that occurs around E15, revealing a two-step mechanism underlying epidermal maturation.

Group II and group III metabotropic glutamate (mGlu) receptors are G protein-coupled receptors (GPCRs) that inhibit adenylyl cyclase via activation of Gαi/o. The purpose of this study was to design a universal method that overcomes previous challenges in consistently measuring group II and group III mGlu-receptor (mGluR) activation in stably transfected systems. In Chinese hamster ovary (CHO) cells stably transfected with the GloSensor cAMP biosensor, we optimized conditions for simple and highly reproducible (<5% S.E.M.) measurements of cAMP in real time. The GloSensor cAMP biosensor is a recombinant firefly luciferase conjugated to a cAMP-binding domain, where cAMP binding promotes a conformational shift within the GloSensor protein, inducing luciferase activity; cAMP levels are positively correlated with light output resulting from the luciferase-mediated breakdown of d-luciferin. Each group II and group III mGluR was then stably transfected into the CHO-GloSensor cell line, and experimental conditions were optimized for each receptor. During assay optimization, we observed ion sensitivity of several receptors and inverse agonist activity of the antagonist, LY341495 [2-[(1S,2S)-2-carboxycyclopropyl]-3-(9H-xanthen-9-yl)-d-alanine]. Although these phenomena have been previously reported, they remain poorly understood, emphasizing the GloSensor assay as an important tool with which to study group II and group III mGlu receptors. Our results highlight many advantages of using the GloSensor method for measuring activation of group II and group III mGlu receptors, and they further suggest that corresponding methods designed to measure activation of any Gαi/o- or Gαs-coupled GPCR will be similarly advantageous.

Cholinergic signaling plays an important role in regulating the growth and regeneration of axons in the nervous system. The α7 nicotinic receptor (α7) can drive synaptic development and plasticity in the hippocampus. Here, we show that activation of α7 significantly reduces axon growth in hippocampal neurons by coupling to G protein-regulated inducer of neurite outgrowth 1 (Gprin1), which targets it to the growth cone. Knockdown of Gprin1 expression using RNAi is found sufficient to abolish the localization and calcium signaling of α7 at the growth cone. In addition, an α7/Gprin1 interaction appears intimately linked to a Gαo, growth-associated protein 43, and CDC42 cytoskeletal regulatory pathway within the developing axon. These findings demonstrate that α7 regulates axon growth in hippocampal neurons, thereby likely contributing to synaptic formation in the developing brain., (© 2013 International Society for Neurochemistry.)

C-Atrial natriuretic peptide (ANP)4-23, a ring deleted analog of ANP that specifically interacts with natriuretic peptide receptor-C (NPR-C), has been shown to decrease the enhanced expression of Giα proteins implicated in the pathogenesis of hypertension. In the present study, we investigated whether in vivo treatment of spontaneously hypertensive rats (SHRs) with C-ANP4-23 could attenuate the development of high blood pressure (BP) and explored the underlying mechanisms responsible for this response. Intraperitoneal injection of C-ANP4-23 at the concentration of 2 or 10 nmol/kg body weight to prehypertensive SHRs attenuated the development of high BP, and at 8 weeks it was decreased by ≈20 and 50 mm Hg, respectively; however, this treatment did not affect BP in Wistar-Kyoto rats. C-ANP4-23 treatment of adult SHRs for 2 weeks also attenuated high BP, heart rate, and restored the impaired vasorelaxation toward control levels. In addition, the enhanced levels of superoxide anion (O2(-)), peroxynitrite, NADPH oxidase activity, and the enhanced expression of Giα proteins, NOX4, p47(phox), nitrotyrosine, and decreased levels of endothelial nitric oxide synthase (eNOS or NOS3) and NO in SHRs were attenuated by C-ANP4-23 treatment; however, the altered levels of NPR-A/NPR-C were not affected by this treatment. In conclusion, these results indicate that NPR-C activation by C-ANP4-23 attenuates the development of high BP in SHRs through the inhibition of enhanced levels of Giα proteins and nitroxidative stress and not through eNOS/cGMP pathway and suggest that NPR-C ligand may have the potential to be used as therapeutic agent in the treatment of cardiovascular complications including hypertension.

G protein signaling is an extremely complex event that is involved in almost every cellular process. As such, G protein-coupled receptors are the most commonly found type of transmembrane receptors used by cells to initiate intracellular signaling events. However, the widely accepted model of cyclical GDP-GTP exchange in response to ligand binding to 7TMRs, followed by dissociation of the G protein subunits and activation of intracellular signaling cascades, has repeatedly been challenged in recent years. Some of the exceptions that have been brought forth include signaling by a non-dissociated, rearranged heterotrimer and the existence of "reverse-mode", active G proteins that interact with active receptors. Here, we focus on G(αi/o), one of the common G(α) classes, and outline a major exception to the classical model, that of G protein coupling to RTKs. We then describe a novel concept in G(αi/o) signaling, namely that the pathways induced by agonist binding circumvent the typical signaling pathways responsive to decreases in the second messenger cAMP, via adenylyl cyclase inhibition.

Study Objectives: Identifying the neurochemistry and neural circuitry of sleep regulation is critical for understanding sleep and various sleep disorders. Fruit flies display sleep-like behavior, sharing essential features with sleep of vertebrate. In the fruit fly's central brain, the mushroom body (MB) has been highlighted as a sleep center; however, its neurochemical nature remains unclear, and whether it promotes sleep or wake is still a topic of controversy., Design: We used a video recording system to accurately monitor the locomotor activity and sleep status. Gene expression was temporally and regionally manipulated by heat induction and the Gal4/UAS system., Measurements and Results: We found that expressing pertussis toxin (PTX) in the MB by c309-Gal4 to block Go activity led to unique sleep defects as dramatic sleep increase in daytime and fragmented sleep in nighttime. We narrowed down the c309-Gal4 expressing brain regions to the MB α/β core neurons that are responsible for the Go-mediated sleep effects. Using genetic tools of neurotransmitter-specific Gal80 and RNA interference approach to suppress acetylcholine signal, we demonstrated that these MB α/β core neurons were cholinergic and sleep-promoting neurons, supporting that Go mediates an inhibitory signal. Interestingly, we found that adjacent MB α/β neurons were also cholinergic but wake-promoting neurons, in which Go signal was also required., Conclusion: Our findings in fruit flies characterized a group of sleep-promoting neurons surrounded by a group of wake-promoting neurons. The two groups of neurons are both cholinergic and use Go inhibitory signal to regulate sleep.

Negative affect is critical for conferring vulnerability to opiate addiction as reflected by the high comorbidity of opiate abuse with major depressive disorder (MDD). Rodent models implicate amygdala prodynorphin (Pdyn) as a mediator of negative affect; however, evidence of PDYN involvement in human negative affect is limited. Here, we found reduced PDYN mRNA expression in the postmortem human amygdala nucleus of the periamygdaloid cortex (PAC) in both heroin abusers and MDD subjects. Similar to humans, rats that chronically self-administered heroin had reduced Pdyn mRNA expression in the PAC at a time point associated with a negative affective state. Using the in vivo functional imaging technology DREAMM (DREADD-assisted metabolic mapping, where DREADD indicates designer receptors exclusively activated by designer drugs), we found that selective inhibition of Pdyn-expressing neurons in the rat PAC increased metabolic activity in the extended amygdala, which is a key substrate of the extrahypothalamic brain stress system. In parallel, PAC-specific Pdyn inhibition provoked negative affect-related physiological and behavioral changes. Altogether, our translational study supports a functional role for impaired Pdyn in the PAC in opiate abuse through activation of the stress and negative affect neurocircuitry implicated in addiction vulnerability.

In macrophages autophagy assists antigen presentation, affects cytokine release, and promotes intracellular pathogen elimination. In some cells autophagy is modulated by a signaling pathway that employs Gαi3, Activator of G-protein Signaling-3 (AGS3/GPSM1), and Regulator of G-protein Signaling 19 (RGS19). As macrophages express each of these proteins, we tested their importance in regulating macrophage autophagy. We assessed LC3 processing and the formation of LC3 puncta in bone marrow derived macrophages prepared from wild type, Gnai3(-/-), Gpsm1(-/-), or Rgs19(-/-) mice following amino acid starvation or Nigericin treatment. In addition, we evaluated rapamycin-induced autophagic proteolysis rates by long-lived protein degradation assays and anti-autophagic action after rapamycin induction in wild type, Gnai3(-/-), and Gpsm1(-/-) macrophages. In similar assays we compared macrophages treated or not with pertussis toxin, an inhibitor of GPCR (G-protein couple receptor) triggered Gαi nucleotide exchange. Despite previous findings, the level of basal autophagy, autophagic induction, autophagic flux, autophagic degradation and the anti-autophagic action in macrophages that lacked Gαi3, AGS3, or RGS19; or had been treated with pertussis toxin, were similar to controls. These results indicate that while Gαi signaling may impact autophagy in some cell types it does not in macrophages.

The migration of effector or memory T cells to the graft is a critical event in the rejection of transplanted organs. The prevailing view is that the key steps involved in T cell migration - integrin-mediated firm adhesion followed by transendothelial migration - are dependent on the activation of Gαi-coupled chemokine receptors on T cells. In contrast to this view, we demonstrated in vivo that cognate antigen was necessary for the firm adhesion and transendothelial migration of CD8+ effector T cells specific to graft antigens and that both steps occurred independent of Gαi signaling. Presentation of cognate antigen by either graft endothelial cells or bone marrow-derived APCs that extend into the capillary lumen was sufficient for T cell migration. The adhesion and transmigration of antigen-nonspecific (bystander) effector T cells, on the other hand, remained dependent on Gαi, but required the presence of antigen-specific effector T cells. These findings underscore the primary role of cognate antigen presented by either endothelial cells or bone marrow-derived APCs in the migration of T cells across endothelial barriers and have important implications for the prevention and treatment of graft rejection.

Taste is an essential sense for the survival of most organisms. In insects, taste is particularly important as it allows to detect and avoid ingesting many plant toxins, such as L-canavanine. We previously showed that L-canavanine is toxic for Drosophila melanogaster and that flies are able to detect this toxin in the food. L-canavanine is a ligand of DmXR, a variant G-protein coupled receptor (GPCR) belonging to the metabotropic glutamate receptor subfamily that is expressed in bitter-sensitive taste neurons of Drosophila. To transduce the signal intracellularly, GPCR activate heterotrimeric G proteins constituted of α, β and γ subunits. The aim of this study was to identify which Gα protein was required for L-canavanine detection in Drosophila. By using a pharmacological approach, we first demonstrated that DmXR has the best coupling with Gαo protein subtype. Then, by using genetic, behavioral assays and electrophysiology, we found that Gαo47A is required in bitter-sensitive taste neurons for L-canavanine sensitivity. In conclusion, our study revealed that Gαo47A plays a crucial role in L-canavanine detection.

Hedgehog (Hh) signaling plays fundamental roles in morphogenesis, tissue repair, and human disease. Initiation of Hh signaling is controlled by the interaction of two multipass membrane proteins, patched (Ptc) and smoothened (Smo). Recent studies identify Smo as a G-protein coupled receptor (GPCR)-like protein that signals through large G-protein complexes which contain the Gαi subunit. We hypothesize Regulator of G-Protein Signaling (RGS) proteins, and specifically RGS5, are endogenous repressors of Hh signaling via their ability to act as GTPase activating proteins (GAPs) for GTP-bound Gαi, downstream of Smo. In support of this hypothesis, we demonstrate that RGS5 over-expression inhibits sonic hedgehog (Shh)-mediated signaling and osteogenesis in C3H10T1/2 cells. Conversely, signaling is potentiated by siRNA-mediated knock-down of RGS5 expression, but not RGS4 expression. Furthermore, using immuohistochemical analysis and co-immunoprecipitation (Co-IP), we demonstrate that RGS5 is present with Smo in primary cilia. This organelle is required for canonical Hh signaling in mammalian cells, and RGS5 is found in a physical complex with Smo in these cells. We therefore conclude that RGS5 is an endogenous regulator of Hh-mediated signaling and that RGS proteins are potential targets for novel therapeutics in Hh-mediated diseases.

The plasma membrane of the California poppy is known to harbour a PLA2 (phospholipase A2) that is associated with the Gα protein which facilitates its activation by a yeast glycoprotein, thereby eliciting the biosynthesis of phytoalexins. To understand the functional architecture of the protein complex, we titrated purified plasma membranes with the Gα protein (native or recombinant) and found that critical amounts of this subunit keep PLA2 in a low-activity state from which it is released either by elicitor plus GTP or by raising the Gα concentration, which probably causes oligomerization of Gα, as supported by FRET (fluorescence resonance energy transfer)-orientated fluorescence imaging and a semiquantitative split-ubiquitin assay. All effects of Gα were blocked by specific antibodies. A low-Gα mutant showed elevated PLA2 activity and lacked the GTP-dependent stimulation by elicitor, but regained this capability after pre-incubation with Gα. The inhibition by Gα and the GTP-dependent stimulation of PLA2 were diminished by inhibitors of peptidylprolyl cis-trans isomerases. A cyclophilin was identified by sequence in the plasma membrane and in immunoprecipitates with anti-Gα antibodies. We conclude that soluble and target-associated Gα interact at the plasma membrane to build complexes of varying architecture and signal amplification. Protein-folding activity is probably required to convey conformational transitions from Gα to its target PLA2.

Background and Purpose: Recent studies demonstrated that the sympathetic nervous system regulates bone metabolism via β(2) -adrenoceptors. Although α-adrenoceptors are also expressed in osteogenic cells, their functions in bone metabolism have been less studied. We previously demonstrated that noradrenaline suppressed potassium currents via α(1B) -adrenoceptors in the human osteoblast SaM-1 cell line. The aim of this study was to investigate the signal transduction pathway and the physiological role of noradrenaline in human osteoblasts in more detail., Experimental Approach: To investigate signal transduction through α(1B) -adrenoceptors, we used whole-cell patch clamp recording and Ca fluorescence imaging. Potassium channels regulate membrane potential and cell proliferation activity in non-excitable cells, so we evaluated cell proliferation activity by BrdU incorporation and WST assay., Key Results: In SaM-1 cells, bath-applied noradrenaline elevated intracellular Ca(2+) concentration and this effect was abolished by both chloroethylclonidine, an α(1B) -adrenoceptor antagonist, and U73122, a PLC inhibitor. However, the inhibitory effect of noradrenaline on whole-cell current was unaffected by U73122. In contrast, in cells pretreated with either Pertussis toxin, a G(i/o) -protein-coupled receptor inhibitor, or gallein, a Gβγ-protein inhibitor, the inhibitory effect of noradrenaline on whole-cell current was significantly suppressed. Noradrenaline-induced enhancement of cell proliferation was inhibited by CsCl, a non-selective potassium channel blocker, gallein and H89, a PKA inhibitor, but not by U73122., Conclusions and Implications: Noradrenaline facilitated cell proliferation by regulation of potassium currents in human osteoblasts via G(i/o) -protein-coupled α(1B) -adrenoceptors, not via coupling to Gq-proteins., (© 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.)

Heterotrimeric G proteins of the Gα(i) family have been implicated in signaling pathways regulating cell migration in immune diseases. The Gα(i)-protein-coupled C5a receptor is a critical regulator of IgG FcR function in experimental models of immune complex (IC)-induced inflammation. By using mice deficient for Gα(i2) or Gα(i3), we show that Gα(i2) is necessary for neutrophil influx in skin and lung Arthus reactions and agonist-induced neutrophilia in the peritoneum, whereas Gα(i3) plays a less critical but variable role. Detailed analyses of the pulmonary IC-induced inflammatory response revealed several shared functions of Gα(i2) and Gα(i3), including mediating C5a anaphylatoxin receptor-induced activation of macrophages, involvement in alveolar production of chemokines, transition of neutrophils from bone marrow into blood, and modulation of CD11b and CD62L expression that account for neutrophil adhesion to endothelial cells. Interestingly, C5a-stimulated endothelial polymorphonuclear neutrophil transmigration, but not chemotaxis, is enhanced versus reduced in the absence of neutrophil Gα(i3) or Gα(i2), respectively, and knockdown of endothelial Gα(i2) caused decreased transmigration of wild-type neutrophils. These data demonstrate that Gα(i2) and Gα(i3) contribute to inflammation by redundant, overlapping, and Gα(i)-isoform-specific mechanisms, with Gα(i2) exhibiting unique functions in both neutrophils and endothelial cells that appear essential for polymorphonuclear neutrophil recruitment in IC disease.

Tolbutamide and gliclazide block the K(ATP) channel K(ir)6.2/Sur1, causing membrane depolarization and stimulating insulin secretion in pancreatic beta cells. We examined the ability of the EPAC-selective cAMP analog 8-pCPT-2'-O-Me-cAMP-AM to potentiate the action of these drugs and the mechanism that might account for it. Insulin secretion stimulated by both 200 μM tolbutamide and 20 μM gliclazide, concentrations that had equivalent effects on membrane potential, was inhibited by thapsigargin (1 μM) or the L-type Ca(2+) channel blocker nicardipine (2 μM) and was potentiated by 8-pCPT-2'-O-Me-cAMP-AM at concentrations ≥2 μM in INS-1 cells. Ca(2+) transients stimulated by either tolbutamide or gliclazide were inhibited by thapsigargin or nicardipine and were significantly potentiated by 8-pCPT-2'-O-Me-cAMP-AM at 5 μM but not 1 μM. Both tolbutamide and gliclazide stimulated phospholipase C activity; however, only gliclazide did so independently of its activity at K(ATP) channels, and this activity was partially inhibited by pertussis toxin. 8-pCPT-2'-O-Me-cAMP-AM alone (5 μM) did not stimulate insulin secretion, but did increase intracellular Ca(2+) concentration significantly, and this activity was inhibited by 25 μM 2-aminoethoxydiphenylborate (2-APB) or the removal of extracellular Ca(2+). 8-pCPT-2'-O-Me-cAMP-AM potentiation of insulin secretion stimulated by tolbutamide was markedly inhibited by 2-APB (25 μM) and enhanced by the PKC inhibitor bisindolylmaleimide I (1 μM). Our data demonstrate that the actions of both tolbutamide and gliclazide are strongly potentiated by 8-pCPT-2'-O-Me-cAMP-AM, that gliclazide can stimulate phospholipase C activity via a partially pertussis toxin-sensitive mechanism, and that 8-pCPT-2'-O-Me-cAMP-AM potentiation of tolbutamide action may involve activation of a 2-APB-sensitive Ca(2+) influx.

To identify molecular mechanisms that function in G-protein signaling, we have performed molecular genetic studies of a simple behavior of the nematode Caenorhabditis elegans, egg laying, which is driven by a pair of serotonergic neurons, the hermaphrodite-specific neurons (HSNs). The activity of the HSNs is regulated by the G(o)-coupled receptor EGL-6, which mediates inhibition of the HSNs by neuropeptides. We report here that this inhibition requires one of three inwardly rectifying K(+) channels encoded by the C. elegans genome: IRK-1. Using ChannelRhodopsin-2-mediated stimulation of HSNs, we observed roles for egl-6 and irk-1 in regulating the excitability of HSNs. Although irk-1 is required for inhibition of HSNs by EGL-6 signaling, we found that other G(o) signaling pathways that inhibit HSNs involve irk-1 little or not at all. These findings suggest that the neuropeptide receptor EGL-6 regulates the potassium channel IRK-1 via a dedicated pool of G(o) not involved in other G(o)-mediated signaling. We conclude that G-protein-coupled receptors that signal through the same G-protein in the same cell might activate distinct effectors and that specific coupling of a G-protein-coupled receptor to its effectors can be determined by factors other than its associated G-proteins.

Background: Takotsubo cardiomyopathy is an acute heart failure syndrome characterized by myocardial hypocontractility from the mid left ventricle to the apex. It is precipitated by extreme stress and can be triggered by intravenous catecholamine administration, particularly epinephrine. Despite its grave presentation, Takotsubo cardiomyopathy is rapidly reversible, with generally good prognosis. We hypothesized that this represents switching of epinephrine signaling through the pleiotropic β(2)-adrenergic receptor (β(2)AR) from canonical stimulatory G-protein-activated cardiostimulant to inhibitory G-protein-activated cardiodepressant pathways., Methods and Results: We describe an in vivo rat model in which a high intravenous epinephrine, but not norepinephrine, bolus produces the characteristic reversible apical depression of myocardial contraction coupled with basal hypercontractility. The effect is prevented via G(i) inactivation by pertussis toxin pretreatment. β(2)AR number and functional responses were greater in isolated apical cardiomyocytes than in basal cardiomyocytes, which confirmed the higher apical sensitivity and response to circulating epinephrine. In vitro studies demonstrated high-dose epinephrine can induce direct cardiomyocyte cardiodepression and cardioprotection in a β(2)AR-Gi-dependent manner. Preventing epinephrine-G(i) effects increased mortality in the Takotsubo model, whereas β-blockers that activate β(2)AR-G(i) exacerbated the epinephrine-dependent negative inotropic effects without further deaths. In contrast, levosimendan rescued the acute cardiac dysfunction without increased mortality., Conclusions: We suggest that biased agonism of epinephrine for β(2)AR-G(s) at low concentrations and for G(i) at high concentrations underpins the acute apical cardiodepression observed in Takotsubo cardiomyopathy, with an apical-basal gradient in β(2)ARs explaining the differential regional responses. We suggest this epinephrine-specific β(2)AR-G(i) signaling may have evolved as a cardioprotective strategy to limit catecholamine-induced myocardial toxicity during acute stress.

Various heterotrimeric G(i) proteins are considered to be involved in cell migration and effector function of immune cells. The underlying mechanisms, how they control the activation of myeloid effector cells, are not well understood. To elucidate isoform-redundant and -specific roles for Gα(i) proteins in these processes, we analyzed mice genetically deficient in Gα(i2) or Gα(i3). First, we show an altered distribution of tissue macrophages and blood monocytes in the absence of Gα(i2) but not Gα(i3). Gα(i2)-deficient but not wild-type or Gα(i3)-deficient mice exhibited reduced recruitment of macrophages in experimental models of thioglycollate-induced peritonitis and LPS-triggered lung injury. In contrast, genetic ablation of Gα(i2) had no effect on Gα(i)-dependent peritoneal cytokine production in vitro and the phagocytosis-promoting function of the Gα(i)-coupled C5a anaphylatoxin receptor by liver macrophages in vivo. Interestingly, actin rearrangement and CCL2- and C5a anaphylatoxin receptor-induced chemotaxis but not macrophage CCR2 and C5a anaphylatoxin receptor expression were reduced in the specific absence of Gα(i2). Furthermore, knockdown of Gα(i2) caused decreased cell migration and motility of RAW 264.7 cells, which was rescued by transfection of Gα(i2) but not Gα(i3). These results indicate that Gα(i2), albeit redundant to Gα(i3) in some macrophage activation processes, clearly exhibits a Gα(i) isoform-specific role in the regulation of macrophage migration.

L-type Ca(2+) channels (LTCCs) play an essential role in the excitation-contraction coupling of ventricular myocytes. We previously found that t-tubular (TT) LTCC current density was halved by the activation of protein phosphatase (PP)1 and/or PP2A, whereas surface sarcolemmal (SS) LTCC current density was increased by the inhibition of PP1 and/or PP2A activity in failing ventricular myocytes of mice chronically treated with isoproterenol (ISO mice). In the present study, we examined the possible involvement of inhibitory heterotrimeric G proteins (G(i/o)) in these abnormalities by chronically administrating pertussis toxin (PTX) to ISO mice (ISO + PTX mice). Compared with ISO mice, ISO + PTX mice exhibited significantly higher fractional shortening of the left ventricle. The expression level of Gα(i2) proteins was not altered by the treatment of mice with ISO and/or PTX. ISO + PTX myocytes had normal TT and SS LTCC current densities because they had higher and lower availability and/or open probability of TT and SS LTCCs than ISO myocytes, respectively. A selective PKA inhibitor, H-89, did not affect LTCC current densities in ISO + PTX myocytes. A selective PP2A inhibitor, fostriecin, did not affect SS or TT current density in control or ISO + PTX myocytes but significantly increased TT but not SS LTCC current density in ISO myocytes. These results indicate that chronic receptor-mediated activation of G(i/o) in vivo decreases basal TT LTCC activity by activating PP2A and increases basal SS LTCC activity by inhibiting PP1 without modulating PKA in heart failure.

Aims: Natriuretic peptide receptor-A (NPR-A) knockout mice exhibited an increased blood pressure that may also be attributed to the up-regulation of NPR-C and associated signalling; however, the interaction between the two receptors has not been investigated. In the present study, we investigated the effect of knockdown of NPR-A using NPR-A antisense (AS) on the expression of NPR-C and adenylyl cyclase (AC) signalling in A10 vascular smooth muscle cells (VSMC)., Methods and Results: The receptor and G protein expression was determined by western blotting, and AC activity was determined by measuring [(32)P]cAMP formation from [α-(32)P]ATP. Treatment of A10 VSMC with NPR-A AS decreased NPR-A and enhanced NPR-C expression without altering the levels of angiotensin II AT1 and muscarinic M2 receptors. In addition, siRNA-NPR-A also resulted in the up-regulation of NPR-C. The re-expression of NPR-A in AS-treated cells reversed the enhanced expression of NPR-C to control levels. In addition, NPR-C-, AT1, and M2 receptor-mediated inhibition of AC and Giα protein expression was enhanced in AS-treated cells, whereas NPR-A-mediated cyclic GMP (cGMP) formation and Gsα-mediated stimulation of AC were significantly reduced. Pertussis toxin treatment attenuated the AS-induced enhanced inhibition of AC to control levels. Furthermore, the enhanced levels of NPR-C and Giα proteins were reversed to control levels by 8-bromo-cGMP (8Br-cGMP) and PD98059, an MEK inhibitor. In addition, 8Br-cGMP also attenuated AS-induced enhanced ERK1/2 phosphorylation to control levels., Conclusion: These results demonstrate that knockdown of NPR-A up-regulates the expression of NPR-C, Giα proteins, and NPR-C-linked AC signalling and suggests a cross-talk between NPR-A and NPR-C.

Interest in how the gut microbiome can influence the metabolic state of the host has recently heightened. One postulated link is bacterial fermentation of "indigestible" prebiotics to short-chain fatty acids (SCFAs), which in turn modulate the release of gut hormones controlling insulin release and appetite. We show here that SCFAs trigger secretion of the incretin hormone glucagon-like peptide (GLP)-1 from mixed colonic cultures in vitro. Quantitative PCR revealed enriched expression of the SCFA receptors ffar2 (grp43) and ffar3 (gpr41) in GLP-1-secreting L cells, and consistent with the reported coupling of GPR43 to Gq signaling pathways, SCFAs raised cytosolic Ca2+ in L cells in primary culture. Mice lacking ffar2 or ffar3 exhibited reduced SCFA-triggered GLP-1 secretion in vitro and in vivo and a parallel impairment of glucose tolerance. These results highlight SCFAs and their receptors as potential targets for the treatment of diabetes.