MiR-590 suppresses the progression of non-small cell lung cancer by regulating YAP1 and Wnt/B-catenin signaling

ObjectiveAccumulating evidence has been revealed that miR-590 is involved in the progression and carcinogenesis of various cancers. However, the molecular mechanism of miR-590 in non-small-cell lung cancer (NSCLC) remains unclear.MethodsQuantitative reverse transcription-PCR (qRT-PCR), western blot, MTT, and transwell assay were applied to investigate the functional role of miR-590 in this study. Dual luciferase reporter assay was utilized to investigate the interaction between YAP1 and miR-590 expression. Cells transfected with miR-590 mimic or inhibitor were subjected to western blot to investigate the role of Wnt/β-catenin signaling in NSCLC modulated by miR-590.ResultsMiR-590 was down-regulated in NSCLC tissues and cells. Kaplan–Meier analysis found that the higher expression of miR-590 in NSCLC patients, the more improved survival rate of NSCLC patients. Over-expression of miR-590 inhibited NSCLC cell proliferation, migration, and invasion. Moreover, increasing miR-590 suppressed Yes-associated protein 1 (YAP1) expression and inhibited the Wnt/β-catenin pathway in NSCLC cells. Furthermore, miR-590 was negatively correlated with YAP1 expression.ConclusionThese findings demonstrated that the miR-590/YAP1 axis exerted an important role in the progression of NSCLC, suggesting that miR-590 might be the appealing prognostic marker for NSCLC treatment.

RGS proteins, GRKs, and beta-arrestins modulate G protein-mediated signaling pathways in asthma.

Asthma is a highly prevalent disorder characterized by chronic lung inflammation and reversible airways obstruction. Pathophysiological features of asthma include episodic and reversible airway narrowing due to increased bronchial smooth muscle shortening in response to external and host-derived mediators, excessive mucus secretion into the airway lumen, and airway remodeling. The aberrant airway smooth muscle (ASM) phenotype observed in asthma manifests as increased sensitivity to contractile mediators (EC50) and an increase in the magnitude of contraction (Emax); collectively these attributes have been termed "airways hyper-responsiveness" (AHR). This defining feature of asthma can be promoted by environmental factors including airborne allergens, viruses, and air pollution and other irritants. AHR reduces airway caliber and obstructs airflow, evoking clinical symptoms such as cough, wheezing and shortness of breath. G-protein-coupled receptors (GPCRs) have a central function in asthma through their impact on ASM and airway inflammation. Many but not all treatments for asthma target GPCRs mediating ASM contraction or relaxation. Here we discuss the roles of specific GPCRs, G proteins, and their associated signaling pathways, in asthma, with an emphasis on endogenous mechanisms of GPCR regulation of ASM tone and lung inflammation including regulators of G-protein signaling (RGS) proteins, G-protein coupled receptor kinases (GRKs), and ß-arrestin.

A Global Map of G Protein Signaling Regulation by RGS Proteins.

The control over the extent and timing of G protein signaling is provided by the regulator of G protein signaling (RGS) proteins that deactivate G protein α subunits (Gα). Mammalian genomes encode 20 canonical RGS and 16 Gα genes with key roles in physiology and disease. To understand the principles governing the selectivity of Gα regulation by RGS, we examine the catalytic activity of all canonical human RGS proteins and their selectivity for a complete set of Gα substrates using real-time kinetic measurements in living cells. The data reveal rules governing RGS-Gα recognition, the structural basis of its selectivity, and provide principles for engineering RGS proteins with defined selectivity. The study also explores the evolution of RGS-Gα selectivity through ancestral reconstruction and demonstrates how naturally occurring non-synonymous variants in RGS alter signaling. These results provide a blueprint for decoding signaling selectivity and advance our understanding of molecular recognition principles.

SPARC promotes insulin secretion through down-regulation of RGS4 protein in pancreatic ß cells.

SPARC-deficient mice have been shown to exhibit impaired glucose tolerance and insulin secretion, but the underlying mechanism remains unknown. Here, we showed that SPARC enhanced the promoting effect of Muscarinic receptor agonist oxotremorine-M on insulin secretion in cultured mouse islets. Overexpression of SPARC down-regulated RGS4, a negative regulator of ß-cell M3 muscarinic receptors. Conversely, knockdown of SPARC up-regulated RGS4 in Min6 cells. RGS4 was up-regulated in islets from sparc -/- mice, which correlated with decreased glucose-stimulated insulin secretion (GSIS). Furthermore, inhibition of RGS4 restored GSIS in the islets from sparc -/- mice, and knockdown of RGS4 partially decreased the promoting effect of SPARC on oxotremorine-M-stimulated insulin secretion. Phosphoinositide 3-kinase (PI3K) inhibitor LY-294002 abolished SPARC-induced down-regulation of RGS4. Taken together, our data revealed that SPARC promoted GSIS by inhibiting RGS4 in pancreatic ß cells.

Regulator of G protein signaling 10: Structure, expression and functions in cellular physiology and diseases.

Regulator of G protein signaling 10 (RGS10) belongs to the superfamily of RGS proteins, defined by the presence of a conserved RGS domain that canonically binds and deactivates heterotrimeric G-proteins. RGS proteins act as GTPase activating proteins (GAPs), which accelerate GTP hydrolysis on the G-protein α subunits and result in termination of signaling pathways downstream of G protein-coupled receptors. RGS10 is the smallest protein of the D/R12 subfamily and selectively interacts with Gαi proteins. It is widely expressed in many cells and tissues, with the highest expression found in the brain and immune cells. RGS10 expression is transcriptionally regulated via epigenetic mechanisms. Although RGS10 lacks multiple of the defined regulatory domains found in other RGS proteins, RGS10 contains post-translational modification sites regulating its expression, localization, and function. Additionally, RGS10 is a critical protein in the regulation of physiological processes in multiple cells, where dysregulation of its expression has been implicated in various diseases including Parkinson's disease, multiple sclerosis, osteopetrosis, chemoresistant ovarian cancer and cardiac hypertrophy. This review summarizes RGS10 features and its regulatory mechanisms, and discusses the known functions of RGS10 in cellular physiology and pathogenesis of several diseases.

Regulator of G-Protein Signaling 5 Maintains Brain Endothelial Cell Function in Focal Cerebral Ischemia.

Background Regulator of G-protein signaling 5 (RGS5) is a negative modulator of G-protein-coupled receptors. The role of RGS5 in brain endothelial cells is not known. We hypothesized that RGS5 in brain microvascular endothelial cells may be an important mediator of blood-brain barrier function and stroke severity after focal cerebral ischemia. Methods and Results Using a transient middle cerebral artery occlusion model, we found that mice with global and endothelial-specific deletion of Rgs5 exhibited larger cerebral infarct size, greater neurological motor deficits, and increased brain edema. In our in vitro models, we observed increased Gq activity and elevated intracellular Ca2+ levels in brain endothelial cells. Furthermore, the loss of endothelial RGS5 leads to decreased endothelial NO synthase expression and phosphorylation, relocalization of endothelial tight junction proteins, and increased cell permeability. Indeed, RGS5 deficiency leads to increased Rho-associated kinase and myosin light chain kinase activity, which were partially reversed in our in vitro model by pharmacological inhibition of Gq, metabotropic glutamate receptor 1, and ligand-gated ionotropic glutamate receptor. Conclusions Our findings indicate that endothelial RGS5 plays a novel neuroprotective role in focal cerebral ischemia. Loss of endothelial RGS5 leads to hyperresponsiveness to glutamate signaling pathways, enhanced Rho-associated kinase- and myosin light chain kinase-mediated actin-cytoskeleton reorganization, endothelial dysfunction, tight junction protein relocalization, increased blood-brain barrier permeability, and greater stroke severity. These findings suggest that preservation of endothelial RGS5 may be an important therapeutic strategy for maintaining blood-brain barrier integrity and limiting the severity of ischemic stroke.

Genetic deletion of Rgs12 in mice affects serotonin transporter expression and function in vivo and ex vivo.

BACKGROUND: Regulator of G protein Signaling (RGS) proteins inhibit G protein-coupled receptor (GPCR) signaling, including the signals that arise from neurotransmitter release. We have shown that RGS12 loss diminishes locomotor responses of C57BL/6J mice to dopamine transporter (DAT)-targeting psychostimulants. This diminution resulted from a brain region-specific upregulation of DAT expression and function in RGS12-null mice. This effect on DAT prompted us to investigate whether the serotonin transporter (SERT) exhibits similar alterations upon RGS12 loss in C57BL/6J mice. AIMS: Does RGS12 loss affect (a) hyperlocomotion to the preferentially SERT-targeting psychostimulant 3,4-methylenedioxymethamphetamine (MDMA), (b) SERT expression and function in relevant brain regions, and/or (c) serotonergically modulated behaviors? METHODS: Open-field and spontaneous home-cage locomotor activities were quantified. 5-HT, 5-HIAA, and SERT levels in brain-region homogenates, as well as SERT expression and function in brain-region tissue preparations, were measured using appropriate biochemical assays. Serotonergically modulated behaviors were assessed using forced swim and tail suspension paradigms, elevated plus and elevated zero maze tests, and social interaction assays. RESULTS: RGS12-null mice displayed no hyperlocomotion to 10 mg/kg MDMA. There were brain region-specific alterations in SERT expression and function associated with RGS12 loss. Drug-naïve RGS12-null mice displayed increases in both anxiety-like and anti-depressive-like behaviors. CONCLUSION: RGS12 is a critical modulator of serotonergic neurotransmission and serotonergically modulated behavior in mice; lack of hyperlocomotion to low dose MDMA in RGS12-null mice is related to an alteration of steady-state SERT expression and 5-HT uptake.

RGS2: A multifunctional signaling hub that balances brown adipose tissue function and differentiation.

OBJECTIVE: Recruitment of brown adipose tissue (BAT) is a potential new strategy for increasing energy expenditure (EE) to treat obesity. G protein-coupled receptors (GPCRs) represent promising targets to activate BAT, as they are the major regulators of BAT biological function. To identify new regulators of GPCR signaling in BAT, we studied the role of Regulator of G protein Signaling 2 (RGS2) in brown adipocytes and BAT. METHODS: We combined pharmacological and genetic tools to investigate the role of RGS2 in BAT in vitro and in vivo. Adipocyte progenitors were isolated from wild-type (WT) and RGS2 knockout (RGS2-/-) BAT and differentiated to brown adipocytes. This approach was complemented with knockdown of RGS2 using lentiviral shRNAs (shRGS2). Adipogenesis was analyzed by Oil Red O staining and by determining the expression of adipogenic and thermogenic markers. Pharmacological modulators and fluorescence staining of F-acting stress fibers were employed to identify the underlying signaling pathways. In vivo, the activity of BAT was assessed by ex vivo lipolysis and by measuring whole-body EE by indirect calorimetry in metabolic cages. RESULTS: RGS2 is highly expressed in BAT, and treatment with cGMP-an important enhancer of brown adipocyte differentiation-further increased RGS2 expression. Loss of RGS2 strongly suppressed adipogenesis and the expression of thermogenic genes in brown adipocytes. Mechanistically, we found increased Gq/Rho/Rho kinase (ROCK) signaling in the absence of RGS2. Surprisingly, in vivo analysis revealed elevated BAT activity in RGS2-deficient mice that was caused by enhanced Gs/cAMP signaling. CONCLUSION: Overall, RGS2 regulates two major signaling pathways in BAT: Gq and Gs. On the one hand, RGS2 promotes brown adipogenesis by counteracting the inhibitory action of Gq/Rho/ROCK signaling. On the other hand, RGS2 decreases the activity of BAT through the inhibition of Gs signaling and cAMP production. Thus, RGS2 might represent a stress modulator that protects BAT from overstimulation.

Regulator of G-protein signaling 5 protein protects against anxiety- and depression-like behavior.

Anxiety and depression are a major health burden. Angiotensin II, via activation of angiotensin II type 1 receptor (AT1R)-mediated brain oxidative stress and inflammation may contribute to these emotional abnormalities. In this study, we investigated the role of a regulator of G-protein signaling 5 (RGS5) protein, which regulates AT1R activity, in angiotensin II-induced brain oxidative stress, inflammation and anxiety-, and depression-like behavior. We hypothesized that deletion of the RGS5 protein would worsen angiotensin II-induced anxiety- and depression-like behavior, cerebral vascular oxidative stress, and brain inflammation. Adult male wild-type and RGS5-deficient mice were implanted with osmotic minipumps delivering either vehicle (saline) or angiotensin II (1 mg/kg/d) for three weeks. Subsequently, mice were tested for locomotor activity, anxiety-like behavior (using the elevated plus maze), and depression-like behavior (using the tail suspension test). After behavioral testing, brain tissue was collected to assess oxidative stress and inflammatory proteins. RGS5 deletion resulted in anxiety-like but not depression-like behavior when compared to wild-type mice. Combined deletion of RGS5 and angiotensin II treatment did not further worsen anxiety-like behavior observed in RGS5-deficient mice. In contrast, depression-like behavior was worsened in RGS5-deficient mice treated with angiotensin II. Interestingly, RGS5 deficiency and angiotensin II treatment had no effect on cerebral vascular oxidative stress, or on expression of the inflammatory marker vascular cell adhesion molecule-1 in the brain. RGS5 deficiency was also associated with decreased blood pressure and an enhanced pressor response to angiotensin II. These data suggest that RGS5 protects against anxiety-like behavior and against angiotensin II-induced depression-like behavior.

A role for Regulator of G protein Signaling-12 (RGS12) in the balance between myoblast proliferation and differentiation.

Regulators of G Protein Signaling (RGS proteins) inhibit G protein-coupled receptor (GPCR) signaling by accelerating the GTP hydrolysis rate of activated Gα subunits. Some RGS proteins exert additional signal modulatory functions, and RGS12 is one such protein, with five additional, functional domains: a PDZ domain, a phosphotyrosine-binding domain, two Ras-binding domains, and a Gα·GDP-binding GoLoco motif. RGS12 expression is temporospatially regulated in developing mouse embryos, with notable expression in somites and developing skeletal muscle. We therefore examined whether RGS12 is involved in the skeletal muscle myogenic program. In the adult mouse, RGS12 is expressed in the tibialis anterior (TA) muscle, and its expression is increased early after cardiotoxin-induced injury, suggesting a role in muscle regeneration. Consistent with a potential role in coordinating myogenic signals, RGS12 is also expressed in primary myoblasts; as these cells undergo differentiation and fusion into myotubes, RGS12 protein abundance is reduced. Myoblasts isolated from mice lacking Rgs12 expression have an impaired ability to differentiate into myotubes ex vivo, suggesting that RGS12 may play a role as a modulator/switch for differentiation. We also assessed the muscle regenerative capacity of mice conditionally deficient in skeletal muscle Rgs12 expression (via Pax7-driven Cre recombinase expression), following cardiotoxin-induced damage to the TA muscle. Eight days post-damage, mice lacking RGS12 in skeletal muscle had attenuated repair of muscle fibers. However, when mice lacking skeletal muscle expression of Rgs12 were cross-bred with mdx mice (a model of human Duchenne muscular dystrophy), no increase in muscle degeneration was observed over time. These data support the hypothesis that RGS12 plays a role in coordinating signals during the myogenic program in select circumstances, but loss of the protein may be compensated for within model syndromes of prolonged bouts of muscle damage and repair.

Modulation of CA2 neuronal activity increases behavioral responses to fear conditioning in female mice.

Activity of hippocampal pyramidal cells is critical for certain forms of learning and memory, and work from our lab and others has shown that CA2 neuronal activity is required for social cognition and behavior. Silencing of CA2 neurons in mice impairs social memory, and mice lacking Regulator of G-Protein Signaling 14 (RGS14), a protein that is highly enriched in CA2 neurons, learn faster than wild types in the Morris water maze spatial memory test. Although the enhanced spatial learning abilities of the RGS14 KO mice suggest a role for CA2 neurons in at least one hippocampus-dependent behavior, the role of CA2 neurons in fear conditioning, which requires activity of hippocampus, amygdala, and possibly prefrontal cortex is unknown. In this study, we expressed excitatory or inhibitory DREADDs in CA2 neurons and administered CNO before the shock-tone-context pairing. On subsequent days, we measured freezing behavior in the same context but without the tone (contextual fear) or in a new context but in the presence of the tone (cued fear). We found that increasing CA2 neuronal activity with excitatory DREADDs during training resulted in increased freezing during the cued fear tests in males and females. Surprisingly, we found that only females showed increased freezing during the contextual fear memory tests. Using inhibitory DREADDs, we found that inhibiting CA2 neuronal activity during the training phase also resulted in increased freezing in females during the subsequent contextual fear memory test. Finally, we tested fear conditioning in RGS14 KO mice and found that female KO mice had increased freezing on the cued fear memory test. These three separate lines of evidence suggest that CA2 neurons are actively involved in both intra- and extra-hippocampal brain processes and function to influence fear memory. Finally, the intriguing and consistent findings of enhanced fear conditioning only among females is strongly suggestive of a sexual dimorphism in CA2-linked circuits.

RGS2 promotes the translation of stress-associated proteins ATF4 and CHOP via its eIF2B-inhibitory domain.

Regulator of G protein signaling 2 (RGS2) is upregulated by multiple forms of stress and can augment translational attenuation associated with the phosphorylation of the initiation factor eIF2, a hallmark of several stress-induced coping mechanisms. Under stress-induced translational inhibition, key factors, such as ATF4, are selectively expressed via alternative translation mechanisms. These factors are known to regulate molecular switches that control cell fate by regulating pro-survival and pro-apoptotic signals. The molecular mechanisms that balance these opposing responses to stresses are unclear. The present results suggest that RGS2 may be an important regulatory component in the cellular stress response through its translational control abilities. Previously, we have shown that RGS2 can interact with the translation initiation factor, eIF2B, and inhibit de novo protein synthesis. Here, we demonstrate that the expression of either full length RGS2 or its eIF2B-interacting domain (RGS2eb) significantly increases levels of ATF4 and CHOP, both of which are linked to stress-induced apoptosis. Furthermore, we show that these effects are translationally regulated and independent of eIF2 phosphorylation. The present results thus point to a novel function of RGS2 in the stress response directly related to its ability to reduce global protein synthesis.

Inhibitory Signaling to Ion Channels in Hippocampal Neurons Is Differentially Regulated by Alternative Macromolecular Complexes of RGS7.

The neuromodulatory effects of GABA on pyramidal neurons are mediated by GABAB receptors (GABABRs) that signal via a conserved G-protein-coupled pathway. Two prominent effectors regulated by GABABRs include G-protein inwardly rectifying K+ (GIRK) and P/Q/N type voltage-gated Ca2+ (CaV2) ion channels that control excitability and synaptic output of these neurons, respectively. Regulator of G-protein signaling 7 (RGS7) has been shown to control GABAB effects, yet the specificity of its impacts on effector channels and underlying molecular mechanisms is poorly understood. In this study, we show that hippocampal RGS7 forms two distinct complexes with alternative subunit configuration bound to either membrane protein R7BP (RGS7 binding protein) or orphan receptor GPR158. Quantitative biochemical experiments show that both complexes account for targeting nearly the entire pool of RGS7 to the plasma membrane. We analyzed the effect of genetic elimination in mice of both sexes and overexpression of various components of RGS7 complex by patch-clamp electrophysiology in cultured neurons and brain slices. We report that RGS7 prominently regulates GABABR signaling to CaV2, in addition to its known involvement in modulating GIRK. Strikingly, only complexes containing R7BP, but not GPR158, accelerated the kinetics of both GIRK and CaV2 modulation by GABABRs. In contrast, GPR158 overexpression exerted the opposite effect and inhibited RGS7-assisted temporal modulation of GIRK and CaV2 by GABA. Collectively, our data reveal mechanisms by which distinctly composed macromolecular complexes modulate the activity of key ion channels that mediate the inhibitory effects of GABA on hippocampal CA1 pyramidal neurons.SIGNIFICANCE STATEMENT This study identifies the contributions of distinct macromolecular complexes containing a major G-protein regulator to controlling key ion channel function in hippocampal neurons with implications for understanding molecular mechanisms underlying synaptic plasticity, learning, and memory.

Selective Role of RGS9-2 in Regulating Retrograde Synaptic Signaling of Indirect Pathway Medium Spiny Neurons in Dorsal Striatum.

In the striatum, medium spiny neurons (MSNs) are heavily involved in controlling movement and reward. MSNs form two distinct populations expressing either dopamine receptor 1 (D1-MSN) or dopamine receptor 2 (D2-MSN), which differ in their projection targets and neurochemical composition. The activity of both types of MSNs is shaped by multiple neuromodulatory inputs processed by GPCRs that fundamentally impact their synaptic properties biasing behavioral outcomes. How these GPCR signaling cascades are regulated and what downstream targets they recruit in D1-MSN and D2-MSN populations are incompletely understood. In this study, we examined the cellular and molecular mechanisms underlying the action of RGS9-2, a key GPCR regulator in MSNs implicated in both movement control and actions of addictive drugs. Imaging cultured striatal neurons, we found that ablation of RGS9-2 significantly reduced calcium influx through NMDARs. Electrophysiological recordings in slices confirmed inhibition of NMDAR function in MSNs, resulting in enhanced AMPAR/NMDAR ratio. Accordingly, male mice lacking RGS9-2 displayed behavioral hypersensitivity to NMDAR blockade by MK-801 or ketamine. Recordings from genetically identified populations of striatal neurons revealed that these changes were selective to D2-MSNs. Surprisingly, we found that these postsynaptic effects resulted in remodeling of presynaptic inputs to D2-MSNs increasing the frequency of mEPSC and inhibiting paired-pulse ratio. Pharmacological dissection revealed that these adaptations were mediated by the NMDAR-dependent inhibition of retrograde endocannabinoid signaling from D2-MSNs to CB1 receptor on presynaptic terminals. Together, these data demonstrate a novel mechanism for pathway selective regulation of synaptic plasticity in MSNs controlled by GPCR signaling.SIGNIFICANCE STATEMENT This study identifies a role for a major G-protein regulator in controlling synaptic properties of striatal neurons in a pathway selective fashion.

Regulator of G protein signaling 5 restricts neutrophil chemotaxis and trafficking.

Neutrophils are white blood cells that are mobilized to damaged tissues and to sites of pathogen invasion, providing the first line of host defense. Chemokines displayed on the surface of blood vessels promote migration of neutrophils to these sites, and tissue- and pathogen-derived chemoattractant signals, including N-formylmethionylleucylphenylalanine (fMLP), elicit further migration to sites of infection. Although nearly all chemoattractant receptors use heterotrimeric G proteins to transmit signals, many of the mechanisms lying downstream of chemoattractant receptors that either promote or limit neutrophil motility are incompletely defined. Here, we show that regulator of G protein signaling 5 (RGS5), a protein that modulates G protein activity, is expressed in both human and murine neutrophils. We detected significantly more neutrophils in the airways of Rgs5-/- mice than WT counterparts following acute respiratory virus infection and in the peritoneum in response to injection of thioglycollate, a biochemical proinflammatory stimulus. RGS5-deficient neutrophils responded with increased chemotaxis elicited by the chemokines CXC motif chemokine ligand 1 (CXCL1), CXCL2, and CXCL12 but not fMLP. Moreover, adhesion of these cells was increased in the presence of both CXCL2 and fMLP. In summary, our results indicate that RGS5 deficiency increases chemotaxis and adhesion, leading to more efficient neutrophil mobilization to inflamed tissues in mice. These findings suggest that RGS5 expression and activity in neutrophils determine their migrational patterns in the complex microenvironments characteristic of inflamed tissues.

RGS14 Restricts Plasticity in Hippocampal CA2 by Limiting Postsynaptic Calcium Signaling.

Pyramidal neurons in hippocampal area CA2 are distinct from neighboring CA1 in that they resist synaptic long-term potentiation (LTP) at CA3 Schaffer collateral synapses. Regulator of G protein signaling 14 (RGS14) is a complex scaffolding protein enriched in CA2 dendritic spines that naturally blocks CA2 synaptic plasticity and hippocampus-dependent learning, but the cellular mechanisms by which RGS14 gates LTP are largely unexplored. A previous study has attributed the lack of plasticity to higher rates of calcium (Ca2+) buffering and extrusion in CA2 spines. Additionally, a recent proteomics study revealed that RGS14 interacts with two key Ca2+-activated proteins in CA2 neurons: calcium/calmodulin and CaMKII. Here, we investigated whether RGS14 regulates Ca2+ signaling in its host CA2 neurons. We found that the nascent LTP of CA2 synapses caused by genetic knockout (KO) of RGS14 in mice requires Ca2+-dependent postsynaptic signaling through NMDA receptors, CaMK, and PKA, revealing similar mechanisms to those in CA1. We report that RGS14 negatively regulates the long-term structural plasticity of dendritic spines of CA2 neurons. We further show that wild-type (WT) CA2 neurons display significantly attenuated spine Ca2+ transients during structural plasticity induction compared with the Ca2+ transients from CA2 spines of RGS14 KO mice and CA1 controls. Finally, we demonstrate that acute overexpression of RGS14 is sufficient to block spine plasticity, and elevating extracellular Ca2+ levels restores plasticity to RGS14-expressing neurons. Together, these results demonstrate for the first time that RGS14 regulates plasticity in hippocampal area CA2 by restricting Ca2+ elevations in CA2 spines and downstream signaling pathways.

Genetic Analysis of Rare Human Variants of Regulators of G Protein Signaling Proteins and Their Role in Human Physiology and Disease.

Regulators of G protein signaling (RGS) proteins modulate the physiologic actions of many neurotransmitters, hormones, and other signaling molecules. Human RGS proteins comprise a family of 20 canonical proteins that bind directly to G protein-coupled receptors/G protein complexes to limit the lifetime of their signaling events, which regulate all aspects of cell and organ physiology. Genetic variations account for diverse human traits and individual predispositions to disease. RGS proteins contribute to many complex polygenic human traits and pathologies such as hypertension, atherosclerosis, schizophrenia, depression, addiction, cancers, and many others. Recent analysis indicates that most human diseases are due to extremely rare genetic variants. In this study, we summarize physiologic roles for RGS proteins and links to human diseases/traits and report rare variants found within each human RGS protein exome sequence derived from global population studies. Each RGS sequence is analyzed using recently described bioinformatics and proteomic tools for measures of missense tolerance ratio paired with combined annotation-dependent depletion scores, and protein post-translational modification (PTM) alignment cluster analysis. We highlight selected variants within the well-studied RGS domain that likely disrupt RGS protein functions and provide comprehensive variant and PTM data for each RGS protein for future study. We propose that rare variants in functionally sensitive regions of RGS proteins confer profound change-of-function phenotypes that may contribute, in newly appreciated ways, to complex human diseases and/or traits. This information provides investigators with a valuable database to explore variation in RGS protein function, and for targeting RGS proteins as future therapeutic targets.

Regulator of G-Protein Signaling 4 (RGS4) Controls Morphine Reward by Glutamate Receptor Activation in the Nucleus Accumbens of Mouse Brain.

Crosstalk between G-protein signaling and glutamatergic transmission within the brain reward circuits is critical for long-term emotional effects (depression and anxiety), cravings, and negative withdrawal symptoms associated with opioid addiction. A previous study showed that Regulator of G-protein signaling 4 (RGS4) may be implicated in opiate action in the nucleus accumbens (NAc). However, the mechanism of the NAc-specific RGS4 actions that induce the behavioral responses to opiates remains largely unknown. The present study used a short hairpin RNA (shRNA)-mediated knock-down of RGS4 in the NAc of the mouse brain to investigate the relationship between the activation of ionotropic glutamate receptors and RGS4 in the NAc during morphine reward. Additionally, the shRNA-mediated RGS4 knock-down was implemented in NAc/striatal primary-cultured neurons to investigate the role that striatal neurons have in the morphine-induced activation of ionotropic glutamate receptors. The results of this study show that the NAc-specific knockdown of RGS4 significantly increased the behaviors associated with morphine and did so by phosphorylation of the GluR1 (Ser831) and NR2A (Tyr1325) glutamate receptors in the NAc. Furthermore, the knock-down of RGS4 enhanced the phosphorylation of the GluR1 and NR2A glutamate receptors in the primary NAc/striatal neurons during spontaneous morphine withdrawal. These findings show a novel molecular mechanism of RGS4 in glutamatergic transmission that underlies the negative symptoms associated with morphine administration.

A gene-based review of RGS4 as a putative risk gene for psychiatric illness.

Considerable efforts have been made to characterize RGS4 as a potential candidate gene for schizophrenia. Investigations span across numerous modalities and include explorations of genetic risk associations, mRNA and protein levels in the brain, and functionally relevant interactions with other candidate genes as well as links to schizophrenia relevant neural phenotypes. While these lines of investigations have yielded partially inconsistent findings, they provide a perspective on RGS4 as an important part of a larger biological system contributing to schizophrenia risk. This gene-based review aims to provide a comprehensive overview of published data from different experimental modalities and discusses the current knowledge of RGS4's systems-biological impact on the schizophrenia pathology.

Proteolytic degradation of regulator of G protein signaling 2 facilitates temporal regulation of Gq/11 signaling and vascular contraction.

Regulator of G protein signaling 2 (RGS2) controls signaling by receptors coupled to the Gq/11 class heterotrimeric G proteins. RGS2 deficiency causes several phenotypes in mice and occurs in several diseases, including hypertension in which a proteolytically unstable RGS2 mutant has been reported. However, the mechanisms and functions of RGS2 proteolysis remain poorly understood. Here we addressed these questions by identifying degradation signals in RGS2, and studying dynamic regulation of Gq/11-evoked Ca2+ signaling and vascular contraction. We identified a novel bipartite degradation signal in the N-terminal domain of RGS2. Mutations disrupting this signal blunted proteolytic degradation downstream of E3 ubiquitin ligase binding to RGS2. Analysis of RGS2 mutants proteolyzed at various rates and the effects of proteasome inhibition indicated that proteolytic degradation controls agonist efficacy by setting RGS2 protein expression levels, and affecting the rate at which cells regain agonist responsiveness as synthesis of RGS2 stops. Analyzing contraction of mesenteric resistance arteries supported the biological relevance of this mechanism. Because RGS2 mRNA expression often is strikingly and transiently up-regulated and then down-regulated upon cell stimulation, our findings indicate that proteolytic degradation tightly couples RGS2 transcription, protein levels, and function. Together these mechanisms provide tight temporal control of Gq/11-coupled receptor signaling in the cardiovascular, immune, and nervous systems.