Function Of RGS2 Research Articles

Modulation of G-protein-coupled receptor 55-mediated signaling by regulator of G-protein signaling 2

Activation of seven-transmembrane G-protein coupled receptor (GPCR) mediates extracellular signals into intracellular responses. G-protein coupled receptor 55 (GPR55) is one of GPCRs and activated by endogenous cannabinoids. A family of regulators of G-protein signaling (RGS) stimulates GTP hydrolysis of alpha subunit of G-protein (Gα) and inhibits GPCR/Gα-mediated signaling. RGS2 is member of R4 RGS family and mainly attenuates GPCR/Gαq signaling. Although RGS2 is known to modulate some GPCR signaling, the specific effects of RGS2 on GPR55-mediated signaling are not fully understood at present. Previously, we reported some RGS proteins interact with protease-activated receptors, one of GPCRs, and modulate their functions. Here, we investigated whether GPR55 interacts with RGS2, employing bioluminescence resonance energy transfer and co-immunoprecipitation analyses. Interestingly, GPR55 interacted with RGS2 alone and also formed a ternary complex with RGS2 and either Gαq or Gα12. In the presence of GPR55 alone and together with Gαq or Gα12, RGS2 translocated from the cytoplasm to plasma membrane while RGS1 remained in the cytoplasm. GPR55 activation significantly induced ERK phosphorylation and intracellular calcium mobilization, which were markedly inhibited by RGS2 in HCT116 colon cancer cell line. Furthermore, GPR55-mediated cell proliferation and migration of HCT116 cells, was significantly attenuated by RGS2. Our collective findings highlight a novel physiological function of RGS2, supporting its utility as a therapeutic target to control GPR55-induced pathophysiology.

Emerging Roles for Regulator of G Protein Signaling 2 in (Patho)physiology.

Since their discovery in the mid-1990s, regulator of G protein signaling (RGS) proteins have emerged as key regulators of signaling through G protein-coupled receptors. Among the over 20 known RGS proteins, RGS2 has received increasing interest as a potential therapeutic drug target with broad clinical implications. RGS2 is a member of the R4 subfamily of RGS proteins and is unique in that it is selective for Gα q Despite only having an RGS domain, responsible for the canonical GTPase activating protein activity, RGS2 can regulate additional processes, such as protein synthesis and adenylate cyclase activity, through protein-protein interactions. Here we provide an update of the current knowledge of RGS2 function as it relates to molecular mechanisms of regulation as well as its potential role in regulating a number of physiologic systems and pathologies, including cardiovascular disease and central nervous system disorders, as well as various forms of cancer. SIGNIFICANCE STATEMENT: Regulator of G protein signaling (RGS) proteins represent an exciting class of novel drug targets. RGS2, in particular, could have broad clinical importance. As more details are emerging on the regulation of RGS2 in various physiological systems, the potential utility of this small protein in therapeutic development is increasing.

Regulators of G-protein signaling, RGS2 and RGS4, inhibit protease-activated receptor 4-mediated signaling by forming a complex with the receptor and G\u03b1 in live cells

BackgroundProtease-activated receptor 4 (PAR4) is a seven transmembrane G-protein coupled receptor (GPCR) activated by endogenous proteases, such as thrombin. PAR4 is involved in various pathophysiologies including cancer, inflammation, pain, and thrombosis. Although regulators of G-protein signaling (RGS) are known to modulate GPCR/Gα-mediated pathways, their specific effects on PAR4 are not fully understood at present. We previously reported that RGS proteins attenuate PAR1- and PAR2-mediated signaling through interactions with these receptors in conjunction with distinct Gα subunits.MethodsWe employed a bioluminescence resonance energy transfer technique and confocal microscopy to examine potential interactions among PAR4, RGS, and Gα subunits. The inhibitory effects of RGS proteins on PAR4-mediated downstream signaling and cancer progression were additionally investigated by using several assays including ERK phosphorylation, calcium mobilization, RhoA activity, cancer cell proliferation, and related gene expression.ResultsIn live cells, RGS2 interacts with PAR4 in the presence of Gαq while RGS4 binding to PAR4 occurs in the presence of Gαq and Gα12/13. Co-expression of PAR4 and Gαq induced a shift in the subcellular localization of RGS2 and RGS4 from the cytoplasm to plasma membrane. Combined PAR4 and Gα12/13 expression additionally promoted translocation of RGS4 from the cytoplasm to the membrane. Both RGS2 and RGS4 abolished PAR4-activated ERK phosphorylation, calcium mobilization and RhoA activity, as well as PAR4-mediated colon cancer cell proliferation and related gene expression.ConclusionsRGS2 and RGS4 forms ternary complex with PAR4 in Gα-dependent manner and inhibits its downstream signaling. Our findings support a novel physiological function of RGS2 and RGS4 as inhibitors of PAR4-mediated signaling through selective PAR4/RGS/Gα coupling.EjpkaY-cVsUCvUePDRpq7pVideo

Loss-of-Function Mutations in Human Regulator of G Protein Signaling RGS2 Differentially Regulate Pharmacological Reactivity of Resistance Vasculature.

Regulator of G protein signaling 2 (RGS2) plays a role in reducing vascular contraction and promoting relaxation due to its GTPase accelerating protein activity toward Gαq. Previously, we identified four human loss-of-function (LOF) mutations in RGS2 (Q2L, D40Y, R44H, and R188H). This study aimed to investigate whether those RGS2 LOF mutations disrupt the ability of RGS2 to regulate vascular reactivity. Isolated mesenteric arteries (MAs) from RGS2-/- mice showed an elevated contractile response to 5 nM angiotensin II and a loss of acetylcholine (ACh)-mediated vasodilation. Reintroduction of a wild-type (WT) RGS2-GFP plasmid into RGS2-/- MAs suppressed the vasoconstrictor response to angiotensin II. RGS2 LOF mutants failed to suppress the angiotensin II constriction response compared with RGS2 WT. In contrast, ACh-mediated vasoconstriction was restored by expression of RGS2 WT, D40Y, and R44H but not by RGS2 Q2L or R188H. Phosphorylation of RGS2 D40Y and R44H by protein kinase G (PKG) may explain their maintained function to support relaxation in MAs. This is supported by phosphomimetic mutants and suppression of vasorelaxation mediated by RGS2 D40Y by a PKG inhibitor. These results demonstrate that RGS2 attenuates vasoconstriction in MAs and that RGS2 LOF mutations cannot carry out this effect. Among them, the Q2L and R188H mutants supported less relaxation to ACh, whereas relaxation mediated by the D40Y and R44H mutant proteins was equal to that with WT protein. Phosphorylation of RGS2 by PKG appears to contribute to this vasorelaxation. These results provide insights for precision medicine targeting the rare individuals carrying these RGS2 mutations. SIGNIFICANCE STATEMENT: Regulator of G protein signaling 2 (RGS2) has been implicated in the control of blood pressure; rare mutations in the RGS2 gene have been identified in large-scale human gene sequencing studies. Four human mutations in RGS2 that cause loss of function (LOF) in cell-based assays were examined in isolated mouse arteries for effects on both vasoconstriction and vasodilation. All mutants showed the expected LOF effects in suppressing vasoconstriction. Surprisingly, the D40Y and R44H mutant RGS2 showed normal control of vasodilation. We propose that this is due to rescue of the mislocalization phenotype of these two mutants by nitric oxide-mediated/protein kinase G-dependent phosphorylation. These mechanisms may guide drug discovery or drug repurposing efforts for hypertension by enhancing RGS2 function.

RGS2 Modulation of Angiotensin II Receptor Type 1 Trafficking and Signaling in Neuroblastoma Cells

IntroductionAltered expression and function of brain angiotensin II receptor type 1 (AT1R) has been implicated in the pathophysiology of various neuropsychiatric diseases such as drug addiction and Alzheimer's disease. However, there is a knowledge gap in understanding the molecular mechanisms of altered AT1R trafficking and function. Regulators of G‐protein signaling (RGS) proteins are negative modulators of G‐protein coupled receptors (GPCRs). Mechanistically, RGS proteins terminate signaling by accelerating the hydrolysis of GTP bound to active G‐alpha subunits. Among all the RGS subtypes, our lab found that RGS2 co‐localizes with AT1R in the brain, suggesting a potential role of RGS2 regulation of AT1R signaling. Additionally, our lab previously found that RGS2 regulates the constitutive and agonist‐induced trafficking of dopamine D2 receptors. Therefore, this project assessed the role of RGS2 in regulation of AT1R trafficking and signaling using a neuroblastoma 2a (N2A) cell model. Knowledge learned from this project may be relevant to the physiological function of RGS2 in the brain.MethodsWe generated stable expression of the human Angiotensin Type I receptor (HA‐AT1R) in N2A cells. N2A‐AT1R cells were then transiently transfected with RGS2 siRNA to knock down RGS2 expression. Cells were then treated with vehicle or angiotensin II (AngII, 10 μM) for 2, 5, or 15 minutes for western blotting of phosphorylated Akt and total Akt. Immunocytochemistry and confocal microscopy were performed on cells treated with AngII for various times to measure the trafficking of AT1R.ResultsWe found that the knockdown of RGS2 enhanced AT1R‐mediated signaling by increasing Akt phosphorylation. RGS2 knockdown also showed to increase AngII‐induced internalization of AT1R. This is the first report on the role of RGS2 in regulation of AT1R trafficking. Further study is necessary to investigate the molecular mechanisms underlying RGS2 modulation of the signaling and trafficking of AT1R in neuronal cells.Support or Funding InformationThis work is supported by NIH R01DA042862, P50DA006634, F31AA025532This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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.

Inhibition of the Binding between RGS2 and β-Tubulin Interferes with Spindle Formation and Chromosome Segregation during Mouse Oocyte Maturation In Vitro.

RGS2 is a negative regulator of G protein signaling that contains a GTPase-activating domain and a β-tubulin binding region. This study aimed to determine the localization and function of RGS2 during mouse oocyte maturation in vitro. Immunofluorescent staining revealed that RGS2 was widely expressed in the cytoplasm with a greater abundance on both meiotic spindles and first/second polar bodies from the fully-grown germinal vesicle (GV) stage to the MII stages. Co-expression of RGS2 and β-tubulin could also be detected in the spindle and polar body of mouse oocytes at the MI, AI, and MII stages. Inhibition of the binding site between RGS2 and β-tubulin was accomplished by injecting anti-RGS2 antibody into GV-stage oocytes, which could result in oocytes arrest at the MI or AI stage during in vitro maturation, but it did not affect germinal vesicle breakdown. Moreover, injecting anti-RGS2 antibody into oocytes resulted in a significant reduction in the rate of first polar body extrusion and abnormal spindle formation. Additionally, levels of phosphorylated MEK1/2 were significantly reduced in anti-RGS2 antibody injected oocytes compared with control oocytes. These findings suggest that RGS2 might play a critical role in mouse oocyte meiotic maturation by affecting β-tubulin polymerization and chromosome segregation.

Regulator of G Protein Signaling 2: A Versatile Regulator of Vascular Function.

Regulators of G protein signaling (RGS) proteins of the B/R4 family are widely expressed in the cardiovascular system where their role in fine-tuning G protein signaling is critical to maintaining homeostasis. Among members of this family, RGS2 and RGS5 have been shown to play key roles in cardiac and smooth muscle function by tightly regulating signaling pathways that are activated through Gq/11 and Gi/o classes of heterotrimeric G proteins. This chapter reviews accumulating evidence supporting a key role for RGS2 in vascular function and the implication of changes in RGS2 function and/or expression in the pathogenesis of blood pressure disorders, particularly hypertension. With such understanding, RGS2 and the signaling pathways it controls may emerge as novel targets for developing next-generation antihypertensive drugs/agents.

Abstract 640: RGS2 Deficiency Impairs Renal Hemodynamics and Function

Regulator of G protein signaling 2 (RGS2) regulates signaling by acting as a GTPase-activating protein for Gq/11α and Gi/oα subunits of heterotrimeric G proteins. Certain single nucleotide polymorphisms and loss-of-function mutations of RGS2 have been linked to human hypertension. RGS2 deficiency in the kidney is sufficient to cause hypertension in mice; however, the renal pathophysiological mechanisms are unknown. Here, we assessed renal hemodynamics and function using renal blood flow (RBF) and glomerular filtration rate (GFR) measurements, and assessment of pressure natriuresis in wild type (WT) and RGS2 null (RGS2-/-) mice. GFR was measured by plasma clearance of FITC-inulin. Pressure natriuresis was determined by increasing renal perfusion pressure (RPP) stepwise by clamping the superior mesenteric and celiac arteries, and abdominal aorta, while total RBF was simultaneously recorded with flow probe. Baseline GFR was markedly decreased in RGS2-/- mice compared to WT controls (1.8 ± 0.1 vs. 1.1 ± 0.1 ml/min/g kidney weight, p=0.004). Renal conductance was reduced (70 ± 9 vs. 51 ± 3 μl/min/g kidney weight, p=0.006) while renal vascular resistance (RVR; 14.9 ± 2.2 vs. 20.2 ± 1.3 mmHg/ml/min/g kidney weight, p=0.002) was elevated in RGS2-/- compared to WT mice. The absence of RGS2 resulted in decreased sensitivity and magnitude of change in RVR (WT: 11.17 ± 1.94 vs. RGS2-/-: 6.87 ± 0.90 mmHg/ml/min/g) and conductance (WT: -25.69 ± 3.22 vs. RGS2-/-: -13.87 ± 2.40 ml/min/mmHg/g) after a step increase in RPP. The acute pressure-natriuresis curve shifted rightward in RGS2-/- relative to WT, with no change in the slope. Fractional sodium excretion was unaffected by RGS2 deficiency. We conclude that RGS2 deficiency impairs renal function and autoregulation by increasing renal vascular resistance and reducing effective renal blood flow. The findings provide a new line of evidence for renal vascular dysfunction as a primary cause of hypertension. Mutations or downregulation of RGS2 in the kidney may contribute to human hypertension by causing renal hypoperfusion and decreased GFR. Signaling pathways regulated by RGS2 may provide therapeutic targets in hypertension patients harboring mutations that potentially decrease expression and/or function of RGS2.

Ischemia induces regulator of G protein signaling 2 (RGS2) protein upregulation and enhances apoptosis in astrocytes

Regulator of G protein signaling (RGS) family members, such as RGS2, interact with Galpha subunits of heterotrimeric G proteins, accelerating the rate of GTP hydrolysis and attenuating the intracellular signaling triggered by the G protein-coupled receptor-ligand interaction. They are also reported to regulate G protein-effector interactions and form multiprotein signaling complexes. Ischemic stress-induced changes in RGS2 expression have been described in astrocytes, and these changes are associated with intracellular signaling cascades, suggesting that RGS2 upregulation may be an important mechanism by which astrocytes may regulate RGS2 function in response to physiological stress. However, information on the functional roles of stress-induced modulation of RGS2 protein expression in astrocyte function is limited. We report the role of ischemic stress in RGS2 protein expression in rat C6 astrocytoma cells and primary mouse astrocytes. A marked increase in RGS2 occurred after ischemic stress induced by chemicals (sodium azide and 2-deoxyglucose) or oxygen-glucose deprivation (OGD, real ischemia). RGS2 mRNA expression was markedly enhanced by 1 h of exposure to chemical ischemia or 6 h of OGD followed by 2 or 6 h of recovery, respectively. This enhanced expression in primary astrocytes and C6 cells was restored to baseline levels after 12 h of recovery from chemically induced ischemic stress or 4-6 h of recovery from OGD. RGS2 protein was also significantly expressed at 12-24 h of recovery from ischemic insult. Ischemia-induced RGS2 upregulation was associated with enhanced apoptosis. It significantly increased annexin V-positive cells, cleaved caspase-3, and enhanced DNA ladder formation and cell cycle arrest. However, a small interfering RNA (siRNA)-mediated RGS2 knockdown reversed the apoptotic cell death associated with ischemia-induced RGS2 upregulation. Upregulated RGS2 was significantly inhibited by SB-203580, a p38 MAPK inhibitor. Rottlerin, a potent inhibitor of PKCdelta, completely abrogated the increased RGS2 expression. We also examine whether ischemia-induced RGS2-mediated apoptosis is affected by siRNA-targeted endogenous PKCdelta downregulation or its phosphorylation. Although RGS2 upregulation was not affected, siRNA transfection significantly suppressed endogenous PKCdelta mRNA and protein expressions. Ischemia-induced PKCdelta phosphorylation and caspase-3 cleavage were dose dependently inhibited by PKCdelta knockdown, and this endogenous PKCdelta suppression reversed ischemia-induced annexin V-positive cells. This study suggests that ischemic stress increases RGS2 expression and that this condition contributes to enhanced apoptosis in C6 cells and primary astrocytes. The signaling it follows may involve PKCdelta and p38 MAPK pathways.

Abstract 5785: A Novel Function of Regulator of G-protein Signaling 2: Stabilization of Signal Transducer and Activator of Transcription 3 Phosphorylation

Background: Regulator of G protein signaling proteins (RGS) are known to regulate G protein coupled receptor (GPCR) signaling at the plasma membrane through their GTPase-activating protein activity. We have previously identified RGS2 as a downstream target of unphosphorylated Signal Transducer and Activator of Transcription 3 (U-STAT3). U-STAT3 mediated Angiotensin II (AngII) type 1 receptor (AT1R), a GPCR, induced cardiac hypertrophy but the mechanism is unclear. Hypothesis: RGS2 is the mediator of U-STAT3 regulated Ang II/AT1R signaling. Methods and Results: In AT1R overexpressing HEK293 cells and HL-1 cells (a mouse cardiomyocyte cell line), Ang II stimulation induced RGS2 expression in parallel with U-STAT3. The localization, distribution and expression of RGS2 were changed upon Ang II stimulation. RGS2 was mainly localized in the nucleus, which was also confirmed in human vascular smooth muscle cells. Gene transfection of RGS2 dose-dependently increased phosphorylated STAT3 (P-STAT3), and maintained a high level of P-STAT3 up to 24 hrs. Co-immunoprecipitation assay identified a complex formed between RGS2 and P-STAT3, suggesting that RGS2 plays a role in maintaining P-STAT3 stability. Treatment with AT1R antagonist, losartan, decreased RGS2/P-STAT3 interaction, suggesting that this interaction is dependent on AT1R activation. On the other hand, in cells overexpressing AT1R and Y705F mutant STAT3, which cannot be phosphorylated on residue 705 (U-STAT3), chromatin immunoprecipitation assay demonstrated that Y705F mutant STAT3 binds to a consensus GAS motif located in the RGS2 promoter, providing direct evidence that RGS2 is a downstream target of U-STAT3. siRNA-mediated gene knockdown of STAT3 consistently caused decreased RGS2 expression in AT1R overexpressing cells. Conclusions: Chronic activation of AT1R induced U-STAT3 overexpression and subsequently induced overexpression of RGS2, which interacted with and stabilized P-STAT3. P-STAT3 further increases the transcription of stat3 gene, forming a positive feedback loop. We reported for the first time a novel function of RGS2, stabilization of P-STAT3, in mediating U-STAT3 regulated Ang II/AT1R signaling. Further studies are now underway utilizing engineered mice model. This research has received full or partial funding support from the American Heart Association, Great Rivers Affiliate (Delaware, Kentucky, Ohio, Pennsylvania & West Virginia).

Translational control by RGS2

The regulator of G protein signaling (RGS) proteins are a family of guanosine triphosphatase (GTPase)-accelerating proteins. We have discovered a novel function for RGS2 in the control of protein synthesis. RGS2 was found to bind to eIF2Bepsilon (eukaryotic initiation factor 2B epsilon subunit) and inhibit the translation of messenger RNA (mRNA) into new protein. This effect was not observed for other RGS proteins tested. This novel function of RGS2 is distinct from its ability to regulate G protein-mediated signals and maps to a stretch of 37 amino acid residues within its conserved RGS domain. Moreover, RGS2 was capable of interfering with the eIF2-eIF2B GTPase cycle, which is a requisite step for the initiation of mRNA translation. Collectively, this study has identified a novel role for RGS2 in the control of protein synthesis that is independent of its established RGS domain function.

Loss of Gs Activity in Platelets from Carriers with a Heterozygous Missense Mutation in the Regulator of G Protein Signaling 2 (RGS2)

Regulator of G protein signaling (RGS) proteins stimulate the GTPase activity of Gα subunits of heterotrimeric G proteins, thereby negatively regulating G protein signaling. In this way, RGS2 acts as a negative regulator of Gq and Gi signaling. It has also been described as a negative regulator of Gs signaling, but via a different mechanism. It inhibits the activation of adenylyl cyclase (AC), the target molecule of Gs, by interacting with it. In olfactory neurons, it was shown that RGS2 attenuates activation of AC type III (Sinnarajah et al., Nature 2001), the main AC subtype in platelets. In this study, we describe the first human genetic defect in RGS2 and provide evidence that RGS2 influences the cAMP level in platelets after Gs stimulation. The proposita is an obese 16-year-old girl with borderline IQ, hirsutism and an increased bone alkaline phosphatase. These symptoms are similar to features of Albright hereditary osteodystrophy, due to heterozygous inactivating mutations in the Gsα gene. The Gsα gene of the proposita is normal, but she carries a missense mutation in the RGS2 gene, resulting in a Gly to Asp substitution in the conserved residue 23 (G23D). This substitution could also be found in her mother and brother, but not in 200 unrelated normal controls. The family members carrying this mutation present with a relatively low number of platelets (+/−150.000/μL) and an increased mean platelet volume (+/−13 fL) and platelet distribution width (+/−17.5 %). Also, platelet function is affected by the mutation. Platelet aggregation is normal in response to all standard agonists, but when the Gs pathway is challenged in their platelets, high levels of different Gs agonists are needed to get inhibition of aggregation in comparison to controls or the father. We also measured cAMP levels in platelets and found that stimulation of the Gs coupled receptors with Gs agonists produced less cAMP in the affected family members. The functional relevance of the mutation was further studied in vitro in HEK293 and MEG-01 cells transfected with wildtype RGS2 and RGS2-G23D. cAMP levels were measured at different time points after stimulation of these cells with Gs agonists. These measurements show that cAMP levels are lower in cells transfected with RGS2-G23D, compared to wildtype RGS2. This indicates that the reduction in cAMP levels found in the platelets of the affected members, is a functional consequence of the mutation. To understand why this mutation leads to an altered function of RGS2, we studied the effect of the mutation at the protein level. Recently, it was shown that there are 4 different translation initiation sites in the RGS2 mRNA, giving rise to 4 proteins with different functional characteristics (Gu et al., Mol Pharmacol 2008). An in vitro transcriptiontranslation assay showed that the presence of the mutation results in a different protein expression profile. We excluded a difference in posttranslational modifications to be the cause of this divergent pattern. The G23D mutation is located in the proximity of 2 of the different translation initiation sites and its presence alters the use of these sites. This results in a different expression profile of the functionally different RGS2 proteins. In conclusion, we present the first platelet Gs signaling defect due to an RGS2 mutation associated with aberrant RGS2 translation.

Influence of RGS2 on Anxiety-Related Temperament, Personality, and Brain Function

Although anxiety disorders are heritable, their genetic and phenotypic complexity has made the identification of susceptibility genes difficult. Well-validated animal models and intermediate phenotypes provide crucial tools for genetic dissection of anxiety. The gene encoding regulator of G protein signaling 2 (Rgs2) is a quantitative trait gene that influences mouse anxiety behavior, making its human ortholog (RGS2) a compelling candidate gene for human anxiety phenotypes. To examine whether variation in RGS2 is associated with intermediate phenotypes for human anxiety disorders. Family-based and case-control association analysis of single-nucleotide polymorphisms at the RGS2 locus in 3 independent samples. Massachusetts General Hospital, University of California, San Diego, and San Diego State University. Study participants included a family-based sample (n = 119 families) of children who underwent laboratory-based assessments of temperament (behavioral inhibition), a sample of 744 unrelated adults who completed assessments of extraversion and introversion, and 55 unrelated adults who underwent functional magnetic resonance imaging measures of response to emotional faces. Laboratory-based behavioral measures of childhood temperament, self-report measure of personality, and functional magnetic resonance imaging response to emotion processing. Markers spanning RGS2 were associated with childhood behavioral inhibition, a temperamental precursor of social anxiety disorder (haplotype P = 3 x 10(-5); odds ratio, 2.99 in complete trios). In independent samples, RGS2 markers, including rs4606, which has previously been associated with RGS2 expression, were also associated with introversion (a core personality trait in social anxiety disorder) and with increased limbic activation (insular cortex and amygdala) during emotion processing (brain phenotypes correlated with social anxiety). The genotype at rs4606 explained 10% to 15% of the variance in amygdala and insular cortex activation to emotional faces. These results provide the first evidence that a gene that influences anxiety in mice is associated with intermediate phenotypes for human anxiety disorders across multiple levels of assessment, including childhood temperament, adult personality, and brain function. This translational research suggests that some genetic influences on anxiety are evolutionarily conserved and that pharmacologic modulation of RGS2 function may provide a novel therapeutic approach for anxiety disorders.

The RGS2 Gene Product from a Candidate Hypertension Allele Shows Decreased Plasma Membrane Association and Inhibition of Gq

Hypertension is a leading risk factor for the development of cardiovascular disease. Data from human and animal studies suggest that RGS2, a potent inhibitor of G(q) signaling, is important for blood pressure regulation. Several RGS2 mutations in the Japanese population have been found to be associated with hypertension. The product of one of these alleles, R44H, is mutated within the amino terminal amphipathic alpha-helix domain, the region responsible for plasma membrane-targeting. The functional consequence of this mutation and its potential link to the development of hypertension, however, are not known. In this study, we showed that R44H was a weaker inhibitor of receptor-mediated G(q) signaling than wild-type RGS2. Confocal microscopy revealed that YFP-tagged R44H bound to the plasma membrane less efficiently than wild-type RGS2. R44 is one of the basic residues positioned to stabilize lipid bilayer interaction of the RGS2 amphipathic helix domain. Tryptophan fluorescence and circular dichroism studies of this domain showed that the R44H mutation prevented proper entrenchment of hydrophobic residues into the lipid bilayer without disrupting helix-forming capacity. Together, these data suggest that decreasing the side-chain length and flexibility at R44 prevented proper lipid bilayer association and function of RGS2. Finally, the R44H protein did not behave as a dominant-negative interfering mutant. Thus, our data are consistent with the notion that a R44H missense mutation in human RGS2 produces a hypomorphic allele that may lead to altered receptor-mediated G(q) inhibition and contribute to the development of hypertension in affected subjects.

RGS2 Determines Short-Term Synaptic Plasticity in Hippocampal Neurons by Regulating Gi/o- Mediated Inhibition of Presynaptic Ca2+ Channels

RGS2, one of the small members of the regulator of G protein signaling (RGS) family, is highly expressed in brain and regulates G(i/o) as well as G(q)-coupled receptor pathways. RGS2 modulates anxiety, aggression, and blood pressure in mice, suggesting that RGS2 regulates synaptic circuits underlying animal physiology and behavior. How RGS2 in brain influences synaptic activity is unknown. We therefore analyzed the synaptic function of RGS2 in hippocampal neurons by comparing electrophysiological recordings from RGS2 knockout and wild-type mice. Our study provides a general mechanism of the action of the RGS family containing RGS2 by demonstrating that RGS2 increases synaptic vesicle release by downregulating the G(i/o)-mediated presynaptic Ca(2+) channel inhibition and therefore provides an explanation of how regulation of RGS2 expression can modulate the function of neuronal circuits underlying behavior.

Tonic plasma membrane association increases the function of RGS proteins as inhibitors of M1 muscarinic receptor signaling

RGS2 is a more potent attenuator of M1 muscarinic receptor signaling than other small RGS proteins. To explain differences, the function of RGS2 and RGS5 were compared in HEK293 cells stably expressing the M1 muscarinic receptor. Results show that the carboxyl terminal GAP domains of RGS2 and RGS5 show comparable inhibitory function. Plasma membrane-targeted RGS GAP domains show enhanced function relative to non-targeted proteins suggesting that membrane association is a determinant of M1 muscarinic receptor inhibition. Amino terminal domain swaps of RGS2 and RGS5 reveal that the RGS2 amino terminus is sufficient to confer increased plasma membrane association and function. Mutation of a single hydrophobic residue in the amino terminal amphipathic helix of RGS2 and the RGS2/5 chimera reduce their plasma membrane association and inhibitory function without disrupting interaction with the third intracellular loop of the M1 muscarinic receptor. Replacement of two unique leucine residues adjacent to the amphipathic helix of RGS2 with the corresponding residues from RGS5 disrupts membrane localization and function. Together, these data suggest that the amphipathic alpha helix in RGS2 displays an elongated hydrophobic surface that facilitates its increased tonic plasma membrane targeting and greater inhibition of M1 muscarinic signaling relative to RGS5.

Developmentally regulated expression of the regulator of G-protein signaling gene 2 ( Rgs2) in the embryonic mouse pituitary

During the development of the anterior pituitary gland, five distinct hormone-producing cell types emerge in a spatially and temporally regulated pattern from an invagination of oral ectoderm termed Rathke's Pouch. Evidence from mouse knockout and ectopic expression studies indicates that 12.5 days post coitum (dpc) to 14.5 dpc is a critical period for the expansion of the progenitor cell pool and the determination of most hormone-secreting cell types. While signaling proteins and transcription factors have been identified as having key roles in pituitary cell differentiation, little is known about the identity and function of proteins that mediate signal transduction in progenitor cells. To identify genes that are enriched in the embryonic pituitary gland, we compared gene expression in 14.5 dpc pituitary and 14.5 dpc embryo minus pituitary tissues using the NIA 15K microarray. Analysis of the data using the R program revealed that the Regulator of G Protein Signaling 2 ( Rgs2) gene was 3.9-fold more abundant in the 14.5 dpc pituitary. In situ hybridisation confirmed this finding, and showed that Rgs2 expression in midline tissues was restricted to the pituitary and discrete regions of the nervous system. Within the pituitary, Rgs2 was expressed in undifferentiated cells, and was downregulated at the completion of the hormone cell differentiation. To investigate Rgs2 function in the pituitary, we examined hormone cell differentiation in Rgs2 null neonate mice. Pituitary cell differentiation and morphology appeared normal in the Rgs2 mutant animals, suggesting that other Rgs family members with similar activities may be present in the developing pituitary.

RGS2-Mediated Regulation of Gqα

Regulators of G-protein signaling (RGS) proteins are GTPase-activating proteins (GAPs) that attenuate signaling by heterotrimeric G proteins. In RGS2, three unique residues within the G-protein-binding (RGS) domain have been shown to direct its selective inhibition of Gqalpha function. The function of RGS2 as a regulator of Gq is also dependent on regulatory sequences in its amino-terminal domain that direct its localization to the plasma membrane. This work details various approaches that have been used to characterize the relative contribution of the RGS and regulatory domains in RGS2 to its function as a regulator of Gq signaling. Specifically, assays describing (i) the identification of alpha subunit binding partners for RGS2 (ii) the characterization of RGS2-mediated inhibition of Gq-dependent phosphatidylinositol signaling in tissue culture models, and (iii) the measurement of Gq-dependent calcium responses in vascular smooth muscle cells from RGS2-deficient mice are presented. Results from these studies have been used to demonstrate the high relative potency of RGS2 for the regulation of Gq signaling at the biochemical, cellular, and physiologic level.

Identification of RGS2 and Type V Adenylyl Cyclase Interaction Sites

The production of cAMP is controlled on many levels, notably at the level of cAMP synthesis by the enzyme adenylyl cyclase. We have recently identified a new regulator of adenylyl cyclase activity, RGS2, which decreases cAMP accumulation when overexpressed in HEK293 cells and inhibits the in vitro activity of types III, V, and VI adenylyl cyclase. In addition, RGS2 blocking antibodies lead to elevated cAMP levels in olfactory neurons. Here we examine the nature of the interaction between RGS2 and type V adenylyl cyclase. In HEK293 cells expressing type V adenylyl cyclase, RGS2 inhibited Galpha(s)-Q227L- or beta(2)-adrenergic receptor-stimulated cAMP accumulation. Deletion of the N-terminal 19 amino acids of RGS2 abolished its ability to inhibit cAMP accumulation and to bind adenylyl cyclase. Further mutational analysis indicated that neither the C terminus, RGS GAP activity, nor the RGS box domain is required for inhibition of adenylyl cyclase. Alanine scanning of the N-terminal amino acids of RGS2 identified three residues responsible for the inhibitory function of RGS2. Furthermore, we show that RGS2 interacts directly with the C(1) but not the C(2) domain of type V adenylyl cyclase and that the inhibition by RGS2 is independent of inhibition by Galpha(i). These results provide clear evidence for functional effects of RGS2 on adenylyl cyclase activity that adds a new dimension to an intricate signaling network.