RGS12 controls pain/mechanical sensitivity in a sex-dependent manner via regulating the estrogen/GPER-calcium signaling pathway.

The RGS protein controls the magnitude and duration of GPCR signaling by acting as a GTPase activating protein of the G subunit, and this activity is governed by the RGS structural domain.

G protein-coupled receptors (GPCRs) play a pivotal role in maintaining the stemness of both normal and cancer stem cells. However, the function of the regulator of G protein signaling (RGS) family, particularly in tumor stem cells, remains poorly under-stood. Through bioinformatics analysis of clinical data, we identified RGS20 as a potential regulator of glioma stemness and temozolomide (TMZ) resistance, which may significantly influence patient prognosis. Subsequent in vitro and in vivo experiments demonstrated that RGS20 inhibition markedly enhanced tumor sphere formation and upregulated stem cell markers by intrinsically activating the WNT/β-catenin signaling pathway, thereby promoting tumorigenesis and ultimately leading to TMZ resistance. Furthermore, in human glioblastoma specimens, β-catenin signaling associated with low RGS20 expression was significantly enriched in hypoxic regions, suggesting that this mechanism may support the maintenance of glioma stem cells (GSCs) and drive TMZ resistance within the hypoxic niche. Our findings reveal that low RGS20 expression sustains WNT/β-catenin signaling in a ligand-reduced manner within hypoxic niches, unveiling a novel intracellular mechanism that drives glioma progression. Targeting this mechanism could provide new therapeutic strategies for glioma treatment.

Regulators of G protein signaling (RGSs) are key modulators of β-cell function and stress adaptation. Similarly, circadian clock components are intricately implicated in the regulation of insulin secretion and β-cell physiology. However, their responses to sustained cellular stimulation under depolarizing conditions remain incompletely understood. We used MIN6 cells subjected to prolonged potassium chloride (KCl) exposure to induce sustained membrane depolarization, mimicking conditions of chronic β-cell stimulation. We analyzed the expression levels of Rgs and core clock genes, and assessed associated changes in cellular stress and differentiation markers. KCl treatment led to the upregulation of endoplasmic reticulum (ER) stress markers, including Chop and Atf4, without inducing oxidative stress. Expression of Rgs2, Rgs4, and Rgs16 was elevated. RGS2 partially co-localized with eIF2α, suggesting a role in translational control during stress. Furthermore, KCl-induced depolarization was associated with characteristic changes in β-cell differentiation markers and disallowed genes, indicative of a dedifferentiation-like state. Transcript levels of several circadian genes were altered, including significant downregulation of Dbp and upregulation of its repressor E4bp4. Notably, Dec1, a clock gene known to be inducible by various external stimuli, was also upregulated, suggesting broader circadian disruption under depolarizing conditions. Sustained membrane depolarization induces ER stress and transcriptional remodeling in MIN6 β-cells, including the modulation of RGS proteins and key circadian regulators such as DBP, E4BP4, and DEC1. These alterations may contribute to functional impairment and a dedifferentiation-like state of β-cells under chronic stimulatory conditions.

Abstract Background Perioperative myocardial infarction is a deadly complication for a million patients per year worldwide. Cytokines induce atherosclerotic plaque inflammation, thus vulnerability and progression. G-Protein Coupled Receptors (GPCR) and regulators of G-protein signalling, here RGS5, regulate signalling processes contributing to atherosclerosis. Cytokines involved in the inflammatory response to surgery were measured in a model of inducible RGS5 deficiency in atheroprone mice. Purpose To assess the role of RGS5 for the humoral perioperative stress response in vivo. Methods ApoE-/-CreERT2/ERT2RGS5flox/flox mice were fed a high fat diet for 7 weeks. Tamoxifen was administered (2mg ip/d for 5d) to induce RGS5 knockout (KO) before the animals were subjected to a perioperative double-hit (DBH) stress model, combining laparotomy with significant blood loss. Animals were sacrificed at 4- and 72-hours after DBH. RGS5 gene deletion and RNA expression were evaluated by real-time quantitative PCR. Plasma cytokine levels were measured using FACS based multiplex ELISA. Data were analyzed using Wilcoxon test using R in R studio. Results RGS5 DNA copies were reduced to 23% in tamoxifen-induced animals (TaqMan-gDNA-PCR) in bone marrow, leukocytes and spleen (ΔΔCT, mean±SD: 1.03±0.07 vs 0.24±0.12, p<0.001). RGS5-mRNA level in spleen was 45% (ΔΔCT: 1.42±1.24 vs 0.78±0.65, p<0.05) when compared to untreated controls. Plasma levels of IL6 increased significantly at 4h (median (CI95) in pg/mL, 3.7 (2.95-23.93) vs 62.8 (26.01-130.97), p<0.01) and then significantly decreased after 72h (5.5 (2.95-24.08), p<0.01) post-surgery. Similarly, CXCL1 levels significantly increased at 4h (in pg/mL, 54.76 (32.45-75.22) vs 124.9 (97.78-257.19), p<0.01) and then dropped back to baseline levels at 72h (40.9 (28.58-70.16), p<0.01) post DBH. RGS5 KO significantly reduced IL6 at 4h (in pg/mL, 34.3 (13.74-48.76), p<0.05) and CXCL1 at 72h (29.6 (19.73-41.20), p<0.05). Additionally, RGS5 KO significantly reduced the concentration of CCL11 at 72h (in pg/mL, 745.6 (366.9-964.4) vs 530.6 (259.8-675.3), p<0.05) post DBH. Similarly, CCL2 and TNF-α levels were reduced at 72h in Tamoxifen-treated mice. Conclusion RGS5 knockout was efficient with satisfactory penetrance in immune relevant tissues. Elevated post-surgery cytokine levels were significantly reduced by RGS5 KO indicating that lack of RGS5 in immune cells deregulates downstream signaling pathways of GPCRs which hampers cytokine release. RGS5 has a crucial role in immune cell activation and vascular inflammation and further research could deliver potential RGS5-targeted therapeutic strategies to prevent inflammation related atherosclerotic cardiovascular accidents.

Background: Pulmonary arterial hypertension (PAH) is a fatal disease characterized by progressive vascular remodeling, leading to increased pulmonary arterial pressure and right ventricular failure. Our previous studies identified regulator of G-protein signaling 5 ( Rgs5 ) as a key negative regulator of G protein-coupled receptor ( GPCR ) signaling, uniquely expressed in pulmonary pericytes ( PCs ). Rgs5 has been implicated in the development of pulmonary hypertension ( PH ), and its deficiency has been linked to cardiac fibrosis. However, the specific role of Rgs5 in PC function is underinvestigated. Therefore, we hypothesize that Rgs5 deficiency promotes PC differentiation into contractile protein-enriched cells by activating the GPCR pathway, contributing to vascular remodeling during PH development. Methods: To directly examine the role of Rgs5 in PCs during PH development, novel Rgs5 PC-KO ( Higd1b -CreERT2::Rosa26- Rgs5 fl/fl ) transgenic mice were generated and subjected to hypoxia (FiO 2 :10%) for 3 weeks. PH and right ventricle hypertrophy were evaluated by measuring right ventricle systolic pressure (RVSP) and Fulton index (FI). Vascular remodeling in Rgs5 PC-KO and WT were evaluated by immunofluorescence (IF) staining in precision cut lung slices using a high resolution confocal microscope. RGS5 function in human healthy PCs was evaluated by overexpressing RGS5 using the pcDNA3-RGS5 plasmid. Results: Overexpression of RGS5 in healthy PCs resulted in the downgulation of smooth muscle cell-specific contractile protein expression such as SMA, Vimentin, and SM22, indicating RGS5 negatively regulates contractile protein expression in PCs. Additionally, hypoxic Rgs5 PC-KO mice developed severe PH with RVSP reaching up to 38.4 mmHg ( Fig 1B ) and RVH (FI: 34.1%, Fig 1C ). IF analyses revealed that PCs integrated into muscularized vessels with coexpression of SMA, and exhibited abnormal stromal cell outgrowth along distal pulmonary arterioles in Rgs5 PC-KO under normoxic and hypoxic conditions ( Fig 1D ). Conclusions: Loss of Rgs5 in PCs is a key contributor to PC differentiation into contractile protein-enriched cells, abnormal vascular remodeling, and the PH development ( Fig 1A ). These findings provide novel insights into the role of RGS5 in PCs and could serve as a new treatment strategy for PAH.

Preeclampsia is a multifaceted pregnancy-associated hypertensive disorder that poses a major threat to maternal and fetal health. Though the etiology is not fully understood, syncytiotrophoblast stress is postulated to be a major driver of maternal symptomology. We previously demonstrated that regulator of G protein signaling-2 (RGS2) expression decreases in human preeclamptic placenta and has a transcriptional dependence on histone deacetylase 9 (HDAC9) in trophoblast cells. Furthermore, experimental reductions of Rgs2 expression in the mouse fetoplacental unit are sufficient to induce preeclampsia-like features, including placental stress, in C57BL/6J dams. Here, we examined the hypotheses that HDAC9 and RGS2 are both expressed within syncytiotrophoblasts, that HDAC9 and RGS2 expression are positively correlated within these cells, and that expression of each is reduced within syncytiotrophoblasts during preeclampsia. HDAC9 and RGS2 mRNA were localized and quantified in syncytiotrophoblast cells of human placental samples from pregnancies with and without preeclampsia, using laser-capture microdissection and in situ hybridization methods. Expression of Hdac9 and Rgs2 was similarly localized in the syncytiotrophoblast of the mouse placenta. Throughout, HDAC9/Hdac9 and RGS2/Rgs2 were detected and positively correlated in syncytiotrophoblasts, but expression of each was substantially reduced during preeclampsia. These results document reduced HDAC9 and RGS2 expression specifically in syncytiotrophoblast cells during preeclampsia and provide additional correlative support of HDAC9-mediated control of RGS2 expression within this population of trophoblasts. This work provides rationale to further explore cell-specific disruptions in HDAC9 and RGS2 control and function as a cause of syncytiotrophoblast stress and ultimately preeclampsia.NEW & NOTEWORTHY Syncytiotrophoblast stress contributes to the pathogenesis of preeclampsia, but many of the underlying causes remain undetermined. Previous work has implicated the loss of placental HDAC9-mediated Rgs2 transcription in the disorder. Extending these findings, we report that HDAC9 and RGS2 were abundant and localized primarily to syncytiotrophoblast cells of the control placenta. Expression of both targets was attenuated in these cells during preeclampsia and thus may be an underappreciated source of syncytiotrophoblast stress, warranting further investigation.

RGS proteins: Potential regulators of mouse melanopsin's signaling phototransduction cascade across ipRGC subtypes.

ABSTRACT Purpose To report multimodal imaging findings and natural history of clinical features in two probands with bradyopsia harboring homozygous variants (c.895T>C) in Regulator of G-protein Signaling 9 (RGS9). Methods Ophthalmic history, clinical examination, fundus autofluorescence (FAF), optical coherence tomography (OCT), microperimetry, flood-illuminated adaptive optics (AO) imaging, and electroretinogram (ERG) were obtained. Results A 37-year-old male and a 67-year-old female from non-consanguineous parents had normal fundus examination, FAF, and OCT. ERGs for the male case between the age of 4 and 38 years showed no progression. Both probands had flat International Society for Clinical Electrophysiology of Vision (ISCEV) Standard full-field ERG light-adapted (LA) responses but dark-adapted (DA) red x-wave and S-cone responses were present. DA 30 Hz flicker was present after 2 but not after 10 seconds. The reduced amplitude and b:a ratio of the DA10 response improved with increasing interstimulus interval. AO and microperimetry demonstrated preservation of foveal cone density and subnormal retinal sensitivity, respectively. Both measures remained stable over 3 years. The c.895T>C variant was classified as pathogenic. Conclusions Bradyopsia associated with homozygous RGS9 c.895T>C variants is characterized by normal retinal structure but subnormal macular sensitivity. Extended ERG protocols can be used to confirm delayed phototransduction recovery.

RGS1 stabilized by METTL3-mediated m6A modification promotes the tumorigenicity and macrophage M2 polarization in osteosarcoma.

Dissociation of SYNGAP1 Enzymatic and Structural Roles: Intrinsic Excitability and Seizure Susceptibility Brill J, Clarke B, Hong I, Huganir R. Proc Natl Acad Sci USA . 2025;122(18): e2427288122. SYNGAP1 is a key Ras-GAP protein enriched at excitatory synapses, with mutations causing intellectual disability and epilepsy in humans. Recent studies have revealed that in addition to its role as a negative regulator of G-protein signaling through its GAP enzymatic activity, SYNGAP1 plays an important structural role through its interaction with postsynaptic density proteins. Here, we reveal that intrinsic excitability deficits and seizure phenotypes in heterozygous Syngap1 knockout (KO) mice are differentially dependent on Syngap1 GAP activity. Cortical excitatory neurons in heterozygous KO mice displayed reduced intrinsic excitability, including lower input resistance, and increased rheobase, a phenotype recapitulated in GAP-deficient Syngap1 mutants. However, seizure severity and susceptibility to pentylenetetrazol (PTZ)-induced seizures were significantly elevated in heterozygous KO mice but unaffected in GAP-deficient mutants, implicating the structural rather than enzymatic role of Syngap1 in seizure regulation. These findings highlight the complex interplay between SYNGAP1 structural and catalytic functions in neuronal physiology and disease.

Protective role of RGS2 in PM2.5-induced ovarian injury in rats: Modulation of Gq/11 signaling to maintain granulosa cell [Ca2 + ]i stability.

Developing inhibitors of the guanosine triphosphate hydrolysis accelerating activity of Regulator of G protein Signaling-14

Selected proteins containing an N-terminal cysteine (Nt-Cys) are subjected to rapid, O2-dependent proteolysis via the Cys/Arg-branch of the N-degron pathway. Cysteine dioxygenation is catalyzed in mammalian cells by 2-aminoethanethiol dioxygenase (ADO), an enzyme that manifests extreme O2 sensitivity. The canonical substrates of this pathway in mammalia are the regulators of G-protein signaling 4, 5, and 16, as well as interleukin-32. In addition to operating as an O2-sensing mechanism, this pathway has previously been described as a sensor of nitric oxide (NO), with robust effects on substrate stability upon modulation of NO bioavailability being widely demonstrated. Despite this, no mechanism to describe the action of NO on the Cys/Arg N-degron pathway has yet been substantiated. We demonstrate that NO can regulate the stability of Cys N-degron substrates indirectly via the regulation of ADO cosubstrate availability. Through competitive, O2-dependent inhibition of cytochrome C oxidase, NO can substantially modify cellular O2 consumption rate and, in doing so, alter the availability of O2 for Nt-Cys dioxygenation. We show that this increase in O2 availability in response to NO exposure is sufficient to alter both dynamic and steady-state ADO substrate levels. It is likely that this mechanism operates to couple O2 supply and mitochondrial respiration with responses to G-protein-coupled receptor stimulation.

Regulator of G protein Signaling-14 (RGS14), an intracellular inactivator of G protein-coupled receptor (GPCR) signaling, is considered an undruggable protein given its shallow and relatively featureless protein-protein interaction interface combined with a distal allosteric site prone to nonspecific inhibition by thiol-reactive compounds. Here, we identify and validate a tractable chemotype that selectively and non-covalently inhibits RGS14 GTPase-accelerating protein (GAP) activity. Combining structure-guided virtual screening, ligand docking across multiple receptor conformers, and enrichment validation, we progressed from a first-generation active, Z90276197, to over 40 second-generation analogs with improved potency. These inhibitors are predicted to engage the solvent-exposed "canyon" in the RGS14 RGS-box that interacts with the Gα switch I region. Binding pose predictions underscored the importance of non-polar interactions and shape complementarity over polar interactions in engaging this Gα-binding canyon and revealed an "ambidextrous" pattern of R1- and R2-group orientations. GAP inhibition was confirmed in fluorescence-based and gold-standard radioactive GTP hydrolysis assays. Two second-generation analogs, Z55660043 and Z55627844, inhibited RGS14 GAP activity in both assays and without measurable cytotoxicity. Deep learning-based scoring of predicted docking poses further supported observed affinity gains from R3-group additions. One analog demonstrated favorable in vivo pharmacokinetics and CNS penetration. Collectively, our findings establish tractable, non-covalent, small molecule inhibition of a G protein regulatory interface and illustrate how machine learning-enhanced docking can guide ligand optimization for shallow protein surfaces. This work opens the door to future development of RGS14 inhibitors as potential therapeutics for central nervous system and metabolic disorders.

A new angle on RGS protein modulation.

Introduction: Inflammatory diseases like rheumatoid arthritis, inflammatory bowel disease, metabolic syndrome, often coincide with cardiac autonomic neuropathies (CAN). CAN is characterized by attenuated heart rate variability and baroreflex sensitivity and linked to a higher risk for cardiovascular disease and mortality. Despite an attenuated vagal tone, cholinergic hyperreactivity (CHR) has been described during CAN. Here we aim to determine the mechanism of inflammation induced CHR and its link to atrial arrhythmia. Methods: Inflammation was induced in mice through dextran sulfate sodium (DSS: 3.5%, 7 days (d)) supplemented drinking water or treatment with IL-17 (250 ng/d, 7d). DSS induces active colitis and increased serum levels of IL-17 accompany the acute inflammatory phase. Cardiac electrophysiological properties were quantified in vivo (ECGs), in the isolated heart (epicardial mapping), and on the single cell level (voltage clamp) under basal condition and during carbachol (CCh) stimulation. Expression of proteins linked to CHR were quantified by Western Blotting. Results: In vivo (0.15mg/kg i.p.) and in the isolated heart (100nM) CCh attenuated the heart rate more significantly in DSS-treated than CTL animals (in vivo: CTL: -11.44±1.63%, n=10; DSS: -39.76±3.06%, n=14; p<0.0001) (ex vivo: CTL: -28.71±2.97%, n=16; DSS: -39.39±2.67%, n=15; p=0.013). CHR did not depend on increased expression of the muscarinic acetylcholine receptor 2 (M2AChR; CTL: 0.94±0.02, n=4; DSS: 0.72±0.09, n=4: p=0.0435) or the G-protein-gated K + channel (GIRK1/4) or I K,ACh current. However, termination of CCh signaling was delayed in DSS in vivo (0.5mg/kg Atropine i.p.; CTL: 107.93±12.99 Dbpm/ms, n=6; DSS: 66.80±7.33 Dbpm/ms, n=5; p=0.026) and ex vivo (1µM; CTL: 15.23±0.65Dbpm/ms, n=3; DSS: 11.24±0.82 Dbpm/ms, n=3; p=0.019). The latter was consistent with downregulation of regulator of G-protein signaling 7 (CTL: 1.28±0.09, n=8; DSS: 0.76±0.16, n=8; p=0.012). Atrial RNAseq and pathway analysis revealed upregulation of atrial IL-17 signaling and CHR was mimicked in IL-17 treated mice (in vivo, CCh: IL-17: -42.0±4%, n=4). Conclusion: The data suggest that colitis induced inflammation promotes CHR by attenuating the termination of M2AChR activity through IL-17 induced atrial remodeling.

Post-translational modifications of RGS protein family: Pivotal switches in cellular signaling transduction and diseases.

Introduction and Objective: Recent advances in single-cell technologies have uncovered intricate cellular heterogeneity, yet its biological implications remain unclear. Key questions include whether functional heterogeneity exists and its impact on tissue activity. Brown and beige adipocytes (BAs) are thermogenic cells critical for energy homeostasis, and a decline in thermogenic activity is linked to age-related metabolic disorders. We previously identified a BA subpopulation with low uncoupling protein 1 (UCP1) expression (BA-L) that coexists with the classical high UCP1-expressing BAs (BA-H). Methods: Utilizing multiple lineage-tracing and gene knockout mouse models, single-cell RNA sequencing, and tissue imaging. Results: Here, we report that BA-Ls have spatial proximity to interleukin-1 beta (IL-1β) positive cells, as IL-1β is “cooling down” the thermogenesis of surrounding BAs, stabilizing these cells in the low-thermogenic states. Specifically, IL-1β stabilizes hexose-6-phosphate dehydrogenase (H6PD) protein from degradation, which promotes glucocorticoid receptor (GR) signaling to suppress UCP1 expression via GR target regulator of G-protein signaling 2 (RGS2). Cold exposure reduces IL-1β levels, depending on the sympathetic innervation, leading to H6PD degradation, and thus converting BA-Ls into BA-Hs to enhance thermogenesis. Rising environmental temperatures trigger the opposite events. BA-specific, inducible deletion of GR during cold exposure prevents the switch of BA-Hs to BA-Ls when the environmental temperature rises, leading to persistent thermogenesis. Critically, IL-1β level is elevated in aged BAT, leading to low BA-H/BA-L ratio and cold intolerance. Suppressing GR in aged mice restores the BA-H/BA-L ratio and increases thermogenesis. Conclusion: Our findings unveil a spatiotemporal regulation of BA heterogeneity governing thermogenesis that integrates neural and immune signals and a fundamental mechanism of thermogenic fat tissue aging. Disclosure A. Song: None. Y. Wang: None. G. Wang: None. J. Yu: None. L. Jiang: None. Y. Zhu: None. Q. Wang: None. Funding National Institutes of Health (R01AG063854); National Institutes of Health (R01HD096152); National Institutes of Health (R01DK128907); California Institute for Regenerative Medicine (DISC0-15689); American Diabetes Association Junior Faculty Development Award (1-19-JDF-023)

Introduction and Objective: Beta-cell function and mass are under control of G protein-coupled receptors (GPCRs) which are themselves subject to intracellular regulation by regulator of G protein signaling (RGS) proteins. Although the importance of GPCRs to beta-cell biology is well documented, the role of RGS proteins is largely unknown. Recent evidence suggests that RGS9 may have a role in the control of beta-cell function. The aim of this study was to better understand how RGS9 regulates insulin secretion. Methods: Quantitative (q) PCR and RNA in situ hybridization were performed on isolated male mouse islets and pancreatic sections, respectively. Male mouse pseudoislets were infected with adenoviruses encoding short-hairpin (sh) RNAs against RGS9 or control viruses. Glucose-stimulated insulin secretion (GSIS) was assessed in 1h-static incubations and is expressed as mean of the percent of insulin content ± SEM. Significance was tested using a two-way ANOVA with post hoc adjustment for multiple comparisons. Results: RGS9 transcripts were detected in beta, alpha and delta cells in mouse pancreatic sections and both RGS9-1 and -2 isoforms were detected in whole islet extracts. RGS9 knockdown with two distinct shRNAs decreased KCl-stimulated insulin secretion (Control: 4.9±1.0 versus shRNA1: 1.3±0.3 p<0.0001 and shRNA2: 2.8±0.4 p<0.05, n=5-6). Surprisingly, in RGS9 knockout (RGS9Δexon2-4) islets GSIS was not affected (wild-type: 0.8±0.1 versus mutant: 0.7±0.06, n=6, ns). However, expression of alternative, truncated RGS9 transcripts were detected in RGS9Δexon2-4 islets, and GSIS was reduced upon RGS9 knockdown in RGS9Δexon2-4 islets (Control: 1.1±0.4 versus shRNA1: 0.3±0.1 p<0.01, n=4). Conclusion: RGS9 positively controls insulin secretion. Preserved GSIS in RGS9Δexon2-4 islets may be due to compensation from truncated RGS9. Disclosure S. Ferragne: None. S.A. Campbell: Employee; Applied Pharmaceutical Innovation, Hepion Pharmaceuticals. L. Reininger: None. C. Tremblay: None. J. Ghislain: None. V. Poitout: Research Support; Biodexa. Funding Natural Sciences and Engineering Research Council of CanadaFonds de recherche du Québec - Nature et technologie