Regulator Of G Protein Signaling Research Articles

Gq Signaling in the Placental Syncytiotrophoblast Layer During Preeclampsia

Preeclampsia is a pregnancy‐associated cardiovascular disorder characterized by hypertension and proteinuria after 20 weeks of gestation. Generalized cellular ‘stress’ (oxidative, mitochondrial, etc.) within the syncytiotrophoblast (STB) layer of placenta is theorized to drive its pathogenesis. Hormones implicated in preeclampsia such as angiotensin, endothelin, and vasopressin signal via receptors that typically couple to the Gq second‐messenger cascade, and Regulator of G protein Signaling‐2 (RGS2) buffers this signaling. We have published that RGS2 expression is decreased in human preeclamptic placenta, and reducing RGS2 expression in placenta is sufficient to cause development of key features of this disorder in mice. Unpublished in situ hybridization data indicate that in both humans and mice, RGS2 is present in the STB layer – and that RGS2 expression in the STB layer is reduced during preeclampsia in humans. Collectively, these findings lead us to generally hypothesize that excess Gq signaling specifically within the STB layer, whether due to excessive hormonal activation or loss of RGS2‐mediated buffering, causes STB‐Stress and thereby contributes to the pathogenesis of preeclampsia. Therefore, the objective of this study was to test whether artificial, exogenous activation of Gq signaling only within STB cells is sufficient to induce placental and maternal phenotypes of preeclampsia in mice.CAG‐FLEX‐hM3Dq+/+ (Gαq DREADD, Jax 026943) or C57BL/6J dams were bred with Gcm1‐Cre+/‐ sires (Jax 026625) or non‐transgenic littermates to elicit STB‐restricted hM3Dqexpression in ≍50% or 0% of placentas. The synthetic DREADD ligand clozapine N‐oxide (CNO) or saline vehicle was administered (2 mg/kg/d, ip) mid‐gestation (GD 12.5‐14.5). Placental and maternal phenotypes were compared between Cre– and Cre+ placentas within a dam, CNO‐ vs saline‐injected double‐transgenic dams, and double‐ vs single/non‐transgenic dams treated with CNO. Activation of Gq in the STB layer caused maternal proteinuria (n=4‐9, 49±6 mg/day vs 28±4 mg/day; p=0.01). Labyrinth vascularization, assessed by percentage of CD31+ area, was reduced with Gq activation (n=5, Cre+ 21±1%, Cre– 27±2%; p=0.01), while other regions remained unaffected (Decidua: Cre+ 14±4%, Cre– 14±3%; Junctional Zone: Cre+ 11±4%, Cre– 12±3%). Possible reductions in placental mass (n=5 Cre+ 0.12±0.01g, Cre– 0.15±0.01g; p=0.06), and placental vascular endothelial growth factor protein levels (n=6‐7 117±67 ng/g vs 151±12 ng/g; p=0.06) were noted, but no obvious trend in fetal growth restriction was observed (n=5 Cre+ 0.21±0.06g, Cre– 0.29±0.04g). These data support the pathological significance of excess Gq signaling specifically within the STB layer. Ongoing studies are aimed at further characterizing maternal phenotypes (blood pressure, circulating factors) in these models, and the synergistic effects of simultaneous STB‐specific deletion of RGS2 upon sensitivity to Gq stimulation in this cell type.

RGS4 Mediates Catecholaminergic Inhibition of Short‐Chain Fatty Acid Receptor FFAR3 Signaling & Function in Cardiomyocytes

BackgroundFree fatty acids (FFAs) are essential cell nutrients but also important signaling molecules regulating various cellular processes. Short‐chain fatty acids (SCFAs), like propionate and butyrate, activate two different types of G protein‐coupled receptors (GPCRs), FFAR2 and FFAR3. FFAR3 (also known as GPR41) couples to Gi/o proteins lowering cyclic 3’,5’‐adenosine monophosphate (cAMP) levels and activating mitogen‐activated protein kinases (MAPKs). In addition to adipose tissue, FFAR3 is highly expressed in peripheral sympathetic ganglia augmenting sympathetic nervous system (SNS) outflow. Regulator of G protein Signaling (RGS)‐4 belongs to the RGS superfamily of proteins and inactivates (terminates) both Gi/o‐ and Gq‐protein signaling. Cardiac RGS4 has been implicated in atrial fibrillation (AFib) prevention via calcium signaling attenuation. Finally, β‐adrenergic receptors (ARs), the major mediators of catecholaminergic and SNS actions in cardiac myocytes, stimulate cAMP synthesis and activate protein kinase A (PKA), which is known to activate RGS4 via phosphorylation.Study objectiveSince catecholamines/SNS and RGS4 play major roles in AFib pathophysiology and FFAR3 promotes SNS activity, we examined whether RGS4 regulates cardiac FFAR3 signaling.MethodsWe used H9c2 cardiomyocytes transfected to express the human FFAR3. After treatment with catecholaminergic agonists and/or propionic acid plus various pharmacological inhibitors and siRNA to deplete RGS4, we performed signaling assays (western blotting, cAMP accumulation, GTP binding, ELISA) to delineate the precise involvement of RGS4 in the cardiac βAR‐FFAR3 signaling crosstalk.ResultsRGS4 is essential for the dampening of propionate/FFAR3‐dependent signaling in H9c2 cardiomyocytes since siRNA‐mediated RGS4 depletion significantly enhances the effect of propionic acid on cAMP lowering, Gi/o activation, and p38 MAPK activation. Notably, concomitant isoproterenol or salbutamol treatment blocks propionic acid‐induced signaling via the FFAR3 in H9c2 cardiomyocytes, indicating negative regulation of FFAR3 by catecholamines via the β2AR. Mechanistically, this is mediated by RGS4, since neither isoproterenol, nor salbutamol can inhibit propionate signaling upon siRNA‐mediated RGS4 depletion. Finally, this β2AR trans‐inhibition of FFAR3 signaling via RGS4 is dependent on β2AR‐activated PKA, because pre‐treatment with the PKA inhibitor H89 abrogated the catecholaminergic inhibition of FFAR3 signaling (cAMP lowering, downstream p38 MAPK activation, interleukin (IL)‐6 production) in H9c2 cardiomyocytes.Conclusionsβ2AR‐activated RGS4 abrogates SCFA/FFAR3 signaling and function in cardiac myocytes. This may represent part of the mechanism via which RGS4 protects against both AFib pathogenesis and FFAR3‐promoted SNS hyperactivity and inflammation in the myocardium.

Deletion of RGS2 Results in Increased Blood Pressure and Depression‐Like Behavior in the Presence of Elevated Ang II Levels in Female Mice

Hypertension and depression are major health problems in the US. Overactivity of the renin angiotensin system (RAS) plays an important role in the pathophysiology of hypertension and depression. The actions of the main effector peptide of the RAS, angiotensin II (Ang II), are mediated by the Ang II type 1 receptor whose activity is modulated by regulator of G‐protein signaling (RGS) proteins, including RGS2. In this study, we assessed the effects of genetic deletion of RGS2 on Ang II‐induced increases in blood pressure (using tail cuff plethysmography) and depression‐like behavior (using the tail suspension test) in adult male and female mice. Adult male and female RGS2 wild‐type (RGS2+/+) and RGS2‐deficient (RGS2‐/‐) mice were treated with osmotic minipumps containing either vehicle (saline) or Ang II (1 mg/kg/d) for 21 days. In males, baseline SBP was lower in RGS2‐/‐ (SBP = 103±5 mmHg, n=14) vs RGS2+/+ (SBP = 120±6 mmHg, n=13, P<0.05 vs RGS2‐/‐) mice. By contrast, in females there was no difference in baseline SBP between RGS2+/+ and RGS2‐/‐ mice, suggesting that deletion of RGS2 results in lower baseline SBP in females only. Chronic treatment with Ang II increased SBP in both RGS2+/+ and RGS2‐/‐ mice compared to saline controls, irrespective of sex. There was no difference in Ang II‐induced increases in SBP in RGS2+/+ and RGS2‐/‐ male mice, suggesting that RGS2 does not protect against Ang II‐induced increases in SBP in male mice. By contrast, in female mice, Ang II‐induced increases in SBP were greater in RGS2‐/‐ compared to RGS2+/+ mice (for example, after 21 days of Ang II treatment, change in SBP from baseline in RGS2+/+ mice was 47±11 mmHg, n=5, and in RGS2‐/‐ mice it was 73±15 mmHg n=4, P<0.05) suggesting that RGS2 protects against Ang II‐induced increases in SBP in female, but not male mice. Chronic Ang II treatment also resulted in decreased immobilization time in RGS2+/+ female mice, but not RGS2‐/‐ mice compared to respective saline controls (immobilization time in vehicle‐treated RGS2+/+= 187±14 sec, n=5; Ang II‐treated RGS2+/+= 124±27 sec, n=5, P<0.05 vs vehicle‐treated RGS2+/+; vehicle‐treated RGS2‐/‐= 196±16 sec, n=5; Ang II‐treated RGS2‐/‐ = 192±13 sec, n=4, P<0.05 vs Ang II‐treated RGS2+/+). These data suggest that RGS2 protects against depression‐like behavior in the presence of elevated Ang II levels in female mice. In contrast to female mice, Ang II treatment and RGS2 deficiency had no effect on depression‐like behavior in adult male mice. Overall, these data suggest that RGS2 protects against blood pressure increases and depression‐like behavior after exposure to elevated Ang II levels in female mice, suggesting that RGS2 activation is a promising therapeutic target to treat elevated blood pressure and depression during conditions of elevated Ang II levels in females.

Advanced Age and Sex Modulate Multiple Mu Opioid Receptor Signaling Mechanisms in the Rat Midbrain Periaqueductal Gray: Implications for Analgesia

Chronic pain is exceedingly prevalent in individuals over 65 years of age. Still, it is under-managed due in large part to a dearth of knowledge regarding the suitable dosing of opioids for chronic pain management. We have shown that advanced age and sex alter morphine modulation of persistent inflammatory pain (induced by intraplantar Complete Freund's adjuvant (CFA)). Specifically, morphine potency was highest in adult male rats (2mos), with a 2-fold rightward shift in the dose-response curve for aged males (18mos) and females regardless of age. In this study, we tested the hypothesis that advanced age attenuates μ-opioid receptor (MOR) signaling in the ventrolateral periaqueductal gray (vlPAG), a critical region for the modulation of pain. We find that aged males exhibit reduced vlPAG MOR expression (Bmax) compared to adult males, and both aged males and aged females exhibit reduced vlPAG MOR affinity (Kd) compared to adult males. Further, aged males and females, regardless of age, show reduced vlPAG MOR binding potential and reduced G-protein activation efficiency compared to adult males. Western blots of phosphorylated MOR at serine 375 revealed no age or sex differences in MOR phosphorylation. DAMGO-induced cAMP inhibition was 2-fold higher in adult males than aged males or females. The regulation of opioid-induced G-protein signaling was further assessed using RNAscope to analyze mRNA expression of Regulator of G-Protein Signaling (RGS) family members RGS4 and RGS9-2. We found increased expression of RGS4 and RGS9-2 in the vlPAG of aged animals compared to adults, indicating that MOR signaling is subjected to greater negative regulation in the aged vlPAG. The observed age-related reductions in vlPAG MOR expression, MOR binding potential, G-protein activation, and cAMP inhibition, along with the observed increase in vlPAG RGS4 and RGS9-2, have significant implications in pain management in the aged population. Grant support from NIH R01DA041529 (AZM), NIH R01DA052340 (JS) NIH P50MH100023 (LJY).

Structural analysis of G protein β5 subunit folding by the cytosolic chaperonin CCT/TRiC and its co‐chaperone phosducin‐like protein 1 reveal molecular mechanisms of chaperone‐mediated β‐propeller protein folding

The cytosolic chaperonin CCT and its co‐chaperone phosducin‐like protein 1 (PhLP1) play important roles in G protein complex assembly by folding G protein β subunits (Gβ) into β‐propeller structures. To understand this process at the molecular level, we have isolated the CCT‐Gβ5‐PhLP1 folding intermediate in both the open and closed CCT conformations and determined its structure by high resolution cryo‐electron microscopy (cryo‐EM). In the open structures, Gβ5 interact with the N‐ and C‐termini of the CCT subunits deep inside the folding chamber between the CCT rings in a closed β‐propeller conformation. PhLP1 allosterically enhances Gβ5 binding to CCT by interacting with the CCT apical domains at the rim of the folding chamber without contacting Gβ5 directly. In the closed CCT structures, Gβ5 move from between the CCT rings into one of the folding chambers, suggesting a path for release of Gβ5 from CCT during its ATPase cycle. These findings provide a molecular explanation for CCT‐dependent folding and release of Gβ5 to interact with RGS proteins and perform their essential functions in G protein signaling.

Role of the Gβ5/R7‐RGS complex in the regulation of pain transmission in sensory ganglia

Neuropathic pain is a chronic disorder resulting from damage to the afferent nerve fibers or central pain pathways and is often a complication in pathological conditions such as diabetes, shingles, multiple sclerosis, and stroke. The opioid epidemic has elucidated the need for more efficacious treatments for neuropathic pain. In 2019 alone, nearly 1.6 million people were diagnosed with an opioid use disorder and 48,000 people died from a synthetic opioid overdose. Despite the addictive properties, opioids are still the most frequently prescribed pain medication, even for chronic neuropathic pain. Heterotrimeric G‐proteins consisting of the α, β, and γ subunits convey extracellular signals sensed by G‐protein coupled receptors (GPCRs) to intracellular effectors. The Gβ5 subunit is a divergent member of the G‐protein β subunit family as it does not bind to traditional γ subunits. Instead, Gβ5 complexes with the R7 subfamily of the regulators of G‐protein signaling (R7‐RGS) containing 4 members: RGS6, 7, 9 and 11. The Gβ5/R7‐RGS complex acts as a GTPase accelerating protein (GAP) for G‐protein αi/o (Gαi/o) subunits. Previous studies have established the integral role of R7‐RGS proteins in pain transmission via their interactions with Gαi/o‐coupled receptors including opioid and metabotropic gamma‐aminobutyric acid (GABA‐B) receptors. Our lab has shown the lack of Gβ5 in sensory ganglia diminishes mechanical, thermal, and chemical nociception. However, the conditional knockout of Gβ5 in Rgs7 expressing neurons reduces only mechanical nociception. This Gβ5/RGS7‐dependent mechanical nociception relies on GABA‐B receptor signaling as indicated by the rescue of mechanical nociception in Rgs7‐Cre; Gnb5 fl/fl mice after treatment with 2‐hydroxysaclofen, a GABA‐B antagonist. We also established that Rgs9 expressing neurons regulate thermal nociception via a Gβ5‐dependent pathway as assayed by the hotplate test in Rgs9‐Cre; Gnb5 fl/fl mice. The purpose of this project has been to understand the molecular role of each R7‐RGS member in the regulation of pain transmission. First, we confirmed co‐localization between the Gnb5 transcript and all four R7‐RGS mRNA transcripts in murine dorsal root ganglia (DRG) using the RNAscope HiPlex assay, a novel in situ hybridization technique. We then established the co‐localization patterns between each R7‐RGS member and various pain related receptors including Mrgprd, Trpa1, and Trpv1. Our RNAscope results support the behavioral tests since Rgs7 transcripts highly co‐express with Mrgprd, a mechanical nociceptor, while Rgs9 transcripts most frequently co‐express with Trpv1, a thermosensitive receptor. These results suggest that each R7‐RGS member might regulate unique types of nociception. We have also shown that Rgs11 transcripts co‐localize with Trpv1 and Trpa1 receptor transcripts which indicates Rgs11 might regulate the chemical nociception as tested by capsaicin and mustard oil administration in the eye‐wipe test. Next, we aim to study the possible roles of Rgs6 and Rgs11 in regulating chemical nociception using conditional Gβ5 knockout mice mediated by Rgs6‐cre and Rgs11‐cre, respectively.

Palmitoylation of RGS20 Affects its Cellular Function

Regulator of G protein signaling 20 (RGS20) is a member of the RZ family of the RGS protein superfamily, which serve as key negative regulators of G protein‐mediated signal transduction. Previous studies showed that overexpression of RGS20 is correlated with progression of triple‐negative breast cancer (TNBC) in tumor tissues and contributes to breast cancer cell migration and invasion. However, there is still a large gap relating to the regulation and function of RGS20 due to its late discovery. RGS20 contains a poly cysteine string motif, which is assumed to be heavily palmitoylated. Palmitoylation, an important post‐translational protein modification, plays an important role in cells by changing cellular functions of proteins. Consequently, in this study we determined palmitoylation regulation of RGS20, which could provide more information for new potential treatment strategies for TNBC. We used click chemistry to show that RGS20 is palmitoylated in transfected HEK‐293T cells and MDA‐MB‐231 triple negative breast cancer cells. We also showed that constitutively activated Gαo, GαoQ205L, significantly increases palmitoylation levels of RGS20. Furthermore, palmitoylated RGS20 associates much more with GαoQ205L, while de‐palmitoylated RGS20 lost the association with GαoQ205L. Next, we detected how palmitoylation affects RGS20 GAP activity, as well as cAMP signaling downstream of Gαo. We mapped the palmitoylation site crucial for all of these effects to a conserved cysteine in the RGS domain. Mutating this cysteine residue resulted in a significant decrease in both palmitoylation signal and the association with GαoQ205L. Overall, we showed that palmitoylation affects functions of RGS20 in cells and that a conserved cysteine in the RGS domain is a critical site for its palmitoylation.

Effect of Regulator of G Protein Signaling Proteins on Bone.

Regulator of G protein signaling (RGS) proteins are critical negative molecules of G protein-coupled receptor (GPCR) signaling, which mediates a variety of biological processes in bone homeostasis and diseases. The RGS proteins are divided into nine subfamilies with a conserved RGS domain which plays an important role in regulating the GTPase activity. Mutations of some RGS proteins change bone development and/or metabolism, causing osteopathy. In this review, we summarize the recent findings of RGS proteins in regulating osteoblasts, chondrocytes, and osteoclasts. We also highlight the impacts of RGS on bone development, bone remodeling, and bone-related diseases. Those studies demonstrate that RGS proteins might be potential drug targets for bone diseases.

Clinical and molecular implications of RGS2 promoter genetic variation in severe asthma

Regulator of G protein signaling (RGS) 2 terminates bronchoconstrictive Gαq signaling; murine RGS2 knockout demonstrate airway hyperresponsiveness. While RGS2 promoter variants rs2746071 and rs2746072 associate with a clinical mild asthma phenotype, their impact on human airway smooth muscle (HASM) contractility and asthma severity outcomes is unknown. We sought to determine whether reductions in RGS2 expression seen with these 2 RGS2 promoter variants augment HASM contractility and associate with an asthma severity phenotype. We transfected HASM with a range of RGS2-specific small interfering RNA (siRNA) concentrations and determined RGS2 protein expression by Western blot analysis and intracellular calcium flux induced by histamine (a Gαq-coupled H1 receptor bronchoconstrictive agonist). We conducted regression-based genotype association analyses of RGS2 variants from 611 patients from the National Heart, Lung, and Blood Institute Severe Asthma Research Program 3. RGS2-specific siRNA caused dose-dependent increases in histamine-stimulated bronchoconstrictive intracellular calcium signaling (2-way ANOVA, P< .0001) with a concomitant decrease in RGS2 protein expression. RGS2-specific siRNA did not affect Gαq-independent ionomycin-induced intracellular calcium signaling (P= .42). The minor allele frequency of rs2746071 and rs2746072 was 0.46 and 0.28 among African American/non-Hispanic Black patients and was 0.28 and 0.27 among non-Hispanic White patients, among whom these single nucleotide polymorphisms were in stronger linkage disequilibrium (r2= 0.97). Among non-Hispanic White patients, risk allele homozygotes for rs2746072 and rs2746071 each had nearly 2-fold greater asthma exacerbation rates relative to alternative genotypes with wild-type alleles (Padditive= 2.86×10-5/Precessive= 5.22×10-6 and Padditive= 3.46×10-6/Precessive= 6.74×10-7, respectively) at baseline, which was confirmed by prospective longitudinal exacerbation data. RGS2 promoter variation associates with a molecular and clinical phenotype characterized by enhanced bronchoconstrictive stimulation invitro and higher asthma exacerbations rates in non-Hispanic White patients.

Regulator of G-Protein Signaling 5 Protein Deficiency Differentially Influences Blood Pressure, Vascular and Behavioral Effects in Aged Male Mice.

Aging and elevated activity of the renin-angiotensin-system (RAS) are associated with hypertension, vascular and emotional behavioral abnormalities, like anxiety and depression. Many actions of the main effector hormone of the RAS, angiotensin II (Ang II), are mediated by Ang II type 1 receptor whose activity is modulated by the regulator of G-protein signaling 5 (RGS5) protein. We assessed the role of RGS5 on blood pressure, vascular and emotional behavioral outcomes in aged male mice in the presence and absence of chronically elevated Ang II levels. We used aged (∼21-month old) male RGS5-deficient (RGS5 -/- ) and wild-type (RGS5 +/+ ) mice treated with vehicle (saline) or Ang II (1 mg/kg/d for 21 days). RGS5 deficiency increased baseline and cerebral vascular superoxide levels in the presence of chronically elevated Ang II levels, suggesting that RGS5 deficiency leads to elevated blood pressure and deleterious cerebral vascular outcomes in aged mice. RGS5 deletion had no effect on Ang II-induced increases in systolic blood pressure. Chronically elevated Ang II levels increased spontaneous locomotor activity in RGS5 +/+ but not RGS5 -/- mice. RGS5 deficiency and Ang II treatment had no effect on anxiety- and depression-like behavior. This is the first study to assess the effects of deficiency of an RGS protein in the vasculature or on emotional behavioral outcomes in aged mice. We report that RGS5 has protective effects on blood pressure and the cerebral vasculature in aged mice. Clinically, these data suggest that RAS blockers may significantly reduce cerebrovascular disease risk in aged males lacking RGS5.

Distribution and the evolutionary history of G-protein components in plant and algal lineages.

Heterotrimeric G-protein complexes comprising Gα-, Gβ-, and Gγ-subunits and the regulator of G-protein signaling (RGS) are conserved across most eukaryotic lineages. Signaling pathways mediated by these proteins influence overall growth, development, and physiology. In plants, this protein complex has been characterized primarily from angiosperms with the exception of spreading-leaved earth moss (Physcomitrium patens) and Chara braunii (charophytic algae). Even within angiosperms, specific G-protein components are missing in certain species, whereas unique plant-specific variants-the extra-large Gα (XLGα) and the cysteine-rich Gγ proteins-also exist. The distribution and evolutionary history of G-proteins and their function in nonangiosperm lineages remain mostly unknown. We explored this using the wealth of available sequence data spanning algae to angiosperms representing extant species that diverged approximately 1,500 million years ago, using BLAST, synteny analysis, and custom-built Hidden Markov Model profile searches. We show that a minimal set of components forming the XLGαβγ trimer exists in the entire land plant lineage, but their presence is sporadic in algae. Additionally, individual components have distinct evolutionary histories. The XLGα exhibits many lineage-specific gene duplications, whereas Gα and RGS show several instances of gene loss. Similarly, Gβ remained constant in both number and structure, but Gγ diverged before the emergence of land plants and underwent changes in protein domains, which led to three distinct subtypes. These results highlight the evolutionary oddities and summarize the phyletic patterns of this conserved signaling pathway in plants. They also provide a framework to formulate pertinent questions on plant G-protein signaling within an evolutionary context.

Discovery of Novel Circulating Immune Complexes in Lupus Nephritis Using Immunoproteomics.

ObjectiveThe goal is to discover novel circulating immune complexes (ICx) in the serum of lupus nephritis (LN) as potential biomarkers.MethodsProtein A/G magnetic beads or C1q-coated plates were used to capture ICx in the serum of LN, followed by the identification of immunoglobulin-binding proteins using liquid chromatography and tandem mass spectrometry (LC-MS/MS). Bioinformatic approaches and single-cell RNA sequencing (scRNA Seq) databases were used to select potential candidate ICx markers in LN. The selected ICx markers were further validated using ELISA.ResultsA total of 300 immunoglobulin-binding proteins were discovered in the screening, among which 77 proteins were detectable only in LN samples. Bioinformatics-assisted selection allowed us to further identify 10 potential immunoglobulin-binding proteins, which form ICx as potential biomarkers in LN. In a validation cohort of 62 LN patients and 21 healthy controls (HC), we found that prolyl 3-hydroxylase 1 (P3H1), phosphatase and actin regulator 4 (PHACTR4), and regulator of G-protein signaling 12 (RGS12) ICx exhibited discriminative capability in distinguishing LN from HC, with an area under the curve (AUC) values of 0.82, 0.99, and 0.90, respectively. Furthermore, a biomarker panel comprising CD14, CD34, cystatin A, myocyte enhancer factor 2C (MEF2C), RGS12, and ubiquitin C (UBC) ICx could distinguish active LN from inactive LN with an AUC value of 0.85, which is comparable to or better than pathological parameters such as renal activity index (AI) and renal chronicity index (CI).ConclusionImmunoproteomics-based discovery studies have enabled us to identify circulating immune complexes as potential biomarkers of LN.

SymRK-dependent phosphorylation of Gα protein and its role in signaling during soybean (Glycine max) nodulation.

Heterotrimeric G proteins, comprised of Gα, Gβ and Gγ subunits, influence signaling in most eukaryotes. In metazoans, G proteins are activated by G protein-coupled receptor (GPCR)-mediated GDP to GTP exchange on Gα; however, the role(s) of GPCRs in regulating plant G-protein signaling remains equivocal. Mounting evidence suggests the involvement of receptor-like kinases (RLKs) in regulating plant G-protein signaling, but their mechanistic details remain scarce. We have previously shown that during Glycine max (soybean) nodulation, the nod factor receptor 1 (NFR1) interacts with G-protein components and indirectly affects signaling. We explored the direct regulation of G-protein signaling by RLKs using protein-protein interactions, receptor-mediated in vitro phosphorylations and the effects of such phosphorylations on soybean nodule formation. Results presented in this study demonstrate a direct, phosphorylation-based regulation of Gα by symbiosis receptor kinase (SymRK). SymRKs interact with and phosphorylate Gα at multiple residues in vitro, including two in its active site, which abolishes GTP binding. Additionally, phospho-mimetic Gα fails to interact with Gβγ, potentially allowing for constitutive signaling by the freed Gβγ. These results uncover an unusual mechanism of G-protein cycle regulation in plants where the receptor-mediated phosphorylation of Gα not only affects its activity but also influences the availability of its signaling partners, thereby exerting a two-pronged check on signaling.

Clinical and molecular implications of RGS2 promoter genetic variation in severe asthma

Regulator of G-Protein Signaling (RGS)-2 terminates pro-bronchoconstrictive Gαq-signaling; murine RGS2 knockout exhibit airway hyperresponsiveness. While RGS2 promoter variants rs2746071 and rs2746072 associate with a clinical mild asthma phenotype, their impact on human airway smooth muscle (HASM) contractility and asthma severity outcomes is unknown. We hypothesized that reductions in RGS2 expression seen with these two RGS2 promoter variants would amplify HASM contractility and associate with an asthma severity phenotype.

Circular RNA hsa_circ_0006091 as a novel biomarker for hepatocellular carcinoma

ABSTRACT Circular RNAs (circRNAs) are stable and extensively distributed non-coding RNA molecules that are differentially expressed in liver cancer tissues in the human body. In this study, we aimed to investigate circRNA as a novel candidate biomarker for hepatocellular carcinoma (HCC). For three groups of HCC and neighboring healthy tissues, the differentially expressed circRNAs were identified through high-throughput sequencing analysis. Reverse transcription PCR (RT-PCR) and quantitative polymerase chain reaction (qPCR) were employed for the evaluation of circRNAs that show an elevated expression level in HCC. The obtained results revealed the significantly differential expression of hsa_circ_0006091 in HCC. Then we obtained their target genes through biological analysis, followed by verifying the underlined target genes, and the regulator of G-protein signaling 12 (RGS12) showed an elevated expression level in HCC tissues. Finally, receiver operating characteristic (ROC) curve analysis was conducted on AFP, RGS12, and hsa_circ_0006091, and combined analysis was performed. Furthermore, hsa_circ_0006091 is a novel candidate biomarker for HCC and could improve the diagnostic strategies, prediction, and follow-up of HCC patients. The joint diagnosis of the hsa_circ_0006091&AFP and hsa_circ_0006091&RGS12 has diagnostic significance and can be used as a molecular marker for HCC diagnosis. Abbreviations: AUC:area under the ROC curve; ROC:Receptor Operating Characteristics; bp:base pair;mRNA:Messenger Ribonucleic acid;ceRNA:Competing endogenous RNA; RT-qPCR: Real time-quantitativen PCR technology; circRNA: circular RNA; HCC:Hepatocellular carcinoma;miRNA:microRNA;KEGG:Kyoto Encyclopedia of Genes and Genomes; RGS12:regulator of G-protein signaling 12; AFP:alpha fetoprotein; ncRNAs:non-coding RNAs; GEO:Gene Expression Omnibus; FDR:false discovery rate

Differential methamphetamine-induced behavioral effects in male and female mice lacking regulator of G Protein signaling 4

Methamphetamine-induced behavioral effects are mediated by several neurotransmitters that act via the G-protein coupled receptors (GPCRs). The functioning of GPCRs are negatively regulated by regulators of G-protein signaling (RGS) proteins. The goal of this study was to assess the role of two specific RGS proteins namely the RGS2 and the RGS4 proteins in methamphetamine-induced behaviors. The effects of methamphetamine (1 mg/kg; i.p.) on conditioned place preference (CPP) and locomotor activity were assessed in genetically modified male and female mice lacking either RGS2 or RGS4 and their wildtype littermates to achieve the above goal. Locomotor activity after methamphetamine administration was assessed in both methamphetamine-naïve and -experienced mice. Methamphetamine-induced CPP at the tested dose was blocked in male, but not female, mice lacking RGS4 compared to respective controls. Interestingly, methamphetamine-induced increase in locomotor activity at the tested dose was observed in methamphetamine-experienced, but not in the methamphetamine-naïve, male mice lacking RGS4. However, methamphetamine-induced increase in locomotor activity at the tested dose was blocked in both methamphetamine-naïve and -experienced female mice lacking RGS4. Interestingly, methamphetamine-induced rewarding effects and methamphetamine-induced increase in locomotor activity at the tested dose were observed in mice lacking RGS2, irrespective of sex and/or history of methamphetamine exposure. Together, the data suggest that RGS4 plays a role in methamphetamine-induced behaviors and could serve as a potential target for medications intended to treat the acute effects of methamphetamine.

Attenuation of nicotine-induced rewarding and antidepressant-like effects in male and female mice lacking regulator of G-protein signaling 2

Nicotine-induced rewarding and mood altering effects contribute to the continued use of nicotine and the subsequent development of nicotine dependence. The goal of this study was to assess the role of two specific regulators of G-protein signaling (RGS) proteins namely RGS2 and RGS4 in the above described effects of nicotine. Male and female mice lacking either RGS2 (RGS2 KO) or RGS4 (RGS4 KO), and their respective wildtype (WT) littermates were used in this study. The rewarding effects of nicotine (0.5 mg/kg, base; s.c.) were assessed using the conditioned place preference model. Nicotine-induced anxiolytic-like (0.1 mg/kg, base; i.p.) and antidepressant-like (1 mg/kg, base; i.p.) effects were assessed using the elevated plus maze and tail suspension test, respectively. We also assessed effects of nicotine (0, 0.05, 0.1 & 0.5 mg/kg, base; s.c.) on spontaneous locomotor activity. Nicotine-induced rewarding and antidepressant-like effects were observed in both male and female RGS2 WT mice, but not in mice lacking RGS2 compared to respective controls. In contrast, nicotine-induced rewarding and antidepressant-like effects were observed in both male and female mice lacking RGS4 and their WT littermates. Interestingly, deletion of RGS4 facilitated antidepressant-like effect of nicotine in male, but not female mice compared to respective WT littermates. Nicotine-induced anxiolytic-like effect was not influenced by deletion of either RGS2 or RGS4, irrespective of sex. Nicotine (0.5 mg/kg) decreased locomotor activity in both WT and KO mice compared to respective saline, irrespective of genotype and sex. Taken together, these data provide evidence that RGS2, but not RGS4, plays a role in mediating the rewarding and antidepressant-like effects of nicotine. Further research is required to explore the role of RGS2 after chronic exposure to nicotine.

Regulator of G-Protein Signalling 4 (RGS4) negatively modulates nociceptin/orphanin FQ opioid receptor signalling: Implication for l-Dopa-induced dyskinesia.

Regulator of G-protein signalling 4 (RGS4) is a signal transduction protein that accelerates intrinsic GTPase activity of Gαi/o and Gαq subunits, suppressing GPCR signalling. Here, we investigate whether RGS4 modulates nociceptin/orphanin FQ (N/OFQ) opioid (NOP) receptor signalling and if this modulation has relevance for l-Dopa-induced dyskinesia. HEK293T cells transfected with NOP, NOP/RGS4 or NOP/RGS19 were challenged with N/OFQ and the small-molecule NOP agonist AT-403, using D1-stimulated cAMP levels as a readout. Primary rat striatal neurons and adult mouse striatal slices were challenged with either N/OFQ or AT-403 in the presence of the experimental RGS4 chemical probe, CCG-203920, and D1-stimulated cAMP or phosphorylated extracellular signal regulated kinase 1/2 (pERK) responses were monitored. In vivo, CCG-203920 was co-administered with AT-403 and l-Dopa to 6-hydroxydopamine hemilesioned rats, and dyskinetic movements, striatal biochemical correlates of dyskinesia (pERK and pGluR1 levels) and striatal RGS4 levels were measured. RGS4 expression reduced NOFQ and AT-403 potency and efficacy in HEK293T cells. CCG-203920 increased N/OFQ potency in primary rat striatal neurons and potentiated AT-403 response in mouse striatal slices. CCG-203920 enhanced AT-403-mediated inhibition of dyskinesia and its biochemical correlates, without compromising its motor-improving effects. Unilateral dopamine depletion caused bilateral reduction of RGS4 levels, which was reversed by l-Dopa. l-Dopa acutely up-regulated RGS4 in the lesioned striatum. RGS4 physiologically inhibits NOP receptor signalling. CCG-203920 enhanced NOP responses and improved the antidyskinetic potential of NOP receptor agonists, mitigating the effects of striatal RGS4 up-regulation occurring during dyskinesia expression. This article is part of a themed issue on Advances in Opioid Pharmacology at the Time of the Opioid Epidemic. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v180.7/issuetoc.

Cardiovascular GPCR regulation by regulator of G protein signaling proteins.

G protein-coupled receptors (GPCRs) play pivotal roles in regulation of cardiovascular homeostasis across all vertebrate species, including humans. In terms of normal cellular function, termination of GPCR signaling via the heterotrimeric G proteins is equally (if not more) important to its stimulation. The Regulator of G protein Signaling (RGS) protein superfamily are indispensable for GPCR signaling cessation at the cell membrane, and thus, for cellular control of GPCR signaling and function. Perturbations in both activation and termination of G protein signaling underlie many examples of cardiovascular dysfunction and heart disease pathogenesis. Despite the plethora of over 30 members comprising the mammalian RGS protein superfamily, each member interacts with a specific set of second messenger pathways and GPCR types/subtypes in a tissue/cell type-specific manner. An increasing number of studies over the past two decades have provided compelling evidence for the involvement of various RGS proteins in physiological regulation of cardiovascular GPCRs and, consequently, also in the pathophysiology of several cardiovascular ailments. This chapter summarizes the current understanding of the functional roles of RGS proteins as they pertain to cardiovascular, i.e., heart, blood vessel, and platelet GPCR function, with a particular focus on their implications for chronic heart failure pathophysiology and therapy.

Structural Studies Reveal Unique Non-Canonical Regulators of G Protein Signaling (RGS) Domains in Sorting Nexins

Structural Studies Reveal Unique Non-Canonical Regulators of G Protein Signaling (RGS) Domains in Sorting Nexins