Abstract 144: Reduced Placental Regulator of G-Protein Signaling-2 (RGS2) and Preeclampsia
Katherine J PerschbacherGuorui DengJeremy A SandgrenLeslie Carillo-SaenzDonna A. SantillanEric J. DevorGary L. PierceMark K. SantillanRory A. FisherKatherine N. Gibson‐CorleyJustin L. Grobe
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The early mechanisms and genetic risk factors driving the pathogenesis of preeclampsia (PE), a cardiovascular disorder of pregnancy, remain largely unclear. Various hormone activators of Gα q second-messenger signaling pathways have been implicated in PE. Regulator of G-protein Signaling 2 (RGS2) acts as an endogenous terminator of Gα q signaling and previous studies identified a SNP (rs4606), which results in reduced RGS2 mRNA, as a risk factor for development of PE and its sequelae. We hypothesized reduced placental expression of RGS2 may precipitate the development of PE due to disinhibited Gα q signaling. In silico reanalysis of publically available dataset GSE75010 revealed RGS2 mRNA is reduced in placentas from pre-term PE pregnancies compared to normal pre-term pregnancies (con: 8.73 ± 0.07 n=35, PE: 8.37 ± 0.055 n=49, p<0.05). Using human placental tissue samples from the University of Iowa Maternal-Fetal Tissue Bank, we confirmed RGS2 mRNA is reduced in PE placentas (19% of control, p<0.05), despite a lack of correlation between the rs4606 SNP and PE. Additionally, in further reanalysis of other datasets, RGS2 mRNA is among the highest-expressed RGS member in normal human placenta, and appears to be selectively reduced in syncytio- and invasive cytotrophoblasts during PE (GSE93839, -26.3%, -23.3% of control). We next examined RGS2 expression in mouse placenta by FISH and found RGS2 mRNA colocalizes with markers of syncytiotrophoblast II (GCMA) and spongiotrophoblast (Tpbpα) layers. To test the effect of RGS2 loss during pregnancy, wildtype C57BL/6J female mice were mated with RGS2-deficient sires and developed diastolic hypertension, placental hypoxia by HIF1α ELISA (con 0.144±0.004, RGS2-KO 0.155±0.004 AU, p<0.05, n=5 each), and reduced placental PlGF mRNA (fold; con=1.0 n=7, RGS2-KO=0.23 n=12, p<0.05), compared to females mated with RGS2 littermate sires. These data support the concept that loss of RGS2 may contribute to the pathogenesis of PE rather than simply correlating with the disorder. Taken together, we have shown placental RGS2 is suppressed in PE, RGS2 is present in cytoplasm of specific layers of trophoblasts, and loss of feto-placental RGS2 is sufficient to cause placental hypoxia and maternal diastolic hypertension.Keywords:
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Regulator of G protein Signaling, or RGS, proteins serve an important regulatory role in signaling mediated by G protein-coupled receptors (GPCRs). They all share a common RGS domain that directly interacts with active, GTP-bound Gα subunits of heterotrimeric G proteins. RGS proteins stabilize the transition state for GTP hydrolysis on Gα and thus induce a conformational change in the Gα subunit that accelerates GTP hydrolysis, thereby effectively turning off signaling cascades mediated by GPCRs. This GTPase accelerating protein (GAP) activity is the canonical mechanism of action for RGS proteins, although many also possess additional functions and domains. RGS proteins are divided into four families, R4, R7, R12 and RZ based on sequence homology, domain structure as well as specificity towards Gα subunits. For reviews on RGS proteins and their potential as therapeutic targets, see e.g. [183, 411, 446, 450, 451, 558, 566, 345, 9].
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Fine‐tuning of signaling via heterotrimeric G proteins by regulators of G protein signaling (RGS) proteins is critical to maintaining physiological homeostasis. Abnormal G protein signaling due to the loss of G protein regulation by RGS proteins is implicated in several diseases including cardiovascular disorders such as hypertension, cardiac hypertrophy, and heart failure. RGS proteins act as GTPase activating proteins (GAPs) to control the kinetics and amplitude of G protein signaling. Multiple RGS proteins are prominently expressed in the cardiovascular system; however, whether their activities/functions are coordinated to control G protein signaling and cardiovascular function is unknown. Here, we generated mice concurrently lacking RGS2 and 5 ( Rgs2/5 dbKO) to determine how the dual absence of potent GAPs for Gq/11 and Gi/oclass G proteins affects cardiovascular function. Blood pressure and heart rate were monitored in conscious, freely moving mice via radiotelemetry. Surgical implantation of the radiotelemetry device induced marked systolic blood pressure increase in Rgs2/5 dbKO mice (WT: 140 ± 6 vs. dbKO: 170 ± 2 mmHg; p <0.01) at baseline, which gradually declined but remained elevated in Rgs2/5 dbKO relative to wild type (WT) mice, several days later. Whereas all WT mice survived the surgery, ~70–80% of male Rgs2/5 dbKO mice surprisingly died 72–96 hr post‐surgery. When subjected to cardiac stress test using acute dobutamine infusion and echocardiography or the invasive pressure‐volume loop analysis of cardiac function, male Rgs2/5 dbKO mice showed hypocontractile response and decreased ejection fraction relative to WT mice. Freshly isolated ventricular cardiomyocytes from male Rgs2/5 dbKO mice showed decreased fractional shortening (WT: 16.1 ± 4.3 vs. dbKO: 7.4 ± 1.1 %; p <0.01) but high calcium transients (WT: 117 ± 20 vs. dbKO: 198 ± 50 au; p =0.07) at baseline, and application of electrical field stimulation or low concentration of the non‐selective β‐adrenergic receptor agonist, isoproterenol (ISO), triggered premature calcium transients, tachyarrhythmia and death of cells from Rgs2/5 dbKO mice. Interestingly, cells from mice harboring just one copy of Rgs2 ( Rgs2+/−, Rgs5−/− ) but not Rgs5 ( Rgs2−/−, Rgs5+/− ) were resistant to low‐dose ISO‐induced arrhythmia. These results together suggest that RGS2 and 5 coordinate their activity to control cardiomyocyte excitation‐contraction coupling and normal cardiac rhythm. Support or Funding Information NIH – NHLBI (1R01 HL139754‐01) and AHA Scientist Development Grant (16SDG27260276) This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .
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"Regulator of G-protein signaling" (RGS) proteins facilitate the termination of G protein-coupled receptor (GPCR) signaling via their ability to increase the intrinsic GTP hydrolysis rate of Galpha subunits (known as GTPase-accelerating protein or "GAP" activity). RGS2 is unique in its in vitro potency and selectivity as a GAP for Galpha(q) subunits. As many vasoconstrictive hormones signal via G(q) heterotrimer-coupled receptors, it is perhaps not surprising that RGS2-deficient mice exhibit constitutive hypertension. However, to date the particular structural features within RGS2 determining its selectivity for Galpha(q) over Galpha(i/o) substrates have not been completely characterized. Here, we examine a trio of point mutations to RGS2 that elicits Galpha(i)-directed binding and GAP activities without perturbing its association with Galpha(q). Using x-ray crystallography, we determined a model of the triple mutant RGS2 in complex with a transition state mimetic form of Galpha(i) at 2.8-A resolution. Structural comparison with unliganded, wild type RGS2 and of other RGS domain/Galpha complexes highlighted the roles of these residues in wild type RGS2 that weaken Galpha(i) subunit association. Moreover, these three amino acids are seen to be evolutionarily conserved among organisms with modern cardiovascular systems, suggesting that RGS2 arose from the R4-subfamily of RGS proteins to have specialized activity as a potent and selective Galpha(q) GAP that modulates cardiovascular function.
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Regulator of G protein Signaling, or RGS, proteins serve an important regulatory role in signaling mediated by G protein-coupled receptors (GPCRs). They all share a common RGS domain that directly interacts with active, GTP-bound Gα subunits of heterotrimeric G proteins. RGS proteins stabilize the transition state for GTP hydrolysis on Gα and thus induce a conformational change in the Gα subunit that accelerates GTP hydrolysis, thereby effectively turning off signaling cascades mediated by GPCRs. This GTPase accelerating protein (GAP) activity is the canonical mechanism of action for RGS proteins, although many also possess additional functions and domains. RGS proteins are divided into four families, R4, R7, R12 and RZ based on sequence homology, domain structure as well as specificity towards Gα subunits. For reviews on RGS proteins and their potential as therapeutic targets, see e.g. [160, 377, 411, 415, 416, 512, 519, 312, 6].
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Abstract ID 16497 Poster Board 121 Regulators of G protein Signaling (RGS) proteins are negative regulators of G-protein-coupled receptors (GPCRs). They reduce the amplitude and duration of GPCR signaling by accelerating the hydrolysis of GTP to GDP through their GTPase-accelerating protein (GAP) activity. Altered RGS protein function is involved in numerous diseased states. However, the therapeutic regulation of RGS proteins is challenging as they lack binding pockets for small molecules. Therefore, identifying mechanisms that control RGS protein activity and expression could be a potential targeting strategy. A notable example where altering RGS protein activity would be beneficial is asthma, a long-term respiratory disease, characterized by inflammation of the airways, resulting in the obstruction of airflow to the lungs. Aberrant activation of Gαq and downstream signaling contribute to chronic symptoms of asthma. RGS2 is known to be selective for Gαq over other G protein subtypes and has reduced expression levels in asthmatic patients. Low RGS2 protein levels are also implicated in many other diseases such as hypertension, cancer, and heart failure. Indeed, we previously showed that pharmacologically stabilizing RGS2 protein levels inhibits Gαq-mediated signaling and are cardioprotective in a mouse model of cardiac injury, suggesting that enhanced RGS2 protein levels correlate with increased function. Therefore, targeting the mechanisms that regulate RGS2 protein levels would be a feasible therapeutic strategy. RGS2 is rapidly degraded through the ubiquitin-proteasomal system (UPS). In the UPS, ubiquitin molecules are covalently linked to the substrate through the cascade of enzymatic reactions (facilitated by E1: ubiquitin-activating enzyme; E2: ubiquitin-conjugating enzyme; E3 ligase). The ubiquitinated substrate is then recognized and degraded by the 26S proteasomal complex. We recently identified the E3 ligase that recognizes RGS2. This E3 ligase complex consists of Cullin 4B (Cul4B), DNA Damage Binding Protein 1 (DDB1), and F-box Only Protein 44 (FBXO44), where FBXO44 acts as the substrate recognition site for RGS2. Therefore, we hypothesize that inhibiting the RGS2-FBXO44 interaction will lead to enhanced RGS2 levels. To identify small molecule RGS2-FBXO44 interaction inhibitors, we utilized NanoLuc® Binary Technology (NanoBiT) to detect the interaction between RGS2 and FBXO44 in a high-throughput screen (HTS). We developed a HEK-293T cell line stably expressing the RGS2-SmBit and LgBit-FBXO44 and optimized the NanoBit assay for HTS. Using this assay, we screened 1600 compounds (Life Chemicals PPI fragment library) at the Purdue Chemicals Genomics Facility. Following hit confirmation and chemical clustering, the top 20 hits that inhibited the RGS2-FBXO44 protein-protein interaction at least 50% were selected for follow-up. The concentration-dependent activity of these 20 hits was performed to only select compounds inhibiting at least 50% of the interaction and having acceptable inhibition curves (Hill slope 0.5 – 2.0) and potencies (IC50<50μM). We are currently working to further validate these compounds as RGS2 stabilizers via inhibiting the RGS2-FBXO44 protein-protein interaction. We will also detect the effects of the selected hits on Gq-mediated signaling. These hit compounds will be optimized by applying iterative rational drug-design strategies to deliver future lead candidates to stabilize RGS2 protein levels.
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