scholarly journals G Protein-Coupled Receptor 30 Expression Is Required for Estrogen Stimulation of Primordial Follicle Formation in the Hamster Ovary

Endocrinology ◽  
2008 ◽  
Vol 149 (9) ◽  
pp. 4452-4461 ◽  
Author(s):  
Cheng Wang ◽  
Eric R. Prossnitz ◽  
Shyamal K. Roy
Keyword(s):  
G Protein ◽  
Fluorescein Isothiocyanate ◽  
Primordial Follicle ◽  
Mrna Levels ◽  
G Protein Coupled Receptor ◽  
Ovary Cells ◽  
Primordial Follicles ◽  
Protein Levels ◽  
G Protein Coupled

Estradiol-17β (E2) plays an important role in the formation and development of primordial follicles, but the mechanisms remain unclear. G protein-coupled receptor 30 (GPR30) can mediate a rapid and transcription-independent E2 signaling in various cells. The objectives of this study were to examine whether GPR30 was expressed in the neonatal hamster ovary and whether it could mediate estrogen action during the formation of primordial follicles. GPR30 mRNA levels decreased from the 13th day of gestation (E13) through the second day of postnatal (P2) life, followed by steady increases from P3 through P6. Consistent with the changes in mRNA levels, GPR30 protein expression decreased from E13 to P2 followed by a significant increase by P7, the day before the first appearance of primordial follicles in the hamster ovary. GPR30 was expressed both in the oocytes and somatic cells, although the expression in the oocytes was low. GPR30 protein was located primarily in the perinuclear endoplasmic reticulum, which was also the site of E2-BSA-FITC (E2-BSA-fluorescein isothiocyanate) binding. E2 or E2-BSA increased intracellular calcium in neonatal hamster ovary cells in vitro. Exposure to GPR30 small interfering RNA in vitro significantly reduced GPR30 mRNA and protein levels in cultured hamster ovaries, attenuated E-BSA binding to cultured P6 ovarian cells, and markedly suppressed estrogen-stimulated primordial follicle formation. These results suggest that a membrane estrogen receptor, GPR30, is expressed in the ovary during perinatal development and mediates E2 action on primordial follicle formation.

Abstract 466: Protein Phosphatase 2A and c-Myc as Intracellular Messengers in the Natriuretic Renal Dopaminergic System

Hypertension ◽  
2013 ◽  
Vol 62 (suppl_1) ◽  
Author(s):  
Hanh T Tran ◽  
John J Gildea ◽  
Robin A Felder
Keyword(s):  
G Protein ◽  
Protein Phosphatase ◽  
Sodium Excretion ◽  
Protein Phosphatase 2A ◽  
Camp Accumulation ◽  
Receptor Kinase ◽  
G Protein Coupled Receptor ◽  
Protein Levels ◽  
Phosphatase 2A ◽  
G Protein Coupled

G protein-coupled receptor kinase 4 (GRK4) is known to negatively regulate the dopamine-1 receptor (D 1 R) in human renal proximal tubule cells (RPTC) leading to reduced sodium excretion. c-Myc is a transcription factor involved in positive regulation of G protein-coupled receptor kinase 4 (GRK4). Protein phosphatase 2A (PP2A) inhibits c-Myc by dephosphorylating a residue that normally stabilizes c-Myc. We have previously shown that stimulation of the natriuretic D 1 R in RPTC led to an increased ratio of PP2A/c-Myc binding. Treatment with PMA (protein kinase C inhibitor) led to a decreased PP2A/c-Myc ratio and a lack of cAMP accumulation after stimulation with fenoldopam (FEN, D 1 R agonist). We hypothesized that PP2A plays a key role in regulating natriuresis and that perturbation of PP2A would directly have effects on protein levels of c-Myc, the ratio of PP2A/c-Myc, and the accumulation of cAMP. We used normal RPTCs (nRPTC) and RPTCs that have an uncoupled D 1 R that no longer stimulates adenylyl cyclase (uRPTC). Inhibition of PP2A in uRPTCs with okadaic acid (OA, 100nM, 3 hr) caused an increase in c-Myc protein levels (97.8% ± 18.9 SEM; n=6; p<0.05 (1.44 / 0.73 RFU)), a decrease in the PP2A/c-Myc ratio (-81.8% ± 1.5 SEM; n=6; p<0.05 (1.42 /7.82 RFU)), and a lack of cAMP accumulation upon treatment with SKF38393 (a D 1 R agonist similar to FEN). Activation of PP2A with FTY720 (PP2A activator, 10μM, 3hr) caused a decrease in c-Myc protein levels (- 85.4% ± 2.3 SEM; n=6; p<0.005 (0.11/ 0.73 RFU)), an increase in the PP2A/c-Myc binding ratio by 345.3% ± 90.3 SEM; n=6; p<0.01 (34.82/ 7.82 RFU), and an increase in cAMP accumulation upon stimulation with SKF38393 (94.0% ± 12.4 SEM; n=3; p<0.05 (9.04/4.66 pmole cAMP/mg protein) compared to VEH. In summary, the D 1 R coupling defect found in uRPTCs was restored through activation of PP2A and inhibition of c-Myc. We conclude that PP2A interacts with c-Myc to regulate the natriuretic effect of the D 1 R providing additional insight into the intracellular regulatory events surrounding sodium excretion.

scholarly journals Vascular Abnormalities in Mice Deficient for the G Protein-Coupled Receptor GPR4 That Functions as a pH Sensor

2006 ◽  
Vol 27 (4) ◽  
pp. 1334-1347 ◽  
Author(s):  
Li V. Yang ◽  
Caius G. Radu ◽  
Meenakshi Roy ◽  
Sunyoung Lee ◽  
Jami McLaughlin ◽  
...  
Keyword(s):  
G Protein ◽  
Mesangial Cells ◽  
Ex Vivo ◽  
Extracellular Ph ◽  
Ph Sensing ◽  
G Protein Coupled Receptor ◽  
Vascular Abnormalities ◽  
G Protein Coupled

ABSTRACT GPR4 is a G protein-coupled receptor expressed in the vasculature, lung, kidney, and other tissues. In vitro ectopic overexpression studies implicated GPR4 in sensing extracellular pH changes leading to cyclic AMP (cAMP) production. To investigate its biological roles in vivo, we generated GPR4-deficient mice by homologous recombination. Whereas GPR4-null adult mice appeared phenotypically normal, neonates showed a higher frequency of perinatal mortality. The average litter size from GPR4−/− intercrosses was ∼30% smaller than that from GPR4+/+ intercrosses on N3 and N5 C57BL/6 genetic backgrounds. A fraction of knockout embryos and neonates had spontaneous hemorrhages, dilated and tortuous subcutaneous blood vessels, and defective vascular smooth muscle cell coverage. Mesangial cells in kidney glomeruli were also significantly reduced in GPR4-null neonates. Some neonates exhibited respiratory distress with airway lining cell metaplasia. To examine whether GPR4 is functionally involved in vascular pH sensing, an ex vivo aortic ring assay was used under defined pH conditions. Compared to wild-type aortas, microvessel outgrowth from GPR4-null aortas was less inhibited by acidic extracellular pH. Treatment with an analog of cAMP, a downstream effector of GPR4, abolished microvessel outgrowth bypassing the GPR4-knockout phenotype. These results suggest that GPR4 deficiency leads to partially penetrant vascular abnormalities during development and that this receptor functions in blood vessel pH sensing.

scholarly journals Deficiency of Proton-Sensing Ovarian Cancer G Protein-Coupled Receptor 1 Attenuates Glucose-Stimulated Insulin Secretion

Endocrinology ◽  
2012 ◽  
Vol 153 (9) ◽  
pp. 4171-4180 ◽  
Author(s):  
Takashi Nakakura ◽  
Chihiro Mogi ◽  
Masayuki Tobo ◽  
Hideaki Tomura ◽  
Koichi Sato ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Insulin Secretion ◽  
G Protein ◽  
Normal Glucose Tolerance ◽  
Extracellular Ph ◽  
G Protein Coupled Receptor ◽  
Glucose Stimulated Insulin Secretion ◽  
G Protein Coupled

Ovarian cancer G protein-coupled receptor 1 (OGR1) has been shown as a receptor for protons. In the present study, we aimed to know whether OGR1 plays a role in insulin secretion and, if so, the manner in which it does. To this end, we created OGR1-deficient mice and examined insulin secretion activity in vivo and in vitro. OGR1 deficiency reduced insulin secretion induced by glucose administered ip, although it was not associated with glucose intolerance in vivo. Increased insulin sensitivity and reduced plasma glucagon level may explain, in part, the unusual normal glucose tolerance. In vitro islet experiments revealed that glucose-stimulated insulin secretion was dependent on extracellular pH and sensitive to OGR1; insulin secretion at pH 7.4 to 7.0, but not 8.0, was significantly suppressed by OGR1 deficiency and inhibition of Gq/11 proteins. Insulin secretion induced by KCl and tolbutamide was also significantly inhibited, whereas that induced by several insulin secretagogues, including vasopressin, a glucagon-like peptide 1 receptor agonist, and forskolin, was not suppressed by OGR1 deficiency. The inhibition of insulin secretion was associated with the reduction of glucose-induced increase in intracellular Ca2+ concentration. In conclusion, the OGR1/Gq/11 protein pathway is activated by extracellular protons existing under the physiological extracellular pH of 7.4 and further stimulated by acidification, resulting in the enhancement of insulin secretion in response to high glucose concentrations and KCl.

scholarly journals High-Throughput Fluorescence Polarization Assay to Identify Ligands Using Purified G Protein-Coupled Receptor

2019 ◽  
Vol 24 (9) ◽  
pp. 915-927
Author(s):  
P. Heine ◽  
G. Witt ◽  
A. Gilardi ◽  
P. Gribbon ◽  
L. Kummer ◽  
...  
Keyword(s):  
G Protein ◽  
High Throughput ◽  
Fluorescence Polarization ◽  
Binding Pocket ◽  
G Protein Coupled Receptor ◽  
Library Screen ◽  
A Cell ◽  
Fluorescence Polarization Assay ◽  
G Protein Coupled

The development of cell-free high-throughput (HT) methods to screen and select novel lead compounds remains one of the key challenges in G protein-coupled receptor (GPCR) drug discovery. Mutational approaches have allowed the stabilization of GPCRs in a purified and ligand-free state. The increased intramolecular stability overcomes two major drawbacks for usage in in vitro screening, the low receptor density on cells and the low stability in micelles. Here, an HT fluorescence polarization (FP) assay for the neurotensin receptor type 1 (NTS1) was developed. The assay operates in a 384-well format and is tolerant to DMSO. From a library screen of 1272 compounds, 12 (~1%) were identified as primary hits. These compounds were validated in orthogonal assay formats using surface plasmon resonance (SPR), which confirmed binding of seven compounds (0.6%). One of these compounds showed a clear preference for the orthosteric binding pocket with submicromolar affinity. A second compound revealed binding at a nonorthosteric binding region and showed specific biological activity on NTS1-expressing cells. A search of analogs led to further enhancement of affinity, but at the expense of activity. The identification of GPCR ligands in a cell-free assay should allow the expansion of GPCR pharmaceuticals with antagonistic or agonistic activity.

scholarly journals Identification of the first surrogate agonists for the G protein-coupled receptor GPR132

RSC Advances ◽  
2015 ◽  
Vol 5 (60) ◽  
pp. 48551-48557 ◽  
Author(s):  
Mohamed A. Shehata ◽  
Hanna Belcik Christensen ◽  
Vignir Isberg ◽  
Daniel Sejer Pedersen ◽  
Andreas Bender ◽  
...  
Keyword(s):  
G Protein ◽  
Binding Mode ◽  
Orphan Receptor ◽  
G Protein Coupled Receptor ◽  
Structure Activity Relationships ◽  
Structure Activity ◽  
G Protein Coupled

We report the first pharmacological tool agonist for in vitro characterization of the orphan receptor GPR132, preliminary structure–activity relationships based on 32 analogs and a suggested binding mode from docking.

scholarly journals Prognostic Value of Lymphocyte G Protein-Coupled Receptor Kinase-2 Protein Levels in Patients With Heart Failure

2016 ◽  
Vol 118 (7) ◽  
pp. 1116-1124 ◽  
Author(s):  
Giuseppe Rengo ◽  
Gennaro Pagano ◽  
Pasquale Perrone Filardi ◽  
Grazia Daniela Femminella ◽  
Valentina Parisi ◽  
...  
Keyword(s):  
Heart Failure ◽  
G Protein ◽  
Prognostic Value ◽  
Receptor Kinase ◽  
G Protein Coupled Receptor ◽  
Protein Levels ◽  
Patients With Heart Failure ◽  
G Protein Coupled

scholarly journals Expression of Bone Morphogenetic Protein Receptor (BMPR) during Perinatal Ovary Development and Primordial Follicle Formation in the Hamster: Possible Regulation by FSH

Endocrinology ◽  
2008 ◽  
Vol 150 (4) ◽  
pp. 1886-1896 ◽  
Author(s):  
Cheng Wang ◽  
Shyamal K. Roy
Keyword(s):  
Bone Morphogenetic Protein ◽  
Somatic Cells ◽  
Primordial Follicle ◽  
Ovary Development ◽  
Mrna Levels ◽  
Primordial Follicles ◽  
Bone Morphogenetic Protein Receptor ◽  
Protein Receptor ◽  
Morphogenetic Protein

To understand whether bone morphogenetic protein plays any role in the formation of primordial follicles in the hamster, we examined the temporal and spatial expression of bone morphogenetic protein receptor (BMPR) mRNA and protein in embryonic (E) 13 through postnatal day (P) 15 ovarian cells and a possible regulation by FSH during the formation of primordial follicles on P8. BMPRIA and BMPRII mRNA levels were significantly higher than that of BMPR1B throughout ovary development. BMPRIA and BMPRII mRNA levels increased significantly on E14 and declined by P5 through P6. Whereas BMPRII mRNA increased again by P7, BMPRIA mRNA levels increased through P8 concurrent with primordial follicle formation. In contrast, BMPRIB mRNA levels increased greater than 10-fold on P7-9, with a further 3-fold increase by P10. BMPR proteins were low in the somatic cells and oocytes on E13 but increased progressively during postnatal development. BMPR expression in somatic cells increased markedly on P8. Whereas BMPRII expression declined by P10 and remained steady thereafter, BMPRIA protein expression fluctuated until P15 when it became low and steady. Overall, BMPRIB immunoreactivity also declined by P10 and then remained low in the interstitial cells through P15. FSH antiserum treatment on E12 significantly attenuated receptor mRNA and protein levels by P8, but equine chorionic gonadotropin replacement on P1 reversed the inhibition. Furthermore, FSH in vitro up-regulated BMPR levels in P4 ovaries. This unique pattern of BMPR expression in the oocytes and somatic cells during perinatal ovary development suggests that BMP may play a regulatory role in primordial follicle formation. Furthermore, FSH may regulate BMP action by modulating the expression of its receptors.

scholarly journals G-protein-coupled-receptor kinases mediate TNFα-induced NF-κB signalling via direct interaction with and phosphorylation of IκBα

2009 ◽  
Vol 425 (1) ◽  
pp. 169-180 ◽  
Author(s):  
Sonika Patial ◽  
Jiansong Luo ◽  
Katie J. Porter ◽  
Jeffrey L. Benovic ◽  
Narayanan Parameswaran
Keyword(s):  
G Protein ◽  
Direct Interaction ◽  
Signalling Pathway ◽  
Raw 264.7 ◽  
G Protein Coupled Receptor ◽  
Raw 264.7 Macrophages ◽  
Iκb Kinase ◽  
Receptor Kinases ◽  
G Protein Coupled

TNFα (tumour necrosis factor α) is a multifunctional cytokine involved in the pathophysiology of many chronic inflammatory diseases. TNFα activation of the NF-κB (nuclear factor κB) signalling pathway particularly in macrophages has been implicated in many diseases. We demonstrate in the present study that GRK2 and GRK5 (G-protein-coupled-receptor kinases 2 and 5) regulate TNFα-induced NF-κB signalling in Raw 264.7 macrophages. RNAi (RNA interference) knockdown of GRK2 or GRK5 in macrophages significantly inhibited TNFα-induced IκBα (inhibitory κBα) phosphorylation and degradation, NF-κB activation and expression of the NF-κB-regulated gene MIP1β (macrophage inflammatory protein 1β). Consistent with these results, overexpression of GRK2 or GRK5 enhanced TNFα-induced NF-κB activity. In addition, we show that GRK2 and GRK5 interacted with IκBα via the N-terminal domain of IκBα and that IκBα is a substrate for GRK2 and GRK5 in vitro. Furthermore, we also found that GRK5, but not GRK2, phosphorylated IκBα at the same amino acid residues (Ser32/Ser36) as that of IKKβ (IκB kinase β). Interestingly, associated with these results, knockdown of IKKβ in Raw 264.7 macrophages did not affect TNFα-induced IκBα phosphorylation. Taken together, these results demonstrate that both GRK2 and GRK5 are important and novel mediators of a non-traditional IκBα/NF-κB signalling pathway.

scholarly journals G-protein-coupled receptor 30 mediates the effects of estrogen on endothelial cell tube formation in vitro

2017 ◽  
Vol 39 (6) ◽  
pp. 1461-1467 ◽  
Author(s):  
Liyuan Zhou ◽  
Hong Chen ◽  
Xun Mao ◽  
Hongbo Qi ◽  
Philip N. Baker ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
G Protein ◽  
Tube Formation ◽  
G Protein Coupled Receptor ◽  
G Protein Coupled
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