scholarly journals In vitro profiling of orphan G protein coupled receptor (GPCR) constitutive activity

2021 ◽  
Author(s):  
Lyndsay R. Watkins ◽  
Cesare Orlandi
Keyword(s):  
G Protein ◽  
Constitutive Activity ◽  
G Protein Coupled Receptor ◽  
G Protein Coupled

scholarly journals Mutational analysis of the R33-encoded G protein-coupled receptor of rat cytomegalovirus: identification of amino acid residues critical for cellular localization and ligand-independent signalling

2004 ◽  
Vol 85 (4) ◽  
pp. 897-909 ◽  
Author(s):  
Yvonne K. Gruijthuijsen ◽  
Erik V. H. Beuken ◽  
Martine J. Smit ◽  
Rob Leurs ◽  
Cathrien A. Bruggeman ◽  
...  
Keyword(s):  
G Protein ◽  
Cellular Localization ◽  
Mutational Analysis ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Constitutive Activity ◽  
G Protein Coupled Receptor ◽  
Human Cmv ◽  
G Protein Coupled

The rat cytomegalovirus (RCMV) R33 gene encodes a G protein-coupled receptor (GPCR), pR33, which possesses agonist-independent, constitutive signalling activity. To characterize this activity further, we generated a series of point and deletion mutants of pR33. Both expression of and signalling by the mutants was evaluated. Several point mutants were generated that contained modifications in the NRY motif. This motif, at aa 130–132 of pR33, is the counterpart of the common DRY motif of GPCRs, which is known to be involved in G protein coupling. We found that mutation of the asparagine residue within the NRY motif of pR33 (N130) to aspartic acid resulted in a mutant (N130D) with similar signalling characteristics to the wild-type (WT) protein, indicating that N130 is not the determinant of constitutive activity of pR33. Interestingly, a mutant carrying an alanine at aa 130 (N130A) was severely impaired in Gq/11-mediated, constitutive activation of phospholipase C, whereas it displayed similar levels of activity to pR33 in Gi/0-mediated signalling. Another protein that contained a modified NRY motif, R131A, did not show constitutive activity, whereas mutants Y132F and Y132A displayed similar activities to the WT receptor. This indicated that residue R131 is critical for pR33 function in vitro, whereas Y132 is not. Finally, we identified two consecutive arginines within the C-terminal tails of both pR33 and its homologue from human CMV, pUL33, which are important for correct cell-surface expression of these receptors.

Involvement of the constitutive activity of the GHS-R1a (ghrelin G-protein coupled receptor) in the tumorigenesis of somatotroph adenomas

2013 ◽  
Author(s):  
Yves Louis Mear ◽  
Xavier Come Donato ◽  
Marie Pierre Blanchard ◽  
Celine Defilles ◽  
Christophe Lisbonis ◽  
...  
Keyword(s):  
G Protein ◽  
Constitutive Activity ◽  
G Protein Coupled Receptor ◽  
G Protein Coupled

scholarly journals Roles of GP33, a guinea pig cytomegalovirus-encoded G protein-coupled receptor homolog, in cellular signaling, viral growth and inflammation in vitro and in vivo

2018 ◽  
Vol 14 (12) ◽  
pp. e1007487 ◽  
Author(s):  
Miei Takeda ◽  
Shinji Watanabe ◽  
Harutaka Katano ◽  
Kazuma Noguchi ◽  
Yuko Sato ◽  
...  
Keyword(s):  
Guinea Pig ◽  
G Protein ◽  
Cellular Signaling ◽  
G Protein Coupled Receptor ◽  
Viral Growth ◽  
Guinea Pig Cytomegalovirus ◽  
G Protein Coupled

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 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

G protein-coupled receptor 120 signaling regulates ghrelin secretion in vivo and in vitro

2014 ◽  
Vol 306 (1) ◽  
pp. E28-E35 ◽  
Author(s):  
Zhi Gong ◽  
Makoto Yoshimura ◽  
Sayaka Aizawa ◽  
Reiko Kurotani ◽  
Jeffrey M. Zigman ◽  
...  
Keyword(s):  
G Protein ◽  
Cell Lines ◽  
G Protein Coupled Receptor ◽  
Growth Hormone Secretagogue Receptor ◽  
Growth Hormone Secretagogue ◽  
Expression Levels ◽  
Ghrelin Secretion ◽  
G Protein Coupled

Ghrelin, an endogenous ligand for the growth hormone secretagogue receptor, is produced predominantly in the stomach. It has been reported that endogenous ghrelin levels are increased by fasting and decreased immediately after feeding and that fasting-induced ghrelin release is controlled by the sympathetic nervous system. However, the mechanisms of plasma ghrelin decrement after feeding are poorly understood. Here, we studied the control of ghrelin secretion using ghrelin-producing cell lines and found that these cells express high levels of mRNA encoding G-protein coupled receptor 120 (GPR120). Addition of GW-9508 (a GPR120 chemical agonist) and α-linolenic acid (a natural ligand for GPR120) inhibited the secretion of ghrelin by ∼50 and 70%, respectively. However, the expression levels of preproghrelin and ghrelin O-acyltransferase (GOAT) mRNAs were not influenced by GW-9508. In contrast, the expression levels of prohormone convertase 1 were decreased significantly by GW-9508 incubation. Moreover, we observed that the inhibitory effect of GW-9508 on ghrelin secretion was blocked by a small interfering RNA (siRNA) targeting the sequence of GPR120. Furthermore, pretreatment with GW-9508 blocked the effect of the norepinephrine (NE)-induced ghrelin elevation in ghrelin cell lines. In addition, we showed that GW-9508 inhibited ghrelin secretion via extracellular signal-regulated kinase activity in ghrelin cell lines. Finally, we found that GW-9508 decreased plasma ghrelin levels in mice. These results suggest that the decrease of ghrelin secretion after feeding is induced partially by long-chain fatty acids that act directly on gastric GPR120-expressing ghrelin cells.

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