scholarly journals G-Protein-Coupled Receptor-Mediated MAPK and PI3-Kinase Signaling Is Maintained in Chinese Hamster Ovary Cells after γ-Irradiation

2011 ◽  
Vol 17 (3) ◽  
pp. 361-369 ◽  
Author(s):  
Ronald I. W. Osmond ◽  
M. Henry Martin-Harris ◽  
Michael F. Crouch ◽  
Janet Park ◽  
Eric Morreale ◽  
...  
Keyword(s):  
G Protein ◽  
Cell Lines ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Calcium Flux ◽  
Kinase Signaling ◽  
G Protein Coupled Receptor ◽  
Screening Programs ◽  
Cellular Models ◽  
G Protein Coupled

To expedite G-protein-coupled receptor (GPCR) drug screening studies, cell lines amenable to transfection (e.g. CHO cells) have been widely used as cellular models. These cells can be frozen in a ready-to-use format, allowing screening of a single batch of cells and validation of the cellular material prior to the screening run. A common method used to deliver frozen cells to screening programs is to γ-irradiate the cells, abrogating cell division after thawing and ensuring consistency in the number of cells analyzed per well. With the recognition that signaling proteins such as ERK and Akt are important markers of GPCR activation, along with the availability of suitable assays for their measurement, these outputs have become important for GPCR screening programs. Here we show that several γ-irradiated and frozen CHO-K1 cell lines expressing transfected GPCRs, initially optimized for performing cAMP or AequoScreen calcium flux assays, can be used for the measurement of GPCR-mediated ERK and Akt phosphorylation. Furthermore, CHO-K1 cells transfected with NOP or GAL1 receptors show pharmacology for a number of agonists and antagonists that is consistent with non-irradiated cultured lines. These data indicate that γ-irradiated CHO-K1 cells can be reliably used for the measurement of GPCR-mediated kinase signaling outputs.

scholarly journals Lipid Receptor GPR31 (G-Protein–Coupled Receptor 31) Regulates Platelet Reactivity and Thrombosis Without Affecting Hemostasis

2020 ◽  
Author(s):  
Layla Van Doren ◽  
Nga Nguyen ◽  
Christopher Garzia ◽  
Elizabeth Fletcher ◽  
Ryan Stevenson ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Carotid Artery ◽  
G Protein ◽  
Arterial Thrombosis ◽  
Platelet Reactivity ◽  
Human Platelets ◽  
Calcium Flux ◽  
G Protein Coupled Receptor ◽  
Carotid Artery Injury ◽  
G Protein Coupled

Objective: 12-LOX (12-lipoxygenase) produces a number of bioactive lipids including 12(S)-HETE that are involved in inflammation and platelet reactivity. The GPR31 (G-protein–coupled receptor 31) is the proposed receptor of 12(S)-HETE; however, it is not known whether the 12(S)-HETE-GPR31 signaling axis serves to enhance or inhibit platelet activity. Approach and Results: Using pepducin technology and biochemical approaches, we provide evidence that 12(S)-HETE-GPR31 signals through Gi to enhance PAR (protease-activated receptor)-4–mediated platelet activation and arterial thrombosis using both human platelets and mouse carotid artery injury models. 12(S)-HETE suppressed AC (adenylyl cyclase) activity through GPR31 and resulted in Rap1 and p38 activation and low but detectable calcium flux but did not induce platelet aggregation. A GPR31 third intracellular (i3) loop–derived pepducin, GPR310 (G-protein–coupled receptor 310), significantly inhibited platelet aggregation in response to thrombin, collagen, and PAR4 agonist, AYPGKF, in human and mouse platelets but relative sparing of PAR1 agonist SFLLRN in human platelets. GPR310 treatment gave a highly significant 80% protection ( P =0.0018) against ferric chloride–induced carotid artery injury in mice by extending occlusion time, without any effect on tail bleeding. PAR4-mediated dense granule secretion and calcium flux were both attenuated by GPR310. Consistent with these results, GPR310 inhibited 12(S)-HETE–mediated and PAR4-mediated Rap1-GTP and RASA3 translocation to the plasma membrane and attenuated PAR4-Akt and ERK activation. GPR310 caused a right shift in thrombin-mediated human platelet aggregation, comparable to the effects of inhibition of the Gi-coupled P2Y 12 receptor. Co-immunoprecipitation studies revealed that GPR31 and PAR4 form a heterodimeric complex in recombinant systems. Conclusions: The 12-LOX product 12(S)-HETE stimulates GPR31-Gi–signaling pathways, which enhance thrombin-PAR4 platelet activation and arterial thrombosis in human platelets and mouse models. Suppression of this bioactive lipid pathway, as exemplified by a GPR31 pepducin antagonist, may provide beneficial protective effects against platelet aggregation and arterial thrombosis with minimal effect on hemostasis.

scholarly journals Role of the G-Protein-Coupled Receptor Signaling Pathway in Insecticide Resistance

2019 ◽  
Vol 20 (17) ◽  
pp. 4300 ◽  
Author(s):  
Ting Li ◽  
Nannan Liu
Keyword(s):  
Insecticide Resistance ◽  
Cell Line ◽  
Signaling Pathway ◽  
G Protein ◽  
Cell Lines ◽  
Sf9 Cells ◽  
Camp Production ◽  
G Protein Coupled Receptor ◽  
G Protein Coupled

The G-protein-coupled receptor (GPCR) regulated intracellular signaling pathway is known to be involved in the development of insecticide resistance in the mosquito, Culex quinquefasciatus. To elucidate the specific role of each effector in the GPCR regulating pathway, we initially expressed a GPCR, G-protein alpha subunit (Gαs), adenylate cyclase (AC), and protein kinase A (PKA) in insect Spodoptera frugiperda (Sf9) cells and investigated their regulation function on cyclic AMP (cAMP) production and PKA activity. GPCR, Gαs, and AC individually expressed Sf9 cells showed higher cAMP production as the expression of each effector increased. All the effector-expressed cell lines showed increased PKA activity however. Moreover, Sf9 cytochrome P450 gene expression and cell tolerance to permethrin were examined. The relative expression of CYP9A32gene in Sf9 cells tested was significantly increased in all effector-expressed cell lines compared to a control cell line; these effector-expressed cell lines also showed significantly higher tolerance to permethrin. Inhibitor treatments on each effector-expressed cell line revealed that Bupivacaine HCl and H89 2HCl robustly inhibited cAMP production and PKA activity, respectively, resulting in decreased tolerance to permethrin in all cell lines. The synergistic functions of Bupivacaine HCl and H89 2HCl with permethrin were further examined in Culex mosquito larvae, providing a valuable new information for mosquito control strategies.

The obestatin/G-protein coupled receptor 39 (GPR39) system in pancreatic cancer: proliferation studies in pancreatic cancer cell lines

Pancreatology ◽  
2019 ◽  
Vol 19 ◽  
pp. S162
Author(s):  
Lara Estevez-Perez ◽  
Saul Leal-Lopez ◽  
Vanesa Veloso-Noya ◽  
Begoña Otero-Alen ◽  
Carlos Seoane-Mosteiro ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
G Protein ◽  
Cell Lines ◽  
Cancer Cell ◽  
Pancreatic Cancer Cell ◽  
Cancer Cell Lines ◽  
G Protein Coupled Receptor ◽  
Cancer Proliferation ◽  
G Protein Coupled

Pathways for internalization and recycling of the chemokine receptor CCR5

Blood ◽  
2002 ◽  
Vol 99 (3) ◽  
pp. 785-791 ◽  
Author(s):  
Anja Mueller ◽  
Eamonn Kelly ◽  
Philip G. Strange
Keyword(s):  
Cell Surface ◽  
G Protein ◽  
Chemokine Receptor ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Coated Pits ◽  
Early Endosomes ◽  
Chemokine Receptor Ccr5 ◽  
G Protein Coupled ◽  
Receptor Ccr5

Abstract M-tropic human immunodeficiency virus (HIV-1) strains enter the cell after interaction with their receptors, CD4 and the G-protein–coupled chemokine receptor CCR5. The number of cell surface CCR5 molecules is thought to be important in determining the infection rate for HIV. Cell surface CCR5 is dependent on the rate of receptor internalization and recycling. Internalization of G-protein–coupled receptors after agonist activation is thought to occur either through clathrin-coated pits or through caveolae. In this study, the role of these different pathways was investigated in Chinese hamster ovary cells expressing CCR5 using specific inhibitors. Internalization of CCR5 after chemokine treatment was inhibited by sucrose, indicating a role for the clathrin-coated pit pathway. Activation of CCR5 leads to arrestin-2 movement in the cells, providing further evidence for the involvement of clathrin-coated pits. Nystatin and filipin also affected the rate of internalization of CCR5, indicating a role for caveolae. Using inhibitors of vesicle transport in the cell, it was found that the CCR5 recycling pathway is independent of the Golgi apparatus and late endosomes. Protein synthesis is not involved in receptor recovery. It seems likely that after internalization, CCR5 is directed to early endosomes and subsequently recycled to the cell surface.

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.

Aberrantly expressed recoverin is functionally associated with G-protein-coupled receptor kinases in cancer cell lines

2003 ◽  
Vol 300 (3) ◽  
pp. 669-673 ◽  
Author(s):  
Yasuhiro Miyagawa ◽  
Hiroshi Ohguro ◽  
Hiroki Odagiri ◽  
Ikuyo Maruyama ◽  
Tadao Maeda ◽  
...  
Keyword(s):  
G Protein ◽  
Cell Lines ◽  
Cancer Cell ◽  
Cancer Cell Lines ◽  
G Protein Coupled Receptor ◽  
Receptor Kinases ◽  
G Protein Coupled
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