β-Arrestin 2 Is a Mediator of GnRH-(1–5) Signaling in Immortalized GnRH Neurons

We have previously demonstrated that the cleavage product of the full-length GnRH, GnRH-(1–5), is biologically active, binds G protein-coupled receptor 173 (GPR173), and inhibits the migration of cells in the immortalized GnRH-secreting GN11 cell. In this study, we attempted to characterize the GnRH-(1–5) intracellular signaling mechanism. To determine whether the signaling pathway mediating GnRH-(1–5) regulation of migration involves a G protein-dependent mechanism, cells were treated with a generic G protein antagonist in the presence and absence of GnRH-(1–5), and a wound-healing assay was conducted to measure migration. G Protein antagonist 2 treatment abolished the GnRH-(1–5) inhibition of migration, indicating that the mechanism of GnRH-(1–5) is G protein coupled. To identify the potential Gα-subunit recruited by GnRH-(1–5) binding GPR173, we measured the second messengers cAMP and inositol triphosphate levels. GnRH-(1–5) treatment did not alter cAMP levels relative to cells treated with vehicle or forskolin, suggesting that GnRH-(1–5) does not couple to the Gαs or Gαi subunits. Similarly, inositol triphosphate levels remained unchanged with GnRH-(1–5) treatment, indicating a mechanism not mediated by the Gαq/11 subunit. Therefore, we also examined whether GnRH-(1–5) activating GPR173 deviated from the canonical G protein-coupled receptor signaling pathway by coupling to β-arrestin 1/2 to regulate migration. Our coimmunoprecipitation studies indicate that GnRH-(1–5) induces the rapid interaction between GPR173 and β-arrestin 2 in GN11 cells. Furthermore, we demonstrate that this association recruits phosphatase and tensin homolog to mediate the downstream action of GnRH-(1–5). These findings suggest that the GnRH-(1–5) mechanism deviates from the canonical G protein-coupled receptor pathway to regulate cell migration in immortalized GnRH neurons.

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The Metabolite GnRH-(1-5) Inhibits the Migration of Immortalized GnRH Neurons

Endocrinology ◽

10.1210/en.2012-1746 ◽

2013 ◽

Vol 154 (2) ◽

pp. 783-795 ◽

Cited By ~ 18

Author(s):

Darwin O. Larco ◽

Madelaine Cho-Clark ◽

Shaila K. Mani ◽

T. John Wu

Keyword(s):

G Protein ◽

Signal Transduction Pathway ◽

Neuronal Cell ◽

Cellular Migration ◽

Biologically Active ◽

G Protein Coupled Receptor ◽

Gnrh Neurons ◽

Dependent Signal ◽

And Function ◽

G Protein Coupled

The decapeptide GnRH is an important regulator of reproductive behavior and function. In the extracellular matrix, GnRH is metabolized by the endopeptidase EC3.4.24.15 (EP24.15) to generate the pentapeptide GnRH-(1-5). In addition to its expression in the adult hypothalamus, EP24.15 is expressed along the migratory path of GnRH-expressing neurons during development. Although we have previously demonstrated a role for EP24.15 in the generation of the biologically active pentapeptide GnRH-(1-5) in regulating GnRH expression and mediating sexual behavior during adulthood in rodents, the modulatory role of GnRH-(1-5) in the migration of GnRH neurons during development remains unknown. To address this information gap, we examined the effect of GnRH-(1-5) on the cellular migration of a premigratory GnRH-secreting neuronal cell line, the GN11 cell, using a wound-healing assay. Dose- and time-response studies demonstrated that GnRH-(1-5) significantly delayed wound closure. We then sought to identify the mechanism by which GnRH-(1-5) inhibits migration. Because the cognate GnRH receptor is a G protein-coupled receptor, we examined whether GnRH-(1-5) regulates migration by also activating a G protein-coupled receptor. Using a high-throughput β-arrestin recruitment assay, we identified an orphan G protein-coupled receptor (GPR173) that was specifically activated by GnRH-(1-5). Interestingly, small interfering RNA to GPR173 reversed the GnRH-(1-5)−mediated inhibition on migration of GN11 neurons. Furthermore, we also demonstrate that the GnRH-(1-5)−activated GPR173-dependent signal transduction pathway involves the activation of the signal transducer and activator of transcription 3 in GnRH migration. These findings indicate a potential regulatory role for GnRH-(1-5) in GnRH neuronal migration during development.

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Baicalein suppresses 17-β-estradiol-induced migration, adhesion and invasion of breast cancer cells via the G protein-coupled receptor 30 signaling pathway

Oncology Reports ◽

10.3892/or.2015.3786 ◽

2015 ◽

Vol 33 (4) ◽

pp. 2077-2085 ◽

Cited By ~ 34

Author(s):

DANDAN SHANG ◽

ZHENG LI ◽

ZHUXIA ZHU ◽

HUAMEI CHEN ◽

LUJUN ZHAO ◽

...

Keyword(s):

Breast Cancer ◽

Cancer Cells ◽

Signaling Pathway ◽

G Protein ◽

Breast Cancer Cells ◽

G Protein Coupled Receptor ◽

G Protein Coupled ◽

Adhesion And Invasion

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

International Journal of Molecular Sciences ◽

10.3390/ijms20174300 ◽

2019 ◽

Vol 20 (17) ◽

pp. 4300 ◽

Cited By ~ 2

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.

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Involvement of Nuclear Factor κB (NF-κB) Signaling Pathway in Regulation of Cardiac G Protein-coupled Receptor Kinase 5 (GRK5) Expression

Journal of Biological Chemistry ◽

10.1074/jbc.m111.324566 ◽

2012 ◽

Vol 287 (16) ◽

pp. 12771-12778 ◽

Cited By ~ 26

Author(s):

Kazi N. Islam ◽

Walter J. Koch

Keyword(s):

Signaling Pathway ◽

G Protein ◽

Nuclear Factor Κb ◽

Nuclear Factor ◽

Receptor Kinase ◽

G Protein Coupled Receptor ◽

G Protein Coupled

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Activation of Intracellular Signaling Pathways by the Murine Cytomegalovirus G Protein-Coupled Receptor M33 Occurs via PLC-β/PKC-Dependent and -Independent Mechanisms

Journal of Virology ◽

10.1128/jvi.02116-08 ◽

2009 ◽

Vol 83 (16) ◽

pp. 8141-8152 ◽

Cited By ~ 15

Author(s):

Joseph D. Sherrill ◽

Melissa P. Stropes ◽

Olivia D. Schneider ◽

Diana E. Koch ◽

Fabiola M. Bittencourt ◽

...

Keyword(s):

Transcription Factor ◽

Protein Kinase ◽

Signaling Pathways ◽

G Protein ◽

Intracellular Signaling ◽

Murine Cytomegalovirus ◽

Dependent Manner ◽

G Protein Coupled Receptor ◽

G Protein Coupled

ABSTRACT The presence of numerous G protein-coupled receptor (GPCR) homologs within the herpesvirus genomes suggests an essential role for these genes in viral replication in the infected host. Such is the case for murine cytomegalovirus (MCMV), where deletion of the M33 GPCR or replacement of M33 with a signaling defective mutant has been shown to severely attenuate replication in vivo. In the present study we utilized a genetically altered version of M33 (termed R131A) in combination with pharmacological inhibitors to further characterize the mechanisms by which M33 activates downstream signaling pathways. This R131A mutant of M33 fails to support salivary gland replication in vivo and, as such, is an important tool that can be used to examine the signaling activities of M33. We show that M33 stimulates the transcription factor CREB via heterotrimeric Gq/11 proteins and not through promiscuous coupling of M33 to the Gs pathway. Using inhibitors of signaling molecules downstream of Gq/11, we demonstrate that M33 stimulates CREB transcriptional activity in a phospholipase C-β and protein kinase C (PKC)-dependent manner. Finally, utilizing wild-type and R131A versions of M33, we show that M33-mediated activation of other signaling nodes, including the mitogen-activated protein kinase family member p38α and transcription factor NF-κB, occurs in the absence of Gq/11 and PKC signaling. The results from the present study indicate that M33 utilizes multiple mechanisms to modulate intracellular signaling cascades and suggest that signaling through PLC-β and PKC plays a central role in MCMV pathogenesis in vivo.

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