Visualization and Quantitative Analysis of G Protein-Coupled Receptor−β-Arrestin Interaction in Single Cells and Specific Organs of Living Mice Using Split Luciferase Complementation

2012 ◽  
Vol 7 (5) ◽  
pp. 901-910 ◽  
Author(s):  
Hideo Takakura ◽  
Mitsuru Hattori ◽  
Masaki Takeuchi ◽  
Takeaki Ozawa
Keyword(s):  
Quantitative Analysis ◽  
G Protein ◽  
Single Cells ◽  
G Protein Coupled Receptor ◽  
Split Luciferase Complementation ◽  
G Protein Coupled ◽  
Split Luciferase

scholarly journals Quantitative Analysis of the Bidirectional Viral G-Protein-Coupled Receptor and Lytic Latency-Associated Nuclear Antigen Promoter of Kaposi's Sarcoma-Associated Herpesvirus

2012 ◽  
Vol 86 (18) ◽  
pp. 9683-9695 ◽  
Author(s):  
Isaac B. Hilton ◽  
Dirk P. Dittmer
Keyword(s):  
Quantitative Analysis ◽  
G Protein ◽  
Binding Sites ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Nuclear Antigen ◽  
G Protein Coupled Receptor ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus ◽  
G Protein Coupled

Kaposi's sarcoma-associated herpesvirus (KSHV) establishes sustained latent persistence in susceptible cells. This is dependent on the latency-associated nuclear antigen (LANA). Understanding how LANA transcription is regulated thus aids our fundamental understanding of KSHV biology. Two hundred ninety-four base pairs are sufficient to regulate LANA transcription in response to the viral RTA protein and RBPjκ. The same region controls K14/viral G-protein-coupled receptor (vGPCR) transcription in the opposite direction. We used a quantitative analysis in conjunction with specific nucleotide substitutions and defined gain-of-function and loss-of-function RTA mutants to dissect this region. We used a bidirectional reporter driving red and green luciferase to study the LANApi and K14p promoters simultaneously. This established that LANApi/K14p functions as a canonical bidirectional promoter. Both were TATA dependent. K14p was favored by ∼50-fold in this context. Eliminating the distal LANApi TATA box increased maximal output and lowered the induction threshold (T) of K14p even further. Two RBPjκ binding sites were independently required; however, at high concentrations of RTA, direct interactions with an RTA-responsive element (RRE) could complement the loss of one RBPjκ binding site. Intracellular Notch (ICN) was no longer able to activate RBPjκ in the viral context. This suggests a model whereby KSHV alters ICN-RBPjκ gene regulation. When the architecture of this pair of head-to-head RBPjκ binding sites is changed, the sites now respond exclusively to the viral transactivator RTA and no longer to the host mediator ICN.

scholarly journals Regulation of Oxytocin Receptor Responsiveness by G Protein-Coupled Receptor Kinase 6 in Human Myometrial Smooth Muscle

2009 ◽  
Vol 23 (8) ◽  
pp. 1272-1280 ◽  
Author(s):  
Jonathon M. Willets ◽  
Paul J. Brighton ◽  
Rajendra Mistry ◽  
Gavin E. Morris ◽  
Justin C. Konje ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Cell Line ◽  
G Protein ◽  
Inositol Phosphate ◽  
Single Cells ◽  
Oxytocin Receptor ◽  
Small Interfering Rnas ◽  
G Protein Coupled Receptor ◽  
G Protein Coupled

Abstract Oxytocin plays an important role in the progression, timing, and modulation of uterine contraction during labor and is widely used as an uterotonic agent. We investigated the mechanisms regulating oxytocin receptor (OTR) signaling in human primary myometrial smooth muscle cells and the ULTR cell-line. Oxytocin produced concentration-dependent increases in both total [3H]inositol phosphate accumulation and intracellular Ca2+ concentration ([Ca2+]i); however, responses were greater and more reproducible in the ULTR cell line. Assessment of phospholipase C activity in single cells revealed that the OTR desensitizes rapidly (within 5 min) in the presence of oxytocin (100 nm). To characterize OTR desensitization further, cells were stimulated with a maximally effective concentration of oxytocin (100 nm, 30 sec) followed by a variable washout period and a second identical application of oxytocin. This brief exposure to oxytocin caused a marked decrease (>70%) in OTR responsiveness to rechallenge and was fully reversed by increasing the time period between agonist challenges. To assess involvement of G protein-coupled receptor kinases (GRKs) in OTR desensitization, cells were transfected with small interfering RNAs to cause specific ≥75% knockdown of GRKs 2, 3, 5, or 6. In both primary myometrial and ULTR cells, knockdown of GRK6 largely prevented oxytocin-induced OTR desensitization; in contrast, selective depletion of GRKs 2, 3, or 5 was without effect. These data indicate that GRK6 recruitment is a cardinal effector of OTR responsiveness and provide mechanistic insight into the likely in vivo regulation of OTR signaling in uterine smooth muscle.

Development of a novel DnaE intein-based assay for quantitative analysis of G-protein-coupled receptor internalization

2011 ◽  
Vol 417 (1) ◽  
pp. 65-72 ◽  
Author(s):  
Yaping Zhang ◽  
Wen Yang ◽  
Linjie Chen ◽  
Ying Shi ◽  
Guo Li ◽  
...  
Keyword(s):  
Quantitative Analysis ◽  
G Protein ◽  
Receptor Internalization ◽  
G Protein Coupled Receptor ◽  
G Protein Coupled

scholarly journals An Update on Glioblastoma Biology, Genetics, and Current Therapies: Novel Inhibitors of the G Protein-Coupled Receptor CCR5

2021 ◽  
Vol 22 (9) ◽  
pp. 4464
Author(s):  
Tamara Lah Turnšek ◽  
Xuanmao Jiao ◽  
Metka Novak ◽  
Sriharsha Jammula ◽  
Gina Cicero ◽  
...  
Keyword(s):  
Stem Cells ◽  
G Protein ◽  
Stromal Cells ◽  
Single Cells ◽  
Therapy Response ◽  
G Protein Coupled Receptor ◽  
Glioblastoma Stem Cells ◽  
Current Therapies ◽  
G Protein Coupled ◽  
Receptor Ccr5

The mechanisms governing therapeutic resistance of the most aggressive and lethal primary brain tumor in adults, glioblastoma, have increasingly focused on tumor stem cells. These cells, protected by the periarteriolar hypoxic GSC niche, contribute to the poor efficacy of standard of care treatment of glioblastoma. Integrated proteogenomic and metabolomic analyses of glioblastoma tissues and single cells have revealed insights into the complex heterogeneity of glioblastoma and stromal cells, comprising its tumor microenvironment (TME). An additional factor, which isdriving poor therapy response is the distinct genetic drivers in each patient’s tumor, providing the rationale for a more individualized or personalized approach to treatment. We recently reported that the G protein-coupled receptor CCR5, which contributes to stem cell expansion in other cancers, is overexpressed in glioblastoma cells. Overexpression of the CCR5 ligand CCL5 (RANTES) in glioblastoma completes a potential autocrine activation loop to promote tumor proliferation and invasion. CCL5 was not expressed in glioblastoma stem cells, suggesting a need for paracrine activation of CCR5 signaling by the stromal cells. TME-associated immune cells, such as resident microglia, infiltrating macrophages, T cells, and mesenchymal stem cells, possibly release CCR5 ligands, providing heterologous signaling between stromal and glioblastoma stem cells. Herein, we review current therapies for glioblastoma, the role of CCR5 in other cancers, and the potential role for CCR5 inhibitors in the treatment of glioblastoma.

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