A cell-based time-resolved fluorescence assay for selection of antibody reagents for G protein-coupled receptor immunohistochemistry

2004 ◽  
Vol 291 (1-2) ◽  
pp. 123-135 ◽  
Author(s):  
Jui-Lan Su ◽  
Jim Fornwald ◽  
Philip Rivers ◽  
Susan Goldsworthy ◽  
Noeleen A. Looney ◽  
...  
Keyword(s):  
G Protein ◽  
Fluorescence Assay ◽  
Time Resolved Fluorescence ◽  
G Protein Coupled Receptor ◽  
Time Resolved ◽  
A Cell ◽  
G Protein Coupled ◽  
Selection Of

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.

Selection of Active ScFv to G-Protein-Coupled Receptor CCR5 Using Surface Antigen-Mimicking Peptides†

Biochemistry ◽  
2004 ◽  
Vol 43 (39) ◽  
pp. 12575-12584 ◽  
Author(s):  
Ying Zhang ◽  
Chadler Pool ◽  
Kristen Sadler ◽  
He-ping Yan ◽  
Jennifer Edl ◽  
...  
Keyword(s):  
G Protein ◽  
Surface Antigen ◽  
G Protein Coupled Receptor ◽  
G Protein Coupled ◽  
Mimicking Peptides ◽  
Receptor Ccr5 ◽  
Selection Of

scholarly journals The oligodendrocyte-specific G protein–coupled receptor GPR17 is a cell-intrinsic timer of myelination

2009 ◽  
Vol 12 (11) ◽  
pp. 1398-1406 ◽  
Author(s):  
Ying Chen ◽  
Heng Wu ◽  
Shuzong Wang ◽  
Hisami Koito ◽  
Jianrong Li ◽  
...  
Keyword(s):  
G Protein ◽  
G Protein Coupled Receptor ◽  
A Cell ◽  
G Protein Coupled

scholarly journals Real-Time Detection of Interactions between the Human Oxytocin Receptor and G Protein-Coupled Receptor Kinase-2

2004 ◽  
Vol 18 (5) ◽  
pp. 1277-1286 ◽  
Author(s):  
Ahmed Hasbi ◽  
Dominic Devost ◽  
Stéphane A. Laporte ◽  
Hans H. Zingg
Keyword(s):  
Real Time ◽  
G Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Oxytocin Receptor ◽  
Dominant Negative ◽  
Receptor Kinase ◽  
G Protein Coupled Receptor ◽  
Time Resolved ◽  
G Protein Coupled

Abstract Although the oxytocin receptor (OTR) mediates many important functions including uterine contractions, milk ejection, and maternal behavior, the mechanisms controlling agonist-induced OTR desensitization have remained unclear, and attempts to demonstrate involvement of a G protein-coupled receptor kinase (GRK) have so far failed. Using the OTR as a model, we demonstrate here directly for the first time the dynamics of agonist-induced interactions of a GRK with a G protein-coupled receptor in real time, using time-resolved bioluminescence resonance energy transfer. GRK2/receptor interactions started within 4 sec, peaked at 10 sec, and decreased to less than 40% within 8 min. By contrast, β-arrestin/OTR interactions initiated only at 10 sec, reached plateau levels at 120 sec, but remained stable with little decrease thereafter. Physical GRK2/OTR association was further demonstrated by coimmunoprecipitation of endogenous GRK2 with activated OTR. In COS-7 cells, which express low levels of GRK2 and β-arrestin, overexpression of GRK2 and β-arrestin increased receptor phosphorylation, desensitization, and internalization to the high levels observed in human embryonic kidney 293 cells. By contrast, specific inhibition of endogenous GRK2 by dominant-negative mutants robustly inhibited OTR phosphorylation and internalization as well as arrestin/OTR interactions. These data characterize the temporal and causal relationship of GRK-2/OTR and β-arrestin/OTR interactions and establish GRK/OTR interaction as a prerequisite for β-arrestin-mediated OTR desensitization.

NPY, ET-1, and Ang II nuclear receptors in human endocardial endothelial cellsThis paper is one of a selection of papers published in this Special Issue, entitled The Nucleus: A Cell Within A Cell.

2006 ◽  
Vol 84 (3-4) ◽  
pp. 299-307 ◽  
Author(s):  
Danielle Jacques ◽  
Sawsan Sader ◽  
Claudine Perreault ◽  
Dima Abdel-Samad ◽  
Farah Jules ◽  
...  
Keyword(s):  
G Protein ◽  
G Protein Coupled Receptors ◽  
Membrane Receptors ◽  
Ang Ii ◽  
Special Issue ◽  
A Cell ◽  
Endocardial Endothelial Cells ◽  
Plasma Membrane Receptors ◽  
G Protein Coupled ◽  
Selection Of

Neuropeptide Y (NPY), endothelin-1 (ET-1), and angiotensin II (Ang II) are peptides that are known to play many important roles in cardiovascular homeostasis. The physiological actions of these peptides are thought to be primarily mediated by plasma membrane receptors that belong to the G-protein-coupled receptor superfamily. However, there is increasing evidence that suggests the existence of functional G-protein-coupled receptors at the level of the nucleus and that the nucleus could be a cell within a cell. Here, we review our work showing the presence in the nucleus of the NPY Y1 receptor, the ETA and ETB receptors, as well as the AT1 and AT2 receptors and their respective ligands. This work was carried out in 20-week-old fetal human endocardial endothelial cells. Our results demonstrate that nuclear Y1, AT1, and ETA receptors modulate nuclear calcium in these cells.

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