A Simple and Powerful Flow Cytometric Method for the Simultaneous Determination of Multiple Parameters at G Protein-Coupled Receptor Subtypes

ChemBioChem ◽  
2006 ◽  
Vol 7 (9) ◽  
pp. 1400-1409 ◽  
Author(s):  
Erich Schneider ◽  
Matthias Mayer ◽  
Ralf Ziemek ◽  
Liantao Li ◽  
Christoph Hutzler ◽  
...  
Keyword(s):  
Simultaneous Determination ◽  
G Protein ◽  
Receptor Subtypes ◽  
G Protein Coupled Receptor ◽  
Multiple Parameters ◽  
Flow Cytometric Method ◽  
Flow Cytometric ◽  
G Protein Coupled

Development of a Novel SNAP-Epitope Tag/Near-Infrared Imaging Assay to Quantify G Protein-Coupled Receptor Degradation in Human Cells

2021 ◽  
pp. 247255522097979
Author(s):  
Kyung-Soon Lee ◽  
Edelmar Navaluna ◽  
Nicole M. Marsh ◽  
Eric M. Janezic ◽  
Chris Hague
Keyword(s):  
G Protein ◽  
Adrenergic Receptor ◽  
Near Infrared ◽  
Human Cells ◽  
Receptor Subtypes ◽  
G Protein Coupled Receptor ◽  
Functional Responses ◽  
Epitope Tag ◽  
Nir Imaging ◽  
G Protein Coupled

We have developed a novel reporter assay that leverages SNAP-epitope tag/near-infrared (NIR) imaging technology to monitor G protein-coupled receptor (GPCR) degradation in human cell lines. N-terminal SNAP-tagged GPCRs were subcloned and expressed in human embryonic kidney (HEK) 293 cells and then subjected to 24 h of cycloheximide (CHX)-chase degradation assays to quantify receptor degradation half-lives ( t1/2) using LICOR NIR imaging–polyacrylamide gel electrophoresis (PAGE) analysis. Thus far, we have used this method to quantify t1/2 for all nine adrenergic (ADRA1A, ADRA1B, ADRA1D, ADRA2A, ADRA2B, ADRA2C, ADRB1, ADRB2, ADRB3), five somatostatin (SSTR1, SSTR2, SSTR3, SSTR4, SSTR5), four chemokine (CXCR1, CXCR2, CXCR3, CXCR5), and three 5-HT2 (5HT2A, 5HT2B, 5HT2C) receptor subtypes. SNAP-GPCR-CHX degradation t1/2 values ranged from 0.52 h (ADRA1D) to 5.5 h (SSTR3). On the contrary, both the SNAP-tag alone and SNAP-tagged and endogenous β-actin were resistant to degradation with CHX treatment. Treatment with the proteasome inhibitor bortezomib produced significant but variable increases in SNAP-GPCR protein expression levels, indicating that SNAP-GPCR degradation primarily occurs through the proteasome. Remarkably, endogenous β2-adrenergic receptor/ADRB2 dynamic mass redistribution functional responses to norepinephrine were significantly decreased following CHX treatment, with a time course equivalent to that observed with the SNAP-ADRB2 degradation assay. We subsequently adapted this assay into a 96-well glass-bottom plate format to facilitate high-throughput GPCR degradation screening. t1/2 values quantified for the α1-adrenergic receptor subtypes (ADRA1A, ADRA1B, ADR1D) using the 96-well-plate format correlated with t1/2 values quantified using NIR-PAGE imaging analysis. In summary, this novel assay permits precise quantitative analysis of GPCR degradation in human cells and can be readily adapted to quantify degradation for any membrane protein of interest.

Recent advances in the determination of G protein-coupled receptor structures

2018 ◽  
Vol 51 ◽  
pp. 28-34 ◽  
Author(s):  
David M Thal ◽  
Ziva Vuckovic ◽  
Christopher J Draper-Joyce ◽  
Yi-Lynn Liang ◽  
Alisa Glukhova ◽  
...  
Keyword(s):  
G Protein ◽  
G Protein Coupled Receptor ◽  
Recent Advances ◽  
G Protein Coupled

scholarly journals Probe dependency in the determination of ligand binding kinetics at a prototypical G protein-coupled receptor

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Reggie Bosma ◽  
Leigh A. Stoddart ◽  
Victoria Georgi ◽  
Monica Bouzo-Lorenzo ◽  
Nick Bushby ◽  
...  
Keyword(s):  
Ligand Binding ◽  
G Protein ◽  
Binding Kinetics ◽  
G Protein Coupled Receptor ◽  
G Protein Coupled

scholarly journals Structural determination of the mechanism of domain separation of G‐protein‐coupled receptor kinase 4g

2019 ◽  
Vol 33 (S1) ◽  
Author(s):  
Jae Kyung Yeon ◽  
Garima Sharma ◽  
Wesley M. Botello‐Smith ◽  
Yun Lyna Luo ◽  
Bradley T. Andresen
Keyword(s):  
G Protein ◽  
Structural Determination ◽  
Receptor Kinase ◽  
G Protein Coupled Receptor ◽  
G Protein Coupled ◽  
Domain Separation

scholarly journals Label-Free Analysis with Multiple Parameters Separates G Protein-Coupled Receptor Signaling Pathways

2020 ◽  
Vol 92 (21) ◽  
pp. 14509-14516
Author(s):  
Teemu Suutari ◽  
Sabrina N. Rahman ◽  
Henry F. Vischer ◽  
Dick van Iperen ◽  
Arto Merivaara ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
G Protein ◽  
Receptor Signaling ◽  
Label Free ◽  
G Protein Coupled Receptor ◽  
Multiple Parameters ◽  
G Protein Coupled
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