The Conserved Seven-Transmembrane Sequence NP(X)2,3Y of the G-Protein-Coupled Receptor Superfamily Regulates Multiple Properties of the .beta.2-Adrenergic Receptor

Biochemistry ◽  
1995 ◽  
Vol 34 (47) ◽  
pp. 15407-15414 ◽  
Author(s):  
Larry S. Barak ◽  
Luc Menard ◽  
Stephen S. G. Ferguson ◽  
Anne-Marie Colapietro ◽  
Marc G. Caron
Keyword(s):  
G Protein ◽  
Adrenergic Receptor ◽  
G Protein Coupled Receptor ◽  
Transmembrane Sequence ◽  
Beta 2 ◽  
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.

Members of the G Protein-Coupled Receptor Kinase Family That Phosphorylate the β2-Adrenergic Receptor Facilitate Sequestration†

Biochemistry ◽  
1996 ◽  
Vol 35 (13) ◽  
pp. 4155-4160 ◽  
Author(s):  
Luc Ménard ◽  
Stephen S. G. Ferguson ◽  
Larry S. Barak ◽  
Lucie Bertrand ◽  
Richard T. Premont ◽  
...  
Keyword(s):  
G Protein ◽  
Adrenergic Receptor ◽  
Receptor Kinase ◽  
G Protein Coupled Receptor ◽  
Kinase Family ◽  
Β2 Adrenergic Receptor ◽  
G Protein Coupled

scholarly journals Regulation of β-Adrenergic Receptor Signaling by S-Nitrosylation of G-Protein-Coupled Receptor Kinase 2

Cell ◽  
2007 ◽  
Vol 129 (3) ◽  
pp. 511-522 ◽  
Author(s):  
Erin J. Whalen ◽  
Matthew W. Foster ◽  
Akio Matsumoto ◽  
Kentaro Ozawa ◽  
Jonathan D. Violin ◽  
...  
Keyword(s):  
G Protein ◽  
Adrenergic Receptor ◽  
Receptor Signaling ◽  
Receptor Kinase ◽  
G Protein Coupled Receptor ◽  
G Protein Coupled

scholarly journals G Protein-coupled Receptor Kinase 5 Regulates β1-Adrenergic Receptor Association with PSD-95

2001 ◽  
Vol 277 (2) ◽  
pp. 1607-1613 ◽  
Author(s):  
Liaoyuan A. Hu ◽  
Wei Chen ◽  
Richard T. Premont ◽  
Mei Cong ◽  
Robert J. Lefkowitz
Keyword(s):  
G Protein ◽  
Adrenergic Receptor ◽  
Receptor Kinase ◽  
G Protein Coupled Receptor ◽  
G Protein Coupled
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