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