Abstract
Although G protein-coupled receptor (GPCR) internalization has long been considered as a major aspect of the desensitization process that tunes ligand responsiveness, internalization is also involved in receptor resensitization and signaling, as well as the ligand scavenging function of some atypical receptors. Internalization thus contributes to the diversity of GPCR-dependent signaling, and its dynamics and quantification in living cells has generated considerable interest. We developed a robust and sensitive assay to follow and quantify ligand-induced and constitutive-induced GPCR internalization but also receptor recycling in living cells. This assay is based on diffusion-enhanced resonance energy transfer (DERET) between cell surface GPCRs labeled with a luminescent terbium cryptate donor and a fluorescein acceptor present in the culture medium. GPCR internalization results in a quantifiable reduction of energy transfer. This method yields a high signal-to-noise ratio due to time-resolved measurements. For various GPCRs belonging to different classes, we demonstrated that constitutive and ligand-induced internalization could be monitored as a function of time and ligand concentration, thus allowing accurate quantitative determination of kinetics of receptor internalization but also half-maximal effective or inhibitory concentrations of compounds. In addition to its selectivity and sensitivity, we provided evidence that DERET-based internalization assay is particularly suitable for characterizing biased ligands. Furthermore, the determination of a Z′-factor value of 0.45 indicates the quality and suitability of DERET-based internalization assay for high-throughput screening (HTS) of compounds that may modulate GPCRs internalization.
Highlights
G protein-coupled receptors (GPCRs) are membrane proteins that respond to a wide variety of extracellular stimuli and play critical roles in intercellular communication [1]
diffusion-enhanced resonance energy transfer (DERET) internalization assay relies on the use of a luminescent terbium cryptate derivative (SNAP-Lumi4-Tb) to label irreversibly the cells expressing the ST-GPCRs
We found that the glutamate agonist was able to promote a time (Figure 8A) and concentration dependent (Figure 8B) internalization of mGluR5 (EC50 = 193.3 ± 0.03 μM) extending the DERET assay to class C GPCR
Summary
G protein-coupled receptors (GPCRs) are membrane proteins that respond to a wide variety of extracellular stimuli and play critical roles in intercellular communication [1] They are central to many physiological and pathological processes and represent one of the most important classes of drug targets. Desensitization is typically controlled by receptor phosphorylation mediated by kinases, including the family of GPCR kinases, which promotes the recruitment of β-arrestins to the receptor. In many cases, these processes lead to receptor internalization, which generally proceeds by either a clathrin-coated pit or caveolae-mediated pathway [2]. Alternative strategies based on the quenching of internalized receptor tagged with a pH-sensitive fluorescent protein probes [11], NanoLuc® luciferase [12], or fluorogen activating protein (FAP) have been used to monitor internalization [13]
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