Profiling of basal and ligand-dependent GPCR activities by means of a polyvalent cell-based high-throughput platform

Abstract

Representing the most attractive and successful druggable receptors of the proteome, GPCRs regulate a myriad of physiological and pathophysiological functions. Although over half of present pharmaceuticals target GPCRs, the advancement of drug discovery is hampered by a lack of adequate screening tools, the majority of which are limited to probing agonist-induced G-protein and β-arrestin-2-mediated events as a measure of receptor activation. Here, we develop Tango-Trio, a comprehensive cell-based high-throughput platform comprising cumate-inducible expression of transducers, capable of the parallelized profiling of both basal and agonist-dependent GPCR activities. We capture the functional diversity of GPCRs, reporting β-arrestin-1/2 couplings, selectivities, and receptor internalization signatures across the GPCRome. Moreover, we present the construction of cumate-induced basal activation curves at approximately 200 receptors, including over 50 orphans. Overall, Tango-Trio’s robustness is well-suited for the functional characterization and screening of GPCRs, especially for parallel interrogation, and is a valuable addition to the pharmacological toolbox.

Development of a V5-tag–directed nanobody and its implementation as an intracellular biosensor of GPCR signaling

“…Subsequently, the translocated tTA engages the nucleus and activates a reporter gene under the control of the tTA-response element. Our study selected an optimized version of the TEV protease, TEV219, which exhibits enhanced efficiency and avoids self-inactivation ( 69 ), to improve further the GPCR-TANGO platform’s performance ( 70 ).…”

“…HTTL (HEK293T stably expressing a luciferase reporter gene under the tTA-response element-Tight promoter) cells, an in-house developed reporter cell line ( 70 ), were seeded in 6-well plates at 1.2 x 10 6 cells and were transfected with Tango-ized constructs using the PEI precipitation method. Twenty hours later, the transfected cells were plated in DMEM supplemented with 1% dialyzed FBS into PLL-coated 384-well white clear bottom cell culture plates at a density of 30,000 cells/well in a total volume of 40 μl for 5 h to ensure proper attachment of cells.…”

The MRGPR family of receptors in immunity

“…2) The genes encoding Mgrprs are highly polymorphic, and the function of these mutations on immune cell function and disease is largely unknown, 3) What is the role of immune-expressed Mrgprs in developing immune cell lineages and 4) It is important to appreciate that Mrgprs can function independently of their ligands, as they exhibit elevated basal activity. 129 What is the functional consequence of this basal activity?…”

ClickArr: a novel, high-throughput assay for evaluating β-arrestin isoform recruitment

“…Alternative β-arrestin recruitment architectures also include enzyme-induced cleavage of transcription factors and bioluminescent resonance energy transfer (BRET)-based assays (Kroning and Wang, 2022). For example, the cleavage-dependent Tango assay system was recently extended to include β-arrestin 1 (Zeghal et al, 2023). However, the Tango system requires unique cell lines for each isoform and cannot report β-arrestin dynamics.…”

“…In contrast, the relative indifference of the ClickArr assay to the choice of linker on the δOR (Figure 1) suggests that there is a high degree of tolerability in this architecture for orientation and spacing of the luciferase fragments. Thus, while there are advantages and disadvantages to each approach, in particular that the BRET and Tango architectures are already available for a wide range of GPCRs (Avet et al, 2022;Zeghal et al, 2023), the ClickArr architecture may be the optimal ClickArr reports arrestin dynamics in 384-well plate format, and TAN67 β-arrestin isoform bias is confirmed by reaction velocities. (A) Following a 5minute equilibration, either leu-enkephalin or TAN67 was added to wells (triangle) and the evolution in the signal for β-arrestin 1 and 2 recorded over time.…”