Luminescence-based methods for the investigation of ligand binding and GRK2 recruitment to GPCRs

The development of novel ligands for G protein-coupled receptors (GPCRs), representing important drug targets, requires the determination of ligand-receptor affinities. This has primarily been done by radioligand competition binding experiments. However, the use of radioligands is disadvantageous with respect to safety concerns, legal handling regulations and waste disposal. Thus, alternative and radioactivity-free methods for studying ligand-receptor binding are needed. Over the last decades, techniques using fluorescent ligands have emerged as a promising option. A recently reported luminescence-based binding assay makes use of the phenomenon of BRET (bioluminescence resonance energy transfer). The N-terminus of the receptor of interest was tagged with a small luciferase, which generates bioluminescence upon the addition of its substrate (e.g. furimazine). When a fluorescent ligand binds to the receptor, BRET can occur due to the close proximity of the luciferase and the fluorescent ligand. The BRET signal correlates with the amount of receptor-bound ligand and, in contrast to radioligand binding assays, ligand binding can be measured in real time with high temporal resolution. The aim of this thesis was the development of BRET-based binding assays for different GPCRs as alternative methods to radioligand binding assays. First, a BRET-based binding assay was developed for the histamine H2 receptor (H2R). For this purpose, the H2R, N-terminally fused to the NanoLuc® (NLuc), was stably expressed in HEK293T cells. Out of three differently labeled fluorescent H2R ligands, which were investigated in BRET saturation binding experiments, the Py-1-labeled ligand 2.1 turned out to be the most favorable in terms of ligand affinity and signal-to-background (S/B) ratio. The receptor binding kinetics of 2.1 were studied in real time and competition binding experiments with 2.1 and several reported H2R ligands afforded H2R binding data in accordance with literature values. Furthermore, a BRET-based binding assay was developed for the M2 muscarinic acetylcholine receptor (M2R). Two TAMRA-labeled M2R ligands (3.1 and 3.2) were characterized by saturation binding, association and dissociation kinetic (in real time) and, for 3.1, competition binding experiments with standard MR ligands. As the fluorophore TAMRA is also suited for fluorescence anisotropy (FA) studies, the BRET-based M2R binding assay was compared with an FA-based real-time binding assay, also using 3.1 and 3.2 as probes. Both methods, representing different methodologies, yielded similar results (ligand binding affinities from saturation binding and competition binding studies). Kinetic data from both assays revealed a complex binding behavior for 3.1 and 3.2 (e.g. biphasic dissociation kinetics), being most pronounced for 3.2. In the case of the neuropeptide Y Y1 receptor (Y1R) or the neurotensin receptor 1 (NTS1R), both exhibiting long N-termini, application of the reported concept (N-terminal fusion of the GPCR to NLuc) failed to establish BRET-based binding assays for these receptors. Obviously, their longer N-termini led to an increased distance or an unfavorable position of the luciferase relative to the fluorescent ligand, precluding efficient BRET. Therefore, an alternative strategy was developed by inserting the luciferase into the second extracellular loop of the Y1R and the NTS1R. This novel approach resulted in feasible BRET binding assays. The same strategy, which is considered a widely applicable method, was applied to two other GPCRs with comparably short N-termini, the angiotensin II receptor type 1 (AT1R) and the M1 muscarinic acetylcholine receptor (M1R). When compared with the N-terminally NLuc-tagged AT1R and M1R, insertion of NLuc into ECL2 of the receptors led to ligand affinities in better accordance with literature data, and/or higher S/B ratios. Finally, a split luciferase-based assay was developed to assess the recruitment of the G protein coupled receptor kinase 2 (GRK2) to three different receptors. Besides the possibility of generating concentration-response curves, the novel assay allowed analyzing GRK2 recruitment in a time-dependent manner. It revealed different recruitment kinetics for the NTS1R, which showed a more sustained interaction with GRK2, when compared with the M1R and the M5R, both showing a short-lasting interaction with GRK2. This suggested a potential classification of GPCRs based on their differential interaction with GRKs