Untersuchungen zur Selektivität der Wechselwirkungen von ausgewählten G-Protein-gekoppelten Rezeptoren mit nachgeschalteten Effektoren und deren pharmakologische Beeinflussung

With more than 800 individuals GPCRs are known to be largest group of membrane spanning receptors in the human genome. They are involved in various physiological processes and are therefore a significant drug target, represented by more than 30 percent of approved drugs binding to GPCRs. The development of selective drugs is a considerable challenge when targeting the orthosteric binding pocket, being evolutionary highly conserved within a given receptor family. Hence, there has been an increasing interest on the part of research and the pharmaceutical industry in the examination and development of allosteric modulators on the one hand, which target alternative less conserved binding pockets and modulate receptor activity, and biased agonists on the other hand, which lead to a selective and preferred activation of a signalling pathway. Both kinds of ligands are supposed to increase the selectivity of drugs and reduce side effects. The first parts of this thesis focused on the allosteric modulation of GPCRs. The goal was to identify allosteric ligands targeting the endothelinB receptor (ETB) and examine their impact on receptors function, cooperating with different working groups. For that purpose, the through docking identified small compounds were analysed regarding a potential modulation of receptor function using FRET-based assays. The tested compounds were docked to the G protein binding pocket of the ETB receptor. Up until now no compound with a sufficient modulation of receptor activity has been discovered, depicting the complexity of allosteric drug development. For the AW12 compound, which was modelled to allosterically modulate the M2 receptor by binding to the intracellular cavity, a decrease in G protein binding to the M2 receptor was detected. In another part of this thesis the functional relevance of newly identified, previously untargeted binding pockets at the M3 receptor was proven. Point mutations were inserted into the receptor in the regions of the two binding pockets. A change in the affinity of acteylcholin as well as a decrease in efficacy of arecoline in the G protein activation was measured. In addition, the mutations led to a reduction in efficacy of acetylcholine in arrestin recruitment. All mutations in the regions of both binding pockets at the M3 receptor resulted in considerable changes in receptor function, suggesting that receptor function can be influenced by the binding of allosteric ligands into these previously untargeted sites. In another chapter the orphan receptor GPRC5B was investigated further. In FRAP-based measurements an interaction of the receptors was observed, suggesting a dimerization. However, a potential G protein binding under nucleotide free conditions could not be detected. The more than 800 GPCRs in humans couple to merely 16 different Gα proteins, divided into 4 families. Despite of long-lasting research no mechanism of coupling selectivity has been identified up until now. The increased crystal and cryo-EM structures of receptor-G protein complexes of recent years provided an insight into the interaction of G protein and receptor. However, they are only snapshots of a complex mechanism. Understanding receptor structures, which are considerably involved in the interaction with the G protein and the coupling selectivity, is of notable advantage when addressing the G protein binding pocket with allosteric modulators. For this thesis M2- and M3-based receptor chimeras were generated and investigated in FRET- and BRET-based assays, in order to identify crucial receptor structures involved in the selective binding of Gαo and Gαq. As a result, the lower parts of transmembrane helices 5 and 6, the polybasic cluster at the proximal c terminus and the distal c terminus of the M3 receptor were detected as important structures for coupling selectivity. The final part of this thesis focused on the agonist bias at the M3 receptor. In this regard, the direct recruitment of effector proteins to the receptor in FRET-based assays was examined, under conditions which allowed a clear separation between the signalling pathways. This led to a detection of a bias in arrestin recruitment, which, however, could not be found on the level of GRK2 recruitment. As a result, new insights were gained into the previously poorly researched GRK2 recruitment in the context of bias. Overall, this thesis contributes further insights into the complex mechanisms of allosteric modulation, the selective binding of G proteins and the agonist induced functional selectivity of GPCRs.