Recruitment of ß-arrestin 1 and 2 to the ß2-adrenoceptor: analysis of 65 ligands.

UNLABELLED: Beyond canonical signaling via Gαs and cAMP, the concept of functional selectivity at ß2-adrenoceptors (ß2ARs) describes the ability of adrenergic drugs to stabilize ligand-specific receptor conformations to initiate further signaling cascades comprising additional G-protein classes or ß-arrestins (ßarr). A set of 65 adrenergic ligands including 40 agonists and 25 antagonists in either racemic or enantiopure forms was used for ßarr recruitment experiments based on a split-luciferase assay in a cellular system expressing ß2AR. Many agonists showed only (weak) partial agonism regarding ßarr recruitment. Potencies and/or efficacies increased depending on the number of chirality centers in (R) configuration; no (S)-configured distomer was more effective at inducing ßarr recruitment other than the eutomer. ßarr2 was recruited more effectively than ßarr1. The analysis of antagonists revealed no significant effects on ßarr recruitment. Several agonists showed preference for activation of Gαs GTPase relative to ßarr recruitment, and no ßarr-biased ligand was identified. IN CONCLUSION: 1) agonists show strong bias for Gαs activation relative to ßarr recruitment; 2) agonists recruit ßarr1 and ßarr2 with subtle differences; and 3) there is no evidence for ßarr recruitment by antagonists.

Computational and experimental analysis of the transmembrane domain 4/5 dimerization interface of the serotonin 5-HT(1A) receptor.

Experimental evidence suggests that most members of class A G-protein coupled receptors (GPCRs) can form homomers and heteromers in addition to functioning as single monomers. In particular, serotonin (5-HT) receptors were shown to homodimerize and heterodimerize with other GPCRs, although the details and the physiological role of the oligomerization has not yet been fully elucidated. Here we used computational modeling of the 5-HT(1A) receptor monomer and dimer to predict residues important for dimerization. Based on these results, we carried out rationally designed site-directed mutagenesis. The ability of the mutants to dimerize was evaluated using different FRET-based approaches. The reduced levels of acceptor photobleaching-Förster resonance energy transfer (FRET) and the lower number of monomers participating in oligomers, as assessed by lux-FRET, confirmed the decreased ability of the mutants to dimerize and the involvement of the predicted contacts (Trp175(4.64), Tyr198(5.41), Arg151(4.40), and Arg152(4.41)) at the interface. This information was reintroduced as constraints for computational protein-protein docking to obtain a high-quality dimer model. Analysis of the refined model as well as molecular dynamics simulations of wild-type (WT) and mutant dimers revealed compensating interactions in dimers composed of WT and W175A mutant. This provides an explanation for the requirement of mutations of Trp175(4.64) in both homomers for disrupting dimerization. Our iterative computational-experimental study demonstrates that transmembrane domains TM4/TM5 can form an interaction interface in 5-HT(1A) receptor dimers and indicates that specific amino acid interactions maintain this interface. The mutants and the optimized model of the dimer structure may be used in functional studies of serotonin dimers.

Interaction of fenoterol stereoisomers with ß2-adrenoceptor-G sα fusion proteins: antagonist and agonist competition binding.

The specific interaction between G-protein-coupled receptors and ligand is the starting point for downstream signaling. Fenoterol stereoisomers were successfully used to probe ligand-specific activation (functional selectivity) of the ß2-adrenoceptor (ß2AR) (Reinartz et al. 2015). In the present study, we extended the pharmacological profile of fenoterol stereoisomers using ß2AR-Gsα fusion proteins in agonist and antagonist competition binding assays. Dissociations between binding affinities and effector potencies were found for (R,S')- and (S,S')-isomers of 4'-methoxy-1-naphthyl-fenoterol. Our data corroborate former studies on the importance of the aminoalkyl moiety of fenoterol derivatives for functional selectivity.

Structure-bias relationships for fenoterol stereoisomers in six molecular and cellular assays at the ß2-adrenoceptor.

Functional selectivity is well established as an underlying concept of ligand-specific signaling via G protein-coupled receptors (GPCRs). Functionally, selective drugs could show greater therapeutic efficacy and fewer adverse effects. Dual coupling of the ß2-adrenoceptor (ß2AR) triggers a signal transduction via Gsα and Giα proteins. Here, we examined 12 fenoterol stereoisomers in six molecular and cellular assays. Using ß2AR-Gsα and ß2AR-Giα fusion proteins, (R,S')- and (S,S')-isomers of 4'-methoxy-1-naphthyl-fenoterol were identified as biased ligands with preference for Gs. G protein-independent signaling via ß-arrestin-2 was disfavored by these ligands. Isolated human neutrophils constituted an ex vivo model of ß2AR signaling and demonstrated functional selectivity through the dissociation of cAMP accumulation and the inhibition of formyl peptide-stimulated production of reactive oxygen species. Ligand bias was calculated using an operational model of agonism and revealed that the fenoterol scaffold constitutes a promising lead structure for the development of Gs-biased ß2AR agonists.

Dissociations in the effects of ß2-adrenergic receptor agonists on cAMP formation and superoxide production in human neutrophils: support for the concept of functional selectivity.

In neutrophils, activation of the ß2-adrenergic receptor (ß2AR), a Gs-coupled receptor, inhibits inflammatory responses, which could be therapeutically exploited. The aim of this study was to evaluate the effects of various ß2AR ligands on adenosine-3',5'-cyclic monophosphate (cAMP) accumulation and N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)-induced superoxide anion (O2(•-)) production in human neutrophils and to probe the concept of ligand-specific receptor conformations (also referred to as functional selectivity or biased signaling) in a native cell system. This is an important question because so far, evidence for functional selectivity has been predominantly obtained with recombinant systems, due to the inherent difficulties to genetically manipulate human native cells. cAMP concentration was determined by HPLC/tandem mass spectrometry, and O2(•-) formation was assessed by superoxide dismutase-inhibitable reduction of ferricytochrome c. ß2AR agonists were generally more potent in inhibiting fMLP-induced O2(•-) production than in stimulating cAMP accumulation. (-)-Ephedrine and dichloroisoproterenol were devoid of any agonistic activity in the cAMP assay, but partially inhibited fMLP-induced O2(•-) production. Moreover, (-)-adrenaline was equi-efficacious in both assays whereas the efficacy of salbutamol was more than two-fold higher in the O2(•-) assay. Functional selectivity was visualized by deviations of ligand potencies and efficacies from linear correlations for various parameters. We obtained no evidence for involvement of protein kinase A in the inhibition of fMLP-induced O2(•-) production after ß2AR-stimulation although cAMP-increasing substances inhibited O2(•-) production. Taken together, our data corroborate the concept of ligand-specific receptor conformations with unique signaling capabilities in native human cells and suggest that the ß2AR inhibits O2(•-) production in a cAMP-independent manner.