BRET-Based Biosensors to Measure Agonist Efficacies in Histamine H1 Receptor-Mediated G Protein Activation, Signaling and Interactions with GRKs and ß-Arrestins.

The histamine H1 receptor (H1R) is a G protein-coupled receptor (GPCR) and plays a key role in allergic reactions upon activation by histamine which is locally released from mast cells and basophils. Consequently, H1R is a well-established therapeutic target for antihistamines that relieve allergy symptoms. H1R signals via heterotrimeric Gq proteins and is phosphorylated by GPCR kinase (GRK) subtypes 2, 5, and 6, consequently facilitating the subsequent recruitment of ß-arrestin1 and/or 2. Stimulation of a GPCR with structurally different agonists can result in preferential engagement of one or more of these intracellular signaling molecules. To evaluate this so-called biased agonism for H1R, bioluminescence resonance energy transfer (BRET)-based biosensors were applied to measure H1R signaling through heterotrimeric Gq proteins, second messengers (inositol 1,4,5-triphosphate and Ca2+), and receptor-protein interactions (GRKs and ß-arrestins) in response to histamine, 2-phenylhistamines, and histaprodifens in a similar cellular background. Although differences in efficacy were observed for these agonists between some functional readouts as compared to reference agonist histamine, subsequent data analysis using an operational model of agonism revealed only signaling bias of the agonist Br-phHA-HA in recruiting ß-arrestin2 to H1R over Gq biosensor activation.

Homogeneous, Real-Time NanoBRET Binding Assays for the Histamine H3 and H4 Receptors on Living Cells.

Receptor-binding affinity and ligand-receptor residence time are key parameters for the selection of drug candidates and are routinely determined using radioligand competition-binding assays. Recently, a novel bioluminescence resonance energy transfer (BRET) method utilizing a NanoLuc-fused receptor was introduced to detect fluorescent ligand binding. Moreover, this NanoBRET method gives the opportunity to follow fluorescent ligand binding on intact cells in real time, and therefore, results might better reflect in vivo conditions as compared with the routinely used cell homogenates or purified membrane fractions. In this study, a real-time NanoBRET-based binding assay was established and validated to detect binding of unlabeled ligands to the histamine H3 receptor (H3R) and histamine H4 receptor on intact cells. Obtained residence times of clinically tested H3R antagonists were reflected by their duration of H3R antagonism in a functional receptor recovery assay.

Synthesis and Characterization of a Bidirectional Photoswitchable Antagonist Toolbox for Real-Time GPCR Photopharmacology.

Noninvasive methods to modulate G protein-coupled receptors (GPCRs) with temporal and spatial precision are in great demand. Photopharmacology uses photons to control in situ the biological properties of photoswitchable small-molecule ligands, which bodes well for chemical biological precision approaches. Integrating the light-switchable configurational properties of an azobenzene into the ligand core, we developed a bidirectional antagonist toolbox for an archetypical family A GPCR, the histamine H3 receptor (H3R). From 16 newly synthesized photoswitchable compounds, VUF14738 (28) and VUF14862 (33) were selected as they swiftly and reversibly photoisomerize and show over 10-fold increased or decreased H3R binding affinities, respectively, upon illumination at 360 nm. Both ligands combine long thermal half-lives with fast and high photochemical trans-/ cis conversion, allowing their use in real-time electrophysiology experiments with oocytes to confirm dynamic photomodulation of H3R activation in repeated second-scale cycles. VUF14738 and VUF14862 are robust and fatigue-resistant photoswitchable GPCR antagonists suitable for spatiotemporal studies of H3R signaling.

Identification of TSPAN4 as Novel Histamine H4 Receptor Interactor.

The histamine H4 receptor (H4R) is a G protein-coupled receptor that is predominantly expressed on immune cells and considered to be an important drug target for various inflammatory disorders. Like most GPCRs, the H4R activates G proteins and recruits ß-arrestins upon phosphorylation by GPCR kinases to induce cellular signaling in response to agonist stimulation. However, in the last decade, novel GPCR-interacting proteins have been identified that may regulate GPCR functioning. In this study, a split-ubiquitin membrane yeast two-hybrid assay was used to identify H4R interactors in a Jurkat T cell line cDNA library. Forty-three novel H4R interactors were identified, of which 17 have also been previously observed in MYTH screens to interact with other GPCR subtypes. The interaction of H4R with the tetraspanin TSPAN4 was confirmed in transfected cells using bioluminescence resonance energy transfer, bimolecular fluorescence complementation, and co-immunoprecipitation. Histamine stimulation reduced the interaction between H4R and TSPAN4, but TSPAN4 did not affect H4R-mediated G protein signaling. Nonetheless, the identification of novel GPCR interactors by MYTH is a starting point to further investigate the regulation of GPCR signaling.

Differential Role of Serines and Threonines in Intracellular Loop 3 and C-Terminal Tail of the Histamine H4 Receptor in ß-Arrestin and G Protein-Coupled Receptor Kinase Interaction, Internalization, and Signaling.

The histamine H4 receptor (H4R) activates Gαi-mediated signaling and recruits ß-arrestin2 upon stimulation with histamine. ß-Arrestins play a regulatory role in G protein-coupled receptor (GPCR) signaling by interacting with phosphorylated serine and threonine residues in the GPCR C-terminal tail and intracellular loop 3, resulting in receptor desensitization and internalization. Using bioluminescence resonance energy transfer (BRET)-based biosensors, we show that G protein-coupled receptor kinases (GRK) 2 and 3 are more quickly recruited to the H4R than ß-arrestin1 and 2 upon agonist stimulation, whereas receptor internalization dynamics toward early endosomes was slower. Alanine-substitution revealed that a serine cluster at the distal end of the H4R C-terminal tail is essential for the recruitment of ß-arrestin1/2, and consequently, receptor internalization and desensitization of G protein-driven extracellular-signal-regulated kinase (ERK)1/2 phosphorylation and label-free cellular impedance. In contrast, alanine substitution of serines and threonines in the intracellular loop 3 of the H4R did not affect ß-arrestin2 recruitment and receptor desensitization, but reduced ß-arrestin1 recruitment and internalization. Hence, ß-arrestin recruitment to H4R requires the putative phosphorylated serine cluster in the H4R C-terminal tail, whereas putative phosphosites in the intracellular loop 3 have different effects on ß-arrestin1 versus ß-arrestin2. Mutation of these putative phosphosites in either intracellular loop 3 or the C-terminal tail did not affect the histamine-induced recruitment of GRK2 and GRK3 but does change the interaction of H4R with GRK5 and GRK6, respectively. Identification of H4R interactions with these proteins is a first step in the understanding how this receptor might be dysregulated in pathophysiological conditions.