BRET-Based Biosensors to Measure Agonist Efficacies in Histamine H-1 Receptor-Mediated G Protein Activation, Signaling and Interactions with GRKs and beta-Arrestins

The histamine H-1 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 G(q)( )proteins and is phosphorylated by GPCR kinase (GRK) subtypes 2, 5, and 6, consequently facilitating the subsequent recruitment of beta-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 Eli R, bioluminescence resonance energy transfer (BRET)-based biosensors were applied to measure H1R signaling through heterotrimeric G(q) proteins, second messengers (inositol 1,4,5-triphosphate and Ca2+), and receptor-protein interactions (GRKs and beta-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 beta-arrestin2 to H1R over G(q) biosensor activation.