Recruitment ofβ-Arrestin 1 and 2 to theβ₂-Adrenoceptor: Analysis of 65 Ligands

Abstract

Beyond canonical signaling via G a s and cAMP, the concept of functional selectivity at b 2 -adrenoceptors (b 2 ARs) describes the ability of adrenergic drugs to stabilize ligand-specific receptor conformations to initiate further signaling cascades comprising additional G-protein classes or b-arrestins (barr). A set of 65 adrenergic ligands including 40 agonists and 25 antagonists in either racemic or enantiopure forms was used for barr recruitment experiments based on a split-luciferase assay in a cellular system expressing b 2 AR. Many agonists showed only (weak) partial agonism regarding barr 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 barr recruitment other than the eutomer. barr2 was recruited more effectively than barr1. The analysis of antagonists revealed no significant effects on barr recruitment. Several agonists showed preference for activation of G a s GTPase relative to barr recruitment, and no barr-biased ligand was identified. In conclusion: 1) agonists show strong bias for G a s activation relative to barr recruitment; 2) agonists recruit barr1 and barr2 with subtle differences; and 3) there is no evidence for barr recruitment by antagonists.

The Regulator of G Protein Signaling Homologous Domain of G Protein-Coupled Receptor Kinase 2 Mediates Short-Term Desensitization of β3-Adrenergic Receptor

“…We initiated the study of the desensitization mechanisms of b3AR in a heterologous expression model, b3AR transfected HEK293T cells. In HEK293T cells transiently transfected with Mock or pCDNA3.1HA-b3AR (HEK293T-b3AR cells) we evaluated receptor expression by binding experiments using [ 3 H]CGP-12177 a ligand for b1/2/3ARs commonly used as a general b blocker (Vrydag and Michel, 2007;Suarez et al, 2014;Gherbi et al, 2015;Littmann et al, 2015). We found a three-fold increase in [ 3 H]CGP-12177 binding sites in HEK293T-b3AR (3024 ± 172) respect to Mock transfected cells (902 ± 50) ( Figure 1A) and a right shift in Kd from 12.8 ± 3.2 to 85.9 ± 14.3.…”

Discovery of β-arrestin-biased β2-adrenoceptor agonists from 2-amino-2-phenylethanol derivatives

“…A research group has studied 65 β 2 -agonists for biased agonism in the signaling pathways downstream of the β 2 -adrenoceptor, namely, G s , β-arrestin-1, and β-arrestin-2 [42][43][44]. These series of studies have included many FEN derivatives including (R,R')-FEN and (R,R')-MNF.…”

G Protein–Coupled Receptors in Asthma Therapy: Pharmacology and Drug Action

“…Studies from Clark and others (Carter and Hill, 2005;Moore et al, 2007;Gimenez et al, 2015;Masureel et al, 2018) demonstrated that salmeterol behaved as a biased ligand against b-arrestin recruitment and receptor desensitization relative to formoterol or epinephrine in assays using transfected cells, although another study suggested similar efficacies of salmeterol for G s -dependent signaling and b-arrestin-associated signaling (Drake et al, 2008). Littmann et al (2015) tested 40 b 2 -agonists in transfected HEK293 cells and reported a bias toward G s signaling over b-arrestin recruitment for many agonists, including salmeterol and fenoterol, relative to isoproterenol and formoterol. However, whether commonly used b 2 -agonists such as salmeterol exhibit functional bias in native systems still needs further investigation.…”