BRET-based β-arrestin2 recruitment to the histamine H₁ receptor for investigating antihistamine binding kinetics

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• 作者：Reggie Bosma ; Ryo Moritani ; Rob Leurs ; Henry F. Vischer ; ^{h.f.vischer@vu.nl" class="auth_mail" title="E-mail the corresponding author}
• 关键词：H1R ; histamine H1 receptor ; GPCR ; G protein-coupled receptor ; NFAT ; nuclear factor of activated T-cells ; ERK ; extracellular signal-regulated kinases ; BRET ; bioluminescence resonance energy transfer ; HEK293T ; Human embryonic kidney cells transformed with large T antigen ; PBZ ; phenoxybenzamine ; CTZ-h ; coelenterazine-h
• 刊名：Pharmacological Research
• 出版年：2016
• 出版时间：September 2016
• 年：2016
• 卷：111
• 期：Complete
• 页码：679-687
• 全文大小：1817 K

文摘

Ligand residence time is thought to be a critical parameter for optimizing the in vivo efficacy of drug candidates. For the histamine H₁ receptor (H₁R) and other G protein-coupled receptors, the kinetics of ligand binding are typically measured by low throughput radioligand binding experiments using homogenized cell membranes expressing the target receptor. In this study, a real-time proximity assay between H₁R and β-arrestin2 in living cells was established to investigate the dynamics of antihistamine binding to the H₁R. No receptor reserve was found for the histamine-induced recruitment of β-arrestin2 to the H₁R and the transiently recruited β-arrestin2 therefore reflected occupancy of the receptor by histamine. Antihistamines displayed similar kinetic signatures on antagonizing histamine-induced β-arrestin2 recruitment as compared to displacing radioligand binding from the H₁R. This homogeneous functional method unambiguously determined the fifty-fold difference in the dissociation rate constant between mepyramine and the long residence time antihistamines levocetirizine and desloratadine.