Differential role of serines and threonines in intracellular loop 3 and C-terminal tail of the histamine H4 receptor in β-arrestin and G protein-coupled receptor kinase interaction, internalization, and signaling

Verweij, E. W. E., Al Araaj, B., Prabhata, W. R., Prihandoko, R., Nijmeijer, S., Tobin, A. B. , Leurs, R. and Vischer, H. F. (2020) Differential role of serines and threonines in intracellular loop 3 and C-terminal tail of the histamine H4 receptor in β-arrestin and G protein-coupled receptor kinase interaction, internalization, and signaling. ACS Pharmacology and Translational Science, 3(2), pp. 321-333. (doi: 10.1021/acsptsci.0c00008) (PMID:32296771) (PMCID:PMC7155198)

Abstract

The histamine H4 receptor (H4R) activates Gαi-mediated signaling and recruits β-arrestin2 upon stimulation with histamine. β-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 β-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 β-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 β-arrestin2 recruitment and receptor desensitization, but reduced β-arrestin1 recruitment and internalization. Hence, β-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 β-arrestin1 versus β-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.

• Item Type: Articles
• Additional Information: This research was supported by The Netherlands Organisation for Scientific Research (NWO) ECHO project 711.013.014 (R.L. and H.F.V).
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Prihandoko, Dr Rudi and Tobin, Andrew
• Authors: Verweij, E. W. E., Al Araaj, B., Prabhata, W. R., Prihandoko, R., Nijmeijer, S., Tobin, A. B., Leurs, R., and Vischer, H. F.
• College/School: College of Medical Veterinary and Life Sciences > School of Molecular Biosciences
• Journal Name: ACS Pharmacology and Translational Science
• Publisher: American Chemical Society
• ISSN: 2575-9108
• ISSN (Online): 2575-9108
• Published Online: 16 March 2020
• Copyright Holders: Copyright © 2020 American Chemical Society
• First Published: First published online in ACS Pharmacology and Translational Science 3(2): 321-333
• Publisher Policy: Reproduced under a Creative Commons Licence