Verweij Eléonore W E, Al Araaj Betty, Prabhata Wimzy R, Prihandoko Rudi, Nijmeijer Saskia, Tobin Andrew B, Leurs Rob, Vischer Henry F
Division of Medicinal Chemistry, Amsterdam Institute for Molecules, Medicines and Systems, Faculty of Science, Vrije Universiteit Amsterdam, De Boelelaan 1108, 1081 HZ Amsterdam, The Netherlands.
Centre for Translational Pharmacology, Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, United Kingdom.
ACS Pharmacol Transl Sci. 2020 Mar 16;3(2):321-333. doi: 10.1021/acsptsci.0c00008. eCollection 2020 Apr 10.
The histamine H receptor (HR) activates Gα-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 HR 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 HR 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 HR did not affect β-arrestin2 recruitment and receptor desensitization, but reduced β-arrestin1 recruitment and internalization. Hence, β-arrestin recruitment to HR requires the putative phosphorylated serine cluster in the HR 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 HR with GRK5 and GRK6, respectively. Identification of HR interactions with these proteins is a first step in the understanding how this receptor might be dysregulated in pathophysiological conditions.
组胺H受体（HR）在受到组胺刺激时激活Gα介导的信号传导并招募β-抑制蛋白2。β-抑制蛋白通过与G蛋白偶联受体（GPCR）C末端尾巴和细胞内环3中的磷酸化丝氨酸和苏氨酸残基相互作用，在GPCR信号传导中发挥调节作用，导致受体脱敏和内化。使用基于生物发光共振能量转移（BRET）的生物传感器，我们发现，在激动剂刺激下，G蛋白偶联受体激酶（GRK）2和3比β-抑制蛋白1和2更快地被招募到HR，而受体向早期内体的内化动力学较慢。丙氨酸取代显示，HR C末端尾巴远端的丝氨酸簇对于β-抑制蛋白1/2的招募至关重要，因此，对于G蛋白驱动的细胞外信号调节激酶（ERK）1/2磷酸化的受体内化和脱敏以及无标记细胞阻抗也是如此。相比之下，HR细胞内环3中的丝氨酸和苏氨酸的丙氨酸取代并不影响β-抑制蛋白2的招募和受体脱敏，但减少了β-抑制蛋白1的招募和内化。因此，β-抑制蛋白招募到HR需要HR C末端尾巴中假定的磷酸化丝氨酸簇，而细胞内环3中的假定磷酸位点对β-抑制蛋白1和β-抑制蛋白2有不同的影响。细胞内环3或C末端尾巴中这些假定磷酸位点的突变不影响组胺诱导的GRK2和GRK3的招募，但分别改变了HR与GRK5和GRK6的相互作用。鉴定HR与这些蛋白质的相互作用是理解该受体在病理生理条件下可能如何失调的第一步。
