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

The histamine H₄ receptor (H₄R) activates Gα_i-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.