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

Abstract

The histamine H 4 receptor (H 4 R) 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 H 4 R 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 H 4 R 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 H 4 R did not affect β-arrestin2 recruitment and receptor desensitization, but reduced β-arrestin1 recruitment and internalization. Hence, β-arrestin recruitment to H 4 R requires the putative phosphorylated serine cluster in the H 4 R 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 H 4 R with GRK5 and GRK6, respectively. Identification of H 4 R interactions with these proteins is a first step in the understanding how this receptor might be dysregulated in pathophysiological conditions.

Differential Involvement of ACKR3 C-Tail in β-Arrestin Recruitment, Trafficking and Internalization

“…Since recruitment of the β-arrestins to ACKR3 upon activation is triggered by a change in the phosphorylation pattern of the C-tail of the receptor [ 23 ], we investigated whether GRKs, proteins involved in GPCR phosphorylation, play a role in ACKR3 activation [ 22 ]. We developed BRET sensors to test this hypothesis by fusing the GRK isoforms (GRK2, GRK3, GRK5 and GRK6) to the mVenus fluorescent protein (GRK-mVenus) [ 35 ]. Upon addition of CXCL12, ACKR3 clearly recruited GRK2 and GRK3 ( Figure 2 a,b).…”

“…Interestingly, ACKR3 also recruits GRK5, a plasma membrane-bound kinase [ 41 ]. On the other hand, ACKR3 does not affect trafficking of GRK6 (see [ 35 ] for a positive control of the GRK6 sensor), another plasma membrane-bound kinase. The pEC 50 values for recruitment of each of the GRKs are in a similar range as the pEC 50 values for recruitment of the β-arrestins ( Table 1 ).…”

“…0.4 µg of pcDEF 3 -hACKR3-RLuc [ 19 ] or pcDEF 3 -hACKR3mut-RLuc and 1.6 µg of the completing BRET pair (pcDEF 3 -β-arrestin1-eYFP, pcDEF 3 -β-arrestin2-mVenus [ 34 ], pcDEF 3 -GRK2-mVenus, pcDEF 3 -GRK3-mVenus, pcDEF 3 -GRK5-mVenus, pcDEF 3 -GRK6-mVenus [ 35 ], Rab5a-mVenus, Rab7a-mVenus, Rab11-mVenus, K-Ras-mVenus [ 36 , 37 ]) plasmids were combined to 12 µg of PEI in a total volume of 250 µL 150 mM NaCl and incubated for 20 min at room temperature. One million resuspended HEK293T or CRISPR HEK293 β-arrestin1/β-arrestin2 KO cells were added to the DNA/PEI mix, and cells were subsequently seeded (30,000 cells per well) in a poly-L-lysine coated (Sigma, Saint-Louis, MO, USA) 96-well white plate (PS, F-bottom, white; Greiner Bio One, Kremsmünster, Austria).…”

The Function of the Histamine H4 Receptor in Inflammatory and Inflammation-Associated Diseases of the Gut

“…As a consequence, calcium ([Ca 2+ ] i ) is mobilized from intracellular stores and the concentration of cytosolic cAMP is diminished. Independent of G protein-signaling, β-arrestin 2 is recruited to agonist-bound H 4 R, initiating mitogen-activated protein kinase (MAPK) cascade activation as well as receptor desensitization and internalization [ 84 , 85 , 86 , 87 ]. Activation of the H 4 R signaling pathways lead to induction of pro-inflammatory AP-1 in Th2 cells and monocyte-derived dendritic cells (MoDC) [ 27 ].…”

Identification of TSPAN4 as Novel Histamine H4 Receptor Interactor

“…The H 4 R is a G protein-coupled receptor (GPCR) that activates heterotrimeric Gα i protein-mediated intracellular signal transduction resulting in decreased cAMP production [3][4][5][6][7], increased Ca 2+ mobilization [8][9][10], activation of extracellular-signal regulated kinase (ERK)1/2 [11][12][13][14][15], as well as cytoskeletal changes [8]. Similar to most other GPCRs, the H 4 R can interact with G protein receptor kinases (GRKs) and β-arrestins that are involved in receptor internalization and consequently the regulation of agonist-induced G protein signaling [14,15].…”