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Differential Role of Serines and Threonines in Intracellular Loop 3 and C-Terminal Tail of the Histamine H₄ Receptor in ?-Arrestin and G Protein-Coupled Receptor Kinase Interaction, Internalization, and Signaling.

ABSTRACT: The histamine H₄ receptor (H₄R) activates G?_i-mediated signaling and recruits ?-arrestin2 upon stimulation with histamine. ?-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 ?-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 ?-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 ?-arrestin2 recruitment and receptor desensitization, but reduced ?-arrestin1 recruitment and internalization. Hence, ?-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 ?-arrestin1 versus ?-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.

SUBMITTER: Verweij EWE

PROVIDER: S-EPMC7155198 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:G-protein-coupled receptors (GPCRs) are critically regulated by ?-arrestins, which not only desensitize G-protein signalling but also initiate a G-protein-independent wave of signalling. A recent surge of structural data on a number of GPCRs, including the ?2 adrenergic receptor (?2AR)-G-protein complex, has provided novel insights into the structural basis of receptor activation. However, complementary information has been lacking on the recruitment of ?-arrestins to activated GPCRs, primarily owing to challenges in obtaining stable receptor-?-arrestin complexes for structural studies. Here we devised a strategy for forming and purifying a functional human ?2AR-?-arrestin-1 complex that allowed us to visualize its architecture by single-particle negative-stain electron microscopy and to characterize the interactions between ?2AR and ?-arrestin 1 using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and chemical crosslinking. Electron microscopy two-dimensional averages and three-dimensional reconstructions reveal bimodal binding of ?-arrestin 1 to the ?2AR, involving two separate sets of interactions, one with the phosphorylated carboxy terminus of the receptor and the other with its seven-transmembrane core. Areas of reduced HDX together with identification of crosslinked residues suggest engagement of the finger loop of ?-arrestin 1 with the seven-transmembrane core of the receptor. In contrast, focal areas of raised HDX levels indicate regions of increased dynamics in both the N and C domains of ?-arrestin 1 when coupled to the ?2AR. A molecular model of the ?2AR-?-arrestin signalling complex was made by docking activated ?-arrestin 1 and ?2AR crystal structures into the electron microscopy map densities with constraints provided by HDX-MS and crosslinking, allowing us to obtain valuable insights into the overall architecture of a receptor-arrestin complex. The dynamic and structural information presented here provides a framework for better understanding the basis of GPCR regulation by arrestins.

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Differential Role of Serines and Threonines in Intracellular Loop 3 and C-Terminal Tail of the Histamine H4 Receptor in ?-Arrestin and G Protein-Coupled Receptor Kinase Interaction, Internalization, and Signaling.

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Differential Role of Serines and Threonines in Intracellular Loop 3 and C-Terminal Tail of the Histamine H₄ Receptor in ?-Arrestin and G Protein-Coupled Receptor Kinase Interaction, Internalization, and Signaling.