OBJECTIVE: Temporomandibular joint osteoarthritis (TMJOA) is a chronic degenerative joint disorder characterized by extracellular matrix degeneration and inflammatory response of condylar cartilage. β-arrestin2 is an important regulator of inflammation response, while its role in TMJOA remains unknown. The objective of this study was to investigate the role of β-arrestin2 in the development of TMJOA at the early stage and the underlying mechanism.
METHODS: A unilateral anterior crossbite (UAC) model was established on eight-week-old wild-type (WT) and β-arrestin2 deficiency mice to simulate the progression of TMJOA. Hematoxylin-eosin (HE) staining and microcomputed tomography (micro-CT) analysis were used for histological and radiographic assessment. Immunohistochemistry was performed to detect the expression of inflammatory and degradative cytokines, as well as autophagy related factors. Terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) assay was carried out to assess chondrocyte apoptosis.
RESULTS: The loss of β-arrestin2 aggravated cartilage degeneration and subchondral bone destruction in the model of TMJOA at the early stage. Furthermore, in UAC groups, the expressions of degradative (Col-X) and inflammatory (TNF-α and IL-1β) factors in condylar cartilage were increased in β-arrestin2 null mice compared with WT mice. Moreover, the loss of β-arrestin2 promoted apoptosis and autophagic process of chondrocytes at the early stage of TMJOA.
CONCLUSIONS: In conclusion, we demonstrated for the first time that β-arrestin2 plays a protective role in the development of TMJOA at the early stage, probably by inhibiting apoptosis and autophagic process of chondrocytes. Therefore, β-arrestin2 might be a potential therapeutic target for TMJOA, providing a new insight for the treatment of TMJOA at the early stage.

β-arrestin2 is a versatile protein for signaling transduction in brain physiology and pathology. Herein, we investigated the involvement of β-arrestin2 in pharmacological effects of fluoxetine for depression. A chronic mild stress (CMS) model was established using wild-type (WT) and β-arrestin2-/- mice. Behavioral results demonstrated that CMS mice showed increased immobility time in the tail suspension test and forced swimming test, elevated concentrations of pro-inflammatory factors in peripheral blood, increased expression of pyroptosis-related proteins, and increased co-labeling of glial fibrillary acidic protein and Caspase1 p10 in the hippocampus compared to the CON group. Treatment with fluoxetine (FLX) ameliorated these conditions. However, compared with the β-arrestin2-/- CMS group, these results of the β-arrestin2-/- CMS + FLX group showed no significant changes. These results suggested that the above effects of FLX could be eliminated by knocking out β-arrestin2. Mass spectrometry implying that FLX promoted the binding of β-arrestin2 to the NLRP2 inflammasome of depressed mice. Subsequently, the results of the cellular experiments suggested that the 5HT2B receptor antagonist may attenuate L-kynurenine + ATP-induced cell pyroptosis by attenuating NLRP2 binding to β-arrestin2. We further found that the lack of β-arrestin2 eliminated the anti-pyroptosis effect of fluoxetine. In conclusion, β-arrestin2 is an essential protein for fluoxetine to alleviate pyroptosis in the hippocampal astrocytes of CMS mice. Mechanistically, we found that the 5-HT2BR-β-arrestin2-NLRP2 axis is vital for maintaining the antidepressant effects of fluoxetine.

The binding and function of β-arrestins are regulated by specific phosphorylation motifs present in G protein-coupled receptors (GPCRs). However, the exact arrangement of phosphorylated amino acids responsible for establishing a stable interaction remains unclear. We employ a 1D sequence convolution model trained on GPCRs with established β-arrestin-binding properties. With this approach, amino acid motifs characteristic of GPCRs that form stable interactions with β-arrestins can be identified, a pattern that we name \"arreSTick.\" Intriguingly, the arreSTick pattern is also present in numerous non-receptor proteins. Using proximity biotinylation assay and mass spectrometry analysis, we demonstrate that the arreSTick motif controls the interaction between many non-receptor proteins and β-arrestin2. The HIV-1 Tat-specific factor 1 (HTSF1 or HTATSF1), a nuclear transcription factor, contains the arreSTick pattern, and its subcellular localization is influenced by β-arrestin2. Our findings unveil a broader role for β-arrestins in phosphorylation-dependent interactions, extending beyond GPCRs to encompass non-receptor proteins as well.

G protein-coupled receptors (GPCRs) are a family of cell membrane receptors that couple and activate heterotrimeric G proteins and their associated intracellular signalling processes after ligand binding. Although the carboxyl terminal of the receptors is essential for this action, it can also serve as a docking site for regulatory proteins such as the β-arrestins. Prokineticin receptors (PKR1 and PKR2) are a new class of GPCRs that are able to activate different classes of G proteins and form complexes with β-arrestins after activation by the endogenous agonists PK2. The aim of this work was to define the molecular determinants within PKR2 that are required for β-arrestin-2 binding and to investigate the role of β-arrestin-2 in the signalling pathways induced by PKR2 activation. Our data show that PKR2 binds constitutively to β-arrestin-2 and that this process occurs through the core region of the receptor without being affected by the carboxy-terminal region. Indeed, a PKR2 mutant lacking the carboxy-terminal amino acids retains the ability to bind constitutively to β-arrestin-2, whereas a mutant lacking the third intracellular loop does not. Overall, our data suggest that the C-terminus of PKR2 is critical for the stability of the β-arrestin-2-receptor complex in the presence of PK2 ligand. This leads to the β-arrestin-2 conformational change required to initiate intracellular signalling that ultimately leads to ERK phosphorylation and activation.

UNASSIGNED: ANG (angiotensin II) elicits dipsogenic and pressor responses via activation of the canonical Gαq (G-protein component of the AT1R [angiotensin type 1 receptor])-mediated AT1R in the subfornical organ. Recently, we demonstrated that ARRB2 (β-arrestin 2) global knockout mice exhibit a higher preference for salt and exacerbated pressor response to deoxycorticosterone acetate salt. However, whether ARRB2 within selective neuroanatomical nuclei alters physiological responses to ANG is unknown. Therefore, we hypothesized that ARRB2, specifically in the subfornical organ, counterbalances maladaptive dipsogenic and pressor responses to the canonical AT1R signaling.
UNASSIGNED: Male and female Arrb2FLOX mice received intracerebroventricular injection of either adeno-associated virus (AAV)-Cre-GFP (green fluorescent protein) to induce brain-specific deletion of ARRB2 (Arrb2ICV-Cre). Arrb2FLOX mice receiving ICV-AAV-GFP were used as control (Arrb2ICV-Control). Infection with ICV-AAV-Cre primarily targeted the subfornical organ with few off targets. Fluid intake was evaluated using the 2-bottle choice paradigm with 1 bottle containing water and 1 containing 0.15 mol/L NaCl.
UNASSIGNED: Arrb2ICV-Cre mice exhibited a greater pressor response to acute ICV-ANG infusion. At baseline conditions, Arrb2ICV-Cre mice exhibited a significant increase in saline intake compared with controls, resulting in a saline preference. Furthermore, when mice were subjected to water-deprived or sodium-depleted conditions, which would naturally increase endogenous ANG levels, Arrb2ICV-Cre mice exhibited elevated saline intake.
UNASSIGNED: Overall, these data indicate that ARRB2 in selective cardiovascular nuclei in the brain, including the subfornical organ, counterbalances canonical AT1R responses to both exogenous and endogenous ANG. Stimulation of the AT1R/ARRB axis in the brain may represent a novel strategy to treat hypertension.

Arrestins are key negative regulators of G Protein-Coupled Receptors (GPCRs) through mediation of G protein desensitisation and receptor internalisation. Arrestins can also contribute to signal transduction by scaffolding downstream signalling effectors for activation. GPCR kinase (GRK) enzymes phosphorylate the intracellular C-terminal domain, or intracellular loop regions of GPCRs to promote arrestin interaction. There are seven different GRK subtypes, which may uniquely phosphorylate the C-terminal tail in a type of \'phosphorylation barcode,\' potentially differentially contributing to arrestin translocation and arrestin-dependent signalling. Such contributions may be exploited to develop arrestin-biased ligands. Here, we examine the effect of different GRK subtypes on the ability to promote translocation of arrestin-2 and arrestin-3 to the cannabinoid CB1 receptor (CB1) with a range of ligands. We find that most GRK subtypes (including visual GRK1) can enhance arrestin-2 and -3 translocation to CB1, and that GRK-dependent changes in arrestin-2 and arrestin-3 translocation were broadly shared for most agonists tested. GRK2/3 generally enhanced arrestin translocation more than the other GRK subtypes, with some small differences between ligands. We also explore the interplay between G protein activity and GRK2/3-dependent arrestin translocation, highlighting that high-efficacy G protein agonists will cause GRK2/3 dependent arrestin translocation. This study supports the hypothesis that arrestin-biased ligands for CB1 must engage GRK5/6 rather than GRK2/3, and G protein-biased ligands must have inherently low efficacy.

The glucagon-like peptide 1 receptor (GLP-1R) is a validated clinical target for the treatment of type 2 diabetes and obesity. Unlike most G protein-coupled receptors (GPCRs), the GLP-1R undergoes an atypical mode of internalisation that does not require β-arrestins. While differences in GLP-1R trafficking and β-arrestin recruitment have been observed between clinically used GLP-1R agonists, the role of G protein-coupled receptor kinases (GRKs) in affecting these pathways has not been comprehensively assessed. In this study, we quantified the contribution of GRKs to agonist-mediated GLP-1R internalisation and β-arrestin recruitment profiles using cells where endogenous β-arrestins, or non-visual GRKs were knocked out using CRISPR/Cas9 genome editing. Our results confirm the previously established atypical β-arrestin-independent mode of GLP-1R internalisation and revealed that GLP-1R internalisation is dependent on the expression of GRKs. Interestingly, agonist-mediated GLP-1R β-arrestin 1 and β-arrestin 2 recruitment were differentially affected by endogenous GRK knockout with β-arrestin 1 recruitment more sensitive to GRK knockout than β-arrestin 2 recruitment. Moreover, individual overexpression of GRK2, GRK3, GRK5 or GRK6 in a newly generated GRK2/3/4/5/6 HEK293 cells, rescued agonist-mediated β-arrestin 1 recruitment and internalisation profiles to similar levels, suggesting that there is no specific GRK isoform that drives these pathways. This study advances mechanistic understanding of agonist-mediated GLP-1R internalisation and provides novel insights into how GRKs may fine-tune GLP-1R signalling.

Glucagon-like peptide-1 receptor (GLP-1R) is a prime drug target for type 2 diabetes and obesity. The ligand initiated GLP-1R interaction with G protein has been well studied, but not with β-arrestin 1/2. Therefore, bioluminescence resonance energy transfer (BRET), mutagenesis and an operational model were used to evaluate the roles of 85 extracellular surface residues on GLP-1R in β-arrestin 1/2 recruitment triggered by three representative GLP-1R agonists (GLP-1, exendin-4 and oxyntomodulin). Residues selectively regulated β-arrestin 1/2 recruitment for diverse ligands, and β-arrestin isoforms were identified. Mutation of residues K130-S136, L142 and Y145 on the transmembrane helix 1 (TM1)-extracellular domain (ECD) linker decreased β-arrestin 1 recruitment but increased β-arrestin 2 recruitment. Other extracellular loop (ECL) mutations, including P137A, Q211A, D222A and M303A selectively affected β-arrestin 1 recruitment while D215A, L217A, Q221A, S223A, Y289A, S301A, F381A and I382A involved more in β-arrestin 2 recruitment for the ligands. Oxyntomodulin engaged more broadly with GLP-1R extracellular surface to drive β-arrestin 1/2 recruitment than GLP-1 and exendin-4; I147, W214 and L218 involved in β-arrestin 1 recruitment, while L141, D215, L218, D293 and F381 in β-arrestin 2 recruitment for oxyntomodulin particularly. Additionally, the non-conserved residues on β-arrestin 1/2 C-domains contributed to interaction with GLP-1R. Further proteomic profiling of GLP-1R stably expressed cell line upon ligand stimulation with or without β-arrestin 1/2 overexpression demonstrated both commonly and biasedly regulated proteins and pathways associated with cognate ligands and β-arrestins. Our study offers valuable information about ligand induced β-arrestin recruitment mediated by GLP-1R and consequent intracellular signaling events.

Previous study showed that higher expression of prolactin (PRL) was found in CRPC samples compared with hormone-naive prostate cancer (HNPC) and benign prostatic hyperplasia (BPH) samples. We further investigate the function of PRL in prostate cancer (PCa) and explored its downstream effects. We found heterogeneous expression of the PRLR in clinical prostate samples. The VCaP and 22Rv1 cells exhibited PRLR expression. Among the downstream proteins, STAT5B was the dominant subtype in clinical samples and cell lines. Human recombinant PRL stimulation of PCa cells with PRLR expression resulted in increased phosphorylation of STAT5B(pSTAT5B) and progression of PCa in vitro and in vivo, and STAT5B knockdown can suppress the malignant behavior of PCa. To understand the mechanism further, we performed Bioinformatic analysis, ChIP qPCR, and luciferase reporter gene assay. The results revealed that ARRB2 was the transcription target gene of STAT5B, and higher expression of ARRB2 was related to higher aggression and poorer prognosis of PCa. Additionally, Gene set enrichment analysis indicated that higher expression of ARRB2 was significantly enriched in the MAPK signaling pathway. Immunohistochemistry (IHC) demonstrated elevated pSTAT5B, ARRB2, and pERK1/2 expression levels in CRPC tissues compared to HNPC and BPH. Mechanically, ARRB2 enhanced the activation of the MAPK pathway by binding to ERK1/2, thereby promoting the phosphorylation of ERK1/2 (pERK1/2). In conclusion, our study demonstrated that PRL stimulation can promote the progression of PCa through STAT5B/ARRB2 pathway and activation of MAPK signaling, which can be suppressed by intervention targeting STAT5B. Blockade of the STAT5B can be a potential therapeutic target for PCa.

The μ-opioid receptor-biased agonist theory holds that Gio protein signaling mediates the analgesic effect of opioids and the related side effects via the β-arrestin2 signaling pathway. A series of μ-opioid-biased agonists have been developed in accordance with this theory, and the FDA has approved TRV130 (as a representative of biased agonists) for marketing. However, several reports have raised the issue of opioid side effects associated with the use of agonists. In this study, five permeable peptides were designed to emulate 11 S/T phosphorylation sites at the μ-opioid receptor (MOR) carboxyl-terminal. In vitro experiments were performed to detect the activation level of G proteins from the cAMP inhibition assay and the β-arrestin2 recruitment by the BRET assay. Designed peptides might effectively interfere with the activation of the Gio and β-arrestin2 pathways when combined with morphine. The resulting morphine-induced tolerance, respiratory inhibition, and constipation in mice showed that the β-arrestin2 pathway was responsible for morphine tolerance while the Gio signaling pathway was involved with respiratory depression and constipation and that these side effects were significantly related to phosphorylation sites S363 and T370. This study may provide new directions for the development of safer and more effective opioid analgesics, and the designed peptides may be an effective tool for exploring the mechanism by which μ-opioid receptors function, with the potential of reducing the side effects that are associated with clinical opioid treatment.