Loss of ß-arrestin2 aggravated condylar cartilage degeneration at the early stage of temporomandibular joint osteoarthritis.

OBJECTIVE: Temporomandibular joint osteoarthritis (TMJOA) is a chronic degenerative joint disorder characterized by extracellular matrix degeneration and inflammatory response of condylar cartilage. ß-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 ß-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 ß-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 ß-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-1ß) factors in condylar cartilage were increased in ß-arrestin2 null mice compared with WT mice. Moreover, the loss of ß-arrestin2 promoted apoptosis and autophagic process of chondrocytes at the early stage of TMJOA. CONCLUSION: In conclusion, we demonstrated for the first time that ß-arrestin2 plays a protective role in the development of TMJOA at the early stage, probably by inhibiting apoptosis and autophagic process of chondrocytes. Therefore, ß-arrestin2 might be a potential therapeutic target for TMJOA, providing a new insight for the treatment of TMJOA at the early stage.

ArreSTick motif controls ß-arrestin-binding stability and extends phosphorylation-dependent ß-arrestin interactions to non-receptor proteins.

The binding and function of ß-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 ß-arrestin-binding properties. With this approach, amino acid motifs characteristic of GPCRs that form stable interactions with ß-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 ß-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 ß-arrestin2. Our findings unveil a broader role for ß-arrestins in phosphorylation-dependent interactions, extending beyond GPCRs to encompass non-receptor proteins as well.

Genetic Deletion of ß-Arrestin 2 From the Subfornical Organ and Other Periventricular Nuclei in the Brain Alters Fluid Homeostasis and Blood Pressure.

BACKGROUND: 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 (ß-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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Mapping the interaction site for ß-arrestin-2 in the prokineticin 2 receptor.

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 ß-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 ß-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 ß-arrestin-2 binding and to investigate the role of ß-arrestin-2 in the signalling pathways induced by PKR2 activation. Our data show that PKR2 binds constitutively to ß-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 ß-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 ß-arrestin-2-receptor complex in the presence of PK2 ligand. This leads to the ß-arrestin-2 conformational change required to initiate intracellular signalling that ultimately leads to ERK phosphorylation and activation.

GPCR kinase subtype requirements for arrestin-2 and -3 translocation to the cannabinoid CB1 receptor and the consequences on G protein signalling.

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 role of G protein-coupled receptor kinases in GLP-1R ß-arrestin recruitment and internalisation.

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 ß-arrestins. While differences in GLP-1R trafficking and ß-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 ß-arrestin recruitment profiles using cells where endogenous ß-arrestins, or non-visual GRKs were knocked out using CRISPR/Cas9 genome editing. Our results confirm the previously established atypical ß-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 ß-arrestin 1 and ß-arrestin 2 recruitment were differentially affected by endogenous GRK knockout with ß-arrestin 1 recruitment more sensitive to GRK knockout than ß-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 ß-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.

Distinct roles of the extracellular surface residues of glucagon-like peptide-1 receptor in ß-arrestin 1/2 signaling.

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 ß-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 ß-arrestin 1/2 recruitment triggered by three representative GLP-1R agonists (GLP-1, exendin-4 and oxyntomodulin). Residues selectively regulated ß-arrestin 1/2 recruitment for diverse ligands, and ß-arrestin isoforms were identified. Mutation of residues K130-S136, L142 and Y145 on the transmembrane helix 1 (TM1)-extracellular domain (ECD) linker decreased ß-arrestin 1 recruitment but increased ß-arrestin 2 recruitment. Other extracellular loop (ECL) mutations, including P137A, Q211A, D222A and M303A selectively affected ß-arrestin 1 recruitment while D215A, L217A, Q221A, S223A, Y289A, S301A, F381A and I382A involved more in ß-arrestin 2 recruitment for the ligands. Oxyntomodulin engaged more broadly with GLP-1R extracellular surface to drive ß-arrestin 1/2 recruitment than GLP-1 and exendin-4; I147, W214 and L218 involved in ß-arrestin 1 recruitment, while L141, D215, L218, D293 and F381 in ß-arrestin 2 recruitment for oxyntomodulin particularly. Additionally, the non-conserved residues on ß-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 ß-arrestin 1/2 overexpression demonstrated both commonly and biasedly regulated proteins and pathways associated with cognate ligands and ß-arrestins. Our study offers valuable information about ligand induced ß-arrestin recruitment mediated by GLP-1R and consequent intracellular signaling events.

PRL-mediated STAT5B/ARRB2 pathway promotes the progression of prostate cancer through the activation of MAPK signaling.

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.

Arrestin beta-2 deficiency exacerbates periodontal inflammation by mediating activating transcription factor 6 activation and abnormal remodelling of the extracellular matrix.

AIM: To investigate the specific role of arrestin beta-2 (ARRB2) in the progression of periodontitis and the underlying mechanisms. MATERIALS AND METHODS: Single-cell RNA sequencing data were used to analyse gene expression in periodontal tissues from healthy controls and patients with periodontitis. Real-time quantitative polymerase chain reaction, Western blotting and immunohistochemical staining were performed to detect the expression of ARRB2. Furthermore, a ligature-induced periodontitis model was created. Using radiographic and histological methods, RNA sequencing and luciferase assay, the role of ARRB2 in periodontitis and the underlying mechanisms were explored. Finally, the therapeutic effect of melatonin, an inhibitor of activating transcription factor 6 (ATF6), on periodontitis in mice was assessed in both in vivo and in vitro experiments. RESULTS: ARRB2 expression was up-regulated in inflammatory periodontal tissue. In the ligature-induced mouse model, Arrb2 knockout exacerbated alveolar bone loss (ABL) and extracellular matrix (ECM) degradation. ARRB2 exerted a negative regulatory effect on ATF6, an essential targeted gene. Melatonin ameliorated ABL and an imbalance in ECM remodelling in Arrb2-deficient periodontitis mice. CONCLUSIONS: ARRB2 mediates ECM remodelling via inhibition of the ATF6 signalling pathway, which ultimately exerts a protective effect on periodontal tissues.

The µ-opioid receptor-mediated Gi/o protein and ß-arrestin2 signaling pathways both contribute to morphine-induced side effects.

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 ß-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 ß-arrestin2 recruitment by the BRET assay. Designed peptides might effectively interfere with the activation of the Gio and ß-arrestin2 pathways when combined with morphine. The resulting morphine-induced tolerance, respiratory inhibition, and constipation in mice showed that the ß-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.

Insights Into the Role of Angiotensin-II AT1 Receptor-Dependent ß-Arrestin Signaling in Cardiovascular Disease.

ß-arrestins are a family of intracellular signaling proteins that play a key role in regulating the activity of G protein-coupled receptors. The angiotensin-II type 1 receptor is an important G protein-coupled receptor involved in the regulation of cardiovascular function and has been implicated in the progression of cardiovascular diseases. In addition to canonical G protein signaling, G protein-coupled receptors including the angiotensin-II type 1 receptor can signal via ß-arrestin. Dysregulation of ß-arrestin signaling has been linked to several cardiovascular diseases including hypertension, atherosclerosis, and heart failure. Understanding the role of ß-arrestins in these conditions is critical to provide new therapeutic targets for the treatment of cardiovascular disease. In this review, we will discuss the beneficial and maladaptive physiological outcomes of angiotensin-II type 1 receptor-dependent ß-arrestin activation in different cardiovascular diseases.

Basal interaction of the orphan receptor GPR101 with arrestins leads to constitutive internalization.

GPR101 is an orphan G protein-coupled receptor that promotes growth hormone secretion in the pituitary. The microduplication of the GPR101 gene has been linked with the X-linked acrogigantism, or X-LAG, syndrome. This disease is characterized by excessive growth hormone secretion and abnormal rapid growth beginning early in life. Mechanistically, GPR101 induces growth hormone secretion through constitutive activation of multiple heterotrimeric G proteins. However, the full scope of GPR101 signaling remains largely elusive. Herein, we investigated the association of GPR101 to multiple transducers and uncovered an important basal interaction with Arrestin 2 (ß-arrestin 1) and Arrestin 3 (ß-arrestin 2). By using a GPR101 mutant lacking the C-terminus and cell lines with an Arrestin 2/3 null background, we show that the arrestin association leads to constitutive clathrin- and dynamin-mediated GPR101 internalization. To further highlight GPR101 intracellular fate, we assessed the colocalization of GPR101 with Rab protein markers. Internalized GPR101 was mainly colocalized with the early endosome markers, Rab5 and EEA-1, and to a lesser degree with the late endosome marker Rab7. However, GPR101 was not colocalized with the recycling endosome marker Rab11. These findings show that the basal arrestin recruitment by GPR101 C-terminal tail drives the receptor constitutive clathrin-mediated internalization. Intracellularly, GPR101 concentrates in the endosomal compartment and is degraded through the lysosomal pathway. In conclusion, we uncovered a constitutive intracellular trafficking of GPR101 that potentially represents an important layer of regulation of its signaling and function.

Development of Ligands for the Super Conserved Orphan G Protein-Coupled Receptor GPR27 with Improved Efficacy and Potency.

The orphan G protein-coupled receptor GPR27 appears to play a role in insulin production, secretion, lipid metabolism, neuronal plasticity, and l-lactate homeostasis. However, investigations on the function of GPR27 are impaired by the lack of potent and efficacious agonists. We describe herein the development of di- and trisubstituted benzamide derivatives 4a-e, 7a-z, and 7aa-ai, which display GPR27-specific activity in a ß-arrestin 2 recruitment-based assay. Highlighted compounds are PT-91 (7p: pEC50 6.15; Emax 100%) and 7ab (pEC50 6.56; Emax 99%). A putative binding mode was revealed by the docking studies of 7p and 7ab with a GPR27 homology model. The novel active compounds exhibited no GPR27-mediated activation of G proteins, indicating that the receptor may possess an atypical profile. Compound 7p displays high metabolic stability and brain exposure in mice. Thus, 7p represents a novel tool to investigate the elusive pharmacology of GPR27 and assess its potential as a drug target.

Distinct activation mechanisms of ß-arrestin-1 revealed by 19F NMR spectroscopy.

ß-Arrestins (ßarrs) are functionally versatile proteins that play critical roles in the G-protein-coupled receptor (GPCR) signaling pathways. While it is well established that the phosphorylated receptor tail plays a central role in ßarr activation, emerging evidence highlights the contribution from membrane lipids. However, detailed molecular mechanisms of ßarr activation by different binding partners remain elusive. In this work, we present a comprehensive study of the structural changes in critical regions of ßarr1 during activation using 19F NMR spectroscopy. We show that phosphopeptides derived from different classes of GPCRs display different ßarr1 activation abilities, whereas binding of the membrane phosphoinositide PIP2 stabilizes a distinct partially activated conformational state. Our results further unveil a sparsely-populated activation intermediate as well as complex cross-talks between different binding partners, implying a highly multifaceted conformational energy landscape of ßarr1 that can be intricately modulated during signaling.

GLP-1 and GIP receptors signal through distinct ß-arrestin 2-dependent pathways to regulate pancreatic ß cell function.

Glucagon-like peptide 1 (GLP-1R) and glucose-dependent insulinotropic polypeptide (GIPR) receptors are G-protein-coupled receptors involved in glucose homeostasis. Diabetogenic conditions decrease ß-arrestin 2 (ARRB2) levels in human islets. In mouse ß cells, ARRB2 dampens insulin secretion by partially uncoupling cyclic AMP (cAMP)/protein kinase A (PKA) signaling at physiological doses of GLP-1, whereas at pharmacological doses, the activation of extracellular signal-related kinase (ERK)/cAMP-responsive element-binding protein (CREB) requires ARRB2. In contrast, GIP-potentiated insulin secretion needs ARRB2 in mouse and human islets. The GIPR-ARRB2 axis is not involved in cAMP/PKA or ERK signaling but does mediate GIP-induced F-actin depolymerization. Finally, the dual GLP-1/GIP agonist tirzepatide does not require ARRB2 for the potentiation of insulin secretion. Thus, ARRB2 plays distinct roles in regulating GLP-1R and GIPR signaling, and we highlight (1) its role in the physiological context and the possible functional consequences of its decreased expression in pathological situations such as diabetes and (2) the importance of assessing the signaling pathways engaged by the agonists (biased/dual) for therapeutic purposes.

Signal transduction at GPCRs: Allosteric activation of the ERK MAPK by ß-arrestin.

ß-arrestins are multivalent adaptor proteins that bind active phosphorylated G protein-coupled receptors (GPCRs) to inhibit G protein signaling, mediate receptor internalization, and initiate alternative signaling events. ß-arrestins link agonist-stimulated GPCRs to downstream signaling partners, such as the c-Raf-MEK1-ERK1/2 cascade leading to ERK1/2 activation. ß-arrestins have been thought to transduce signals solely via passive scaffolding by facilitating the assembly of multiprotein signaling complexes. Recently, however, ß-arrestin 1 and 2 were shown to activate two downstream signaling effectors, c-Src and c-Raf, allosterically. Over the last two decades, ERK1/2 have been the most intensely studied signaling proteins scaffolded by ß-arrestins. Here, we demonstrate that ß-arrestins play an active role in allosterically modulating ERK kinase activity in vitro and within intact cells. Specifically, we show that ß-arrestins and their GPCR-mediated active states allosterically enhance ERK2 autophosphorylation and phosphorylation of a downstream ERK2 substrate, and we elucidate the mechanism by which ß-arrestins do so. Furthermore, we find that allosteric stimulation of dually phosphorylated ERK2 by active-state ß-arrestin 2 is more robust than by active-state ß-arrestin 1, highlighting differential capacities of ß-arrestin isoforms to regulate effector signaling pathways downstream of GPCRs. In summary, our study provides strong evidence for a new paradigm in which ß-arrestins function as active "catalytic" scaffolds to allosterically unlock the enzymatic activity of signaling components downstream of GPCR activation.

Gαs is dispensable for ß-arrestin coupling but dictates GRK selectivity and is predominant for gene expression regulation by ß2-adrenergic receptor.

ß-arrestins play a key role in G protein-coupled receptor (GPCR) internalization, trafficking, and signaling. Whether ß-arrestins act independently of G protein-mediated signaling has not been fully elucidated. Studies using genome-editing approaches revealed that whereas G proteins are essential for mitogen-activated protein kinase activation by GPCRs., ß-arrestins play a more prominent role in signal compartmentalization. However, in the absence of G proteins, GPCRs may not activate ß-arrestins, thereby limiting the ability to distinguish G protein from ß-arrestin-mediated signaling events. We used ß2-adrenergic receptor (ß2AR) and its ß2AR-C tail mutant expressed in human embryonic kidney 293 cells wildtype or CRISPR-Cas9 gene edited for Gαs, ß-arrestin1/2, or GPCR kinases 2/3/5/6 in combination with arrestin conformational sensors to elucidate the interplay between Gαs and ß-arrestins in controlling gene expression. We found that Gαs is not required for ß2AR and ß-arrestin conformational changes, ß-arrestin recruitment, and receptor internalization, but that Gαs dictates the GPCR kinase isoforms involved in ß-arrestin recruitment. By RNA-Seq analysis, we found that protein kinase A and mitogen-activated protein kinase gene signatures were activated by stimulation of ß2AR in wildtype and ß-arrestin1/2-KO cells but absent in Gαs-KO cells. These results were validated by re-expressing Gαs in the corresponding KO cells and silencing ß-arrestins in wildtype cells. These findings were extended to cellular systems expressing endogenous levels of ß2AR. Overall, our results support that Gs is essential for ß2AR-promoted protein kinase A and mitogen-activated protein kinase gene expression signatures, whereas ß-arrestins initiate signaling events modulating Gαs-driven nuclear transcriptional activity.

Competing Engagement of ß-arrestin Isoforms Balances IGF1R/p53 Signaling and Controls Melanoma Cell Chemotherapeutic Responsiveness.

Constraints on the p53 tumor suppressor pathway have long been associated with the progression, therapeutic resistance, and poor prognosis of melanoma, the most aggressive form of skin cancer. Likewise, the insulin-like growth factor type 1 receptor (IGF1R) is recognized as an essential coordinator of transformation, proliferation, survival, and migration of melanoma cells. Given that ß-arrestin (ß-arr) system critically governs the anti/pro-tumorigenic p53/IGF1R signaling pathways through their common E3 ubiquitin-protein ligase MDM2, we explore whether unbalancing this system downstream of IGF1R can enhance the response of melanoma cells to chemotherapy. Altering ß-arr expression demonstrated that both ß-arr1-silencing and ß-arr2-overexpression (-ß-arr1/+ß-arr2) facilitated nuclear-to-cytosolic MDM2 translocation accompanied by decreased IGF1R expression, while increasing p53 levels, resulting in reduced cell proliferation/survival. Imbalance towards ß-arr2 (-ß-arr1/+ß-arr2) synergizes with the chemotherapeutic agent, dacarbazine, in promoting melanoma cell toxicity. In both 3D spheroid models and in vivo in zebrafish models, this combination strategy, through dual IGF1R downregulation/p53 activation, limits melanoma cell growth, survival and metastatic spread. In clinical settings, analysis of the TCGA-SKCM patient cohort confirms ß-arr1-/ß-arr2+ imbalance as a metastatic melanoma vulnerability that may enhance therapeutic benefit. Our findings suggest that under steady-state conditions, IGF1R/p53-tumor promotion/suppression status-quo is preserved by ß-arr1/2 homeostasis. Biasing this balance towards ß-arr2 can limit the protumorigenic IGF1R activities while enhancing p53 activity, thus reducing multiple cancer-sustaining mechanisms. Combined with other therapeutics, this strategy improves patient responses and outcomes to therapies relying on p53 or IGF1R pathways. IMPLICATIONS: Altogether, ß-arrestin system bias downstream IGF1R is an important metastatic melanoma vulnerability that may be conductive for therapeutic benefit.

Understanding the Molecular Regulation of Serotonin Receptor 5-HTR1B-ß-Arrestin1 Complex in Stress and Anxiety Disorders.

The serotonin receptor subtype 5-HTR1B is widely distributed in the brain with an important role in various behavioral implications including neurological conditions and psychiatric disorders. The neuromodulatory action of 5-HTR1B largely depends upon its arrestin mediated signaling pathway. In this study, we tried to investigate the role of unusually long intracellular loop 3 (ICL3) region of the serotonin receptor 5-HTR1B in interaction with ß-arrestin1 (Arr2) to compensate for the absence of the long cytoplasmic tail. Molecular modeling and docking tools were employed to obtain a suitable molecular conformation of the ICL3 region in complex with Arr2 which dictates the specific complex formation of 5-HTR1B with Arr2. This reveals the novel molecular mechanism of phosphorylated ICL3 mediated GPCR-arrestin interaction in the absence of the long cytoplasmic tail. The in-cell disulfide cross-linking experiments and molecular dynamics simulations of the complex further validate the model of 5-HTR1B-ICL3-Arr2 complex. Two serine residues (Ser281 and Ser295) within the 5-HTR1B-ICL3 region were found to be occupying the electropositive pocket of Arr2 in our model and might be crucial for phosphorylation and specific Arr2 binding. The alignment studies of these residues showed them to be conserved only across 5-HTR1B mammalian species. Thus, our studies were able to predict a molecular conformation of 5-HTR1B-Arr2 and identify the role of long ICL3 in the signaling process which might be crucial in designing targeted drugs (biased agonists) that promote GPCR-Arr2 signaling to deter the effects of stress and anxiety-like disorders.

Profiling of basal and ligand-dependent GPCR activities by means of a polyvalent cell-based high-throughput platform.

Representing the most attractive and successful druggable receptors of the proteome, GPCRs regulate a myriad of physiological and pathophysiological functions. Although over half of present pharmaceuticals target GPCRs, the advancement of drug discovery is hampered by a lack of adequate screening tools, the majority of which are limited to probing agonist-induced G-protein and ß-arrestin-2-mediated events as a measure of receptor activation. Here, we develop Tango-Trio, a comprehensive cell-based high-throughput platform comprising cumate-inducible expression of transducers, capable of the parallelized profiling of both basal and agonist-dependent GPCR activities. We capture the functional diversity of GPCRs, reporting ß-arrestin-1/2 couplings, selectivities, and receptor internalization signatures across the GPCRome. Moreover, we present the construction of cumate-induced basal activation curves at approximately 200 receptors, including over 50 orphans. Overall, Tango-Trio's robustness is well-suited for the functional characterization and screening of GPCRs, especially for parallel interrogation, and is a valuable addition to the pharmacological toolbox.