Conformational dynamics underlying atypical chemokine receptor 3 activation.

Atypical Chemokine Receptor 3 (ACKR3) belongs to the G protein-coupled receptor family but it does not signal through G proteins. The structural properties that govern the functional selectivity and the conformational dynamics of ACKR3 activation are poorly understood. Here, we combined hydrogen/deuterium exchange mass spectrometry, site-directed mutagenesis, and molecular dynamics simulations to examine the binding mode and mechanism of action of ACKR3 ligands of different efficacies. Our results show that activation or inhibition of ACKR3 is governed by intracellular conformational changes of helix 6, intracellular loop 2, and helix 7, while the DRY motif becomes protected during both processes. Moreover, we identified the binding sites and the allosteric modulation of ACKR3 upon ß-arrestin 1 binding. In summary, this study highlights the structure-function relationship of small ligands, the binding mode of ß-arrestin 1, the activation dynamics, and the atypical dynamic features in ACKR3 that may contribute to its inability to activate G proteins.

AMPK Attenuation of ß-Adrenergic Receptor-Induced Cardiac Injury via Phosphorylation of ß-Arrestin-1-ser330.

BACKGROUND: ß-adrenergic receptor (ß-AR) overactivation is a major pathological cue associated with cardiac injury and diseases. AMPK (AMP-activated protein kinase), a conserved energy sensor, regulates energy metabolism and is cardioprotective. However, whether AMPK exerts cardioprotective effects via regulating the signaling pathway downstream of ß-AR remains unclear. METHODS: Using immunoprecipitation, mass spectrometry, site-specific mutation, in vitro kinase assay, and in vivo animal studies, we determined whether AMPK phosphorylates ß-arrestin-1 at serine (Ser) 330. Wild-type mice and mice with site-specific mutagenesis (S330A knock-in [KI]/S330D KI) were subcutaneously injected with the ß-AR agonist isoproterenol (5 mg/kg) to evaluate the causality between ß-adrenergic insult and ß-arrestin-1 Ser330 phosphorylation. Cardiac transcriptomics was used to identify changes in gene expression from ß-arrestin-1-S330A/S330D mutation and ß-adrenergic insult. RESULTS: Metformin could decrease cAMP/PKA (protein kinase A) signaling induced by isoproterenol. AMPK bound to ß-arrestin-1 and phosphorylated Ser330 with the highest phosphorylated mass spectrometry score. AMPK activation promoted ß-arrestin-1 Ser330 phosphorylation in vitro and in vivo. Neonatal mouse cardiomyocytes overexpressing ß-arrestin-1-S330D (active form) inhibited the ß-AR/cAMP/PKA axis by increasing PDE (phosphodiesterase) 4 expression and activity. Cardiac transcriptomics revealed that the differentially expressed genes between isoproterenol-treated S330A KI and S330D KI mice were mainly involved in immune processes and inflammatory response. ß-arrestin-1 Ser330 phosphorylation inhibited isoproterenol-induced reactive oxygen species production and NLRP3 (NOD-like receptor protein 3) inflammasome activation in neonatal mouse cardiomyocytes. In S330D KI mice, the ß-AR-activated cAMP/PKA pathways were attenuated, leading to repressed inflammasome activation, reduced expression of proinflammatory cytokines, and mitigated macrophage infiltration. Compared with S330A KI mice, S330D KI mice showed diminished cardiac fibrosis and improved cardiac function upon isoproterenol exposure. However, the cardiac protection exerted by AMPK was abolished in S330A KI mice. CONCLUSIONS: AMPK phosphorylation of ß-arrestin-1 Ser330 potentiated PDE4 expression and activity, thereby inhibiting ß-AR/cAMP/PKA activation. Subsequently, ß-arrestin-1 Ser330 phosphorylation blocks ß-AR-induced cardiac inflammasome activation and remodeling.

Roles of the gate loop in ß-arrestin-1 conformational dynamics and phosphorylated receptor interaction.

Arrestins interact with phosphorylated G protein-coupled receptors (GPCRs) and regulate the homologous desensitization and internalization of GPCRs. The gate loop in arrestins is a critical region for both stabilization of the basal state and interaction with phosphorylated receptors. We investigated the roles of specific residues in the gate loop (K292, K294, and H295) using ß-arrestin-1 and phosphorylated C-tail peptide of vasopressin receptor type 2 (V2Rpp) as a model system. We measured the binding affinity of V2Rpp and analyzed conformational dynamics of ß-arrestin-1. Our results suggest that K294 plays a critical role in the interaction with V2Rpp without influencing the overall conformation of the V2Rpp-bound state. The residues K292 and H295 contribute to the stability of the polar core in the basal state and form a specific conformation of the finger loop in the V2Rpp-bound state.

ARRB1 inhibits extracellular matrix degradation and apoptosis of nucleus pulposus cells by promoting autophagy and attenuates intervertebral disc degeneration.

OBJECTIVE: Intervertebral disc degeneration (IVDD) is the leading cause of lower back pain (LBP). ß-arrestin 1 (ARRB1) is a multifunctional protein that regulates numerous pathological processes. The aim of this study was to investigate the role of ARRB1 in IVDD. METHODS: The expression of ARRB1 in nucleus pulposus (NP) of rats with IVDD was assayed. Next, rat nucleus pulposus cells (NPCs) were infected with lentiviruses containing shArrb1 (LV-shArrb1) and overexpressing Arrb1 (LV-oeArrb1). The roles of Arrb1 in serum-deprived NPCs were investigated by measuring apoptosis, extracellular matrix degradation, and autophagic flux. For experiments in vivo, LV-oeArrb1 lentivirus was injected into the NP tissues of IVDD rats to evaluate the effects of Arrb1 overexpression on NP. RESULTS: In the NP tissues of IVDD rats, ARRB1 and cleaved caspase-3 expression increased, and the ratio of LC3II/LC3I protein expression was upregulated. Arrb1 knockdown aggravated extracellular matrix degradation, cellular apoptosis, and impairment of autophagic flux in rat NPCs under serum-deprived conditions, whereas Arrb1 overexpression significantly reversed these effects. ARRB1 interacted with Beclin 1, and Arrb1 knockdown suppressed the formation of the Beclin1-PIK3C3 core complex. The autophagy inhibitor 3-methyladenine (3-MA) offset the protective effects of Arrb1 overexpression in serum-deprived NPCs. Furthermore, Arrb1 overexpression inhibited apoptosis and extracellular matrix degradation, promoted autophagy in NP, and delayed the development of IVDD in rats. CONCLUSION: ARRB1 prevents extracellular matrix degradation and apoptosis of NPCs by upregulating autophagy and ameliorating IVDD progression, presenting an innovative strategy for the treatment of IVDD.

The ß-arrestin1/endothelin axis bolsters ovarian fibroblast-dependent invadosome activity and cancer cell metastatic potential.

Recruitment of fibroblasts to tumors and their activation into cancer-associated fibroblasts (CAFs) is a strategy used by tumor cells to direct extracellular matrix (ECM) remodeling, invasion, and metastasis, highlighting the need to investigate the molecular mechanisms driving CAF function. Endothelin-1 (ET-1) regulates the communication between cancer and stroma and facilitates the progression of serous ovarian cancer (SOC). By binding to Endothelin A (ETA) and B (ETB) receptors, ET-1 enables the recruitment of ß-arrestin1 (ß-arr1) and the formation of signaling complexes that coordinate tumor progression. However, how ET-1 receptors might "educate" human ovarian fibroblasts (HOFs) to produce altered ECM and promote metastasis remains to be elucidated. This study identifies ET-1 as a pivotal factor in the activation of CAFs capable of proteolytic ECM remodeling and the generation of heterotypic spheroids containing cancer cells with a propensity to metastasize. An autocrine/paracrine ET-1/ETA/BR/ß-arr1 loop enhances HOF proliferation, upregulates CAF marker expression, secretes pro-inflammatory cytokines, and increases collagen contractility, and cell motility. Furthermore, ET-1 facilitates ECM remodeling by promoting the lytic activity of invadosome and activation of integrin ß1. In addition, ET-1 signaling supports the formation of heterotypic HOF/SOC spheroids with enhanced ability to migrate through the mesothelial monolayer, and invade, representing metastatic units. The blockade of ETA/BR or ß-arr1 silencing prevents CAF activation, invadosome function, mesothelial clearance, and the invasive ability of heterotypic spheroids. In vivo, therapeutic inhibition of ETA/BR using bosentan (BOS) significantly reduces the metastatic potential of combined HOFs/SOC cells, associated with enhanced apoptotic effects on tumor cells and stromal components. These findings support a model in which ET-1/ß-arr1 reinforces tumor/stroma interaction through CAF activation and fosters the survival and metastatic properties of SOC cells, which could be counteracted by ETA/BR antagonists.

Ligand efficacy modulates conformational dynamics of the µ-opioid receptor.

The µ-opioid receptor (µOR) is an important target for pain management1 and molecular understanding of drug action on µOR will facilitate the development of better therapeutics. Here we show, using double electron-electron resonance and single-molecule fluorescence resonance energy transfer, how ligand-specific conformational changes of µOR translate into a broad range of intrinsic efficacies at the transducer level. We identify several conformations of the cytoplasmic face of the receptor that interconvert on different timescales, including a pre-activated conformation that is capable of G-protein binding, and a fully activated conformation that markedly reduces GDP affinity within the ternary complex. Interaction of ß-arrestin-1 with the µOR core binding site appears less specific and occurs with much lower affinity than binding of Gi.

NPAS2, transcriptionally activated by ARRB1, promotes the malignant behaviours of lung adenocarcinoma cells and regulates the reprogramming of glucose metabolism.

Lung adenocarcinoma (LUAD) is a serious threat to public health and is accompanied by increased morbidity and mortality worldwide. Neuronal PAS domain protein2 (NPAS2) has been confirmed as an oncogene in LUAD; however, little is known about its molecular mechanism. Here, the expression level of NPAS2 was detected in LUAD cell lines and 16HBE cells. Gain- and loss-of-function experiments were performed. Cell Counting Kit-8, colony formation, flow cytometry, wound-healing and Transwell assays were conducted to assess cell proliferation, apoptosis, migration and invasion, respectively. Reprogramming of glucose metabolism was evaluated via oxygen consumption rate (OCR), complexes activities, lactic production and glucose consumption. The expression of critical proteins was examined by western blot. We demonstrated aberrant upregulation of NPAS2 and ß-arrestin-1 (ARRB1) in LUAD cell lines. ARRB1 was found to be a critical transcription factor of NPAS2 with binding sites within the promoter region of NPAS2, thereby causing its transcriptional activation. Functional experiments revealed that NPAS2 depletion significantly inhibited the malignant behaviours of A549 cells by suppressing cell proliferation, migration, invasion and epithelial-mesenchymal transition and promoting cell apoptosis. Meanwhile, NPAS2 depletion increased OCR and activities of complexes (I, II, III and V), and reduced lactic acid production and glucose uptake in A549 cells, indicating that NPAS2 depletion inhibited aerobic glycolysis, accompanied by reduced expression of glycolytic enzymes. However, the changes caused by NPAS2 knockdown were partly restored by ARRB1 overexpression. In conclusion, our study suggests that ARRB1 could transcriptionally activate NPAS2, facilitating malignant activities and glycolysis, and ultimately promoting the progression of LUAD, proving a novel therapeutic strategy for the treatment of LUAD.

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.

The Opioid Receptor Influences Circadian Rhythms in Human Keratinocytes through the ß-Arrestin Pathway.

The recent emphasis on circadian rhythmicity in critical skin cell functions related to homeostasis, regeneration and aging has shed light on the importance of the PER2 circadian clock gene as a vital antitumor gene. Furthermore, delta-opioid receptors (DOPrs) have been identified as playing a crucial role in skin differentiation, proliferation and migration, which are not only essential for wound healing but also contribute to cancer development. In this study, we propose a significant association between cutaneous opioid receptor (OPr) activity and circadian rhythmicity. To investigate this link, we conducted a 48 h circadian rhythm experiment, during which RNA samples were collected every 5 h. We discovered that the activation of DOPr by its endogenous agonist Met-Enkephalin in N/TERT-1 keratinocytes, synchronized by dexamethasone, resulted in a statistically significant 5.6 h delay in the expression of the core clock gene PER2. Confocal microscopy further confirmed the simultaneous nuclear localization of the DOPr-ß-arrestin-1 complex. Additionally, DOPr activation not only enhanced but also induced a phase shift in the rhythmic binding of ß-arrestin-1 to the PER2 promoter. Furthermore, we observed that ß-arrestin-1 regulates the transcription of its target genes, including PER2, by facilitating histone-4 acetylation. Through the ChIP assay, we determined that Met-Enkephalin enhances ß-arrestin-1 binding to acetylated H4 in the PER2 promoter. In summary, our findings suggest that DOPr activation leads to a phase shift in PER2 expression via ß-arrestin-1-facilitated chromatin remodeling. Consequently, these results indicate that DOPr, much like its role in wound healing, may also play a part in cancer development by influencing PER2.

Macrophage ß-arrestin-1 deteriorates DSS-induced colitis through interaction with NF-κB signaling.

ß-arrestin-1 has been demonstrated to participate in the regulation of inflammatory reactions in several diseases. Thus, this study aimed to investigate the role of macrophage ß-arrestin-1 in the pathogenesis and progression of ulcerative colitis (UC). A myeloid ß-arrestin-1 conditional knockout mouse model was generated to explore the role of macrophage ß-arrestin-1. DSS was employed for the establishment of an ulcerative colitis mouse model, using TNF-α as an inflammatory stressor in vitro. The expression level of ß-arrestin-1 was detected via western blot and immunofluorescence assays, whilst disease severity was evaluated by clinical score and H&E staining in the DSS-induced colitis model. In the in vitro experiments, the levels of inflammatory cytokines were examined using real-time PCR. NF-κB activation was detected through the double luciferase reporter system, western blot, and electrophoretic mobility shift assay (EMSA). BAY11-7082 was used to inhibit NF-κB activation. Our results exposed that the level of ß-arrestin-1 was increased in monocytes/macrophages derived from DSS-induced colitis mice or under the TNF-α challenge. Moreover, conditionally knocking out the expression of myeloid ß-arrestin-1 alleviated disease severity, while knocking out the expression of ß-arrestin-1 decreased the levels of inflammatory cytokines. Additionally, NF-κB was identified as a central regulatory element of ß-arrestin-1 promoter, and using BAY11-7082 to inhibit NF-κB activation lowered the level of ß-arrestin-1 under TNF-α challenge. ß-arrestin-1 led to the activation of the NF-κB signaling pathway by enhancing binding to IκBα and IKK under the TNF-α challenge. Taken together, our findings demonstrated macrophage ß-arrestin-1 contributes to the deterioration of DSS-induced colitis through the interaction with NF-κB signaling, thus highlighting a novel target for the treatment of UC.

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.

Investigating selectivity and bias for G protein subtypes and ß-arrestins by synthetic cannabinoid receptor agonists at the cannabinoid CB1 receptor.

The cannabinoid CB1 receptor (CB1) is a G protein-coupled receptor (GPCR) with widespread expression in the central nervous system. This canonically G⍺i/o-coupled receptor mediates the effects of Δ9-tetrahydrocannabinol (THC) and synthetic cannabinoid receptor agonists (SCRAs). Recreational use of SCRAs is associated with serious adverse health effects, making pharmacological research into these compounds a priority. Several studies have hypothesised that signalling bias may explain the different toxicological profiles between SCRAs and THC. Previous studies have focused on bias between G protein activation measured by cyclic adenosine monophosphate (cAMP) inhibition and ß-arrestin translocation. In contrast, the current study characterises bias between G⍺ subtypes of the G⍺i/o family and ß-arrestins; this method facilitates a more accurate assessment of ligand bias by assessing signals that have not undergone major amplification. We have characterised G protein dissociation and translocation of ß-arrestin 1 and 2 using real-time BRET reporters. The responses produced by each SCRA across the G protein subtypes tested were consistent with the responses produced by the reference ligand AMB-FUBINACA. Ligand bias was probed by applying the operational analysis to determine biases within the G⍺i/o family, and between G protein subtypes and ß-arrestins. Overall, these results confirm SCRAs to be balanced, high-efficacy ligands compared to the low efficacy ligand THC, with only one SCRA, 4CN-MPP-BUT7IACA, demonstrating statistically significant bias in one pathway comparison (towards ß-arrestin 1 when compared with G⍺oA/oB). This suggests that the adverse effects caused by SCRAs are due to high potency and efficacy at CB1, rather than biased agonism.

Role of Formyl Peptide Receptors and ß-Arrestin-1 in suPAR Signal Transduction in Mouse Podocytes: Interactions with αVß3-Integrin.

The soluble urokinase plasminogen activator receptor (suPAR) has been implicated in a wide range of pathological conditions including primary nephrotic syndromes and acute kidney injuries. suPAR can trigger transduction cascades in podocytes by outside-in activation of αVß3-integrin, but there is evidence that the functional cell surface response element is actually a complex of different types of receptors, which may also include the receptor for advanced glycation end-products (RAGE) and formyl peptide receptors (FPRs). Here we observed that ROS accumulation and Src activation could be evoked by continuous 24 h exposure to either suPAR or the FPR agonist fMLF. Responses to suPAR and fMLF were completely blocked by either the FPR antagonist WRW4 or by the αV-integrin inhibitor cilengitide. Moreover, endogenous podocyte mouse Fpr1 co-immunoprecipitates with ß3-integrin, suggesting that these receptors occur as a complex on the cell surface. suPAR- and fMLF-evoked activation of Src and ROS differed in time course. Thus, robust pertussis toxin (PTX)-sensitive responses were evoked by 60 min exposures to fMLF but not to suPAR. By contrast, responses to 24 h exposures to either suPAR or fMLF were PTX-resistant and were instead abolished by knockdown of ß-arrestin-1 (BAR1). FPRs, integrins, and RAGE (along with various Toll-like receptors) can all function as pattern-recognition receptors that respond to "danger signals" associated with infections and tissue injury. The fact that podocytes express such a wide array of pattern-recognition receptors suggests that the glomerular filter is designed to change its function under certain conditions, possibly to facilitate clearance of toxic macromolecules.

In vivo identification of Drosophila rhodopsin interaction partners by biotin proximity labeling.

Proteins exert their function through protein-protein interactions. In Drosophila, G protein-coupled receptors like rhodopsin (Rh1) interact with a G protein to activate visual signal transduction and with arrestins to terminate activation. Also, membrane proteins like Rh1 engage in protein-protein interactions during folding within the endoplasmic reticulum, during their vesicular transport and upon removal from the cell surface and degradation. Here, we expressed a Rh1-TurboID fusion protein (Rh1::TbID) in Drosophila photoreceptors to identify in vivo Rh1 interaction partners by biotin proximity labeling. We show that Rh1::TbID forms a functional rhodopsin that mediates biotinylation of arrestin 2 in conditions where arrestin 2 interacts with rhodopsin. We also observed biotinylation of Rh1::TbID and native Rh1 as well as of most visual signal transduction proteins. These findings indicate that the signaling components in the rhabdomere approach rhodopsin closely, within a range of ca. 10 nm. Furthermore, we have detected proteins engaged in the maturation of rhodopsin and elements responsible for the trafficking of membrane proteins, resembling potential interaction partners of Rh1. Among these are chaperons of the endoplasmic reticulum, proteins involved in Clathrin-mediated endocytosis as well as previously unnoticed contributors to rhodopsin transportation, such as Rab32, Vap33, or PIP82.

ARRB1 downregulates acetaminophen-induced hepatoxicity through binding to p-eIF2α to inhibit ER stress signaling.

Acetaminophen (APAP) stands as the predominant contributor to drug-induced liver injury (DILI), and limited options are available. ß-Arrestin1 (ARRB1) is involved in numerous liver diseases. However, the role of ARRB1 in APAP-induced liver injury remained uncertain. Wild-type (WT) and ARRB1 knockout (KO) mice were injected with APAP and sacrificed at the indicated times. The histological changes, inflammation, endoplasmic reticulum (ER) stress, and apoptosis were then evaluated. Hepatic cell lines AML-12 and primary hepatocytes were used for in vitro analyses. Systemic ARRB1-KO mice were susceptible to APAP-induced hepatotoxicity, as indicated by larger areas of centrilobular necrosis area and higher levels of ALT, AST, and inflammation level. Moreover, ARRB1-KO mice exhibited increased ER stress (indicated by phosphorylated α subunit of eukaryotic initiation factor 2 (p-eIF2α)-activating transcription factor 4 (ATF4)-CCAAT-enhancer-binding protein homologous protein (CHOP)) and apoptosis (indicated by cleaved caspase 3). Further rescue experiments demonstrated that the induction of apoptosis was partially mediated by ER stress. Overexpression of ARRB1 alleviated APAP-induced ER stress and apoptosis. Moreover, co-IP analysis revealed that ARRB1 directly bound to p-eIF2α and eIF2α. ARRB1 protected against APAP-induced hepatoxicity through targeting ER stress and apoptosis. ARRB1 is a prospective target for treating APAP-induced DILI.

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.

ß-Arrestin-independent endosomal cAMP signaling by a polypeptide hormone GPCR.

Many G protein-coupled receptors (GPCRs) initiate a second phase of stimulatory heterotrimeric G protein (Gs)-coupled cAMP signaling after endocytosis. The prevailing current view is that the endosomal signal is inherently ß-arrestin-dependent because ß-arrestin is necessary for receptor internalization and, for some GPCRs, to prolong the endosomal signal. Here we revise this view by showing that the vasoactive intestinal peptide receptor 1 (VIPR1), a secretin-family polypeptide hormone receptor, does not require ß-arrestin to internalize or to generate an endosomal signal. ß-Arrestin instead resolves the plasma membrane and endosomal signaling phases into sequential cAMP peaks by desensitizing the plasma membrane phase without affecting the endosomal phase. This appears to occur through the formation of functionally distinct VIPR1-ß-arrestin complexes at each location that differ in their phosphorylation dependence. We conclude that endosomal GPCR signaling can occur in the absence of ß-arrestin and that ß-arrestin sculpts the spatiotemporal profile of cellular GPCR-G protein signaling through location-specific remodeling of GPCR-ß-arrestin complexes.

T-2 Toxin-Mediated ß-Arrestin-1 O-GlcNAcylation Exacerbates Glomerular Podocyte Injury via Regulating Histone Acetylation.

T-2 toxin causes renal dysfunction with proteinuria and glomerular podocyte damage. This work explores the role of metabolic disorder/reprogramming-mediated epigenetic modification in the progression of T-2 toxin-stimulated podocyte injury. A metabolomics experiment is performed to assess metabolic responses to T-2 toxin infection in human podocytes. Roles of protein O-linked-N-acetylglucosaminylation (O-GlcNAcylation) in regulating T-2 toxin-stimulated podocyte injury in mouse and podocyte models are assessed. O-GlcNAc target proteins are recognized by mass spectrometry and co-immunoprecipitation experiments. Moreover, histone acetylation and autophagy levels are measured. T-2 toxin infection upregulates glucose transporter type 1 (GLUT1) expression and enhances hexosamine biosynthetic pathway in glomerular podocytes, resulting in a significant increase in ß-arrestin-1 O-GlcNAcylation. Decreasing ß-arrestin-1 or O-GlcNAc transferase (OGT) effectively prevents T-2 toxin-induced renal dysfunction and podocyte injury. Mechanistically, O-GlcNAcylation of ß-arrestin-1 stabilizes ß-arrestin-1 to activate the mammalian target of rapamycin (mTOR) pathway as well as to inhibit autophagy during podocyte injury by promoting H4K16 acetylation. To sum up, OGT-mediated ß-arrestin-1 O-GlcNAcylation is a vital regulator in the development of T-2 toxin-stimulated podocyte injury via activating the mTOR pathway to suppress autophagy. Targeting ß-arrestin-1 or OGT can be a potential therapy for T-2 toxin infection-associated glomerular injury, especially podocyte injury.

Functional profiling of the G protein-coupled receptor C3aR1 reveals ligand-mediated biased agonism.

G protein-coupled receptors (GPCRs) are leading druggable targets for several medicines, but many GPCRs are still untapped for their therapeutic potential due to poor understanding of specific signaling properties. The complement C3a receptor 1 (C3aR1) has been extensively studied for its physiological role in C3a-mediated anaphylaxis/inflammation, and in TLQP-21-mediated lipolysis, but direct evidence for the functional relevance of the C3a and TLQP-21 ligands and signal transduction mechanisms are still limited. In addition, C3aR1 G protein coupling specificity is still unclear, and whether endogenous ligands, or drug-like compounds, show ligand-mediated biased agonism is unknown. Here, we demonstrate that C3aR1 couples preferentially to Gi/o/z proteins and can recruit ß-arrestins to cause internalization. Furthermore, we showed that in comparison to C3a63-77, TLQP-21 exhibits a preference toward Gi/o-mediated signaling compared to ß-arrestin recruitment and internalization. We also show that the purported antagonist SB290157 is a very potent C3aR1 agonist, where antagonism of ligand-stimulated C3aR1 calcium flux is caused by potent ß-arrestin-mediated internalization. Finally, ligand-mediated signaling bias impacted cell function as demonstrated by the regulation of calcium influx, lipolysis in adipocytes, phagocytosis in microglia, and degranulation in mast cells. Overall, we characterize C3aR1 as a Gi/o/z-coupled receptor and demonstrate the functional relevance of ligand-mediated signaling bias in key cellular models. Due to C3aR1 and its endogenous ligands being implicated in inflammatory and metabolic diseases, these results are of relevance toward future C3aR1 drug discovery.

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.