G Protein-coupled Receptor Kinase Research Articles

In vitro and in vivo exploitation of cell stress pathways using methanolic extracts of Phlomis stewartii in diabetic rat’s model

Various bioactive constituents present in plants are used to treat metabolic disorders linked to oxidative stress and obesity. Low-grade to chronic inflammation and IR are considered as main causes of obesity, which is strongly correlated with the prevalence and incidence of Type-2 Diabetes Mellitus (T2DM). In this study, the biological influence of Phlomis stewartii extracts was evaluated in vitro and in vivo using rat diabetic models. The biological effects included anti-inflammatory, anti-proliferative, anti-apoptotic, metabolic hormonal via molecular signaling pathways, and antioxidant potential. The extracts from P. stewartii exhibited the strongest DPPH and FRAP potential for LM (38.92 µg/mL) and (45.28 µg/mL) respectively. Moreover, the high absorbance for reducing power was observed in leaves methanol (LM) 1.36 which is greater than flower methanol (FM) 1.12, and whole plant methanol (WPM) 1.21 at a concentration of 500 µg/mL when comparing treated groups to the negative control (NC), and positive control (PC) groups. The results of an Enzyme-linked Immunosorbent Assay (ELISA) study measuring a metabolic hormonal profile that includes Triiodothyronine (T3), Thyroxine (T4), Thyroid Stimulating Hormone (TSH), insulin, leptin, and glucokinase showed that LM has been greatly recovered. The study examined the gene expression analysis of cell stress pathways, and it found that the PC groups had higher levels of Janus kinase (JAK), Signal Transducer and Activator of Transcription (STATS) pathways through JAK (JAK-2 and STAT-1), G protein-Coupled Receptor Kinase 2 (GRK-2), Calmodulin 2 (CALM-2), Proteins Inhibitor of Activated STAT 1 (PIAS-1), Forkhead Box 01, 03, 04, (FOXO-1, FOXO-3, FOXO-4), Bcl-2 Associated X-protein (BAX), B-cell Lymphoma 2 (BCL-2), Interleukins (IL-6, IL-1β), and Tumour Necrosis Factor-α (TNF-α). On the other hand, downregulation was observed in NC groups and P. stewartii extract-treated groups. The outcome of this study suggests a strong relationship towards ameliorative effects.

GRK5 is required for adipocyte differentiation through ERK activation.

Previous studies have identified G protein-coupled receptor (GPCR) kinase 5 (GRK5) as a genetic factor contributing to obesity pathogenesis, but the underlying mechanism remains unclear. We demonstrate here that Grk5 mRNA is more abundant in stromal vascular fractions of mouse white adipose tissue, the fraction that contains adipose progenitor cells, or committed pre-adipocytes, than in adipocyte fractions. Thus, we generated a GRK5 knockout (KO) 3T3-L1 pre-adipocyte to further investigate the mechanistic role of GRK5 in regulating adipocyte differentiation. During adipogenic stimulation, GRK5 KO pre-adipocytes were unable to achieve mature adipocyte morphology and lipid accumulation compared to wildtype cells coupled with suppressed adipogenic and lipogenic gene expression. Beside GPCR signaling, RNA sequencing and pathway analysis identified insulin-like growth factor 1 (IGF-1) signaling to be one of the top 5 significantly dysregulated pathways in GRK5 KO cells. GRK5 KO cells also displayed decreased insulin-stimulated ERK phosphorylation, a downstream target of insulin-stimulated IGF-1 receptor activation, suggesting that GRK5 acts through this critical pathway to impact 3T3-L1 adipocyte differentiation. To find a more translational approach, we identified a new small molecule GRK5 inhibitor that was able to reduce 3T3-L1 adipogenesis. These data suggest that GRK5 is required for adipocyte differentiation through IGF-1 receptor/ERK activation and may be a promising translational target for obesity.

Differential Modulation of Catecholamine and Adipokine Secretion by the Short Chain Fatty Acid Receptor FFAR3 and α2-Adrenergic Receptors in PC12 Cells.

Sympathetic nervous system (SNS) hyperactivity is mediated by elevated catecholamine (CA) secretion from the adrenal medulla, as well as enhanced norepinephrine (NE) release from peripheral sympathetic nerve terminals. Adrenal CA production from chromaffin cells is tightly regulated by sympatho-inhibitory α2-adrenergic (auto)receptors (ARs), which inhibit both epinephrine (Epi) and NE secretion via coupling to Gi/o proteins. α2-AR function is, in turn, regulated by G protein-coupled receptor (GPCR)-kinases (GRKs), especially GRK2, which phosphorylate and desensitize them, i.e., uncouple them from G proteins. On the other hand, the short-chain free fatty acid (SCFA) receptor (FFAR)-3, also known as GPR41, promotes NE release from sympathetic neurons via the Gi/o-derived free Gβγ-activated phospholipase C (PLC)-β/Ca2+ signaling pathway. However, whether it exerts a similar effect in adrenal chromaffin cells is not known at present. In the present study, we examined the interplay of the sympatho-inhibitory α2A-AR and the sympatho-stimulatory FFAR3 in the regulation of CA secretion from rat adrenal chromaffin (pheochromocytoma) PC12 cells. We show that FFAR3 promotes CA secretion, similarly to what GRK2-dependent α2A-AR desensitization does. In addition, FFAR3 activation enhances the effect of the physiologic stimulus (acetylcholine) on CA secretion. Importantly, GRK2 blockade to restore α2A-AR function or the ketone body beta-hydroxybutyrate (BHB or 3-hydroxybutyrate), via FFAR3 antagonism, partially suppress CA production, when applied individually. When combined, however, CA secretion from PC12 cells is profoundly suppressed. Finally, propionate-activated FFAR3 induces leptin and adiponectin secretion from PC12 cells, two important adipokines known to be involved in tissue inflammation, and this effect of FFAR3 is fully blocked by the ketone BHB. In conclusion, SCFAs can promote CA and adipokine secretion from adrenal chromaffin cells via FFAR3 activation, but the metabolite/ketone body BHB can effectively inhibit this action.

Adrenal G protein-coupled Receptor Kinase (GRK)-2 Downregulation And Regulator of G protein Signaling (RGS)-4 Upregulation Mediate The Sympatholytic Effect Of Dapagliflozin

Dapagliflozin is a sodium/­glucose co-­transporter (SGLT)-­2 inhibitor with beneficial cardiovascular effects independent of its anti­diabetic actions. Among these is sympatholysis, i.e., norepinephrine (NE) lowering, beneficial in chronic human heart failure (HF). Adrenal G protein­-coupled receptor kinase (GRK)-­2 upregulation is a major driver of circulating catecholamine (CA) elevation and sympathetic nervous system (SNS) hyperactivity in HF due to dysregulation of adrenal sympatho-­inhibitory α2­-adrenergic receptors (ARs). On the other hand, we recently reported that Regulator of G protein Signaling (RGS)-­4 inhibits free fatty acid receptor (FFAR)-­3-stimulated NE release from sympathetic neurons, while it is also known to inhibit adrenal CA secretion induced by acetylcholine. Thus, we hypothesized herein that dapagliflozin may lower SNS hyperactivity by enhancing adrenal α2AR and RGS4 functions. We used the rat pheochromocytoma PC12 cell line expressing human α2A­AR, as well as freshly isolated adrenal glands from rats treated with dapagliflozin in vivo. Dapagliflozin treatment for 7 consecutive days (20 mg/kg/d in drinking water) led to a significant reduction in blood circulating NE levels (217±67 pg/ml, n=6), compared to vehicle-­treated rats (363±77 pg/ml, n=6, p<.05), suggesting reduced SNS activity. This was accompanied by reduced GRK2 and tyrosine hydroxylase (TH) mRNA and protein levels in dapagliflozin-­treated vs. vehicle­-treated adrenal glands. In contrast, RGS4 mRNA and protein levels were increased by dapagliflozin ­treatment. Adrenal α2AR density was higher in dapagliflozin- ­vs. vehicle-­treated rats (51.3±7.3 vs. 26.1±8.1 fmol/mg of protein, respectively; n=12 glands from 6 animals per group, p<.05). These results (i.e., GRK2 & TH downregulation with RGS4 upregulation) were completely recapitulated in PC12 cells in culture, treated with 5 μM dapagliflozin (or vehicle) for 24 hours. Importantly, α2AR signaling towards inhibition of CA secretion was markedly enhanced, while the cholinergic agonist carbachol-induced CA secretion was reduced post-­dapagliflozin treatment of PC12 cells. GRK2 overexpression and RGS4 siRNA-mediated knockdown partially reversed dapagliflozin‘s effect on CA secretion, confirming the involvement of GRK2 and RGS4 in dapagliflozin‘s sympatholytic effects in PC12 cells. In conclusion, dapagliflozin exerts adrenal sympatholysis via: a) downregulation of TH, which reduces CA biosynthesis, and GRK2, which reduces α2AR desensitization (i.e., enhances α2AR sympatho-inhibitory signaling); and b) upregulation of RGS4, which directly opposes cholinergic-stimulated CA secretion. 1) NIH/NHLBI #HL155718-01. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Unveiling G-protein coupled receptor kinase-5 inhibitors for chronic degenerative diseases: Multilayered prioritization employing explainable machine learning-driven multi-class QSAR, ligand-based pharmacophore and free energy-inspired molecular simulation

GRK5 holds a pivotal role in cellular signaling pathways, with its overexpression in cardiomyocytes, neuronal cells, and tumor cells strongly associated with various chronic degenerative diseases, which highlights the urgent need for potential inhibitors. In this study, multiclass classification-based QSAR models were developed using diverse machine learning algorithms. These models were built from curated compounds with experimentally derived GRK5 inhibitory activity. Additionally, a pharmacophore model was constructed using active compounds from the dataset. Among the models, the SVM-based approach proved most effective and was initially used to screen DrugBank compounds within the applicability domain. Compounds showing significant GRK5 inhibitory potential underwent evaluation for key pharmacophoric features. Prospective compounds were subjected to molecular docking to assess binding affinity towards GRK5's key active site amino acid residues. Stability at the binding site was analyzed through 200 ns molecular dynamics simulations. MM-GBSA analysis quantified individual free energy components contributing to the total binding energy with respect to binding site residues. Metadynamics analysis, including PCA, FEL, and PDF, provided crucial insights into conformational changes of both apo and holo forms of GRK5 at defined energy states. The study identifies DB02844 (S-Adenosyl-1,8-Diamino-3-Thiooctane) and DB13155 (Esculin) as promising GRK5 inhibitors, warranting further in vitro and in vivo validation studies.

Rapid elucidation of agonist-driven regulation of the neurokinin 1 receptor using a GPCR phosphorylation immunoassay

Agonist-induced phosphorylation is a crucial step in the activation/deactivation cycle of G protein-coupled receptors (GPCRs), but direct determination of individual phosphorylation events has remained a major challenge. We have recently developed a bead-based immunoassay for the quantitative assessment of agonist-induced GPCR phosphorylation that can be performed entirely in 96-well plates, thus eliminating the need for western blot analysis. In the present study, we adapted this assay to three novel phosphosite-specific antibodies directed against the neurokinin 1 (NK1) receptor, namely pS338/pT339-NK1, pT344/pS347-NK1, and pT356/pT357-NK1. We found that substance P (SP) stimulated concentration-dependent phosphorylation of all three sites, which could be completely blocked in the presence of the NK1 receptor antagonist aprepitant. The other two endogenous ligands of the tachykinin family, neurokinin A (NKA) and neurokinin B (NKB), were also able to induce NK1 receptor phosphorylation, but to a much lesser extent than substance P. Interestingly, substance P promoted phosphorylation of the two distal sites more efficiently than that of the proximal site. The proximal site was identified as a substrate for phosphorylation by protein kinase C. Analysis of GPCR kinase (GRK)-knockout cells revealed that phosphorylation was mediated by all four GRK isoforms to similar extents at the T344/S347 and the T356/T357 cluster. Knockout of all GRKs resulted in abolition of all phosphorylation signals highlighting the importance of these kinases in agonist-mediated receptor phosphorylation. Thus, the 7TM phosphorylation assay technology allows for rapid and detailed analyses of GPCR phosphorylation.

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.

Chemoattractant receptor signaling in humoral immunity.

Efficient induction of humoral immune responses depends on orchestrated migration of B cells within lymphoid organs, which is governed by G protein-coupled receptors (GPCRs) responding to chemoattractants, represented by chemokines. After ligand binding, GPCRs are phosphorylated by different GPCR kinases (GRKs) at distinct sites on the receptor C termini, which dictates functional outcomes of β-arrestin-mediated signaling, ranging from receptor inactivation to effector molecule activation. However, the molecular mechanisms by which individual GRKs are selectively targeted to GPCRs have been poorly understood. Our recent study revealed that a protein complex consisting of copper metabolism MURR1 domain-containing (COMMD) 3 and 8 (COMMD3/8 complex) functions as an adaptor that recruits a specific GRK to chemoattractant receptors and plays an important role in the control of B cell migration during humoral immune responses. In this review, we summarize the current understanding of chemoattractant receptor signaling in the context of humoral immunity and discuss the potential of the COMMD3/8 complex as a therapeutic target for autoimmune diseases.

Quantitative proteomics assay reveals G protein-coupled receptor kinase 4-induced HepG2 cell growth inhibition

Background and aimTo investigate the biological effects and putative biological mechanism of G protein-coupled receptor kinase 4 (GRK4) on HepG2 cells. Materials and methodsCell proliferation, cycle, and apoptosis were evaluated by Cell Counting Kit-8 and flow cytometry (FCM) in HepG2 cells infected with either the GRK4-overexpressing lentivirus vector (OE) or the negative control lentivirus vector (NC). The protein profiles and differentially expressed proteins (DEPs) of the OE and NC cells were analyzed and compared using the quantitative proteomics technique, and their function, expression, and probable mechanism were investigated using bioinformatic assays and parallel reaction monitoring (PRM). ResultsHepG2 cells that received the OE grew more slowly than those that received the NC. FCM revealed that, when compared to the NC cells, the OE cells had undergone S-phase cycle arrest, and neither the OE nor NC cells underwent apoptosis. Among the 7006 proteins that were identified by quantitative proteomics, 403 DEPs were examined based on the filtering parameters, with the expressions of 135 being downregulated and 268 being upregulated. In addition to being involved in the peroxisome proliferator-activated receptor (PPAR) signaling pathway, the DEPs were implicated in the biological processes of cell proliferation, cycle, and metabolism. PRM verified the expressions of DEPs that were connected to the PPAR pathway. ConclusionsThis study shows that GRK4 prevents HepG2 cells from proliferating and causes cell cycle arrest in the S-phase, while the PPAR pathway is involved in the regulation of HepG2 cells via GRK4.

The selective serotonin reuptake inhibitor paroxetine improves right ventricular systolic function in experimental pulmonary hypertension

BackgroundPulmonary hypertension (PH) often leads to right ventricle (RV) failure, a significant cause of morbidity and mortality. Despite advancements in PH management, progression to RV maladaptation and subsequent failure remain a clinical challenge. This study explored the effect of paroxetine, a selective serotonin reuptake inhibitor (SSRI), on RV function in a rat model of PH, hypothesizing that it improves RV function by inhibiting G protein-coupled receptor kinase 2 (GRK2) and altering myofilament protein phosphorylation. MethodsThe Su5416/hypoxia (SuHx) rat model was used to induce PH. Rats were treated with paroxetine and compared to vehicle-treated and control groups. Parameters measured included RV morphology, systolic and diastolic function, myofilament protein phosphorylation, GRK2 activity, and sympathetic nervous system (SNS) markers. ResultsParoxetine treatment significantly improved RV systolic function, evidenced by increased stroke volume, cardiac output, and ejection fraction, without significantly affecting RV hypertrophy, myosin heavy chain/titin isoform switching, or fibrosis. Enhanced phosphorylation of titin and myosin light chain-2 was observed, correlating positively with improved systolic function. Contrary to the hypothesis, improvements occurred independently of GRK2 inhibition or SNS modulation, suggesting an alternate mechanism, potentially involving antioxidant properties of paroxetine. ConclusionParoxetine improves RV systolic function in PH rats, likely through mechanisms beyond GRK2 inhibition, possibly related to its antioxidant effects. This highlights the potential of paroxetine in managing RV dysfunction in PH, warranting further investigation into its detailed mechanisms of action and clinical applicability.

Abstract 303: Revealing molecular mechanism of G protein-coupled receptor kinase 2 (GRK2)-mediated extracellular matrix remodeling in ovarian cancer

Abstract G protein-independent pathway (β-arrestin dependent signaling cascade) is initiated by activated GPCRs phosphorylated by G protein-coupled receptor kinases (GRKs), triggering multiple signaling pathways. The purpose of this research is to reveal the role of GRK2 on the tumor microenvironment (TME). Through in vitro assays, we showed that GRK2 overexpression led to loss of cell-matrix and cell-cell adhesion. The cell spreading assay further indicated that the loss of focal adhesion (FA) assembly and cell size decreased in GRK2-overexpressing cells. In vivo experiments demonstrated that tumor formation and metastasis were suppressed in xenograft mouse models implanted with GRK2-overexpressing ovarian cancer cells. In addition, label-free imaging detection of the collagen pattern suggested that GRK2 overexpression caused bundle loss but the formation of thicker, twisted collagen fibers in xenograft tumor samples. Together with the transcriptome and proteomic data, we hypothesize that GRK2 suppresses cell motility and metastasis by reducing FA and extracellular matrix (ECM) remodeling in ovarian cancer. This research revised the mechanisms underlying GRK2-mediated ovarian cancer metastasis and suggested the potential to target the GRK2-meditated pathway for cancer suppression. Citation Format: Weng I Lei, Shan Yu, Chi Teng Lei, Zhiming Zhang, Tzu Ming Liu, Wakam Chang, Leo Tsz On Lee. Revealing molecular mechanism of G protein-coupled receptor kinase 2 (GRK2)-mediated extracellular matrix remodeling in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 303.

The role of G protein-coupled receptor kinases in GLP-1R β-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 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.

Cryo-electron microscopy for GPCR research and drug discovery in endocrinology and metabolism.

G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors, with many GPCRs having crucial roles in endocrinology and metabolism. Cryogenic electron microscopy (cryo-EM) has revolutionized the field of structural biology, particularly regarding GPCRs, over the past decade. Since the first pair of GPCR structures resolved by cryo-EM were published in 2017, the number of GPCR structuresresolved by cryo-EM has surpassed the number resolved by X-ray crystallography by 30%, reaching >650, and the number has doubled every ~0.63 years for the past 6 years. At this pace, it is predicted that the structure of 90% of all human GPCRs will be completed within the next 5-7 years. This Review highlights the general structural features and principles that guide GPCR ligand recognition, receptor activation, Gprotein coupling, arrestin recruitment and regulation by GPCR kinases. The Review also highlights the diversity of GPCR allosteric binding sites and how allosteric ligands could dictate biased signalling that is selective for a G protein pathwayor an arrestin pathway. Finally, the authors use the examples of glycoprotein hormone receptors and glucagon-like peptide 1 receptor to illustrate the effect of cryo-EM on understanding GPCR biology in endocrinology and metabolism, as well as on GPCR-related endocrine diseases and drug discovery.

GPCR kinases differentially modulate biased signaling downstream of CXCR3 depending on their subcellular localization.

Some G protein-coupled receptors (GPCRs) demonstrate biased signaling such that ligands of the same receptor exclusively or preferentially activate certain downstream signaling pathways over others. This phenomenon may result from ligand-specific receptor phosphorylation by GPCR kinases (GRKs). GPCR signaling can also exhibit location bias because GPCRs traffic to and signal from subcellular compartments in addition to the plasma membrane. Here, we investigated whether GRKs contributed to location bias in GPCR signaling. GRKs translocated to endosomes after stimulation of the chemokine receptor CXCR3 or other GPCRs in cultured cells. GRK2, GRK3, GRK5, and GRK6 showed distinct patterns of recruitment to the plasma membrane and to endosomes depending on the identity of the biased ligand used to activate CXCR3. Analysis of engineered forms of GRKs that localized to either the plasma membrane or endosomes demonstrated that biased CXCR3 ligands elicited different signaling profiles that depended on the subcellular location of the GRK. Each GRK exerted a distinct effect on the regulation of CXCR3 engagement of β-arrestin, internalization, and activation of the downstream effector kinase ERK. Our work highlights a role for GRKs in location-biased GPCR signaling and demonstrates the complex interactions between ligands, GRKs, and cellular location that contribute to biased signaling.

Mechanisms of biased agonism by Gαi/o-biased stapled peptide agonists of the relaxin-3 receptor.

The neuropeptide relaxin-3 is composed of an A chain and a B chain held together by disulfide bonds, and it modulates functions such as anxiety and food intake by binding to and activating its cognate receptor RXFP3, mainly through the B chain. Biased ligands of RXFP3 would help to determine the molecular mechanisms underlying the activation of G proteins and β-arrestins downstream of RXFP3 that lead to such diverse functions. We showed that the i, i+4 stapled relaxin-3 B chains, 14s18 and d(1-7)14s18, were Gαi/o-biased agonists of RXFP3. These peptides did not induce recruitment of β-arrestin1/2 to RXFP3 by GPCR kinases (GRKs), in contrast to relaxin-3, which enabled the GRK2/3-mediated recruitment of β-arrestin1/2 to RXFP3. Relaxin-3 and the previously reported peptide 4 (an i, i+4 stapled relaxin-3 B chain) did not exhibit biased signaling. The staple linker of peptide 4 and parts of both the A chain and B chain of relaxin-3 interacted with extracellular loop 3 (ECL3) of RXFP3, moving it away from the binding pocket, suggesting that unbiased ligands promote a more open conformation of RXFP3. These findings highlight roles for the A chain and the N-terminal residues of the B chain of relaxin-3 in inducing conformational changes in RXFP3, which will help in designing selective biased ligands with improved therapeutic efficacy.

GRK2-YAP signaling is implicated in pulmonary arterial hypertension development.

Pulmonary arterial hypertension (PAH) is characterized by excessive proliferation of small pulmonary arterial vascular smooth muscle cells (PASMCs), endothelial dysfunction, and extracellular matrix remodeling. G protein-coupled receptor kinase 2 (GRK2) plays an important role in the maintenance of vascular tone and blood flow. However, the role of GRK2 in the pathogenesis of PAH is unknown. GRK2 levels were detected in lung tissues from healthy people and PAH patients. C57BL/6 mice, vascular smooth muscle cell-specific Grk2 -knockout mice ( Grk2ΔSM22 ), and littermate controls ( Grk2flox/flox ) were grouped into control and hypoxia mice ( n =8). Pulmonary hypertension (PH) was induced by exposure to chronic hypoxia (10%) combined with injection of the SU5416 (cHx/SU). The expression levels of GRK2 and Yes-associated protein (YAP) in pulmonary arteries and PASMCs were detected by Western blotting and immunofluorescence staining. The mRNA expression levels of Grk2 and Yes-associated protein ( YAP ) in PASMCs were quantified with real-time polymerase chain reaction (RT-PCR). Wound-healing assay, 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay, and 5-Ethynyl-2'-deoxyuridine (EdU) staining were performed to evaluate the proliferation and migration of PASMCs. Meanwhile, the interaction among proteins was detected by immunoprecipitation assays. The expression levels of GRK2 were upregulated in the pulmonary arteries of patients with PAH and the lungs of PH mice. Moreover, cHx/SU-induced PH was attenuated in Grk2ΔSM22 mice compared with littermate controls. The amelioration of PH in Grk2ΔSM22 mice was accompanied by reduced pulmonary vascular remodeling. In vitro study further confirmed that GRK2 knock-down significantly altered hypoxia-induced PASMCs proliferation and migration, whereas this effect was severely intensified by overexpression of GRK2 . We also identified that GRK2 promoted YAP expression and nuclear translocation in PASMCs, resulting in excessive PASMCs proliferation and migration. Furthermore, GRK2 is stabilized by inhibiting phosphorylating GRK2 on Tyr86 and subsequently activating ubiquitylation under hypoxic conditions. Our findings suggest that GRK2 plays a critical role in the pathogenesis of PAH, via regulating YAP expression and nuclear translocation. Therefore, GRK2 serves as a novel therapeutic target for PAH treatment.

GRK5 promoted renal fibrosis via HDAC5/Smad3 signaling pathway.

Renal fibrosis is a common pathological feature of chronic kidney diseases (CKD), poses a significant burden in the aging population, and is a major cause of end-stage renal disease (ESRD). In this study, we investigated the role of G protein-coupled receptor kinases (GRKs) 5 in the pathogenesis of renal fibrosis. GRK5 is a serine/threonine kinase that regulates G protein-coupled receptor (GPCR) signaling. GRK5 has been shown to play a role in various diseases including cardiac disorders and cancer. However, the role of GRK5 in renal fibrosis remains largely unknown. Our finding revealed that GRK5 was significantly overexpressed in renal fibrosis. Specifically, GRK5 was transferred into the nucleus via its nuclear localization sequence to regulate histone deacetylases (HDAC) 5 expression under renal fibrosis. GRK5 acted as an upstream regulator of HDAC5/Smad3 signaling pathway. HDAC5 regulated and prevented the transcriptional activity of myocyte enhancer factor 2A (MEF2A) to repress the transcription of Smad7 which leading to the activation of Smad3. These findings first revealed that GRK5 may be a potential therapeutic target for the treatment of renal fibrosis. Inhibition of GRK5 activity may be a promising strategy to attenuate the progression of renal fibrosis.

Biased Signaling in Mutated Variants of β2-Adrenergic Receptor: Insights from Molecular Dynamics Simulations.

The molecular basis of receptor bias in G protein-coupled receptors (GPCRs) caused by mutations that preferentially activate specific intracellular transducers over others remains poorly understood. Two experimentally identified biased variants of β2-adrenergic receptors (β2AR), a prototypical GPCR, are a triple mutant (T68F, Y132A, and Y219A) and a single mutant (Y219A); the former bias the receptor toward the β-arrestin pathway by disfavoring G protein engagement, while the latter induces G protein signaling explicitly due to selection against GPCR kinases (GRKs) that phosphorylate the receptor as a prerequisite of β-arrestin binding. Though rigorous characterizations have revealed functional implications of these mutations, the atomistic origin of the observed transducer selectivity is not clear. In this study, we investigated the allosteric mechanism of receptor bias in β2AR using microseconds of all-atom Gaussian accelerated molecular dynamics (GaMD) simulations. Our observations reveal distinct rearrangements in transmembrane helices, intracellular loop 3, and critical residues R1313.50 and Y3267.53 in the conserved motifs D(E)RY and NPxxY for the mutant receptors, leading to their specific transducer interactions. Moreover, partial dissociation of G protein from the receptor core is observed in the simulations of the triple mutant in contrast to the single mutant and wild-type receptor. The reorganization of allosteric communications from the extracellular agonist BI-167107 to the intracellular receptor-transducer interfaces drives the conformational rearrangements responsible for receptor bias in the single and triple mutants. The molecular insights into receptor bias of β2AR presented here could improve the understanding of biased signaling in GPCRs, potentially opening new avenues for designing novel therapeutics with fewer side-effects and superior efficacy.

G protein-coupled receptor-biased signaling: potential drug discovery to facilitate treatment of metabolic diseases

G protein-coupled receptors (GPCRs) are important, potential drug targets for the treatment of metabolic disorders, such as obesity. GPCRs crosstalk with several transducers, including heterotrimeric G proteins, GPCR kinases (GRKs), and β-arrestins. GPCR-biased agonism has raised the potential of novel drug development to preferentially activate therapeutic signaling pathways over pathways that lead to unwanted side effects. The obesity epidemic and its metabolic complications continue to be a major global public health threat but effective treatments are limited. The accelerated development of structural techniques, like X-ray crystallography and cryo-electron microscopy, has paved the way to understanding how biased agonism measured at GPCRs results in specific downstream physiologic responses. Herein some well-validated GPCR targets are briefly summarized and several new and promising receptors for obesity treatment are outlined. This review highlights the significance of deciphering the role of GPCRs in obesity pathology and biased signaling for drug development. We anticipate the review will facilitate the development of novel GPCR-targeted anti-obesity drugs that lead to heightened therapeutic efficacy with decreased side effect profiles.

Nicotine Diminishes Alpha2-Adrenergic Receptor-Dependent Protection Against Oxidative Stress in H9c2 Cardiomyocytes.

Nicotine is a major component of cigarette smoke with various detrimental cardiovascular effects, including increased oxidative stress in the heart. Agonism of α2-adrenergic receptors (ARs), such as with dexmedetomidine, has been documented to exert cardioprotective effects against oxidative stress and related apoptosis and necroptosis. α2-ARs are membrane-residing G protein-coupled receptors (GPCRs) that primarily activate Gi/o proteins. They are also subjected to GPCR-kinase (GRK)-2-dependent desensitization, which entails phosphorylation of the agonist-activated receptor by GRK2 to induce its decoupling from G proteins, thus terminating α2AR-mediated G protein signaling. In the present study, we sought to examine the effects of nicotine on α2AR signaling and effects in H9c2 cardiomyocytes exposed to H2O2 to induce oxidative cellular damage. As expected, treatment of H9c2 cardiomyocytes with H2O2 significantly decreased cell viability and increased oxidative stress, as assessed by reactive oxygen species (ROS)-associated fluorescence levels (DCF assay) and superoxide dismutase activity. Both H2O2 effects were partly rescued by α2AR activation with brimonidine in control cardiomyocytes but not in cells pretreated with nicotine for 24 hours, in which brimonidine was unable to reduce H2O2-induced cell death and oxidative stress. This was due to severe α2AR desensitization, manifested as very low Gi protein activation by brimonidine, and accompanied by GRK2 upregulation in nicotine-treated cardiomyocytes. Finally, pharmacological inhibition of adenylyl cyclase (AC) blocked H2O2-dependent oxidative damage in nicotine-pretreated H9c2 cardiomyocytes, indicating that α2AR activation protects against oxidative injury via its classic coupling to Gai-mediated AC inhibition. Nicotine can negate the cardioprotective effects of α2AR agonists against oxidative injury, which may have important implications for patients treated with this class of drugs that are chronic tobacco smokers.