Protein-coupled Receptor Kinase Research Articles

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.

Phototoxicity avoidance is a potential therapeutic approach for retinal dystrophy caused by EYS dysfunction.

Inherited retinal dystrophies (IRDs) are progressive diseases leading to vision loss. Mutation in the eyes shut homolog (EYS) gene is one of the most frequent causes of IRD. However, the mechanism of photoreceptor cell degeneration by mutant EYS has not been fully elucidated. Here, we generated retinal organoids from induced pluripotent stem cells (iPSCs) derived from patients with EYS-associated retinal dystrophy (EYS-RD). In photoreceptor cells of RD organoids, both EYS and G protein-coupled receptor kinase 7 (GRK7), one of the proteins handling phototoxicity, were not in the outer segment, where they are physiologically present. Furthermore, photoreceptor cells in RD organoids were vulnerable to light stimuli, and especially to blue light. Mislocalization of GRK7, which was also observed in eys-knockout zebrafish, was reversed by delivering control EYS into photoreceptor cells of RD organoids. These findings suggest that avoiding phototoxicity would be a potential therapeutic approach for EYS-RD.

Fungal alkaloid malbrancheamide reorients the lipid binding domain of GRK5

G protein-coupled receptors (GPCRs) are the largest group of receptors involved in various types of signaling. GPCR signaling is regulated via receptor phosphorylation by G protein-coupled receptor kinases 5 (GRK5). Calmodulin (CaM), a universal Ca2+ sensor, inhibits receptor phosphorylation by binding to GRK5. However, the inhibitor malbrancheamide (MBC), which binds at CaM C-lobe, allows for receptor phosphorylation. To understand the phosphorylation mechanism by GRK5, we carried out a MD simulation of the CaM/GRK5 complex in the presence and absence of the MBC inhibitor. The lipid binding domain (LBD) of GRK5 adopted different positions in the presence and absence of inhibitor. Furthermore, the inhibitor MBC restricted the movement of the N-lobe tether (NLT) loop, probably blocking the autophosphorylation of GRK5. Communicated by Ramaswamy H. Sarma

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.

Abstract 5772: Structural insights and rational design, optimization of highly potent and selective novel GRK5/6 inhibitors for cancer therapy

Abstract G protein-coupled receptor kinases (GRKs) regulate cell signaling by triggering receptor desensitization via phosphorylation on G protein-coupled receptors (GPCRs). There are seven human GRKs (GRK1−GRK7) where GRK5 and GRK6 are structurally similar. GRK5 is ubiquitous and required for cancer progression in various cancer types. Knock-down of GRK5 has been shown to suppress prostate cancer and non-small-cell lung cancer growth. GRK6 is highly abundant in immune cells and is overexpressed in multiple myeloma (MM). Knocking down GRK6 has been shown to cause apoptosis of MM cells. Therefore, targeting GRK5 or 6 is a potential chemotherapeutic strategy. Currently, we are developing a series of inhibitors for GRK5/6 based on a lead compound derived from sunitinib and utilizing Cys474 residue unique in GRK5/6 to enhance selectivity by covalent capture. We tested a series of novel inhibitor warheads to improve potency and selectivity without potentially toxic functional groups. So far, we have identified several inhibitors with low nanomolar IC50 for GRK5 and more than 100,000-fold selectivity over GRK2, another closely related member expressed in the heart. I have developed a pipeline of X-ray crystallography of GRK5 available for soaking various ligands and have solved several structures of novel inhibitor-bound GRK5. The structure elucidated the binding mode of this new class of GRK5 inhibitors, where the core interacts with the adenine site, a fluorophenyl group stabilizes the P loop and density suggests in some cases covalent bond forming between the reversible covalent warhead and Cys474. Our ongoing studies focus on further compound optimizations based on SAR. Overall, the discovery of the inhibitors can significantly facilitate understanding and treatment and cancers. Citation Format: Yueyi Chen, Amol D. Sonawane, Rajesh Manda, Ranjith K. Gadi, Arun K. Ghosh, John J. Tesmer. Structural insights and rational design, optimization of highly potent and selective novel GRK5/6 inhibitors for cancer therapy [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 5772.

Histamine H1 Receptor-Mediated JNK Phosphorylation Is Regulated by Gq Protein-Dependent but Arrestin-Independent Pathways.

Arrestins are known to be involved not only in the desensitization and internalization of G protein-coupled receptors but also in the G protein-independent activation of mitogen-activated protein (MAP) kinases, such as extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), to regulate cell proliferation and inflammation. Our previous study revealed that the histamine H1 receptor-mediated activation of ERK is dually regulated by Gq proteins and arrestins. In this study, we investigated the roles of Gq proteins and arrestins in the H1 receptor-mediated activation of JNK in Chinese hamster ovary (CHO) cells expressing wild-type (WT) human H1 receptors, the Gq protein-biased mutant S487TR, and the arrestin-biased mutant S487A. In these mutants, the Ser487 residue in the C-terminus region of the WT was truncated (S487TR) or mutated to alanine (S487A). Histamine significantly stimulated JNK phosphorylation in CHO cells expressing WT and S487TR but not S487A. Histamine-induced JNK phosphorylation in CHO cells expressing WT and S487TR was suppressed by inhibitors against H1 receptors (ketotifen and diphenhydramine), Gq proteins (YM-254890), and protein kinase C (PKC) (GF109203X) as well as an intracellular Ca2+ chelator (BAPTA-AM) but not by inhibitors against G protein-coupled receptor kinases (GRK2/3) (cmpd101), β-arrestin2 (β-arrestin2 siRNA), and clathrin (hypertonic sucrose). These results suggest that the H1 receptor-mediated phosphorylation of JNK is regulated by Gq-protein/Ca2+/PKC-dependent but GRK/arrestin/clathrin-independent pathways.

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.

Structural insights into a new class of noncovalent, potent, and highly selective G protein-coupled receptor kinase 5 inhibitors

Structural insights into a new class of noncovalent, potent, and highly selective G protein-coupled receptor kinase 5 inhibitors

The Role of G Protein-Coupled Receptors and Receptor Kinases in Pancreatic β-Cell Function and Diabetes.

Type 2 diabetes (T2D) mellitus has emerged as a major global health concern that has accelerated in recent years due to poor diet and lifestyle. Afflicted individuals have high blood glucose levels that stem from the inability of the pancreas to make enough insulin to meet demand. Although medication can help to maintain normal blood glucose levels in individuals with chronic disease, many of these medicines are outdated, have severe side effects, and often become less efficacious over time, necessitating the need for insulin therapy. G protein-coupled receptors (GPCRs) regulate many physiologic processes, including blood glucose levels. In pancreatic β cells, GPCRs regulate β-cell growth, apoptosis, and insulin secretion, which are all critical in maintaining sufficient β-cell mass and insulin output to ensure euglycemia. In recent years, new insights into the signaling of incretin receptors and other GPCRs have underscored the potential of these receptors as desirable targets in the treatment of diabetes. The signaling of these receptors is modulated by GPCR kinases (GRKs) that phosphorylate agonist-activated GPCRs, marking the receptor for arrestin binding and internalization. Interestingly, genome-wide association studies using diabetic patient cohorts link the GRKs and arrestins with T2D. Moreover, recent reports show that GRKs and arrestins expressed in the β cell serve a critical role in the regulation of β-cell function, including β-cell growth and insulin secretion in both GPCR-dependent and -independent pathways. In this review, we describe recent insights into GPCR signaling and the importance of GRK function in modulating β-cell physiology. SIGNIFICANCE STATEMENT: Pancreatic β cells contain a diverse array of G protein-coupled receptors (GPCRs) that have been shown to improve β-cell function and survival, yet only a handful have been successfully targeted in the treatment of diabetes. This review discusses recent advances in our understanding of β-cell GPCR pharmacology and regulation by GPCR kinases while also highlighting the necessity of investigating islet-enriched GPCRs that have largely been unexplored to unveil novel treatment strategies.

The mechanism of 25-hydroxycholesterol-mediated suppression of atrial β1-adrenergic responses.

25-Hydroxycholesterol (25HC) is a biologically active oxysterol, whose production greatly increases during inflammation by macrophages and dendritic cells. The inflammatory reactions are frequently accompanied by changes in heart regulation, such as blunting of the cardiac β-adrenergic receptor (AR) signaling. Here, the mechanism of 25HC-dependent modulation of responses to β-AR activation was studied in the atria of mice. 25HC at the submicromolar levels decreased the β-AR-mediated positive inotropic effect and enhancement of the Ca2+ transient amplitude, without changing NO production. Positive inotropic responses to β1-AR (but not β2-AR) activation were markedly attenuated by 25HC. The depressant action of 25HC on the β1-AR-mediated responses was prevented by selective β3-AR antagonists as well as inhibitors of Gi protein, Gβγ, G protein-coupled receptor kinase 2/3, or β-arrestin. Simultaneously, blockers of protein kinase D and C as well as a phosphodiesterase inhibitor did not preclude the negative action of 25HC on the inotropic response to β-AR activation. Thus, 25HC can suppress the β1-AR-dependent effects via engaging β3-AR, Gi protein, Gβγ, G protein-coupled receptor kinase, and β-arrestin. This 25HC-dependent mechanism can contribute to the inflammatory-related alterations in the atrial β-adrenergic signaling.

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.

Caveolin-1, a Determinant of the Fate of MCF-7 Breast Cancer Cells.

The scaffolding protein, caveolin-1 (Cav-1), participates in multiple cellular functions including promotion of sodium excretion from the kidney. Loss of expression of Cav-1 is associated with tumorigenesis of various types of cancer. We have shown the potential link between hypertension and breast cancer via abnormal function of the G protein-coupled receptor kinase type 4 (GRK4). The current studies tested the hypothesis that Cav-1 acts as a tumor-suppressive factor in breast cancer cells and enhances the sensitivity to the inhibitory effect of the type 1 dopaminergic receptor (D1R). Michigan Cancer Foundation (MCF) MCF-7 cells stably expressing a Cav-1/mCherry fusion protein or mCherry alone were used as models to examine the effect of Cav-1 on cell growth, apoptosis, and senescence. Cell proliferation was determined by cell counting, cell cycle analysis (flow cytometry), and BrdU incorporation. Apoptosis was determined using the Cell Death Detection ELISA kit from Roche Diagnosis. Senescence was determined using the senescence associated beta galactosidase (SA-β-gal) assay. Reactive oxygen species (ROS) was measured using 2',7'-dichlorodihydrofluorescein diacetate. Western blot analysis was used to measure activation of signaling pathway molecules. All statistical analyses were conducted with Microsoft Excel. Overexpression of Cav-1 in MCF-7 cells reduced cellular growth rate. Both inhibition of proliferation and induction of cell death are contributing factors. Multiple signaling pathways were activated in Cav-1-expressing MCF-7 cells. Activation of Akt was prominent. In MCF-7-expressing Cav-1 (MCF-7 Cav-1) cells, the levels of phosphorylated Akt at S473 and T308 were increased 28- and 8.7-fold, respectively. Instead of protecting cells from apoptosis, extremely high levels of activated Akt resulted in increased levels of ROS which led to apoptosis and senescence. The tumor-suppressive effect plus downregulation of GRK4 makes Cav-1-expressing MCF-7 cells significantly more sensitive to the inhibitory effect of the D1R agonist, SKF38393. Caveolin-1 acts as a tumor-suppressing factor via extreme activation of Akt and down regulation of survival factors such as GRK4, survivin, and cyclin D1.

Pulsed radiofrequency on DRG inhibits hippocampal neuroinflammation by regulating spinal GRK2/p38 expression and enhances spinal autophagy to reduce pain and depression in male rats with spared nerve injury

Evidence indicates that microglial G protein-coupled receptor kinase 2 (GRK2) is a key regulator of the transition from acute to chronic pain mediated by microglial products via the p38 mitogen-activated protein kinase (MAPK) pathway in the spinal cord dorsal horn (SCDH). Increasing studies have shown that autophagic dysfunction in the SCDH and neuroinflammation in the hippocampus underlie NeP. However, whether GRK2/p38MAPK and autophagic flux in the SCDH and hippocampal neuroinflammation are involved in NeP and depression comorbidity has not been determined. Here, we explored the effects of high-voltage pulsed radiofrequency (PRF) (85 V-PRF; HV-PRF) to the dorsal root ganglion (DRG) on pain phenotypes in Wistar male rats with spared nerve injury (SNI) and the underlying mechanisms. The exacerbation of pain phenotypes was markedly relieved by PRF-DRG. The SNI-induced reduction in GRK2 expression, elevation of p-p38 MAPK levels in the SCDH, and increase in IL-1β and TNF-α levels in the hippocampus were reversed by PRF, which was accompanied by an increase in autophagic flux in spinal microglia. The beneficial effect of 85 V-PRF was superior to that of 45 V-PRF. In addition, the improvements elicited by 85 V-PRF were reversed by intrathecal injection of GRK2 antisense oligonucleotide, and these changes were accompanied by GRK2 downregulation and p-p38 upregulation in the SCDH, increased pro-inflammatory factor levels in the hippocampus, and excessive autophagy in spinal microglia. In conclusion, our data indicate that the application of HV-PRF to the DRG could serve as an excellent therapeutic technique for regulating neuroimmunity and neuroinflammation to relieve pain phenotypes.

GRK2 mediated degradation of SAV1 initiates hyperplasia of fibroblast-like synoviocytes in rheumatoid arthritis

Hyperplasia and migration of fibroblast-like synoviocytes (FLSs) are the key drivers in the pathogenesis of rheumatoid arthritis (RA) and joint destruction. Abundant Yes-associated protein (YAP), which is a powerful transcription co-activator for proliferative genes, was observed in the nucleus of inflammatory FLSs with unknown upstream mechanisms. Using Gene Expression Omnibus database analysis, it was found that Salvador homolog-1 (SAV1), the pivotal negative regulator of the Hippo-YAP pathway, was slightly downregulated in RA synovium. However, SAV1 protein expression is extremely reduced. Subsequently, it was revealed that SAV1 is phosphorylated, ubiquitinated, and degraded by interacting with an important serine-threonine kinase, G protein-coupled receptor (GPCR) kinase 2 (GRK2), which was predominately upregulated by GPCR activation induced by ligands such as prostaglandin E2 (PGE2) in RA. This process further contributes to the decreased phosphorylation, nuclear translocation, and transcriptional potency of YAP, and leads to aberrant FLSs proliferation. Genetic depletion of GRK2 or inhibition of GRK2 by paroxetine rescued SAV1 expression and restored YAP phosphorylation and finally inhibited RA FLSs proliferation and migration. Similarly, paroxetine treatment effectively reduced the abnormal proliferation of FLSs in a rat model of collagen-induced arthritis which was accompanied by a significant improvement in clinical manifestations. Collectively, these results elucidate the significance of GRK2 regulation of Hippo-YAP signaling in FLSs proliferation and migration and the potential application of GRK2 inhibition in the treatment of FLSs-driven joint destruction in RA.

CP-25 inhibits the hyperactivation of rheumatic synoviocytes by suppressing the switch in Gαs-Gαi coupling to the β2-adrenergic receptor

In essence, the β2 adrenergic receptor (β2AR) plays an antiproliferative role by increasing the intracellular cyclic 3’,5’-adenosine monophosphate (cAMP) concentration through Gαs coupling, but interestingly, β2AR antagonists are able to effectively inhibit fibroblast-like synoviocytes (FLSs) proliferation, thus ameliorating experimental RA, indicating that the β2AR signalling pathway is impaired in RA FLSs via unknown mechanisms. The local epinephrine (Epi) level was found to be much higher in inflammatory joints than in normal joints, and high-level stimulation with Epi or isoproterenol (ISO) directly promoted FLSs proliferation and migration due to impaired β2AR signalling and cAMP production. By applying inhibitor of receptor internalization, and small interfering RNA (siRNA) of Gαs and Gαi, and by using fluorescence resonance energy transfer and coimmunoprecipitation assays, a switch in Gαs-Gαi coupling to β2AR was observed in inflammatory FLSs as well as in FLSs with chronic ISO stimulation. This Gαi coupling was then revealed to be initiated by G protein coupled receptor kinase 2 (GRK2) but not β-arrestin2 or protein kinase A-mediated phosphorylation of β2AR. Inhibiting the activity of GRK2 with the novel GRK2 inhibitor paeoniflorin-6′-O-benzene sulfonate (CP-25), a derivative of paeoniflorin, or the accepted GRK2 inhibitor paroxetine effectively reversed the switch in Gαs-Gαi coupling to β2AR during inflammation and restored the intracellular cAMP level in ISO-stimulated FLSs. As expected, CP-25 significantly inhibited the hyperplasia of FLSs in a collagen-induced arthritis (CIA) model (CIA FLSs) and normal FLSs stimulated with ISO and finally ameliorated CIA in rats. Together, our findings revealed the pathological changes in β2AR signalling in CIA FLSs, determined the underlying mechanisms and identified the pharmacological target of the GRK2 inhibitor CP-25 in treating CIA.7HWufKwpH5ZfY_hx8fyY6aVideo

Development of a new class of potent and highly selective G protein-coupled receptor kinase 5 inhibitors and structural insight from crystal structures of inhibitor complexes

G protein-coupled receptor kinase 5 (GRK5) is an important drug development target for heart failure, cardiac hypertrophy, and cancer. We have designed and developed a new class of highly selective, potent, and non-covalent GRK5 inhibitors. One of the inhibitors displayed GRK5 IC50 value of 10 nM and exhibited >100,000-fold selectivity over GRK2. The X-ray structure of a ketoamide-derived inhibitor-bound GRK5 showed the formation of a hemithioketal intermediate with active site Cys474 in the GRK5 active site and provided new insights into the ligand-binding site interactions responsible for high selectivity. The current studies serve as an important guide to therapeutic GRK5 inhibitor drug development.

Dapagliflozin downregulates G protein-coupled receptor Kinase-2 and upregulates regulator of g protein signaling-4 in adrenals to exert sympatholysis in heart failure

Abstract Dapagliflozin is a sodium-glucose co-transporter (SGLT)-2 inhibitor with beneficial cardiovascular effects independent of its anti-diabetic actions. Among these is sympatholysis, i.e., norepinephrine (NE) lowering which is 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) [1]. On the other hand, we recently reported that signaling by neuronal free fatty acid receptor (FFAR)-3, which stimulates NE release from sympathetic neurons, is blocked by Regulator of G protein Signaling (RGS)-4 [2,3]. We hypothesized herein that dapagliflozin may lower SNS hyperactivity in the adrenal gland by improving α2AR, and antagonizing FFAR3 signaling in adrenal chromaffin cells. We used the rat pheochromocytoma PC12 cell line expressing human α2A-AR, 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 control, 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 rat adrenals vs. vehicle-treated animal-derived glands. In contrast, RGS4 mRNA levels were increased in dapagliflozin-treated adrenals. 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 microM dapagliflozin (or vehicle) for 24 hours. Importantly, α2AR-dependent G protein-mediated signaling towards inhibition of CA secretion was markedly enhanced, whereas FFAR3-dependent phospholipase C/calcium signaling was reduced, at 24 hrs post-dapagliflozin treatment of PC12 cells. GRK2 overexpression partially reversed dapagliflozin`s effect on cholinergic-induced CA secretion. In conclusion, dapagliflozin exerts a sympatholytic action in the adrenal medulla via a) downregulation of TH, which reduces CA biosynthesis, and GRK2, which reduces α2AR desensitization; and b) upregulation of RGS4 to block FFAR3-stimulated CA secretion (Figure).Figure