Cardioprotective Effects of the GRK2 Inhibitor Paroxetine on Isoproterenol-Induced Cardiac Remodeling by Modulating NF-κB Mediated Prohypertrophic and Profibrotic Gene Expression.

Pathological cardiac remodeling is associated with cardiovascular disease and can lead to heart failure. Nuclear factor-kappa B (NF-κB) is upregulated in the hypertrophic heart. Moreover, the expression of the G-protein-coupled receptor kinase 2 (GRK2) is increased and linked to the progression of heart failure. The inhibitory effects of paroxetine on GRK2 have been established. However, its protective effect on IκBα/NFκB signaling has not been elucidated. This study investigated the cardioprotective effect of paroxetine in an animal model of cardiac hypertrophy (CH), focusing on its effect on GRK2-mediated NF-κB-regulated expression of prohypertrophic and profibrotic genes. Wistar albino rats were administered normal saline, paroxetine, or fluoxetine, followed by isoproterenol to induce CH. The cardioprotective effects of the treatments were determined by assessing cardiac injury, inflammatory biomarker levels, histopathological changes, and hypertrophic and fibrotic genes in cardiomyocytes. Paroxetine pre-treatment significantly decreased the HW/BW ratio (p < 0.001), and the expression of prohypertrophic and profibrotic genes Troponin-I (p < 0.001), BNP (p < 0.01), ANP (p < 0.001), hydroxyproline (p < 0.05), TGF-ß1 (p < 0.05), and αSMA (p < 0.01) as well as inflammatory markers. It also markedly decreased pIκBα, NFκB(p105) subunit expression (p < 0.05) and phosphorylation. The findings suggest that paroxetine prevents pathological cardiac remodeling by inhibiting the GRK2-mediated IκBα/NF-κB signaling pathway.

GRK2 expression and catalytic activity are essential for vasoconstrictor/ERK-stimulated arterial smooth muscle proliferation.

Prolonged vasoconstrictor signalling found in hypertension, increases arterial contraction, and alters vessel architecture by stimulating arterial smooth muscle cell (ASMC) growth, underpinning the development of re-stenosis lesions and vascular remodelling. Vasoconstrictors interact with their cognate G protein coupled receptors activating a variety of signalling pathways to promote smooth muscle proliferation. Here, angiotensin II (AngII) and endothelin 1 (ET1), but not UTP stimulates ASMC proliferation. Moreover, siRNA-mediated depletion of endogenous GRK2 expression, or GRK2 inhibitors, compound 101 or paroxetine, prevented AngII and ET1-promoted ASMC growth. Depletion of GRK2 expression or inhibition of GRK2 activity ablated the prolonged phase of AngII and ET-stimulated ERK signalling, while enhancing and prolonging UTP-stimulated ERK signalling. Increased GRK2 expression enhanced and prolonged AngII and ET1-stimulated ERK signalling, but suppressed UTP-stimulated ERK signalling. In ASMC prepared from 6-week-old WKY and SHR, AngII and ET1-stimulated proliferation rates were similar, however, in cultures prepared from 12-week-old rats AngII and ET1-stimulated growth was enhanced in SHR-derived ASMC, which was reversed following depletion of GRK2 expression. Furthermore, in ASMC cultures isolated from 6-week-old WKY and SHR rats, AngII and ET1-stimulated ERK signals were similar, while in cultures from 12-week-old rats ERK signals were both enhanced and prolonged in SHR-derived ASMC, and were reversed to those seen in age-matched WKY-derived ASMC following pre-treatment of SHR-derived ASMC with compound 101. These data indicate that the presence of GRK2 and its catalytic activity are essential to enable pro-proliferative vasoconstrictors to promote growth via recruitment and activation of the ERK signalling pathway in ASMC.

GPCR activation and GRK2 assembly by a biased intracellular agonist.

Phosphorylation of G-protein-coupled receptors (GPCRs) by GPCR kinases (GRKs) desensitizes G-protein signalling and promotes arrestin signalling, which is also modulated by biased ligands1-6. The molecular assembly of GRKs on GPCRs and the basis of GRK-mediated biased signalling remain largely unknown owing to the weak GPCR-GRK interactions. Here we report the complex structure of neurotensin receptor 1 (NTSR1) bound to GRK2, Gαq and the arrestin-biased ligand SBI-5537. The density map reveals the arrangement of the intact GRK2 with the receptor, with the N-terminal helix of GRK2 docking into the open cytoplasmic pocket formed by the outward movement of the receptor transmembrane helix 6, analogous to the binding of the G protein to the receptor. SBI-553 binds at the interface between GRK2 and NTSR1 to enhance GRK2 binding. The binding mode of SBI-553 is compatible with arrestin binding but clashes with the binding of Gαq protein, thus providing a mechanism for its arrestin-biased signalling capability. In sum, our structure provides a rational model for understanding the details of GPCR-GRK interactions and GRK2-mediated biased signalling.

Ubiquitination of GRK2 Is Required for the ß-Arrestin-Biased Signaling Pathway of Dopamine D2 Receptors to Activate ERK Kinases.

A class-A GPCR dopamine D2 receptor (D2R) plays a critical role in the proper functioning of neuronal circuits through the downstream activation of both G-protein- and ß-arrestin-dependent signaling pathways. Understanding the signaling pathways downstream of D2R is critical for developing effective therapies with which to treat dopamine (DA)-related disorders such as Parkinson's disease and schizophrenia. Extensive studies have focused on the regulation of D2R-mediated extracellular-signal-regulated kinase (ERK) 1/2 signaling; however, the manner in which ERKs are activated upon the stimulation of a specific signaling pathway of D2R remains unclear. The present study conducted a variety of experimental techniques, including loss-of-function experiments, site-directed mutagenesis, and the determination of protein interactions, in order to investigate the mechanisms underlying ß-arrestin-biased signaling-pathway-mediated ERK activation. We found that the stimulation of the D2R ß-arrestin signaling pathway caused Mdm2, an E3 ubiquitin ligase, to move from the nucleus to the cytoplasm and interact with tyrosine phosphorylated G-protein-coupled receptor kinase 2 (GRK2), which was facilitated by Src, a non-receptor tyrosine kinase. This interaction led to the ubiquitination of GRK2, which then moved to the plasma membrane and interacted with activated D2R, followed by the phosphorylation of D2R as well as the mediation of ERK activation. In conclusion, Mdm2-mediated GRK2 ubiquitination, which is selectively triggered by the stimulation of the D2R ß-arrestin signaling pathway, is necessary for GRK2 membrane translocation and its interaction with D2R, which in turn mediates downstream ERK signaling. This study is primarily novel and provides essential information with which to better understand the detailed mechanisms of D2R-dependent signaling.

Overexpression of GRK2 in vascular smooth muscle leads to inappropriate hypertension and acute heart failure as in clinical scenario 1.

Clinical scenario 1 (CS1) is acute heart failure (HF) characterized by transient systolic blood pressure (SBP) elevation and pulmonary congestion. Although it is managed by vasodilators, the molecular mechanism remains unclear. The sympathetic nervous system plays a key role in HF, and desensitization of cardiac ß-adrenergic receptor (AR) signaling due to G protein-coupled receptor kinase 2 (GRK2) upregulation is known. However, vascular ß-AR signaling that regulates cardiac afterload remains unelucidated in HF. We hypothesized that upregulation of vascular GRK2 leads to pathological conditions similar to CS1. GRK2 was overexpressed in vascular smooth muscle (VSM) of normal adult male mice by peritoneally injected adeno-associated viral vectors driven by the myosin heavy chain 11 promoter. Upregulation of GRK2 in VSM of GRK2 overexpressing mice augmented the absolute increase in SBP (+ 22.5 ± 4.3 mmHg vs. + 36.0 ± 4.0 mmHg, P < 0.01) and lung wet weight (4.28 ± 0.05 mg/g vs. 4.76 ± 0.15 mg/g, P < 0.01) by epinephrine as compared to those in control mice. Additionally, the expression of brain natriuretic peptide mRNA was doubled in GRK2 overexpressing mice as compared to that in control mice (P < 0.05). These findings were similar to CS1. GRK2 overexpression in VSM may cause inappropriate hypertension and HF, as in CS1.

GRK2 differentially regulates FcεRI and MRGPRB2-mediated responses in mast cells.

In addition to high-affinity IgE receptor (FcεRI), a subtype of mouse mast cells (MCs) expresses a G protein-coupled receptor known as Mas-related G protein-coupled receptor (GPCR)-B2 (MRGPRB2; human ortholog MRGPRX2). GPCR kinase 2 (GRK2) is a Serine/Threonine kinase that phosphorylates GPCRs to promote their desensitization and internalization. We previously showed that silencing GRK2 expression in mouse bone marrow-derived MCs (BMMCs) blocks IgE-mediated degranulation. Compound 48/80 (C48/80), substance P (SP) and LL-37 cause degranulation in human and mouse MCs via MRGPRX2 and MRGPRB2, respectively. We also reported that C48/80 and SP cause desensitization and internalization of MRGPRX2, but LL-37 does not. Here, we generated mice with MC-specific deletion of Grk2 (Cpa3Cre+/Grk2fl/fl ) to determine its role on IgE-mediated responses and to assess whether it differentially regulates degranulation in response to LL-37, C48/80 and SP. Absence of GRK2 substantially inhibited IgE-mediated tyrosine phosphorylation of STAT5, calcium mobilization, and degranulation in mouse primary lung-derived MCs (PLMCs). By contrast, peritoneal MCs (PMCs) from Cpa3Cre+/Grk2fl/fl mice demonstrated significant enhancement of degranulation in response to C48/80 and SP, but not LL-37. Deletion of Grk2 in MCs attenuated IgE-mediated passive cutaneous anaphylaxis (PCA) and itch but not passive systemic anaphylaxis (PSA). Surprisingly, PSA was significantly reduced in Mrgprb2-/- mice. These findings suggest that GRK2 contributes to PCA and itch but not PSA. By contrast, GRK2 desensitizes MRGPRX2/B2-mediated responses to C48/80 and SP but not LL-37. However, IgE-mediated PSA likely involves the activation of MRGPRB2 by LL-37 or a similar agonist, whose function is resistant to modulation by GRK2.

A novel GRK2 inhibitor alleviates experimental arthritis through restraining Th17 cell differentiation.

T helper type 17 (Th17) cell which is induced by interleukine-6 (IL-6)-signal transducers and activators of transcription 3 (STAT3) signaling is a central pro-inflammatory T cell subtype in rheumatoid arthritis (RA) and could be significantly reduced by paeoniflorin-6'-O-benzene sulfonate (CP-25) treatment with unclear mechanisms. This study was aimed to found out the mechanism of CP-25 in hampering Th17 cells differentiation in arthritic animals thus explore more therapeutic targets for RA. In mice with collagen-induced arthritis (CIA), both circulating and splenic Th17 subsets were expanded with increased STAT3 phosphorylation and decreased Src homology 2 domain-containing protein tyrosine phosphatase 1 (SHP1)-ß-arrestin2 (arrb2)-STAT3 interaction in CD4+ helper T (Th) cells. Either CP-25 or paroxetine (PAR), an established G protein coupled receptor kinase 2 (GRK2) inhibitor treatment effectively relieved the joints inflammation of CIA mice with substantially reduced Th17 cell population through inhibiting STAT3 and restoring the SHP1-arrb2-STAT3 complex. Knockout of arrb2 exacerbated the clinical manifestations of collagen antibody-induced arthritis with upregulated Th17 cells. In vitro studies revealed that depletion of arrb2 or inhibition of SHP1 promoted Th17 cell differentiation. Moreover, stimulation of adenosine A3 receptor (A3AR) simultaneously promoted Th17 cell differentiation via accelerating abbr2-A3AR binding, which could be prevented through inhibiting GRK2 phosphorylation by CP-25 or PAR, or genetically reducing GRK2. This work has demonstrated that CP-25 or PAR treatment recovers the SHP1-arrb2-STAT3 complex which prevents STAT3 activation in Th cells through reducing arrb2 recruitment to A3AR by inhibiting GRK2 phosphorylation, leading to the reduction in Th17 cell differentiation and arthritis attenuation.

Selectivity mechanism of GRK2/5 inhibition through in silico investigation.

As two representative isoforms of G protein-coupled receptor kinases family, the largest known membrane receptor family, GRK2 and GRK5 are ubiquitously distributed in human heart, brain, lung, kidney, skeletal muscle and other tissues. GRK2 and GRK5 have common functions implicated in the regulation of heart failure, though GRK5 has also been involved in diseases like hypertension, cancer, diabetes and Alzheimer's disease. Therefore, to clarify the selectivity mechanism towards GRK2 and GRK5 will be of great significance for the discovery of effective and selective inhibitors. To this end, the structures and chemical properties of key residues were analyzed among GRK2 and GRK5 derived from their respective protein crystal structures. Furthermore, a combination of multiple computational strategies, including sequence superposition, receptor-ligand docking, molecular dynamics, MM-GBSA calculation, QM/MM approach and pharmacological modeling, were integrated to validated and elucidate their unique binding modes towards highly selective inhibitors. In addition, the specific amino acid distribution within the GRK2/5 target site is also analyzed in this paper, which can guide future research and development of selective inhibitors in a more targeted manner. Overall, our study comprehensively clarifies the selectivity mechanism of GRK2/5 inhibition, thereby providing guidance for further rational design of selective inhibitors targeting GRK2/5.

The tyrosine phosphorylation of GRK2 is responsible for activated D2R-mediated insulin resistance.

Dopamine D2 receptor (D2R) plays a key role in the regulation of glucose homeostasis by stimulating the secretion of many glucoregulatory hormones. Insulin resistance (IR) is associated with the pathogenesis of metabolic disorders which occurs when PI3K/Akt signaling pathway is downregulated. However, the potential involvement of D2R in insulin resistance remains unclear. In the present study, we investigated the regulation of glucose transport by D2-like receptors and discovered that activation of D2R, but not D3R or D4R, suppressed insulin-induced 2-DOG uptake and Glut4 membrane translocation in a GRK2- and Src-dependent manner. Further study revealed that activation of D2R inhibits insulin-induced phosphorylation of Akt at Thr308 and Ser473, which are hallmarks of its kinase activity, by increasing the interaction of tyrosine phosphorylated GRK2 with Akt and then preventing Akt from interacting with PDK1. Thus, this study demonstrates that Src mediated GRK2 tyrosine phosphorylation is an essential physiological event that mediates the roles of D2R in insulin resistance.

Targeting GRK2 and GRK5 for treating chronic degenerative diseases: Advances and future perspectives.

The development of small-molecule inhibitors targeting G protein-coupled receptor kinase 2 (GRK2) and G protein-coupled receptor kinase 5 (GRK5) for the treatment of chronic degenerative diseases has attracted wide attention. GRK2 and GRK5 can regulate essential physiological processes by phosphorylating G protein-coupled receptor (GPCR). Alterations in the functional levels of GRK2 and GRK5 have been found in a variety of chronic degenerative diseases, such as cardiovascular diseases, neurodegenerative diseases, cancers, type 2 diabetes, and rheumatoid arthritis (RA). Abnormal GRK2 and GRK5 expression contribute to the development of chronic degenerative diseases through environmental molecular mechanisms, making them promising molecular targets for treating chronic degenerative diseases. To date, many novel GRK2 and GRK5 inhibitors have been reported for the treatment of chronic degenerative diseases. We focus on the recent progress of single and dual-target inhibitors of GRK2/GRK5. This review summarizes the structural optimization rationale, structure-activity relationships (SARs), and the latest application in the treatment of chronic degenerative diseases. We believe it will shed light on the future development of small molecule inhibitors of GRK2 and GRK5, as well as the clinical applications in chronic degenerative diseases.

Early Gßγ-GRK2 Inhibition Ameliorates Osteoarthritis Development by Simultaneous Anti-Inflammatory and Chondroprotective Effects.

The G-protein-coupled receptor kinase 2 (GRK2) is an important regulator of inflammation and pathological macrophage phenotype in a variety of diseases. We hypothesize that Gßγ-GRK2 signaling promotes the early inflammatory response and chondrocyte loss in osteoarthritis (OA). Using the destabilization of the medial meniscus (DMM) model in 12-week-old male C57BL/6 mice, we determined the role of Gßγ-GRK2 signaling in synovitis, macrophage activation, and OA development. We achieved Gßγ-GRK2 inhibition at the time of DMM by administering the Gßγ inhibitor "gallein" and the GRK2 inhibitor "paroxetine" daily, starting from 2 days before DMM surgery, for a duration of 1 or 12 weeks. Synovial and cartilage structural changes were evaluated by histomorphometry, and molecular events and macrophage activation were examined. We studied the direct role of Gßγ-GRK2 in synovitis and macrophage activation in vitro using SW982 and THP1 cells. Continuous Gßγ-GRK2 inhibition initiated at the time of DMM attenuated OA development and decreased chondrocyte loss more effectively than delayed treatment. GRK2 expression and the M1 macrophage phenotype were elevated in the inflamed synovium, while early gallein and paroxetine treatment for 1 and 12 weeks following DMM resulted in their reduction and an upregulated M2 macrophage phenotype. In vitro experiments showed that Gßγ-GRK2 inhibition attenuated synoviocyte inflammation and the M1 phenotype. We show that early Gßγ-GRK2 inhibition is of higher therapeutic efficacy in OA than delayed inhibition, as it prevents OA development by inhibiting the early inflammatory response.

GRK2 in cardiovascular disease and its potential as a therapeutic target.

Cardiovascular diseases (CVDs) represent the leading cause of death globally. Despite major advances in the field of pharmacological CVD treatments, particularly in the field of heart failure (HF) research, case numbers and overall mortality remain high and have trended upwards over the last few years. Thus, identifying novel molecular targets for developing HF therapeutics remains a key research focus. G protein-coupled receptors (GPCRs) are critical myocardial signal transducers which regulate cardiac contractility, growth, adaptation and metabolism. Additionally, GPCR dysregulation underlies multiple models of cardiac pathology, and most pharmacological therapeutics currently used in HF target these receptors. Currently-approved treatments have improved patient outcomes, but therapies to stop or reverse HF are lacking. A recent focus on GPCR intracellular-regulating proteins such as GPCR kinases (GRKs) has uncovered GRK2 as a promising target for combating HF. Current literature strongly establishes increased levels and activity of GRK2 in multiple models of CVD. Additionally, the GRK2 interactome includes numerous proteins which interact with differential domains of GRK2 to modulate both beneficial and deleterious signaling pathways in the heart, indicating that these domains can be targeted with a high level of specificity unique to various cardiac pathologies. These data support the premise that GRK2 should be at the forefront of a novel investigative drug search. This perspective reviews cardiac GPCRs, describes the structure and functions of GRK2 in cardiac function and maladaptive pathology, and summarizes the ongoing and future research for targeting this critical kinase across cellular, animal and human models of cardiac dysfunction and HF.

G protein-coupled receptor kinase type 2 and ß-arrestin2: Key players in immune cell functions and inflammation.

G protein-coupled receptor kinase type 2 (GRK2) and ß-arrestin2 are representative proteins that regulate the transduction and trafficking of G protein-coupled receptor (GPCR) signaling. The kinase GRK2 and the multifunctional scaffolding protein ß-arrestin2 are key integrated signaling nodes in various biological processes, and both of them regulate cell proliferation and promote cell invasion and migration. GRK2/ß-arrestin2 play multiple roles in the pathological mechanisms of a wide range of diseases including heart failure, cancer, and inflammatory diseases. This review summarizes the roles of GRK2/ß-arrestin2 in immune cell function and focuses on the pathological implications of GRK2/ß-arrestin2 in various inflammatory diseases.

GRK2 selectively attenuates the neutrophil NADPH-oxidase response triggered by ß-arrestin recruiting GPR84 agonists.

In order to avoid a prolonged pro-inflammatory neutrophil response, signaling downstream of an agonist-activated G protein-coupled receptor (GPCR) has to be rapidly terminated. Among the family of GPCR kinases (GRKs) that regulate receptor phosphorylation and signaling termination, GRK2, which is highly expressed by immune cells, plays an important role. The medium chain fatty acid receptor GPR84 as well as formyl peptide receptor 2 (FPR2), receptors expressed in neutrophils, play a key role in regulating inflammation. In this study, we investigated the effects of GRK2 inhibitors on neutrophil functions induced by GPR84 and FPR2 agonists. GRK2 was shown to be expressed in human neutrophils and analysis of subcellular fractions revealed a cytosolic localization. The GRK2 inhibitors enhanced and prolonged neutrophil production of reactive oxygen species (ROS) induced by GPR84- but not FPR2-agonists, suggesting a receptor selective function of GRK2. This suggestion was supported by ß-arrestin recruitment data. The ROS production induced by a non ß-arrestin recruiting GPR84 agonist was not affected by the GRK2 inhibitor. Termination of this ß-arrestin independent response relied, similar to the response induced by FPR2 agonists, primarily on the actin cytoskeleton. In summary, we show that GPR84 utilizes GRK2 in concert with ß-arrestin and actin cytoskeleton dependent processes to fine-tune the activity of the ROS generating NADPH-oxidase in neutrophils.

Myocardial GRK2 Reduces Fatty Acid Metabolism and ß-Adrenergic Receptor-Mediated Mitochondrial Responses.

G-protein coupled receptor (GPCR) kinase 2 (GRK2) is upregulated in heart failure (HF) patients and mouse models of cardiac disease. GRK2 is a regulator of ß-adrenergic receptors (ßARs), a GPCR involved in ionotropic and chronotropic responses. We and others have recently reported GRK2 to be localized in the mitochondria, although its function in the mitochondria and/or metabolism remain not clearly defined. We hypothesized that upregulation of GRK2 reduced mitochondrial respiratory function and responses to ßAR activation. Utilizing isolated mouse primary adult cardiomyocytes (ACMs), we investigated the role of glucose, palmitate, ketone bodies, and BCAAs in mediating cell survival. Our results showed that myocyte upregulation of GRK2 promotes palmitate-induced cell death. Isotopologue labeling and mass spectrometry showed that the upregulation of GRK2 reduces ß-hydroxybutyryl CoA generation. Next, using isoproterenol (ISO), a non-selective ßAR-agonist, we determined mitochondrial function in mouse and human primary ACMs. Upregulation of GRK2 impaired ISO-mediated mitochondrial functional responses, which we propose is important for metabolic adaptations in pathological conditions. Increased cardiac levels of GRK2 reduced fatty acid-specific catabolic pathways and impaired ISO-stimulated mitochondrial function. Our data support the notion that GRK2 participates in bioenergetic remodeling and may be an important avenue for the development of novel pharmacological strategies in HF.

Novel inhibitors of phosphorylation independent activity of GRK2 modulate cAMP signaling.

G protein-coupled receptors kinase 2 (GRK2) plays a major role in receptor regulation and, as a consequence, in cell biology and physiology. GRK2-mediated receptor desensitization is performed by its kinase domain, which exerts receptor phosphorylation promoting G protein uncoupling and the cessation of signaling, and by its RGS homology (RH) domain, able to interrupt G protein signaling. Since GRK2 activity is exacerbated in several pathologies, many efforts to develop inhibitors have been conducted. Most of them were directed toward GRK2 kinase activity and showed encouraging results on in vitro systems and animal models. Nevertheless, limitations including unspecific effects or pharmacokinetics issues prevented them from advancing to clinical trials. Surprisingly, even though the RH domain demonstrated the ability to desensitize GPCRs, this domain has been less explored. Herein, we show in vitro activity of a series of compounds that, by inhibiting GRK2 RH domain, increase receptor cAMP response, avoid GRK2 translocation to the plasma membrane, inhibit coimmunoprecipitation of GRK2 with Gαs subunit of heterotrimeric G protein, and prevent receptor desensitization. Also, we preliminarily evaluated candidates' ADMET properties and observed suitable lipophilicity and cytotoxicity. These novel inhibitors of phosphorylation-independent actions of GRK2 might be useful in elucidating other RH domain roles and lay the foundation for the development of innovative pharmacologic therapy for diseases where GRK2 activity is exacerbated.

The ROS/GRK2/HIF-1α/NLRP3 Pathway Mediates Pyroptosis of Fibroblast-Like Synoviocytes and the Regulation of Monomer Derivatives of Paeoniflorin.

Hypoxia is an important factor in the development of synovitis in rheumatoid arthritis (RA). The previous study of the research group found that monomeric derivatives of paeoniflorin (MDP) can alleviate joint inflammation in adjuvant-induced arthritis (AA) rats by inhibiting macrophage pyroptosis. This study revealed increased levels of hypoxia-inducible factor- (HIF-) 1α and N-terminal p30 fragment of GSDMD (GSDMD-N) in fibroblast-like synoviocytes (FLS) of RA patients and AA rats, while MDP significantly inhibited their expression. Subsequently, FLS were exposed to a hypoxic environment or treated with cobalt ion in vitro. Western blot and immunofluorescence analysis showed increased expression of G protein-coupled receptor kinase 2 (GRK2), HIF-1α, nucleotide-binding oligomerization segment-like receptor family 3 (NLRP3), ASC, caspase-1, cleaved-caspase-1, and GSDMD-N. Electron microscopy revealed FLS pyroptosis after exposure in hypoxia. Next, corresponding shRNAs were transferred into FLS to knock down hypoxia-inducible factor- (HIF-) 1α, and in turn, NLRP3 and western blot results confirmed the same. The enhanced level of GSDMD was reversed under hypoxia by inhibiting NLRP3 expression. Knockdown and overexpression of GRK2 in FLS revealed GRK2 to be a positive regulator of HIF-1α. Levels of GRK2 and HIF-1α were inhibited by eliminating excess reactive oxygen species (ROS). Furthermore, MDP reduced FLS pyroptosis through targeted inhibition of GRK2 phosphorylation. According to these findings, hypoxia induces FLS pyroptosis through the ROS/GRK2/HIF-1α/NLRP3 pathway, while MDP regulates this pathway to reduce FLS pyroptosis.

The RAF Kinase Inhibitor Protein (RKIP): Good as Tumour Suppressor, Bad for the Heart.

The RAF kinase inhibitor protein, RKIP, is a dual inhibitor of the RAF1 kinase and the G protein-coupled receptor kinase 2, GRK2. By inhibition of the RAF1-MAPK (mitogen-activated protein kinase) pathway, RKIP acts as a beneficial tumour suppressor. By inhibition of GRK2, RKIP counteracts GRK2-mediated desensitisation of G protein-coupled receptor (GPCR) signalling. GRK2 inhibition is considered to be cardioprotective under conditions of exaggerated GRK2 activity such as heart failure. However, cardioprotective GRK2 inhibition and pro-survival RAF1-MAPK pathway inhibition counteract each other, because inhibition of the pro-survival RAF1-MAPK cascade is detrimental for the heart. Therefore, the question arises, what is the net effect of these apparently divergent functions of RKIP in vivo? The available data show that, on one hand, GRK2 inhibition promotes cardioprotective signalling in isolated cardiomyocytes. On the other hand, inhibition of the pro-survival RAF1-MAPK pathway by RKIP deteriorates cardiomyocyte viability. In agreement with cardiotoxic effects, endogenous RKIP promotes cardiac fibrosis under conditions of cardiac stress, and transgenic RKIP induces heart dysfunction. Supported by next-generation sequencing (NGS) data of the RKIP-induced cardiac transcriptome, this review provides an overview of different RKIP functions and explains how beneficial GRK2 inhibition can go awry by RAF1-MAPK pathway inhibition. Based on RKIP studies, requirements for the development of a cardioprotective GRK2 inhibitor are deduced.

Lycium barbarum polysaccharide antagonizes cardiomyocyte apoptosis by inhibiting the upregulation of GRK2 induced by I/R injury, and salvage mitochondrial fission/fusion imbalance and AKT/eNOS signaling.

Ischemia-reperfusion (I/R) injury is the main reason why infarct size continues to progress during the process of restoring myocardial perfusion, and it significantly increases the risk of death. At present, the therapeutic effects of clinically used drugs are limited. Therefore, it is particularly necessary to explore myocardial-protective agents that effectively prevent I/R injury. Lycium barbarum polysaccharide (LBP) is a water-soluble polysaccharide extracted from wolfberry fruit. In this study, we found that LBP limited myocardial infarct size, improved adverse remodeling, and reduced cell death and oxidative stress. G protein-coupled receptor kinase-2 (GRK2) is a key molecule involved in myocardial I/R injury. In vivo and in vitro experiments showed that LBP inhibited the upregulation of GRK2 expression induced by I/R injury, which was related to the antiapoptotic effect of LBP. In addition, we found that LBP partially restored I/R-induced mitochondrial fission/fusion imbalance, as well as levels of phosphorylated protein kinase B (p-AKT) and phosphorylated endothelial cell nitric oxide synthase (p-eNOS), and this restorative effect could be attenuated by overexpression of GRK2. Overall, our findings suggest that LBP antagonizes cardiomyocyte apoptosis by inhibiting the upregulation of GRK2 induced by I/R injury and saves mitochondrial fission/fusion imbalance and AKT/eNOS signaling. This study may provide new ideas for the study of I/R injury and the rational application of the herbal medicine LBP.

Heterotrimeric Gq proteins act as a switch for GRK5/6 selectivity underlying ß-arrestin transducer bias.

Signaling-biased ligands acting on G-protein-coupled receptors (GPCRs) differentially activate heterotrimeric G proteins and ß-arrestins. Although a wealth of structural knowledge about signaling bias at the GPCR level exists (preferential engagement of a specific transducer), little is known about the bias at the transducer level (different functions mediated by a single transducer), partly due to a poor understanding of GPCR kinase (GRK)-mediated GPCR phosphorylation. Here, we reveal a unique role of the Gq heterotrimer as a determinant for GRK-subtype selectivity that regulates subsequent ß-arrestin conformation and function. Using the angiotensin II (Ang II) type-1 receptor (AT1R), we show that ß-arrestin recruitment depends on both GRK2/3 and GRK5/6 upon binding of Ang II, but solely on GRK5/6 upon binding of the ß-arrestin-biased ligand TRV027. With pharmacological inhibition or genetic loss of Gq, GRK-subtype selectivity and ß-arrestin functionality by Ang II is shifted to those of TRV027. Single-molecule imaging identifies relocation of AT1R and GRK5, but not GRK2, to an immobile phase under the Gq-inactive, AT1R-stimulated conditions. These findings uncover a previously unappreciated Gq-regulated mechanism that encodes GRK-subtype selectivity and imparts distinct phosphorylation-barcodes directing downstream ß-arrestin functions.